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http://www.nature.com/nature/about/editors/

INDEX

S.

N.

Title Authors Page

No. 1 AWARENESS, ATTITUDE AND PRACTICE OF

PHARMACOVIGILANCE AMONG

HEALTHCARE PROFESSIONALS AND

STUDENTS IN A TERTIARY CARE

TEACHING HOSPITAL

Apurva Agrawal,

Parul Chaturvedi

1

2 INFLUENCE OF MICROBIAL INOCULATION

ON PLANT HEIGHT OF BLUE PINE (Pinus

Wallichiana) AND HIMALAYAN CYPRESS

(Cupressus Torulosa) UNDER NURSERY

CONDITIONS

Malik A. A,

Zargar M. Y,

Najar G. R,

Mir S. A ,

Agha F.

11

3

AN OBSERVATIONAL STUDY ON

SENSORY BASED OUTDOOR PLAY

PREFERENCES IN CHILDREN AGED

BETWEEN 3 - 12 YEARS : A PRELIMINARY

STUDY

K. R. Banumathe, Vineeta

I. Ram, Alzeena Pinto,

Samuel Jonathan

20

4

TESTING INDEX VOLATILITY OF INDIAN

STOCK MARKET IN THE CONTEXT OF

FOREIGN INSTITUTIONAL INVESTOR’S

INVESTMENT

Himanshu Barot,

V. K.Sapovadia

29

5

STUDY OF LIPID PEROXIDATION,

ANTIOXIDANT STATUS AND LIPID

PROFILE IN BREAST CANCER

G.S.R.Kedari,

G.S.R.Hareesh,

A. Saseekala

46

6 PSYCHOLOGICAL STRESS AND ORAL

DISEASES – A LINK?

Little Mahendra,

Ravi David Austin,

Jaideep Mahendra,

S. Senthil Kumar

52

7 AN IN VITRO SCREENING OF GROWTH

INHIBITORY POTENTIAL OF ALLIUM

SATIVUM TOWARDS SOME MICROBES OF

SPOILAGE AND HEALTH SIGNIFICANCE

Mamta Bhatia,

Alka Sharma

61

8 IN-VITRO ESTIMATION OF FREE RADICAL

SCAVENGING ACTIVITY OF FRUIT JUICES

BY DPPH ASSAY

Singh Meenu,

Pragzna Yanamadala,

Dharmadev Bommi

68

9 CAREER, JOB SATISFACTION AND ITS

EVALUATION METHODS

Muhammad Farzanjou

75

10

DRUG RESISTANCE PROFILE OF NEW PTB

PATIENTS WITH OR WITHOUT HIV

INFECTION

Niladri Sekhar Das

81

11 BACTERIOLOGICAL APPLICATIONS OF

QUANTUM DOTS

Nithish.U.S,

Sarah Sunitha

86

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INDEX

S.

N.

Title Authors Page

No.

12 EFFECT OF EARLY DEFOLIATION ON THE

NODULATION AND SOME AGRONOMIC TRAITS OF SOME SELECTED COWPEA

(Vigna Unguiculata l. Walp) VARIETIES

Ogundele O. E., Awosanya

A. O. 99

13

INTERESTING FACTS ON THYMUS GLAND

CHANGES - A REVIEW

Praful S. Jevoor, Mathada V.

Ravishankar, SandhyaDharwadkar,

Amit Magadum, Somashekhar Biradar

106

14

INDIGENOUS UNCOATED AND

HYDROXYAPATITE COATED

COMMERCIALLY PURE TITANIUM FOILS

FOR GUIDED BONE REGENERATION IN

DEFECT SITES OF IMPLANTS – AN IN VITRO STUDY

Ranzani R.,

Abby Abraham,

Chakravarthy R., Lakshmi S.

112

15

OCCUPATIONAL AWARENESS OF RURAL

WOMEN WORKERS IN HEN POULTRY

FARM, COIMBATORE DISTRICT, SOUTH INDIA

Sharmila Banu A,

Gunasekaran C, SelvaKumar

V, Mohana P, Kandappan K, Elanchezhian

M

121

16 FUSION OF FIRST AND SECOND

THORACIC RIB NEAR STERNAL END

Londhe Shashikala, Kori

Rohini,

Panjakash Samreen

127

17 CONSTRUCTION OF THE MATHEMATICAL

MODEL FOR EVALUATION AND

FORECAST OF INTERURBAN PASSENGERS TRAVEL

Shkelqim Gjevori

130

18 INFECTION RISK CONTROL IN

“COMPUTER RADIOGRAPHY

IMAGING PLATE” IN DIAGNOSTIC

RADIOLOGY DEPARTMENT

Suresh sukumar,

Sushil Yadav

139

19 STRESS ANALYSIS OF SPUR GEAR USING

FINITE ELEMENT METHOD – A REVIEW

Sushil Kumar Tiwari,

Upendra Kumar Joshi

144

20 FORMULATION AND EVALUATION OF

XANTHAN GUM BASED FLOATING TABLET OF TRAMADOL

HYDROCHLORIDE

Somnath Patil,

Swati Jagdale, Shailendra Kela, Varsha Divekar

153

21 VIBRATION THRESHOLD OF UPPER LIMB

DURING ULNT1 IN INDIVIDUALS WITH TYPE II DIABETES MELLITUS AND NON

DIABETIC INDIVIDUALS

Mamta Mohan,

Ravi Shankar Reddy, Ganesh B. M.

162

22 CARDIOVASCULAR CO-MORBIDITY AND ROLE OF EXERCISE IN RHEUMATOID

ARTHRITIS: A REVIEW

Shahnawaz Anwer, Ameed Equebal

168

23 NON-HEALING SKIN ULCER IN HIV /

TUBERCULOSIS CO-INFECTION: A CASE

REPORT

Monali N. Rajurkar, Silpi

Basak

175

24 GROWTH AND CHARACTERIZATION OF

PICRIC ACID MIXED ZTS SINGLE CRYSTALS

N. Balasundari,

P. Selvarajan, S. Lincy Mary Ponmani,

D. Jencylin

182

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Apurva Agrawal et al Awareness, attitude and practice of Pharmacovigilance among healthcare professionals and

students in a tertiary care teaching hospital

Int J Cur Res Rev, Sept 2012 / Vol 04 (17) , Page 1

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research

Received on:28/06/12

Revised on:15/07/12

Accepted on:28/08/12

AWARENESS, ATTITUDE AND PRACTICE OF PHARMACOVIGILANCE

AMONG HEALTHCARE PROFESSIONALS AND STUDENTS IN A TERTIARY

CARE TEACHING HOSPITAL

Apurva Agrawal1, Parul Chaturvedi

2

1Department of Pharmacology, Geetanjali Medical College, Udaipur, Rajasthan.

E-mail of Corresponding Author: [email protected]

ABSTRACT

Aims: To find out the level of awareness and attitude towards pharmacovigilance and extent of

ADR reporting in healthcare professionals & medical students.

Materials and method: A total of 799 participants including healthcare professionals and students

were asked to fill a predesigned questionnaire. It consisted of questions regarding awareness,

attitude & practice of pharmacovigilance. Data collected were analyzed using relevant statistical

tests. Awareness between healthcare professionals & students was compared using chi square

test.

Results: 70.46% of participants responded to the questionnaire. 22% of doctors and 37% of

nurses had reported ADR to any authority in last 2 years. Lack of awareness about the ADR

reporting system was the most common reason for non-reporting. Majority of healthcare

professionals and students considered ADR reporting as very important and recommended active

involvement of pharmacovigilance in medical curriculum.

Conclusion: Overall level of awareness was low both among healthcare professionals and

students. There is a great need to increase the awareness and improve the attitude of healthcare

professionals and students towards pharmacovigilance and its national programme. Regular

training sessions and awareness campaigns need to be conducted. Pharmacovigilance should be

included in the undergraduate training of MBBS, pharmacy, nursing and physiotherapy students.

Key Words: ADR reporting, healthcare professionals, medical students, Pharmacovigilance

INTRODUCTION

Pharmacovigilance as described by WHO is

detection, assessment, understanding and

prevention of adverse effects or any other drug

related problem.[1]

Adverse drug reactions

(ADRs) are associated with significant

morbidity and mortality, and are an important

cause of hospitalizations.[2]

Studies have

shown ADRs as fourth major cause of death in

USA, but such data are lacking in India.[3]

As

health care professionals are the first one who

are in contact with patients taking drugs,

spontaneous reporting by them is an effective

way to generate early signals of ADRs. It is

the most practical way to detect rare adverse

events, adverse events caused by prolonged

use of drugs and many drug-drug interactions. [4]

Thus, awareness among healthcare workers




and their attitude towards pharmacovigilance

are important determinants of ADR reporting

rate.

WHO has developed a system for reporting of

ADRs by establishment of International Drug

Monitoring Programme, coordinated by

Uppsala Monitoring Centre, Sweden. In India

also National Pharmacovigilance Programme

(NPP) was started in 2004.[5]

This programme

was relaunched in 2010 as Pharmacovigilance

Programme of India (PvPI), and is now

coordinated by the Indian Pharmacopoeia

Commission, Ghaziabad.[6]

Still

pharmacovigilance is in its infancy phase in

India and under reporting is a major problem.

Studies done in other countries also reveal

under reporting of ADRs.[7-10]

Lack of

awareness among health care professionals is

one of the reasons for under reporting. Thus,

to improve the ADR reporting rate, it is

important to improve the awareness, attitude

and practices of the healthcare professionals

regarding pharmacovigilance. The best time to

do this is during undergraduate training of

students of MBBS, pharmacy and nursing.

This will help by developing a culture of ADR

reporting in healthcare workers. Though

studies reporting the level of awareness &

practices of pharmacovigilance have been

done in other countries, [7, 8, 11, 12]

very few

studies have focused this aspect in India, and

during literature search no such study was

found from Rajasthan. Further most of the

studies have included healthcare workers, but

studies on awareness among undergraduate

students are limited. [9]

Moreover, there was

no peripheral centre of NPP in Rajasthan.

Recently a new ADR Monitoring Center

under PvPI has been established in a Medical

College of Rajasthan. [6]

Hence, the present

study was conducted to develop a baseline

data of awareness, attitude and practice of

pharmacovigilance in health care

professionals and medical students in a

Tertiary Healthcare Teaching Hospital in

Rajasthan.

MATERIALS AND METHODS

This was a cross sectional, observational,

questionnaire based study conducted in a

Tertiary Care Teaching Hospital of Rajasthan.

Duration of study was of 2 months from 1st

May 2011 to 30th

June 2011. Total 799

participants were approached, which included

health care professionals and medical

students. All healthcare professionals working

in the Hospital including clinicians,

pharmacists and nursing staff were included.

Medical students of MBBS, students of

Nursing College, Pharmacy College and

Physiotherapy College attached to the

Hospital were included in the study. All those

who denied participation in the study and

students who have not been introduced to

Pharmacology (e.g. students who were in 1st

year of their course) were excluded from the

study.

Procedure:

Approval from Institutional Ethics Committee

was taken before starting the study. All health

care professionals and students were contacted

personally. The study was explained to them

in brief. A predesigned questionnaire was

provided to them which consisted of ten

questions for assessment of awareness,

attitude and practice of pharmacovigilance and

ADR reporting. Consent of participants was

taken in written informed consent form. They

were asked to fill the questionnaire without




any assistance. It required approximately 10 to

15 minutes filling the questionnaire.

Out of total 799 participants who were asked

to fill the questionnaire, 565 (70.7%)

responded to the questionnaire. There were 11

pharmacists, only 2 responded. Therefore,

pharmacists were not included for analysis.

Thus total 563 questionnaires were analyzed.

All questionnaires were completely filled.

Data collected were analyzed and percentages

were calculated. Awareness between

healthcare professionals and students was

compared using chi square test. Practice of

ADR reporting and reasons for non-reporting

were assessed only for healthcare

professionals as Pharmacovigilance

Programme of India (PvPI) mentions only

healthcare professionals who can report the

ADR. [6]

RESULTS

Among healthcare professionals, 73%

(73/100) doctors and 57% (115/202) nurses

responded to the questionnaire. Among

students, 72.4% (210/290) MBBS students,

96% (100/104) pharmacy students, 61%

(33/54) nursing students and 84.2% (32/38)

physiotherapy students responded to the

questionnaire. 65.4% of healthcare

professionals (83.5% of doctors and 53.9% of

nurses), while 83.5% of students have ever

heard of the term “Pharmacovigilance”.

(Table 1) Awareness about this term was

different between the two groups with

statistical significance (p value < 0.001).

41.5% of healthcare professionals (58.9% of

doctors and 30.43%of nurses) and 39% of

students were able to define

Pharmacovigilance. (Table 2) No statistically

significant difference between the two groups

(p value > 0.005) was found. 39.4% of

healthcare professionals (43.8% of doctors

and 36.5% of nurses) and 31.7% of students

were aware of National Programme for

Pharmacovigilance, (Table 2) with no

statistically significant difference (p value >

0.005).

53.7% of healthcare professionals (60.3% of

doctors and 49.6% of nurses) and 57.1% of

students had the knowledge of ADR reporting.

(Table 2) No statistically significant difference

(p value > 0.005) was found. The awareness

that any healthcare professional can report

ADR was present in 43.1% of healthcare

professionals (67.1% of doctors and 27.8% of

nurses) and 48.3% of students, (Table2) with

no statistically significant difference (p value

> 0.005). 72.3% of healthcare professionals

(91.8% of doctors and 60% of nurses) and

60.8% of students were aware how to report

ADR. (Table 2) There was statistically

significant difference between two groups

with p value < 0.01. 78.1% of doctors and

62.6% of nursing staff have not reported any

ADR to any authority in last two years. Only

21.9% of doctors and 37.4% of nursing staff

have reported any ADR to any authority in

last two years. (Figure 1)

About 56% of doctors and 46% of nurses

reported lack of awareness about the reporting

system as the major cause of non-reporting of

ADR. 20.5% of doctors and 23.5% of nurses

were not sure about the reason for non-

reporting. (Table 3) 68% of healthcare

professionals (75% of doctors and 63% of

nurses) and 76.8% of students opined that

ADR reporting is very important. Only 2.6%

of healthcare professionals and 1.6% of

students considered it as waste of time.




(Figure2) 84% of healthcare professionals

(97% of doctors and 75.6% of nurses) and

84.3% of students accepted that

pharmacovigilance should be an active part of

medical curriculum. Only 5.8% of healthcare

professionals and 6.4% of students denied.

(Figure 3)

DISCUSSION

The overall awareness about

pharmacovigilance and ADR reporting was

low, both in healthcare professionals and

students. Though majority of students (83.5%)

had heard the term “Pharmacovigilance”, less

than half (39%) could define it. These students

are told about it in pharmacology but not

actively discussed. This shows that there is a

need to stress on pharmacovigilance during

undergraduate teaching. On the other hand

65% of healthcare professionals had heard this

term which is significantly lower than that of

students, but only 41% could define it. More

doctors (59%) were able to define than

nursing staff (30%). Healthcare professionals

are not exposed to pharmacology after II year

of undergraduate course, which may be

responsible for their low awareness about the

term “Pharmacovigilance”. A Nigerian study

done on community pharmacists also reported

similar results in which 55% of responders

were aware of the term “pharmacovigilance”,

but only 18% could define it. [8]

More alarming was the lack of awareness

about the national programme. More than half

of healthcare professionals and students were

not aware about the national programme

which is running since 2004 in India. If they

do not know about it, how could they be

expected to participate in it? To make the

national programme successful, it is important

to aware the healthcare professionals about it

and how it functions. This can be done by

awareness campaigns, information leaflets etc.

About half of healthcare professionals (54%)

and students (57%) were aware about ADR

reporting. The awareness that every healthcare

professional including doctor, nurse,

pharmacist and physiotherapist can report

ADR was very low, which is in accordance

with the findings of Gupta P et al.[13]

This

awareness was minimum among nurses, both

nursing staff (28%) as well as nursing students

(21%). Active involvement of paramedical

staff in spontaneous reporting is very

important, since they are in close contact with

the patients and for a longer duration as

compared to doctors.

The ultimate goal of Pharmacovigilance is that

the benefits of medicine use outweighs the

risks and thus safeguard the health of patients.

Spontaneous ADR reporting by healthcare

professionals can play an important role to

achieve this goal. In present study only about

22% of doctors and 37% of nurses had

reported ADR to any authority in last two

years. Other studies have also reported such

low levels of ADR reporting. [7-9]

Under-

reporting is a major and worldwide problem

associated with spontaneous reporting system.

A systematic review by Hazell L et al has

stated significant and widespread under-

reporting of ADRs to spontaneous reporting

systems including serious or severe ADRs. [10]

Aggressive interventions are required to

encourage the healthcare professionals for

ADR reporting. They need to be informed that

why ADR reporting is necessary and how it

can help in ensuring safe and rational use of




medicines. Prescribers can be encouraged by

providing feedback on their ADR reports,

discussion on ADR reports in academic

meetings and publishing bulletins on ADRs.

Oreagba et al have suggested that

remuneration for ADR reporting may increase

the reporting rate. [8]

Lack of awareness about the reporting system

was the most common reason stated by

responders (56% of doctors & 46% of nurses)

for non-reporting. This finding is in

accordance with that reported by other studies, [8, 9, 13]

and is understandable as there is no

well established reporting system in this

hospital and healthcare professionals are not

well aware about the national programme.

Thus to improve the reporting rate, ADR

monitoring centers should be established in all

healthcare institutions, at least at tertiary care

level. The recent Pharmacovigilance

Programme of India targets to establish such

centre in most of the Medical Colleges of

India in coming years. [6]

This may help to

improve the existing scenario. Further to

increase the awareness about ADR reporting

system, regular training sessions and

awareness campaigns should be conducted

which may help in improving ADR reporting

rate. Ramesh M et al have reported 63%

increase in ADR reporting in one year after

the launch of continuous awareness campaign. [14]

As mentioned by other studies lack of

time, complex procedure and non significant

ADR, were among the other reasons for non-

reporting in present study also. [8, 13]

Both healthcare professionals and students had

positive attitude toward ADR reporting and

majority of them considered it as very

important. Only 2.6% of healthcare

professionals and 1.6% of students considered

it as waste of time. Further majority of

responders (84%) opined that

pharmacovigilance should be taught as an

active part of medical curriculum. As medical

students are future healthcare professionals,

there is a need to actively train them, so that

pharmacovigilance becomes a part of their

medical practice. As recommended by Rehan

et al knowledge and awareness of

pharmacovigilance among prescribers can be

improved by a reinforcement training

programme at the commencement of

internship and thereafter through continuous

education programmes. [9]

The major limitation of the present study is

that the study findings could not be applied to

wider medical community as the study was

restricted to hospital setup. Therefore it is

recommended that several studies of similar

kind especially in community setup need to be

conducted to know the awareness and attitude

of healthcare professionals in community and

their practice of pharmacovigilance. This will

help to find out the present status and to

develop strategies to improve the ADR

reporting system in India.

CONCLUSION

The results of present study show lack of

awareness about pharmacovigilance in

healthcare professionals and students. ADR

reporting rate is also very low among

healthcare professionals. There is a great need

to create awareness and promote ADR

reporting among healthcare professionals.

Regular training sessions to stress the

importance of ADR reporting and the

functioning of reporting system are required.

Further awareness about the national




programme need to be increased, this will

ensure greater participation from healthcare

professionals and success of such

programmes. Pharmacovigilance should be a

part of undergraduate curriculum of not only

MBBS students but also of students of

nursing, physiotherapy and pharmacy. As

these students are future healthcare

professionals, this will help in developing a

culture of ADR reporting in the country.

ACKNOWLEDGEMENT:

We acknowledge the ICMR (Indian Council

of Medical Research), as this study was

conducted under STS 2011 (short term

studentship).

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4.

Table No. 1: Frequency of responders who ever heard the term “Pharmacovigilance”

S. No. Group Subgroup (n) Ever heard

‘Pharmacovigilance’

Percentage

1. Healthcare

Professionals

Doctors (73) 61 83.56

Nurses (115) 62 53.9

2. Students MBBS (210) 198 94.28

Nursing (33) 19 57.57

Pharmacy (100) 68 68

Physiotherapy (32) 28 87.5

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Iribhogbe%20OI%22%5BAuthor%5D




Table No. 2: Level of awareness about Pharmacovigilance and ADR reporting

S.

No.

Awareness Healthcare

Professionals (%)

Students (%)

Doctor

s

(n=73)

Nurses

(n=115)

MBB

S

(n=21

0)

Nursin

g

(n=33)

Pharmac

y

(n=100)

Physiotherap

y

(n=32)

1. What is

Pharmacovigilance

58.9 30.4 48.6 18.2 23 46.9

2. National Programme 43.8 36.5 31.4 27.3 35 28.1

3. ADR Reporting 60.3 49.6 59 42.4 56 62.5

4. Who can report 67.1 27.8 60.5 21.2 33 43.7

5. How to report ADR 91.8 60 63.3 36.4 63 62.5

Table No. 3: Reasons for non-reporting of ADRs

S. No. Reasons Healthcare Professionals (%)

Doctors (n=73) Nurses (n=115)

1. Lack of awareness 56.2 46.1

2. Lack of time 8.2 8.7

3. Complex procedure 8.2 9.5

4. No significance 6.9 12.2

5. Not sure 20.5 23.5




Figure No. 1: Frequency of ADR reported by Healthcare Professionals in last 2 years

Figure No. 2: Importance of ADR reporting in views of Healthcare Professionals & Students




Figure No. 3: Opinion of responders regarding inclusion of Pharmacovigilance in medical

curriculum

Malik A. A. et al INFLUENCE OF MICROBIAL INOCULATION ON PLANT HEIGHT OF BLUE PINE (Pinus

Wallichiana) AND HIMALAYAN CYPRESS (Cupressus Torulosa) UNDER NURSERY CONDITIONS


IJCRR

Vol 04 issue 17

Section: General Science

Category: Research


Revised on:13/07/12


INFLUENCE OF MICROBIAL INOCULATION ON PLANT HEIGHT

OF BLUE PINE (Pinus Wallichiana) AND HIMALAYAN CYPRESS

(Cupressus Torulosa) UNDER NURSERY CONDITIONS

Malik A . A1, Zargar M.Y

2, Najar G . R

3, Mir S. A

4 , Agha F.

5

1 Division of Environmental Sciences, Skuast-Kashmir, India

2 Regional Research Station (RRS ),Wadura,Kashmir, India

3 Division of Soil Science ( FAO ) Wadura,Kashmir, India

4 Division of Agri-Stat, Skuast-Kashmir, India

5 Division of Agronomy, Skuast-Kashmir, India

ABSTRACT

A pot experiment was carried out during 2009 -2010 to study the impact of microbial inoculants

on plant height of Blue pine (Pinus wallichiana A.B. Jackson) and Himalayan cypress

(Cupressus torulosa Don) under nursery conditions. The experiment was laid in Completely

Randomized Design with three replications which comprised forty-two treatment combinations

of seven inoculants (Azotobacter sp., Azospirillum sp., Pseudomonas fluorescens, Bacillus

subtilis, Pisolithus tinctorius, Laccaria laccata and control). The growth character viz., plant

height at various intervals responded significantly to all the microbial inoculants. Maximum

plant height was recorded in Himalayan cypress (38.72 cm) as compared to Blue pine seedlings

(24.78 cm). Among microbial inoculants the two ectomycorrhizae viz., Pisolithus tinctorius and

Laccaria laccata gave best results than rest of the inoculants. It was followed by Azotobacter sp.,

Azospirillum sp., Pseudomonas fluorescens and Bacillus subtillis. Thus the two treatments viz.,

Pisolithus tinctorius and Laccaria laccata proved to be the best for the studied parameter viz.,

plant height of both the species.

Key words : Pinus wallichiana, Cupressus torulosa, Microbial inoculation, Azotobacter,

Azospirillum, Pseudomonas, Bacillus, Pisolithus, Laccaria

INTRODUCTION

The geographical area of Jammu and Kashmir

is 1,01,387 km2 excluding the area under

Pakistan and China with recorded forest area

of 20,230 km2 inside line of control. However,

recorded forest cover as per Forest Survey of

India is only 16,309 km2 (Anonymous,

2009a). Commercial forests of the state

occupy only 8,26,939 ha (Anonymous, 2005).

As per inventory records, very dense forests in

Jammu and Kashmir occupy 2,95,800 ha,

moderately dense 6,50,700 ha, open forests

have now spread over 6,84,400 ha and rest of

the recorded forests have been engulfed by

blanks and scrubs (Anonymous, 2009b). The




growing stock of our commercial forests is

132.9 million m3 with average annual yield of

1.65 m3 ha

-1 (Anonymous, 2009b). With this

productivity annual yield of timber from

commercial forest area alone must be 27.25

million cubic feet. Contrary to this fact, we

presently import timber. Due to timber mining

more than 60 per cent of our demarcated

forests have been declared as

uncommercial/degraded (Anonymous, 2005).

Natural regeneration does not practically take

place in forests where crown density is less

than 40 per cent. Relying on natural

succession, it will take us hundreds of years to

regenerate the degraded forests to climax

stage with species like Pinus wallichiana

Jackson (kail), Cedrus deodara (Roxb.), G.

Don (Deodar), Abies pindrow Spach (silver

fir), Picea smithiana Wall. (spruce) and

Cupressus torulosa Don (Himalayan cypress)

which dominate vegetation of our forests.To

meet the huge demand and supply of timber,

fuelwood and firewood, raising of blue pine

and Himalayan cypress forests on degraded

forest patches can be a good and viable option

in future. The indiscriminate use of inorganic

fertilizers and pesticides is neither

environmentally safe nor economically

feasible. There is pressing demand for

microbial inoculants for quality seedling

production in nursery and also the

establishment of plantation to increase the

forest productivity. Bioinoculants are cost

effective, ecofriendly, cheaper and renewable

sources of plant nutrients and play a vital role

in maintaining long-term soil fertility and

sustainability. Thus, to meet the challenges

like poor regeneration, deforestation and

spread of wastelands, introduction of

microbial inoculants at the nursery stage of

forest trees has become inevitable. Although

various aspects of mycorrhizal impact of the

forest trees have been studied, no work has

been done on the impact of other microbial

inoculants on the regeneration of forest trees.

Therefore, the present study was undertaken to

determine the role of microbial inoculation on

growth attribute viz.,plant height of Blue pine

and Himalayan cypress under nursery

conditions.


The present investigations were undertaken at

the Forest Nursery of Department of Forestry,

Faculty of Agriculture and Regional Research

Station, SKUAST-Kashmir, Wadura, Sopore

during 2009-2010.Microbial inoculants

isolated from rhizosphere of blue pine and

Himalayan cypress forest stands were used in

the studies.

Mass production of microbial inoculants

The two free living aerobic nitrogen fixing

bacteria viz., Azotobacter sp. and Azospirillum

sp. were mass cultured using nutrient medium

enriched with glucose and peptone. Plant

growth promoting rhizobacteria (PGPR) viz.,

Pseudomonas fluorescens and Bacillus subtilis

were mass propagated in King’s B nutrient

broth. The two ectomycorrhizae viz.,

Pisolithus tinctorius and Laccaria laccata

were mass multiplied in Melin Norkran’s

nutrient broth and Potato Dextrose Agar,

respectively.

Field operations

For the microbial inoculation, one year old

seedlings of blue pine and Himalayan cypress

of uniform heights and collar diameter

growing in polyethylene bags (9 x 7)

containing 1 kg potting material of soil and




sand mixture in the ratio of 1:1 were selected.

Microbial inoculation

For inoculation, the different broth cultures of

N-fixers, P-solubilizers and ectomycorrhizal

inoculants isolated from local forest stands

were applied to the potting material (25

ml/seedling) in the month of March, 2010,

without disturbing the root system of the

seedlings.

Nursery operations

The seedlings were irrigated with rose-cans as

and when needed and maintained virtually

weed free by manual weeding

Plant growth measurement

The growth parameter viz., plant height (cm)

of both the species were measured by using

measuring tape at an interval of 2 months upto

12 months.The plant height of the seedlings at

the initial stage of the experiment were also

recorded.

Statistical analysis

The data was statistically analysed by using

O.P Stat software developed by Haryana

Agriculture University, Hisar.

Results and Discussion

Plant height

The data on impact of various microbial

inoculants on plant height of Blue pine

seedlings indicates that mean plant height was

significantly more in response to various

treatments as compared to control (Table-

1;Fig 1, Plate-1). Azotobacter and

Azospirillum inoculation exhibited 37.17 and

36.83 per cent more plant height over control.

Similarly Pseudomonas flourescens and

Bacillus subtilis inoculation resulted in 35.56

and 33.91 per cent more plant height while as

the inoculation with two ectomycorrhizal

fungi viz., Pisolithus tinctorius and Laccaria

laccata resulted in 42.97 and 40.77 per cent

more plant height as compared to control

respectively. However, the application of

Pisolithus tinctorius showed maximum

increase (42.97%) in plant height over control,

thus proved superior over all the individual

inoculants. Moreover, there was an increasing

trend in plant height from April to October

and from December onwards till February

there was a slight increase. The interactions

between inocula and months were significant

till October and from December to February it

was non-significant. Perusal of the data

presented in Table-2,Fig 2,Plate-1 shows that

the application of various microbial inoculants

significantly enhanced the mean plant height

of the Himalayan cypress seedlings as

compared to control. Amongst various

microbial inoculants, Pisolithus tinctorius

resulted in maximum increase in plant height

over control (38.27 %). It was followed by

Laccaria laccata (35.66%), Azotobacter sp.

(28.29%), Azospirillum sp. (25.91%),

Pseudomonas fluorescens (21.76%) and

Bacillus subtilis (19.36%), respectively.

Treatment of seedlings with ectomycorrhizal

fungi viz., Pisolithus tinctorius was

significantly superior over all other

treatments. Plant height revealed a significant

increase from April to October and from

October on wards till February there was a

slight increase. Moreover, the interactions

between inocula and month’s were significant

till October and thereafter it was non-

significant. The increase in shoot height by P.

tinctorius and L. laccata could be attributed to

the production of growth promoting




substances like auxins (Dehn, 1982) and

enhancement of water absorption and nutrient

mobilization (Dar et al., 1997) by vastly

increased surface area network of the fungal

mycelia (Myer, 1992). In case of Azotobacter

and Azospirillum sp. inoculation the increase

in shoot height could be ascribed to nitrogen

fixing ability, synthesis of growth promoting

substances like cytokinens, gibberellins,

auxins (Reynders and Vlassak, 1979;

Hartmann et al., 1983; Jain and Patriquin,

1985) and production of antifungal antibiotics

(Chahal and Chahal, 1988). However, increase

in shoot height by inoculation with

Pseudomonas fluorescens and Bacillus subtilis

could be through iron chelating siderophores

(Schippers, 1988) by releasing

phytohormones, solubilizing P and reduction

in population of deleterious microorganisms

(Weller, 1988). Further our findings are in

close conformity with the results of Oh and

Park (1989), Jeffries and Dodd (1991),

Natarajan et al. (1995) who reported that P.

tinctorius and L. laccata inoculation resulted

in enhancement of plant height of Acaccia

nilotica, Quercus serrata, Eucalyptus

camaldulensis and E. deglupta seedlings

respectively. similarly, the enhancement in

plant height with respect to Azotobacter and

Azospirillum sp. has also been reported in

Quercus serrata (Pandey et al., 1986) in peach

(Awasthi et al., 1996). Moreover, the

inoculation of clover plants with

Pseudomonas putida has also been reported to

enhance the plant height (Meyer and

Linderman, 1986). However, the maximum

increase in shoot height of Himalayan cypress

seedlings lies in the fact that Himalayan

cypress being a fast growing species, has got

an efficient root system as compared to kail

which is comparatively a slow growing

species. Moreover, the gradual decline in plant

height of both the species in the later half of

study period could be due to below freezing

soil temperatures and short growing season of

conifers.

References

1. Anonymous, 2005. Handbook of

Forest Statistics. Jammu and Kashmir

Government; Forest Department,

Srinagar, J&K, pp 10.

2. Anonymous, 2009a. Forest Survey of

India. Indian State of Forest Report

2009, 5 : 44.

3. Anonymous, 2009b. Digest of Forest

Statistics. Jammu and Kashmir

Government; Forest

4. Department, Srinagar, Jammu and

Kashmir.

5. Awasthi, R.P., Godara, R.K. and

Kainth, N.S. 1996. Interaction effect of

VA-mycorrhizae

6. and Azotobacter inoculation on peach

seedlings. Indian Journal of

Horticulture 53(1) : 8-13.

7. Chahal, P.P.K. and Chahal, V.P.S.

1988. Biological control of root-knot

nematode of brinjal

8. with Azotobacter chroococcum. In :

Advances in Plant Nematology [Eds.

M.A. Maqbool,

9. A.M. Golden, A.M. Gaffar and A.

Krusberg]. Research Centre,

University of Karachi, pp. 257-263.

10. Dar. G.H., Zargar, M.Y. and Beigh,

G.M. 1997. Biocontrol of Fusarium




root rot in common bean (Phaseolus

vulgaris L.) by using symbiotic

Glomus masseae and Rhizobium

leguminosarum. Microbial Ecology 34

: 74-80.

11. Dehn, H.W. 1982. Interaction between

vasicular-arbuscular mycorrhizal fungi

and plant pathogens. Phytopathology

72 : 115-119.

12. Hartmann, A., Singh, M. and

Klingmuller, W. 1983. Isolation and

characterization of Azospirillum

mutants excreating high amounts of

indoleacetic acid. Canadian Journal of

Microbiology 29 : 916-923.

13. Jain, D.K. and Patriquin, D.G. 1985.

Characterization of a substance

produced by Azospirillum which

causes branching of wheat root hairs.

Canadian Journal of Microbiology 31

: 206-210.

14. Jeffries, P. and Dodd, J.C. 1991. The

use of mycorrhizal inoculants in

forestry and agriculture. In : Handbook

of Applied Mycology. [Eds D.K. Arora,

Bharat Raj, K.G. Mukerji and G.R.

Knudsen]. Marcel Dekker, New York,

USA, pp. 155-185.

15. Meyer, J.R. and Liderman, R.G. 1986.

Response of subterraneum clover to

dual inoculation with VAM fungi and

a plant growth promoting bacterium

Pseudomonas putida. Soil Biology and

Biochemistry 18 : 185-190.

16. Myer, M. 1992. Mycorrhizas, their

use as biofertilziers. The horticulturists

2 : 8-12.

17. Natarajan, K., Nagarajan, G. and

Reddy, M.S. 1995. In vitro

mycorrhization and growth response of

Acacia nilotica seedlings by

inoculation with ectomycorrhizal

fungi. Indian Journal of Microbiology

35 : 35-38.

18. Oh, K.I. and Park, W.S. 1989. The

effect of ectomycorrhizae and nitrogen

levels on the

19. growth of Quercus serrata seedlings.

Journal of Korean Forestry Society 78

: 160-167

20. Pandey, P.K., Bahl, R.K. and Rao,

P.R.T. 1986. Growth stimulating

effects of nitrogen fixing bacteria

(biofertilizer) on oak seedlings. Indian

Forester 112(1) : 75-79.

21. Reynders, L. and Vlassak, K. 1979.

Conversion of tryptophan to

indoleacetic acid by Azospirillum

brasiliense. Soil Biology and

Biochemistry 11 : 547-548.

22. Schippers, B. 1988. Biological control

of pathogens with rhizobacteria.

Phiols. Trans. R. Soc. Land. B. 318 :

283-292.

23. Weller, D.M. 1988. Biological control

of soil borne plant pathogens in the

rhizosphere with bacteria. Annual

Review of Phytopathology 26 : 379-

407.




Table-1 : Impact of microbial inoculation on plant height (cm) of Blue pine (Pinus

wallichiana A.B. Jackson) at nursery stage

Treatment 2009 2010 Mean

April June August October December February

Control 11.30 12.70 14.82 15.31 15.33 15.33 14.13

Azotobacter sp. 14.95 18.97 22.98 26.90 22.94 22.94 22.49

Azospirillum sp. 14.20 18.35 22.42 26.40 26.43 26.43 22.37

Pseudomonas fluorescens

13.90 17.95 21.97 25.92 25.94 25.94 21.93

Bacillus subtilis 13.30 17.42 21.50 25.35 25.37 25.37 21.38

Pisolithus tinctorius

16.40 20.82 24.92 28.85 28.87 28.87 24.78

Laccaria laccata 15.87 19.90 23.94 27.82 27.84 27.84 23.86

Mean 14.27 18.01 21.79 25.22 25.24 25.24

Treatment (T) Month (M) T x M

CD (p 0.05) 0.013 0.012 0.032

SEm 0.047 0.043 0.011

Initial plant height = 9.70 cm




Fig-1 : Diagram showing treatment effects on plant height of cypress

Table-2 : Impact of microbial inoculation on plant height (cm) of Himalayan cypress

(Cupressus torulosa Don) at nursery stage

Treatment 2009 2010 Mean

April June August October December February

Control 18.33 21.40 24.26 26.46 26.48 26.48 23.90

Azotobacter sp. 22.66 27.23 32.30 39.26 39.28 39.28 33.33

Azospirillum sp. 21.75 26.17 31.10 38.18 38.19 38.19 32.26

Pseudomonas fluorescens

20.58 25.10 29.20 36.13 36.15 36.15 30.55

Bacillus subtilis 19.58 24.23 28.43 35.20 35.21 35.21 29.64

Pisolithus tinctorius

24.40 31.06 38.13 46.23 46.25 46.25 38.72

Laccaria laccata 23.15 30.06 36.96 44.23 44.25 44.27 37.15

Mean 21.49 26.46 31.48 37.95 37.97 37.97

Treatment (T) Month (M) T x M

CD (p 0.05) 0.067 0.062 0.016

SEm 0.024 0.022 0.058

Initial plant height = 17.00 cm




Fig-2 : Diagram showing treatment effects on plant height of blue pine

ControlBSPSASAZLLPT

Blue pine inoculated seedlings




ControlBSPSASAZLLPT

Himalayan cypress inoculated seedlings

Plate-1 :Growth of Pinus wallichiana and Cupress torulosa seedlings in response to

microbial inoculations at nursery stage

PT = Pisolithus tinctorius; LL = Laccaria laccata; AZ = Azotobacter sp.;

AS = Azospirillum sp.; PS = Pseudomonas fluorescens; BS = Bacillus subtilis

K. R. Banumathe et al AN OBSERVATIONAL STUDY ON SENSORY BASED OUTDOOR PLAY PREFERENCES IN

CHILDREN AGED BETWEEN 3 - 12 YEARS : A PRELIMINARY STUDY

Int J Cur Res Rev, Sept 2012 / Vol 04 (17) ,

Page 20

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research


Revised on:15/07/12


AN OBSERVATIONAL STUDY ON SENSORY BASED

OUTDOOR PLAY PREFERENCES IN CHILDREN AGED

BETWEEN 3 - 12 YEARS : A PRELIMINARY STUDY 1K. R. Banumathe,

2Vineeta I. Ram,

3Alzeena Pinto,

4Samuel Jonathan

1Assistant Professor, Department of Occupational Therapy, Manipal

College of Allied Health Sciences, Manipal University, Manipal, KA,

India. 2,3,4

Occupational Therapist, Manipal, KA, India.

E-mail of corresponding authors: [email protected]

ABSTRACT

Aim: To find sensory based outdoor play preferences of children between the ages 3 -12 years.

Method: 90 normal children were observed by 3 raters for 30 minutes minimum, while engaged

in free play at outdoor playgrounds. Play preferences were observed for the affinity towards

certain sensory components; tactile, auditory, vestibular and proprioception. The most

observable sensory based play preferences were noted for quality and frequency and the children

were scored on a 7 point Likert scale. The data was then grouped by age into 3 groups, 1, 2 and

3; 3.1 to 6 years, 7.1 to 9 years and 10.1 to 12 years respectively and analyzed using SPSS,

version 16. Results: On analysis of data between the groups showed that there was significant

difference at p<0.05 levels for tactile, auditory and proprioceptive except vestibular based play

preferences at p>0.05 levels. Auditory based play preferences showed a significant difference at

p<0.05 level to other preferences for group 1. Group 2 showed significant difference between all

sensory based play preferences at p< 0.05 levels and for group 3, no significant difference

between auditory and tactile based play preferences at p>0.05 levels and significant difference

between the rest of the components at p<0.05 levels. It also inferred tactile and auditory based

play preferences decrease while proprioceptive and vestibular based play preferences increase

with the increase in age. Conclusion: These findings proved that sensory based outdoor play

preferences of children exist and differ between the ages 3 to 12 years.

Key Words: Play preferences, Sensory Based Play, Outdoor Play Preferences

Introduction

Play is a child’s primary and most important

occupation1.It has historically been regarded

by occupational therapists as both an indicator

of development and a means of intervention2.

Many occupational therapists feel that the

common themes in play include intrinsic

motivation, internal reality, and internal locus

of control, are needed for a child to engage in

playful behaviors and interactions. When

these are present, a child is self-motivated to

engage in a play activity, is free from rules,

procedures or guidelines to follow during the

play, and is able to self-direct play3. If a child

has a deficit in his or her ability to be

intrinsically motivated, to suspend reality, to

have an internal locus of control, to be happy,

energetic or playful, or has an inability to




Page 21

process sensory information from play

experiences, then a child’s development may

be stifled4. Taking this into consideration, how

children make play choices and assign

meaning to the experience of this occupation

is an important area of study for occupational

therapists, to better understand this occupation

and the use of play within practice5.

Significant research has been accumulated on

play preference with regard to gender and age;

less research exists on the relation of play

preferences to the ability4.Research on the

meaning of play for children or on children’s

perspective and rationale for their play choices

remains scarce6.

A study on Children’s perceptions of play

experiences and the development of play

preferences, found that it would be beneficial

for therapists to understand the long-term

implications of play choices in children and

their impact on development over time. Also

suggested the relationship between a

children’s sensory processing and his or her

specific play choices could be an important

area for further study4.

Based on this, and related literature, this

preliminary study was undertaken to further

understand, explore and describe the

relationship between sensory processing and

play preferences which in turn will improve

the use of play in pediatrics evaluation and

intervention with the following research

question:

Do children between the ages 3 and 12 years

have sensory based play preferences?

Aim

The aim of this study is to find sensory based

outdoor play preferences of children between

3 and 12 years of age.

Objectives

To observe children between 3 -12 years

playing in an outdoor playground.

To identify the sensory based play

preferences among these children.

Research Hypothesis

Null Hypothesis

There will not be any significant difference in

sensory based play preferences for children

between the ages 3 and12.

Methodology

Pilot Study

A pilot study was conducted initially to check

if sensory based play preferences are present

and can be assessed. It is also to assess the

reliability of scores of the three raters. This

was done by each rater observing the same

five children individually in an open

playground with common play equipment

such as swings, slides; see saw, monkey bars,

the merry go round and sand pits for duration

of 30 minutes each. Observations were noted

by the 3 raters separately and were scored

using the 7 point Likert scale.

Inclusion Criteria

Normal children of both boys and girls

aged between 3 to 12 years.

Exclusion Criteria

Children with any physical or mental

disability.

Children who played for less than 30

minutes.




Page 22

Setting

Two outdoor playgrounds with similar

types of play equipment such as slides,

swings, see-saw, monkey bars, sand pits

and merry go rounds.

Sample Size

90 children

Study Design

Observational Study

Tool

A stop watch

Scoring Criteria

7 point Likert’s scale

Procedure

Children who came with their parents

were selected randomly for the study;

oral consent was taken prior to the

study from the children’s parents along

with the child’s details demographic,

physical and mental health.

Three investigators directly observe

the spontaneous play of an individual

child in a playground for 30 minutes

minimum from a suitable vantage

point with the children being unaware

of the observers.

Using the 7- point Likert scale as the

tool of measure, each observer

evaluated the same individual child

independently using the definitions

mentioned before in the Operational

definitions as a reference.

The average of each observer’s score

for each variable was taken following

which the data was analyzed.

Data Analysis

The results were analyzed at p<0.05 level of

significance. Using SPSS version 16, the

following tests such as Descriptive analysis in

order to summarize the data and compute

means and standard deviations, ANOVA to

compare the means of play preferences among

the age group and within the age group, POST

HOC to analyze which type of play preference

is significantly difference among the age

group and within the age group was used.

Results

As mentioned in the procedure, sensory based

play preferences were observed for normal

children for the affinity towards certain

sensory components such as Tactile, Auditory,

Vestibular and Proprioception. These

components were observed and noted for the

type of play they engaged in, the time period

for which they took part in such, and joy

manifestation. The most observable sensory

based behaviors were noted and the quality

and frequency at which they occurred. Based

on these factors, the raters scored the children

using Likert’s scale.

Three raters examined and grouped the data

under three groups 1,2 and 3 according to age,

3.1 to 6, 7.1 to 9 and 10.1 to 12 years

respectively and calculated the mean for each

sample which was rounded off to the nearest

whole number, the and the results were then

analyzed as follows:

Tactile based play preferences between the

3 age groups

For tactile based play preferences, the mean

scores obtained were 6.1892, ±.84452 for

group 1, 5.322, ±.79108 for group 2 and

5.0909, ±.52636, for group 3. The mean

difference obtained with the group was tested

for significance using ANOVA and the ‘F’

score 18.075 which was significant at p< 0.05

levels. The Post HOC further revealed a

significant difference between the groups 1

and 2 and groups 1 and 3 at p<0.05 levels;




Page 23

however no significant difference was seen at

p>0.05 levels when the groups 2 and 3 were

compared.

Auditory based play preferences between

the 3 age groups

For auditory based play preferences, the mean

scores obtained were 5.3514, ± .75337 for

group1, 4.8065, ±.47745 for group 2 and,




score 9.052 which was significant at p<0.05






compared.

Proprioceptive based play preferences

between the 3 age groups

For proprioceptive based play preferences, the

mean scores obtained were 6.3243, ± .70923

for group1, 6.8710, ±.34078 for group 2 and,




score 12.844 which was significant at p<0.05






compared.

Vestibular based play preferences between

the 3 age groups

For vestibular based play preferences, the

mean scores obtained were 6.2973, ± .61756

for group1, 6.3548, ±.66073 for group 2 and,

6.5000, ±.51177 for group 3. The mean



score .772 which was not significant at p>0.05

levels. The Post HOC revealed no significant

difference between the groups 1 and 2 and 3 at

p>0.05 levels.

Play preferences within group 1

The mean scores obtained for tactile based

play preferences 6.1892,±.84452,auditory

based play preferences

5.3514,±.75337,proprioceptive based play

6.3243,.70923 and vestibular based play

preferences 6.2937,.61756.We used ANOVA

to find the significant difference between

these mean values, with the ‘F’ score being

14.663,we found that they were significantly

different at the p<0.05 levels. On further

analysis using the Post HOC revealed that

auditory based play preferences when

compared to the other sensory play

preferences considered for this project shows

a significant difference at p<0.05 levels. There

was no significant difference between tactile,

proprioceptive and vestibular based play

preferences at p>0.05 levels.



play preferences 5.3226, ±.79108,auditory

based play preferences 4.8065,±.47745,

proprioceptive based play 6.8710, .34078 and

vestibular based play preferences

6.3548,±.66073. We used ANOVA to find the

significant difference between these mean

values, with the ‘F’ score being 72.287, we

found that they were significantly different at

p <0.05 levels. On further analysis using the

Post HOC shows a significant difference

between all sensory based play preferences

considered at p<0.05 levels.




Page 24



play preferences 5.0909, ±.52636, auditory

based play preferences 4.8182, ±.39477,

proprioceptive based play 6.9091, ±29424 and

vestibular based play preferences 6.5000,

±.51177. We used ANOVA to find the

significant difference between these mean

values, with the ‘F’ score being 119.504,we

found that they were significantly different at

P<0.05 levels. On further analysis using the

Post HOC shows no significant difference

between auditory and tactile based play

preferences at P>0.05 levels however there is

a significant difference between the rest of the

components at P<0.05 levels.

Discussion

The purpose of our study was to better

understand play and play preferences and as a

preliminary study to assess the feasibility of

studying sensory based play preferences.

From these results we may infer that play

preferences may be sensory based in children

between the ages 3-12 years. This clearly

answers our research question and satisfies

our aim and objectives as mentioned before.

Our inference that sensory based play

preferences change with age may be further

supported by these statements ‘Outdoor play

preferences have been shown to change with

age’, as reported by Scarlett et al., 2005 and

‘Children’s play preferences are influenced by

age and developmental status’ as found by

Case Smith and Kuhaneck, 2008 in their

studies.

We observed that tactile and auditory based

play preferences decrease with the increase in

age, while proprioceptive and vestibular based

play preferences increase with the increase in

age. We also had the opportunity to observe

free play and the changes that influence play

in terms of social, cognitive and physical

components with regard to age and

development. As mentioned in the literature

review supported by statements from

Morrison CD et al., 2001 and Bundy AC,

2002, we chose to observe free play in order

to proceed with our study with regard to

common themes in play, which included

intrinsic motivation. While we believe these

statements hold true, we also feel, on

observation that we must also consider other

influences such as social influences that may

motivate children to engage in play, especially

in older children. As this was a preliminary

study, we feel there is a further need to

explore this area using more standardized

methods.

Implications

The implications of this study are threefold;

1. Our results show play preferences may be

sensory based.

2. The results may support giving sensory

based play across the ages according to the

found age group preferences in clinical

practice in order to increase the quality of play

as therapy, by increasing intrinsic motivation

and the internal locus of control and as Ayers

suggested, to support children enhance their

development through active exploration of the

environment and receiving various sensory

inputs through manipulation of materials.

3. As this study was a preliminary one, and

our results support our research question. This

study may be used as a baseline or guide for

further studies in this important area.

Limitations and Recommendations

As this study is a preliminary study, the results

open a broad window for future studies to give




Page 25

a better perspective on the relationship

between sensory based play preferences of

children with regard to their developmental

age. Population for the study was taken only

based on the parental information and no

standardized screening tool was used. For

future studies, more standardized screening

tool can be used.

