Sri Shivarathreeshwara Nagara Mysore

J.S.S. COLLEGE OF PHARMACY

Sri Shivarathreeshwara Nagara Mysore - 570015

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation/thesis entitled “PREPARATION AND

EVALUATION OF AN ANTACID AND ANTIULCER SUSPENSION

CONTAINING HERBAL DRUGS” is a bonafide and genuine research

work carried out by me under the guidance of Mr.R.S.Bhat, Assistant

Professor, Department of Pharmaceutics, J.S.S.College of Pharmacy, Mysore-15.

Date: Signature of the Candidate Place: Mr.Murthy Sudarshan Dakshina

J.S.S. COLLEGE OF PHARMACY

Sri Shivarathreeshwara Nagara Mysore - 570015

CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled “PREPARATION AND


CONTAINING HERBAL DRUGS” is a bonafide research work done by

Mr.Murthy Sudarshan Dakshina in partial fulfillment of the requirement

for the degree of Master of Pharmacy (Industrial Pharmacy).

Date: Signature of the guide Place: Mysore Mr.R.S.Bhat Assistant Professor Dept. of Pharmaceutics

J.S.S.College of Pharmacy Mysore-15.

CERTIFICATE

ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE INSTITUTION This is to certify that the dissertation entitled “PREPARATION AND


CONTAINING HERBAL DRUGS” is a bonafide research work done by

Mr.Murthy Sudarshan Dakshina under the guidance of Mr.R.S.Bhat,

Assistant Professor, Dept. of Pharmaceutics, J.S.S.College of Pharmacy,

Mysore-15.

Seal & Signature of the HOD Seal & Signature of the Principal Dr. H.G. SHIVAKUMAR Dr. B.G.NAGAVI

Vice Principal & Head, Principal

Department of Pharmaceutics, J.S.S. College of Pharmacy

J.S.S. College of Pharmacy, Mysore.

Mysore. Date:

COPYRIGHT

DECLARATION BY THE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health Sciences,

Karnataka shall have the rights to preserve, use and disseminate this

dissertation / thesis in print or electronic format for academic / research

purpose.

Date: Signature of the Candidate Place: Mr. Murthy Sudarshan Dakshina

© Rajiv Gandhi University Of Health Sciences, Karnataka

ACKNOWLEDGEMENT

I am grateful to my esteemed guide Mr.R.S.Bhat, Asst.Professor, Dept.of

Pharmaceutics, for his generous support, valuable suggestions, inspiring encouragement,

and keen interest that greatly eased the task of completing my work.

My sincere thanks to Dr.B.G.Nagavi, Professor and Principal, J.S.S.College of

Pharmacy, Mysore and to Dr.H.G.Shivakumar, Professor and Head of Department of

Pharmaceutics, J.S.S.College of pharmacy, Mysore for providing the necessary facilities

required and moral support for carrying out this work.

I express my sincere gratitude to Mr.V.S.Datta Murthy, Head of the Department

of Pharmaceutics, PES College of Pharmacy, Bangalore and Dr.T.N.Nagraja, vice

principal J.S.S.Ayurveda Medical College, Mysore for providing invaluable guidance on

herbal drugs.

I sincerely thank M/S. Sami Labs Ltd, Mysore for providing me gift sample of

Glycyrrhetic acid.

I am thankful to Mr.P.K.Kulkarni, Asst.Professor, Dept.of Pharmaceutics,

J.S.S.College of pharmacy, Mysore for his valuable suggestions that helped to complete

my dissertation work without difficulty.

I am also very thankful to Mr.Mahadevan, Asst.Professor, Dept. of

Pharmacognosy, J.S.S.College of pharmacy, ooty for carrying out the HPTLC analysis of

the samples for the purpose of stability studies.

I am especially thankful to the teaching staff Mr.D.Vishakantegowda,

Mr.T.M.Pramodkumar, Mr.C.S.Satish, Mr. N.M.Mahesh, Mr.Madhusudhan Purohit and

I

Mr.D.H.P.Gowda and non-teaching staff Ms. Namitha shivanna, Mr.Somanna, Mr. Suresh

and Mr.Shankar for their cooperation towards my work.