The sample size was small, and was restricted

to Manipal area alone, so further studies can

be considered by including other different

regions.

The raters for this study were not blinded and

the unavailability of an expert in the field of

observation which could influence the study

results. Prospective studies in this field can

improvise on the above mentioned limitations.

If the play behavior was videotaped and

assessed it would have been more

standardized. Time constraints on the children

playing may also influence the nature of the

play in turn influence the inclusion of the

subject and quality of play. This can be

considered for future research as it will

influence the study results. Further study can

be focused on indoor play preferences as it is

feasible for practice in indoor based clinical

settings.

Conclusion

From the outcome of this study, we can reject

the null hypothesis. The results have proved

that there is a significant difference in the

sensory based outdoor play preferences of

children between the ages 3 to 12 years. The

tactile and auditory based play preferences

decrease with the increase in age, while

proprioceptive and vestibular based play

preferences increase with the increase in age.

This implies the need for considering the play

preferences in clinical practice to improve the

quality of play as therapy.

Acknowledgements

We would like to take this opportunity to offer

our sincere gratitude to Mr. Shovan Saha,

HOD and Mr. Shashidhar Rao, Dept. of

Occupational Therapy, & Mr. Hari Prakash,

Dept. of Speech & Hearing, Manipal for their

constant support and encouragement. Authors

acknowledge the immense help received from

the scholars whose articles are cited and

included in references of this manuscript. The

authors are also grateful to authors / editors /

publishers of all those articles, journals and

books from where the literature for this article

has been reviewed and discussed.

References

1. Bundy AC. Play Theory and Sensory

Integration. In: Bundy AC, Lane SJ, Murray

EA editors. Sensory Integration Theory and

Practice. 2nd ed. Philadelphia: FA Davis;

2002. p. 227-40.

2. Parham L, Primeau L. Play and occupational

therapy. In: Parham LD, Fazio L, editors. Play

in occupational therapy for children. St. Louis:

Mosby; 1997. p. 02–21.

3. Morrison CD, Metzger P. Play. In: Schrefer J,

White K, Mosby L, editors. Occupational

Therapy for Children. 4th ed. St. Louis:

Mosby; 2001. p.528-40.

4. Miller E, Kuhaneck H. Children’s perceptions

of play experiences and the development of

play preferences: A qualitative study.

American Journal of Occupational Therapy

2008; 62:407-15.

5. Couch KJ, Deitz JC, Kanny EM. The role of

play in pediatric Occupational therapy. Am J

of Occupational Therapy1998; 52: 111–17.

6. Smith CJ, Kuhaneck H. Play preferences of

typically developing children and children

with developmental delays between the ages 3

and 7 years. Occupational Therapy J of

Research 2008; 1:19-20.




Page 26

Tables

Table 1: Descriptive Analysis of Play Preferences among All Age Groups

Type of Play

Age groups

(in years)

n

Mean

Standard

Deviation

Tactile 3.1-6 37 6.1892 .84452

7.1-9 31 5.3226 .79108

10.1-12 22 5.0909 .52636

Total 90 5.6222 .89415

Auditory 3.1-6 37 5.3514 .75337

7.1-9 31 4.8065 .47745

10.1-12 22 4. 8182 .39477

Total 90 5.0333 .64390

Proprioceptive 3.1-6 37 6.3243 .70923

7.1-9 31 6.8710 .34078

10.1-12 22 6.9091 .29424

Total 90 6.6556 .58369

Vestibular 3.1-6 37 6.2973 .61756

7.1-9 31 6.3548 .66073

10.1-12 22 6.5000 .51177

Total 90 6.3667 .60800

There are differences between the mean scores for each sensory based play preferences with

relationship to the 3 age groups.

Graph 1 – Comparison of the mean scores of all play preferences among all 3 age groups

Table 2: Comparison of Play Preferences among the Age Groups (ANOVA)




Page 27

Type of Play Age Group F p

Tactile

3.1 to 6 years

7.1 to 9 years

10.1 to 12

years.

18.075 .000*

Auditory 9.052 .000*

Proprioceptive 12.844 .000*

Vestibular .772 .465

*- significant at p< 0.05 level

There is significant difference between the 3 age groups with relation to sensory based play

preferences

Table 3: Comparison of Play Preferences between the 3 Age Groups (Post Hoc).

Type of Play Age Group P

Tactile

Group 1-2 .000*

Group 2-3 .832

Group 3-1 .000*

Auditory

Group 1-2 .000*

Group 2-3 1.000

Group 3-1 .004*

Proprioceptive

Group 1-2 .000*

Group 2-3 1.000

Group 3-1 .000*

Vestibular

Group 1-2 .465

Group 2-3 1.000

Group 3-1 .660


Note: Group: 1 - 3.1 to 6 years, 2 - 7.1 to 9 years, 3- 10.1 to 12 years.

There are differences in sensory based play preferences between the 3 age groups.

Table 4: Comparison of Play Preferences within the Age Groups (ANOVA)

Age

Groups Play Preferences F p

3.1 – 6

years

Tactile

14.663 .000* Auditory

Proprioceptive

Vestibular

7.1 – 9

years

Tactile

78.287 .000* Auditory

Proprioceptive

Vestibular

10.1 – 12

years

Tactile

119.504 .000* Auditory

Proprioceptive

Vestibular





Page 28

There is significant difference among the play preferences within each age group.

Table 5: Comparison between Play Preferences within Each Age Group (Post Hoc).

Age group Play Preference P

3.1 – 6 years

Tactile – Auditory .000*

Tactile – Proprioceptive 1.000

Tactile – Vestibular 1.000

Auditory – Proprioceptive .000*

Auditory – Vestibular .000*

Vestibular – Proprioceptive 1.000

7.1 – 9 years

Tactile – Auditory .005*

Tactile – Proprioceptive .000*

Tactile – Vestibular .000*



Vestibular – Proprioceptive .005*

10.1 – 12

years

Tactile – Auditory .263

Tactile – Proprioceptive .000*

Tactile – Vestibular .000*



Vestibular – Proprioceptive .017*


There are differences in sensory based play preferences for each component between each of the

3 age groups

Himanshu Barot et al TESTING INDEX VOLATILITY OF INDIAN STOCK MARKET IN THE CONTEXT OF FOREIGN

INSTITUTIONAL INVESTOR’S INVESTMENT


Page 29

IJCRR

Vol 04 issue 17

Section: Management

Category: Research


Revised on:17/07/12


TESTING INDEX VOLATILITY OF INDIAN STOCK MARKET IN

THE CONTEXT OF FOREIGN INSTITUTIONAL INVESTOR’S

INVESTMENT

1Himanshu Barot,

2V.K.Sapovadia

1Asst. Professor, Faculty of Management, SRI Campus, Mehsana, India

2Executive Director, Shanti Business School, Ahmedabad, India

E-mail of corresponding authors: [email protected]

ABSTRACT

Indian stock market has seen an unprecedented growth in the last few years. Since year 2002,

Indian market has grown from a much volatile conditions to growth phenomena; this has been

due to not only the domestic market but also the international investors. FIIs investment is

considered to be one of the key influencing factors after the economic fundamentals. From 1993

reforms in the Indian Capital market on liberalisation of the FII flows has a considerable impact

on Indian stock market. The main objective of the study is to test index volatility of Indian stock

market in the contest of FII’s net investment in equity and for this two net change in indices have

been considered i.e. BSE Sensex and S&P CNX Nifty on annual basis from January 2000 to

December 2011. The empirical result found through statistical tests like correlation, regression

and t-test which reveals that significant relation between all three variables and result found that

2008 and 2011 were one of the worst years for Indian markets which make it the worst performer

in Asia. Global financial recession in 2007 and slowing growth, rising inflation, and policy

paralysis have blown the Indian markets off course in 2011. In 2008, the sub-prime crisis and

Lehman brothers collapse crippled the global markets when 2011 saw high inflation rate

dampening the market spirit. The Great Indian Dream seems to be coming to a close as most of

the FIIs are pulling out of Indian markets. The FIIs were net sellers in year 2008 and 2011,

pulling out their positions form the Indian market which leads to conclude that the FII is one of

the key movers on volatility of Indian stock market and FII’s investment is not negligible for

Indian Capital Market.

Keywords: FII, Index, volatility, BSE Sensex, S&P CNX Nifty, Inflation

Introduction

A well-developed stock market has its impact

on the development of economy. It provides

investors with an array of assets with varying

degree of risk, return and liquidity. This

increased choice of assets and the existence of

a vibrant stock market provide savers with

more liquidity and options, thereby inducing

more savings. Increased competition from

foreign financial institutions also paves the

way for the derivatives’ market. All this,

according to the mainstream belief,

encourages more savings in equity related

instruments. This, in turn, raises the domestic

savings rate and improves capital formation.




Page 30

Foreign Institutional Investors

FII or the Foreign Institutional Investors are

basically referred to investors who are

organized in the form of an institution or

entity and indulge in investing funds in the

financial market of a foreign country, i.e.

different from where the entity was originally

registered or incorporated.

In India, FII can invest their funds in the

country only under the norms prescribed by

Security and Exchange Board of India (SEBI).

FII’s investment includes mutual funds,

investment trust, asset management company,

nominee company, bank, university funds,

endowments, foundations, charitable trusts,

charitable societies, overseas pension funds

etc.

It was in July 1991 that the New Economic

Policy was unveiled that ushered in an era of

Liberalization Privatization and Globalization

(LPG) for the Indian economy. In September

1992 and 1993, India opened its stock market

for foreign investors, which facilitated receipt

of funds from foreign institutional investors in

the form of equities. The enactment led to

sweeping changes where various restrictions

imposed on investments by the foreign

investors in India were eased. SEBI opened a

new path for the foreigners to invest in India

by simplifying many terms and conditions due

to which a large number of foreign investors

flocked towards India.

Number of Foreign Institutional Investors

(FIIs)

One of the most important features of the

development of stock market in India in the

last 20 years has been the growing

participation of FIIs. Since September, 1992

when FIIs were allowed to invest in India, the

no. of FIIs has grown over a period of time.

The net addition in SEBI registered FIIs failed

to keep up the momentum seen in 2007-08

and 2008-09 wherein there was addition of

322 and 316 FIIs respectively. There was a net

addition of 78 SEBI registered FIIs in 2009-10

which took their total number to 1,713 at end

March 2010 compared to that of 1,635 at the

end of March 2009 (Table I and Chart II).

Trends in Investment of Foreign

Institutional Investors

This is not unusual as most of the developing

economies might be experiencing the same

patterns. The increasing role of FIIs has

brought in the development of our stock

markets as well such as expansion of the

business in securities, increased depth and

breadth of the market, etc.

FIIs contribute to almost 13% of the entire

market capitalization at National Stock

Exchange in India. If we talk of FIIs

investment, this has been continuously grown

over years except 1998-99 and 2008-09 when

FIIs sold more than they purchased in Indian

stock market. (Table II)

The gross purchases of debt and equity by FIIs

increased by 17.27 percent to Rs. 9,92,599

crore in 2010-11 from Rs. 8,46,438 crore in

2009-10 (Table II). The combined gross sales

by FIIs increased by 20.23 percent Rs.

8,46,161 crore from Rs. 7,03,780 crore during

the same period in previous year. The total net

inflow of FII was increased by 2.65 percent

Rs. 1,46,438 crore in 2010-11 as against an

inflow of FII was Rs. 1,42,658 crore in 2009-




Page 31

10. This was the highest net inflow for any

financial year so far.

Cumulative investment by FIIs at acquisition

cost, which was USD 89.3 billion at the end of

March 2010, increased to USD 121.5 billion at

the end of March 2011 (Figure 2). Because of

their war chests of money, the role of FIIs

can’t be ignored. FIIs have dynamic portfolios

across countries which they use to restructure

and rebalance depending on the market

conditions, definitely, with a motive to

increase their gains. Because of their size of

investment in any market, they have the

ability to make or break the fortunes of any

market.

Foreign Institutional Investments- Equity

and Debt

FIIs were allowed to invest in the Indian

Capital Market from September 1992.

Investments by them, however, were first

made in January 1993. Till December 1998,

investments were related to equity only as the

Indian gilts market opened up for FII

investment in April 1998. FIIs’ investment in

debt started from January 1999. Foreign

Institutional Investors (FIIs) continued to

invest large funds in the Indian securities

market. For two consecutive years in 2004-05

and 2005-06, net investment in equity showed

year-on-year increase of 10%.

After experiencing a net outflow of Rs

477,070 million in equity instruments in 2008-

09, FIIs scaled record equity investment in

2010-11 which stood at Rs 1,105,297 million.

(Table III) The net investments by FIIs in the

debt segment also bounced back in 2010-11

with a staggering all-time high of Rs 421,451

million compared to that of Rs 18,950 and Rs

324,380 in 2008-09 and 2009-10 respectively.

Review of Literature

Many empirical studies have been conducted

to examine the relationship between stock

prices and buying of equity by FIIs in Indian

stock market.

Purnendra Verma (2001)2 studied the impact

of FII on Indian Capital Market from 1993 to

2001 and found that there is significant effect

of FIIs on Nifty and no significant effect on

BSE Sensex, but in both the cases the co-

efficient of correlation is low and very low so

the effect are less and very less respectively.

Study concluded that FII did not have any

significant impact on the Indian Capital

Market.

T. N. Aravind, et al. (2008)3 examined the

study on FII’s influence on Indian stock

market for the period of 2003 to 2008 and

concluded that in Oct. 2007, speculation about

governments plan to control P-Notes had

caused the biggest fall in Indian stock market.

And they have proved that there is a direct

relationship between the FII’s money flow and

the movement of Sensex.

Prasanna P.K. (2008)4 examined the

contribution of FIIs in SENSEX base

companies for the period of 2001 to 2006 and

found that foreign investors invested more in

companies with a higher volume of shares

owned by the general public. The promoters’

holdings and the foreign investments are

inversely related. Study argued that the

foreign institutional investors withdraw their

money when the stock market performance

starts slowing down.

Dr. Rahul Singh, et al. (2008)5 studied of

FIIs investment flow and SENSEX movement

for period from January, 2004 to December,

2007 and proved with empirically that there is

a negative correlation between the net FIIs and




Page 32

volatility in SENSEX. And with respect to net

FIIs inflows and returns on SENSEX have a

positive and significant correlation.

Soumita Patra, et al. (2009)6 conducted the

study on impact of FIIs flow on the BSE

SENSEX and Nifty between 1997 and 2008

and proved that there is no significant

relationship between FII and SENSEX

excluding years 2003 and 2004 where FII and

Nifty there is no significant relation.

Gaurav Agrawal, et al. (2010)7 investigated

the causal relationship between Nifty and FIIs

net investment for the period January, 1999

to February, 2009 using daily data and

through application of correlation test, found

that Nifty is positively correlated to FIIs.

Madan Sabnavis, et al. (2010)8 analyzed

implications of FIIs inflow in Economic

Studies from January to September 2010 and

empirically proved that the coefficient of

correlation is quite strong between FIIs flows

and SENSEX movement which means there is

a significant relationship.

A.Q.Khan and Sana Ikram (2010)9 tested

the efficiency in relation to the impact of FIIs

largely on the Indian Capital Market during

the period 2000 to 2010 and proved that there

is a significant relation between the movement

of FII and the two major stock exchanges of

India that is the BSE and NSE. However the

coefficient of correlation is a low degree of

positive correlation indicating that the effect is

not very strong. The study concluded that the

FIIs investment has a significant impact on

Indian Capital Market and the Indian Capital

Market is Semi-strong form efficient in

relation to the impact of FIIs investment on

Indian Capital Market.

Narendra Singh Bohra and Akash Dutt

(2011)10

studied data from 2000 to 2009 and

found that there is a positive correlation

between stock market and investment of FII’s

in a relation that Sensex follows the

investment behavior of FII’s and in the case of

individual group securities FII’s had shown a

positive correlation in less regulated and high

capitalized securities in the market to earn

high equity yield. And study suggest to the

policy implication that the authorities can

focus on domestic economic policies to

stabilize the stock market.

M. Anuradha Reddy (2011)11

examined the

FIIs investment behavior and its relationship

between SENSEX movement during years

2000 to 2011 (May, 31) and found that the

FIIs are influencing the Sensex movement and

proved evidently there is a significant relation

between FIIs flow and Sensex movement.

Ravi Akula (2011)12

conducted the study on

trends in foreign institutional investment in

India for the period of 2006 to 2010, and

observed that the FIIs investment has shown

significant improvement in the liquidity of

stock prices of both BSE and NSE. Study

argued that there is a high degree of positive

co-efficient of correlation between FIIs

investment and market capitalisation which

reveals that the liquidity and volatility in

indices are highly influenced by FIIs flows.

Dr. Ambuj Gupta (2011)13

examined the

relationship between Indian Stock Market and

FIIs investment in India for the period from

April 2006 to February, 2011 and proved

through tests that there is a positive

relationship between stock market and FIIs

investment which means when FIIs

purchase/sell, there is an influence on the

stock market. Consequently, either the stock

market rise or fall on account of FIIs

activities.




Page 33

Statement of Problem

The growing participation of FIIs in Indian

stock market has raised eyebrows of many

Indians. Their influence on stock markets in

India has been widely debated and remained a

hot topic in media. Some of the market

pundits believe that FIIs are responsible for

rise or fall in the Indian stock market. This

raises a question as to whether FIIs are really a

cause or effect of the rise or fall in the Indian

stock market. In this paper the Researchers

make an earnest attempt to study the

relationship between FII’s Investment &

Indian Stock Market. For the purpose, two

major stock indices viz. NSE and BSE have

been selected. There are certain other

significant factors also, which influence Stock

market like inflation, government policies,

budgets, economic factors etc. However, in

this paper only one independent variable i.e.

FII’s investment has been taken.

Research Gap

There are contradictory findings by various

researchers regarding the causal relationship

between FIIs investment and stock market

movement in India. Therefore, there is a need

to investigate whether FIIs are the cause or

effect on stock market volatility in India.

Scope of the Study

The present study tests impact of FII’s

investment on Indian Stock Market. With

India emerging to be one of the leading

destinations for Foreign Investments, the role

and importance of FII’s in the last few years

has increased manifold as a result of

globalization of the markets. This study covers

the period of 12 years i.e.; from 2000 to 2011.

The FII’s are emerging to be a key driver in

the movement of stock index and it is

imperative to study the relation in order to

develop an understanding about the efficiency

of the market. In the last decade FII had a

significant impact on the unprecedented

growth in the Sensex and also in its downfall

due to the financial recession of 2007. An

attempt is made to carve out a clear picture of

the impact of FII’s investment on Indian

bourses and also determine the market trend

relating to inflows and outflows of FIIs.

Objectives

1. To assess the growth and development of

Indian Stock Market

2. To develop an understanding about the

concept and role of Foreign Institutional

Investors (FIIs) in India.

3. To study the relationship between FII’s

investment and Indian Stock Market

4. To test the impact of FIIs investment on

Indian Capital Market

Hypothesis

A. Testing Index Volatility of BSE Sensex

with FIIs investment

H0: FII’s investment has significant relation

with Volatility of BSE Sensex

H1: FII’s investment has no significant

relation with Volatility of BSE Sensex

B. Testing Index Volatility of S&P CNX

Nifty with FIIs investment

H0: FII’s investment has significant relation

with Volatility of S&P CNX Nifty

H1: FII’s investment has no significant

relation with Volatility of S&P CNX

Nifty

C. Testing the impact of FIIs investment on

Indian Capital Market

H0: FIIs investment has significant impact on

the Indian Capital Market




Page 34

H1: FIIs investment has no significant impact

on the Indian Capital Market

RESEARCH METHODOLOGY

Data Collection Method

The data analyzed in this paper has been

collected from the reliable source i.e. from the

Handbook of Statistics and Bulletin published

by the Securities Exchange Board of India

(SEBI) and Reserve Bank of India (RBI),

Indian Securities Market Review, NSE fact

book from 2000 to 2010 and internet. The

sample consists of yearly net changes in two

major stock indices of India viz. BSE Sensex

and S&P CNX Nifty, and yearly FII’s Net

investment in India as on January, 2000 to

December, 2011. The data collected is

compiled in the form of tables and graphs and

scrutinized through statistical tools and

techniques.

Hypothesis Testing Technique

In this paper, researchers test index volatility

of Indian stock market. And for this two major

stock indices have been taken i.e. BSE Sensex

and S&P CNX Nifty. There may be other

factors also on which stock market may

depend like inflation, government policies,

budgets, FDI, economic and political

conditions etc. But in this study only one

variable i.e. FII have been selected in order to

study its relation with stock market volatility.

In this paper the concept of correlation and

regression is being. Moreover, t-test is being

employed here (as the numbers of

observations are less than 30).

Analysis and Result

Analysis and Result based on Graph

The Charts or Graphical analysis are based on

Table-IV. Charts are the best medium through

which the study shows the trends of the

market. Here with the help of the graphs the

study has analyzed the comparative trend of

Net FII’s investment in equity with net

changes in two major stock indices of Indian

Stock Market i.e. Sensex and S&P CNX Nifty

and the market’s prime movers spent to move

each point in Sensex and Nifty. These graphs

also show the relationship between all the

three variables i.e. NSE, BSE and FII.

Through this study can analyze the impact of

FII’s investment on Indian Stock Market as

well as Indian Capital Market.

The figures in Table - IV have been depicted

in the Figure 4, 5 and 6. From the Figure 4, 5

and 6 study can analyze the comparative trend

of FII’s net investment in equity and net

change in Indian Stock Market indices for the

period 2000- 2011. A business Standard

Research Bureau study of monthly inflows

data since 2001 shows the market’s prime

movers have spent between Rs 5 crore to Rs

50 crore to move each point in Sensex.14

The

charts shows that there is an increase in net

investment in 2002-05 due to which Sensex

and Nifty also rises, then there was a sharp fall

in the year 2006. After that there was a steep

increase in net investment in the year 2007.

This was the best period in Indian Stock

Market where stock prices were at a record

high and the market was bullish.

Foreign investors have been net buyers in 10

of 12 calendars years since 2000. They have

been net sellers in 2008, when the sub-prime

crisis and Lehman brothers collapse crippled




Page 35

the global markets. That year they spent Rs 5

crore and Rs 16.78 crore for each of the

10667.96 points the Sensex and 3177.60

points the Nifty lost. And in calendar year

2011, Delivery-based buying in the secondary

market has hit a four-year low, down 25 per

cent over the past 12 months, as investors

preferred to stay away from a gloomy equity

market. So, too, for risk appetite, with average

trading volume on the BSE and NSE down 23

per cent over the level a year before. The

market was volatile and the reduction in the

FII’s investment was one of the causes of

volatility. FIIs has pulled out around Rs

53,309.7 crore and Rs 3417.70 crore from the

Indian stock market and the withdrawal led to

a fall by approximately 51 percent and 24.64

percent in Sensex and 20 percent and 24.62

percent in Nifty in 2008 and 2011

respectively. The post-Lehman recovery that

started in April 2009 and got an upward push

with return of the UPA government at the

center, was even more expensive, at Rs 22.7

crore per point in the Sensex.14

In terms of cost per point in Sensex and Nifty,

2004 was the most expensive for FII, when

they spent Rs. 38.05 crore and Rs. 138.83

crore for every point in Sensex and Nifty

respectively. In year, 2010, was next on the

list, with every Sensex and Nifty point costing

Rs. 34.94 crore and Rs. 113.60 crore

respectively when in last year, 2011 again it

bounce backed Rs. 0.68 crore and Rs. 2.26

crore for cost per point in Sensex and Nifty

respectively.

2011 was one of the worst years for Indian

markets as it fell more than 20% on a Year on

Year basis, which makes it the worst

performer of 2011 in Asia. Slowing growth,

rising inflation, and policy paralysis have

blown the Indian markets off course in 2011.

2011 saw high inflation rate dampening the

market spirit. Headline inflation, as measured

by wholesale price index (WPI), has been

above the 9% mark since December 2010.

This in turn led the RBI action of hiking the

repo and reverse repo rates consistently 11

times. And now the Interest rate cycle in India

is at the verge of peaking out. However, even

if this action by RBI could not tame inflation

immediately it helped in reducing the growth

story of India. The hike in interest rate,

inflation and lack of demand in global markets

put huge pressure on Indian markets. The IIP

data too saw some shameful numbers with

October 2011 IIP registering a negative

growth of 5.1%. The GDP growth of India

was revised down to 7.6 % for the FY 12 by

RBI. Indian Rupee too witnessed huge

depreciation in value this year. USD/INR

crossed 54 levels in December 2011. RBI's

move to bring down speculation in the

FOREX market has imparted some stability to

the rupee. But the move has negatively

affected the foreign inflows. The Great Indian

Dream seems to be coming to a close as most

of the FIIs are pulling out of Indian markets.

The FIIs were net sellers this year pulling out

their positions.

In Figure 4an interesting correlation can be

observed between FII and Sensex as Sensex

rises with the increase in FII inflows and falls

with the decrease in FII inflows. Also the gap

between the FII and Sensex is very low

indicating the huge impact and contribution of

FII in the movement of the BSE Sensex.

Figure 5 also shows co-relation between FII’s

investment and S&P CNX Nifty as the index

of Nifty rises with the rise in FII’s investment




Page 36

and falls with the fall in FII’s investment. This

shows that the FII’s investment is having a

significant effect over the indices of Nifty.

Figure 6 shows the relationship of FII’s

investment with the BSE Sensex and S&P

CNX Nifty. From this it can be seen very

clearly that the peaks and the troughs of FII

coincide with the peaks and troughs of Sensex

and S&P CNX Nifty. From all this it can be

analyzed that the FII influence the Indian

Stock as well as Indian capital market. The

graphical analysis indicates the relation is

positively correlated and all three appear to be

moving in tandem. The rise and peak of all the

three curves appear closely related. While the

curves for BSE and Nifty are overlapping each

other that of FII investments seems to be

following the same pattern or trend. It

highlights the significant role and proportion

of FII on the movement of stock market and

also raises certain questions on the basis of

past performance. The role is so prominent

that withdrawal leads to a massive fall and

increase in investment leads to a

corresponding rise. Hence, our markets

seemed to be reliant and dependent on FII’s

raising questions about stability and reliability

from the point of view of domestic investors.

Analysis and Result based on Hypothesis

This study employs the technique of

correlation and regression between FII &

Sensex and FII and S&P CNX Nifty in order

to test the index volatility of Indian stock

market in the context of FII’s investment. First

of all, the relationship between these three

variables viz; NSE, BSE and FII should be

analyzed. In the present study, data has been

taken on annual basis and it may be so that the

data on monthly or daily basis may provide

different result.

Correlation, Regression and T-Test

Analysis

Correlation and regression is being calculated

in this study in order to analyze the result and

t-test is being employed here in order to test

the statistical significance of the results

calculated which is depicted in Table V, VI

and VII.

In Table- V Karl-Pearsons’ Product Moment

Correlation is being calculated which is a

simple correlation and shows the relationship

between one dependent variable and one

independent variable. Here, FII is taken as an

independent variable and Sensex and S&P

CNX Nifty are being taken as dependent

variables, which are taken one by one for the

purpose of calculation.

The above table indicates that the correlation

between Sensex and FII’s investment is 0.839

which shows high degree of positive

correlation. The R-square is 0.704 which

means 70.4% of the variance in the variables

is explained by this relationship. According to

t-test the calculated value at 5% significance

level if 0.053 which lies between the critical

values + 2.20 reveals that study is not able to

reject the Hull Hypothesis. Hence there is

significant relation between FII and Sensex.

The above table also shows the correlation of

coefficient 0.844 of Nifty with FII which

indicates there is strong positive correlation.

The R-square is 0.712 which implies that

71.2% of the variance in the variables is

explained by this relationship. The calculated

t-value is 0.048 which lies within the critical

values + 2.20 leads not able to reject the Null

Hypothesis. Hence there is relation between

Nifty and FII.




Page 37

It is further observed from above table that

correlation coefficient between BSE Sensex

and S&P CNX Nifty is 0.997 that is a very

strong positive correlation. And R-square is

0.995 which reveals 99.5% of the variance in

the variable is examined. The calculated t

value is 0.712 which lies between critical

values + 2.16 which empirically proved that

the study can not able to accept the

Alternative Hypothesis. Hence there is a

significant relation of Sensex with Nifty.

Table VI highlights that there is a linear

relationship between the variables. It is

observed that the value of the slope is 0.098

signifying that for every unit change in X that

stands for FII there is a 0.098 unit change in Y

or Sensex. On the other hand the intercept is at

-1907.21 which seem that FII is playing

hottest role in the volatility of Sensex. There

are numbers of indicators which influence in

the index volatility of Sensex but FII is one of

most influencing factor towards index

volatility. Significance value is calculated as

0.000649, which is less than the critical value

of 0.05 which means not able to accept the

Alternative Hypothesis. Hence there is an

impact of the FII on the movement of the

Sensex.

Table VII highlights that there is linear

relationship between the variables analyzed. It

is observed that the value of slope is 0.030,

which reveals that for every unit change in FII

the value of Nifty is moved by 0.030. On the

other hand the intercept at -561.31 indicating

the role of FII in the movement of Nifty. It

means that if the value of FII is zero then the

value of NSE or Nifty would be affected by -

561.31 units. The significance value is

calculated at 0.000555, which is less than the

critical value of 0.05. Again it leads to the

acceptance of Null Hypothesis. Hence there is

an impact of FII on the volatility of Nifty

Index.

The both the tables VI & VII support fail to

reject the Null Hypothesis which signifies that

there is strong relation of FII with volatility of

BSE Sensex and S&P CNX Nifty, the effect is

slightly more pronounced Nifty. It leads to

conclusion that FII’s investment has

significant impact on Indian Capital Market.

Conclusion

Through the statistical tests, data analysis and

graphical presentation it has been found that

there is significant relation between the FII’s

net investment in equity and volatility in BSE

Sensex and S&P CNX Nifty as well as

between two indices BSE Sensex and S&P

CNX Nifty. Hence it is referred that every

movement of FII’s investment there is an

instant reaction in the Indian Capital Market.

The other factors are also influencing on

movement of the stock exchanges. The macro

factors in the form of the change in the interest

rate, inflation and demand and supply in the

global market which Indian market has faced

in year 2011. Moreover the sovereign debt

crisis in Europe took the center stage in

pulling the global market down. The factor

might be micro in terms to relating

profitability and operations of the listed

companies or the economic health of the

nation. While exploring the impact on

volatility in the stock exchanges it should be

kept in mind that FII flows are major drivers

of stock markets in India and hence a sudden

reversal of flows may harm the stability of its

markets it was seen in financial recession in

year 2007 and 2011 where the FII is the net

buyer from 2000 to 2011 excluding two years




Page 38

2008 and 2011 which leads the heavy

investment and selling attitude of FII’s

causing a major hurdle in stabilization of

market sentiments.

Suggestions

The investment scenario in India has been

witnessing turbulent times due to high

inflation, poor policy implementation by the

government. The problems were further

accentuated by the Euro zone crisis and pull

backed investment by the FIIs leading to a

more than 24% decline in Indian Equities in

2011. Strong policy decisions from the

government’s side will make India an

attractive investment destination which may

attract more FII investments for growth of

Indian stock market and making the rupee

stronger. There is no doubt FIIs are

influencing the volatility on indices of stock

exchanges to a greater extent. But the role and

effect of the FII on the Indian economy should

be duly monitored and regulated by

government agencies as our economy is still in

the developing stage. It is imperative to ensure

that the domestic investors are protected from

the established foreign players.

Acknowledgment

Authors acknowledge the immense help

received from the scholars whose articles are

cited and included in references of this

manuscript. The authors are also grateful to

authors / editors / publishers of all those

articles, journals and books from where the

literature for this article has been reviewed

and discussed.

References

[1] Link Model, developed by Narendra

Singh Bohra available in “FIIs Investment

in Indian Capital Market: A Study of Last

one Decade”, International Research

Journal of Finance and Economics, Issue

68 (2011), pp .103

[2] Purnendra Verma (2001), “Impact of FII

on Capital Market – An Empirical study

on Indian Capital Market”, pp. 1-19,

retrieved from

www.scribd.comdoc6988166Impact-of-

Fii-On (07/12/2011)

[3] Aravind, T. N, et al. (2008) “FII’s

Influence in Indian Stock Market”,

retrieved from

http://www.coolavenues.com/know/fin/sub

y-stock-1.php (10/12/2011)

[4] Prasanna P.K. (2008), “Foreign

Institutional Investors: Investment

Preference in India”, Journal of

Administration & Governance, Vol. 3, No.

2, pp. 40-51

[5] Dr. Rahul Singh, et al. (2008), “A Study

of FII Investment Flow and SENSEX

Movement”, pp. 1-11, retrieved from

www.google.com (07/12/2011)

[6] Soumita Patra, et al. (2009), “Impact of

FII Flow on the BSE SENSEX and Nifty”,

pp. 1-42, retrieved from

http://www.slideshare.net/skyranger_007/i

mpact-of-fii-on-sensex-nifty/download

(07/12/2011)

[7] Gaurav Agrawal, et al. (2010),

“Investigation of Causality between FIIs

Investment and Stock Market Return”,

International Research Journal of Finance

and Economics, Issue 40, pp. 100-112

[8] Madan Sabnavis, et al. (2010),

“Implication of FII Inflows”, Economic

Studies: CARE Ratings, pp. 1-5, retrieved




Page 39

from

http://www.careratings.comarchive38915.

pdf (07/12/2011)

[9] A.Q.Khan and Sana Ikram (2010),

“Testing Semi-Strong Form of Efficient

Market Hypothesis in Relation to the

Impact of FIIs Investments in Indian

Capital Market”, International Journal of

Trade, Economics and Finance, Vol. 1,

No. 4, pp. 373-379

[10 Narendra Singh Bohra and Akash Dutt

(2011), “FIIs Investment in Indian Capital

Market: A Study of Last one Decade”,

International Research Journal of Finance

and Economics, Issue 68, pp .103-116

[11] M. Anuradha Reddy (2011), “SENSEX

Movement and FII Flows – A Study”, pp.

1-12, retrieved from

http://www.wbiconpro.com306-

Venkat.pdf (07/12/2011)

[12] Ravi Akula (2011), “An Overview of

Foreign Institutional Investment in

India”, Indian Journal of Commerce &

Management Studies, Vol. 2, Issue 1, pp.

100-104

[13] Dr. Ambuj Gupta (2011), “Does the

Stock Market Rise or Fall Due to FIIs in

India”, International Referred Research

Journal, Vol. 2, Issue 2, pp. 99-107

[14] N S Subramanian & Sameer

Mulgaonkar (2011), “FIIS get more

bang for bug this year”, Business

Standard: Smart Investor, pp. 1 and 4

(14/12/2011)

[15] http://www.sebi.gov.in

[16] http://www.nseindia.com

[17] http://www.bseindia.com

[18] http://www.rbi.org.in

[19] http://www.moneycontrol.com

[20] http://www.business-standard.com

[21] http://www.google.com

[22] SEBI handbook of statistics on the Indian

Securities Market 2009, 2010

[23] RBI handbook of statistics on the Indian

Economy 2009-10, 2010-11

[24] SEBI Annual Report 2009-10, 2010-11

[25] Indian Securities Market Review, from

2000 to 2010

[26] NSE factbook from 2000 to 2011




Page 40

Figure 1: Link Model: Portfolio Investment and Economic Development1

Table - I: SEBI Registered FIIs in India

Year FII at end of March Net Additions of FIIs

(1) (2) (3)

1993-94 3 3

1994-95 156 153

1995-96 353 197

1996-97 439 86

1997-98 496 57

1998-99 450 -46

1999-00 506 56

2000-01 527 21

2001-02 490 -37

2002-03 502 12

2003-04 540 38

2004-05 685 145

2005-06 882 197

2006-07 997 115

2007-08 1,319 322

2008-09 1,635 316

2009-10 1,713 78

Source: ISMR 2010




Page 41

Table - II: Trends in Investment of Foreign Institutional Investors

Year Purchases

(Rs Cr.)

Sales

(Rs Cr.)

Net Investment

(Rs Cr.)

Net Investment

(US$ mn.)

Cumulative

Net Investment

(US$ mn.)

(1) (2) (3) (4) (5) (6)

1992-93 18 4 13 4 4

1993-94 5,593 467 5,127 1,634 1,638

1994-95 7,631 2,835 4,796 1,528 3,167

1995-96 9,694 2,752 6,942 2,036 5,202

1996-97 15,554 6,980 8,575 2,432 7,635

1997-98 18,695 12,737 5,958 1,650 9,285

1998-99 16,116 17,699 -1,584 -386 8,899

1999-00 56,857 46,735 10,122 2,474 11,373

2000-01 74,051 64,118 9,933 2,160 13,532

2001-02 50,071 41,308 8,763 1,839 15,372

2002-03 47,062 44,372 2,689 566 15,937

2003-04 1,44,855 99,091 45,764 10,005 25,943

2004-05 2,16,951 1,71,071 45,880 10,352 36,294

2005-06 3,46,976 3,05,509 41,467 9,363 45,657

2006-07 5,20,506 4,89,665 30,841 6,821 52,478

2007-08 9,48,018 8,81,839 66,179 16,442 68,919

2008-09 6,14,576 6,60,386 -45,811 -9,837 59,082

2009-10 8,46,438 7,03,780 1,42,658 30,253 89,335

2010-11 9,92,599 8,46,161 1,46,438 32,226 1,21,561

Source: SEBI. Annual Report 2010-11

Figure 2: Trends in Foreign Institutional Investment




Page 42

Source: SEBI, Annual Report 2010-11

Figure 3: Number of FIIs and Net Investments

Source: ISMR 2010

Table – III: Net Investments by FIIs in Equity and Debt (Rs million)

Year Net Investment in Equity Net Investment in Debt

(1) (2) (3)

2001-02 80,670 6,850

2002-03 25,280 600

2003-04 399,600 58,050

2004-05 441,230 17,590

2005-06 488,010 -73,340

2006-07 252,360 56,050

2007-08 534,040 1,27,750

2008-09 -4,77,060 18,950

2009-10 1,102,200 3,24,380

2010-11 1,105,297 4,21,451

Source: ISMR 2010, www.moneycontrol.com




Page 43

Table - IV: Movement of Net BSE Sensex, Net S&P CNX Nifty and Net FII in Equity

Year Net BSE

Sensex

Net S&P

CNX Nifty

Net FII in

Equity

(Rs. in Crores)

Cost of 1 point

of Sensex Rs.

cr

Cost of 1 point

of Nifty Rs. cr

(1) (2) (3) (4) (5) (6)

2000 -1,237.42 -218.60 6,386 -5.16 -29.21

2001 -728.32 -204.45 13,026.5 -17.89 -63.71

2002 115.27 34.65 3,629 31.48 104.73

2003 2,455.11 786.15 30,458 12.41 38.74

2004 730.21 200.15 27,787 38.05 138.83

2005 2,771.44 756.55 30,845 11.13 40.77

2006 4,364.42 1,129.60 18,586 4.26 16.45

2007 6,459.22 2,172.35 64,836 10.04 29.85

2008 -10,677.96 -3,177.60 -53,309.7 4.99 16.78

2009 7,744.26 2,237.75 89,574 11.57 40.03

2010 3,035.64 933.60 1,06,053.1 34.94 113.60

2011 -5,054.09 -1,510.20 -3,417.70 0.68 2.26

Source: www.bseindia.com, www.nseindia.com, ISMR 2000 to 2011 and SEBI handbook 2010

Figure 4: Net FII V/s Net BSE Sensex

Source: Data compiled from table IV




Page 44

Figure 5: Net FII V/s Net S&P CNX Nifty


Figure 6: Net FII V/s Net BSE Sensex and Net S&P CNX Nifty


Table – V: Karl-Pearsons’ Product Moment Correlation Coefficient (2000-2011)

Indices Coefficient of

Correlation (r) R-square

t- Value

(Significance at 5%)

Sensex & FII 0.839 0.704 0.053

S&P CNX Nifty & FII 0.844 0.712 0.048

Sensex & S&P CNX

Nifty

0.997 0.995 0.712

Source: Data compiled from Appendix 1




Page 45

Table – VI: Regression Analysis of BSE Sensex with FII

Linear Model Coefficient p-value

(at 5% significance) Values Std. Error

Intercept

Slope

-1907.21

0.098

1000.598

0.020

0.0858

0.000649


Table – VII: Regression Analysis of S&P CNX Nifty with FII

Linear Model Coefficient p-value

(at 5% significance) Values Std. Error

Intercept

Slope

-561.31

0.030

294.3346

0.006

0.0856

0.000555


G.S.R.Kedari et al STUDY OF LIPID PEROXIDATION, ANTIOXIDANT STATUS AND LIPID PROFILE IN

BREAST CANCER


Page 46

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research


Revised on:23/07/12


STUDY OF LIPID PEROXIDATION, ANTIOXIDANT STATUS

AND LIPID PROFILE IN BREAST CANCER

G.S.R.Kedari1, G.S.R.Hareesh

2, A. Saseekala

3

1Department of Biochemistry, Saveetha Medical College, Thandalam,

Chennai, India 2Department of Surgery, Rajiv Gandhi Institute of Medical Sciences,

Kadapa, A.P., India 3Department of Biochemistry, Sri Venkateswara Medical College,

Tirupati, A.P.,India E-mail of corresponding authors: [email protected]

ABSTRACT

Objective: Breast cancer is the commonest malignancy in women in India and western

countries. Increased production of oxygen free radicals exhaust the antioxidant levels in

the body leading to the development of oxidative stress which results in the production

of cancer. The reasons for alteration of lipid profile in breast cancer is not clearly

understood. The aim of our present study is to evaluate the role of oxidative stress and

also the status of serum lipid profile in breast cancer patients.

Methods: The role of oxidative stress in breast cancer patients were evaluated by

estimating the levels of lipid peroxidation assessing plasma Malondialdehyde(MDA)

levels and antioxidant status by reduced glutathione(GSH), Vitamin-C in blood. We also

tried to assess lipid profile by estimating Serum total cholesterol, triglycerides, HDL

cholesterol and LDL cholesterol. For this, we have taken 50 cases of breast cancer

patients compared with 50 age matched control women.

Results: There were significant increase in the levels of MDA and significant decreases

in the levels of antioxidants like GSH and Vitamin-C in breast cancer patients when

compared with controls. There were also significant increase in the levels of Serum total

cholesterol, triglycerides and LDL cholesterol and no statistical significant difference was

observed with HDL cholesterol in cases when compared with controls.

Conclusion: Our results indicate that oxidative stress and alterations in lipid profile are

associated with the development of breast cancer and the need for modification of

lifestyle for the reduction of breast cancer development.

Keywords: Oxidative stress, Malondialdehyde, Reduced glutathione (GSH) and Vitamin-C

INTRODUCTION

Breast cancer is one of the most common

neoplasm’s in women and is a leading

cause of cancer related deaths worldwide.

The aetiology of breast cancer is

multifactorial. Epidemiologic studies have

identified many risk factors that increase

the chance of a woman developing breast

cancer include early age at menarche, late

age of menopause, null parity, obesity, oral


BREAST CANCER


Page 47

contraception, hormone replacement therapy,

diet, family history, prior history of benign

breast disease and lactation. The common

denominator for many of these risk factors

is their effect on the level and duration of

exposure to endogenous or exogenous

estrogens.

Oxidative stress is implicated in the

pathogenesis of a variety of human

diseases(1). Oxidative damage occurs to

biomolecules like lipids, proteins,

carbohydrates and nucleic acids and other

extracellular components like collagen and

hyaluronic acid which are very

deleterious(2 )resulting in lipid peroxidation,

mutagenesis and carcinogenesis. However,

the body’s defense mechanisms play an

important role in the form of antioxidants

that help to minimize the damages which

are caused by oxidative stress. Antioxidants

are compounds that dispose, scavenge and

suppress the formation of free radicals or

oppose their actions. Oxidative stress

occurs when there is an imbalance

between reactive oxygen species(ROS) and

antioxidants reaction capacity which

stimulate the development of a disease

such as breast cancer(3).Several case

control studies have reported a relationship

with antioxidant status(4,5) and a reduction

in antioxidant level due to the presence of

free radicals may increase risk of breast

cancer. The neoplastic disease is related to

new growth where there is greater

utilization of lipids including total

cholesterol, lipoproteins, and triglycerides for

new membrane biogenesis. Cells fulfill

these requirements either from circulation,

by synthesis through the metabolism or

from degradation of major lipoprotein

fractions like VLDL, LDL or HDL(6). The

aim of our study was to evaluate the role

of lipid peroxidation in breast cancer by

estimating the plasma MDA levels and the

role of enzymatic and non enzymatic

antioxidants by estimating reduced

glutathione and vitamin-C and also to

evaluate the relationship between the lipid

profile and breast cancer.


The present study was conducted in the

department of biochemistry and department

of general surgery in Saveetha medical

college and S.V. Medical college,

Tirupati.50 newly diagnosed cases of

breast cancer belonging to age group of

30-70 years were included in this study.

Out of cases,43 were having ductal

carcinoma and 6 patients were having

lobular carcinoma and 1 was having mixed

carcinoma(both ductal and lobular).75 age

matched women who have no history of

breast diseases were taken as controls. All

the subjects were not using any kind of

hormonal therapy and they had no any

other diseases like Diabetes mellitus, liver

diseases and thyroid disorders. Informed

consent was obtained from all the subjects.

Due permission was obtained from Ethical

clearance committee for this study. 10ml of

fasting blood samples were collected by

venipuncture and for the separation of

sera, 5ml of blood was centrifuged at

3000rpm for 5min and the remaining 5ml

of blood was taken into a plain vial

containing EDTA and was centrifuged at

3000rpm for 10min for the separation of

plasma. The plasma MDA levels were

estimated by using thiobarbituric acid

reacting substances(TBARS) by the method


BREAST CANCER


Page 48

of Yagi(7) and Sinnhuber et al(8).Reduced

glutathione was determined by the method

of Beutler et al(9). The activity of

Ascorbic acid was determined by the

method of Tietz(10).Serum was used for

the estimation of lipid profile. Total

cholesterol and triglycerides were estimated

by enzymatic methods(11,12).HDL

cholesterol(HDL-C) was estimated by

phosphotungstic acid precipitation followed

by enzymatic analysis in supernatant

fraction(13) and LDL-cholesterol was

determined by using Friedwald’s

equation(14). All the results were

expressed as mean ± SD and statistical

comparisons were done using student t-test

using the SPSS package.

RESULTS

Comparison of levels of MDA, enzymatic

and non-enzymatic antioxidants in cases

and controls discussed in table 1. Comparison

of levels of Serum lipid profile in cases

and controls discussed in table 2.

Evaluation of oxidative stress is done

based on the levels of MDA and

statistically significant increase in the level

of MDA was observed in breast cancer

patients when compared to controls.

Statistically significant decreases were

observed in the levels of antioxidants like

GSH and Vitamin-C in cases when

compared to controls. There were also

significant increases in the levels of serum

total cholesterol, triglycerides and LDL

cholesterol in cases when compared to

controls. There was no statistical

significant difference in the levels of HDL

in cases when compared with controls.

DISCUSSION

Damage to the breast epithelium by

oxygen free radicals can lead to fibroblast

proliferation, epithelial hyperplasia, cellular

atypia and breast cancer. Studies have

shown increased lipid peroxidation in solid

tumors(15,16).The significant rise in the

MDA levels in breast cancer confirms that

it is associated with an increased

production of reactive oxygen

species(ROS) and free radicals. Lipid

peroxidation is a chain reaction which

provides a continuous supply of free

radicals that initiate further

peroxidation(17). We also observed a

significant decrease in the levels of

reduced glutathione in the cases as

compared to the controls. The intracellular

depletion of reduced glutathione can be

either due to the formation of a direct

complex with a electrophilic agent or due

to the inhibition of synthesis or due to the

subjection of the cell to oxidative

stress(18). When a cell is subjected to

oxidative stress, there is increased

utilization of glutathione, thus leading to its

depletion. Many enzymes are GSH

dependent and their activity may be

regulated by the thiol disulphide exchange.

They are thus dependent on the GSH

status. Glutathione –S- transferase (GST) is

reduced in diabetics which are dimeric,

mainly cytosolic enzymes that have

extensive ligand binding properties in

addition to their catalytic role in

detoxification(19,20). This reduction is due

to the reduced levels of GSH.

There is also a decrease in the levels of

non-enzymatic anti-oxidants such as Vit-C,

which states that there is an increased


BREAST CANCER


Page 49

defense mechanism against oxidative

damage in breast

cancer. The decrease in the levels of these

non-enzymatic antioxidant parameters may

be due to an increased turnover for

preventing oxidative damage in these

patients, thus suggesting an increased

defense against oxidative damage. Our

results support the researchers who

reported decreases in the antioxidant level

and increases in lipid peroxidation

level(21,22).Several other researchers

showed over expression of

antioxidants(16,23).

In the present study, there is significant

increase in the levels of serum total

cholesterol, LDL cholesterol and

triglycerides in the breast cancer patients

when compared with normal subjects and

there is no significant difference in the

levels of HDL cholesterol . The patho

physiological mechanism for lipid

alterations underlying is not well

understood. Lipids are major cell membrane

components essential for various biological

functions including cell growth and

division of normal and malignant tissues.

Low levels of cholesterol in the

proliferating tissues and in blood

compartments could be due to the process

of carcinogenesis. The raised plasma

concentrations of theses parameters in

patients with breast cancer may be due to

an increased rate of lipid absorption as the

fat splitting enzymes, lipases, were also

found to be increased in the patients(24).

In conclusion, the present study

demonstrated high oxidative stress, low

antioxidant status with rise in plasma lipid

levels except HDL in breast cancer

patients which shows the need to adopt a

healthy lifestyle to reduce oxidative stress

with consumption of diet rich in

antioxidant nutrients to prevent breast

cancer.

ACKNOWLEDGEMENTS


received from the scholars whose articles

are cited and included in references of this


authors/editors/publishers of all those



and discussed.

REFERENCES

1. Beck MM, Levander OA. Dietary

oxidative stress and potentiation of viral

infection. Annu Rev Nutr,1998; 18: 93-

116.

2. Frei B. Reactive oxygen species and

antioxidant vitamins: mechanisms of

action. Am J Med,1994; 97: S5-S13.

3. Aghvami T, Djalali M, Kesharvarz A, et

al. Plasma level of antioxidant vitamins

and lipid peroxidation in breast cancer

patients. Iran J Publ Health,2006; 35: 42-

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4. Ching S, Ingra D, Hahnel R, Beilby J,

Rossie E. Serum levels of micronutrients,

antioxidants and total antioxidant status

predict risk of breast cancer in a case

control study. J Nutr, 2002; 132: 303-6.

5. Do MH, Lee SS, Jung PJ, Lee MH. Intake

of dietary fat and vitamin in relation to

breast cancer risk in Korean women: a

case control study. J Korean Med

Sci,2003; 18: 534-40.

6. M.I.Qadir, S.A.Malik. Plasma lipid profile

in gynecologic cancers.

Eur.J.Gynaec.oncol, 2008; 29: 158-161.


BREAST CANCER


Page 50

7. Yagi K. Lipid peroxides and human

diseases. Chem Phys Lipids, 1978; 45:

337-351.

8. Sinnhuber RO, Yu TC. Characterization

of red pigment formed in thiobarbituric

acid determination of oxidative rancidity.

Food Res,1958; 23: 626-630.

9. Beutler E, Duron O, Kelly BM. Improved

method for determination of blood

glutathione. J Lab Clin Med, 1963;61:882-

888.

10. Tietz NW(Ed). Textbook of Clinical

Chemistry. W.B.Saunders Company,

Philadelphia, London, Toronto.2004;

pp.960-962.

11. Alian CC,Poon LS, Chan CSG. Enzymatic

determination of total serum cholesterol.

Clinical Chemistry.1974; 20; 470-475.

12. Bucolo G, David H. Quantitative

determination of serum triglycerides by

the use of enzymes. Clinical chemistry.

1973;19:476-482.

13. Burstein M, Scholnick HR, Morfin R.

Rapid method for the isolation of

lipoproteins from human serum by

precipitation with polyamines. Journal of

lipid research,1970;2:583-595.

14. Friedewald WT, Levy RI, Fredrickson DS.

Estimation of the concentration of low

density lipoprotein cholesterol in plasma

without the use of preparatory

centrifuge. Clin Chem 1972; 18: 499-503.

15. Zieba M, Nowak D, Suwalski M,et al.

Enhanced lipid peroxidation in cancer

tissue homogenates in non small cell

lung cancer.Monaldi Arch Chest Dis

2001;56:110-4.

16. Skrzydlewska E, Stankiewicz A,

Sulkowska M, Sulkowski S, Kasacka I.

Antioxidant status and lipid peroxidation

in colorectal cancer. J Toxicol Environ

Health 2001;64:213-22.

17. Murray RK, Granner DK, Rodwell VW.

Harper’s Illustrated Biochemistry. 27th

Edn.2006.Mc Graw Hill Lange

International Edition.pp128-129.

18. Deneke SM, Farberg TZ. Regulation of

cellular glutathione lung cells.

Mol.Physiol,1994 ;1163-1173.

19. Listowsky I,Abramovitz M, Homma H et

al. Intracellular binding and transport of

hormones and Xenobiotics by

glutathione-S-transferase. Drug Metab

Res,1988;19:305-318.

20. Ketley JN, Habig WH, Jacoby WB.

Binding of non substrate ligands to

glutathione-s-transferases. J Biol

Chem,1976;250:8670-8673.

21. Gonenc A, Erten D, Aslan S, et al. Lipid

peroxidation andantioxidant status in

blood and tissue of malignant breast

tumor and benign breast disease. Cell

Biol Int 2006;30:376-80.

22. Yeh CC, Hou MF, Tsai SM, et al.

Superoxide anion radical, lipid peroxides

and antioxidant status in the blood of

patients with breast cancer. Clin Chim

Acta 2005;361:104-11.

23. Iscan M, Coban T, Cok I, et al. The

organochlorine pesticide residues and

antioxidant enzyme activities in human

breast tumors: is there any association?

Breast Cancer Res Treat, 2002;72:173.

24. Basu T.K and Williams D.C. Plasma

and body lipids in patients with

carcinoma of the

breast.Oncol.,1975;31:172.


BREAST CANCER


Page 51

TABLE 1

Comparison of levels of MDA, enzymatic and non-enzymatic antioxidants in cases and

controls

Parameters Cases(n=50) Controls(n=50) P value

MDA

(nmol /ml)

7.98±0.13 2.16±0.12 <0.001

(highly significant)

GSH

(mg/g Hb)

8.46±0.21 14.76±0.13 <0.001

(highly significant

Vitamin-C

(mg/dl)

0.58±0.16 2.14±0.19 <0.001


TABLE 2

Comparison of levels of Serum lipid profile in cases and controls

Parameters Cases(n=50) Controls(n=50) P value

Total cholesterol

(mg/dl)

213.72±9.31 154.15±6.68 <0.001


Triglycerides

(mg/dl)

186.64±5.61 158.26±6.31 <0.05

(significant)

LDL Cholesterol

(mg/dl)

112.42±4.12 76.86±4.05 <0.05

(significant)

HDL Cholesterol

(mg/dl)

52.24±3.32 51.62±4.23 >0.05

( not significant)

Little Mahendra et al PSYCHOLOGICAL STRESS AND ORAL DISEASES – A LINK?


Page 52

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Review


Revised on:30/07/12


PSYCHOLOGICAL STRESS AND ORAL DISEASES – A LINK?

Little Mahendra1, Ravi David Austin

2, Jaideep Mahendra

3, S. Senthil

Kumar4

1A. P., Dept. of Periodontology, Raja Muthaiah Dental College and

Hospital, Annamalai University, Tamil Nadu, India. 2Dean, Raja Muthaiah Dental College and Hospital, Annamalai University,

Chidambaram, Tamil Nadu, India 3Department of Periodontology, Meenakshi Ammal Dental College,

Meenakshi Academy of Higher Education and Research, Chennai, India 4Dept. of Periodontology, Raja Muthaiah Dental College and Hospital

Annamalai University, India E-mail of corresponding authors: [email protected]

ABSTRACT

The relationship between stress and any disease is explained by hormonal modifications and behavioural

changes induced by the stress. Periodontitis is a chronic bacterial infection that involves the gingiva and

the periodontal supporting structures of the tooth. Several studies have shown significant associations

between stress factors and periodontal disease. The purpose of the present review article is to present

findings, which show that a lot of prospective studies are still needed for a more defined role of stress and

periodontal disease.

Keywords: Immune system; periodontal disease; Stress.

Introduction:

Epidemiologic studies and clinical

observations suggest that negative life events

and psychological factors may contribute to an

increased susceptibility to periodontal disease.

Stress a term continually being redefined in

the scientific study of disease and illness, is

nevertheless a confirmed and important factor

in the etiology and maintenance of many

inflammatory diseases, including periodontal

disease. stress is an attempt to understand how

the body regulates itself to maintain smooth,

adaptive and homeostatic functioning when

confronted with disruptive endogenous or

exogenous forces. Hippocrates thought of

health as a harmonious balance of the

elements comprising the quality of life while

disease represented disruption or disharmony

among those elements. In the seventeenth

century, Sydenharm suggested that

pathological states represented diseases of

adaptation failure of the adaptive processes to

restore wellbeing. Cannon elaborated how

fight or flight mechanisms represented

adaptive efforts by the body to re-establish

homeostasis, a term he introduced to describe

a dynamic internal physiological equilibrium

which the body sought to maintain along both

physical and emotional dimensions.

Hans Selye, the father of stress theory,

describes stress as, “Everybody knows what it

is, no one knows what it is. It is the

nonspecific response of the body to any

demand put upon it and stress is the spice of

life” (1). While Stress is defined as a state

induced by a stimulus that manifests itself by

virtue of one’s cognitive interpretation (2), the



Page 53

stimulus itself is considered a stressor.

Accordingly, a stressor is any stimulus that

evokes stress, whereas stress reactions are the

observable consequences of the stressors.

Several studies have demonstrated the

relationship between psychological stress and

diseases, from common cold (3) to

cardiovascular disorders (4); from asthma (5)

to rheumatoid arthritis (6). Periodontal disease

being multifactoral with a complex interaction

between bacterial infection and host

responses, there is a reasonable amount of

research indicating the association between

periodontitis with psychosocial stress, distress,

and depression (7, 8).

When demands imposed by events exceed a

person’s ability to cope, a psychological stress

response composed of negative cognitive and

emotional state is elicited (9). Substantial

literature both in humans and animals support

association between psychological stress and

suppression of immune responses (10). While

stress can directly influence immune function

through the activation of neuro-endocrinal

pathways that lead to release of hormones and

neurotransmitters, such as cortisol and

catecholamines (11); it can also alter immune

responses through the adoption of coping

behaviors, such as smoking or drinking

alcohol, which are known to compromise

immunity (12).

Pathways between Stress and the Immune

System:

How can stress affect the immune response?

In 1936, Hans Selye defined stress

physiologically as the state in which the

sympatho-adreno-medullary system and the

limbic-hypothalamic-pituitary-adrenal axis

(HPA) are co-activated (13). A bidirectional

communication pathway exits between the

CNS and the immune system that modulates

both the cellular and humoral immunity (14).

First, stress induces the sympathetic fibers

which descend from the brain into both

primary (bone marrow and thymus) and

secondary (spleen and lymph nodes) lymphoid

tissues (15) to release a wide variety of

substances that influence immune responses

by binding to receptors on white blood cells

(14, 15). Though all lymphocytes have

adrenergic receptors, differential density and

sensitivity of adrenergic receptors on

lymphocytes may affect responsiveness to

stress among cell subsets. For example,

natural killer cells have both high-density and

high affinity β2-adrenergic receptors, B cells

have high density but lower affinity, and T

cells have the lowest density (16, 17).Second,

the stress activates the hypothalamic–

pituitary–adrenal axis, the sympathetic–

adrenal–medullary axis or locus coeruleus-

norepinephrine system, to induce secretion of

adrenal hormones epinephrine,

norepinephrine, and cortisol (13, 15); and also

the pituitary hormones prolactin and growth

hormone; and the brain peptides melatonin, β-

endorphin, and enkephalin. These substances

bind to specific receptors on white blood cells

and have diverse regulatory effects on their

distribution and function (18).Communication

between the neuro-endocrine (hypothalamic-

pituitary-adrenal) and immune inflammatory

systems functions as a feedback loop that

regulates the immune components of the

inflammatory response. For example, a

negative feed back loop functions such that

activation of the immune system, associated

with increases in levels of circulating

cytokines (e.g. interleukin-1 and interleukin-

6), increases activity in the corticotrophin



Page 54

releasing hormone/hypothalamic-pituitary-

adrenal system yielding increased levels of

circulating adreno-corticotropic hormone and

cortisol – major modulators of the stress

system (19).

Connection between Stress and

Periodontitis:

Stress can be viewed as a process with both

psychological and physiological components.

Pollman and Dietrich (1979), Moulton et al

(1952) pointed out that stress may affect

periodontium directly or indirectly (7). While

the psychosocial stressors initiate cascade of

events through the hypothalamic-pituitary-

adrenal axis, the autonomic nervous system

and the central nervous system, it enhances

the likelihood of infection and specifically,

periodontal disease (8, 20).While cortisol has

been called the hormone of stress, serum

cortisol level increases during challenging or

unpleasant situation (21, 22). Cortisol being

an immunosuppressant influences not only the

inflammatory cells, chemotaxis etc., but also

the pro-inflammatory cytokines like

interleukin-6 (IL-6), interleukin-1 receptor

antagonist (IL-1ra) (23, 24, 25). It is always

being studied that stress causes activation of

neuro-endocrinal pathways with release of

cortisol, which in turn suppresses the

immunity. And in such an environment the

progression of periodontal infection by the

pathogen is unhampered (7). Though this

relation gives a link between stress and

periodontal disease, this holds good for other

inflammatory diseases as well. Though the

periodontitis is initiated by the pathogens, the

mediators of connective tissue breakdown are

produced by host-derived enzymes called

Matrix metalloproteinases (MMPs) (26). They

are zinc and calcium dependent proteolytic

enzymes responsible for remodeling and

degradation of extracellular matrix (27). The

homeostasis of extracellular matrices depends

on the release of MMPs from cells such as

fibroblasts and macrophages, and the presence

of tissue inhibitors of matrix

metalloproteinases (TIMPs), which are

distributed widely in tissues and fluids (28).

While the MMP gene family encodes nine or

more metal-dependent endopeptidases, eight

human MMPs have been cloned and

sequenced, of which MMP-1 cleaves fibrillar

collagen types I, II, and III (27) that constitute

the gingival and periodontal connective tissue.

While MMP-1 is implicated to play an

important role in the initiation of collagen

degradation in periodontal disease, MMP-8 is

thought to play an important role in

periodontal tissue destruction (29).

Very few in-vitro studies, using exogenous

steroids, have shown the effects of

corticosteroids on the expression of MMPs

and TIMPs in fibroblasts (30, 31). A study by

Cury PR et al. (31) showed that

hydrocortisone produced a dose-dependent

regulation of MMP and TIMP expression, by

significantly up-regulating mRNA expression

of MMP-1, -2, -7, and -11 and TIMP-1 in

human gingival fibroblasts. When Pubmed

was searched for studies where endogenous

cortisol, due to stress, induced expression of

MMPs and TIMPs in fibroblasts, none were

found.

Significant Reviews:

Several studies have demonstrated a

relationship between psychological stress and

inflammatory diseases such as rheumatoid

arthritis, osteoarthritis etc., (20, 32, 33) and

periodontitis (7, 8).Gerard J. Linden et al.



Page 55

(34) examined the association between

occupational stress and the progression of

periodontitis in employed adults and

suggested that occupational stress may have a

relationship to the progression of

periodontitis. Torbjørn Breivik et al. (35)

reviewed studies to find effects of emotional

stress on immunity, gingivitis and

periodontitis, and noted that emotional

stressors and the nervous and neuro-endocrine

responses to psychological stressors may

modulate the immune response to bacteria,

and thus expected stress to influence the

progression and course of gingivitis and

periodontitis.In a case-control study, Moss et

al. (36) explored the association between

social factors and adult periodontitis by

comparing self-reported information for daily

strains and symptoms of depression. They

found that an elevated Depression score may

be a marker for social isolation, which could

play a role in immune function during periods

of social strain. Croucher R et al. (37) also in

a case-control study investigated the role of

life-events in periodontitis. The results

showed that periodontitis was associated with

the negative impact of life-events, the number

of negative life-events, high levels of dental

plaque, tobacco smoking and unemployment.

Genco RJ, Ho AW, Jeffrey Kopman et al. (38),

Genco RJ, Ho AW, Grossi SG et al. (8)

evaluated the association of stress, distress,

and coping behaviors with periodontal

disease. They found that subjects with

financial strain and inadequate coping had

greater attachment and alveolar bone loss, in

contrast to subjects with financial strain and

good coping. Salivary cortisol levels were

higher in a test group exhibiting severe

periodontitis, as compared to a control group

consisting of those with little or no periodontal

disease. Renate Deinzer et al. (39, 40)

analyzed the effects of academic stress on

periodontal health of medical students and

found that psychological stress was a

significant risk factor for periodontal

inflammation. Elter et al. (41) found that

clinical depression may have a negative effect

on periodontal treatment outcome in health

maintenance organization (HMO) patients.

Study by Hugoson A, Ljungquist B, Breivik T

(42) revealed that, in addition to increased

age, oral hygiene status, and smoking, the

negative life events like, loss of a spouse

(being a widow or widower) and the

personality trait of exercising extreme external

control were also associated with severe

periodontal disease. Aleksejuniené t al. (43)

tested the hypothesis that psychosocial stress

and lifestyle are related to periodontal status.

Although the pathway between psychosocial

stress and remaining periodontal support was

not empirically supported, the researchers

concluded that there was reason to believe that

such link was likely. Wimmer G et al. (44)

examined the influence of different coping

behaviors on a non-surgical periodontal

therapy and on the course of periodontal

disease. They found patients with a defensive

coping style had statistically significant poorer

attachment values (P= 0.000) after 2 years

compared to patients with other coping

behaviors. The number of sites with severe

advanced CAL (>5mm) was significantly

correlated with a suppressive coping style

(P=0.0001).Alexander Saletu et al. (45)

investigated the relationship between

periodontitis and psychopathology utilizing

psychometry, both observer-rating scales and

self-rating scales. Partial correlation analyses



Page 56

between psychometric measures and dental

variables revealed positive correlations of

periodontal disease severity/CAL with the

depression/anxiety, subjective well-being and

complaints scores, and a negative correlation

with quality of life. R. Akhter et al. (46)

designed a study to identify possible

relationship between stress and periodontal

disease in residents of a rural Japan and found

subjects who felt job stress and those who felt

stress due to self health were more prone to

have periodontal disease than were those who

never or only rarely felt such stress. Vettore et

al. (47) in a case-control study investigated

the relationship of stress and anxiety with

periodontal clinical characteristics. They

found the frequency of moderate clinical

attachment loss (4-6 mm) and moderate

probing pocket depth (4-6 mm) significantly

associated with higher trait anxiety scores,

after adjusting for socioeconomic data and

cigarette consumption. J.B. Hilgert, F.N.

Hugo D.R. Bandeira and M.C. Bozzetti (48)

evaluated the extent and severity of chronic

periodontitis and its association with the levels

of salivary cortisol and the scores obtained

with a stress questionnaire in a population

aged 50 years. They found that the cortisol

levels were positively associated with the

extent and severity of Periodontitis. Fernando

N. Hugo, Juliana B. Hilgert et al (49)

evaluated the effects of stress, depression, and

cortisol levels in dental plaque accumulation

and gingivitis in a population of individuals

aged ≥50 years. They found that stress was a

significant risk indicator of elevated levels of

plaque and gingivitis, whereas cortisol was a

risk indicator of plaque. Amy E Rosania (50)

did a cross-sectional pilot study to explore the

associations between psychological factors,

markers of periodontal disease, psycho-neuro-

immunologic variables, and behavior. She

found that stress, depression, and salivary

cortisol correlated with measures of

periodontal disease. In addition, oral care

neglect during periods of stress and depression

was associated with loss of attachment and

missing teeth.

Conclusion:

Stress can be a risk factor for periodontitis, on

one hand in stress the person’s oral hygiene

habits are altered causing accumulation of

plaque and on the other it reduces the

immunity of person through its endocrinal

connections. Many studies have shown a

positive relationship was observed between

stress and periodontal disease, further

representative research is needed to determine

the impact of stress/psychological factors as

risk factors for periodontal disease.

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37. Croucher R, Marcenes WS, Torres MC,

Hughes F, Sheiham A ., The relationship

between life-events and periodontitis A

case-control study., J Clin Periodontol.

1997:24(1):39-43.

38. Genco RJ, Ho AW, Jeffrey Kopman,

Grossi SG, Dunford RG, Tedesco LA.,

Models to evaluate the role of stress in

periodontal disease. Ann Periodontol.

1998: 3(1):288-302.



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39. Renate Deinzer, Stefan Rüttermann, Ole

Möbes Armin Herforth., Increase in

gingival inflammation under academic

stress. J Clin Periodontol. 1998:

25(5):431–433.

40. Renate Deinzer, Daniel Hilpert, Katharina

Bach, Michael Schawacht and Armin

Herforth., Effects of academic stress on

oral hygiene – a potential link between

stress and plaque-associated disease? J

Clin Periodontol. 200:8(5): 459 – 464.

41. Elter JR, White BA, Gaynes BN, Bader

JD. Relationship of clinical depression to

periodontal treatment outcome. J

Periodontol.2002:73(4):441-9.

42. Hugoson A, Ljungquist B, Breivik T., The

relationship of some negative events and

psychological factors to periodontal

disease in an adult Swedish population 50

to 80 years of age. J Clin Periodontol.

2002:29(3):247-53.

43. Aleksejuniené J, Holst D, Eriksen HM,

Gjermo P., Psychosocial stress, lifestyle

and periodontal health. J Clin Periodontol.

2002:29(4):326-35

44. Wimmer G, Köhldorfer G, Mischak I,

Lorenzoni M, Kallus KW., Coping with

stress: its influence on periodontal

therapy. J Periodontol. 2005:76(1):90-8.

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Franziska Saletu, Leopold Linzmayer,

Peter Anderer and Michael Matejka.,

Controlled clinical and psychometric

studies on the relation between

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Periodontol. 2005:32(12):1219–1225.

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and periodontal disease in a rural area

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relationship of stress and anxiety with

chronic periodontitis. J Clin Periodontol.

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Page 60

(FIGURE- 1) Pathways between Stress and the Immune System

Mamta Bhatia et al AN IN VITRO SCREENING OF GROWTH INHIBITORY POTENTIAL OF ALLIUM SATIVUM

TOWARDS SOME MICROBES OF SPOILAGE AND HEALTH SIGNIFICANCE


Page 61

IJCRR

Vol 04 issue 17

Section: Technology

Category: Research


Revised on:23/07/12


AN IN VITRO SCREENING OF GROWTH INHIBITORY POTENTIAL

OF ALLIUM SATIVUM TOWARDS SOME MICROBES OF SPOILAGE

AND HEALTH SIGNIFICANCE

Mamta Bhatia, Alka Sharma

Department of Food Technology, Guru Jambheshwar University of Science

and Technology, Hisar, Haryana, India.

Email of corresponding author:[email protected]

ABSTRACT

The use of Allium sativum (garlic) as a cure and condiment predates written history. In present

study aqueous extract, crude juice, essential oil and powdered form of Allium sativum were

screened for their inhibitory potential towards some food borne pathogens, in culture media. Test

microbes included: Bacillus cereus, Enterococcus faecalis, Escherichia coli, Psuedomonas

aeruginosa, Psuedomonas alkaligenes, Shigella sonnei and Staphylococcus aureus. Spice agar

method was opted for investigating antibacterial activity of powdered spice samples. Agar well

assay and broth dilution techniques were followed for determining growth inhibitory potentials

of aqueous extract, crude juice and essential oil. Results revealed that essential oil most

effectively inhibited bacterial strains followed by crude juice, while aqueous extract and

powdered forms remained ineffective in arresting the growth of test bacteria.

Keywords: Antibacterial, Antimicrobial, Allium sativum, essential oil, garlic, spices.

Introduction

Foods, by their very nature need to be

nutritious and microbiologically stable. To

ensure that food is safe and can be stored in a

satisfactory state, it is necessary to either

destroy the microorganisms present, or

manipulate the food so that microbial growth

is prevented or hindered. Resurgence in the

use of natural herbal alternatives instead of

synthetic preservatives to increase the shelf

life of food commodoties has brought the use

of aromatic plants to the forefront of

investigations. Allium sativum, is one of the

world’s most popular spices, and is used

extensively from India to America, in French

aioli, Greek skordalia, Indian korma, Turkish

cacik and Vietnamese pho bo. Since ancient

times, it has been used as a cure as well as

food. Pliny the elder, a Roman naturalist,

described in his Historia Naturalis how A.

sativum could be used for gastrointestinal

disorders, dog and snake bites, scorpion

stings, asthama, madness, convulsions and

tumours. Components from A. sativum

modulate the cardiovascular and immune

systems. Alongwith medicinal properties, it is

known to have antiviral1,2

and antiprotozoal3,4

activities. Encouraged by these results, an in

vitro trial was carried out to evaluate different

forms of A. sativum viz. aqueous extract,

crude juice, essential oil and powdered form,

for their antimicrobial potencies, against seven

food borne pathogens.




Page 62

Materials and Methods

Procurement of spice samples

Fresh bulbs of A. sativum purchased in the

amounts of 1 kg, from grocery shop, local

market, Hisar, India. The spice samples were

washed with clean water followed by distilled

water to remove extraneous matter. The outer

coverings of A. sativum bulbs/clove were

peeled off manually with the help of knife.

For the extraction of crude juice, peeled

cloves of A. sativum were sliced into thin

pieces and were crushed in pestle-mortar to

get a thick paste. Thick pastes of spice

samples were passed through sieve cloth.

Filtrates thus obtained were sterilized by

passing through syringe filter assembly having

membrane filters of pore size 0.45 um under

aseptic conditions. Crude extract thus obtained

was stored in sterilized glass vials at 4+1° C

and was used at various concentration levels

within the 2 h. of their preparation.

To get the powdered form, peeled cloves of A.

sativum were dried in shade for 5 days

followed by their grinding in the laboratory

grinder and were kept in airtight containers till

further use.

Essential oil of A.sativum was procured from

Aroma Chemicals Pvt. Limited, Delhi, India,

stored in the dark amber colored, screw

capped glass bottle and was kept away from

light to avoid physicochemical changes in its

composition. Purity of the essential oil was

assured by the company to be more than

99.0%.

Chemicals and culture media

Ethyl violet azide dextrose agar, Ethyl violet

azide dextrose broth, MacConkey broth,

MacConkey agar, Nutrient agar and Nutrient

broth were obtained from Hi-Media Pvt. Ltd,

India. Dimethylsulphoxide (DMSO) and

Sodium chloride (NaCl) were obtained from

Central drug house Pvt. Limited, India.

Bacterial cultures

All the pure bacterial cultures viz. Bacillus

cereus (MTCC 430), Enterococcus faecalis

(MTCC 439), Escherichia coli (MTCC 1687),

Psuedomonas aeruginosa (MTCC 1688),

Psuedomonas alkaligenes (MTCC 405),

Shigella sonnei (MTCC 2957) and

Staphylococcus aureus (MTCC 5021) were

obtained from Microbial Type Culture

Collection (MTCC), Institute of Microbial

Technology (IMTECH), Chandigarh, India.

The reference bacterial strains were

maintained on their respective media slants,

subcultured bimonthly to maintain their

viability and were stored at 4+1° C. Culture

media, incubation temperatures and duration

of incubation of reference bacterial strains are

presented in Table 1.

Inoculum preparation

A flamed sterile wire loop was used to

dislodge the lawns of test bacterial strains

from their respective pure culture slants (24h.

old) with 10 ml of sterilized normal saline

(NaCl, 0.85% (w/v)) solution under aseptic

conditions. Bacterial suspensions were

adjusted with the same solution to contain

approximately 1×107

cfu /ml and were utilized

the same day.

Prepearation of aqueous extract

Aqueous extract of dried and powdered bulbs

was prepared. Powdered spice sample was

steeped overnight (temperature: 24-27°C) in

sterilized distilled water in a ratio of 1:1 (w:

v), followed by their homogenization in a




Page 63

blender at high speed for 2 min. The

homogenized spice mixture was filtered

through Whatmann No. 1 filter paper. Filtrate

thus obtained, was sterilized by passing

through syringe filters containing 0.45 um

pore size membrane filters under aseptic

conditions, collected in sterilized glass vial

and was stored at 4+1° C. This aqueous

extract was further used within the 2 h. of its

preparation.

Preliminary screening of antibacterial

activities of aqueous extract, crude juice

and essential oil

Agar-well diffusion technique was followed5.

Freshly prepared inoculum (100 ul) of each

reference bacterial strain was poured in plates

with 20 ml of appropriate media. The

petriplates seeded with bacterial strains were

kept undisturbed for 30 min. for proper

solidification and setting of agar to facilitate

uniform digging of wells. Sterile cork borer

(diameter: 8 mm) was used to bore wells in

the solidified media plates previously seeded

with bacterial inocula. Subsequently, different

volumes of test substances were introduced

into the wells of agar plates. Sterilized DMSO,

instead of crude juice and essential oil served

as negative control. These plates were allowed

to stand at room temperature for at least 1 h.

for the even diffusion of poured components

and were incubated without inversion at their

respective incubation temperatures in

incubator for 24-48 h. After incubation, zones

of inhibition formed around the wells were

measured in millimeters (mm) and results

were expressed as the net zone of inhibition

which represented the subtraction of the

diameter of the well (8 mm) from the

measured zone.

Mnimum inhibitory concentrations (MIC)

of crude juice and essential oil

MIC values of crude juice and essential oil

were determined by broth dilution method6.

The media (broth) containing 2000 ul/ml of

test substance was serially diluted twofold

each with the media (broth) to give

concentrations of 1000, 500, 250, 125, 62.50,

31.25, 15.62, 7.81, 3.90, 1.95, 0.97, 0.48, 0.24,

0.12, 0.06 ul/ml. Sterilized DMSO, instead of

crude juice and essential oil, served as

negative control. To the diluted solution, 100

ul of freshly prepared inoculum of each

bacterial strain was added. These mixtures

were incubated in the B.O.D. incubator at

suitable incubation temperatures of microbes,

for appropriate incubation periods. After the

completion of incubation, 100 ul of the above

mixture was evenly spread on the surface of

solidified media petriplates with the help of

sterile bent glass rod. These petriplates were

incubated in an inverted position to observe

the minimum concentration of test substances,

at which visible growth of the reference

microbes was completely inhibited.

Antibacterial activity of powdered form

Antibacterial activity of powdered form of

spice sample was examined in culture media

using spice agar method7. Erelenmeyer flasks

(100 ml capacity) containing 20 ml of

appropriate media (containing agar) and

powdered spice at different concentrations

(0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0,

3.5, 4.0, 4.5, 5.0, 5.5, 6.0 (%, w/v) were

autoclaved at 121° C for 20 minutes. After

autoclaving, spice agar mixtures (cooled but

still molten) were poured into sterilized

petriplates under aseptic conditions and these

plates were kept undisturbed for 30 min. for




Page 64

proper setting of agar. Freshly prepared

inoculum of each test microbe at 100 ul level

was evenly spread over the entire surface of

the respective solidified media in petriplate

using a sterile bent glass rod. Seeded

petriplates were incubated in incubator at

appropriate temperatures and were examined

for bacterial growth at 12 h. intervals,

throughout the incubation period of 30 days.

A similar experiment was carried out without

any spice sample that served as control. The

time for initiation of microbial growth on

control (without spice samples) and media

supplemented with different concentrations of

spice were recorded.

Statistical analysis:

All the experiments were performed in

triplicates with two independent trials and the

results obtained were highly reproducible.

Values of growth inhibitory zones are mean

±SD (n=3) of three replicates.

Results

Zone inhibition assay results (Table 2)

revealed that seeded petriplates with DMSO

and aqueous extract of A. sativum, did not

display growth inhibitory zones towards any

bacterial strain under observation. Crude juice

at 100 ul/well level exhibited inhibitory circles

towards B. cereus, P.aeruginosa and S.

aureus. On the other hand, essential oil of A.

sativum, at 10ul/well exhibited distinct zones

of inhibition towards all the bacterial strains

under investigation. The diameter of

inhibitory zones varied with the type of

bacterial strains and test substances implicated

in the study. Crude juice exhibited widest

diameter of inhibitory zone towards S.aureus,

while essential oil produced broadest

inhibitory circle towards B.cereus. It was also

observed that g+ve bacterial strains gave

wider inhibitory zones towards test substances

as compared to g-ve bacterial strains. On the

basis of diameter of growth inhibitory zones,

sensitivity of microbes in descending order

towards test substances may be put in the

following manner:

Essential oil:

B.cereus>S.aureus>P.aeruginosa>P.alkalige

nes>S.sonnei>E.faecalis>E.coli.

Crude juice:

S.aureus>B.cereus>P.aeruginosa=E.coli=E.f

aecalis=P.alkaligenes=S.sonnei.

Results of broth dilution technique depicted

that crude juice and essential oil of A. sativum

effctively inhibited all the bacterial strains

(Table 3). MIC values of essential oil towards

bacterial strains ranged from 62.50-500.00

(ul/ml), whereas, that of crude juice ranged

from 125.00-500.00 (ul/ml), thereby

indicating the higher susceptibility of the

microbes towards former. It was noticed that

higher concentrations of crude juice and

essential oils were required to inhibit g-ve

bacterial strains.

Dried and powdered bulbs of A. sativum at all

the concentration levels i.e. 0.0, 0.1, 0.2, 0.4,

0.6,0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,

5.5, 6.0 (%, w/v), remained ineffective in

arresting bacterial strains, and visible growth

of all the microbes was noticed on 2nd

day of

incubation, as in control set of petriplates,

without any spice sample.

Discussion

Functional properties of aromatic plant/spices

etc. are contained in their volatile aromatic

secrections commonly known as essential

oils8. A. sativum bulbs have 0.1%-0.25%




Page 65

essential oil, which is composed of 60%

diallyl disulphide (Allicin), 20% diallyl

trisulphide, 6% allyl propyl disulphide and

diallyl sulphide9. The inhibitory activity of

essential oil of A. sativum is widely attributed

to diallyl disulphide (Allicin). Allicin is highly

volatile and is formed by the action of enzyme

allinase on alliin (an odourless precursor),

when fresh cloves/bulbs of A. sativum are cut

or bruised10

. The mode of action of allicin to

inhbit growth of bacterial strains is not yet

well understood however, it may involve:

hydrophobic and hydrogen bonding of active

components of essential oil to membrane

proteins, perturbation of membrane

permeability, leakage of ions and other cell

contents, inhibition of membrane embedded

enzymes, destruction of electrons transport

systems, disruption of proton motive force

(PMF) and coagulation of cell contents

leading to death. The ineffectivity of

aqueous extract and powderd form of A.

sativum bulbs towards test microbes in the

present experiment may be attributed to the

loss of allicin during drying of cloves/bulbs

under ambient conditions.

The greater susceptibility of g+ve bacterial

strains towards crude juice and essential oil of

A. sativum may be due to the absence of an

outer membrane in their cell membrane which

makes them more sensitive to external

environmental changes such as temperature,

pH , natural extracts, essential oils and other

antimicrobial substances. On the other hand,

the lipopolysacharides in the cell membrane of

g-ve bacteria could provide a barrier to many

antimicrobial agents, rendering these bacteria

more resistant to certain agents than g+ve

bacteria. It is worth mentioning here that

crude extract more effectively arrested

microbes during broth dilution technique than

zone inhibition assay. This may be attributed

to the direct contact of the microbes with

liquid media which might have allowed the

easy and quick diffusion of antimicrobial

components of crude juice to the target site.

Conclusion

Present in vitro study indicates that essential

oil of A. sativum inhibited food borne

pathogens most effectively and may be

considered for food preservation. Further

studies should be undertaken to elucidate the

safety, stability and organoleptic aspects of

essential oil and its precise mode of action.

Interactions of essential oil components with

different food matrices during various food

processing treatments must be the focal area

of research before their commercialization as

‘biopreservatives’.

References

1. Weber ND, Anderson DO, North JA,

Murray BK, Lawson LD, Hughes BG. In

vitro virucidal activity of Allium sativum

(garlic) extract and compounds. Planta Med

1992; 58: 417–23.

2. Shoji S, Furuishi K, Yanase R, Miyazaka

T, Kino M. Allyl compounds selectively

killed human deficiency virus-type 1-

infected cells. Biochem Biophys Res

Commun 1993; 194: 610–21.

3. Lun ZR, Burri C, Menzinger M, Kaminsky

R. Antiparasitic activity of diallyl trisulfide




Page 66

(Dasuansu) on human and animal

pathogenic protozoa (Trypanosoma sp.,

Entamoeba histolyica and Giardia lamblia)

in vitro. Ann Soc Belg Med Trop 1994;

74:51–9.

4. Reuter HD, Koch HP, Lawson LD. 1996.

Therapeutic effects and applications of

garlic and its preparations. In: Koch

HP,Lawson LD (eds) Garlic: the science

and therapeutic application of Allium

sativum L. and related species. Williams

and Wilkins, Baltimore, pp 135–213.

5. Iroegbu CU, Nkere. Evaluation of the

antibacterial properties of Picralima nitida

stembark extracts. International J Mol Med

Adv Sci 2005; 1: 182-9.

6. Kim HO, Park SW and Park HD.

Inactivation of Escherichia coli 0157:H7

by cinnamic aldehyde purified from

Cinnamomum cassia shoot. J Food Micro

2004; 21:105-10.

7. Azzouz MA, Bullerman LB. Comparative

antimycotic effects of selected herbs,

spices, plant components and commercial

antifungal agents. J Food Prot 1982; 45:

1298-1301.

8. Pruthi JS. Spices and Condiments. National

Book Trust, New Delhi, India, 1976; pp.

117–21.

9. Stoll V, Seebeck E. Allium compounds. I.

Alliin the true mother compound of garlic

oil. Helv Chem Acta 1948 ; 31:189.

10. Lawson LD. The composition and

chemistry of garlic cloves and processed

garlic. In: Koch HP, Lawson LD (eds)

Garlic: the science and therapeutic

application of Allium sativum L. and related

species. Williams and Wilkins, Baltimore,

1996; pp 37–107.

Table 1: Bacterial strains tested

Bacterial strains Strain

number

Media used Temperature of

incubation

Time period of incubation

Bacillus cereus MTCC 430

Nutrient agar, Nutrient broth

300 C 24-48 h.

Enterococcus

faecalis

MTCC

439

Ethylviolet azide dextrose

agar, Ethylviolet azide

dextrose broth

450 C 24-48 h.

Escherichia coli MTCC 1687

MacConkey agar, MacConkeybroth

450 C 24-48 h.

Psuedomonas

aeruginosa

MTCC

424

Nutrient agar, Nutrient

broth

320 C 24-48 h.

Psuedomonas

alkaligenes

MTCC 405


320 C 24-48 h.

Shigella sonnei MTCC

2957

Nutrient agar, Nutrient

broth

320 C 24-48 h.

Staphylococcus

aureus

MTCC 5021


370 C 24-48 h.




Page 67

Table 2: Inhibitory effect of aqueous extract essential oil and crude juice of A. sativum on

bacterial strains

Results are expressed as mean±SD (n=3); DMSO: Dimethylsulphoxide

Table 3: Minimum inhibitory concentrations of different forms of A. sativum against

bacterial strains

Test

Substances

Bacterial strains

B.cereus E.faecalis E.coli P.aeruginosa P.alkaligenes S.sonnei S.aureus

Essential

oil

(ul/ml)

62.50 250.00 500.00 250.00 250.00 250.00 125.00

Crude

juice

(ul/ml)

500.00 1000.00 2000.00 1000.00 2000.00 1000.00 500.00

DMSO

(ul/ml) ND ND ND ND ND ND ND

ND: Not Detected; DMSO: Dimethylsulphoxide

Bacterial

strains

Zones of Inhibition (mm)

DMS

O

(10

ul)

Essential

oil

(10 ul)

Aqueous extract Crude juice

(80 ul) (100

ul)

(80 ul) (100 ul)

B.cereus 0.00 28.00±0.32 0.00 0.00 0.00 12.00±0.24

E.faecalis 0.00 5.10±0.07

0.00 0.00 0.00 0.00±0.00

E.coli 0.00 4.00±0.23

0.00 0.00 0.00 0.00±0.00

P.aeruginosa 0.00 19.00±0.10

0.00 0.00 0.00 9.00±0.06

P.alkaligenes 0.00 16.00±0.13

0.00 0.00 0.00 0.00±0.00

S.sonnei 0.00 15.10±0.08

0.00 0.00 0.00 0.00±0.00

S.aureus 0.00 19.20±0.17

0.00 0.00 0.00 16.10±0.09

Singh Meenu et al IN-VITRO ESTIMATION OF FREE RADICAL SCAVENGING ACTIVITY OF FRUIT JUICES BY

DPPH ASSAY


Page 68

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research


Revised on:23/07/12


IN-VITRO ESTIMATION OF FREE RADICAL SCAVENGING

ACTIVITY OF FRUIT JUICES BY DPPH ASSAY

Singh Meenu, Pragzna Yanamadala, Dharmadev Bommi

Department of Pharmacology, CMR College of Pharmacy, Kandlakoya

(V), Hyderabad, A.P. India

Email of corresponding author: [email protected]

ABSTRACT

Objective: A number of different beverage products claim to have antioxidant potency due to

their perceived high content of polyphenols. The present study was designed to evaluate the in

vitro estimation of free radical scavenging activity of three different fruit juices as follows:

Grapes, Guava and Pineapple available in local market.

Method: Above mentioned fruit juices were screened for their free radical scavenging property

using diphenyl picryl hydrazyl (DPPH) radicals. The absorbance of these fruit juices at 517nm is

found by using UV-spectroscopy at various time intervals such as 15 minutes, 30 minutes and 24

hours respectively. Then the absorbance was compared with each other and better antioxidant

activity of the fruit juices was estimated.

Results: The results obtained from the DPPH assay demonstrated that Grapes juice had better

antioxidant activity than Pineapple and Guava juices.

Conclusions: The present research demonstrates that although a number of popular beverages

have evidence of antioxidant activity, there are clear differences in antioxidant potency. Some

beverages with lower potency would need to be consumed in much larger amounts to equal the

antioxidant potency of Grapes juice.

Keywords: Anti-oxidant activity, DPPH (diphenyl picryl hydrazyl), Grapes, Guava, Pineapple.

INTRODUCTION

Clinical trials and epidemiological studies have

established an inverse correlation between the

intake of fruits and vegetables and the

occurrence of diseases such as inflammation,

cardiovascular disease, cancer, and aging-

related disorders 1. The defensive effects of

natural antioxidants in fruit and vegetables are

related to three major groups; vitamin,

phenolics and carotenoids and are believed to

be the effective nutrients in the prevention of

these oxidative stress related diseases 2,3

. There

is therefore a parallel increase in the use of

methods for estimating the efficiency of such

substances as antioxidants 4,5

. One such method

that is currently popular is based upon the use

of the stable free radical 2,2-di(4-

tertoctylphenyl)- 1-picrylhydrazyl (DPPH)

which was an easy and accurate method with

regard to measuring the antioxidant capacity of

fruit and vegetable juices or extracts 4.

Pineapple fruit is considered a highly nutritious

fruit because it contains a high level of vitamin

C, a natural antioxidant which may inhibit the


DPPH ASSAY


Page 69

development of major clinical conditions

including heart disease and certain cancers 6.

The fruit also contains phenolic compounds

and β-carotene 7,8

, which constitute natural

sources of antioxidants. Guava (Psidium

guajava L.) fruit is considered a highly

nutritious fruit because it contains a high level

of ascorbic acid (50–300 mg/100 g fresh

weight), which is three to six times higher than

oranges. Phenolic compounds such as

myricetin and apigenin 9, ellagic acid, and

anthocyanins 10

are also at high levels in guava

fruit.

The grapes has been well recognized

worldwide for over 2,000 years as one among

the edible sweet fruits and recognized for its

wide spectrum of biological properties.

Resveratrol (3,5,40-trans-trihydroxystilbene)

is a natural phytoalexin abundantly found in

grapes and red wine, which has potent

antioxidant property 11

. Thus, red fruit juices

such as grapes and others like guava and

pineapple have received attention due to their

antioxidant activity.

Whereas there are numerous phytochemicals

consumed in our diet, polyphenols constitute

the largest group and have attracted much

attention due to their antioxidant properties 12

.

In fact, the potential health benefits of plant

foods are commonly linked to their

polyphenol content. Currently, there are a

number of commercial ready-to-drink (RTD)

polyphenol-rich beverages, which base their

marketing strategies on antioxidant potency.

However, to the best of our knowledge, data

on the direct comparison of antioxidant

activity of these widely available leading

beverage products have not been obtained. It

is of great interest to the general public to

know the antioxidant capacity of the

beverages that they consume. However, it

should be cautioned that because of the

inherent complexity of food matrices, the use

of one antioxidant capacity method to

determine antioxidant potency is ineffective.

This is because antioxidants respond to

different reactive species in different tests,

which is partially attributed to multiple

reaction mechanisms and reaction phases 13,14

.

The aim of the current study was to compare

the antioxidant activity of three marketed fruit

juices i.e., Grapes, Guava and Pineapple juices

by DPPH assay.


Ready-to-Drink Polyphenol-Enriched

Beverages

The following preparations are obtained from

the local provisional market of Hyderabad:

Grape, Guava and Pineapple of “Real

Company”. All fruit juices were analyzed in

late March or early April prior to their

expiration dates as stated on their packages.

All beverages were kept at storage conditions

as specified on their labels prior to analyses.

Analysis of Ascorbic acid content (AAC)

The AAC was determined by the iodine

titration method 15

or the RP-HPLC method:

Waters C-18 column (3.9×150 mm, 5µm

particle size), mobile phase 5% acetic acid (Sd

Fine Chem Limited, Mumbai), flow-rate 0.5

mL/min and 254 nm detection wavelength.

Analysis of Total phenol content (TPC)

TPC was determined using the Folin-

Ciocalteu’s reagent 16

(LOBACHEMIE,

Mumbai). Samples (0.3 mL, triplicate) were


DPPH ASSAY


Page 70

introduced into test tubes followed by 1.5 mL

of Folin-Ciocalteu’s reagent (diluted 10 times

with water) and 1.2 mL of sodium carbonate,

7.5%w/v (Virat Lab, Hyderabad). The tubes

were vortexed, covered with parafilm and

allowed to stand for 30 min. Absorption at 765

nm was measured. If the sample absorbance

exceeded 1, the sample was appropriately

diluted to give reading less than 1. Total

phenol contents were expressed in gallic acid

equivalents (mg per 100 g fresh fruit).

Free Radical Scavenging Capacity:

The free radical scavenging capacity was

analyzed by the DPPH assay 17,18

. 2,2-

diphenyl-1-picrylhydrazy, DPPH (Santa cruz

biotechnology, Inc) is a radical generating

substance that is widely used to monitor the

free radical scavenging abilities (the ability of

a compound to donate an electron) of various

antioxidants. The DPPH radical has a deep

violet color due to its impaired electron, and

radical scavenging can be followed

spectrophotometrically by the loss of

absorbance at 517 nm, as the pale yellow non

radical form is produced 19

. The DPPH assay

was typically run by the following procedure:

In this method five dilutions of each fruit juice

with two replicates were analyzed. Reaction

solution was prepared by mixing 50 µL of

diluted fruit juice with 300 µL of methanolic

DPPH solution (1mM) and the final volume

was brought to 3 mL with methanol (Sd Fine

Chem Limited, Mumbai). The solution was

kept in dark at room temperature for 15

minutes. The absorbance (Ajuice) was read

against the prepared blank (50 µL diluted fruit

juice, 2950 µL methanol) at 517nm. A DPPH

blank solution was prepared (300 µL of 1mM

DPPH solution, 2.7 mL of methanol) and

measured. Percent inhibition of DPPH radical

was calculated for each dilution of juice

according to formula:

% Inhibition = [(ADPPH-Ajuice)/ADPPH×100]

Where ADPPH is the absorbance value of the

DPPH blank solution, Ajuice is the absorbance

value of the sample solution. IC50, the

concentration of antioxidant required for 50%

scavenging of DPPH radical in the specified

time period was derived from the % Inhibition

vs Concentration plot. Results are shown in

table and graphs.

Each category of the juice sample contained

the extract optimized for total phenolics,

ascorbic acid content and IC50 values

respectively. Each value is the mean ±

Standard deviation (n = 3). Means in the same

row bearing different letters are significantly

different (p<0.05) as analysed by the scheffe

test.

RESULTS

The results of total phenolic, ascorbic acid

contents and IC50 of different fruit juices are

summarized in Table 1. From the table the

total phenolic content was found to be highest

in Grapes juice 116.73 ± 0.14 mg/100g

followed by Pineapple juice 95.20 ± 1.10

mg/100g and lowest in Guava 79 ± 0.06

mg/100g. The change in colorization from

violet to yellow and subsequent fall in

absorbance of the stable radical DPPH was

measured at 517nm for various concentrations

i.e. 50-250 μg/mL. The IC50 value for each

fruit extract defined as the concentration of

extract causing 50% inhibition of absorbance

was calculated, since IC50 is a measure of

inhibitory concentration, a lower IC50 value

would reflect greater antioxidant activity of

the sample. Antioxidant activity among the


DPPH ASSAY


Page 71

fruit samples was found to be maximum in

Grapes having lowest IC50 value of 0.79 ±

0.34 mg/mL and minimum in Guava having

highest IC50 1.71 ± 0.61 mg/mL as lower IC50

value would reflect greater antioxidant activity

of the sample.

DISCUSSION

A polyphenol-rich food with health benefits

has become a more common element in food

marketing these days. The public is highly

aware of the term “antioxidant”, which has

been defined by the Institute of Medicine of

the National Academy of Sciences as follows:

“a substance in foods that significantly

decreases the adverse effects of reactive

species, such as reactive oxygen and nitrogen

species, on normal physiologic function in

humans.” Therefore, the marketing of many

so-called “superfoods” is commonly based on

their antioxidant potential. Multiple assays

with different sensitivities and specificities for

antioxidant activity are being used separately

to justify health claims. Consumers have a

difficult time distinguishing among the

various antioxidant claims for widely

available antioxidant beverages. Therefore,

the present study was significant in comparing

the most commonly available brands of

beverages for antioxidant activity using the

well-known and established laboratory

methods for determining antioxidant capacity.

Grapes juice had the highest antioxidant

capacity and the most complete antioxidant

coverage in vitro which may be contributed to

its constituent, phytoalexin. The present

research demonstrates that although a number

of popular beverages have evidence of

antioxidant activity in vitro, there are clear

differences in antioxidant potency.

The bleaching of the DPPH solution increases

regularly with increasing amount of fruit in a

given volume of solution. The bleaching

action is mainly attributed to the presence of

polyphenols and ascorbic acid extracted into

the solution. The total phenolic content, IC50,

and the ascorbic acid content of the fruit juices

are summarized in Table 1, Graphs 1, 2 and 3.

For a given amount of fruit, the higher the

absorbance, the better is the reducing power.