I am extremely grateful to all the albino rats that have laid their life for my work

without which the work would have been half complete.

I remember all my friends Krishna, Sumit, Subhash, Shafiullah, Gunashekhar,

Satish, Shankar, Chetan, Rajesh, Harsha, Naveen Vishwnath, Manohar, Jayadev,

Kumarswamy, Venkatesh, Solanki, who by their honest opinions and diligence kept me

lively and all my juniors who by their honest opinions and diligence kept me lively.

I am always indebted to my family members for their overwhelming enthusiasm,

love and affection.

I wish to express my gratitude to umesh and manju for their invaluable help in

scientific illustrations and printing of manuscript.

I submit my humble pranamas to the lotus feet of Sri Shivarathreeshwara

Deshikendra Mahaswamiji.

I thank the Almighty for giving me courage and confidence when nobody else could

and for being there in every walk my of life.

Place:

Date: Mr. Murthy Sudarshan Dakshina

II

ABSTRACT

Gastric ulcer affects about 60% of the adults and about 80% of the child population

in the tropical countries. The herbs Glycyrrhiza glabra, Terminalia chebula, Terminalia

belerica, Emblica officinalis and mineral Turbinella rapa are reported in classical

Ayurvedic texts to possess anti-phlogistic activity, astringent activity and acid

neutralization activity that is desirable for the treatment of gastric ulcer. Hence they can be

combined in a suitable formulation to provide an effective treatment for gastric ulcer. The

individual herbs were identified by thin layer chromatographic analysis and evaluated for

the foreign organic matter, ash value, acid insoluble ash values, water-soluble and alcohol

soluble extractive values. The values obtained were within the limits prescribed in the

pharmacopoeia. The total tannin contents of the herbs Terminalia chebula, Terminalia

belerica and Emblica officinalis were estimated colorimetrically by Folin-Dennis method.

The mineral drug Shankha Bhasma was evaluated for its calcium carbonate content and

acid neutralization capacity as per the reported methods. The extracts of herbs Terminalia

chebula, Terminalia belerica and Emblica officinalis were obtained by cold maceration

process using 95% ethyl alcohol as the macerating solvent. These herbs and their extracts

were formulated into a suspension with Xanthan gum as the suspending agent to produce

cumulative antacid and anti-gastric ulcer activity. The concentration of the Xanthan gum

was varied as 0.2%, 0.25%, 0.3% and 0.35%.

The suspensions were then evaluated for the pH, Viscosity, sedimentation volume,

redispersibility, antacid and antiulcer activity. All the formulations showed a pH in the

basic range about 8.2 and ANC between 2-3mEq/ml. All the formulation showed high

sedimentation volume and good redispersibility .The formulation containing extracts of the

III

herbs showed significant decrease in the ulcer index as compared to one containing the

powders of these drugs The stability studies on the selected formulations were performed

at 30ºC/ 65%RH and 40ºC /75%RH for 90 days. The formulations were found to be stable

at the end of 90 days.

IV

LIST OF ABBREVIATIONS

λMax Absorption maxima

Conc. Concentration oC Degree centigrade

RH Relative Humidity

% Percentage

mEq Milliequivalents

ANC Acid neutralization

Capacity

hr Hour

R.T. Room temperature

μg Microgram

Rf Retention factor

mg Milligram

ml Milliliter

Min Minute

nm Nano meter

N Normality

% Percent / percentage

g grams

mcg microgram

SD Standard deviation

UV Ultra Violet

NLT Not Less Than

NMT Not more than

W/v Weight /volume

W/w Weight / weight

r.p.m Rotations per minute

V

CONTENTS

Sl.No Chapter Page No

1. Introduction 01

2. Literature Review 05

3. Materials And Methods 16

4. Results And Discussions 35

5. Conclusions 49

6.

7.