Correlation of IC50 with reducing power:

DPPH assay measures the ability of the extract

to donate hydrogen to the radical. In DPPH

assay the lower the IC50 the better it is able to

scavenge the radicals, particularly peroxy

radicals which are the propagators of the

autoxidation of lipid molecules and thereby

break the free radical chain reaction 20

. It is

observed that grapes having low IC50, is a very

potent radical scavenger. In terms of reducing

power, grape rank highest but guava is

significantly lower than that of pineapple. The

high antioxidant potential (as characterized by

low IC50 and high reducing power) of grape is

attributed to its high TPC and AAC. The low

antioxidant potential of guava (IC50 = 1.71 ±

0.61 mg/mL) is due its low TPC and AAC.

Pineapple in spite of its relatively high TPC

has low antioxidant potential (IC50 = 0.83 ±

0.24 mg/mL). Three possible reasons may be

able to account for this: First, it has been

reported that 21

reaction of DPPH with certain

phenols such as eugenol and its derivatives is

reversible, resulting in low readings for

antioxidant activity (% disappearance). The

second possible reason could be due to the

slow rate of the reaction between DPPH and

the substrate molecules 13

.The third possible

explanation (for the relatively low reducing

power) could be that certain phenols in the


DPPH ASSAY


Page 72

pineapple juice have a higher redox potential

than that of other fruit juices. To clarify this

anomaly further work is necessary. Finally, it

is also observed that the antioxidant potential

correlates well with AEAC.

CONCLUSION

For a body to maintain antioxidant level,

external supplementation is necessary for

healthy living. From the present research the

order of the antioxidant activity in the fruit

juices was found to be

Grapes>Pineapple>Guava. These fruits can be

used as alternative source of natural

antioxidant rather than synthetic antioxidant

like BHT (Butylated hydroxytoluene) and

BHA (Butylated hydroxyanisole) because of

carcinogenicity.

ACKNOWLEDGEMENT

We thank everyone who supported this study.

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be Healthy-The HarVard Medical School

Guide to Healthy Eating. Am J Epidemiol

2001;154(12):1160.

2. Ames BN, Gold LS, Willet WC. The

causes and prevention of cancer. Proc Natl

Acad Sci USA 1995;92:5258-5265.

3. Kaur C, Kapoor HC. Antioxidants in fruits

and vegetables-the millennium’s health.

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725.

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used to evaluate the free radical

scavenging activity in foods and biological

systems. Food Sci Tech Int 2002;8(3):121-

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5. Schwarz K, Bertelsen G, Nissen LR,

Gardner PT, Heinonen MI, Hopia A, et al.

Investigation of plant extracts for the

protection of processed foods against lipid

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antioxidant compounds. Eur Food Res

Technol 2001;212:319-328.

6. Diplock AT. Antioxidants and disease

prevention. Molecular Aspects of

Medicine 1994;15:293-376.

7. Gardner PT, White TAC, McPhail DB,

Duthie GG. The relative contributions of

vitamin C, carotenoids and phenolics to

the antioxidant potential of fruit juices.

Food Chemistry 2000;68:471-474.

8. Charoensiri R, Kongkachuichai R,

Suknicom S, Sungpuag P. Betacarotene,

lycopene, and alpha-tocopherol contents of

selected Thai fruits. Food Chemistry

2009;113:202-207.

9. Miean KH, Mohamed S. Flavonoid

(myricetin, quercetin, kaempferol, luteolin,

and apigenin) content of edible tropical

plants. Journal of Agricultural and Food

Chemistry 2001;49:3106–3112.

10. Misra K, Seshadri TR. Chemical

components of the fruits of Psidium

guajava. Phytochemistry 1968;7:641–645.


DPPH ASSAY


Page 73

11. Yadav M, Jain S, Bhardwaj A, Nagpal R,

Puniya M, Tomar R, et al. Biological and

Medicinal Properties of Grapes and Their

Bioactive Constituents: an Update. J Med

Food 2009;12(3):473-484.

12. Proteggente AR, Pannala AS, Paganga G,

Van Buren L, Wagner E, Wiseman S, et

al. The antioxidant activity of regularly

consumed fruit and vegetables reflects

their phenolic and vitamin C composition.

Free Radical Res 2002;36:217-233.

13. Huang D, Ou B, Prior RL. The chemistry

behind antioxidant capacity assays. J

Agric Food Chem 2005;53:1841-1856.

14. Prior RL, Wu X, Schaich K. Standardized

methods for the determination of

antioxidant capacity and phenolics in

foods and dietary supplements. J Agric

Food Chem 2005;53:4290-4302.

15. Suntornsuk L, Kritsanapun W,

Nilkamhank S, Paochom, A. Quantitation

of vitamin C content in herbal juice using

direct titration. Journal of Pharmaceutical

and Biochemical Analysis 2002;28:849-

855.

16. Singleton VL, Rossi JA. Colorimetry of

total phenolics with phosphomolybdic–

phosphotungstic acid reagents. American

Journal of Enology and Viticulture 1965;

16:144-158.

17. Malterud KE, Farbrot TL, Huse AE, Sund

RB. Antioxidant and radical scavenging

effects of anthraquinones and anthrones.

Pharmacology 1993;47:77-85.

18. Nenseter MS, Halvorsen B, Rosvold Q,

Rustan AC, Drevon CA. Paracetamol

inhibits copper ion-induced, azo

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1995;15:1338-1344.

19. Seeram NP, Aviram M, Zhang Y, Henning

SM, Feng L, Dreher M, et al. Comparison

of Antioxidant Potency of Commonly

Consumed Polyphenol-Rich Beverages in

the United States. J Agric Food Chem

2008; 56:1415-1422.

20. Frankel EN. Recent advances in lipid

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and Agriculture 1991; 54:495-511.

21. Bondet V, Brand-Williams W, Berset C.

Kinetic and mechanisms of antioxidant

activity using the DPPH free radical

method. LWT-Food Science and

Technology 1997;30 (6):609-615.


DPPH ASSAY


Page 74

Table 1: Table showing total phenolic content,

ascorbic acid content and IC50 of fruit juices

of the Real company.

Sl.

No.

Fruit

juices

TPC

(mg/100g)

AAC

(mg/100g)

IC50

(mg/mL)

1 Grapes 116.73 ±

0.14a

144 ±

1.02c

0.79 ±

0.34b

2 Guava 79 ± 0.06b

132 ±

0.62a

1.71 ±

0.61a

3 Pineapple 95.20 ±

1.10a

98 ± 0.3b

0.83 ±

0.24c

Graph 1: Total phenolic content of the three

fruit juices of the Real Company

Graph 2: Ascorbic acid content of the three

fruit juices of the Real company

Graph 3: IC50 values of the three fruit juices of

the Real company

Muhammad Farzanjou

CAREER, JOB SATISFACTION AND ITS EVALUATION METHODS


Page 75

IJCRR

Vol 04 issue 17

Section: Management

Category: Review

Received on: 13/06/12

Revised on: 23/07/12

Accepted on: 10/08/12

CAREER, JOB SATISFACTION AND ITS EVALUATION

METHODS

Muhammad Farzanjou

Technical and Vocational Faculty of Shahid Bahonar, Technical and

Vocational University, Zahedan, Iran


ABSTRACT

Job is among the issue that has often been engaged by human mind, governments and nations.

Although job and occupation is apparently associated with economic-subsistence aspect of

humans, it is closely linked to their personal, familial, social, political and cultural dimensions.

Job satisfaction is a filed within which social psychological, sociological, economic and political

and educational sciences perspectives have been mentioned. Today, there are thousands of job

and professions in each country which people are involved in and thereby continue their life.

What always attracts the attention of psychologists and social sciences thinkers are people's job

satisfaction and the effects of this satisfaction in their spirits and working productivity. If one is

not interested in his / her job, one's creativity and talent will not be flourished in one's work field

and then will be affected by fatigue, depression and frustration and his/her work will be

inconclusive, and hereby the community will be affected. This article aims to consider this issue.

Keywords: job, job satisfaction, factors of job satisfaction, methods of assessment

INTRODUCTION

Definition of Job

Job literally means gets someone to work and

what is the cause of engagement. The

individuals will actively participate in the

production processes and services through

employment and then receive cash or material

rewards (1). Job and work is a physical or

intellectual activity which is directed on

production and services. Generally, job is an

activity which is asked for and hence is paid

by (2). In summary, it can be said that job is

the work a person is involved with and

through which perform his/her duties and gain

his livelihood. In another definition, job is a

group of similar positions in an institution,

office or workshop in which the quailed

people are able to take this opportunity and

carry out their duties (3).

Definition of Job Satisfaction

Job satisfaction is a set of feelings and beliefs

that people have about their current jobs (4).

Job satisfaction is one of the important factors

in job success, a factor that increases

efficiency and also feeling of personal

satisfaction (5). Job satisfaction means to be

interested in the situations and requirements of

a job, a situation in which a work is done and

the reward for which is received (6).

According to the above-mentioned contents, it

can be indicated that job satisfaction is

feelings of fulfillment and satisfaction one

have of him/herself and the pleasure is

Muhammad Farzanjou



Page 76

therefore achieved and consequently will be

encouraged and attached to his/her job. Job

satisfaction is a desirable, emotional and

positive state that is resulted from job

assessment or job experiences. It is a concept

that has various dimensions, aspects and

factors which must be taken into consideration

as a whole. Of these factors are the

characteristics of employer and employee,

type of work, work environment and human

relations in working (7).

V. E. Fisher and J.V.Hanna maintain that jib

satisfaction is an internal factor and believes

that it is a type of emotional compatibility

with job and job conditions, that is if the

regarded job provides the satisfaction and

desirable pleasure, he/she will be happy with

his/her job, and conversely, if the regarded job

does not provide the satisfaction and desirable

pleasure, he/she will be disappointed and is

going to change it (8).

R. Hoppock maintains that job satisfaction is a

complex and multi-dimensional concept and is

associated with mental, physical and social

factors. Only one factor is not led to job

satisfaction, but a specific combination of a

set of various factors cause an employer is

satisfied with his/her job in a specific moment

of time, and tell him/herself that he/she is

satisfied with his/her job and enjoys it (9).

it is found out from the above0definitions

about job satisfaction that this concepts

accounts for the positive and feelings and

attitudes which one has towards his/her job.

When it is said that someone has high levels

of job satisfaction (10), it means he/she

generally likes his/her job and considers a

great value for it and takes it into account as a

positive issue, in short, he has a good and

desirable feeling towards it (11).

Factors of Choosing Job

Job is an issue that is not randomly chosen,

but it requires many factors, including:

Physical Condition

Each job requires a certain physical

characteristics. A great size and strength is

required in some jobs, while these features

may prevent doing some tasks in some jobs.

Also, having hand and foot health is necessary

in some jobs, while in others, lacking or

defecting limbs and other organs does not

create any problem.

Talent

One of the important factors in job selection

and continuing a successful employment is

talent. Talent means being equipped with,

readiness and ability to perform a certain job,

that is one's innate ability that helps him learn

and accelerates it. Hence, talent predict the

method and amounts of learning in various

fields in the future, and the one who is gifted

in a certain area will be more benefitted with

his/her experiences in that regard.

Interest

Interest means having desire, willingness and

desire to acquire something. Also, the pleasant

feeling, desire or curiosity towards something

or concept is called interest. Interest is an

important impetus for effort and human's

activity. Success in any job requires having

interest.

Personal and Social Facilities

In addition to the above-mentioned materials,

other factors such as personality, realism,

environmental facilities and community needs

has an important effect in choosing job (12).

In short, it can be said that personal factors

(such as physical status, talent, interest and

personality traits), social factors (such as

family pressure, social and cultural values, the

Muhammad Farzanjou



Page 77

amount of facilities in a community and the

opportunities that are available to individuals),

economic factors (such as poverty and

unemployment) as well as inheritance and

gender are effective in job selection.

Factors of Job Satisfaction

Researchers have long been in search of

fundamental causes of job selection in offices

and organizations. They have been able to

achieve a string of constant factors associated

with job satisfaction; however, not a

comprehensive empirical model has yet been

achieved. Several factors can be briefly

pointed out that are more important in this

field. W. Porter and M. Steers pointed out to

four factors as follows:

Overall factors of organization: i.e.

variables those are widely applicable to

most employees, such as salary and

promotion opportunities.

The immediate causes of occupational

environment: variables representing

occupational groups, such as supervision

method and quality of relationship with

colleagues, working conditions and

workplace.

Content factors or actual occupational

activities, such as occupation territory (of

diversity, autonomy and responsibility)

and role clarity.

Individual factors: the characteristics that

make a person distinct from another as

well as age, duration of service and

character (self-confidence, determination

and maturity) (13).

E.A.Locke summarizes the most important

factors influencing job satisfaction as follows:

Mental precarious work that a person can

be successfully compatible with (success

in coping with the work).

Personal interest to the job itself in that

the more one is interested in a job, the

more would be his/her satisfaction.

A work that is not physically too boring,

that is the more is a person tired, the less

will be his/her satisfaction and the less

one is tired, the more will be his/her

satisfaction.

Reward for performance would be fair,

informative and consistent with one's

demand.

Working conditions that would be

consistent with physical needs and

contribute to career goals.

Feeling of self-esteem from the

employed, the more one feels respect

from others, the more will be his/her

satisfaction.

Factors in workplace that facilitate the

need for occupational values, such as

increased pay and promotion (14).

Results of having job satisfaction

Being aware of significant results of job

satisfaction is as important as being aware of

what leads to satisfaction. These results are

as follows:

Satisfaction and Service Renunciation

Job satisfaction and service renunciation is

related with each other. V.H.Vroom found

out that the correlation range between these

two variables in various studies is from 25%

to 42%.

Revisionists in recent decades, which studied

the relationship between job satisfaction and

job renunciation, reported that there is a

Muhammad Farzanjou



Page 78

negative relationship between them, that is;

if employees are satisfied with their job, they

will not leave their job and vice versa, that is

if they are not satisfied with their job, they

will leave their job. A relatively similar

report was presented by Lock in 1976.

Job Satisfaction and absence in working

The evidence shows that there is a balanced

and an inverse relationship between job

satisfaction and absence of employees from

their workplace. Vroom showed in various

studies that the compatibility range is from

14% to 38%. This study was confirmed by

Porter and Stirz, etc.

Satisfaction and Performance

One of the most controversial issues in the

field of job satisfaction is its relationship

with performance. Three hypotheses have

been proposed in this regard.

1. Satisfaction is led to performance.

2. Performance is led to satisfaction.

3. Reward acts as an intermediate between

performance and satisfaction.

The first two hypotheses are weakly

supported, but the third one, based on which

reward acts as an intermediate between

performance and satisfaction, has been more

strongly supported. Prior performance is

resulted in receiving internal reward (feeling

of personal prosperity) and external reward

(wage and promotion). This reward, by itself,

enhances one's performance in the future and

also is effective in increasing one's job

satisfaction.

Vroom found out in his studies that there is a

positive relationship between job satisfaction

and amount of efficiency and performance.

Steers and Porter mentioned in their books

that the more an employee and worker's

occupational motivation and the more their

attitude towards their jobs (that is, to be more

satisfied with his/her job), the more will be

their performance and vice versa, that is, the

less is motivation and positive attitude

towards one's job (the less job satisfaction),

the less will be one's performance.

The Effect of Satisfaction on Organization

The analyses indicate that when employees of

an organization are satisfied with their jobs,

their organization are faced with positive

effects and acts as an effective and useful

organization.

In addition to above-mentioned issues, job

satisfaction has some other results. The

employees who are entirely satisfied are less

intended to submit complaint and are more

acquired with physical and mental health and

have a more longitude; they learn new duties

related to their job more swiftly and are

acquired with less occupational accidents (15).

Suggestions

According to the above contents, the

following works are suggested for increasing

job satisfaction in an organization:

Making employees interested in their job to

flourish creativity and innovation.

Preventing from depression and frustration

in employees.

Creating factors that increase efficiency

and satisfaction in organization.

Creating significant motivation and interest

for the effort and willingness of people to

successfully perform the job

Being respected from other people to

provide greater satisfaction.

Muhammad Farzanjou



Page 79

CONCLUSION

By reflecting and hesitating on what was

mentioned and by considering the strategic

issues and the coherent guidance of

organization, which reminiscent of

undeniable and important role of managers

which is certainly highly effective in victory

or defeat of programs, creating " job

satisfaction" in a person depends on various

factors which together are achieved to

optimal result, and somehow lack of one

factor in a person is led to one's job

dissatisfaction; factors such as income level,

nature of work and its social status,

organizational prestige and authenticity of

job promotion, job security, lack of role

ambiguity, physical working condition,

structure and organizational culture and

relationship with colleagues, paying

attention to one's personality traits,

performance evaluation, fitness, flexibility

and innovation. Finally, it can be said that

job satisfaction is totally a positive and

pleasant feeling that one has about his/her

job. More generally, scientists maintain that

social factors, working environment and the

nature of working are effective in job

satisfaction. All theories of job satisfaction

somehow emphasize on meeting people's

needs, whether physical or mental and takes

the demands and expectations of employees

as an important thing.

REFERENCES

1. Abdollah Shafiabadi, "professional and

career counseling and guidance", Roshd

Publications, P. 3, Tehran, Iran, 1997.

2. Boures. A. Shertzer, "occupational life

planning and review", translated by

Zandipour, P. 207, Tehran, Iran, 1990.

3. Abdollah Shafiabadi," Professional ad

career guidance and counseling", Roshd


4. George. M. Jennifer & Jones. R.

Gareth,”Organizational Behavior

Understanding and managing”, Addison

Wesley, P. 74, New York,U.S.A,1999.

5. Abdollah Shafiabadi,"Professional ad

career guidance and counseling", Roshd


6. Boures. A. Shertzer, "occupational life

planning and review", translated by

Zandipour, P. 207, Tehran, Iran, 1990.

7. Hellriegel. Don, Woodman. W.

Richard,” Organizational Behavior”

,South - Western College Publishing An

International Thomson Publishing

Company,P. 53-55, 1996.

8. Gholam Abbas Tavasoli, "working and

occupation sociology", SAMT

Publications, PP. 166-170, Tehran, Iran,

1996.

9. Khadijeh Safiri, "sociology of women

employment", Tabyan Publications, P.

76, Tehran, Iran, 1998.

10. Abbas Mahmoudzade, Armen

Mehroujan, "organizational behavior of

contingency perspective", Allameh

Tabatabaie University Press, P. 274,

Tehran, Iran, 1996.

11. Abdollah Shafiabadi, "career and

academic guidance", publication center

and Payam Nour University Press,

Tehran, Iran, 1991.

12. Abbas Mahmoudzade, Armen

Mehroujan, "organizational behavior of

contingency perspective", Allameh

Tabatabaie University Press, P. 374,

Tehran, Iran, 1996.

Muhammad Farzanjou



Page 80

13. Iraj Soltani, "Productivity of Human

Resources", Arkan Publications, Isfahan,

Iran, 2006.

14. Leap. L. Terry, Crion. D. Michael,

“Personal Human Resource

Management”, Macmillan Publishing, P.

366-367, New York, 1989.

15. Norollah Khalilzade, "Study of the

factors effective in job satisfaction and

dissatisfaction of students and teachers of

Payame Nour University of Oroumieh",

Faculty of Psychology and Educational

Sciences, Tehran University, PP. 21-25,

Tehran, Iran, 1997.

Niladri Sekhar Das DRUG RESISTANCE PROFILE OF NEW PTB PATIENTS WITH OR WITHOUT HIV INFECTION


Page 81

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research




DRUG RESISTANCE PROFILE OF NEW PTB PATIENTS WITH

OR WITHOUT HIV INFECTION

Niladri Sekhar Das

College of Medicine and JNM Hospital, West Bengal University of Health

Sciences, Nadia, West Bengal, India


ABSTRACT

Tuberculosis is the commonest opportunistic infection in persons infected with human

immunodeficiency virus (HIV). HIV is the most powerful risk factor for reactivation of latent

tuberculosis infection to active disease. Emergence of drug resistant isolates of M. tuberculosis

(Mtb) highlights the need for continuous monitoring of drug resistance to anti tuberculosis drugs.

The aim of our study was to see the anti tubercular drug resistance profile of M.tb in HIV

seropositive and seronegative patients.

Material and methods: We enrolled 100 new pulmonary tuberculosis (PTB) patients. This study

was carried out at Department of Microbiology MGIMS, Sevagram, Wardha Maharashtra from

2007-2009. For every patient staining and culture was done from sputum sample by conventional

method. After the identification of isolates as M.tb drug sensitivity testing was done. HIV

antibody status was identified in every patient by using rapid and ELISA test. Results were

analyzed using SPSS software.

Results: There were 8 patients who were HIV seropositive out of total 35 culture positive cases.

No resistant patterns were detetected in case of HIV +ve patients with TB infection. Out of 27

HIV seronegative cases with TB infection, 4 cases showed mono-resistant to streptomycin. No

multi drug resistant (MDR) strain was detected, neither in HIV positive nor in HIV negative

cases.

Conclusion: HIV infection may not be associated with drug resistant tuberculosis. However due

to high prevalence of HIV–TB infection in the country, monitoring of drug resistance in M.

tuberculoisis isolates needs prioritization to ensure success in national tuberculosis control

programme.

Key words: Human immunodeficiency virus (HIV), Pulmonary tuberculosis (PTB),

Mycobacterium tuberculosis (Mtb), Multi drug resistant (MDR)

INTRODUCTION

Tuberculosis (TB) is one of the most serious

diseases that physicians have recognized and

grappled with through the millennia due to its

high risk of person to person transmission,

morbidity, and mortality. Currently there are

9.2 million new cases of tuberculosis every

year with 1.7 million deaths occurring

worldwide1. Despite the discovery of the

tubercle bacillus more than a hundred years

ago, and all the advances in our knowledge of

the disease made since then, tuberculosis still



Page 82

remains one of the major health problems

facing mankind, particularly in developing

countries. In India each year, 1.9 million new

cases of TB occur in the country, of which

about 0.8 million are infectious new smear

positive pulmonary TB cases2. The situation is

expected to worsen due to the emergence of

multi-drug resistant tuberculosis (MDRTB).

Development of drug resistance in M.

tuberculosis isolates may adversely impact the

management of the disease. Inadequate

therapies and indiscriminate use of antibiotics

causes such emergence of multi drug resistant

bacilli and HIV/AIDS pandemic resulting in

accumulation of pool of individuals who are

more susceptible to TB have worsened the

TB scenario. A High HIV seroprevalence

among newly diagnosed TB patients has been

reported in India.3, 4

.

On the global situation of drug resistance in

M.tb , first comprehensive study was

conducted by the Research and Surveillance

unit, Global TB Programme, WHO5 and was

observed that, the rates of acquired resistance

were invariably higher than primary resistance

for each drug and multi drug resistance was

more common when resistance was acquired

rather than when it was primary. The

increasing prevalence of multidrug resistance

in several parts of the world including India

has been one of the major reasons for

declaring tuberculosis control as a global

emergency by WHO6,7

.

Several outbreaks of MDR-TB showed the

importance of continuous monitoring of drug

resistance for treatment of TB patients and

initiating adequate public health measures.

The present study was undertaken with the

objective of comparing the anti TB drug

resistance profile of MTB isolates in new PTB

patients with or without HIV infection.

MATERIAL AND METHODS

Among 100 suspected tuberculosis patients

study was done at Microbiology Laboratory in

MGIMS, Sevagram between 2007 to 2009

with due permission from the institutional

ethical committee.

Sputum was collected from each individual,

smear was made and staining was done by

Ziehl–Neelson method. For isolation rest of

the sample was decontaminated by NALC-

NaOH method8 and inoculation was done on 2

LJ slopes. LJ bottles were observed weekly till

the colonies grew sufficiently for

identification whereas negative LJ culture was

noted if there was no growth after incubation

at 370c for 8 wks. Isolates were identified later

by conventional biochemical methods9

After the identification of the strain as MTB,

drug sensitivity testing was performed against

1st line drugs by LJ proportion method

described by Canetti et al 1969 10.

For Drug

sensitivity testing stock and working

concentration of drugs were made according

to Guidelines of TRC manual 200611

. The

concentration of drugs used were

Streptomycin 4 µg/ml, INH 0.2 µg/ml,

Rifampicin 40 µg/ml and Ethambutol 2 µg/ml.

Before drug sensitivity testing by LJ

proportion method, drug containing LJ slopes

was made.

At first preparation of standard suspension 11

and dilution was made. 0.1 ml of each of the

two bacterial dilutions, 10–3

mg/ml and 10–5

mg/ml were inoculated on two slopes of L J

medium without drug and one slope of

medium with drug. The standard strain of

M.tuberculosis, H37Rv was tested with each



Page 83

batch of sensitivity testing. All the LJ slopes

for DST were placed in the incubator at 37 °C.

Reading and reporting of the LJ slopes were

done according to the reference10

Detection of

HIV status in every patient was done by rapid

method (TRIDOT) using serum which was

collected in a plain bulb. If any sample was

found to be positive by rapid method,

confirmation was done by ELISA

(Vironostika) confirmed by second ELISA at

NARI Pune.

RESULTS

We got 35 total MTB isolates no NTM (Non

tubercular mycobacterium) was isolated.

Among 35 MTB isolates 87.9 percent (29/33)

from smear positive and 9 percent (6/67) from

smear negative patients were obtained.

Of these 8 isolates were HIV seropositive and

27 HIV seronegative (Table no 1). Among the

35 total MTB isolates, monoresistant was

found with Streptomycin that was 11.4% only

in HIV seronegative patients (Table no 1).

Resistance to any 1st line drug was seen

(7/35), in 20% isolates. Drug resistance to at

least one drug was found in (7/27), 25.9%

isolates in HIV seronegative cases whereas no

resistant strain was isolated even for a single

drug in HIV+ve cases however in HIV

seropositive cases we got (8/8), 100% isolates

sensitive to all four drugs. The prevalence of

polyresistance (resistance to two or more

drugs, but not both isoniazid and refampicin)

was found to be (3/27), 11.11% only in HIV

seronegative with TB infective patients.

Neither individual drug resistance nor the

MDR or polyresistance was found to differ

significantly in the HIV infected and

uninfected TB patients.

DISCUSSION

Emergence and spread of drug resistance in

M.tuberculosis is a serious threat to

tuberculosis control programme because

patients with drug resistance bacilli respond

less readily to therapy than those with

sensitive bacilli, resulting in preferential

spread of the drug resistant bacilli in the

community.

Drug resistance develops either due to

infection with a resistant strain, or as a result

of inadequate treatment such as when a patient

is exposed to a single drug, or because of

selective drug intake, poor compliance, use of

inappropriate non-standardized treatment

regimens, irregular drug supply, poor drug

quality, or rarely erratic absorption of

medications 12

. Though the development of

drug resistance in India was noted since the

beginning of the chemotherapeutic era, it was

based on clinical perception and several

isolated reports, which failed to give an idea

of the national situation as a whole.

In this direction the first definite step was

taken in 1965-67, when the ICMR conducted

two surveys to estimate the prevalence of drug

resistance13

. In this study resistance to

Isoniazid ranged from 11-20 per cent, to

Streptomycin 8-20 per cent and to both drugs

4-11 per cent. The second study showed

resistance to Isoniazid from 15-69 per cent, to

Streptomycin 12-63 per cent and to both drugs

from 5-58 per cent. However these studies

were carried out in the pre-Rifampicin era.

Subsequently after about two decades

(1980’s), 11 other studies from different parts

of the country reported similar resistance to

Isoniazid and Streptomycin like those in the

earlier studies with the notable exception of

Rifampicin resistance being observed in 10 of



Page 84

the 11 publications and the level of MDRTB

in all the centers was observed but it was less

than 5 per cent. According to third Global

Report total prevalence of drug resistance

among new cases in India (Wardha) was

19.8% and MDR was 0.5 %. Several small

surveys conducted across the country have

shown the prevalence rates of MDR-TB in the

country at around 3% among new cases, and

12% among retreatment cases. A large scale

population based survey in the states of

Gujarat and Maharashtra has also indicated

similar resistance levels (new- 3% and

retreatment- 12-17%) 13.

We found that

prevalence of resistance to isoniazid and

streptomycin was 0 and 11.4 percent and that

to both drugs were 2.8%. The issue of whether

infection with HIV is a risk factor for drug

resistant tuberculosis still remains unanswered

since results of some studies supported this

hypothesis while some did not. A study

conducted in newyork city 14

revealed that

HIV infected TB patients were significantly

more likely to develop resistance to at least

one drug and MDR than those without HIV

infection.

No significant difference was found in drug

resistance between HIV seropositive and HIV

seronegative tuberculosis patients in our

study, which is in accordance with the

findings from other studies in Europe 15, 16

.

In conclusion, we didn’t find any kind of drug

resistance in anti-tubercular drug especially in

case of HIV seropositive cases. In view of the

conflicting results from other studies and high

prevalence of HIV-TB in our country, we feel

that time to time monitoring of anti-

tuberculosis drug resistance pattern in TB

patients in general and HIV seropositive

tuberculosis patients in particular would

provide important data, which may be crucial

for the control of tuberculosis.

ACKNOWLEDGEMENTS

I want to acknowledge the persons who helped

me during the research work.

1. Dr. D.C Thamke, Professor, Department of

microbiology, MGIMS sevagram.

2. Mr Padmakar Dhone, Senior Lab

technician, Department of microbiology,

MGIMS sevagram

I also want to acknowledge the immense help



manuscript and also grateful to

authors/editors/publishers of all those articles,

journals and books from where the literature

for this article has been reviewed and

discussed. We did this research work from the

departmental funding.

REFERENCES

1. Global tuberculosis control: surveillance,

planning, financing: WHO report 2008.

Geneva: World Health Organization

(WHO/HTM/TB/2008.393).

2. TB India 2008. RNTCP Status Report.

3. Paranjape R S ,Tripathy SP, Menon PA,

Mehendale SM, khatavkar P, Joshi DR.et al.

Increasing trend of HIV seroprevalence among

pulmonary tuberculosis patients in Pune, India

.indian J Med res 1997;106: 207-11

4. Tripathy S, Joshi DR, Mehendale SM , Menon

P, Joshi AN, Ghorpade SV, et al.Sentinel

surveillance for HIV infection tuberculosis

patients in India. Indian J tuberc 2002; 49: 17-

20.

5. Cohn DL, Bustreo F, Raviglione MC. (1997).

Drug resistant tuberculosis: review of the

worldwide situation and the WHO/IUATLD

global surveillance project. Clin Infect Dis

1997; 24(1): 121-130.



Page 85

6. Anti tuberculosis drug resistance in the world.

The WHO/ WATLD global project on anti

tuberculosis surveillance

.Geneva,Switzerland;1997.WHO/TB/97.229.

7. Anti tuberculosis drug resistance in the world

,report No.2.WHO/CDS/TB/2000.278

8. Procedure for isolation and identification of

mycobacterium CDC manual1975.

9. Laboratory services in tuberculosis control

.Part 3-Culture /WHO/98.258,1998.

10. Canetti, G., Fox, W., Khomenko, A., Mahler,

H.T., Menon, N.K., Mitchison, D.A., et al.

Advances in techniques of testing

mycobacterial drug sensitivity, and the use of

sensitivity tests in tuberculosis control

programmes. Bulletin of the World Health

Organisation, 1969. 41, 21-43.

11. Bacteriological methods and Laboratory

diagnosis of Tuberculosis TRC Chennai

2006.

12. Behera D. Drug Resistant Tuberculosis In India

– Is it a matter of concern? Ind J Tuberc 2007;

54:105-109.

13. Multi-drug resistant and Extensively drug

resistant TB in India, Consensus statement on

the problem, prevention, management and

control, From the consultative meeting of

national experts organized by the TB

Research Centre, ICMR, Govt. of India, on

14-15 September 2007.

14. Gordin FM, Nelson ET,Matts JP, Cohn DL,

Ernst J,Benator D,et al. The impact of human

immunodeficiency virus infection on drug

resistant tuberculosis. Am J.Respir Crit care

Med 1996; 154: 1478-83.

15. Migliori GB, Centis R, Fattorini L, Besozzi

G, Saltini C, Scarparo C, et al.

Mycobacterium tuberculosis complex drug

resistance in Italy. Emerg Infect Dis

2004;10:752-3

16. Shafer RW, Chirgwin KD, Glat AE, Dahdouh

MA, Landesman SH, Suster B. HIV

prevalence , immunosuppression , and drug

resistance in patients with tuberculosis in an

area endemic for AIDS.AIDS 1991;5:399-

405.

Table No 1.Drug resistance pattern of M.

tuberculosis isolates to primary anti-

tuberculosis drugs

Total

(n=35)

HIV+ve

(n=8)

HIV-ve (n=27)

Any resistance

H 1(2.8%) 0 1(3.7%)

S 7(20%) 0 7(25%)

E 3(8.5%) 0 3(11.11%)

R 0 0 0

Mono resistance

H 0 0 0

S 4(11.4

%)

0 4(14.9%)

E 0 0 0

R 0 0 0

Multidrug resistance

HR

±S±

E

0 0 0

H= INH, S=Streptomycin, E= Ethambutol,

R=Rifampicin.

Nithish.U.S et al. BACTERIOLOGICAL APPLICATIONS OF QUANTUM DOTS


Page 86

IJCRR

Vol 04 issue 17

Section: Technology

Category: Research




BACTERIOLOGICAL APPLICATIONS OF QUANTUM DOTS

Nithish.U.S, Sarah Sunitha

Department of Biotechnology, PES Institute of Technology, Bangalore,

India


ABSTRACT

Quantum Dots are nanocrystals which are fluorescent in nature and their unique optical

properties depend on their size. Quantum Dots have replaced conventional fluorophores which

have disadvantages like photobleaching, narrow absorption spectra, stability and short period of

fluorescence. Due to the possibility of conjugating the Quantum Dots to various types of bio

molecules like streptavidin, antibodies, mannose etc there have been numerous applications in

the detection, enumeration and differentiation of various bacteria. Quantum Dots can be applied

to various matrices like food, water, tissue and blood samples. Quantum Dots have been used to

detect pathogenic bacteria like E.coli, Staphylococcus aureus, Salmonella typhimurium, Bacillus

anthracis, Listeria monocytogenes and Mycobacterium tuberculosis. Quantum dots can detect

bacteria at the levels of 103- 10 CFU/ml in contaminated water samples. The minimum time for

detection of bacteria using Quantum Dots ranges from 15 minutes to 2 hours. The major

hindrance in using the Quantum Dots is its cost of production. This review summarises the

properties, synthesis and applications of Quantum dots in the detection of bacteria which can

have major implications in food and water safety evaluation, diagnosis of bacterial diseases and

environmental enumeration of bacteria.

Keywords: Quantum Dots, Optical properties, Detection, Enumeration

INTRODUCTION

Quantum Dots (QD) are highly crystalline

semiconductor nanocrystals. These are

crystals whose dimension is less than that of

the Exiton Bohr radius [1]. Due to their

nanoscale size; they have discrete electronic

energy state giving rise to unique optical and

electronic properties. They also possess the

property of size dependent photo-emission

which is due to the phenomenon of Quantum

Confinement [2]. Their stability against

photobleaching has attracted many researchers

to develop bio-imaging and Quantum

computing techniques. Due to their unique

properties they have wide range of

applications in electronics, computing and

biology. QDs are made up of elements like

Cadmium, Selenium, Indium, Tellurium,

Phosphorous, Sulphur etc. These elements

make up the core of a Quantum dot, which are

then coated with a shell made of material of

higher band energy. The outer shell also forms

a protective layer on the Quantum dots. To

make QDs biologically compatible one or

more layer of organic polymers are added and

are conjugated with organic ligands. The



Page 87

diameter of QDs varies from 1-20nm which

consists of 100-100,000 atoms [2]. Due to

their distinctive properties, QDs have

improved the sensitivity of cellular analysis

and molecular detection by few folds.

The Quantum Dots were first studied by A.I.

Ekimov and A.A. Onushchenko [3]. The size

of microscopic CuCl crystals grown in

transparent dielectric matrix varied from tens

to hundreds of angstroms. Later high quality

CdSe, CdS and CdTe semiconductor

nanocrystals were synthesized based on

pyrolysis of organometallic reagents [4]. The

particles sizes were as small as 20 angstroms

in diameter. Syntheisis of water soluble

Quantum dots paved way for their conjugation

to bio molecules and thereby opened up

different applications in biology [5, 6].

In Quantum Dots, the energy levels of the

valence electrons are discrete and have large

differences. Thus they show the characteristics

of an atom and hence they are also called

artificial atoms [8]. The reduction in the size

of the crystal brings the valence electrons

closer thereby increasing their overall energy.

This leads to splitting up of different electron

levels giving it a discrete electron energy level

system and also increasing their band gaps.

This results in merging of valence band and

conduction band making it a very good

conductor of electricity. The splitting of

energy level leads to excitement of the

electron by absorption of energy and transition

to higher energy levels. But these electrons

return to their ground state by emitting a

photon of energy either equal or lesser than

that of the absorbed energy. The emitted

photon has lesser energy when the electron

rests in a metastable level for a brief period of

time. This gives QDs their fluorescent

property making them applicable in imaging

and detection of molecules or cells. Thus, as

the size reduces, the gap between energy

levels broadens up and the electron has to

return from a much higher level which

increases the emitted photon energy.

Therefore as the size reduces, the emitted

photon shifts from the red region of visible

light spectra to the blue region. This gives rise

to the unique fluorescent emission of the

Quantum Dots.

PROPERTIES

The exceptional properties of the Quantum

Dots like photo stability, broad range of

absorption spectra, ease in change of emission

spectra, sensitivity, brightness and ability of

coupling with biological molecules, make

them very useful in the field of biological

imaging of different types of cells. Quantum

Dots emitting different colours can be excited

by monochromatic light. The time taken to

fluoresce upon excitation is very less

compared to conventional fluorophores and

this property lasts longer for a given Quantum

Dot. The Quantum Dots are very stable and

resistant to photobleaching and overcome the

disadvantages exhibited by fluorophores,

which are important for imaging applications.

SYNTHESIS

QDs can be synthesized by colloidal synthesis,

E-field method or by fabrication through

etching or self-assembly.

Colloidal chemistry method

This method is also called Hot-Injection

method [2].Here Quantum Dots are prepared

as a batch by quickly adding appropriate

amount of precursors into hot solvent and



Page 88

organic ligand system. First the precursors are

decomposed to monomers by chemical or

physical means like temperature. This results

in nucleation process where the monomers

starts binding to each other. This reaction is

fast in the beginning and slows down in the

end due to change in concentration.

E-field technique

In this method, Quantum Dots are synthesised

between the interfaces of heterojunction of

monomers [8]. On applying an electric field

on the interface, 2D Electron Gas [2DEG] is

created. Later these charges are confined to

the interface by external devices. Thus the

voltage and the 2DEG permit rearrangement

of atoms, finally giving a Quantum Dot at the

interface.

Fabrication

Etching or Lithographic method

In this method, first a Quantum Well or

Double Barrier Heterostructure [DBH] are

grown by the Epitaxial Method. Then pillars

are etched out of it and they are further etched

to give nanocrystals like Quantum Dots [8].

These pillars have the dimension of a

Quantum Wire, which is then metalized on

each terminal. Application of electric current

on the terminals of Quantum Wire leads to the

breakdown of the Quantum Wire and

formation of Quantum Dots.

Self-assembly growth technique

Here Quantum Dots are formed as an out

growth in the Epitaxial technique [8]. The

effect is similar to the condensation of water

droplets on cold surface. Generally, the

epitaxial growth proceeds by forming a layer

of atoms at once. But lattice mismatch and

difference in surface energy between the

atoms induces the formation of islands, thus

Quantum Dots are formed.

It is seen that bare Quantum Dots are

unsuitable for biological application. This is

because; they are insoluble in water and toxic

due to their heavy metal composition, small

size and active surface [9]. To overcome these

problems, the surface of Quantum Dots is

treated with stabilizing and coating

substances. Most of the coating substances are

organic polymers [10] which are amphiphilic

in nature, also making them water soluble and

easy to apply for biological analysis by

conjugating with proteins, antibodies and

other molecules. Additionally, it is observed

that coating provides better photo stability

[11]. It is also seen that some bio molecules

like proteins can be effective nucleating and

stabilizing agents for Quantum Dots [12].

APPLICATIONS OF QUANTUM DOTS

IN BACTERIOLOGY

The superior optical properties of Quantum

Dots entail them for numerous applications in

labelling of cells. They are highly suitable for

labelling, as the intense emission of light helps

in detection of even a small number of the

cells. They find applications in enumeration of

bacterial cells in various matrices, detection of

pathogens, food contaminants, and in the

study of micro flora in bio films.

Enumeration

Here the number of bacterial cells can be

enumerated by relating the intensity of light

emitted by the Quantum Dots to the cell count.

For example, Alteromonas species inhabiting

the surface of small marine animals like



Page 89

Copepods were enumerated using QDs [13].

The CdSe Quantum Dots were conjugated

with streptavidin so that QD can specifically

bind to the anti-antibody against Alteromonas

species. It is also possible to detect wide range

of bacteria from a sample such as sewage

water using water soluble Quantum Dots [14].

Quantum Dots can also be used to detect a

particular strain of a bacterial species as

supported from the result of an experiment

where mannose conjugated CdS Quantum

Dots could detect a strain Escherichia coli

producing FimHlectin which binds

specifically to mannose [15]. In a study on

fluorescence detection of count of Escherichia

coli and Staphylococcus aureus using

Quantum Dots, spectrofluorometer was used

for roughly counting the cells [16]. The low

detection limit for this method was in order of

102CFU/ml. In a different study the same

detection limit was obtained when the

Quantum Dots were labelled to bacteria

covalently using glutaraldehyde [17]. It was

also seen that there was a linear relationship

between fluorescence intensity and total

bacterial count. By using Quantum Dots it is

possible to distinguish between wild type and

auxotrophic strain of a bacteria belonging to

same species. This technique involves

conjugating Quantum Dots with the

biomolecule needed for bacterial growth but

which cannot be synthesized by the bacteria.

This method is applied for detection of purine

auxotrophs of Bacillus subtilis and

Escherichia coli where Quantum Dots

conjugated with AMP were taken up

preferentially by purine-auxotrophs rather

than the wild type [18]. The Quantum Dots

can also be coupled to the antibodies used in

ELISA which can give us an intense signal

even in a minute concentration of antigen.

This method was applied for detection of

Escherichia coli in water samples where the

target bacteria were separated by antibody

coated magnetic beads and then detected by

ELISA using Quantum Dots conjugated to

secondary antibodies [19]. Thus Quantum Dot

based enumeration of bacteria can be rapid

and sensitive compared to the conventional

methods.

Detection of pathogens

Quantum Dots have better optical properties

than other dyes or fluorophores; it is widely

applicable in detecting pathogens from various

clinical samples and to detect a particular

pathogen from a cluster of bacteria. There are

number of studies where Escherichia coli and

Salmonella typhimurium were detected

simultaneously using antibodies against these

two species and conjugating different

antibodies with Quantum Dots emitting light

of different wavelength [20]. Multiplexed

detection was also applied for Escherichia coli

and Salmonella enteritidis [21]. Denatured

Bovine serum albumin stabilized Quantum

Dots were conjugated to secondary antibodies

for detection of Escherichia coli and Listeria

monocytogenes [22]. This type of conjugation

to the secondary antibodies gives us an

advantage of universal usage of the antibody

for the detection, ease in their production and

circumventing the tedious process of labelling

individual primary antibodies. The

interactions between antibody and antigen are

weak and these are further weakened by

conjugation of bulk substance like Quantum

Dot. This reduces the staining property of

Quantum Dots conjugated to antibodies,

hence, it is preferred to conjugate small



Page 90

molecules like biotin to antibody and then

bind them to Quantum Dots conjugated to

streptavidin. The above method was also

adopted for detection of E.coli [23] and

Listeria monocytogenes where the limit of

detection was found to be 2-3 CFU/ml with a

detection time of 1.5 hrs. [24]. In another

study Mycobacterium bovis BCG and

Mycobacterium tuberculosis was detected

using genus specific antibody and biotinylated

anti-antibody which would bind to

streptavidin coated Quantum Dots [25]. The

result gave a limit of detection of 103 bacteria

/ ml. This method was also applied for

detection of Escherichia coliO157:H7 strain

with a limit of detection of 103 CFU/ml with a

detection time less than 2hrs [26]. In one of

the studies Escherichia coli was imaged using

organic acid stabilized Quantum Dots [27].

Here the Quantum Dots were internalized by

the bacteria giving rise to fluorescence signal.

It was found that none of the other bacteria

interfered with the test. Quantum Dot labelling

of the cells also aids Flow cytometric

separation and analysis of pathogenic bacteria

from other non-pathogenic ones. This method

is a fast and effective technique for separation.

In a study pathogenic Escherichia coli strain

O157:H7 were separated from non-pathogenic

bacteria using Quantum Dots and Flow

cytometer [28]. The limit of detection was 1%

of the pathogenic strain in the mixture of cells.

It is also possible for detection of strain and

metabolism specific bacteria using Quantum

Dots conjugated to a biomolecule which might

bind to the receptor on the particular strain or

may be used as a substrate for a metabolic

pathway [29].

In another study different antibodies are

conjugated to Quantum Dots of different

emission peaks for multiplexed analysis of

Bacillus anthracis spores and Yersinia pestis

by Flow cytometry [30]. It also possible to

detect one spores of Bacillus anthracis from

other similar bacterial spores by conjugating

Quantum Dots by BABA peptides [31] or a

short peptide chain from gamma-phage lysine

protein [32]. This analysis can be performed

using methods like Flowcytometry, Confocal

laser scanning microscopy and

Spectrofluorometer with single cell resolution.

An innovative yet specific method of labelling

bacteria is using phage conjugated with

Quantum Dots. This method can be used for

recognising both slow growing and highly

infectious bacteria. This method was studied

for detection of Escherichia coli using

biotinylated phage and streptavidin coated

Quantum Dots [33]. Quantum Dots coated

with zinc [II]-dipicolylamine could detect only

mutant and pathogenic Escherichia coli that

lacks O-antigen and could also facilitate in

vivo optical imaging of the infection in the

animal [34]. It is also possible to identify the

bacterial species by detecting the species

specific DNA sequence. In this method of

detection the Quantum Dots are made to bind

to a probe which is complementary to the

species specific DNA sequence. In a study this

method was used for detection of

Mycobacterium tuberculosis and

Mycobacterium avium subsp paratuberculosis

where the biotinylated probe hybridized with

the DNA was detected by streptavidin coated

Quantum Dots [35]. This method was 70 to

90% accurate compared to real time PCR and

had a limit of detection of 12.5 ng of DNA in

20µl. This technique can be implied in

diagnostic assay for rapid, specific and

sensitive method of pathogen detection. In



Page 91

another study Quantum Dots based molecular

beacons were used to detect the antibiotic

resistant β-lactamase genes in pUC18 of

Escherichia coli [36]. The mechanism of

detection used in the study was a single step

FISH, which gave an excellent signal on genes

in the plasmid. There was an attempt to use

ferrichrome conjugated to Quantum Dots for

detection of bacteria with receptors for ferrous

ions [37]. This method was successful in

detecting Pseudomonas fluorescens having

ferrous receptors.

Detection of bacterial food contaminants

Many bacterial infections are spread by

contaminated food. Thus, detection of

bacterial contaminants in food is important to

monitor outbreaks of food borne infections.

Three food-borne pathogens namely

Salmonella typhimurium, Shigella flexneri and

Escherichia coli were detected by antibodies

against each bacteria which was conjugated to

Quantum Dots of different emission

wavelength [38]. The same method of

antibody conjugated Quantum Dots were

developed for specific detection of

Staphylococcus aureus in food and

environment [39]. The bacteria were detected

under a fluorescent microscope and limit of

detection was found to be 900 CFU/ml. Most

of the contaminants are found in meats, thus

Quantum Dots can be used to analyse

contaminant in meat sample. In a recent study,

Quantum Dots were used to detect pathogens

like Escherichia coli and Salmonella in

ground beef by conjugating Quantum Dots to

specific antibodies against the pathogens [40].

In a study chicken carcass wash water was

used as a sample for detecting Salmonella

typhimurium contaminant in chicken meat

[41]. Here contaminant was separated from

sample with antibody conjugated magnetic

beads and then reacted to biotin tagged

secondary antibody which binds to

streptavidin coated Quantum Dots. In this

method the limit of detection was in order of

103 CFU/ml. Also there has been a report of

use of indirect immunofluorescence coupled

to Quantum Dots for labelling and detection

specific bacterial serotype of pathogen Vibrio

parahaemolyticus attached to small marine

animals which are pathogen carriers [42]. This

method can also be extended for detection of

other Vibrio species like Vibrio cholerae.

With the use of Quantum Dots in biosensors

and microarrays, the size of the instrument

have been reduced and also permitted for

automation thus increasing rapidity and

sensitivity of the test. These techniques have

been used in assaying Salmonellae [43].

Nowadays array systems have been developed

for rapid and sensitive detection of bacteria as

indicated by a study where Escherichia coli

O157:H7 was detected using Quantum Dots

conjugated to antibody and the concept of

sandwich ELISA [44]. The above method

gave a limit of detection of below 10 CFU/ml.

Escherichia coli was also detected using

Pathogen Type of QD Reference

Escherichia coli Primary Antibody-QD, Secondary Antibody-QD, Streptavidin-

QD

21, 23, 24, 28

Salmonella typhimurium,

Salmonella enteritidis

Primary Antibody-QD, 21, 22

Listeria monocytogenes Secondary Antibody-QD, Streptavidin-QD 23, 25

Mycobacterium tuberculosis Streptavidin-QD 26,35

Bacillus anthracis Primary Antibody-QD, Peptide-QD 30,31

Yersinia pestis Primary Antibody-QD 30

Table 1: List of some important pathogenic bacteria detected using Quantum Dots



Page 92

colistin-functionalised Quantum Dots which

gave a limit of detecting as low as 28cells/ml

[45] with a short analysis time of 15 min.

excluding preparation and photoactivation

time.

Detection of bacteria in Oral bio films

It is observed that many bacteria exhibit a

symbiotic relationship between each other.

These bacteria form biofilm which is a

complex cluster of bacteria. These biofilms

are found widely in our environment and also

in our body like mouth etc. It is desirable to

study symbiosis for evolutionary studies.

Quantum Dots can be used for specific

detection of bacteria in oral biofilms. This

method has been used in a study where

specific human oral bacteria namely

Streptococcus gordonii DL1, Streptococcus

mutans UA159 and Veillonella spp. strain R1

were detected in a biofilm in vivo and in vitro

[46]. Here immunofluorescence method of

imaging was used, and this method does not

depend on detection of bacteria based on their

morphology, which makes this rapid

technique amenable to automation, enabling

the detection from numerous samples. These

studies also give an insight on the spatial

relationship between bacteria and interspecies

interaction in biofilm. Presence of specific

gene in the biofilm can give a large insight on

the species of the bacteria and also function of

that gene in the biofilm. In a report Bacillus

spoOA gene was analysed in a biofilm using

DNA-Quantum Dots system [47]. The

hybridization of target DNA to the probe was

detected using flowcytometer and had a limit

of detection of 0.02 nM. It has been observed

that biofilms made of Streptococcus sp. and

Veillonella sp. are formed in early stage of the

plaque. These results were confirmed by using

Quantum Dot based immunofluorescence on

the enamel surface [48].

TOXICITY OF QUANTUM DOTS TO

BACTERIA

We have seen the brighter side of the

Quantum Dots but it has been demonstrated

that these nanoparticles are toxic to living

cells including bacteria. Formation of reactive

oxygen species by the interaction of Quantum

Dots with other bio molecules or release of the

heavy metal ions which constitute the

Quantum Dots have been the mechanism of

inducing toxicity to the cells. The toxicity of

Quantum Dots on four different strains of

bacteria namely Pseudomonas aeruginosa,

Staphylococcus aureus, Bacillus subtilis and

Escherichia coli were studied [49]. The result

indicated that Gram-positive strains were

more resistant to the toxicity than the Gram-

negative bacteria. This might be due to large

amount of peptidoglycan on cell wall of

Gram-positive bacteria. The study also

indicated that Gram-positive bacteria

previously exposed to Quantum Dot showed

lack of reproducibility.

CONCLUSION

Today, the major hindrance for using

Quantum Dots in bacteriological applications

is its toxicity. It is suggested that by

eliminating the heavy elements from Quantum

Dots, the toxicity can be reduced. Thus new

Heavy Metal-free Quantum Dots like carbon

quantum Dots are being produced [50]. But

their effective usage is still under research.

The cost of production of Quantum Dots is

also topic of concern when they have to be

mass produced and used as diagnosis &



Page 93

analysing tools. Many methods for

synthesizing biocompatible Quantum Dots

using bacterial cells like Escherichia coli have

been proposed [51]. The efficacy of these

Quantum Dots in various applications will

have to be proved. In conclusion, it can be

said that Quantum Dot based techniques are

rapid, sensitive and reliable tools for the

enumeration, detection and differentiation of

Bacteria.

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Fig 1: Schematic diagram of a Quantum Dot with an inorganic coating and conjugated bio molecules

Fig 2: Variation of emission wavelength of Quantum Dots with their size. Adapted from [7].



Page 98

Fig 3: Image of Escherichia coli using organic acid stabilized Quantum Dots with emission wavelength ranging from 540-630 nm. [Bar-

2µm] Reproduced with permission [28].

Fig 4: Detection of food-borne pathogens using Quantum Dots antibody conjugation complex, a- Salmonella typhimurium bound

to Quantum Dot emitting 620 nm light, b- Shigellaflexneri bound to Quantum Dot emitting 560 nm light, and c- Escherichia coli

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Ogundele O. E. et al. EFFECT OF EARLY DEFOLIATION ON THE NODULATION AND SOME AGRONOMIC TRAITS OF

SOME SELECTED COWPEA (Vigna Unguiculata l. Walp) VARIETIES


Page 99

IJCRR

Vol 04 issue 17

Section: General Science

Category: Research




EFFECT OF EARLY DEFOLIATION ON THE NODULATION

AND SOME AGRONOMIC TRAITS OF SOME SELECTED

COWPEA (Vigna Unguiculata l. Walp) VARIETIES

Ogundele O. E., Awosanya A. O.

Department of Biological Sciences, Tai Solarin University of Education,

Ijagun, Ijebu-Ode, Ogun State, Nigeria.


ABSTRACT

This study investigated the effect of defoliation at emergence of cotyledons, 2 weeks and 4

weeks after planting on the number of nodules, nodule diameter, total nodule weight, nodule

efficiency, root length and plant height of two improved varieties and one local variety of Vigna

unguiculata (L.) Walp. Defoliation treatments were carried out in triplicates using the CRD.

Plants were raised in pots and data collected after 6 weeks. This study simulated early defoliation

of the crop as evidenced in pest attack, disease infection and/or grazing animals and significantly

reduced nodule number (P<0.001), nodule diameter (P<0.002) and total nodule weight

(P<0.001). Change in nodule pigmentation was also observed. The effect of reduction in nodule

characters was observed to culminate in a corresponding decrease in agronomic traits such as

plant height (P<0.001) and root length (P<0.001). To reduce the effect of early defoliation on

cowpea, farmers should plan the crop’s sowing so that its early growth stage would escape the

season of dominance of its major defoliators.

Keywords:agronomic, defoliation, cotyledons, nodulation, Vigna unguiculata

INTRODUCTION

The problem of maintaining soil fertility in

time past had led to the use of manure and

fertilizers. However, these methods were

constrained by erosion which leads to the

washing away of the nutrients in the top soil.

Alternative methods have since been used by

planting legumes such as cowpea to maintain

soil fertility and at the same time prevent soil

erosion. Crop farmers now focus on legumes

for the aforementioned benefits they attract

and their ability to form association with

bacteria that can fix nitrogen to the soil.

Consequently, productivity is improved in

terms of quality and quantity.

Cowpea, a staple food with high protein,

mineral and vitamin content is also valued for

improving soil fertility through the formation

of root nodules for nitrogen fixation. It plays

an important role in agriculture by providing

soil nitrogen. The symbiosis can help relieve

the requirement for added nitrogenous

fertilizer of other crops that are intercropped

with cowpea. The legume is known for its

symbiotic association with Rhizobium bacteria

enabling it to fix atmospheric nitrogen (Laity

et al., 2003). Bacteria of the genus Rhizobium

play a very important part in agriculture by

inducing nitrogen-fixing nodules on the roots

of legumes such as cowpea.

mailto:[email protected]




Page 100

However, the growth of cowpea is limited by

numerous biotic (diseases, insects and other

pests) and abiotic factors that can cause

serious devastation (Rahman et al., 2008).

Several foliage defoliators, including insect

pests have been reported to cause severe

defoliation of cowpea. Aphids (Aphis

craccivora) attack cowpea especially at the

early vegetative stage (IITA, 2000) while a

considerable number of insects belonging to

Acrididae and Lepidopteran larvae have been

reported feeding on cowpea leaves,

“skeletonizing” and sometimes defoliating the

entire plant. Other foliage defoliators of

cowpea belong to the family Chrysomelidae

(Allen et al., 1996).

Several studies in crop defoliation have

received attention in the last four decades

using various crops. Studies have also been

carried out to determine the effects of

defoliation at the different growth stages on

yields of crops, as it simulates pests and

disease damages. Some studies (DeBeer,

1983; Schneiter et al.,1987; Schneiter and

Johnson, 1994) described an interaction

between the severity of damage (percentage

defoliation) and the growth stage at which this

occur.

Some of the earlier studies on defoliation of

cowpea were particular about defoliation at

the different growth stages (Rahman et al.,

2008) and situations peculiar to America and

Europe. There is probably little or no

information on cowpea defoliation at the

vegetative stage in Nigeria where the crop is

largely grown.

Defoliation as it may be caused by pests and

disease conditions affects the growth of the

entire plant. Perhaps nodulation of the crop is

also affected by the defoliation thereby having

a great impact on nitrogen fixation and

consequently crop productivity. This study

was therefore conducted to determine the

effects of defoliation at the vegetative stage of

growth on the nodulation of some selected

varieties of cowpea Vigna unguiculata (L.)

Walp. in the South-Western part of Nigeria.


Cowpea Varieties

Two improved varieties of Vigna unguiculata

(L.) Walp. (IT89KD-288 and Damila) from

IITA and a local variety (Mata) in Nigeria

were used for the research.

Preparation of Experimental Pots

The experiment was a completely randomised

design with 36 experimental pots arranged in

greenhouse. The experiment was carried out at

the botanical garden of the biological sciences

department in Tai Solarin University of

Education, Ijagun, Nigeria between March and

April.

The experimental pots were perforated at the

base to allow for proper drainage. Loamy soil

from the botanical garden was used.

Planting

Three hand-picked healthy seeds were planted

in each pot and later thinned to one after

germination. Cultural practices such as weed

control were also carried out regularly.

Treatments

There were 4 treatments including the control

for each variety. The treatments were:

Defoliation immediately after emergence of

cotyledons;

Defoliation 2 weeks after planting;

Defoliation 4 weeks after planting;

No defoliation (control)

Each treatment was done in triplicates.




Page 101

Data Collection

Data were collected 6 weeks after planting. In

determining the number of nodules, each pot

was carefully emptied and the soil on the root

was gently washed with water. Root nodules

were physically counted with the aid of hand

lens.

The nodules present on the roots of each plant

were collected and weighed with electric

weighing balance to obtain the total nodule

weight. Nodule diameter was measured with

the aid of a micrometer screw gauge. Nodule

efficiency was determined by examining the

root nodules of each plant to check for the

presence of the reddish-pink pigment

(leghaemoglobin), which indicates a

functional or efficient nodule.

Plant height and root length were measured

using meter rule. Dry mass of shoot was

measured using an electric weighing balance

after shoots had been oven-dried at 60OC for

72hours.

Data Analysis

Analysis of data was done using ANOVA

with Genstat® statistical package.

RESULTS

The effect of defoliation on the number of

nodules was highly significant in the three

varieties used (P<0.001). Similar results were

obtained for total nodule weight, nodule

diameter, root length and plant height with

P<0.001, P<0.002, P<0.001, P<0.001

respectively. In figure 1, earliest defoliation

treatments significantly inhibited nodule

formation and consequently reduced nodule

numbers. Similarly, Defoliation 2 weeks after

planting and 4 weeks after planting still had

significant reduction on the nodule numbers

but not as pronounced as the earliest

defoliation.

Defoliation immediately after emergence of

cotyledons, at week 2 and week 4 reduced

nodule diameter relatively only in IT89KD-

288. Mata and Damila showed significant

reduction of nodule diameter only in those

defoliated at week 4 (Figure 2). Figure 3

shows that defoliation immediately after

emergence of cotyledons reduced plant height

>50% in all the three varieties. However,

week 4 treatments mildly reduced height of

IT89KD-288 and Mata which are the erect

type. The total nodule weight was

significantly reduced in all three varieties

when treatment was applied immediately after

emergence of cotyledons and 2 weeks after

planting. However total nodule weights were

severely reduced in IT89KD-288 when

treatments were applied 4 weeks after planting

(Table 1). Table 2 shows that nodules of the

plants defoliated after emergence of

cotyledons and 2 weeks after planting had

green pigment indicating lack of

leghaemoglobin. In week 4 treatments, only

nodules of the two improved varieties showed

traces of leghaemoglobin with pale pink

pigmentation. There was a relative reduction

in root length in treatments with earlier

defoliations. Only the root length of Mata, the

local variety was slightly reduced in week 4

treatment (Table 3).

DISCUSSION

Early defoliation had negative effect on

nodule characters (such as nodule numbers,

nodule diameter, total nodule weight and

nodule pigmentation), root length and plant

height. However, the severity of the effect

depends on the time of defoliation. Earliest

form of defoliation (immediately after

emergence of cotyledons) produced the most

severe reduction in nodulation, nodule




Page 102

characters and other traits studied. Defoliation

at week 4 produced severe and mild reduction

on all the traits studied. The result of this

study is in agreement with the observations of

Muro et al.,(2001) that showed that the effect

of defoliation depends on foliar surface area

eliminated and on growth periods at which

this takes place.

The significant reduction in nodule characters

as a result of defoliation treatments suggests

that there is a relationship between foliar

surface area and nodulation process in

cowpea. Loss or reduction of foliage parts

hinders photosynthesis. Perhaps, the

cotyledons and leaves supply food and some

necessary precursors for the initiation of

nodulation. Anonymous (2009) also reported

similar observations using soybean seedlings

and clover seedlings when it noted that the

product of photosynthesis and cotyledons

supply essential factors for nodulation and that

cotyledon removal soon after germination

delays, reduces or prevents infection of root

hairs for nodulation.

Foliage removal immediately after emergence

caused stunting in the plants while the

removal of the foliage parts at week 2 and

week 4 only caused slight reduction in plant

height when compared with the control. The

stunting may not be unconnected with the

inhibition of gibberellins production which

stimulates growth in apices of young plants

and their inability to synthesize enough

carbohydrate through photosynthesis, which is

necessary for plant growth and development.

This is corroborated by the findings of Mondal

et al., (1978) and Selter et a.,(1980) that

suggested that defoliation alters hormone

balance, starch, sugar, protein and chlorophyll

concentration of plants.

Change in nodule pigmentation observed in

defoliated plants suggests that photosynthesis

and nitrogen fixation are inter-related

processes. The greenish content of nodules of

defoliated plants at the time of data collection

points to the fact that the nodules, though

formed, were not only reduced in number and

diameter, but also non-functional and

inefficient. This observation is supported by

Hartwig and Nosberger (1994) that defoliation

causes dramatic decrease in nitrogenase

activity.

CONCLUSION AND RECOMMENDATION

Cowpea helps to increase the nitrogen pool of

the soil, improving its fertility and enhancing

agricultural productivity. Early defoliation of

the crop as it may be caused by pest attack,

disease infection and/or grazing animals has

been revealed by this study and others to

severely reduce nodule characters such as

nodule numbers, nodule diameter, total nodule

weight and nodule efficiency. The effect of

this reduction in nodule characters was

observed to culminate in a corresponding

decrease in other traits such as plant height

and root length in similar manner. This in turn

tends to undermine the potential of cowpea to

serve as Africa’s cheapest source of soil

nitrate. To reduce or eliminate the effect of

defoliation on cowpea, farmers are advised to

plan the crop’s sowing such that its early

growth stage should escape the season of

dominance of its major defoliators. Further

research works should be conducted to

discover other ways of preventing foliage loss

apart from the use of pesticides and breeding

for varieties with higher leaf surface area.

Finally, cowpea breeders should make attempt

in their future work to breed varieties that are

tolerant to pest attack and disease-resistant.




Page 103

This may also help to limit the effect of early

defoliation on the nodulation of the crop.

ACKNOWLEDGEMENT

IITA for providing the two improved

varieties of cowpea used for this research

REFERENCES

1. Allen D J, Ampofo J K O, Wortman C S

(1996). Pests, diseases and Nutritional

disorders of the common bean in Africa. A

field guide. The Netherlands, CTA, 132pp.

Anonymous 2009. Rhizobium and Legume

Root Nodulation.

Http://www.microbiologyprocedure.com/r

hizobium-and-legume-root-

Nodulation/temperature-and-light.html.

2. DeBeer J P (1983) Hail damage simulation

by leaf area removal at different growth

stage of sunflower. Gewasproduksie. 12:

110-112.

3. Hartwig U A and Nosberger J (1994). Plant

and soil 16(1): 109-114. IITA, 2000.

Crops and farming systems.

Http://www.iita.org/crop/cowpea.htm

4. Laity F, Diouf D and Fall M A (2003).

Genetic diversity in cowpea varieties

determined by ARA and RAPD

techniques. Afr. J. Biotechnol. 2: 48-50

5. Mondal M H, Brun W A, Brenner M L

(1978). Effect of sink removal on

photosynthesis and senescence in leaves of

soya beans plants. Plant physiology. 61:

394-397

6. Muro J, Irigoyen I, Militino A F, Lamsfus

C (2001). Defoliation effects on sunflower

yield reduction. Agronomy Journal. 93:

634-637.

7. Rahman S A, Ibrahim U and Ajayi F A

(2008). Effect of defoliation at different

growth stages on yield and profitability of

cowpea (Vigna unguiculata L. Walp.) E. J.

Env. Agric. & Food Chem. 7(9): 3248-325

8. Schneiter A A, Johnson B L (1994).

Response of sunflower plants of physical

injury. Can. J. Plant Science. 74: 763-766.

9. Schneiter A A, Jones J M, Hammond J J

(1987). Simulated hail research in

sunflower defoliation. Agronomy Journal.

79: 431-434.

10. Selter T L, Brun W A, Brenner M L

(1980). Effect of obstructed translocation

of leaf abiscic acid and associated stomatal

closure and photosynthesis decline. Plant

physiol. 65: 1111-1115.

11. Shibbles R, Secor J, Ford D M (1987).

Carbon assimilation and metabolism.

Agronomy. 16: 535-588.




Page 104

*WAP – Weeks after planting

Figure 2 Effect of defoliation on nodule diameter

*WAP – Weeks after planting Figure 1 Effect of defoliation on number of nodules




Page 105

Table 1: Total nodule weight at 6th

week (g)

Variety Defoliation at emergence of

cotyledons

Defoliation 2 weeks

after planting

Defoliation 4 weeks

after planting

Control

IT89KD-288 0.067 0.083 0.667 8.063

Mata 0.325 0.400 4.280 6.333

Damila 0.075 0.050 3.737 7.297

Table 2: Nodule pigmentation at 6th

week


cotyledons

Defoliation 2 weeks

after planting

Defoliation 4 weeks

after planting

Control

IT89KD-288 Green Green Pale pink Pink

Mata Green Green Green Pink

Damila Green Green Pale pink Pink

Table 3: Root length at 6th

week (cm)


cotyledons

Defoliation 2 weeks

after planting

Defoliation 4 weeks

after planting

Control

IT89KD-288 5.0 14.1 25.3 37.5

Mata 9.0 10.2 31.9 38.0

Damila 6.7 10.3 15.7 39.5

*WAP – Weeks after planting Figure 3 Effect of defoliation on plant height

Praful S. Jevoor et al. INTERESTING FACTS ON THYMUS GLAND CHANGES-A REVIEW


Page 106

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Review




INTERESTING FACTS ON THYMUS GLAND CHANGES - A REVIEW

Praful S. Jevoor1, Mathada V.Ravishankar

1, Sandhya Dharwadkar

2, Amit

Magadum1, Somashekhar Biradar

2

1Department of Anatomy, J.N. Medical College, K.L.E. University, Belgaum

2Dept. of Anatomy, USM-KLE International Medical Programme, Belgaum


ABSTRACT

In most of the cadavers procured for the dissections in the department of anatomy belongs to

male adult cadavers, which were devoid of active thymus gland and often the gross features of

this gland was ignored because of its remnants were found with variable degree of shape and

sizes. During dissections in most of the cadavers we have often noticed the morphology of most

thymus remnant from simple flat mass to a well defined solid mass of thymic tissue. To test our

curiosity on microscopic studies we have noticed features like, abundant number of discrete

lymphocytes along with Hassall’s corpuscles were appreciated under hematoxylin and eosin

staining. The thymus being a central lymphoid organ develops from endoderm of third

pharyngeal pouch along with the parathyroid gland. It remains active till the age of 14 years and

later shows gradual inclination towards its involution as conventionally it is believed to be an

involuting organ with advancing age. Thymus being an important immune competent organ

which has created a curiosity regarding its changes from advancing age to number of invasive

and non invasive experimental studies, such interesting facts were considered to drag the

attention of involuting organ.

Key Words - Acetylcholine, Adipocytes, Hassall’s corpuscles, Immunosenescence

INTRODUCTION

Thymus is a central lymphoid organ located in

the superior mediastinum; many times it was

ignored during the dissections of adult

cadavers practical point of view its

morphology was less stressed as it is replaced

by the fatty tissue in most of the adult

cadavers. Immunologically it is considered to

be an important gland which programs the

stem cells which are originating from bone

marrow for its specific antigen activity.

Regarding the involution of the thymus gland

and its significance is sited in the texts of

histology, this organ created curiosity in

connection with its versatile changes in

different age, sex and disease conditions, such

as immune deficient ailments varying from

opportunistic susceptibility of body to simple

viral infections to life threatening condition

like acquired immunodeficiency syndrome

(AIDS). The thymus arises from the third

pharyngeal pouch of thymic primordia at the

end of the fourth week in the form of

endodermal proliferations, these endodermal

proliferation forms a hallow tubes that invade

the underlying mesoderm which later

transforms into solid branching cords, these

cords are the primordia of the polyhedral

mailto:[email protected]



Page 107

thymic lobules. The whorl like Hassall’s

corpuscles within the medulla apparently

arises from the ectodermal cells of the third

pharyngeal cleft shortly after the formation of

thymus is subsequently infiltrated by

lymphocytes. As the thymus is developed

along with many structures in the primitive

pharyngeal floor during the fetal life, often it

tend to take a different pathway and

destination with or without influencing its

accompanied structures 1,2

. Such rare

incidence of embedded thymic follicles within

the section of thyroid gland was accidentally

found during the histopathological

investigations showing its diverse destination3.

The congenital anomaly like digeorge

syndrome is often associated with

maldevelopment of thymus gland. The thymus

is highly active during the perinatal period and

continues to grow throughout childhood

reaching its maximum size at puberty and later

on the gland involutes rapidly which is

replaced subsequently with fatty tissue4. The

importance of the thymus often reviewed in

adults with the suspected clinical locomotors

functions, most of such signs and symptoms

are often proved with persistent activity of

thymus. In case of adults the thymus often

start producing its antigens against own body

components which can lead to condition like

myasthenia gravis this condition can be

handled effectively by thymectomy 5. During

routine dissections in most of the cadaveric

gross thymic remnant was noticed as a mass

resembling the original gland as shown in the

photo legend (1) and its microscopic

appearance has shown in the photo legend (2).

DISCUSSION

If we observe the whole life span of an

individual as the age advances some of the

bodily organs are showing obvious changes

from simple atrophy to involution, often these

changes are so subtle that the functional

derangements are correlated with advanced

structural changes in number of organs.

During the early period of post natal life

thymus gland plays a significant role of

programming the cells which are reaching

the gland for their charged specific tasks, such

cells are destined to be involved in immune

mechanism, our body defense activities are

influenced by factors like age, sex,

environment, food, nutritional status, genetic

factor, hormonal status etc. During the

dissections in most of the male cadavers often

we have noticed and collected a lobulated

mass situated at the upper and anterior part of

the pericardium in the region of superior

mediastinum(Photo legend-1), where usual

thymus gland is situated. Such mass was

studied thoroughly from out side and was later

subjected to histological tissue processing,

embedding, microtome sectioning and it was

stained with hematoxylin and eosin, later on

microscopic analysis it has shown a classical

involuntary changes in most of the areas

which resembles the thymic tissue and the

Hassall’s corpuscles (Photo legend-2) were

sparsely spread among dense fatty tissue

along with large number of scattered

lymphocytes. These findings have confirmed

that it is an involuted thymic remnant.

Thymus gland shows a linear increase in its

weight as the age advances till the 16 years of

age, later it undergoes gradual loss in its gross

weight 6. Thymus is being a primary lymphoid

organ plays an important role in the

development of other secondary lymphoid

organs. This fact can be realized by the non-

development or immature development of



Page 108

other secondary lymphoid organs followed by

thymectomy during the early postnatal life,

such dependent activity makes one to become

more susceptible to infections from simple to

complex nature. The activity of thymus begins

in the early fetal life itself as a source of

production of diversified T cell production

which is further boosted in the early stages of

post natal life, but its function declines as the

age advances. The neonatal thymectomy

results in reduced number of T cell counts in

short and long term complications which

shows its indispensable functional

significance through infection prone attitude

in infants with advancing ageing process.7 The

developmental anomalies like digeorge

syndrome often associated with the absence of

thymus gland, during such situations clinical

trials associated with the transplantation of the

thymus gland in early childhood was found

with formation of new T-cells after a span of 5

months suggesting immune reconstitution

which was able to render an essential support

for host to fight against the entry of foreign

bodies 8,9

.It is commonly seen that the

advanced ageing process affects all aspects of

immune system followed by thymectomy

during infancy a particular type of “ T “cell

activity in an individual is often characterized

by lower CD4 +

and CD8+

cell number and

diversity. The removal of thymus in infancy

results in premature onset of decline in

immune competency, often such individuals

are susceptible for infectious diseases, such

clinical findings shows premature signs of

immune aging and Immunosenescence which

was experimentally correlated with the

consequence of thymectomy in the early

postnatal period 10

. There are some

experimental results correlating that the early

thymectomy in case of the female rats are

showing the precautious sexual maturity but

the same procedure in case of the male rats

showing no significant effects 11

.

A prospective randomized study treatment

with growth hormone in HIV infected adults

were showing changes in the body in the form

of increased thymic mass along with increase

T cell receptor rearrangement, such studies are

indicating that the growth hormone is an

important factor which can regulate the T cell

development which can reverses the adverse

influence on thymo-poiesis in rodents. The

thymus being a primary site of de novo cell

production it will modulate the recovery in

immune deficient cells, which supports the

hypothesis of enhancement of thymus activity

by growth hormone administration in immune

deficient conditions. The possibility of the

functional reinstate of the thymus was

observed in a condition where the growth

hormone treatment can revive the thymic

function by enhancing the thymo-poiesis 12

.

Such immune reconstitution studies can

support the highly virulent HIV infections

which are targeting the thymus where it shows

a strong correlation associated with reversible

changes in the remnants of thymus gland 13, 14

.

The age factor of an individual and the

capacity of thymus reversibility are seems to

be interdependent one where the immune

modification process shows difference in

childhood and adulthood 15

. A relatively

common clinical condition where the

derangement in functional activity of the

primary lymphoid organ due to autoimmune

factors targets the normal neuromuscular

mechanisms leading to condition like

myasthenia gravis where the surgical evidence

of thymectomy and its clinical impact on the



Page 109

prognosis of the myasthenia patients have

shown that the thymus is profoundly involved

in pathogenesis by synthesizing

autoantibodies against acetylcholine (Ach)

receptors. In more than 70% of all the

myasthenics the thymus gland shows

lymphofollicular hyperplasia, where the

thymectomy appears to be one of the most

rewarding therapeutic measure16

. Though the

thymus is showing its involution process in

either sex, indeed as the age advances the

gland is obviously showing some

discrimination. These changes could be due to

different hormones involved in driving the

advance age related changes in an individual.

Stage like pregnancy has overall influence on

the body of an individual which was studied in

mammals has shown that the thymus loses its

weight and cellularity with a marked

involuntary changes in the cortical portion.

The increase in the levels of hormones in

pregnancy is generally believed to be one of

the causative factors for its altered

morphology and function. The thymus cortical

part is the site of the cell trafficking in and out

of the thymus, the small MER (Medulla

termed epithelial medullary ring) seen in

virgin mice with the advancing age, where the

MER will enlarge and much more expansion

is seen in its size during pregnancy. In the

later stages of pregnancy the cortical epithelial

cells appears too effete and most of the

macrophages become heavily laden with

apoptotic cells followed by drastic changes in

the thymic microcirculation which could

affect the secretion of cytokines and other

products of the thymus 17

.

The collection of thymus in diversified age

group of people and when they were subjected

to micro anatomical studies have found that

during the biopsies obtained from the patients

immediately prior to partial thyroidectomy for

nontoxic nodular goiter, on its microscopic

examination 50% of the glandular cell activity

declination was recognized especially in late

adolescent individuals and 10% in case of late

middle aged individuals. In men the age

involution is linear whereas in women it is

biphasic. In humans during second and third

decades the proportion of the gland

parenchyma is relatively less in women when

compared to men. But with advanced ageing

process there is a little sex difference where

the medulla shows a continuous linear

involution which is similar in both the sex.

But with respect to cortical part of the thymus

in case of men which has shown accelerated

atrophy in contrast with the changes in age

matched women. Probably the sex related

changes in the thymus attributed to hormonal

levels in the body which were experimentally

correlated 18

. The number of invasive and

investigative studies were showing some

interesting structural changes in many organs

with the help of improved scanning techniques

where the drastic revolution in the field of

radio diagnostics has helped us to understand

and correlate the structural and functional

changes of many more organs19,20

.

CONCLUSION

Thymic parenchyma atrophies with advancing

age and it is replaced by fatty tissue it may be

diffuse or focal. The thymic remnants are

showing wide changes from infancy to

adulthood, in different sex, stressful and

disease status of the body. Unlike our

conventional thoughts its capability of

functional and structural reversibility in

immune deficient adults certainly taps

possibility of its functional revival, where the



Page 110

thymus gland is showing its versatile

morphological and histological pattern in the

body is associated with a wide range of

immune mediated response. Indeed such

peculiar experimental out come makes one to

go further in the direction of the research

related to immune modulation where the

thymus stands as a peculiar and interesting

organ.

COMPETING INTERESTS

Here the authors declare that they don’t have

any competing interests.

ACKNOWLEDGEMENT

The author would like to acknowledge the

support provided by Dept. of Anatomy, JN

medical college, KLE University, Belgaum.

FUNDING

This work has not utilized any funding or

financial aid from any of the sources.

REFERENCE

1. Sadler T W. Langmans medical

embryology,Chapter 16,Head and neck,11th

edition, Lippincott’s Williams and

Wilkins,2010: 271-277.

2. Drake R l, Vogal A W,Mitchell A M.

Conceptional overview: Gray’s anatomy

for students. 2nd Ed. Churchill living stone.

Elsevier, Philadelphia,2009: 709.

3. Maradi K, Sharma J. Ectopic intra thyroidal

thymus-rare finding in an adult. The

internet journal of head and neck surgery.

2009; 3 (1). www.ispub.com/journal/the-

internet-journal-of-head-and-neck-

surgery.(Date of access 14-12-2012)

4. Edwin et al. Age related changes in the

cellular composition of the thymus in

children. The journal of allergy and clinical

immunology. 2005; 115(4):834-840.

5. Papatestas A E et al. Effects of thymectomy

in Mysthenia grevis. Annals of

surgery.1987;206(1): 79-88.

6. Begum M, Paul U K, Alam M J. Age

related changes in weight of the Thymus

Gland of Bangladeshi people. Bangladesh j

anat. 2010; 8(1): 10-12.

7. Afifi A, Raja S G, Pennington D J and

Tsang V T. For neonates undergoing

cardiac surgery does thymectomy as

opposed to thymic preservation have any

adverse immunological consequences

?.Interactive cardiovascular and thoracic

surgery. 2010; 11(3): 287-291.

8. Markert ML,Devlin BH,Mccarthy EA.

Thymus transplantation.Clinical

immunology.2010; 135(2):236-46.

9. Markert ML, Devlin BH, Alexieff MJ, Li J,

McCarthy EA, Gupton SE,Chinn IK, et al.

Review of 54 patients with complete

DiGeorge anomaly enrolled in protocols for

thymus transplantation: outcome of 44

consecutive transplants. Blood. 2007;

109(10):4539-4547.

10. Sauce D, Larsen M,Fastenackels,Duperrier

A,Keller M,Loebenstein BG, et al.

Evidence of premature immune aging in

patients thymectomised during early

childhood. The journal of clinical

immunology.2009; 119(10): 3070-3078.

11. Jr. Ahearn T F. "The Relation of the

Thymus Gland to Sexual Maturity"

http://ecommons.luc.edu/luc_theses/435 (

date of access 26-4-2012)

12. Napolitano L A et al. Growth hormones

enhances the thymic function in HIV

infected adult. J clin investigation, 2008;

18(3): 1085-1098.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Markert%20ML%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Devlin%20BH%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Alexieff%20MJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Li%20J%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22McCarthy%20EA%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gupton%20SE%22%5BAuthor%5D

http://ecommons.luc.edu/luc_theses/435



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13. Hazra R, Mackall C. Thymic functions in

HIV infections. Current HIV/ AIDS report.

2005;(2):24-28.

14. Savino W, Dardenne M, Marche C,

Trophilme D, Dupuy J M, Pekovic D, et al.

Thymic epithelium in AIDS. An immune

histological study. AJP1986;122(2):302-

307.

15. Ping Ye, Denise E, Kirschner and Athena

P, Kourtis, The thymus during HIV disease:

Role in pathogenesis and in immune

recovery. Current HIV research.2004;2(2)

:177-183.

16. Melms A, B.C. Schalke B C,Kirchner T,

Muller-Hermilink H K, Albert E and

Wekerle H. Thymus in mysthenia grevis.

Isolation of T lymphocytes in lines specific

for the nicotinic acetyl choline receptor

from thymuses of myasthenia gravis

patients, journal of clinical investigation.

1988; 81 (3):902-908.

17. Kendall M D and Clarke A G. The thymus

in the mouse changes its activity during

pregnancy, a stage of the

microenvironment, journal of anatomy

2000: 197(3); 393 -411.

18. Simpson J G, Grey E S and Beck J S. Age

involution in the normal human adult

thymus, Clinical experimental immunology

1975;19(2):261-265.

19. Moor A V et al. Age related changes in the

thymus gland CT- Pathologic correlation.

Am J Radiol. 1983; 141: 241-246.

20. Daga B V, Chamangokar V A,

Dhamangaokar V B. Case report-CT

diagnosis of thymic remnant cyst/

thymopharyngeal duct cyst. Ind J radiol

imaging. 2009; 19: 293-295.

Photo legend 1- Gross appearance of thymus

remnant in the cadaver

Number 1 and 2 indicated the lobes of the

thymus gland

Photo legend 2-Histology of thymic ramnent

tissue under H& E staining

Number 1 and 2 is indicating the adipocytes

3 and 4 is showing the Hassall’s corpuscles 5

and 6 is showing dense collection of

lymphocytes

Ranzani. R.et al. INDIGENOUS UNCOATED AND HYDROXYAPATITE COATED COMMERCIALLY PURE

TITANIUM FOILS FOR GUIDED BONE REGENERATION IN DEFECT SITES OF IMPLANTS – AN

IN VITRO STUDY


Page 112

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research




INDIGENOUS UNCOATED AND HYDROXYAPATITE COATED

COMMERCIALLY PURE TITANIUM FOILS FOR GUIDED BONE

REGENERATION IN DEFECT SITES OF IMPLANTS – AN IN VITRO

STUDY

Ranzani R., Abby Abraham, Chakravarthy R., Lakshmi S.

Department of Prosthodontics, Meenakshi Ammal Dental College,

Chennai, India


ABSTRACT

Use of barrier membranes for the regeneration of bone defects has significantly changed implant

dentistry in the past years. The design of the membranes employed in this study plays an important role

in establishing a healing environment by separating tissues during healing and thus providing space for

regeneration of bone in cases of insufficient bone around implants, it acts like a tent in cases of

extraction sites- which is a potential site for future implant placement thus reducing the problems of

irregular ridge defect, and the rigidity of the material prevents tissue ward collapse of the membrane.

The present study was conducted to prepare the titanium in the form of foil, and tested for

biocompatibility using fibroblast cells, hydroxyapatite coating was made on the foil, characterization

by X-ray diffraction for both the uncoated and the coated foils was done, and osteoblasts adhesion test

was done and viewed under scanning electron microscope both before and after the test.

Key words: Titanium foils, Hydroxyapatite coated foils, Fibroblast culture, Osteoblast adhesion test,

Scanning electron micrographs.

INTRODUCTION

“Everything has its time” The dinosaurs had their

time, and so will it once be said that amalgam had

its time too. In dentistry, implantology currently is

in “its time”. Other than the discovery of

osseointegration more than twenty years ago, the

concept of guided bone regeneration represents

the most important progress in implant dentistry.

This material helps in following cases:

In cases of insufficient bone around implants,

Acts like a tent over the graft material,

Preserves the clot formed by acting as a tent in

cases of extraction site, which is a potential site

for future implant placement.

The rigidity of the material prevents tissue

ward collapse of the membrane.

The present study was conducted to obtain an

improved uncoated and hydroxyapatite coated

titanium foils, both of which can be an economic

replacement for their costly counterparts.

Aim of the study:

To prepare the titanium in the form of foil.

To test biocompatibility of the material using

fibroblast cells.

To make hydroxyapatite coating on the foil.


Preparation of titanium foil (fig: 1 a, 2a, 2b, 2c)

The titanium sheets were cut to a dimension of

3cm×3cm. 16% hydrofluoric acid w/v was taken

in a glass beaker, and the cut sheets are dipped in

the acid intermittently, and it is alternatively

dipped in water to wash the acid. For every



IN VITRO STUDY


Page 113

minute of etching, the material is measured with a

micrometer to check the thickness of the foil. This

procedure is carried on until the material is 60μ

thick and uniform pores appear.

Ultrasonic cleaning of the foil:

Foils are ultrasonically cleaned in

a. Acetone for 20 min,

b. 70% ethyl alcohol solution for 20 min and

c. Finally in distilled water for 20 min.

Preparation of hydroxyapatite coating over the

titanium foil (fig: 1b, 2a, 2b, 2c):

The ingredients of the dip-coating solution and

their weight percentages are:

d. Hydroxyapatite – 7.3%

e. Ethanol – 66.2%

f. Polyethyleneglycol 600 – 2.2%

g. Glycerol 10.2%

h. Distilled water – 14.1%

Preparation of dip coating solution (flow chart

no:1):

A mixture of hydroxylapatite and water is made

and maintained for 8 minutes. After 2 minutes,

polyethylene glycol and ethanol are mixed in a

separate beaker and maintained for 8 minutes.

Glycerol is added to the hydroxyapatite and water

mixture and maintained for 2 minutes. After this

time the polyethylene glycol and ethanol mix is

added to the mixture of hydroxyapatite, water and

glycerol. Then the whole solution is maintained

for 4 minutes. The final solution is the dip-coating

solution. Titanium foil is dipped and withdrawn at

45 degree angle in a freshly prepared dip-coating

solution at a constant speed of 10mm/15 seconds.

This procedure was repeated 2-3 times to increase

the coating thickness.

The coated foils were immediately dried in an

oven for 45 minutes. The microwave oven used in

this process is a BPL micro convection system.

X-ray diffraction (fig 3a & 3b):

The hydroxyapatite powder, titanium foil, and

hydroxyapatite-coated foil were characterised by

x-ray diffraction technique. The dip-coated

titanium foils clearly shows the hydroxyapatite

peaks along with the substrate titanium peaks.

Fibroblast culture

Commercially pure titanium foils, Glaxo

modification eagle’s medium (GMEM), Cell line:

VERO cells, Fetal calf serum were procured from

HI media, India and stored at -20 degree Celsius,

Antibiotic stock: Antibiotic stock was prepared

with the following antibiotics in distilled water

and added to the medium in the following

concentration.

Gentamycin : 50 mg/lit

Penicillin : 1,00,000 units/litre

Streptomycin: 50-100 mg/litre

Fungizone : 25μ / litre

Sodium bicarbonate stock: Sodium bicarbonate

8.8% solution (w/v) in phosphate buffered

solution was autoclaved and stored at +4 degree

Celsius until required.

Trypsin versene glucose was sterilized by

filtration through 0.22 μ membrane filters, ale

quoted and stored at -20 degree Celsius until

required.

Phosphate buffered saline (10X) was prepared in

sterile distilled water from this 1 litre of 1X was

prepared and sterilized by autoclaving at 121

degree Celsius for 15 minutes at 15 lbs pressure.

Procedure:

Sterilization of glassware’s:

The glassware’s were washed thoroughly, with

mild detergents and rinsed thoroughly with

distilled water, dried and covered with aluminium

foil and sterilized in autoclave at 121 degree

Celsius for 20 minutes at 15 lbs pressure or in hot

air oven at 180 degree Celsius for 2 hours.

Preparation and sterilization of medium:

The powdered medium was dissolved in 960 ml

of distilled water, tryptose phosphate broth (10%)

was added and the pH was adjusted to 7.4 with

sodium bicarbonate and 2.3 ml/L of antibiotic



IN VITRO STUDY


Page 114

stock was added and sterilized by filtration

through 0.22μ membrane filter and stored at+4

degree Celsius.

Maintenance of VERO cells:

VERO cells were maintained in growth medium

(GMEM 8% FCS and antibiotics). VERO cells

were passaged at a split ratio of 1:3 on every 3-

day. Briefly spent medium from bottles (25 sp

cm), containing confluent monolayer were

decanted and the cell layer washed with trypsin

versene solution twice. Detached cells were

harvested in 3ml of growth medium and

resuspended to make 24 ml of cell suspension in

growth medium. Cell suspension was seeded into

3 bottles (25-sq cm) at the rate of 8 ml per bottle

and incubated at 37 degree Celsius.

Extraction:

The extraction liquid resulted from the incubation

at 37 degree Celsius for 120 hours of the material

in the extraction “vehicle” (minimum essential

medium) under specific conditions.

A blank extraction was done using Glaxo

modification earl’s medium under the same

conditions, except for the absence of material.

The cells were tested by direct contact of

extraction liquid of the test implant material.

VERO Cyto toxicity study:

This was performed in 6 well tissue culture plates

(NUNC). Cells were seeded at the rate of 2×105

cell/ml. 1.5ml of trypsinised cells were added

with equal volume of each of the four metal

extract in growth medium. 1.5ml of this mixture

was seeded into one well of the plate. Four such

extracts and negative controls were tested in a

single plate. Cytopathic effect was observed daily

for four days, at the end of which the cells were

stained with Giemsa.

Result of the test (fig 4a & 4b):

VERO cyto toxicity: none of the metallic extracts

caused any cytopathic changes in the VERO cells,

when the extracts were used as neat.

Osteoblasts adhesion test

Procedure:

Cell culture system:

Osteoblasts were maintained with minimum

essential media supplemented with foetal calf

serum.

Maintenance of cells:

When the cells become confluent, subculture the

cells using trypsin, incubate until the cells become

rounded and begin to detach by checking under

microscope every one minute. Add 4ml of serum

containing medium and disperse the cells

carefully using a Pasteur pipette.

Adhesion test:

Test samples were conditioned with media. Cells

were seeded on to the conditioned test samples,

standardized to 1.5 x 1.5cm, in multiwell plates

with required amount media. Incubated the

culture for 24 hours. After 24 hours, discarded the

medium from the culture dish, rinsed the cells

with phosphate buffered saline, fixed in 2.5%

glutaraldehyde solution in phosphate buffer for

three times. Dehydrated in different grades of

alcohol, immersed in asoamyl acetate for 2

minutes. Then processing is done for critical point

drying, gold coating and observation under

scanning electron microscopy.

Result of the test (fig: 5a, 5b, 5c, 6a, 6b, 6c ):

Osteoblasts adhered on to both coated and

uncoated test samples. Cell covered surface is

seen in scanning electron micrographs of 400x. At

higher magnification spreading of an individual

cell can be seen.

RESULTS

The result in the present study indicates that a

possible new way of thinking about implant

placement may be on the horizon.

The foil prepared was 60μ thick with uniform

distribution of pores, which can act as a passage

for the nutrients to reach the underlying tissues.



IN VITRO STUDY


Page 115

Four samples of 15mm X 15mm dimension were

subjected to fibroblast culture study. Extract of

the material was prepared and tested for

cytotoxicity. None of the metallic extracts caused

any cytopathic changes in VERO cells when

observed for four days. Thus, proving the

biocompatibility of the material.

The hydroxyapatite coating was done by

preparing a dip coating solution. When assessed

by X-ray diffraction the coating material was

identified as hydroxyapatite and the substrate as

titanium.

Scanning electron microscopic study revealed that

the uncoated material had distribution of pores

that were through and through, the area in

between the pores appeared scaly and rough,

proving the ability of the material to retain the

hydroxyapatite coating. In the coated material,

hydroxyapatite was uniformly distributed on the

substrate. The vertical section showed that the

thickness of the hydroxyapatite material was 50μ.

Osteoblast adhesion test was performed for both

coated and uncoated test samples. 1.5 x 1.5 cm

test samples were conditioned with media and

cells were seeded on the samples in multiwell

plates. Step wise procedures were conducted and

observed under scanning electron microscope.

Micrographs showed that the osteoblasts had

adhered onto both the coated and the uncoated

test samples. Cell covered surface was seen, and

at higher magnification and spreading of an

individual cell was seen.

DISCUSSION

The term guided bone regeneration, has been used

in tissue engineering for some years and is

actually a specialized sub-area of tissue

engineering and using this procedure for

treatment of partial and total edentulism with

dental implants has become an accepted treatment

modality. Guided bone regeneration is based on

the concept that most tissues are capable of self-

reconstitution if appropriate conditions exist and

by compartmentalizing wound healing. By

placing a physical barrier between epithelial and

connective tissues on one side and implants and

bone on the other side, guided bone regeneration

procedures aim to create a protected space for the

blood clot to form and organise. The presence of a

cell occlusive membrane is required to prevent the

migration of epithelial and connective tissue cells

into the wound area, thus allowing bone cells to

form from marrow spaces to repopulate the defect

and to mature into new bone. Membranes used in

guided bone regeneration should possibly achieve

a good degree of tissue integration with

neighbouring connective tissues in order to obtain

a mechanically stable environment necessary for

successful bone and soft tissue healing. However

regeneration of the bone tissue may be influenced

by the nature of the membranes themselves and

regeneration takes place in sequence of events

including blood clot formation, angiogenesis,

osteoprogenitor cell migration, woven bone

formation, compaction of woven bone and

secondary remodelling. In this study, we have

made indigenous commercially pure titanium foils

by etching, until it is 60μ thick and uniform pores

are seen. Hydrofluoric acid is used because it

attacks titanium even at very dilute

concentrations. Hydroxyapatite coating is done on

the foil by dip-coating, in a solution prepared by a

combination of hydroxyapatite powder, ethanol,

polyethyleneglycol, glycerol, and distilled water.

Hydroxyapatite is used because of its

osteoconductive and osteophilic properties.

Assessment of fibroblast culture: The titanium

was tested for biocompatibility using fibroblast

cells. Metal extract was prepared by incubation at

37◦ C for 120 hours of the material in the

extraction vehicle. Trypsinised VERO cells were

added with equal volume of the metal extract in

the growth medium. 1.5 ml of this mixture is



IN VITRO STUDY


Page 116

added to 5 wells of the six well plate and negative

control were tested. The cells were observed daily

for four days. None of the metal extract caused

any cytopathic changes, thus proving the

biocompatibility of the material.

Assessment of x-ray diffraction: The

hydroxyapatite powder, titanium foil and

hydroxyapatite-coated foil were characterized by

x-ray diffraction. The uncoated foil clearly shows

the titanium peaks and the coated foil shows the

hydroxyapatite peaks along with the substrate

titanium peaks.

Assessment of osteoblasts adhesion test:

Although the study of fibroblasts provides

valuable information about its response to

exogenous materials, cells of different origin react

differently to foreign bodies. Thus, the results

from fibroblasts should not be transferred to bone

cells. The uncoated and the coated samples were

tested for osteoblasts adhesion. Cells were seeded

on to the surface of the materials. The material

along with the medium was incubated for 24

hours. Later the medium was discarded from the

culture dish and processed for critical point

drying, gold coating and observed under scanning

electron microscope. The surface chemistry of

biomaterials can directly affect cell behaviour and

interactions between the host environment and the

biomaterials may have significant downstream

consequences. Therefore the nature of the

materials used in implantology may drastically

influence surrounding bone regeneration. Cell

attachment and migration are dependent on cell/

substrate interaction. The surface properties of the

material have a strong biophysical influence on

the cell kinetics. Osteoblasts are an anchorage –

dependent cell type and need to attach and spread

so as to divide and become confluent. The

cytoskeletal response (actin filament and focal

contact formation) of osteoblast-like cells to

various substrates has been studied by many

investigators. Bone cells are known to secrete a

great amount of transforming growth factor-β

(TGF - β1) and express cell surface TGF – β1

binding sites. Produced at all stages of bone

remodelling, TGF – β1 is a potent mitogen in

osteoblast-enriched cultures from foetal tissue. It

increases the expression of many genes associated

with osteoblast activity and the production of

extra cellular matrix macromolecules such as type

I collagen, fibronectin and osteonectin. Hasegawa

and coworkers1 demonstrated that the

conformational state of proteins can influence cell

activity and consequently extra cellular matrix

formation. This might be one of the reasons why

cells, because of more favourable spreading

reaction, increased their rate of growth and

supported the formation of mineralized matrix.

The role of TGF-β1 in bone regeneration is

mediated by changes in extra cellular matrix

macromolecules, which, in turn, are regulated by

a balanced secretion of biologically active growth

factors. Human osteoblast synthesis of these

growth factors and extra cellular matrix

macromolecules may be influenced by the

materials used in barrier membranes. Guided bone

regeneration is a means of using the osteogenic

potential of progenitor bone cells, to create new

growth in a variety of osseous defects.

Osteogenesis on bioactive substrates in

characterized by a temporal sequence of biologic

events involving cell morphology, proliferation

and differentiation. Hurzeler and associates2

found a substantial amount of “re-

osseointegration” after treating ligature peri-

implantitis using a combination of guided bone

regeneration and bone grafts.

Assessment of scanning electron micrographs

before osteoblasts adhesion:

The coated samples exhibited a thickness of 50μm

of hydroxyapatite coating in cross section. When

the surface was viewed, the hydroxyapatite



IN VITRO STUDY


Page 117

crystals were uniformly distributed and uniform

in size. The surface of uncoated foil revealed

uniform distribution of pores and the surface in

between the pores had a scaly appearance, which

aided in both adhesion of osteoblasts and

hydroxyapatite coating.

Assessment of scanning electron micrographs

after osteoblasts adhesion: The micrographs

revealed that osteoblasts adhered to both the

uncoated and the hydroxyapatite coated samples.

Cell covered surfaces were seen in the

micrographs of 400x. at higher magnification

spreading of an individual cell was seen. Thus the

material used in this study has proved to be

osseoconductive by promoting the adhesion of

osteoblasts to the substrate. The present study is

an in vitro study. For further clinical application

an in-depth invivo study is of paramount

importance.