References

Appendices

51

54

VI

LIST OF TABLES

Sl.No Tables Page No

1. Formulation chart of the Suspension 30

2. Chart of ulcer score 34

3 Qualitative Analysis of Shankha Bhasma 35

4 Determination of foreign organic matter 36

5 Determination of Total ash value & acid insoluble ash value 37

6 Determination of alcohol &water soluble extractive value 38

7 TLC Identification of Individual Herbs 39

8 Standard curve of tannic acid in distilled water 40

9 Total tannin content in Triphala powder and extract 41

10 PH and viscosity of the suspension 42

11 Redispersibility and Sedimentation volume of suspension 43

12 Acid neutralization capacity of the suspension 44

13 Anti-ulcer activity of the suspension 45

14 Estimation of gallic acid by HPTLC &TLC ofsuspension at 0th Day and 90th Day

46

15

16

Viscosity, pH and ANC of the suspension at 0th, 30th, 60th and 90th day Organoleptic properties of the suspensions at 0th, 30th, 60th and 90th day

47

48

VII

LIST OF FIGURES

Sl.No Figures/Graphs Page No

1. UV Spectra of tannic acid in distilled water. 25

2. TLC identification of the herbs 39(a)

3. Calibration curve of tannic acid in distilled water at 703 nm

40

4. Particle size distribution data of suspension 41

5. Histopathological studies of anti-ulcer activity 45(a)

6. HPTLC graph of gallic acid (standard) 46(a)

7. HPTLC graph of gallic acid (FI-3) at 0th Day 46(b)

8. HPTLC graph of gallic acid (FII-2) at 0th Day 46(c)

9. HPTLC graph of gallic acid (FI-3) at 90th Day at 30°C/65%RH 46(d)

10. HPTLC graph of gallic acid (FI-3) at 90th Day at 30°C/65%RH 46(e)

11. HPTLC graph of gallic acid (FII-2) at 90th Day at 40°C/75%RH 46(f)

12. HPTLC graph of gallic acid (FII-2) at 90th Day at 40°C/75%RH 46(g)

13. TLC of the formulations at 0th Day and 90th Day 46(h)

VIII

Ayurveda the traditional system of medicine continues to be matchless so far its perfection

in health care is concerned.It's time tested theurapeutics based on the use of natural

resources has broken all the barriers and is gradually making inroads in the domain of the

allopathy and rightly gaining more popularity not only at home but abroad too.

The word "Ayurveda" is a combination of the two words "Ayuh" and "Veda"

which literally means knowledge about the life .The origin of the Ayurveda is based on the

vedas which are said to be the oldest classics of the world written between 10,000-4,000

B.C.1

Gastric (mainly peptic) ulcer is one of the major factors affecting about 60% of the

human adults and about 80% of the child population in the tropical countries individuals. It

is interesting to note that nearly 50% of the Indian population harbors the disease.

Peptic ulcer as a disease is characterized by a group of disorders described

lesions of the upper gastrointestinal tract (with stomach and duodenum being the most

affected areas

Peptic ulcer is defined as the pathological lesions and ulcers of any portion of the

gastrointestinal tract exposed to the acid activated pepsin. Even though the acid and pepsin

are the contributing factors, current reasoning incorporates additional factors into the

etiology of the disease.

Gastric ulcers also occur due to the intense irritation of the various reflexogenous

zones and portions of the nervous system as well as due to the unfavorable emotions also.

The dependence of the gastric ulcers on the seasons has also been noted, mostly prevalent

1

in the spring and autumn seasons. Further several studies also indicate that gastric ulcers

result from the malnutrition in the intestinal mucosa.

The appearance of the gastric ulcers has also been attributed to the antral

infection of the gastric mucosa by the organism, Helicobacter pylori.

The various factors that are responsible for causing peptic ulcers can be categorized into

the following: -

General factors

Vagal effects, hormonal effects (histamine), insufficient circulation, shock, general

ischemia etc

Constitutional and environmental factors

Sex, age, temperament, family history, social class, geographical difference, occupation

etc.

Local factors in the stomach

Aggressive factors

Hydrochloric acid, Pepsin, refluxed bile, on-steroidal Anti-inflammatory Drugs,

Alcohol, Proteolytic enzymes, ingested irritants, bacterial toxins, Physicochemical trauma,

etc

Defensive factors

Mucus, bicarbonates, blood flow, restitution of epithelium, prostaglandins,etc.

Microbial factors

Helicobacter pylori infection.

It is generally acknowledged that an ulcer results from an imbalance between the

Aggressive gastric factors and the defensive (resistance) factors.2, 3

2

It is a firm belief that the drugs of the plant origin have very little or no side effects and in

view of this property herbal drugs are preferred over the synthetic drugs. The practitioners

of the Ayurveda since ages have been reaping the advantages of the medicinal plants in

one form or the other, offering comparatively cheaper and safer health remedies to the

masses. The use of the plant derived crude drugs for the treatment and cure drugs for the

treatment and cure of the chronic and refractive diseases is a healthy sign of our great

tradition.

Hence the present study involved preparing an antacid and anti-ulcer

suspension containing these herbal drugs.

3

OBJECTIVES

. To carry identification tests for the herbs and minerals.

. To carry out the monographic analysis of the ingredients.

. To extract the active principles from the herbs.

. To prepare the suspension and carry out the invitro evaluation.

. To evaluate the anti-ulcer activity of the Prepared Suspension.

4

Gastric and duodenal ulcers, gastrooesophageal reflux disease are the gastrointestinal

disorders sharing a common abnormality: too much acid and pepsin activity for the local

tissue resistance. The therapy for these disorders is directed at the correction of an apparent

imbalance between the acid and pepsin activity and the mucosal resistance. The success of

the therapy is measured in terms of ulcer healing, symptom control, relapse rate etc.

The strategy adopted for the treatment of the gastric ulcers may involve one or more of the

following mechanism of action: -

Inhibition or neutralization of the gastric acid.

Inhibition of the pepsin activity.

Resistance to pepsin activity.

Inhibition of the bile reflux or absorption of the bile.

Gastro protective actions like

a) Increased bicarbonate secretion at the mucus gastric fluid interface.

b) Increased mucus secretion.

c) Increased surfactant formation.

d) Increased cell migration (cell restitution).

e) Increased cell division.

f) Normalization of the cell metabolism.

g) Anti-helicobacter pylori compounds.

h) Preventing the degradation of gastro- protective Prostaglandins.3

5

Herbal compounds including have been used for a variety of the disorders of the

gastrointestinal tract. The herbal drugs are considered to be safe for use, easily available at

cheaper cost and produce minimal side effects.

Goel R.K, Pathak N.K.R have studied the anti-ulcer properties of trunk,

bark and wood chips of Tectona grandis on aspirin induced gastric ulcers and histamine

induced duodenal ulcers in rats. They have reported that the plant parts reduced the

increased peptic activity induced by aspirin and also increase in sialicacid and mucin

secretion. The active constituent was reported to be lapachol.4

Banerjee RS et al have studied the antiulcer activity of shilajit a

herbomineral compound on pylorus ligation induced and aspirin induced gastric ulcer and

cystamine induced and histamine induced duodenal ulcers in rats.They have reported that it

exhibited its anti-ulcer activity by reducing acid pepsin secretion and increase in mucin

secretion.5

Sairam K et al have reported the anti-ulcer activity of the centella Asiatica

whole plant on cold restraint stress and aspirin induced gastric ulcers in rats. They have put

forth that the fresh juice of the plant exhibited anti-ulcer activity by increasing the mucin

secretion and increasing the lifespan of the mucosal cells.6

Glycyrrhiza glabra, which is known as the yashtimadhu in Ayurveda, is

known to possess anti-phlogistic activity (increasing the mucus secretion and its viscosity).