SUMMARY

The study was conducted to prepare a titanium

foil, which can be used as a membrane for guided

bone regeneration in defect sites of implants.

Titanium sheets of required dimensions were cut

and immersed in hydrofluoric acid intermittently

and measured every minute of etching using

micrometer until the foil is 60μ thick and

distribution of micropores is present. The material

was subjected to fibroblast culture to evaluate the

biocompatibility. The results indicated that the

material is biocompatible. The prepared foils are

dip-coated, and the uncoated and the coated foils

were characterized by X-ray diffraction

technique. The uncoated foils clearly shows the

titanium peaks and the dip-coated titanium foils

showed the hydroxyapatite peaks along with the

substrate titanium peaks. Scanning electron

microscopic study showed micropores in the

uncoated material and the scaly substrate in

between the pores. And the hydroxyapatite coated

material showed uniform coating of the material.

Osteoblasts adhesion test was conducted on both

the uncoated and coated test samples. The

scanning electron micrographs revealed the

adhered osteoblasts on the surface of both the test

samples.

CONCLUSION

A new membrane material, titanium foil was

prepared; the material was proved biocompatible

in fibroblast culture study. This material was dip-

coated with hydroxyapatite. X-ray diffraction

showed foils with hydroxyapatite peaks along

with hydroxyapatite peaks along with the

substrate titanium peaks. Scanning electron

microscopic study revealed micropores and scaly

appearance of the uncoated foil, and uniform

coating of hydroxyapatite. The surface roughness

and pores has given provision for the attachment

of the hydroxyapatite crystals. Osteoblasts

adhesion test was done and scanning electron

microscopic study was conducted to view the

adhered cells. The osteoblast cells have adhered

to the substrate of both the coated and uncoated

foils. At higher magnification spreading of an

individual cell was seen. The results reveal that

this study could give rise to a new generation of

osseoconductive membranes which can be used in

various clinical conditions.

REFERENCES

1. Hasegawa, Jan-Thorsten Schantz, Dietmar et

al, Evaluation of a tissue-engineered

membrane-cell construct for guided bone

regeneration. INT J ORAL MAXILLOFAC

IMPLANTS 2002; 17: 161 – 174.

2. Hurzeler, Francisco H. Nociti, et al,

Absorbable versus Nonabsorbable membranes

and bone autografts in the treatment of

ligature-induced peri-implantitis defects in

dogs: A histometric investigation. INT J ORAL

MAXILLOFAC IMPLANTS 2001; 16: 646-

652



IN VITRO STUDY


Page 118

Legends

1a. Uncoated commercially pure titanium foil

2c & d At 300 and 500 magnification respectively uniform distribution of hydroxyapatite crystals are seen

2b. SEM study -In cross section 50µ thickness of hydroxyapatite coating is

seen

2a. Hydroxyapatite coated titanium foil

1b. SEM Study - Uniform distribution of pores at 20 magnification

1c & d At 300 and 500 magnification respectively scaly appearance of

material between the pores reveals a rough surface.



IN VITRO STUDY


Page 119

Flow chart No 1 3a

4a . Cell rounding during trypsinization

4b. Cells were well rounded and no sign of cytopathic changes

3b



IN VITRO STUDY


Page 120

Scanning electron micrographs after osteoblast adhesion test – uncoated foils

Scanning electron micrographs after osteoblast adhesion test – hydroxyapatite coated samples

5a .& 5b. At 400 magnification cell covered surfaces is seen

6a

5c. At higher magnification spreading of an individual cell is seen

6b

6c

Sharmila Banu A et al. OCCUPATIONAL AWARENESS OF RURAL WOMEN WORKERS IN HEN POULTRY FARM,

COIMBATORE DISTRICT, SOUTH INDIA


Page 121

IJCRR

Vol 04 issue 17

Section:General Science

Category: Review




OCCUPATIONAL AWARENESS OF RURAL WOMEN WORKERS IN

HEN POULTRY FARM, COIMBATORE DISTRICT, SOUTH INDIA

Sharmila Banu A1, Gunasekaran C

2, SelvaKumar V

3, Mohana P

4,

Kandappan K5, Elanchezhian M

6

1, 2, 4, 6

Conservation Biology Laboratory, Department of Zoology,

Bharathiar University, Coimbatore, TN, India 3, 5

Department of Social Work, Sri Ramakrishna Mission Vidyalaya

College of Arts and Science,Coimbatore,TN, India


ABSTRACT

Frequently the rural area women’s are first-rate the preference to poultry works. Especially

women’s are the majority to work on this field. Rural areas are mainly depends on the women

incomes. The present research focus to scrutiny the occupational awareness in hen poultry farm,

in specific reference to faecal cleaners of women workers. The foremost objective of the

research is to bestow the awareness about work place and mould with them health and safety

decisive factor. The calculation is done by percentage (%) intensity and chi-square test. The

recommendations in addition discuss among women workers.

Key words: occupational awareness, rural, hen faecal cleaning women, livelihood, percentage

and chi-square test.

INTRODUCTION

Even though they are home makers, but they are

forced to work for their family. This is help to

their children future and they have a thought

about empowerment development. The women

are the backbone of agricultural workforce but

worldwide her hard work has mostly been un

paid. She does the most tedious and back-

breaking tasks in agriculture, animal husbandry

and homes (DARE/ICAR ANNUAL REPORT

2003–2004). The choice of occupation is

traditionally (Penny Kane) or time being taken to

commencing of rural women’s and they have the

poor dexterity of occupation effects. Thus, the

rural women’s are randomly selects the hen

poultry farm. In particularly, we have one

question: Why, the research is to selects the

poultry farm and especially in women workers.

Because, POULTRY is by far the largest

livestock group and is estimated to be about 14

000 million, consisting mainly of chickens, ducks

and turkeys (FAO 1999).One third of the world is

moving only the food of chicken. Women are the

back fillet of the whole lot in family. She have to

work for all the relationships like Mother,

Daughter, Sister, Grand mother, worker in

occupation etc. She didn’t feel difficulty with

anything which comes from family or occupation.

The bacterial count in poultry housing systems is

high in comparison to those of pig and cattle.

Little is known about the bacteria present in the

poultry environment such as in poultry litter and

air of poultry house (Saleh et al., 2003, Nasrin

etal). Micro organisms like Bacteria, (USA

Wrestling 2007-08) fungi-Small organisms that

are found everywhere in the environment and on

skin. Where as their population is more high in

pollutants and excrete area like animal faeces.

These organisms move under the skin and start

the problem to the humans. In poultry farm may

cause the problems of infection disease

respiratory problems and so on, these




Page 122

overwhelming effects to the worker when they

have not any idea about basic occupational skill

(safety manners). The possible negative effect of

noise on reproduction is biologically plausible, as

well as amenable to prevention (Irene Figà-

Talamanca). So the research have in mind of these

points of basic health problems and its root will

be spread when the daily habits are not in

following the proper comportment such as clean

dress, wear the gloves and mask, and footwear.

Since, these are the fundamental to hygienic

working people. The present research asked the

question about clean dress, wearing the gloves,

mask, footwear’s and hand washing habits. These

all done by direct interview method. This analysis

is calculated by percentage (%) variation. Based

on the results they urge.

METHODOLOGY

The research work was conducted on the rural

women worker in hen poultry farm in Coimbatore

District. After approval from Institutional Ethics

Committee,(Kartik et al 2005) the research was

on track with conquering an informed permission

from the partaker of poultry farm. Totally 130

samples were selected for this research which

included age group of Up to 25 with 29 samples

(group I), 26-30 with 74 samples (group II), 31-

35with 19 sample (group III), 36 an above with 8

sample (group IV) and the study was conducted

during the epoch 3rd

-24th

May 2012.The study

was vigilance by direct interview method, we

calculated the percentage of women’s worker

awareness through the questionnaire include the 5

basic question, this also designed by the points of

daily activity such as clean dress code, wearing

gloves and mask , footwear then washing habit of

hands, and the answers were corrected with

options of 1.Yes, 2.No then this research was

successfully completed by the percentage and chi-

square test calculations.

The Direct interview Questions are following

A. Are they workers maintaining the dress code (a

clean pair of working dress) to work place?

B. Are they having the habit to wear footwear in

work place?

C. Are they wearing the disposable gloves?

D. Are they use the mask at work place?

E. Are they washing hands before drinking water?

The standard optionally answers were assessed

directly by the research team. Based on the

options the findings were examined.

I.RESULTS BASED ON SOCIO ECONOMIC

CHARACTERISTICS

Educational qualification of the Respondents

Education Frequency (N) Percent (%)

Secondary

education

7

5.4

SSLC 35 26.9

Illiterate 88 67.7

Total 130 100.0

Marital status of the Respondents

Parameters Frequency (N) Percent (%)

Married 85 65.4

Unmarried 39 30.0

Widow 6 4.6

Total 130 100.0

Family type of the respondents

Parameters Frequency Percent

Joint family 32 24.6

Nuclear family 98 75.4

Total 130 100.0

Income of respondents

Parameter Frequency Percent

up to 2200 37 28.5

2201 – 2600 48 36.9

2601 – 3000 43 33.1

3001and above 2 1.5

Total 130 100.0

Experience of respondents


Up to 5 year 78 60.0

6 -10 47 36.2

11 and above 5 3.8

Total 130 100.0




Page 123

Results based on awareness response from

workers


Moderate 38 29.2

Low 92 70.8

Total 130 100.0

Respondents’ age and awareness of the

respondents

Respondents

age

Awareness

of the

respondents

Total

Mo

dera

te

Low

1

2

3

4

Up to 25 Count 9 20 29

%within

respondents

age

31.0

%

69.0

%

100.0%

26 -30 Count 29 45 74

% within

respondents

age

39.2

%

60.8

%

100.0%

31-35 Count 19 19

% within

respondents

age

100.0

%

100.0%

36 and

above

Count 8 8

% within

respondents

age

100.0

%

100.0%

Total Count 38 92 130

% within

respondents

age

29.2

%

70.8

%

100.0%

II. RESULTS BASED ON AWARENESS

QUESTION

The total number of samples was 130 and

analyzed by five types of awareness questions and

calculated by optional answers (yes, no)

A. Clean dress code Frequency Percent

Yes

N

12

(%)

9.2

No 118 90.8

Total 130 100.0

B. Custom of Footwear


Yes 28 21.5

No 102 78.5

Total 130 100.0

C. Practice of disposable gloves


Yes 24 18.5

No 106 81.5

Total 130 100.0

D. Handling of Mask


Yes 58 44.6

No 72 55.4

Total 130 100.0

E. Ritual of washing hands


Yes

N

46

(%)

35.4

No 84 64.6

Total 130 100.0

DISCUSSION

The present research was focused to identified the

occupational awareness in rural area especially

women workers in hen poultry farm. In the

attendance the research encounter only the fecal

cleaners. Why? The study concentrated to faecal

cleaners because they were more infected through

the excrete materials in poultry farm, According

to Mojtaba Yegani (2010)Zoonotic diseases are

transmitted from animals to humans and include

bacterial, viral, fungal, and parasitic diseases.

Salmonellosis, campylobacteriosis, chlamydiosis,

tuberculosis, Newcastle Disease, and avian

influenza are amongst the most common zoonotic




Page 124

diseases transmitted from poultry to humans.

Poultry workers are at a greater risk of being

affected by these diseases. This review also

support to our research. Based on the results the

age group of up to 25 was more aware worker

than the rest of the age group. Why? The research

team asked the question about clean dress means

some worker have the habit to wear pair of dress

for work time, some of the worker have the habit

to wear clean dress in daily basis, but few of the

workers used the dress for two day or three days.

The table A. question only 12 samples answered

and the percentage was 9.2%, High percentage

was 90.8% found from 118 samples. Similar to

the second question about the habit of foot wear,

why? We had chosen this, because any time every

minute whenever we want anything we used only

our travel pack of legs, sometime we may go

hospital for children or elders and reach our home

without footwear means it can spread the growth

of microbial diversity through work place. The

table B shows in the percentage of 21.5 from 28

samples and high percentage from 102 samples.

The third question asked about gloves, why? The

research team set this kind because hands are the

direct conduct to get the infection; through the use

of hand they cleaned the fecal ooze material. This

material has the quality to induce in the skin and

cause diseases and hands are the machine for

everything like eating, cooking, washing etc.

without proper practice in the work place we may

get the foundation of ill. So cleaning is the main

factor of the above. The table C shows the

percentage variation like start from 24 samples of

18.5 and the 81.5 percentage from 106 samples.

Its basic, its simple. The number one way to

improve health in rural areas is not to provide

clean water; that comes in third. It is much easier

than that: teach people how to wash their hands

(Mihelcic, 2003). Third world communities suffer

greatly from preventable water borne diseases and

from diseases spread through poor hygiene

practices. By improving sanitation and

environmental conditions in the communities and

implementing an educational program of health

and hygiene education appropriate for the

community, the numbers of preventable deaths

and illnesses have been shown to decrease

(Panchita Paulete, 2003). According to WHO

guidelines for indoor air quality: dampness and

mould, exposure to microbial contaminants is

clinically associated with respiratory symptoms,

allergies, asthma and immunological reactions.

The microbial indoor air pollutants of relevance to

health are widely heterogeneous, ranging from

pollen and spores of plants coming mainly from

outdoors, to bacteria, fungi, algae and some

protozoa emitted outdoors or indoors. They also

include a wide variety of microbes and allergens

that spread from person to person. There is strong

evidence regarding the hazards posed by several

biological agents that pollute indoor air; however,

the WHO working group convened in October

2006 concluded that the individual species of

microbes and other biological agents that are

responsible for health effects cannot be identified.

This is due to the fact that people are often

exposed to multiple agents simultaneously, to

complexities in accurately estimating exposure

and to the large numbers of symptoms and health

outcomes due to exposure. The exceptions include

some common allergies, which can be attributed

to specific agents, such as house-dust mites and

pets. So, it’s our duty to be safe in work place and

in environment, based on this statement the

research team frames the question about wearing

of mask at work place, this will help to control the

respiratory problems. The table D showed the

percentage of 58 samples 44.6 and 72 samples

55.4.Improved hygiene (hand washing) and

sanitation (latrines) have more impact than

drinking water quality on health outcomes,

specifically reductions in diarrhea, parasitic




Page 125

infections, morbidity and mortality, and increases

in child growth,.(Water, sanitation & hygiene,

2002).the research team included the question

about washing hands before drinking water or any

intervals based on the answered from the samples

the finding were done. The table E shows the 35.4

percentage from 46 samples and 64.6 from 84

samples respectively. The chi-square test valuable

were calculated by the factors of age and

awareness, income and awareness, experience and

awareness. The Pearson chi-square value was

14.75, Degrees of freedom was 3 and p value was

.002(less than .05) these were the values of age

and awareness. The income and awareness was

Pearson chi-square value 20.155, degrees of

freedom 3 and p value was .000 (less than 0.05).

The experience and awareness was Pearson value

0.817, the degrees of freedom was 2 and p value

was 0.665. From the chi-square results the

research find out that experience is not mould the

person directly but some cases it can. Age is the

factor this is have the ability to know that what

we will do or what we didn’t. Because the

research were not get the significance in

experience where as the significance appear in

age and income compared with awareness.

Hygienic and health conscious to support our

immune actions. The research questions such as

wearing footwear, to avoid the microbial infection

in legs and foot and the questions of gloves and

mask were support to the workers from

respiratory problems like sneezing, cough and

cold. Washing hands and wearing clean dress are

important to live good life. These are the basic

questions arise from the research team and

observed by direct interview method. This is the

first stage in a process that helps empower

members of a community. To assess their

knowledge base, investigate the local

environment, visualize a future scenario, analyze

constraints on change, plan for change, and

implement it,. (PHAST, 1998).Based on this

review support, our research was analyzing the

occupational environment on rural women

workers in first stratum.

CONCLUSION

During the month of period the research was

conducted on hen poultry with randomly selected

130 samples in various poultry farm. Whenever,

we interview the women workers by directly that

time on spot awareness created to them its shows

visibly. After two or four hours the awareness was

disappear. This is because of the care less and not

giving the preference to health and long life. But

awareness not only the source to protect them,

apart from this self appraisal with awareness of

each individual concentration is more important

than the voice arise from the researcher. As a

result we entitled the aphorism to their hearts that

is realize your work and know the reality of your

occupation, this will reduce the occupational

problems of health.

ACKNOWLEDGEMENT

I thank to God to have this opportunity to do

something good to society especially women

workers, Our sincere thanks to Dr.Sasikala, head

and professor, Department of Zoology, Bharathiar

University, with her support and encourage

completing this work in smooth way. Gratitude to

Institutional Human Ethical Committee of

Bharathiar University for permit the research

work. Our heartfelt thanks to poultry farm women

workers for their patience and managing director,

supervisor and last but not least Self Help Group

members for their support in this field. Authors

acknowledge the immense help received from the

scholars whose articles are cited and included in

references of this manuscript. The authors are also

grateful to authors / editors / publishers of all

those articles, journals and books from where the

literature for this article has been reviewed and

discussed




Page 126

REFERENCES

1. FAO 1999. Statistical database, www.fao.org.

Food and Agriculture Organisation of the

United Nations. Rome, Italy. Houghton, MI.

2. Irene Figà-Talamanca Occupational risk

factors and reproductive health of women

Department of Hygiene and Industrial Health,

University of Rome ‘La Sapienza’, Piazzale

Aldo Moro 5, 00185 Roma, Italy. e-mail:

[email protected]

3. Jim Porter, USA Wrestling MRSA and other

Infection Facts Making Wrestling Safer Guide

to Recognition of Skin Infections

[email protected]

4. Kartik R Shah and Rajnarayan R Tiwari 2005,

OCCUPATIONAL SKIN PROBLEMS IN

CONSTRUCTION WORKERS Indian J

Dermatol. 2010 Oct-Dec; 55(4): 348–

351.doi: 10.4103/0019-5154.74537

5. M.S. Nasrin, M.J. Islam, K.H.M.N.H. Nazir,

K.A. Choudhury and M.T. Rahman1

Identification of bacteria and determination of

their load in adult layer and its environment

Dept. of Microbiology and Hygiene,

Bangladesh Agricultural University,

Mymensingh-2202, Bangladesh J. Bangladesh

Soc. Agric. Sci. Technol., 4(1 & 2): 69-72,

2007 ISSN 1811-6221

6. Mihelcic, Jim, Ph.D. Lecture. (13 March

2003). Michigan Technological University.

Houghton, MI

7. Mojtaba Yegani, 08 Jul 2010 Prevention /

Control Health hazards: Safety always comes

first, University of Alberta, Canada

8. Panchita Paulete (May 2003), Practice and

importance of combining health/hygiene and

environmental education with water sanitation

for sustainable health benefits in rural areas

School of Forest Resources & Environmental

Science Michigan Technological University,

Houghton, MI

9. Penny Kane, Senior Associate, Editor “women

and occupational health, issues and policy”

paper prepared for the global commission on

women's health The Office for Gender and

Health, The University of Melbourne,

Australia.

10. PHAST Initiatives in East Africa. (1998). The

World Bank. Presented at the Community

Water Supply and Sanitation Conference,

Washington,DC.http://www.wsp.org/english/fo

cus/conference/hyg_phast.pdf SOPAC WRU

Strategies.

11. Saleh, M., Seedorf, J. and Hartung, J. 2003.

Total count of bacteria in the air of three

different laying hen housing systems. Dtsch.

Tierarztl. Wochenschr., 110(9): 94-97.

12. Water, sanitation & hygiene: at a

glance..(March 2002). World Bank Group.

http://wbln0018.worldbank.org/HDNet/hdd

60/9d1422d8016e85d885256b90005e1f76/$FI

LE /5WatSan%20AAG.pdf

13. WHO guidelines for indoor air quality

:dampness and mould This document presents

World Health Organization (WHO) guidelines

for the protection of public health from health

risks due to dampness, associated microbial

growth and contamination of indoor spaces oct

2006

14. Women in Agriculture, (Dare/Icar Annual

Report 2003–2004)

http://occmed.oxfordjournals.org/search?author1=Irene+Fig%C3%A0-Talamanca&sortspec=date&submit=Submit

Londhe Shashikala et al. FUSION OF FIRST AND SECOND THORACIC RIB NEAR STERNAL END


Page 127

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Case Report




FUSION OF FIRST AND SECOND THORACIC RIB NEAR

STERNAL END

Londhe Shashikala, Kori Rohini, Panjakash Samreen

Dept of Anatomy, Al-Ameen Medical College Bijapur, KA, India


ABSTRACT

During routine work in the osteology section in the department of anatomy, Al- Ameen medical

college Bijapur, we got an unusual specimen showing fusion of first and second rib near its

sternal end on left side. Fusion was along the outer margin of first rib and inner margin of

second rib, 2.2 cm and 2.8cm behind the costo-chondral junction, along the inner and outer

margin of first and second ribs respectively. Fused segment was 8.5cm in front of vertebral end

along outer margin of first rib. 8.2cm in front of vertebral end along inner margin of second rib.

Transverse Length of fused part between first and second rib was 3.3cm and length of

intercostal space in front and behind the fusion was 0.6cm and 1.1cm respectively. Both the ends

of both ribs were free so intercostal space at the costochondral junction and at the vertebral end

was open. Because of such type of rib variations there are chances of compression of

neurovascular bundle along the intercostal space. These fused rib affect chest wall expansion,

may result in respiratory complications. Knowledge and awareness of such anatomical variation

of rib is important for physicians, radiologist and surgeons.

Key Words - Fusion, first and second thoracic rib.

INTRODUCTION

There are two types of variations related to ribs,

numerical and structural. In numerical type,

number of ribs either more or less. Cervical or

lumbar rib may be present as extra ribs, one or

more ribs may be absent as in less ribs. In

structural type, there is bifid rib, short rib and

fused rib.1 Each rib originates from the caudal

half of one sclerotome and the cranial half of the

next subjacent sclerotome. Head develops from

sometocele cells from one somite which migrate

with the caudal half of sclerotome, proximal

portion of the rib develops from both caudal and

cranial sclerotomal halves, and there is no mixing

of cells from these origins. Distal portion of rib

developed from caudal and cranial halves of

sclerotome, these cells mix as the rib extends into

the ventral bodywall and segmentation

diminishes .2 Any change or variation at this step

of development may be the reason for fusion of

ribs. Irregular segmentation of primitive vertebral

arches lead to fusion anomalies, recent

experimental studies in mice have implicated

splotch gene mutation for various structural

abnormalities in the ribs3.

Bicipital rib is an unusual anatomical pecularity

which results due to the fusion of shaft of two

distinct ribs into a common body and is seen

exclusively in relation to the first rib, either due to



Page 128

fusion of cervical rib with first rib or more

commonly due to fusion of the first rib with

second rib4, 5

. Its incidence has been reported

o.3% in a study based on chest radiograph. 5

The rib anomalies whether pathological or normal

varients such as cervical rib, pelvic rib, bifid rib

and bicipital rib etc. often indicate an underlying

systemic disorder.6

CASE REPORT

We noticed a left sided bone specimen showing

fusion of first and second ribs, during routine

work in the osteology section of department of

anatomy, Al- Ameen medical college Bijapur

.Specimen was examined in detail for its

anatomical features, measurements were done and

photograph taken from both aspects of rib as in

figure 1. Complete length of first rib along the

outer margin was 13cm, complete length of

second rib, along the inner margin was 14.3cm.

Fusion of two ribs was near the sternal end, fused

segment was 2.2cm and 2.8cm behind the

costochondral junction of sternal end, along the

inner and outer margin of first and second rib

respectively. Fused segment was 8.5cm infront of

vertebral end along outer margin of first rib and

8.2cm infront of vertibral end along inner margin

of second rib .Transverse length of fused segment

was 3.3cm. The remaining part of intercostal

space, before and after the fused segment of rib

was open. The first rib had normal anatomical

features that are head, neck and tubercle of

vertebral end was normal and at the sternal end it

had depression at the tip for costal cartilage.

Along the outer margin of first and second ribs

there were prominent impressions for serratus

anterior muscle towards the vertebral end. The

second rib had rudimentary vertebral end, head,

neck and tubercle was not prominent and sternal

end was cut.

DISCUSSION

The first rib anomalies include the floating rib,

central defects bridged by ligamentous band,

rudimentary structure terminating in a synostosis

or pseudoarthrsis with second rib, bifurcated rib

etc.7Vinita Gupta et al reported partial fusion of

rib in anterior and posterior portion and

completely blended with each other in middle, the

first intercostal space was obliterated8. Rib fusion

causes scoliosis and restriction of chest wall

expansion, which may require surgical correction.

Fused ribs also encountered in Gorlin syndrome.6

Rani Anita et al reported synostosis between the

anterior ends and shafts of 1st and 2

nd ribs of right

side. There was single articular oval facet on head

with scalene tubercle at the inner margin of shaft

of upper segment1.

David Levi reported a case of 8 year boy with

lump at inner end of first right rib. There was

history of injury, the skiagram showed fusion of

first and second rib in the middle third of their

curve, he stated that condition appeared to be

congenital. He also stated that when fusion is at

the sternal end is due to ossification at the

costochondral joint, it spread towards the sternal

end of ribs, when fusion occurs at the vertebral

end, it is due to lesion of intrathoracic content,

especially long standing collapse of lung. Fusion

of middle third is a rare condition for that no

instance is recorded in anatomy textbooks9.

Any scoliosis associated with fused rib may result

in three dimensional thoracic deformity with

adverse effect on thoracic growth and function

with development of thoracic insufficiency

syndrome, which is defined as the inability of the

thorax to support normal respiration or lung

growth. He also stated that children suffering

from thoracic insufficiency syndrome referred to

them were on oxygen or ventilator support, in all

of these patient the common denominator was a



Page 129

small, stiff distorted thorax that could not provide

volume for growth of the underlying lungs and

functioned poorly, with minimal secondary

breathing mechanism because of fused,

malformed or absent rib.10

Baumgarlner F et al reported two cases of fused

first and second ribs associated with thoracic

outlet syndrome in a radiological study.11

Seema

Deepak reported an unusual case of a bicipital rib

in which first and second rib of right side were

fused obliterating the intervening intercostals

space, she concluded that such a specimen cannot

present with thoracic outlet syndrome but can also

be an indicator of an underlying systemic

disorder.12

REFERENCES

1. Rani Anita, Rani Archana, Chopra Jyoti, Manik

Punita.Synostosis of First and Second Rib- Case

Report.J. Anat. Soc. India. 2009; 58(2):189-91.

2. Standrings, Johnson D, Ellis HAND Collin

P.Grays Anatomy, 39th edition, Churchill

Livingstone, London,2005. pp 796.

3. Evans DJR. Contribution of somatic cells to the

avian ribs. Developmental Biology.2003; 256:114-

26.

4. Turner WM. Cervical ribs and the so called

Bicipital ribs in man in relation to corresponding

structures in the Cetacea. Journal of anatomy and

physiology. 1883;17(30)384-400.

5. Terry RJ, Trotter M. The Ribs, In: Schaeffer JP

(eds) Human Anatomy, A Complete Systemic

treatise 11th edition. The Blakiston Company, New

York, Toronto, 1953, pp.192-98.

6. Glass RB, Norlon KI, Milre SA, Kaug E. Pediatric

ribs:a spectrum of abnormalities.

Radiographics.2002; 22: 87-104.

7. White JC, Poppel MH, Adams R. Congenital

malformation of the first thoracic rib: A case of

brachial neuralgia which simulates the cervical rib

syndrome. Surg Gynecol Obstel.1945; 81:643-59.

8. Vanita Gupta, Rajesh Kumar Suri, Gayatri Rath,

Hitendra Loh. Synostosis of first and second

thoracic ribs: Anatomical and radiological

assessment. International journal of anatomical

variations. 2009; 2:131-33.

9. David Levi. Fusion of middle third of the first and

second ribs. Proc R Soc Med. 1932; 25(8)1233.

10. Robert M. Cambell JR, Melvin D. Smith,Thomas

C.Mayes,John A. Mangos,Donna B. Willey-

Courand, Nusret Kose, Ricardo F. Pinero,Marden

E.Alder, DDS,Hoa L. Duong, Jennifer L.Surber,

BS.The characteristics of thoracic insufficiency

syndrome associated with fused ribs and

congenital scoliosis.The journal of bone and joint

surgery, Jbis org.203;85(3):399-408.

11. Baumgarlner F, Nelson RJ, Robertson JM.The

rudimentary first rib :Acause of thoracic outlet

syndrome with arterial compromise. Arch Surg.

1989; 124:1090-92.

12. Seema Deepak, Dikshayani KR. An unusual case

of a bicipital rib- a case report.Anatomica

Karnataka.2011; 5 (1)50-52.

Fig 1: Showing superior and inferior aspect of

fused part between 1st & 2nd rib

Shkelqim Gjevori CONSTRUCTION OF THE MATHEMATICAL MODEL FOR EVALUATION AND FORECAST OF

INTERURBAN PASSENGERS TRAVEL


Page 130

IJCRR

Vol 04 issue 17

Section: General Sciences

Category: Research




CONSTRUCTION OF THE MATHEMATICAL MODEL FOR

EVALUATION AND FORECAST OF INTERURBAN

PASSENGERS TRAVEL

Shkelqim Gjevori

Ministry of Public of Works and Transport. Institute of Transport, Tirana,

Albania.


ABSTRACT

For a more harmonious development between economy and transport and for a close relation

supply and demand for travel, proper scientific studies should be conducted to determine

operational instruments for resolving the contradictions between this report and especially those

technical instruments should provide recommendations on permanent and long-term planning

that preceded the decision and policy-makers. One of these instruments that are in use with much

interest and after 50 years are mathematical models in transportation. They are based on the

assumption that a linear equation could represent the relationship between a dependent variable

“Y “that represents a pointer or economic phenomenon and one or more independent variables

“ pXXX ...., 21 ” and that each of them taken separately can satisfy the assumption of linearity that

is expressed in mathematical form as follows,

eXaXaXaaFy nn ....... 22110 (1.0)

It is important that we have implemented practical use of information which is part of a database

system for collection and data management of travel and road traffic in our country. In particular

and very carefully, we have also defined specific requirements for input data analysis and have

made their selection for the purpose of constructing the model.

Key words: O/D matrix, demand modelling, supply, variables, balance.

INTRODUCTION

Theoretical treatment and research on our

part has been extended by examining all

transport’s components, taking into account

the current information system that forms

the basis of existing data, and providing

other forms of data impossible to ensure up

to now but that are indispensable. On the

other hand refereed to the specific

conditions of Albania's research and

collection of appropriate data is not easy

mainly because of the fragmented

characteristics of existing statistical data

and lack of time series. Basic variables

which we are supported affect the quantity

and quality of travels and we have grouped

into three categories as follows,

demographic, economic, territorial factors.


Based on three categories of the above

variables from an analysis of their

grouping, have deemed to representation

and application of the travel modeling, the

following variables as, resident population

in cities, the number of passenger vehicles,

and the active forces able to work,

economically active enterprises, number of

employees. With travel ensuring O/D

(origin-destination) between the cities of




Page 131

the country and variables expressed above

we have ensured the necessary database for

the travel modeling through the application

of selected mathematical model. To derive

the travel legality in the function of

variables "Xi" selected according to the

recommendations and analysis made by us,

we were focused on some of the country's

main towns which make up about 96 % of

the population and economic activities, of

variables "Xi" taken into consideration.

Starting from the given travel data O/D and

for the 5 selected variables we have draw 5

tables. Starting from the given travel data

O/D and for the 5 selected variables we

have draw 5 tables, travels O/D depending

on 1.the population variable (Xp), 2.number

of economically active enterprises (Xnd),

3.vehicle of transportation for passengers

(Xmj), 4.active work force (Xap) and

5.number of employees.(Xnp) as an example

only one of them (table 1) for,

a) selection of independent variables

classifying by weight occupied by each of

them, and to make a second selection of the

5 variables in the three we have applied the

mathematical model of the shape force,

2

1

cXcY (1.1)

and for,

b) modeling and forecast of interurban trips

we shall apply the mathematical model of

linear form,

eXaXaXaaY nn ...22110 (1.2)

Mathematical model of the selected form Y

= c1X c2

is based on comparing of the

process conduct and this of the model,

especially when the dependence between

the "Y" and "Xi" is not linear and more so

when there is no identity of conduct.

The objective of the method is to modify

the model parameters in order to achieve a

better fit by assuring a very fast

convergence and adjacent to the

redevelopment status and optimal.

The result of the model describes a

relationship between average variables of

the two variables. The results given by the

model are tabular and in graphical form,

showing except of the parameters values

(coefficients before variables "Xi"), and

some important indicators that show the

reliability of the model and extent of links

between the two sizes "X and Y" such as, R,

R2 (or R

2 correlation coefficients, an

indication that express the value of the

model), adjusted R2 (or Adjuster R) is

useful in comparing this model with other

models, F (value of the coefficient of

reliability, P-value (probability value of

reliability of coefficients before variables

"Xi", etc.

Based on the selected mathematical model

we have applied software of this model

(1.1) for nine main cities like Tirana,

Durres, Vlora, Elbasan, Fieri, Korca, Berat,

Shkodra, Lezha, representing cities centers

of the county towns that constitute

the major economic, social and cultural

potentials, taking as the origin one city and

destination eight other cities and so

changing the positions of origin of travels

to eight cities in an order we have received

the following results which express the

legality of dependency between

variables "Xi" and function "Y". In here we

are providing only one of the results of

application of software. Journeys in the

function of the population origination from

Elbasan. (table 1/1) where;

TT(pop)-is variable “Xp” that gives the

number of population for cities Berat,

Durres, Fier, Korca, Lezha, Shkodra, Tirana

and Vlora (which are marked with the

indices according to the vehicles number

plates of the respective cities).




Page 132

UP(O/D)-are current journeys originating

travel between the city of Elbasan and 8

other cities of destination (which in fact is

the value of the function of “Y”

mathematical model that we implemented)

YP(legality), is the result of the travel legality

"Y" according to the application of

mathematical model. From the results of

model we are taking,

Fopt = 3.762306016217445E-002

Copt(1)=2.658623009781310E007

Copt(2)=1.790759920467837

where; Fopt-function value (Y legality)

Copt(1), Copt(2)- values of model parameters.

In that way the function has the form,

790.10000002658.0 pXY

(1.3)

In the same way we extracted the results

and for eight other cities taken by order as

the origin of travels. In this way the

characteristics of transport in urban areas

reflect in a broad way the social, economic,

physical conditions at a given point in a

given time. In Table 2 are given the

variables and the correlation’s coefficient.

Thus from the 200 directions of travel

between 8 cities in the study, about 80-85%

of them express a reliable correlation

between the curve of O/D and that of

legality of the O/D found by modeling the

remaining 15-20% displays a correlation

not very satisfactory. To select which of the

variables “Xi” influence more on trips O/D,

it was applied their simulation “Xp, Xmj, Xnd,

Xfa, Xpu” by increasing them to the extent of

10%.

From the application of mathematical

model of the type power (equation 1.1) we

obtained 200 values of trips O/D between

the couple of cities and we get the

following results. Specifically for

simulation with 10 % of “Xp” we get the

results as table 3. As a conclusion based on

the above two assessments therefore;

1) to the extent of influence after the

simulation with 10% of variables,

2) and coefficient “kO/D/Xi” [travel/unit

variable].

Have made their classification, from which

we selected three of variables that have

significant impact on the generation and

attraction of trips which are,

population (Xp), vehicle of transportation of

passengers (Xmj), and economic enterprises

(Xnd)

Based on these three selected variables in

accord with (equation1.2) "construct" the

model of trips generated or attracted in

nationally rate, where, e-error term,

for X1=Xp,; X2=Xnd and X3=Xmj. Then our

function will have the following form,

Y = a0 + a1Xp + a2Xnd + a3Xmj + e (1.64)

STATISTICAL METHODS

Descriptive statistics, mean and standard

deviation were tabulated using SPSS 16.0

software.

RESULTS

Based on official data of INSTAT and the

values of travel according to the matrix

O/D we have benefited Table 4. To obtain

the mathematical form of the model for

interurban travel in national scale have

applied software SPSS, where we have the

following results and linear form of

mathematical model of inter urban

travel. According to the results of software

and the taken coefficients interurban travel

modeling has the form of general equation

as follows,

Y=1003.578–0.029Xp+1.68Xnd+0.632Xmj

(1.7)

Based on the above model we calculated

the values of the function “Y“ for 22

centering (cities) and the results are given




Page 133

in table 5. In this case, model rejected by

the negative value of coefficient a1 = -0.029

variable that is before "Xp" which is

impossible, because it is in confrontation to

the generation theory and trips attraction

which estimate that the population is the

community of variables that

affect positively in increasing the travel.

This discrepancy is due to multicorelation

among the selected variables by our side

with the desire to include as many variables

in the modeling of travel. To continue to the

realization of the goal of work shall exclude

the variable "Xp" from the table 5 above and

would appreciate the dependence of travels

with two other variables “Xmj,& Xnd”.

Besides the elimination of population

variables we have increased the number of

zone (cities) to have an even greater

representation of spatial interaction. To

obtain the mathematical form of model for

generation interurban trips (table 5) in

nationally scale we have applied again

software SPSS, where we get the following

results and linear form of mathematical

model of generating interurban travel. (table

6,7)

DISCUSSION

According to the results of software and

coefficients of Table 7, are,

a0 = - 329.479; a1= 1.315; a2= 0.535; (1.8)

and interurban travel modeling generation

has the form of general equation as follows

Ygj = - 329.4+1.315*Xnd+ 0.535*Xmj (1.9)

Regarding the reliability of function (1.9)

we halted in the analysis of some

parameters that are evaluated by software

through ANOVA (analysis of variation).

From the results of tables 6 and 7 we see

that the coefficient of determination is

R2=0.97 or 97%, which highlights the

extent of variation of the “Y” from two

variables “Xmj,&Xnd” and from statistical

theory is considered as a criterion to judge

objectively on the quality of the model

because it is the indicator of the degree of

approximation of “Y and Xi” likewise, “R”

corrected (adjusted R Square) and "R"

Multiple, have very high values that attests

to a strong correlation between independent

variables and dependent variable.

The data results of Table ANOVA according

the table OUTPUT SUMMARY results that

Significance F=1.45705E-20 is less

than 05.0 so we are within a specified

condition, while the validity coefficient

(importance) to the 05.0 According the

values of table I the book of Statistics) we

have that probability of regression

coefficient at the variable “Xnd" is 90% as

the P-value=0:06, while the probability of

the coefficient of regression at the variable

"Xmj" is 95% as the P-value =0007 so many

good values which estimate the model of

"construction". Above model parameters

carry a significant meaning so the

regression parameter a1=1.315 before

variable "Xnd" indicates that when the

number of enterprises increased by 1% the

number of trips generated will increase by

1.31% while for the regression parameter

a2=0.535 before variable "Xmj" it shows that

when the number of vehicles increased by

1% the number of trips generated will be

increased by 0535%. Regarding the

parameter a0 = - 329.479 in general have no

much sense and requires great care in

interpretation.

CONCLUSION

The Design of a model for the evaluation of

interurban trips will help decision makers

in policy planning, management and

investment in the transport field.




Page 134

ACKNOWLEDGEMENT

I thank the staff of the Institute of Transport

for the support given in data-base as well as

colleagues of the Faculty of Mechanical

Engineering, Polytechnic University of

Tirana. Also thank the authors and co-

authors for the literature that was useful in

this publication. Authors acknowledge the

immense help received from the scholars

whose articles are cited and included in the

references of this manuscript.

REFERENCES

1. John P. Sammon, Robert J. Caverly

2007. Transportation Systems.

2. Josef Sussman 2005. Introduction to

Transportation System.

3. Kumares C. Sinha, Samuel Labi. 2007.

Transportation Decision Making.

4. Avishai Ceder 2007. Public Transport

Planning and Oparation.

5. C.S. Papacostas. P.D. Prevedours 2005.

Transportation Engineering and

Planning.

6. Prof.Dr. Myslym Osmani. 2005

Statistics.

7. Louis Berger. S.p.a. 2009. Albania

National Transport Plan.

8. INSTAT 2009. Indicators by

Prefectures.

9. Peter R. Stopher. Arnim H. Meyburg.

Urban Transportation Modeling and

Planning. 1975

10. Michael D. Meyer, Eric J. Miller. 2001

Urban Transportation Planning; a

decision-oriented approach.

11. Susan Hanson. The geography of urban

transportation. Aug 6. 2004

12. Ryuichi Kitamura, Satoshi Fujii, and

Eric I. Pas. Time-use data analysis and

modeling: toward the next generation of

transportation planning methodologies.

1997.

13. David A.Hensher, Kenneth J.Button.

2000. Handbook of Transport

Modelling.




Page 135

TABLES

TABELA 1. TRAVELS O/D DEPENDING ON THE POPULATION VARIABLE. (XP)

Destination

cities

population

“Xp”

The cities of origin

Tirana Durresi Fieri Elbasani Vlora

Travels O/D

Berat 127837 2768 721 450 62 827

Durrës 181662 15230 - 1155 1225 626

Elbasan 221635 4224 1150 640 - 681

Fier 199082 3850 1100 - 1050 3560

Korcë 142909 4046 427 73 583 177

Lezhë 67734 2118 171 75 15 50

Shkodër 185395 2247 532 82 54 79

Tiranë 519720 - 14097 4191 4583 3694

Vlorë 147128 4377 565 2045 404 -

TABLE 1/1 RESULTS OF APPLICATION OF SOFTWARE. JOURNEYS IN THE FUNCTION OF THE

POPULATION ORIGINATING FROM ELBASAN.

cities TT

(pop)

UP

(O/D)

YP

(legality)

BR 127837 62 371

DR 181662 1225 696

FR 199082 1050 820

KO 142909 583 453

LE 67734 15 119

SH 185395 54 722

TR 519720 4583 4573

VL 147128 404 477




Page 136

TABELA 2. VARIABEL AND CORELATION’S COEFFICIENT.

City of origin

V A R I A B E L

AND CORRELATION’S COEFFICIENT

Xp Xmj Xnd Xfa Xpu

Correlation coefficients

Elbasan 0.94 0.95 0.98 0.91 0.98

Durrës 0.98 0.99 0.99 0.94 0.99

Fier 0.77 0.80 0.83 0.67 0.79

Vlorë 0.52 0.44 0.58 0.66 0.50

Tirane 0.95 0.80 0.59 0.99 0.89

TABLE 3. TRAVEL INCREASE BY SIMULATION 10 % OF “ XP”

Cities of Travel Origine

TIRANA ELBASAN FIER VLORE

Travel

increase in %

Travel

increase in %

Travel

increase in %

Travel

increase in %

Cities of Travel Destinations

BR 21 BR 18.6 BR 16.3 BR 12.6

DR 21 DR 18.6 DR 16.3 DR 12.6

EL 21 FR 18.6 EL 16.3 EL 12.6

FR 21 KO 18.6 KO 16.3 FR 12.6

KO 21 LE 18.6 LE 16.3 KO12.6

LE 21 SH 18.6 SH 16.3 LE 12.6

SH 21 TR 18.6 TR 16.3 SH 12.6

VL 21 VL 18.6 VL 16.3 TR 12.6

TABLE 4. REAL TRAVELS ACCORDING TO 3 VARIABLES.

No.

Cities Xp Xnd Xmj

O/D

Real travels

1) Tirana 519720 19363 72925 63922

2) Durres 181662 6058 19721 25222

3) Kavaje 78179 1683 4430 6296

4) Kruje 63517 455 5641 5837

5) Elbasan 221635 3422 14594 8390

6) Peqin 32964 267 1170 534

7) Fier 199082 3906 12449 9423

8) Vlore 147128 3947 12143 11574

9) Lushnje 143933 2120 5611 4069

10) Shkoder 185395 1582 13972 4964

11) Sarande 35089 433 3221 1809

12) Lezhe 67734 550 4085 3236

13) Lac 54392 480 3706 3562

14) Korce 124870 3245 11016 5367

15) Pogradec 70471 578 2846 1359

16) Librazhd 72387 131 1081 870




Page 137

17) Erseke 17161 224 682 393

18) Kukes 102037 344 2443 1287

19) Gjirokaster 54647 1327 4713 1756

20) Tepelene 32404 141 1241 1077

21) Permet 25780 168 924 429

22) Diber 132546 723 4039 1048

TABLE 5. THE VALUES OF THE FUNCTION FOR LEGALITY OF TRAVELS “Y’

AND REAL TRAVELS “Y”

a0

a1

Xp

b1

Xnd

c1

Xmj

Legality

of travels

(Y) O/D

Real

travels

O/D (Y)

City

1003.578 0.029 519720 1.68 19363 0.632 72925 64550 63922 TR

1003.578 0.029 181662 1.68 6058 0.632 19721 18376 25222 DR

1003.578 0.029 78179 1.68 1683 0.632 4430 4363 6296 KJ

1003.578 0.029 63517 1.68 455 0.632 5641 3491 5837 KR

1003.578 0.029 221635 1.68 3422 0.632 14594 9548 8390 EL

1003.578 0.029 32964 1.68 267 0.632 1170 1235 534 PE

1003.578 0.029 199082 1.68 3906 0.632 12449 9660 9423 FR

1003.578 0.029 147128 1.68 3947 0.632 12143 11042 11574 VL

1003.578 0.029 143933 1.68 2120 0.632 5611 3937 4069 LU

1003.578 0.029 185395 1.68 1582 0.632 13972 7115 4964 SH

1003.578 0.029 35089 1.68 433 0.632 3221 2749 1809 SR

1003.578 0.029 67734 1.68 550 0.632 4085 2545 3236 LE

1003.578 0.029 54392 1.68 480 0.632 3706 2574 3562 LA

1003.578 0.029 124870 1.68 3245 0.632 11016 9796 5367 KO

1003.578 0.029 70471 1.68 578 0.632 2846 1729 1359 PG

1003.578 0.029 61234 1.68 131 0.632 1081 131 870 LB

1003.578 0.029 17161 1.68 224 0.632 682 1313 393 ER

1003.578 0.029 102037 1.68 344 0.632 2443 166 1287 KU

1003.578 0.029 54647 1.68 1327 0.632 4713 4626 1756 GJ

1003.578 0.029 32404 1.68 141 0.632 1241 1085 1077 TP

1003.578 0.029 25780 1.68 168 0.632 924 1122 429 PR

1003.578 0.029 132546 1.68 723 0.632 4039 927 1048 DI

Table 6

SUMMARY OUTPUT

Regression Statistics

Multiple R 0.9888221

R Square 0.97776914

Adjusted R Square 0.97591657

Standard Error 1959.44908

Observations 27




Page 138

Table 7

ANOVA

df SS MS F

Significance

F

Regression 2 4052837796 2026418898 527.79013 1.45705E-20

Residual 24 92146576.44 3839440.68

Total 26 4144984373

Coefficients

Standard

Error t Stat P-value Lower 95%

Intercept

- 329.4979926 429.6622815 -0.7668767 0.4506332 1216.277351

Xnd 1.31581788 0.677392307 1.94247538 0.0639025 0.082251118

Xmj 0.535742204 0.183371013 2.92162973 0.0074701 0.157283036

Suresh sukumar et al. INFECTION RISK CONTROL IN “COMPUTER RADIOGRAPHY IMAGING PLATE” IN

DIAGNOSTIC RADIOLOGY DEPARTMENT


Page 139

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research




INFECTION RISK CONTROL IN “COMPUTER RADIOGRAPHY

IMAGING PLATE” IN DIAGNOSTIC RADIOLOGY DEPARTMENT

Suresh sukumar, Sushil Yadav

Department of Medical Imaging Technology, Manipal College of Allied

Health Sciences, Manipal University, Manipal, KA, India


ABSTRACT

This study was carried out in order to establish whether infection control measures we’re being

undertaken sufficiently on Computer Radiography Imaging Plate (CRIP) used in Radio

Diagnosis and Imaging Department of our medical college. CRIP is used to obtain the take

computerised radiographic image. This study involved the swabbing of a sample of CRIP used

within different areas of the department. Swabs were taken from the area on the corners and the

centre of the plate. Each plate was firstly swabbed to determine the current level of

microorganism contamination (determination of baseline data) and then again after

recommended cleaning. Comparisons were then made between the number of microorganisms’

present (colony forming units/cm2) pre and post-cleaning at each location. All CRIP were found

to be contaminated with microorganisms. Methylated spirit used in the practice of medicine,

with water and soap is used to clean the CRIP was found to be significantly reduce the amount

of microorganisms present. The results suggest that the All CRIP were not being cleaned

sufficiently which has infection control implications for the department. In order for cross

contamination to be kept to a minimum an effective infection control policy needs to be

employed and this Should be to carry out regular cleaning.

Key word: Infection control, computer Radiography Imaging Plate, Hospital acquired infections,

Radiology Department

INTRODUCTION

Infection within healthcare has been in the

news and has become a high priority

recently, particularly as some infections

are becoming harder to treat. The

resistances of antibiotics and other

antimicrobial agents have been reported on

along with concerns regarding the rise of

methicillin resistant Staphylococcus aureus

(MRSA) (1). Staphylococcus aureus is one

of the most common of all bacteria and can

cause superficial infections of the skin and

serious infections (2).Epidemic strains

exist, which spread easily from person to

person and can cause ward closure and

disrupt hospital services (3). Infection

control in hospitals is concerned with

decontamination; this prevents

microorganisms reaching a susceptible site

in sufficient quantities to cause infection or

potential harm to patients (4). Hospitals

can become contaminated with organic

matter and potentially infectious organisms

and a safe environment can only be

achieved by decontamination in the form

of cleaning, disinfection and sterilization,

breaking the chain of infection (5). A

major reason for the importance of

infection control is to prevent the

occurrence of Nosocomial or Hospital

Acquired Infections. These are infections

that occur during a patient’s stay in




Page 140

hospital which were not present or

incubating at the time of admission (2). In

contrast to community acquired infections

these infections usually occur as a result of

pathogens taking advantage of patients

whose normal defences against infection

are contravened (2).