It is also reported in various literatures that liquorice inhibits the enzymes that degrade the

gastrocyto-protective Prostaglandins E and Fα thus raising their concentration.7 Joshi

V.K.et al have reported the anti-ulcer activity of liquorice on pylorus ligation and cold

6

restraint stress induced gastric ulcers in rats. They have reported that water decoction of

the root enhances the mucosal defensive factors and thus exhibits anti-ulcer activity.8

Terminalia Chebula, Terminalia belerica and Emblica officinalis are astringent

compounds. In Ayurveda these are combined in equal proportions to give an Ayurvedic

preparation “TRIPHALA CHURNA”, which is being used for a variety of the ailments.

These have a property of forming a complex with the proteins. This complex has been

proved to be resistant to the proteolytic enzymes.9 Rege N.N et al have reported the anti-

ulcer activity of fresh juice of the fruits Emblica officinalis on aspirin induced, pylorus

ligation induced and ethanol induced gastric ulcers in rats.They have reported that

antiulcer activity is exhibited by enhancement of mucosal defensive factors10.

Shankha Bhasma, which is the ash of conch shell, contains mainly calcium carbonate has

the property to neutralize the acid.11

The properties that these drugs possess and the strategies that have been put forth for the

treatment of gastric ulcers prompted us to prepare a polyherbal formulation containing

these herbs and minerals and assess the extent of antacid and antiulcer activity.

The anti-ulcer activities of the suspension claiming anti-ulcer activity were

studied. It was found that suspension containing varatika, Dugdhapashana, Mouktika

shukti powders and extract of liquorice and which was processed in amalaki, guduchi and

punarnava showed significant decrease in ulcer index 12.

7

Suspension as a dosage form offers the following advantages13

1) As the disintegration step of the tablets and capsules are absent, it permits quicker

onset of action,

2) As the particle size of the suspension is small, it permits quicker dissolution of the

drug in the gastrointestinal fluids and hence quicker onset of the action.

3) Suspension has the advantage of the flexibility of the dosage that is particularly

advantageous for an antacid and antiulcer suspension since symptoms of acidity

depend upon a variety of conditions and varies from patient to patient.

4) Suspension has the advantage of ease of administration than tablets and capsules.

Requirements of a good suspension14

1) Suspended particles should not settle down to form a hard cake.

2) Suspended materials settled down should be easily redispersible.

3) Suspension should have a very good viscosity so that it can be easily poured out of

the bottle and should have elegant appearance, smell and taste.

4) The particles should remain dispersed for sufficient time for dose to be

administered.

8

Individual monographs of the drugs are briefly described below 15, 16

TERMINALIA CHEBULA (Haritaki)

Biological Source

It consists of the dried, ripe and fully matured fruits of Terminalia chebula Retz.

Belonging to the family Combretaceae.

Other Names

Hindi :- Harara, Bal-har.

Malayalam :- Kadukka.

Telgu :- Karaka, Karakkaya.

Kannada : - Alalekayi.

English :- Chebulic myrobalan , Indian gallnut.

Phytochemistry

Myrobalan fruits are an important source of the tannins. The tannins of the myrobalan

fruits are of Pyrogallol type that on hydrolysis yields chebulic acid and d-galloyl glucose.

Chebulagic acic, chebulinic acid ellagic acid and gallic acid are the other constituents of

the myrobalan.

Marker constituent Gallic Acid.

9

TERMINALIA BELERICA (Vibhitaki)

Biological Source

It consists of the dried ripe fruits of the Plant, Terminalia belerica Roxb.belonging to the

family Combretaceae.

Other Names

Hindi :- Bhaira, Bahera.

Malayalam :- Tannikai, Tanni.

Telgu :- Tani.

Kannada : - Tarekayi.

English :- Beleric myrobalan .

Phytochemistry

The fruit contains about 20-30% of the tannins. It contains gallic acid, ellagic acid,

Phyllemblin, ethyl gallate and galloyl glucose. It also contains most of the sugars as

reported in the myrobalan.

Marker Constituent Gallic Acid

10

EMBLICA OFFICINALIS (Amalaki)

Biological Source

It consists of the fresh or dried fruits of Emblica officinalis Gaerth belonging to the family

Euphorbiaceae.

Other Names

Hindi :- Amla, Aonla.

Malayalam :- Nelli.

Tamil :- Nelli.