AIM

To establish whether CR plate can become

contaminated with microorganisms and

become a potential reservoir for cross

infection and if simple, regular cleaning

can significantly reduce this cross

infection risk

Objectives

1. To determine whether there is currently

a detectable Presence of

microorganisms on a sample of CRIP

2. To determine any presence of

microorganisms after recommended

cleaning (methylated spirit used in the

practice of medicine with water and

soap is used to clean the CRIP).

3. To evaluate the findings and make

suggestions for future practice,

including recommendations for re-audit.

METHOD

20 CRIP were swabbed, from Different

areas within the Radiology department.

These included general X-ray rooms for

inpatients (room number 1, room number

2, and room number 3) and outpatient’s

room. Randomisation of the sample was

not practical, as it was necessary to

ascertain data from each area. It was

possible to swab all of CRIP from general

X-ray rooms for in-patients (room number

1, room number 2, and rom number 3) and

outpatients. Swabbing was carried out with

Tryptic Soy Agar contact plates which are

used to sample flat surfaces of equipment.

They consist of a domed surface which is

placed gently upon the area causing any

microorganisms to be transferred onto the

agar (10) (13). The plates were taken to the

microbiology laboratory for culturing. The

current level of microorganism

contamination on the sample CRIP is

known as baseline data. Following the

collection of baseline data, each CRIP in

the sample was cleaned according to

recommended guidelines and swabbing

was repeated. Data collected from this part

of the audit was to identify a standard to

compare to future practice and any future

infection control carried out on CRIP

After the data is collected the presence and

absence of infection is mentioned as

presence in CRIP and absence in CRIP.

RESULT

From this work all CRIP pre cleaning were

contaminated with microorganisms. Table

shows pre and post-cleaning results for

location general X-ray rooms for in-

patients (room number 1, room number 2,

rom number 3) and out-patients x ray

room. Post-cleaning data demonstrates that

on most of the CRIP the number of colony

forming units was reduced after cleaning.

All plates were inspected by microbiology

staff to identify the range of

microorganisms present. Species of

microorganisms found across the samples

included most significantly Gram positive

cocci in the form of Staphylococci, both

coagulases positive and negative.




Page 141

DISCUSSION

Despite the fact that no Methicillin

resistant strains exist was present upon the

CRIP sampled, microorganism growth was

found on all CRIP. This compares with

other studies (6-9) Coagulase-negative

staphylococci are found as normal skin

flora and include for example

Staphylococci epidermis. These bacteria

rarely cause infection (2, 4, 13). It has

however recently been recognised that

Staphylococci epidermis can be an

important cause of HAIs as it produces an

extracellular polysaccharide, a type of

slime that enables it to adhere to plastics

and metals(2,11). Staphylococcus aureus

was also identified and is coagulase

Positive staphylococci.

One-third of the population carry it on

their skin or in their nose and throat

asymptomatically (3). However, it is an

important pyogenic pathogen, causing pus

to form, which can cause a range of

superficial infections of the skin if it

penetrates the dermis such as septic spots,

boils and abscesses and other more serious

problems such as

osteomyelitis, septicaemia and pneumonia

(2,3,11, 13) It is also an important cause of

HAIs, being responsible for around 40 to

50% of surgical wound infections and

approximately 25% of blood stream

infections(2,13), and is particularly

capable of developing resistance to

antibiotics. Methicillin resistant strains

exist (MRSA), which are found in greatest

abundance. In the hospital setting as many

patients receive antimicrobial therapy and

are vulnerable to serious infection (2), it is

also becoming recognised as an important

pathogen due to its ability to colonise and

cause infection of biomedical devices.1

Staphylococci released in skin scales will

collect in dust and survive for long periods

of time in the environment (2, 13).

Swain and Flint on (7) compared the use

of soap and water with alcohol wipes and

phenolic disinfectant for the infection

control of X-ray cassettes and concluded

that all cleaning methods had a significant

reduction in bacterial numbers. However,

the alcohol wipes were found by the

authors to be 100% effective, because of

this and ease of use they were

recommended as the cleaning method of

choice. Another study shed doubt on

The use of alcohol wipes, forensic tools

were used to look for the presence of blood

on seemingly clean cassettes and results

suggested that if alcohol wipes were used

universally to clean cassettes they are

ineffective in cleaning any that are blood

soiled (12). In our study use of methylated

spirit used in the practice of medicine

(especially for cleansing the skin before

injections or before surgery) with water

and soap is more effective method of

cleaning the CRIP.

CONCLUSION

The results of the audit suggest that the

CRIP were not cleaned effectively.

Although the microorganisms identified

are quite harmless in the majority of cases,

all have the potential to be pathogenic

when coming into contact with the variety

of patients that present for examination in

the Radiology department. This possibility

is increased in cases where for example,

there are damaged sites of skin such as

wounds or cannula insertion sites (2). An

effective infection control policy for the

cleaning of CRIP should be established as

an essential method to reduce cross

contamination. It can be concluded that

cleaning with methylated spirit used in the

practice of medicine (especially for




Page 142

cleansing the skin before injections or

before surgery) with water and soap is

more effective method of cleaning CRIP

can significantly reduce the number of

microorganisms present and it should be

carried out routinely.

ACKNOWLEDGEMENT

Author acknowledges the immense help



manuscript. The authors are also grateful

to authors / editors /publishers of all those



and discussed. The author is highly

thankful to the referees for their very

constructive, valuable suggestions and

useful technical comments, which led to a

significant improvement of the paper.

REFERENCES

1. National Audit Office. The

Management and control of hospital

Acquired infection in acute NHS trusts

in England. London:National Audit

Office; 2000.

2. Wilson J. In: Infection control in

clinical practice. 3rd Ed. Edinburgh:

Elsevier Limited; 2006.

3. Gould D, Brooker C. Applied

Microbiology for Nurses. Basingstoke:

Macmillan Press Limited; 2000

4. McCulloch J, editor. Infection control,

science, management And practice.

London: Whurr Publishers; 2000.

5. Horton R, Parker L. In: Informed

infection control practice. 2nd ed.

Edinburgh: Churchill Livingstone;

2002.

6. Fox M, Harvey J. An investigation of

infection control for X-ray Cassettes in

a Diagnostic Imaging Department.

Radiography 2008; 14:306e11.

7. Swain JA, Flinton DM. X-ray cassettes:

a potential crossinfection Risk? Journal

of Diagnostic Radiography and Imaging

2000; 3:121e5.

8. Smith A, Lodge T. Can radiographic

equipment be contaminated?By micro-

organisms to become a reservoir for

cross Infection? Synergy 2004; Dec:

12e7.

9. Hodges A. Radiographic markers:

friend or fomite? Radiologic

Technology 2001; 73:183e5.

10. Booth C. Microbe monitoring.

Cleanroom Technology 2006; Oct:

18e20.

11. Meers P, Sedgwick J, Worsley M. The

microbiology and epidemiology of

infection for health science students.

London: Chapman and Hall; 1995

12. Study on blood contamination reveals

disturbing results. Society of

Radiographers. Available from:

www.sor.org/members/snnarchive/SNR

Aug03p07.pdf; 2003 [accessed

16/04/09].

13. ‘‘Do lead rubber aprons pose an

infection risk?’’ Helen Boyle, Ruth M.

Strudwick (2010)




Page 143

Table 1: The situation of all the CRIP used in the study.

Room

number

Number

of

cassette

Before cleaning – presence of infection After cleaning – presence of infection

Room

number – 1

(inpatients)

4 Increase number of microorganisms (colony forming

units/cm2)

(CRIP - 1 to 4)

Absent of microorganisms (colony forming

units/cm2) in 1 CRIP and decrease in number

of microorganisms’ present (colony forming

units/cm2) in 3 CRIP Absent – CRIP -4

Decrease – CRIP – 1st ,2nd and 3rd

Room

number – 2

(inpatients)

4 Increase number of microorganisms (colony forming units/cm2) (CRIP - 5 to 8)

decrease in number of microorganisms’ (colony forming units/cm2) in 4 CRIP

Decrease – CRIP – 5th ,6th ,7th and 8th

Room

number – 3

(inpatients)

5 Increase number of microorganisms (colony forming units/cm2)

(CRIP - 9 to 13)


units/cm2) in 2 CRIP and decrease in number

of microorganisms (colony forming units/cm2) in 3 CRIP

Absent – CRIP -10th and 11th

Decrease – CRIP – 9th ,12th and 13th

Out patients 7 Increase number of microorganisms (colony forming

units/cm2)

(CRIP - 14 to 20)


units/cm2) in 5 CRIP and decrease in number of microorganisms (colony forming units/cm2)

in 2 CRIP

Absent – CRIP -15th ,16th ,18th ,19th and 20th Decrease – CRIP – 14th and 17th

Table 2:- The situation of all the CRIP used in the study

Room number CRIP

Room number 1 (inpatients) CRIP - 1st to 4

th

Room number 2 (inpatients) CRIP - 5th to 8

th

Room number 3(inpatients) CPIP – 9th to 13

th

Out patients room CRIP – 14th to 20

th

Sushil Kumar Tiwari et al STRESS ANALYSIS OF SPUR GEAR USING FINITE ELEMENT METHOD – A REVIEW


IJCRR

Vol 04 issue 17

Section: Technology

Category: Review




STRESS ANALYSIS OF SPUR GEAR USING FINITE ELEMENT

METHOD – A REVIEW

Sushil Kumar Tiwari, Upendra Kumar Joshi

Department of Mechanical Engineering JEC Jabalpur (M.P.) India


ABSTRACT

The basic aim of this review paper is to provide the information for calculating the stresses of an

involute gear in meshing. Gears are critical component in the rotating machinery. The

researchers throughout the years had given various research methods such as Theoretical,

Numerical and Experimental. We prefer the Theoretical and Numerical methods because

Experimental testing can be expensive. This study says Finite Element Method is the best

numerical solution for calculating gear stress.

Key words: Spur Gear, Stress Calculation, Bending Stress, Hertz Stress, Finite Element Method.

INTRODUCTION

Gears are use to transmit power and

motion from one shaft to another. Spur

gear is cylindrical in form and has teeth,

which are of involute form in most cases.

The tooth surface elements are parallel to

the gear axis. Each gear tooth may

consider as a cantilever beam. When it

transmits the load, it subjected to bending.

The bending stress is highest at the fillet

and can caused breakage or fatigue failure

of tooth in root region, whereas contact

stresses are on the both side of the tooth

may causes Scoring Wear, Bending and

Pitting fatigue.[1]

Pitting fatigue is a compressive fatigue

occurring at the point of maximum

Hertzian stress. Gear stresses are affected

by surface hardness, carbon content,

metallurgical structure and lubrication

condition. Gear tooth strength is the ability

to resist tooth breakage and the ability to

resist pitting is referred as durability.[1] To

calculating bending stress, Lewis Formula

is used, and for calculating contact stress

Hertz equation can be applied in spur

gear.[2]

BENDING STRESS (LEWIS

FORMULA) [1]

A gear tooth is essentially a stubby

cantilever beam. At the base of the beam,

there is tensile stress on the loaded side

and compressive stress on the opposite

side. The ability of gear tooth to resist

tooth breakage usually referred to as their

‘Beam Strength’. Wilfred Lewis accurately

calculated this in 1893; he conceived the

idea of inscribing a parabola of uniform

strength inside a gear tooth.



Figure (1) shows a rectangular cantilever beam. The stress at the root of this beam given by,

------------------

(1)

If we substitute a gear tooth for the rectangular beam, we can find the critical point in the root

fillet of the gear by inscribing a parabola.

Now, from figure (3) and, from equation (1)

Where, Wt = Transmitted Load (Newton)

F = Face width (m or mm)

m = Module (m or mm) and, Y = Lewis form Factor.

Y is the function of number of teeth, pressure angle and an involute depth of the gear, and the

value of

It is a fact that when teeth mesh, the load is delivered to the teeth with some degree of impact.

To calculate bending stress, the velocity factor must be used in calculation. In the case of cut

or milled profile gears, using Barth equation, [2]

The velocity factor, (Where, V is the Pitch Line Velocity)

And,

Where, n= r.p.m and, d= Pitch diameter of Gear or Pinion (mm)

Now Lewis equation becomes,

--------------- (2)



CONTACT STRESS (HERTZ EQUATION) [1] Following Figure (4) shows the stresses in the region of tooth contact.

In the centre of the band, there is a point of

maximum compressive stress. Directly

underneath this point, there is a maximum

subsurface shear stress.

Just ahead of the band of contact, there is a

narrow band of the compression and just

behind the band of contact; there is a

narrow region of tensile stress.

A bit of metal on the surface of a gear

tooth goes through a cycle of compression

and tension each time, when a mating gear

tooth passes over it. If gear tooth loaded

heavily enough, and there will usually be

evidence of both surface cracks and plastic

flow on the contacting surface, and there

may be a rupturing of the metal due to

surface shear stresses.

The stress on the surface of gear teeth are

usually determined by formula derived

from the work of H. Hertz’s; and these

stresses are termed as Hertz stress. Hertz

determined the width of the contact band

and the stress pattern when various

geometric shapes were loaded against each

other. Considering figure (5),



Where, W = applied force

L = length of cylinders

B = width of the band of contact

The Hertz formula for the width of the band of contact is,

Where,

K1 = and, K2 =

1, 2 = Possion’s Ratio, and, E1, E2 = Modulus of Elasticity,

Now, the maximum compressive stress is,

Putting the value of B, and = 0.3

Above Hertz formula can be applied to Spur Gears quite easily by considering that the

contact condition of gears are equivalent to those of cylinders having the same radius of

curvature at the point of contact as the gear have. Ali Raad Hassan [8] presented formula for

calculating Hertz equation for contact stresses in the pair of mating spur gear teeth. As figure

(6)



Assuming R1 and R2 are as the respective radius of the involute curve at the contact point.

Thus and,

Now the Hertz equation for contact stresses in the teeth becomes,

-------------- (3)

Where and are the pitch radius of

the Pinion and Gear respectively and is

the pressure angle. Equations (2) and (3)

are use for quick calculation of Bending

and Contact stress of a pair of mating gear

efficiently. American Gear Manufacture

Association (AGMA) provides the

standard equations for calculating bending

and contact stresses for a pair of mating

gear including various factors.

Following literature review gives a lot of

information about stress calculation based

on Lewis formula, Hertz theory,

AGMA/ANSI (American National

Standards Institute) equations and Finite

Element Method.

LITERATURE REVIEW

Shuting Li (2002) researcher, performed

loaded tooth contact analysis of a three

dimensional, thin-rimed gear by presenting

a method that combines the Mathematical

Programming Method (MPM) with the 3-

dimentional finite element method and

compare the results with experiment. and

concluded that FEM with MPM efficiently

calculates bending stress in gears.[3]

Andrzej Kawalec (2006) author gives

comparative study of tooth-root strength

evaluation methods used within

International Standard Organization (ISO)

and AGMA standards then verifying them

with Finite Element method. The results

allow for a better understanding of existing

limitation in the current standards applied

in engineering practice as well as provide a

basis for future improvement of gear

standard.[4]

Jose I. Pedrero (2007) researcher used a

nonuniform model of load distribution

along the line of contact. He says if the

load is, assume to be uniformly distributed

along the line of contact, simple equations

given by the linear theory of elasticity and

the Hertzian contact model are not good

agreement with experimental results. In



this paper a non uniform model has been

considered for stress analysis at contact

ratio 2 and 2.5, and concluded that,

1) The critical tooth-root stress

corresponds to contact at the inner

point of the outer interval of the two

pair tooth contact, and teeth loaded

60% of total transmitted load.

2) The critical contact stress corresponds

to either one of the three following

condition.

(a) Contact at the inner point of contact

and teeth loaded with 25% of the total

transmitted load.

(b) Contact at the inner point of the two

pair tooth contact and teeth loaded

with 40% of the total transmitted load.

(c) Contact at the outer point of the inner

interval of the two pair tooth contact,

and teeth loaded with 60% of the total

transmitted load.[5]

Shuting Li (2007) researcher presented 3-

Dimentional Finite Element Method to

conduct Surface contact stress (SCS) and

Root bending stress (RBS) calculations for

a pair of spur gear with Machining Error

(ME), Assembly Error (AE) and Tooth

Modification(TM). Moreover, found that

ME, AE and TM exerts great effects on

SCS and RBS of the gear. Results obtained

by International Standard Organization

(ISO) and Japan Gear Manufacturing

Association (JGMA) standards are

comparing with result of Finite Element

Analysis. [6]

Shuting Li (2008) researcher investigates

the effect of addendum modification on

contact strength, bending strength and

basic performance parameters of spur gear.

A face contact model of teeth and MPM

and 3D finite element methods are used

together to conduct loaded tooth contact

analysis. He concluded that Hertz formula

is not exact enough for the contact stress

calculation because in engagement

position the contact point is for away from

the pitch point, also Hertz formula cannot

be use for contact stress calculation of the

gear at Tip or Root contact. Contact stress

and contact width are changed slightly if

addendum becomes longer and number of

teeth is not changed.[7]

Ali Raad Hassan (2009) author is

analysed contact stress between two spur

gear teeth in different contact position,

between 0º to 30º. Start with an angle of 0º

and end at 30º with an interval of 3º with

corresponding length of contact and

contact ratio. and found that a highest

value of contact stress is at beginning of

contact and then starts reducing until it

reaches the location of single tooth

contact. Result obtained by finite element

analysis is compare with theoretical value

which is found at geometrical contact

point.[8]

Rubin D. Chacon (2010) researcher

analysed the contact stress between spur

gear teeth using a plane model and validate

Hertz stress and AGMA contact stress

with finite element contact stress. Then

concluded that FEM is able to simulate

contact stress in a pair of mating gear, the

contact stress is highest at higher point on

the involute and lower at a single pair of

teeth. Assume the full load transmitted and

minimal at the pitch point of contact.[9]

Konstandinos G. Raptis, (2010) author

performed photo-elasticity test to

determine stresses in a pair of mating gear.

Obtained experimental results were

comparing with the theoretical value of

maximum stress at gear tooth root (when

tooth is loaded at their most unfavorable

contact point i.e. highest point of single



tooth contact). Then concluded that the

result obtained by the applied method is in

reasonable value whereas it rises with

increasing number of teeth on the large

gear.[10]

Wei Yangang,(2010) author provided a

theoretical procedure for obtaining

maximum contact stress at different point

in meshing under the light of Hertz

formula. and concluded that when the

number of teeth on small gear is smaller

than a certain value, the maximum contact

stress in the meshing process is not

generated in the inner critical point in the

single tooth meshing region of the small

gear.[11]

Xianzhang FENG (2011) author use a

precise model in large-scale CAD software

and define the stress and displacement

field to determine the maximum equivalent

stress and maximum displacement. By

defining the quasi-static characteristic of

finite element model, shows that model

can accurately simulate the distribution of

equivalent stress and displacement change

in the process of teeth meshing. The

results are well agree with the actual

meshing law and not only verify the

correctness of model but also expect to

dynamic analysis of gear.[12]

Ignacio Gonzlez-Peraz (2011) researcher

developed a finite element model and

validate in terms of contact area, pressure

distribution and maximum contact

pressure for those cases where the Hertz

theory can be applied and provide partial

crowning to the finite element model

where Hertz theory do not work properly.

And concluded that analysis results gives

good agreement with Hertz theory for

calculating maximum contact pressure,

contact area, and deflection with minute

errors. [13]

Ali Kamil Jabur, (2011) author

investigates the characteristics of an

involute gear system including contact

stress between a pair of the gear from 3-

dimansional analysis and compared the

result with experimental data.

Experimental setup uses DC servomotor

and mounting the strain gauge in the tooth

of the gear made by Polyimide materials.

and concluded that increasing the spur

gear design parameters (number of teeth

with module) leads to improvement in the

tooth strength and increasing the thickness

of critical section and makes it able to

withstand higher load.[14]

S. Sankar, (2011) author used circular

root fillet instead of standard trochoidal

root fillet in gear. and concluded that the

tooth deflection in the circular root fillet is

less when compared to the trochoidal root

fillet, further there is appreciable reduction

in bending stress and contact shear stress

for circular root fillet design in comparison

to that of trochoidal root fillet design.[15]

Seok- Chul Hwang (2011) author

compared the variation of contact stress

during rotation with contact stress at

Lowest Point of Single Tooth Contact

(LPSTC) and concluded that gear design

that consider the contact stress in a pair of

mating gear is more severe than that of the

AGMA standard.[16]

Massimiliano Pau (2012) researcher

performed a tooth contact analysis, and

contact area, contact pressure using an

ultrasonic experimental setup. The results

provide the information about the size and

shape of the nominal contact area and

contact pressure distribution. He

concluded that the ultrasonic method has

good potential as an effective tool in

investigating contact problem in gear.[17]



CONCLUSION

The above literature review presents that

the Finite Element Method is widely used

for stress analysis in a pair of gear. In

addition, FEM software has been use for

performing meshing simulation. Almost in

all of the above cases, contact stress

calculation and Bending Stress calculation

play more significant role in the design of

gear. This study shows that Hertz theory is

the basis of contact stress calculation and

Lewis formula is use for calculating

bending stress in a pair of gear.

Theoretical result obtained by Lewis

formula and Hertz equation and results

found by AGMA/ANSI equations are

comparable with Finite Element Analysis

of spur gear.

REFERENCES 1. Darle W. Dudley, Practical Gear

Design, McGraw-Hill Book Company,

1954

2. Peter R.N. Childs, Mechanical Design,

Second edition, Elsevier Butterworth-

Heinemaan, 2004

3. Shuting Li, ‘Gear Contact Model and

Loaded Tooth Contact Analysis of a

Three-Dimensional Thin-Rimmed

Gear’ Journal of mechanical Design,

ASME, 2002; vol.124/511.

4. Andrzej Kawalec, Jerzy Wiktor,

Dariusz Ceglarek, ‘Comparative

Analysis of Tooth-root Strength Using

ISO and AGMA Standard in Spur and

Helical Gear With FEM-based

Verification’ Journal of mechanical

Design, ASME, 2006; vol. 128/1141.

5. Jose I. Pedrero, Izaskun I.Vallejo,

Miguel Pleguezuelos, ‘Calculation of

Tooth Bending Strength and Surface

Durability of High Transverse Contact

Ratio Spur and Helical Gear Drives’

Journal of mechanical Design, ASME,

2007; vol. 129/69.

6. Shuting Li, ‘Finite Element Analyses

for Contact Strength and bending

Strength of a pair of spur gears with

Machining Errors, Assembly Errors and

Tooth Modifications’ Mechanism and

Machine Theory, Elsevier, 2007; 42:

88-114.

7. Shuting Li, ‘Effect of Addendum on

Contact Strength, Bending Strength and

Basic Performance Parameters of a pair

of Spur Gears’ Mechanism and

Machine Theory, Elsevier, 2008; 430:

1557-1584

8. Ali Raad Hassan,‘Contact Stress

Analysis of Spur Gear Teeth Pair’

WASET 2009; 58

9. Rubin D. Chacon, Luis J. Adueza,

‘Analysis of Stress due to Contact

between Spur Gears’ Wseas.us, 2010

10. Konstandinos G. Raptis,’ Theodore N.

Costopoulos ‘Rating of Spur Gear

Strength using Photo elasticity and the

Finite Element Method’ American

Journal of Engineering and Applied

Sciences, 2010; 3(1): 222-231.

11. Wei Yangang, Zhang Xiujuan, Liu

Yankui, ‘Theoretical research on the

maximum Contact Stress of Involute

spur Cylindrical Gear Pair in the

External Meshing Process’ IEEE,2010

12. Xianzhang FENG, ‘Analysis of field of

Stress and Displacement in process of

Meshing Gears’ vol.5, 2011

13. Ignacio Gonzalez-Perez, Jose L. Iserte,

Alfonso Fuentes, ‘Implementation of

Hertz theory and validation a Finite

Element Model for stress analysis of

gear drives with localized bearing

contact’ Mechanism and Machine

Theory, Elsevier, 2011; 46: 765-783



14. Ali Kamil Jebur, L.A.Khan, Y.Nath,

‘Numerical and Experimental Dynamic

Contact of Rotating Spur Gear’

Canadian Center of Science and

Education, Modern Applied Science

vol.5;2011

15. S. Sankar. Muthusamy Natraj, ‘Profile

Modification- A Design approach for

increasing the Tooth Strength in Spur

Gear’ International Journal of Advance

Manufacturing Technology,

Springer,2011; 55: 1-10.

16. Seok-Chul Hwang, Jin-hwan Lee,

‘Contact Stress Analysis for a pair of

Mating Gears’ Mathematics and

computer modelling, Elsevier, 2011

17. Massimiliano Pau,Bruno leban, Antonio

Baldi, Francesco Ginesu, ‘Experimental

Contact pattern Analysis for a Gear-

Rack system’

Meccanica,Springer,2012; 47: 51-61

Somnath Patil et al FORMULATION AND EVALUATION OF XANTHAN GUM BASED FLOATING TABLET OF

TRAMADOL HYDROCHLORIDE


Page 153

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research




FORMULATION AND EVALUATION OF XANTHAN GUM BASED

FLOATING TABLET OF TRAMADOL HYDROCHLORIDE

Somnath Patil, Swati Jagdale, Shailendra Kela, Varsha Divekar

MAEER’s Maharashtra Institute of Pharmacy, Pune, India.


ABSTRACT

The aim of the present work was to prepare floating tablets of Tramadol HCl (TH) using xanthan

gum as carrier. TH is a centrally acting oral analgesic that blocks pain through opoid receptor

binding and inhibition of nor epinephrine & serotonin reuptake. It has elimination half-life of

about 6 hrs. It has an oral bioavailability of about 70% which is suitable for developing gastro

retentive floating drug delivery system. The formulations were prepared by varying the

concentrations of xanthan gum and sodium bicarbonate. The tablets were prepared by direct

compression technique. Xanthan gum and Hydroxy propyl methyl cellulose (HPMC) were used

as alone and in combination as matrix forming agent and sodium bicarbonate for development of

CO2. The prepared floating tablets were evaluated for tablet properties such as hardness,

thickness, weight variation, floating property. In vitro dissolution was carried out for 8 hrs in

0.1N HCl at 37±0.50C using USP paddle type dissolution apparatus. It was noted that, all the

prepared formulations had desired floating lag time and constantly floated on dissolution

medium by maintaining the matrix integrity. The drug release from prepared tablets was found

to vary with varying concentration of the polymer, xanthan gum and HPMC K4M. From the

study it was concluded that floating drug delivery system can be prepared by using xanthan gum

as a carrier.

Key words: Floating Tablet, HPMC K4M, in- vitro dissolution, Tramadol HCl, Xanthan gum

INTRODUCTION

The real issue in the development of oral

controlled release dosage forms is not just to

prolong the delivery of drugs for more than

12 hours, but to prolong the presence of the

dosage forms in the stomach or upper

gastrointestinal (GI) tract until all the drug is

released for the desire period of time [1]

.

Rapid GI transit could result in incomplete

drug release from the drug delivery device

in the absorption zone leading to diminished

efficacy of the administered dose [2]

. Several

approaches are currently used to retain the

dosage form in the stomach. These include

bio adhesive systems [3]

. swelling and

expanding systems,[4‐5]

floating drug

delivery systems (FDDS),[6‐7]

and other

delayed gastric emptying devices.[8]

FDDS,

also called hydrodynamically balanced

system, is an effective technology to prolong

the gastric residence time in order to

improve the bioavailability of the drug.[9]

This technology is suitable for drugs with an

absorption window in the stomach or in the

upper part of the small intestine,[10]

drugs

acting locally in the stomach,[11]

and for




Page 154

drugs that are poorly soluble or unstable in

the intestinal fluid.[12]

FDDS have a bulk

density lower than the gastric fluid and thus

remain buoyant in the stomach, without

affecting the gastric emptying rate for a

prolonged period of time. While the system

is floating on the gastric contents, the drug is

released slowly. Based on the mechanism of

buoyancy, two distinctly different

technologies, i.e. noneffervescent and

effervescent systems, have been utilized in

the development of FDDS. The effervescent

system utilizes matrices prepared with

swellable polymers and effervescent

components, e.g. sodium bicarbonate and

citric acid or stearic acid. The matrices are

fabricated such that in the stomach carbon

dioxide is liberated by the acidity of the

gastric contents and is entrapped in the

gellified hydrocolloid. This produces an

upward motion of the dosage form and

maintains its buoyancy. In noneffervescent

FDDS, the drug is mixed with a gel‐forming

hydrocolloid, which swells on contact with

the gastric fluid after oral administration and

maintains relative integrity of shape and a

bulk density of less than unity within an

outer gelatinous barrier. The air trapped by

the swollen polymer confers buoyancy to

these dosage forms. [13‐14]

TH is a centrally acting oral analgesic that

blocks pain through opoid receptor binding

and inhibition of nor epinephrine& serotonin

reuptake. TH is having short plasma half life

6 hrs which is suitable for developing gastro

retentive floating drug delivery system.


MATERIALS:

Tramadol hydrochloride was provided as a

gift sample from JCPL, Jalgaon and hydroxy

propyl methyl cellulose K4M and xanthan

gum obtained as a gift sample from Vapi

Care Pharma Pvt Ltd. Vapi. Other excipients

and chemicals were of analytical grade and

purchased from Pure Chem. Laboratories,

Pune.

METHOD

Floating tablets containing TH were

prepared by direct compression technique

using variable concentrations of Xanthan

gum and HPMC K4M with sodium

bicarbonate. Different tablets formulations

were prepared by direct compression

technique. All the powders were passed

through 60 mesh sieve. Required quantity of

drug, and low-density polymer were mixed

thoroughly. Magnesium stearate was finally

added as lubricant. The blend was directly

compressed (9mm diameter punches) using

tablet compression machine. Each tablet

contained 100mg of TH and other

pharmaceutical ingredients as listed in

table1.

Evaluation of powder blend:

The powder blend used for preparation of

tablets was evaluated for angle of repose,

and compressibility index.

Angle of repose:

The angle of repose is a relatively simple

technique for estimation of the flow property

of a powder. Powders with low angle of

repose are free flowing and those with a

high angle of repose are poorly flowing

powders [7]

. 10 gm of powder was passed




Page 155

through funnel and the pile was formed. The

height and weight of the pile was measured

and the angle of repose was calculated by

using the formula:-

Angle of repose (θ) = tan-1 (height /radius)

The angle of repose less than 300 usually

indicate a free- flowing material and more

than

400 suggest a poorly flowing material

[8].

Carr`s compressibility index:

The Carr’s compressibility index was

calculated by calculating the tapped and

bulk density using the 100 ml measuring

cylinder. Compressibility is calculated by

the formula.

Carr`s compressibility index = (TBD-LBD)/

TBD X100

Where, TBD is tapped bulk density and

LBD is loose bulk density. A carr`s index

greater than 25 is considered to be an

indication of poor flowability, and below 15,

of excellent flowability. [9]

Table 2: Characterization of precompresion

parameters of table

Evaluation of Tablets:

All the formulations were evaluated for

various parameters such as hardness,

friability, weight variation, % drug content,

buoyancy lag time, swelling index, in-vitro

drug release, release experiments, IR

spectroscopy and optimized formulation

were evaluated for in-vivo study.

Hardness:

Hardness of tablets was determined using

monsanto hardness tester.

Friability:

For each formulation, the friability of 20

tablets was determined using the Roche

friabilator. In this test tablets were subject to

the combined effect of shock abrasion by

utilizing a plastic chamber which revolves at

a speed of 25 rpm, dropping the tablets to a

distance of 6 inches in each revolution. A

sample of pre weighted 20 tablets was

placed in Roche friabilator which was then

operated for 100 revolutions i.e. 4 mins. The

tablets were then dusted and reweighed.

Percent friability (%F) was calculated as

follows,

% F= (loss in weight / initial weight) x 100

Conventional compressed tablets that lose

less than 0.5 to 1.0% of their weight are

generally considered acceptable.

Thickness:

Thickness of all tablets was measured using

a vernier calliper.

Weight variation:

The weight of 20 tablets was taken on

electronic balance and the weight variation

was calculated. The weight variation

tolerance allowed for tablet weighing 324

mg and more is 5%.

Drug content:

To calculate the drug content, the tablets

were triturated in the mortar. 30mg of the

tablet powder (10mg Tramadol HCL) was

added to 10 ml of distilled water and drug

solution was filtered through Whatman

paper no.1. The sample was analyzed for

drug content by UV spectrophotometer

(Varian Cary 100) at 270 nm after suitable

dilutions. Drug stability in the 0.1 N HCl

and distilled water was checked using UV

spectrophotometer for a period of 10 hours.

Buoyancy Studies:

In vitro buoyancy was determined by

buoyancy lag time. The tablets were placed




Page 156

in a 100 ml beaker containing 0.1N HCl.

The time required for the tablet to rise to the

surface and float was determined as floating

lag time.

Swelling index:

The swelling index of the tablets was

calculated in order to find out the swelling

ability of the tablets. For calculating the

swelling index, the previously weighed

tablets were placed in the 100 mL beaker

containing 0.1 N HCl. The tablets were

removed at the time interval of 1 hr for 8

hours and weighed. The swelling index of

the tablets can be measured by studying its

dimensional changes, weight gain or water

uptake. Hence swelling index was calculated

by the formula;

Swelling index= (Wt-Wo) X 100/Wo

Where, Wt= Final weight of tablets at time‘t’

Wo= Initial Weight of tablets.

In Vitro Dissolution Studies:

The release rate of Tramadol HCl from

floating tablets was determined using United

States Pharmacopeia (USP) 24 dissolution

testing apparatus 2 (paddle method). The

dissolution test was performed using 900

mL of 0.1N HCl, at 37 ± 0.5°C and 60 rpm.

A sample (5 mL) of the solution was

withdrawn from the dissolution apparatus

hourly for 10 hours, and the samples were

replaced with fresh dissolution medium. The

samples were filtered through a 0.45-μ

membrane filter and diluted to a suitable

concentration with 0.1N HCl. Absorbance of

these solutions was measured at 270 nm

using double beam UV spectroscopy.

Cumulative percentage drug release was

calculated using PCP disso software.

RESULTS:

Evaluation of tablets:

Hardness:

Hardness of the formulations F1-F13 was

observed within the range of 7.4-9.3 as

shown in table 3.

Friability:

Friability of the tablets was observed below

0.30% for all batches which was in the

acceptable limit.

Thickness:

The thickness of all the tablets was found

within the range of 3 ± 0.5 mm.

Weight variation:

The weight of all the tablets was found

within the range of 300 mg ± 5mg. Hence

the weight of all formulations was found

within the limit.

Drug content:

The range of % drug content of the

formulations F1-F13 was found between

96.01 and 102.87. The tablets showed

hardness, friability, thickness, weight

variation and % drug content within the

limit. The in-vitro buoyancy study shows

that all the formulations shows good floating

property.

In Vitro Buoyancy Studies:

The in-vitro buoyancy study showed the

good floating ability of the tablets as shown

in the table 3. Buoyancy lag time indicates

the time required for the formulation to float

in the medium. From table 2, it was

observed that formulations F10 and F12

shows comparatively more floating lag time

as compared to other formulations. It was

further observed that formulation F2 shows

lowest floating lag time among the all

formulations.




Page 157

Swelling index:

From the swelling index study of all the

batches, it was observed that the increase in

the concentration of polymers increases the

swelling property of the tablets as shown in

table 3. Further the formulation containing

optimized swelling index was obtained.

From the formulation batches, it was

observed that the formulations F10 and F13

showed maximum swelling index.

In Vitro Dissolution Studies:

The drug release patterns from all the

formulations are shown in table 3. The

percent drug release after 8 hours is as

shown in figure 1, 2 and 3. It was observed

that the drug release was retarded from the

formulations containing more concentration

of Xanthan gum and HPMC K4M. The drug

release profile of formulations F1-F13

indicates that as the concentration of

polymer increases, the drug release was

retarded. From the comparison of release

profile of all the batches, it was observed

that the formulations containing

combination of polymers shows more

retardation in drug release in less

concentration as compared to Xanthan gum

and HPMC K4M alone. This indicates that

combination of polymers is more efficient in

formulating the sustained release dosage

form.

Table 3: Evaluation results of formulations

F1-F13

Fig.1: Effect of Various conc. of Xanthan

gum on drug release

Fig. 2: Effect of Various conc. of HPMC on

drug release

Fig. 3: Effect of Xanthan gum & HPMC in

Various combinations on drug release

DISCUSSION:

The flow properties of the powder blends

were studied and formulation F5 was found

to have comparatively good compressibility

index and hausner ratio than other

formulations. From the results of floating

properties it was shown that all tablets had

good floating properties. From the results of

swelling study it was concluded that

swelling index increases as time passes

because the polymer gradually absorbed

water due to hydrophilic in nature and swell.

The swelling index increases with time up to

2 hours in some batches which might due to

low viscosity of polymer and after 2 hours,

the polymer chain relaxation was

dominating phenomenon as swelling reaches

thresholds resulting in lowering of swelling

index. Thus the viscosity of polymer had a

major role on swelling process, matrix

integrity as well as floating capability. From

the swelling study of the formulations,

formulation F10 was found to have good

swelling properties. The in vitro dissolution

was carried out for all batches. From the

dissolution studies of the formulations,

formulation F10 was found to have better

drug release profile than other formulations.

Formulation F10 was also found to have

better swelling capacity and floating time

than other formulations. The drug release

was extended for more than 8 hours from the

floating drug delivery formulations.

CONCLUSION

Regulated drug release attained in the

current study indicates that the matrix tablets

of Tramadol hydrochloride, prepared using

various polymers, can successfully be

employed as a once-a-day oral controlled




Page 158

release drug delivery system. High floating

ability of the formulation is likely to

increase its GI residence time, and

eventually, improve the extent of

bioavailability. However, appropriate

balancing between various levels of the 2

polymers is imperative to acquire proper

controlled release and flotation of the

formulation. Formulation F10 shows good in

vitro gastro retentive floating drug delivery

of Tramadol HCl.

ACKNOWLEDGEMENTS

Authors are thankful to JCPL, Jalgaon for

providing the drug sample of Tramadol

hydrochloride, also thankful to Vapi care

pharma Ltd. for providing Xanthan gum and

HPMC K4M. Authors are very much

thankful to Principal and management of

MAEER’s Maharashtra Institute of

Pharmacy, Pune for their help and support.





authors / editors / publishers of all those



and discussed.

REFERENCES

1. Bardonnet P, Faivre V, Pugh WJ,

Piffaretti JC, Falson F. [2006] Gastro

retentive dosage forms: Overview

and special case of helicobacter pylori. J.

Control. Release 111:1 – 18.

2. Obaidat AA, Obaidat RM. [2001]

Controlled release of tramadol

hydrochloride from matrices prepared

using glyceryl behenate, Eur. J. Pharm.

Sci. 52: 231-235

3. Brunton L. Parker K. Blumenthal D. I.

Buxton, Goodman and Gilman`s: Manual

of pharmacology and therapeutics, Mc

Graw Hill, New York. 358-367.

4. Tiwari SB, Murthy TK, Pai MR, Mehta

PR, Chowdhary PB. [2003] Controlled

release formulation of Tramadol

hydrochloride using hydrophilic and

hydrophobic matrix system AAPS

PharmSciTech 31: 1-6

5. Narendra C, Srinath MS, Ganesh B.

[2006] Optimization of bilayer floating

tablet containing Metoprolol tartrate as a

model drug for gastric retention. AAPS

PharmSciTech 34:E1-E7.

6. Dave BS, Amin AF, Patel MM. [2004]

Gastroretentive drug delivery system of

ranitidine hydrochloride: formulation and

in vitro evaluation AAPS PharmSciTech

34: 1-6.

7. Aulton ME. [2008] Aulton`s

Pharmaceutics-The design and

manufacture of medicine, second ed.,

Churchill Livingstone Elsevier:. 133,

441-450.

8. Lachman L, Lieberman HA, [2009] the

theory and practice of industrial

pharmacy, CBS publishers and

distributors, special Indian edition, New

Delhi. 300,301,317,299.

9. Allen LV, Popovich NG, Ansel HC.

[2008] Ansel`s pharmaceutical dosage

form & drug delivery system, Lippincott

William & Wilkins: 186-203.

10. Jain NK. Response surface optimization

of drug delivery system, CBS Publishers

and Distributors, New Delhi.




Page 159

11. Singh B. [1998] Comprehensive

computer program for the study of drug

release kinetics from compressed

matrices. Ind. J. Pharm. Sci. 60: 358-362.

12. Singh B, Chakkal SK, Ahuja N. [2006]

Formulation and optimization of

controlled release Mucoadhesive tablets

of atenolol using response surface

methodology. AAPS PharmSciTech 3:

E1-E10.

13. Davis SS, Stockwell AF, Taylor MJ. The

effect of density on the gastric emptying

of single and multiple unit dosage forms.

Pharm Res.1986; 3:208‐213.

14. Urguhart J, Theeuwes F. Drug delivery

system comprising a reservoir containing

a plurality of tiny pills. US patent 4 434

153. February 28, 1994.

15. Mamajek RC, Moyer ES. Drug

dispensing device and method. US Patent

4 207 890. June 17, 1980.

Table Caption:

1. Table 1: Composition of Floating Tablet of TH

2. Table 2: Characterization of precompresion parameters of table

3. Table 3: Evaluation results of formulations F1-F13

Figure Caption:

1. Fig.1: Effect of Various conc. of Xanthan gum on drug release

1. Fig. 2: Effect of Various conc. of HPMC on drug release

2. Fig. 3: Effect of Xanthan gum & HPMC in Various combinations on drug release

Table 1: Composition of Floating Tablet of TH

SN Ingredient F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13

1 Tramadol.

HCL 100 100 100 100 100 100 100 100 100 100 100 100 100

2

Sod.

Bicarbona

te

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20

%)

60

(20%)

3 Citric acid

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10

%)

30

(10%)

4 Xanthan

gum 75 90 - - 45 105 - 70 35 120 - 40 60

5 HPMC

K4 M - - 75 90 45 - 105 35 70 - 120 60 40

6 Mg.

Stearate 5 5 5 5 5 5 5 5 5 5 5 5 5

7 Mannitol 30 15 30 15 15 - - - - - - 5 5

Total Weight 300 300 300 300 300 300 300 300 300 315 315 300 300




Page 160

*All quantities are in mg

Table 2: Characterization of precompresion parameters of tablet: Batch Bulk density(g/cm3) Tapped density(g/cm3) Compressibility index Hausner ratio

F1 0.361 0.537 14.08 1.48

F2 0.300 0.348 20.37 1.26

F3 0.300 0.480 39.63 1.59

F4 0.333 0.526 36.67 1.58

F5 0.482 0.561 14.08 1.06

F6 0.436 0.558 21.86 1.27

F7 0.303 0.397 31.02 1.31

F8 0.462 0.549 15.84 1.18

F9 0.300 0.432 30.55 1.44

F10 0.335 0.546 38.64 1.45

F11 0.394 0.463 14.90 1.17

F12 0.416 0.563 26.11 1.35

F13 0.428 0.519 17.53 1.21

Table 3: Evaluation results of formulations F1-F13 Formulation

No.

% drug release

within 8 Hrs.

%Drug

Content

Swelling

Index

Buoyancy Lag

Time (Sec.)

Hardness

(Kg/cm2)

Thickness

(mm)

F1 99.71 99.04 183.39 41 8.6 2.54±0.07

F2 90.56 97.56 210.85 25 7.4 2.64±0.04

F3 93.21 96.01 182.53 39 8.3 2.58±0.03

F4 85.69 101.90 204.02 38 7.9 2.50±0.05

F5 73.69 97.98 203.46 44 8.8 2.43±0.08

F6 84.04 101.44 210.89 31 7.6 2.36±0.02

F7 81.48 98.70 213.52 40 8.5 2.68±0.10

F8 79.56 102.87 190.93 31 7.4 2.59±0.04

F9 84.91 101.67 161.38 46 8.3 2.52±0.02

F10 87.75 98.46 237.97 48 9.1 2.58±0.04

F11 93.04 99.02 210.04 45 8.7 2.50±0.03

F12 82.37 97.35 218.51 54 9.3 2.43±0.07

F13 78.39 100.30 219.36 35 7.8 2.36±0.03




Page 161

Fig.1: Effect of Various conc. of Xanthan gum on drug release

Fig. 2: Effect of Various conc. of HPMC on drug release

Fig. 3: Effect of Xanthan gum & HPMC in Various combinations on drug release

Mamta Mohan et al VIBRATION THRESHOLD OF UPPER LIMB DURING ULNT1 IN INDIVIDUALS WITH TYPE II

DIABETES MELLITUS AND NON DIABETIC INDIVIDUALS


Page 162

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Research




VIBRATION THRESHOLD OF UPPER LIMB DURING ULNT1 IN

INDIVIDUALS WITH TYPE II DIABETES MELLITUS AND NON

DIABETIC INDIVIDUALS

Mamta Mohan, Ravi Shankar Reddy, Ganesh B. M.

Department of Physiotherapy, MCOAHS, Manipal University, KA, India


ABSTRACT

Background: Type II diabetes mellitus patients have been shown to affect the multimodal

sensory, reflex and motor systems in distal extremities. Studies have examined the

mechanosensitivity and vibration threshold in type II diabetes mellitus patients in the lower limb

and compared it with normal individuals. There is scanty literature available in comparison of

the vibration threshold in the upper limb in type II diabetes mellitus patients with non diabetic

individuals.

Methods: Thirty type II diabetic individuals were included in the diabetic group and thirty

asymptomatic age matched individuals were taken to match the group. Vibration threshold (VT)

was measured by tester 1 at the baseline for both the groups using a bioesthesiometer capable of

deriving a vibration of 100 Hz. After the VT was taken at three levels 1) at the baseline, the

tester performed the upper limb neurodynamic test 1(ULNT1) for each individual. During the

sequence of the ULNT1, 2) vibration threshold was measured at initial onset of pain i.e. P1 and

3) short of maximal pain i.e. P2.

Results: Repeated measures ANOVA was used to compare the VT differences within group

and between groups. There was a statistical significant difference between the vibration

threshold of diabetic and non diabetic group at the three levels with a p value < 0.001.

Conclusion: Vibration threshold of the upper limb is higher in individuals with type II diabetes

mellitus as compared to non diabetic individuals.

Keywords: Vibration threshold, ULNT1, Diabetes mellitus

INTRODUCTION

Diabetes mellitus is a group of metabolic

diseases characterized by hyperglycaemia

resulting from defects in insulin secretion,

insulin action, or both. Type II diabetes

mellitus is most common form which is a

disease of insulin resistance that usually has

relative (rather than absolute) insulin

deficiency1. Earliest change in diabetic

nerve function is alteration in axonal

excitability due to changes in ion

conductance of axon membrane due to

metabolic processes directly affecting the

nerves, microvascular abnormalities of the

endoneurium and auto immune

inflammation. Four main mechanisms have

been postulated to underlie the pathogenesis

of nerve pathology in diabetes mellitus,

which are metabolic processes directly

affecting nerve fibres, endoneurial

microvascular disease, autoimmune

inflammation and deranged neurotrophic




Page 163

support.2 It is due to these effects of

hyperglycemia that peripheral nerve

involvement is highly frequent in type II

diabetes mellitus and it has been

documented that one third of type II diabetic

patients have peripheral neuropathy3.

Among the nerves, there is a tendency of the

large diameter nerve fibers that mediate

sense of vibration to get involved first in

diabetes mellitus.2

Neurodynamic tests involve sequential limb

movements that are employed to include the

link between mechanical and physiological

types of mechanisms. An aim of using these

tests in assessment of a nerve is to stimulate

mechanically and move neural tissues in

order to gain an impression of their mobility

and sensitivity to mechanical stresses so as

to evoke the physiological responses.4 In

order to assess the upper limb nerve

function, the standard upper limb neuro

dynamic test 1 (ULNT1) is usually used as it

evokes symptoms of distribution of the

median nerve because the forces generated

by the test are biased towards this structure.4

There are various techniques of assessing

the conductivity of nerve such as NCV that

basically assesses the motor and sensory

aspects of the nerve whereas the vibration

threshold (VT) reflects particular function of

the peripheral nervous system especially the

somatosensory pathway.5

Type II diabetes mellitus patients have been

shown to affect the multimodal sensory,

reflex and motor systems in distal

extremities. Mechanosensitivity in diabetes

mellitus patients should be considered as an

essential inclusion in the assessment to

predict the extent of involvement of the

nerve.6

Studies have also been done to

determine the vibration threshold in lower

limb in normal individuals but there is

scanty literature available in comparison of

the vibration threshold in the upper limb in

type II diabetes mellitus patients with non

diabetic individuals.

METHODOLOGY

Study Design

This study took place in department of

physiotherapy, Manipal University,

Manipal, India. A cross –sectional 2-group

design was used. Completion of

questionnaires and all measurement

procedures were conducted in the same

room on each occasion.

Subject Selection

Type II diabetic subjects in the study were

selected from all patients presenting for the

first time to physiotherapy outpatient and

inpatient clinic over a one year period. 30

subjects with mean age of 55.60 ± 9.79 were

included in the study. All new patients

completed a simple questionnaire as part of

the inclusion-exclusion procedure. On daily

review of these first stage questionnaires,

the clinical records of patients who

provisionally met the inclusion criteria were

subjected to secondary detailed screening by

an experienced member of the

physiotherapy faculty who is experienced

the in the field of musculoskeletal

physiotherapy and diabetic neuropathy. The

Diabetic subjects with clinical signs of

neuropathy were excluded from the study.

After this screening, subjects who met

inclusion criteria invited to participate in the

study and were given further verbal and




Page 164

written information about the study, and

were asked to read and sign a consent form.

For controlled age matched normal subjects

an advertisement was given in

physiotherapy department and Manipal

University for their voluntary participation

in the study. To be considered for inclusion,

the subjects must have been aged between

30 to 70 years, have had no history of

diabetes, upper limb disorders, Cervico-

brachial pain syndrome, Acute

inflammatory/ demyelinating diseases, Any

recent surgeries in upper limb. Finally,

eligible 30 control subjects were selected by

age to ensure a similar distribution to the

patient group. The mean age of the subjects

was 53.43±9.96. The subjects first session

were to familiarize them with the equipment

and vibration threshold testing tasks. All

participants signed a written consent form

prior to participating in the experiment.

Ethics approval was obtained from the

Manipal University Ethics Committee.

Measurement of Vibration Threshold

(VT)

VT was measured by tester 1 at the baseline

for both the groups using a bioesthesiometer

capable of delivering a vibration of 100 Hz.

The subjects were made to sit comfortably

on a chair with hand and arm completely on

the pillow. The probe of the Vibrometer was

placed at the pulp of the distal phalanx of

the thumb.7 Either right or left hand was

tested. The subjects were shielded from the

Vibrometer display during testing. At

baseline, tester 1 first increased the vibration

to a point where the subject perceived the

stimulus. This was taken as appearance of

vibration. Then the intensity was further

increased and slowly reduced till they

identified the disappearance of the stimulus.

This measurement was done thrice and the

average of the six values was taken as the

vibration threshold.

After the VT was taken at the baseline, the

tester performed the ULNT1 (adopted from

M.Shacklock)6 for each individual. For this

a pressure biofeedback inflated to 50 mm

Hg was used to prevent shoulder elevation.

Then the shoulder was abducted to 90-110

degrees followed by complete external

rotation, forearm supination, wrist and

finger extension. The last component of

ULNT1 was elbow extension and elbow

extension value was recorded using

universal goniometer as a measure of

mechanosensitivity. During the sequence of

the ULNT1, the occurrence of the first

response of elbow extension i.e. pain

considered as P1 was noted. The angle of its

occurrence was measured with the universal

Goniometer and VT at this position in the

same manner as that of baseline was taken

for both the groups by the tester 2. The next

occurrence of the symptom i.e. P2 at which

any further movement was intolerable was

noted. The corresponding elbow extension

angle of P2 was measured. The range of

elbow extension was reduced until the

feeling of discomfort disappeared and VT

was measured at this point for both the

groups by the tester 2. The reduction of

elbow extension was adopted to avoid the

masking of pain for perception of vibration.

The measurement of vibration threshold was

measured for both diabetic individuals and

age matched normal individuals.




Page 165

Data Analysis

The statistical analysis was done using the

SPSS 14.0 for Windows software. The

statistical significance value was set at 0.05

with 95% confidence interval and p value

less than or equal to 0.05 would be

considered as significant. Repeated

measures ANOVA were used to compare

the VT differences within group and

between groups.

RESULTS

Demographic data regarding the age (yrs),

sex and duration of individuals with type II

diabetes mellitus and non diabetic

individuals are shown in table no. 1 Analysis

of repeated measures ANOVA shows the

comparison of the vibration threshold

between the diabetic and the non diabetic

group at 3 levels i.e. VT at baseline, VT at

P1 and VT at less than P2. There was a

statistical significant difference between the

vibration threshold of diabetic and non

diabetic group at the three levels with a p

value < 0.001. This states that the vibration

threshold was found to be raised in the

diabetic group at all the three levels

compared to the non diabetic group. There

was no statistical significant difference

between the three levels within each group

with a p value of 0.755.This states that there

was no difference in vibration threshold

during the ULNT1 procedure within each

group (Table no. 2 and Figure 1) Thus

vibration threshold of the upper limb is

higher in individuals with type II diabetes

mellitus as compared to non diabetic

individuals. However, vibration threshold

did not change within subjects of each group

during the ULNT1 testing.

DISCUSSION

Our study aimed at comparing the vibration

threshold of the upper limb during ULNT1

in individuals with type II diabetes mellitus

and non diabetic individuals. As per the

results, the vibration threshold was found to

be increased in the individuals with type II

diabetes mellitus as compared to the non

diabetic individuals.

Vibration threshold is a measure of

conductivity i.e. a function of the axon in

conducting the impulse from the external

receptor. Thus, alteration of the vibration

threshold in type II diabetic individuals may

be due various reasons. Studies in human

and animal models with diabetes mellitus

have shown reduced nerve perfusion and

endoneurial hypoxia. Investigations on

biopsy materials from patients with mild to

severe neuropathy show graded structural

changes in nerve microvasculature including

basement membrane thickening, pericyte

degeneration and endothelial cell

hyperplasia. Arterio-venous shunting also

contributes to the reduced endoneurial

perfusion. These vascular changes strongly

correlate with clinical defects and nerve

pathology. Early vasa nervorum functional

changes are caused by metabolic insults of

diabetes, the balance between vasodilator

and vasoconstrictor are altered.8

The findings of our study regarding the

vibration threshold are in contrast with the

study done by David A Gebler et al. They

conducted a study to check the vibratory and

thermal thresholds in normal and diabetic

individuals using an Optacon Tactile Tester

(OTT) and Thermal Sensitivity Tester. They




Page 166

did not find the vibratory and thermal

threshold of diabetic subjects to be different

from the normal individuals. But in diabetic

individuals with neuropathy, the thermal and

vibratory thresholds were found to be

increased.9

CONCLUSION

Vibratory threshold of the upper limb in

type II diabetic individuals is higher than the

non diabetic individuals. Vibration threshold

and Mechanosensitivity in diabetes mellitus

patients should be considered as an essential

inclusion in the assessment to predict the

extent of involvement of the nerve.

REFERENCES

1. Fazan V, Vasconcelos C, Valença M,

Nessler R, Moore K. Diabetic Peripheral

Neuropathies: A Morphometric

Overview. Int J Morphol 2010;28:51-64.

2. Raymond AA. Management of Diabetic

Neuropathy. Malaysian Journal of

Medical Sciences 2003;10:27-30.

3. Comi G, Corbo M. Metabolic

neuropathies. Curr Opin Neurol 1998;

11:523-9.

4. Shacklock M. Clinical Neurodynamics -

A new system of musculoskeletal

treatment. London: Elsiever 2005.63-65

5. Lise H, Jepsen JR, Sjogaard G.

Vibrotactile sense in patients with

different upper limb disorders compared

with a control group. Int Arch Occ Env

Hea 2006;79:593-601.

6. Boyd BS, Wanek L, Gray AT, Topp KS.

Mechanosensitivity during lower

extremity neurodynamic testing is

diminished in individuals with Type 2

Diabetes Mellitus and peripheral

neuropathy: a cross sectional study.

BMC Neurology 2010; 10:75.

7. Colette R, Jane G, Nicola J. P. Effect of

straight leg raise examination and

treatment on vibration thresholds in the

lower limb: a pilot study in

asymptomatic subjects. Man Ther 2005;

10:136-143.

8. Cameron NE, Eaton SE. Vascular

factors and metabolic interactions in the

pathogenesis of diabetic neuropathy.

Diabetologia 2001;44:1973-1988.

9. Gelber DA, Pfeifer MA, Broadstone VL,

Munster EW, Peterson M, Arezzo JC.

Components of variance for vibratory

and thermal threshold testing in normal

and diabetic subjects. J Diabetes

Complications 1995;9:170-176

TABLES AND FIGURES

Table no. 1: Study population

characteristics [n = 60]

Demographic

Parameters

Diabetic

group

n= 30

Control

group

n= 30

Age (yrs)

55.60 ± 9.79

(Mean ±

SD)

53.43±9.96

(Mean ±

SD)

Sex

Male: female

19:11

15:15

Duration of

diabetes (yrs)

median (IQR)

5.50 ( 1.75 –

10.50)

-

http://www.sciencedirect.com/science/journal/10568727

http://www.sciencedirect.com/science/journal/10568727

http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235080%231995%23999909996%23190193%23FLP%23&_cdi=5080&_pubType=J&view=c&_auth=y&_acct=C000066081&_version=1&_urlVersion=0&_userid=5010750&md5=c0c3b36c852709b569edf009d8488b57




Page 167

Table no. 2: Comparison of Vibration threshold between and within groups

Outcome

measure

Diabetic group

Mean±SD

Control group

Mean ±SD

p value

VT baseline

6.06 ± 1.98

3.72 ± 1.08

Between

groups:

< 0.001

VT at P1

6.24 ± 2.16

4.04 ± 1.24

VT at less than

P2

6.46 ± 2.13

3.99 ± 1.40

p value

Within groups

0.755

Figure1. Trend line showing Vibration Threshold difference between groups and within

group

0

1

2

3

4

5

6

7

VT baseline VT at P1 VT at P2

Diabetic group

Control group

Vt

in v

olt

s

Shahnawaz Anwer et al CARDIOVASCULAR CO-MORBIDITY AND ROLE OF EXERCISE IN RHEUMATOID ARTHRITIS: A

REVIEW


Page 168

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Review




CARDIOVASCULAR CO-MORBIDITY AND ROLE OF EXERCISE IN

RHEUMATOID ARTHRITIS: A REVIEW

Shahnawaz Anwer, Ameed Equebal

Padmashree Dr. D. Y. Patil College of Physiotherapy, Pune, M.S., India


ABSTRACT

Patients with rheumatoid arthritis have an increased risk of cardiovascular disease (CVD).

Cardiovascular event rates are markedly increased in rheumatoid arthritis (RA). Data from

population- and clinic-based epidemiologic studies of rheumatoid arthritis patients suggest that

individuals with rheumatoid arthritis are at risk for developing clinically evident congestive heart

failure (CHF). The vasculature plays a crucial role in inflammation, angiogenesis, and

atherosclerosis associated with the pathogenesis of inflammatory rheumatic diseases. There is

overwhelming evidence that, in the general population and several at risk subpopulations, exercise

provides significant physical and psychosocial benefits, and facilitates management and

improvements of outcome in Rheumatic disease. There is increased recognition of the need for

structured preventive strategies to reduce the risk of CVD in patients with RA. In this review, the

research agenda for understanding and preventing CVD co-morbidity in patients with rheumatoid

arthritis is discussed.

Key-words: Rheumatoid arthritis, Cardiovascular Disease, Inflammation, Exercise

INTRODUCTION

Cardiovascular disease is an increasingly

recognized contributor to excess morbidity and

mortality in rheumatoid arthritis (RA) [1-3]

. The

most probable cause of cardiac death in

rheumatoid arthritis, as in the general

population, is atherosclerotic coronary artery

disease leading to ischaemic heart disease [4]

.

Rheumatoid arthritis, which is characterized by

inflammatory polyarthritis with progressive

joint damage, occurs in about 0.5%-1% of

adult population in most countries [5]

. Several

studies have shown a higher incidence or

prevalence of ischaemic cardiac pathologies

such as myocardial infarction, congestive heart

failure, and coronary deaths in patients with

rheumatoid arthritis than in the general

population [3,6-7]

. Traditional cardiovascular

risk factors do not adequately account for the

extent of cardiovascular disease in RA [3,8]

.

Although hypertension and age are potential

additional contributors to cardiovascular events

in this disease [6]

, markers of current and

cumulative inflammation (white cell counts

and radiographic joint damage, respectively)

are associated with ultrasonographically

determined subclinical atherosclerosis [9]

– a

predictor of cardiovascular events [10]

. The use

of methotrexate is associated with a

significantly lower risk for cardiovascular (CV)

events in RA patients compared with patients

who had never used disease-modifying

antirheumatic drugs (DMARDs) [11]

. Suissa and

colleagues [12]

found a negative association

between the rate of myocardial infarction and

the current use of any DMARD in a case

control study. A study from Sweden [13]

suggested that the risk for developing first CV

events in RA was lower in patients who were

treated with tumour necrosis factor-alpha

(TNF-α) blockers. In this review we examine

the evidence of risk for CVD in patients with


REVIEW


Page 169

rheumatoid arthritis and the suggested

underlying mechanism and discuss the role of

exercise for the prevention and management of

CVD in such patients.

Epidemiology of Cardiovascular disease in

patients with rheumatoid arthritis

Fewer statistics on incidence and prevalence

rates for Congestive heart failure (CHF) in

patients with RA are available and are derived

from a handful of population-based[7,14]

and

clinic-based RA cohorts[15]

. Gabriel et al.[7]

estimated the incidence of CHF among all RA

patients in Olmsted Country, Minnesota, from

data abstracted from medical records. Between

1955 and 1985, 78 cases of incident CHF were

identified among 450 prevalent cases of RA

compared to 54 cases among the same number

of non-RA community controls matched for

age, sex, and baseline comorbidity, yielding a

relative risk of 1.60 (95% CI 1.12-2.27). In

contrast, the risk of incident CHF in patients

with osteoarthritis (OA), a non-inflammatory

arthritis, was not increased compared to non-

OA community controls [7]

.

In a follow-up retrospective review of the same

cohort extended to 1995, now using the

Framingham diagnostic criteria for CHF,

Nicola et al.[14]

confirmed an increased risk of

incident CHF in both rheumatoid factor (RF)

negative and positive RA patients compared to

non-RA controls adjusted for age, sex, and CV

risk factors. Incident CHF risk remained

elevated after further adjustment for comorbid

ischemic heart disease, although the risk

relationship was no longer statistically

significant for RF negative patients in this

model [14]

.

In a combined cohort of RA patients from

community-based practices and drug safety

monitoring studies (n = 9093), Wolfe et al.[2]

estimated an adjusted lifetime relative risk of

CHF in patients with RA is more as compared

with OA controls. The adjusted lifetime

prevalence of CHF in the RA population was

0.7 % greater as compared to OA controls.

Data were collected via patient survey of self

reported, physician-diagnosed CHF, and

confirmed by review of a random sample of

medical records in 50% of patients reporting

CVD events. In a subsequent analysis [15]

, in

which the drug safety cohort represented a

third (n = 4,307) of the total sample (n =

13,171), Wolfe et al. reported an adjusted

frequency of CHF of 3.9% (95% CI 3.4-4.3%)

in RA patients compared to 2.3% (95% CI 1.6-

3.3%) in controls with knee or hip OA. Factors

associated with prevalent and incident of CHF

were those typically associated with CHF in

the non-RA population (e.g., age, male gender,

hypertension, coronary artery disease, diabetes,

and smoking) while RA-related measures

(patient-reported disability, pain, and RA

global severity) were also associated with

prevalent and incident of CHF.

Risk factors for cardiovascular events in

patients with rheumatoid arthritis:

Traditional risk factors for vascular disease

such as, smoking, hypertension, diabetes and

hyper lipidemia are important for the increased

risk of CVD in subjects with RA [16]

. However,

traditional risk factors alone do not fully

explain the excess CVD risk in RA. Other non-

traditional factors are hypothesized to play a

role, in particular the burden of inflammation

as indicated by the C-reactive protein (CRP)

and/or erythrocyte sedimentation rate (ESR) [17,

18]. In a community-based cohort of patients

with inflammatory polyarthritis, Goodson, et al

also noted that excess CVD mortality was

confined to patients who were rheumatoid

factor positive [19]

. These markers of

inflammation and inflammatory burden confer

additional risk of CVD death in those with RA

after adjusting for traditional CVD risk factors

and comorbidities [20]

. Severity of disease has

consistently been associated with an increased

risk of CVD events in RA. Patient with severe

extra articular RA manifestations are at an


REVIEW


Page 170

increased risk of developing coronary artery

disease [21]

as well as peripheral vascular

disease [22]

; and severe extra articular RA is a

predictor of both overall mortality [23]

and

cardiovascular mortality [20]

, indicating that

systemic inflammation is a major determinants

of vascular comorbidity in RA. In contrast with

the general population, a low body mass index

(BMI), rather than obesity, has been associated

with increased CVD in patients with RA [24, 25]

.

Role of exercise in Rheumatoid arthritis and

Cardiovascular disease:

Exercise is one of the most important

behavioral interventions that can have a major

beneficial impact on the likelihood to develop,

suffer symptomatically or die from CVD. Any

physical activity is better than no, or little,

physical activity. There is overwhelming

evidence that, in the general population and

several at risk subpopulations, exercise

provides significant physical and psychosocial

benefits, and facilitates management and

improvements of outcome in Rheumatic

disease. It helps maintain a healthy life-style,

reduce CVD risk factors including obesity [26]

,

dyslipidaemia [27, 28]

, hypertension [29]

, diabetes

mellitus [30]

and possibly even inflammation [31]

; it is also effective for preventing acute

coronary syndromes [32-34]

. Moreover, exercise

helps the management of established CVD:

both aerobic exercise [35, 36]

and resistance

training [37]

improve myocardial contractility

and quality of life in patients with chronic heart

failure and produce significant functional

benefits in people with intermittent

claudication [38]

. More importantly, cardiac

exercise rehabilitation programmes are an

important part in the management of patients

after an acute coronary syndrome (ACS) [39]

and lead to significantly improved quality of

life and reduced mortality rates [40, 41]

.

The overall physiological adaptations that

occur as a result of exercise [42]

provide

protection against CVD mortality, even in the

presence of well-established CVD risk factors [43, 44]

. CVD mortality is lower in highly fit than

in moderately fit individuals [45]

, while physical

inactivity is an independent risk factor for the

development of CVD [46, 47]

. Even though

cardiorespiratory fitness may have a familial

component [48]

, it can be increased significantly

by exercise training, regardless of age, gender,

race and initial fitness levels [49]

. The required

activity levels can be accrued through formal

training programmes or leisure-time physical

activities [50]

. Moreover, supervised exercise

programmes are more effective compared with

non-supervised exercise [51, 52]

, most likely due

to greater adherence.

Great controversy still exists about the

optimum amount of exercise for eliciting the

greatest cardiovascular benefit. Different

exercise intensity [53]

and duration [54]

, as well

as various combinations of them [55]

, may have

different impacts on the magnitude of

cardiorespiratory fitness improvement. Most

authors agree that there is a dose–response

relation between the amount of exercise, all-

cause and cardiovascular mortality [53, 54]

. The

greatest potential for reduced mortality is in

sedentary individuals (such as many RA

patients), in whom even slight increases in

daily physical activity are beneficial [56,57]

; for

more active individuals, higher levels of

intensity should be pursued [56]

. Depending

primarily on the starting levels of physical

activity, cardiovascular fitness has been

reported to increase by 8–51% following an

exercise intervention [54, 56]

.

Moderate-intensity exercise of long duration

appears to elicit the most benefit on CVD risk

and mortality [53, 55-57]

. Current guidelines by

the American College of Sports Medicine

(ACSM) suggest that an individual should

engage in exercise at least three times a week,

at an intensity of 60–80% of maximum oxygen

uptake (VO2max), for at least 20–30 min, in

order to experience significant improvements


REVIEW


Page 171

in cardiorespiratory fitness and optimum

cardiovascular benefits [58]

. In terms of caloric

expenditure, this can be translated to 1000–

2000 kcal/week [59]

. These calories can be

expended in either continuous exercise or

accumulated from several short bouts of

exercise during a day [59, 60]

. Aerobic exercise is

the most appropriate, but this can be

supplemented by low-to-moderate intensity

resistance training [60]

. The exercise regimen

should be reconsidered regularly, usually every

4–6 weeks, based on the principles of exercise

periodization [61]

so that participants continue

to improve their performance.

SUMMARY

There is strong evidence that persons with

rheumatoid arthritis are at high risk for

developing cardiovascular disease. Several

studies have shown a higher incidence or

prevalence of ischaemic cardiac pathologies

such as myocardial infarction, congestive heart

failure, and coronary deaths in patients with

rheumatoid arthritis than in the general

population. Severity of disease has consistently

been associated with an increased risk of CVD

events in RA. CVD mortality was more

confined to patients who were rheumatoid

factor positive. Role of exercise in the

management and prevention of CVD in RA

patients is very important yet neglected area of

RA patients treatment programmes. As this

review shows, there is accumulating evidence

that in patients with RA, exercise therapy is

effective in improving the prognostic risk

factor profile. There is little has been

investigated and published on the role of

exercise as a means to control risk and manage

CVD in individuals with RA. More research is

required to identify the optimal regimens,

timing and environment for exercise, as well as

educational and behavioural intervention that

will facilitate long term adherence to an active

life style and/or structured exercise.

ACKNOWLEDGMENTS

We acknowledge the immense help received

from the scholars whose articles are cited and

included in references of this manuscript. We

are also grateful to authors/editors/publishers

of all those articles, journals and books from

where the literature for this article has been

reviewed and discussed.

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8. Dessein PH, Joffe BI, Stanwix AE:

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13. Jacobsson LT, Turesson C, Gulfe A,

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14. Nicola PJ, Maradit-Kremers H, Roger VL,

Jacobsen SJ, Crowson CS, Ballman KV et

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15. Wolfe F, Michaud K. Heart failure in

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Rheum 2004;50:3444-9.

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ML, Rantapaa-Dahlqvist S. Extent of

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EM, Silman AJ, Symmons DP. Mortality in

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Monali N. Rajurkar et al. NON-HEALING SKIN ULCER IN HIV / TUBERCULOSIS CO-INFECTION: A CASE REPORT


Page 175

IJCRR

Vol 04 issue 17

Section: Healthcare

Category: Case Report




NON-HEALING SKIN ULCER IN HIV / TUBERCULOSIS CO-

INFECTION: A CASE REPORT

Monali N. Rajurkar, Silpi Basak

Department of Microbiology, Jawaharlal Nehru Medical College,

Sawangi, Wardha, M.S., India


ABSTRACT

Introduction: Tuberculosis (TB) is very common in India, China and other developing

countries. World Health Organisation (WHO) had estimated 9.2 million new cases of TB,

worldwide in 2006 of which 7.7% were positive for Human Immunodeficiency Virus (HIV). In

India, at the end of 2007, there were 2.5 million people living with HIV and AIDS (PLWHA)

whereas incidence of TB was 1.8 million cases per year. Tuberculosis is the most common HIV

related opportunistic infection in India and caring for patients with HIV/TB co-infection is a

major public health challenge. The incidence of tuberculosis is more in people living with HIV

infection. So, WHO has developed the strategy of treating HIV/TB co-infection irrespective of

patient’s CD4 count. If any HIV positive patient is diagnosed to be infected with tuberculosis,

the Antitubercular treatment (ATT) is started along with Antiretroviral therapy (ART). Here, we

report a case of skin ulcer due to Mycobacterium tuberculosis on chest, secondary to pulmonary

tuberculosis in HIV infected person with varied presentation.

Material and Methods: At first pyogenic infection due to Methicillin Resistant Staphylococcus

auereus (MRSA) was diagnosed. As the patient did not improve even after the full course of

Linezolid therapy for Methicillin Resistant Staphylococcus aureus (MRSA) which was

superadded infection, the discharge and also tissue material were collected from the base of ulcer

and cultured on Lowenstein – Jenesen media and Ziehl-Neelsen staining were done. The acid

fast bacilli were present on staining and growth on Lowenstein - Jenesen media was identified as

Mycobacterium tuberculosis.

Outcome of study: As the patient was also HIV positive both ATT and ART was started. A

significant improvement of the cutaneous lesion was noted after one month of treatment and

patient was discharged after another fifteen days.

Conclusion: Tuberculosis is very common in India and sophisticated automated system for

detecting M.tuberculosis is not available in all centers. Any non-healing ulcer not responding to

routine antibiotics must be screened for tuberculosis in developing countries. If tuberculosis is

detected, promptly HIV testing must be done so that treatment strategy can be finalised.

Key Words: HIV/TB co-infection, People living with HIV and AIDS (PLWHA), Skin

tuberculosis

INTRODUCTION

Tuberculosis is one of the oldest diseases,

known to mankind and its evidence is

detected even in Egyptian mummies. It

remains as a leading cause of death

worldwide, especially in developing

countries like India, China etc. In the initial

period of twentieth century, V.A.Moore has

rightly said that as a destroyer of mankind,

tuberculosis has no equal [1]. Almost one

third of world population is infected with

tuberculosis and in 1993 World Health

Organisation (WHO) has declared

tuberculosis an global emergency [2]. The



Page 176

risk of developing tuberculosis is estimated

to be between 20-37 times greater in people

living with HIV than among those without

HIV infection [3]. Similarly, tuberculosis

accelerates the progression of HIV

infection to Acquired Immuno Deficiency

Syndrome (AIDS) and shortens the survival

of such patients. Of 1.8 million HIV related

deaths in 2009, 22% were due to

tuberculosis [4]. Even risk of drug resistant

tuberculosis is higher amongst persons with

HIV infection compared to others (HIV

negative).

Tuberculosis skin ulcers are extremely

unusual. Cutaneous tuberculosis is caused

by Mycobacterium tuberculosis and rarely

by Mycobacterium bovis. Even in India and

China where tuberculosis is quite common,

cutaneous tuberculosis cases are rare i.e.

0.1 to 2.5%[5]. Moreover, Seeman et al in

2008 have reported that cutaneous

tuberculosis is still a difficult disease to

diagnose [6].

Here, we present a case with ulcerative

lesion on right side of chest. The patient

was diagnosed as a case of cutaneous

tuberculosis with HIV and was treated with

proper antiretroviral therapy and

antitubercular drugs.

Word count - 246

CASE STYDY

OBSERVATIONS AND RESULTS:

A 38 years old man presented with multiple

ulcers over right axilla extending to the

right chest wall with purulent discharge,

was admitted to our hospital. The patient

gave the history of small swelling over

right axilla, 6-8 months back which was

gradually increasing in size and was

painful. As the patient was an agricultural

worker, the history of trauma or thorn prick

was specifically asked to rule out any

actinomycotic or fungal infection. The

patient had history of persistent cough 3-4

months back. But there was no history of

weight loss. Patient was treated by many

doctors from time to time but patient did

not respond. The swelling was around 4cm

× 3cm and as it was on the lower part of

right axilla, it was diagnosed as axillary

abscess and incision and drainage was done

two and half month back, in a private

nursing home. The pus was not sent for any

investigation and patient was treated with

antibiotic. As patient was very anaemic, 2

bottles of blood transfusion was given in

the nursing home. Then other axillary

swelling developed and ulcerated within

one and half month. Hence, multiple ulcers

with purulent discharge developed

extending from right axilla to right chest

wall (Photo 1). The investigations done in

our hospital was:

Fasting plasma glucose levels: 90 mg/dl,

Haemoglobin: 6.1 gm/dl, ESR: 138 mm in

first hour, Peripheral smear showed

microcytic hypochromic anaemia, platelets

were adequate and other parameters were in

normal limits and X – ray chest: Lungs

clear.

Fine needle aspiration cytology (FNAC)

from ulcerative lesion showed acute

inflammatory cells. Gram’s staining of the

pus showed plenty of Gram positive cocci

arranged in clusters. On routine culture,

Methicillin Resistant Staphylococcus

aureus (MRSA) was isolated which was

sensitive to Vancomycin and Linezolid and

resistant to Penicillin, Erythromycin,

Ciprofloxacin and Quinapristine,

Dalfopristine. Methicillin resistance was

detected using Cefoxitin (Cx 30μg) disk as

per CLSI guideline [7].The patient was

treated with Linezolid but the ulcers were

not healing.

Considering high ESR and as the ulcers

were not healing, the discharge was

collected from edge and base of the ulcer



Page 177

and Ziehl Neelsen staining with 20%

H2SO4 was done. In the smear plenty of

Acid fast bacilli (AFB) were present and

some were beaded in appearance (Photo 2).

On the same day, patient’s sputum samples

were examined and it was negative for Acid

fast bacilli. On next day morning, the

induced sputum was collected and smear

showed plenty of Acid fast bacilli (3+) and

some were beaded in appearance (Photo 3).

As the patient was AFB positive, patient’s

serum was tested for HIV antibody and the

patient was found to be HIV positive as per

NACO guidelines [8], though the patient

did not give any relevant history.

The tissue collected from base of ulcer was

homogenized and concentrated by Petroff’s

method and the deposit was inoculated into

two bottles of Lowenstein-Jensen media (L-

J). Patient’s sputum was also inoculated

into two bottles of L-J media after Petroff’s

method. The rough, tough and buff

coloured colonies of M.tuberculosis appear

on L-J slant on fourth week from sputum

and from the tissue growth appeared on

sixth week on L-J media (Photo 4). The

AFB staining from the growth revealed

plenty of Acid fast bacilli and the growth

was niacin positive.

As the patient was HIV positive and

sputum and exudate from ulcer was

positive for Mycobacterium tuberculosis,

antiretroviral therapy (ART) and

antitubercular drug regimen was also

started on the same day. The patient

responded to the treatment very well and

after three and half months came for follow

up when skin ulcers healed completely.

DISCUSSION:

Cutaneous tuberculosis is also an ancient

disease and was described long before

Robert Koch identified Mycobacterium

tuberculosis in 1882. Laennec in 1826 first

gave the description of cutaneous

tuberculosis on his own prosector’s wart

which developed after an injury while

performing autopsy on a patient with spinal

tuberculosis [9]. In 1886, Reil and Paltauf

established that the wart was a tubercular

lesion [10]. The clinical varieties of

cutaneous tuberculosis can be divided into

three broad groups – a) patients who were

not previously exposed to M.tuberculosis,

b) patients who were previously sensitized

and c) tuberculids that develops a

hypersensitive response of a tuberculosis

focus elsewhere in the body. As previously

sensitized hosts are very common in

developing countries like India, lupus

vulgaris is the most common variety of

cutaneous tuberculosis reported from India,

followed by TB verrucosa cutis and

scrofuloderma [5]. No systematic survey

for prevalence of cutaneous tuberculosis

has been carried out in India. In one of the

study it was found that cutaneous

tuberculosis was associated with

tuberculosis in other organs in 22.1%

patients and the other organ most

commonly involved were lungs. Even in

the present case, the patient was having

tubercular ulcer along with involvement of

lungs. Most studies also reported that male

are most commonly affected. Cutaneous

tuberculosis sometimes has very diverse

clinical presentation.

The initial presentation may resemble a

common bacterial infection or the

ulcerative lesion may have superadded

bacterial infection [11, 12]. In our case,

initially patient had superadded infection

with Methicillin Resistant Staphylococcus

aureus (MRSA). After taking full course of

Linezolid, the ulcer remained as it is and

that made us to think for doing acid fast

staining from the discharge. In culture, the



Page 178

growth was identified as M.tuberculosis.

Currently the cause of skin ulcers may be

vascular ulcers, squamous cell carcinoma,

rodent ulcers, tubercular ulcers etc [13].

The differential diagnosis of cutaneous

tuberculosis also includes infections with

Mycobacterium ulcerans and

Mycobacterium marium, Cutaneous

anthrax, Cutaneous leishmaniasis,

Sporotrichosis, Cat scratch disease due to

Bartonella henselae etc [14].

In our case, as the patient was suffering

from tuberculosis and HIV seropositive, the

patient was treated with ART and ATT. In

2009, out of 1.7 million people died from

tuberculosis. 4,00,000 (24%) were among

people living with HIV. Tuberculosis is

also one of the leading causes of morbidity

and mortality among PLWHA. Hence,

WHO implemented collaborative HIV /

Tuberculosis activities to decrease the

burden of HIV / Tuberculosis coinfection.

In patients with latent Tuberculosis

infection, the risk of developing active

diseases is several hundred folds higher

among persons who acquire HIV. In 2007 it

was reported from Brazil, 80% reduction in

tuberculosis cases in HAART treated

compared to ART naïve HIV infected

person [15].

CONCLUSION

It has been observed that HIV epidemic

continues to fuel TB epidemics and each

increasing the morbidity and mortality of

the other. WHO recommends the

implementation of the Three I’s for HIV /

Tuberculosis co-infection to reduce the

burden of Tuberculosis among people

living with HIV [16]. The three I’s are – i)

Intensive tuberculosis case finding, ii)

Isoniazid preventive therapy and iii)

Infection control for tuberculosis. There is

strong evidence that Antiretro-viral

Therapy (ART) can lower a person’s viral

load and restore the immune system and

hence, significantly reduces HIV and

Tuberculosis. WHO in 2011 recommends

earlier ART at ≤350 CD4 count and

immediate initiation of ART for all patients

with HIV/TB co-infection irrespective of

CD4 count[17]. Proper training and

continuing medical education of health care

workers is needed for early detection of

cases with HIV/TB co-infection, so that

WHO treatment strategies can be followed

for a better outcome of the patient.

Hence to conclude, in India, China and

other developing countries, any non-healing

skin ulcer, not responding to routine

antibiotics, must be screened for

tuberculosis and if positive, the patient

must be screened for HIV.

ACKNOWLEDGEMENT





authors/ editors/publishers of all those



and discussed.

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Cruz DJ, et al. Cutaneous

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Medicine 1981;60: 95-109.

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Research, 2010; 4: 3561-65.

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Laserson K, Barreire D Silva G, Santos

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010/9789241599764_eng.pdf



Page 180

FIGURES:

Figure 1: Superadded skin ulcer infection

Figure 1: Skin ulcer with superadded

infection

TOP SIDE ↑

Figure 1: Skin ulcer with superadded

infection

Figure 2: Ulcer after traetment with

Linezolid

Figure 2: Tubercular ulcer after treatment

with Linezolide.

TOP SIDE ↑

Figure 2: Tubercular ulcer after treatment

with Linezolide.

Figure 3: AFB from ulcer

Figure 3: Acid fast bacilli from base of the

ulcer.

TOP SIDE ↑

Figure 3: Acid fast bacilli from base of the

ulcer

Figure 4: AFB from sputum

Figure 4: Acid fast bacilli from sputum

TOP SIDE ↑

Figure 4: Acid fast bacilli from sputum



Page 181

Figure 5: M.tuberculosis on L-J media

Figure 5: Growth of M.tuberculosis on L-J

media from tissue exudate

TOP SIDE ↑

Figure 5: Growth of M.tuberculosis on L-J

media from tissue exudate

N. Balasundari et al. GROWTH AND CHARACTERIZATION OF PICRIC ACID MIXED ZTS SINGLE CRYSTALS


Page 182

IJCRR

Vol 04 issue 17

Section: Technology

Category: Research




GROWTH AND CHARACTERIZATION OF PICRIC ACID

MIXED ZTS SINGLE CRYSTALS

N. Balasundari1, P. Selvarajan

2, S. Lincy Mary Ponmani

1, D. Jencylin

1

1Department of Physics, Infant Jesus College of Engineering, Tuticorin,

TN, India 2Physics Department, Aditanar College of Arts and Science, Tuticorin, TN,

India


ABSTRACT

The growth of picric acid-doped Zinc tris (thiourea) sulphate (ZTS) single crystals were grown

from aqueous solution by slow evaporation technique. 1.5 Mol% of picric acid was added in

saturated solution of ZTS. When picric acid was added as dopant, morphological alterations

were noticed in the grown crystals. Cell parameters of the grown crystals were obtained from the

XRD analysis and the presence of functional groups was identified by FTIR study. Its optical

properties were examined by UV-vis analysis. The microhardness values were measured for the

grown crystals and discussed.

Keywords: ZTS; doped, Solubility, Crystal Growth, FTIR, Microhardness, SHG

Introduction

Zinc (tris) thiourea sulphate (ZTS) is a semi-

organic NLO material for second harmonic

generation from metal complexes of thiourea.

High-damage threshold and wide transparency

make it a better alternative for KDP crystals in

frequency-doubling and laser fusion experiments

[1,2]. SHG efficiency of ZTS crystal is 1.2 times

more than that of KDP [3,4]. ZTS crystal belongs

to the orthorhombic system with the space group

Pca21. The growth of ZTS crystals from aqueous

solution and its characterization have been

reported in a number of recent publications [5-9].

Picric acid is one of the organic compounds

having tendency to form the stable picrate

compounds with various organic molecules. It

has been reported that doping NLO crystals with

organic and inorganic additives can alter various

physical properties and doped–NLO crystals may

find wide applications in optoelectronic devices

compared to pure NLO crystals [10, 11]. Keeping

this in mind, pure and picric acid-doped Zinc

(Tris) Thiourea sulphate (ZTS) crystals have been

crystallized and studied in the present work. The

results of the growth, solubility, XRD studies,

FTIR studies, UV-Vis analysis, and

microhardness studies of the grown crystals are

reported in this paper.

Experimental methods and instrumentation

Synthesis and Growth

Pure crystal of Zinc (tris) Thiourea sulphate

(ZTS) was synthesized by dissolving high purity

AR grade Zinc Sulphate heptahydrate and

thiourea in molar ratio 1:3 in double distilled

water. The solution was stirred by a magnetic

stirrer and white crystalline ZTS salt was obtained

on heating at 45 oC for a long time. The salt was

purified by repeated re-crystallization. To obtain

picric acid-doped ZTS salt, 1.5 mol% of picric

acid was added to solution of ZTS and the

solution was heated at 60 oC. Single crystals of



Page 183

ZTS and picric acid-doped ZTS were grown from

saturated solutions of synthesized salts by the

solution growth employing slow evaporation

technique at room temperature (31 oC). The good

quality transparent and colorless pure ZTS crystal

was harvested in 20 -25 days. Transparent and

pale yellow colored picric acid-doped ZTS

crystals were harvested within 20 days. The

photograph of grown crystals is shown fig. 1.

Instrumentation

Single crystal X-ray diffraction studies were

carried out for the grown crystals of this work

using ENRAF NONIUS CAD-4 X-ray

diffractometer with MoKα (λ=0.71069 Å)

radiation to evaluate the structural properties. The

transmission properties of the crystals were

examined using Lambda 35 model Perkin Elmer

double beam UV-vis-NIR spectrometer in the

range from 190 nm to 1100 nm. Optically

polished single crystal of thickness 2 mm was

used for this study. The Fourier transform infrared

spectrum (FTIR) of the sample was recorded in

the region 400-4000 cm-1

with Perkin Elmer

Fourier transform infrared spectrometer (Model :

Spectrum RXI) using KBr pellet. Microhardness

measurement was carried out using Vickers

microhardness tester fitted with a diamond

indentor. The Second Harmonic Generation

(SHG) conversion efficiency was tested using

a set-up of Kurtz and Perry [12] and it was

carried out using Q-switched mode locked

Nd:YAG laser with first harmonic output at 1064

nm.

The grown sample was powdered with uniform

particle size of about 600 nano meter using a ball

mill and the powdered sample was packed

densely between two transparent glass slides. The

fundamental laser beam of 1064 nm wavelength,

8 ns pulse with 10 Hz pulse rate was made to fall

normally on the sample cell. The emitted green

light from the sample was detected by a

photomultiplier tube (PMT) and displayed on a

Cathode Ray Oscilloscope (CRO). KDP crystal

was powdered into identical size as that of the

sample of this work and it was used as reference

material in the SHG measurement.

Results and discussion

Solubility studies

Solubility study for the synthesized salts was

carried out using a hot-plate magnetic stirrer and a

digital thermometer. The procedure for finding

solubility is reported elsewhere [13]. The

variation of solubility with temperature for the

samples is shown in figure 2. It is observed from

the results that the solubility increases with

temperature for both the samples and it is found to

be more for picric acid doped ZTS sample. It is

clear that for the picric acid -doped sample, the

solvent is able to accommodate a marginally

increased amount of solute for the saturation at

the same temperature. Since solubility increases

with temperature, the samples of this work have

positive temperature coefficient of solubility. The

increase in solubility for the picric acid-doped

sample may be responsible for the change in the

growth rate and morphological change observed

in the present work.

Single crystal XRD studies

The grown crystals were subjected to single

crystal XRD analysis and single crystalline data

were obtained. From the data, it is observed that

pure and picric acid-doped ZTS crystals

crystallize in orthorhombic system and the unit

cell parameters are listed in table 1. The obtained

values of lattice parameters for the pure ZTS

crystal are found to be in good agreement with the

reported literature [5]. The changes in the lattice

parameters are due to incorporation of picric acid

in the lattice of ZTS crystal.



Page 184

Powder XRD Analysis

The powder X-ray diffraction (XRD) pattern

picric acid-doped ZTS crystal are shown in

the figure 3. The well-defined peaks at specific 2θ

values show high crystallinity of the grown

crystals. All the reflections of powder XRD

patterns of this work were indexed using the

TREOR software package following the

procedure of Lipson and Steeple [14]. The values

of hkl, relative intensity and 2 theta values for the

reflection peaks of the powder XRD pattern are

given table 2. Using the values in the table 2 and

the UNITCELL software package, the cell

parameters were found and it is observed that the

values of unit cell parameters obtained from

powder XRD method are in comparable with

those obtained from single crystal XRD method.

FTIR Spectral studies

The FTIR analysis was carried out by Perkin

Elmer FTIR spectrometer by KBr pellet technique

in the range 500-4000 cm-1

. The FTIR spectra of

pure and picric acid mixed ZTS samples are

shown in figure 4. In ZTS complex, there are two

possibilities by which the coordination with metal

can occur. It may be either through nitrogen or

through sulfur. From spectra, the N-H, absorption

bands in the high frequency region in thiourea

were not shifted to lower frequencies on

formation of metal thiourea complex, thus

coordination of thiourea occurs through sulfur in

ZTS [11]. The NH, C=S and N-C-N stretching

vibrations were also seen. The comparison of the

samples shows slight shift in characteristic

vibrational frequencies of 1.5 mol% picric acid

doped ZTS with respect to pure ZTS. This

confirms the addition of picric acid in grown

crystal. The spectral assignments for the picric

acid mixed ZTS sample are provided in the table

UV-Visible spectral study

The study of optical transmission range of grown

crystals is important because the crystals are

mainly used for optical applications. The UV–

visible study of grown crystal was carried out by

Varian-Cary 5E UV-vis Spectrometer in a range

200 nm to 1100 nm. The absorption spectrum of

1.5 mol% picric acid–doped ZTS is shown in

fig.6 and the inset figure shows UV spectrum of

pure ZTS for comparison. The absorption

spectrum reveals that the crystal has lower cutoff

wavelength at around 290 nm. The absorption

near UV region is associated electron with

transition within thiourea units of ZTS. Doping of

1.5 mol % of picric acid in ZTS does not destroy

the transparency of the crystal. From spectra it is

observed that the lower cutoff wavelength is

almost the same for picric acid doped ZTS and

pure ZTS crystals. The wide range of

transparency in UV-visible and IR region enables

good transmission of the second harmonic

frequencies of Nd: YAG laser. This is an added

advantage of the grown crystals of this work in

the field of optoelectronic applications.

Measurment of microhardness

Microhardness of a crystal is its capacity to resist

indentation. Physically hardness is the resistance

offered by a material to the localized

deformations caused by scratching or by

indentations [15].The selected smooth surfaces of

the grown crystals were used for microhardness

measurement at room temperature using a Vickers

microhardness tester fitted with a diamond

indenter. The hardness of the crystal is calculated

using the relation The hardness of the crystal is

calculated using the relation Hv = 1.8544 P / d2

kg / mm2

where P is the applied load in kg and d

is the length of indentation impression in

millimeter and 1.8544 is a constant of a

geometrical factor for the diamond pyramid [16].

A plot drawn between the hardness value and



Page 185

corresponding loads is shown in figure 6. It is

noticed that Vickers hardness number (Hv)

increases with the applied load satisfying the

indentation size effect. When a ZTS crystal is

doped with picric acid, the hardness decreases and

this decrease in the hardness value of doped

sample can be attributed to the incorporation of

picric acid in the lattice.

Conclusion

Single crystals of pure and picric acid-doped Zinc

(Tris) Thiourea sulphate(ZTS) were synthesized

and solubility studies were carried out for the

prepared samples in de-ionized water in the

temperature ranging from 30 to 50 oC. In

accordance with the solubility data, saturated

solutions were prepared for growing pure and

picric acid doped ZTS crystals by slow

evaporationtechnique. Morphological alterations

have been observed when ZTS crystals are doped

with picric acid. The powder and single crystal

XRD studies confirms the orthorhombic structure

of the grown crystal..The unit cell parameters of

pure ZTS crystals are in agreement with the

literature values. The FTIR spectrum confirms the

presence of all the functional groups in the grown

crystals. UV-visible study reveals the picric acid

doped ZTS crystal has lower cutoff wavelength at

around 290 nm. Mechanical property of the

grown crystals has been studied by microhardness

test and noticed that there is a decrease of

microhardness number for 1.5 mol% of picric

acid-mixed ZTS crystals.

Acknowledgement

The supports extended in the research by

Sophisticated Analytical Instrumentation Facility

(SAIF), IIT, Chennai, RRL, Trivandrum and

M.K.University, Madurai are gratefully

acknowledged. We thank authorities of Aditanar

College of Arts and Science, Tiruchendur and

Infant Jesus College of Engineering, Keela

Vallanadu, Tuticorin for the encouragement given

to us to carry out the research work.

References

1. P. Kerkoc, V. Venkataraman, S. Lochran,

R.T. Bailey, F.R.Cruickshank, D. Pugh, J.N.

Sherwood, J. Appl. Phys. 80 (1996) 6666.

2. H.O. Marcy, L.F. Warren, M.S. Webb, C.A.

Ebbers, S.P. Velsko, G.C. Kennedy, G.C.

Catella, Appl. Opt. 31 (1992) 5051.

3. V. Venkataraman, C.K. Subramanian, H.L.

Bhat, J. Appl. Phys. 77 (1995) 6049.

4. M. Oussaid, P. Becker, M. Kemiche,

Phys.Stat.Sol. (b) 207 (1998)103.

5. P.M. Ushasree, R. Jayavel, C. Subramanian,

P. Ramasamy, J. Crystal Growth 197 (1999)

216.

6. S.Meenakshisundaram, S. Parthiban, N.

Sarathi, R. Kalavathy, G. Bhagavannarayana,

J Crystal Growth 293(2006) 376.

7. J. Ramajothi,S.Dhanuskodi, K. Nagarajan,

Cryst.Res.Technol. 39(2004)414

8. S.Verma,M.KSingh, V.K.Wadhawan,

C.H.Suresh, Pramana, J. Phys. 54(2000)879

9. P.A. Angalinmary, S. Danushkodi, Cryst. Res.

Technol. 36 (2001) 1231.

10. C.Krishnan, P.Selvarajan, T.H.Freeda,

J.Crystal Growth 311 (2008) 141.

11. P.M. Ushasree, R. Jayavel, P. Ramasamy

Mater. Sci. Eng. B65 (1999) 153.

12. S.K. Kurtz, T.T. Perry, J. Appl. Phys. 39

(1968) 3798.

13. B Helina, P Selvarajan and A S J Lucia Rose,

Phys. Scr. 85 (2012) 055803.

14. H. Lipson, H. Steeple, Interpretation of X-ray

Powder Diffraction Patterns, fifth ed.,

Macmillan, New York, 1970.

15. J. H. Westbrook and H. Conrad (Eds), The

Science of Hardness Testing and its Research

Applications (ASME, Metals Park OH,1973).

16. A.S.J. Lucia Rose, P. Selvarajan, S. Perumal,

Spectrochimica Acta Part A 81(2011)270.



Page 186

(a) (b)

Fig. 1: Photographs of (a) Pure ZTS and

1.5.mol% picric acid added ZTS crystals

30 32 34 36 38 40 42 44 46 48 50 52

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

So

lub

ilit

y(g

m/1

00

ml)

Temperature(oC)

Pure ZTS

ZTS + 1.5 mol % of picric acid

Fig.2: Solubility diagram for pure and picric acid-

doped ZTS samples

Table 1: Lattice parameter values of pure and

picric acid-doped ZTS crystals

Sample Cell

parameters

Volume Z

Pure ZTS

crystal

a = 7.858 Å

b=11.255 Å

c=15.529 Å

α=β=γ=90o

1373.41

4

Picric acid-

mixed ZTS

crystal

a =7.752 Å

b=11.166 Å

c=15.501 Å

=β =γ=90o

1342.10

4



Page 187

10 20 30 40 50 60 70

0

200

400

600

800

1000

1200

10

505

0

11

61

063

01

04

22

30

20

30

15

13

110

4

11

3

10

30

22

11

20

21

10

21

11

01

2

00

2

Inte

ncity

(A

.U)

Two Theta (degrees)

Fig. 3: Powder XRD pattern of picric acid-doped

ZTS crystals.

Table 2: Values of 2- theta, relative intensity and

hkl for picric acid-mixed ZTS sample

Peak No. 2-Theta

(degrees)

Relative

intensity %

hkl

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

11.343

13.883

15.019

15.955

16.891

17.8944

19.498

20.434

22.038

25.648

26.784

28.522

29.792

32.934

34.137

35.340

36.543

39.953

48.174

54.56

26.97

95.60

82.07

100

29.39

34.40

80.01

33.69

41.03

39.69

32.61

56.09

34.31

59.13

24.28

21.86

21.19

19.53

002

012

111

102

021

112

022

103

113

104

131

203

015

230

042

301

106

050

105

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

621

723

773

945

1123

1336

1396

1515

1638

2099

1816

2357

2691

2865

3167

3266

3373

Tra

nsm

itta

nce(

%)

Wavenumber(cm-1

)

Fig.4 (a)

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

33

72

32

75

31

85

2964

2763

2621

1995

2355

589

646792

993

11

59

12

83

14

21

15

33

1624

Tra

nsm

itta

nce

(%

)

Wave number (cm-1)

Fig. 4(b)

Fig.4: FTIR spectrum of (a) pure and (b) picric

acid mixed ZTS samples



Page 188

Table 3: FTIR spectral assignments for picric

acid-doped ZTS sample

Wave number (cm-1

) Assignments

589

646

792

993

1159

1283

1421

1533

1624

3185

3372

δ (C-S-N)

δ (C-S-N)

γ (C=S)

γ(SO4)

γ(N-C-N)

γ(N-C-N)

γ(C=S)

γ(N-C-N)

δ(NH2)

γs(NH2)

γas(NH2)

γ- Stretching, δ–bending, s–symmetric,

as–asymmetric

Fig.5: UV-vis. Absorption spectra of 1.5 mol %

picric acid doped ZTS

20 30 40 50 60 70 80 90 100 110

50

60

70

80

90

100

110

120

130

140

150

Ha

rdn

ess n

um

be

r (k

g/m

m2)

Load (grams)

Pure ZTS crystal

ZTS crystal + 1.5 mol% of picric acid

Fig. 6: Variation of microhardness number with

load for pure and picric acid-doped ZTS crystals.

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