Kannada : - Nelli,Amlalaka.

English :- Indian Gooseberry,Emblic myrobalan.

Phytochemistry

It contains the hydrolysable tannins as the major bioactive constituents along with gallic

acid and ellagic acid. It also contains alkaloids Phyllantidine and Phyllantine,Pectin and

minerals. It also contains Vitamin C.

Marker Constituent Gallic Acid.

11

GLYCYRRHIZA GLABRA (Yashtimadhu)

Biological Source

It consists of dried, peeled or unpeeled roots and stolons of Glycyrrhiza glabra Linn.

belonging to the family Leguminosae.

Other Names

Hindi :- Mulethi.

Sanskrit :- Jyesthamadhu.

Tamil :- Atimadhuram.

Kannada : - Atimadhura.

English :- Liquorice.

Phytochemistry

The chief constituent of liquorice is the triterpenoid saponin glycyrrhizin which is the

potassium and calcium salt of the glycyrrhizinic acid. Other constituents are flavonoids,

Liqueritin and Liquertigenin, Isoliqueritin, glycyramarin resins, asparagin, sucrose.fats and

glucose.

Marker constituent Glycyrrhetic acid.

12

Conch shell ash (Shankha Bhasma)

Biological Source: -

It is the ash of the conch shell of the shell fish Turbinella rapa belonging to the family

mollusca. It is prepared by soaking the shell in the limejuice and calcinating covered

crucible ten to twelve times and finally reducing it to the powder (ash).

Other Names: -

Sanskrit :- Shankha Bhasmam.

Telgu :- Sehkham.

Kannada : - Shankha Bhasma.

English :- Conch Shell Ash.

Constituents: -

It is mainly composed of calcium carbonate (about 94%). In minor amounts it also

contains magnesium, manganese and traces amounts of other minerals.

Marker Constituent Calcium Carbonate

13

Xanthan Gum NF 17

Description

It is a natural anionic biopolysaccharide made up of different monosaccharides, mannose,

and glucose and glucuronic acid.

It is soluble in water but insoluble in organic solvents. This gum can tolerate upto 50% of

water miscible organic solvents.

Xanthan gum exhibits pronounced pseudoplastic rheology with a definite yield point.

Enzymes that degrade other natural polymers such as cellulose do not affect Xanthan gum

that is due to the uniform polymer structure and shielding of the polymer backbone side

chains.

This polymer is resistant to shear depolymerisation.

Xanthan gum has a good stability from pH 1 to 12.

Xanthan gum has good temperature stability. Its viscosity is not affected by temperature

changes.

Other features of the Xanthan gum are that it exhibits high viscosity at low concentration

and compatible with the divalent and polyvalent ions such as calcium salts, with tannins

etc.

14

Sorbitol Solution 70% (Non-Crystallizing) 18,19

Sorbitol solution 70% (Non-Crystallizing) is an aqueous solution of hydrogenated, partly

hydrolyzed starch.

Description

It is clear, colorless or faintly yellow, syrupy liquid and odorless and is miscible with

water.

Uses

It is widely used as a vehicle in sugar free formulations. It is used as a stabilizer in drug

and vitamin formulations. It is widely used in antacid suspensions as a stabilizer,

sweetening vehicle and as an anti-caking agent.

Concentrations to be used

Sorbitol solution (70%) can be used upto a concentration of 70%.

15

INSTRUMENTS

Digital Balance Shimadzu Corporation, Japan.

Electric Furnace Tempo Instruments and Equipments

Pvt. Ltd., Mumbai.

Microscope Labfit.

Digital pH Meter Systronics.

Silica Crucibles

Stage and Eyepiece micrometer Erma, Japan.

TLC developing Chamber and Plates

UV-VIS Spectrophotometer UV-1601 Shimadzu Corporation,

Japan

Brookefields Viscometer model DV-III+ Modrobs Grant,Germany

HPTLC Apparatus Cadrach

Homogeniser Shreeji Chemicals,Mumbai

Hot Air Oven Tempo Instruments and Equipments

Pvt. Ltd.,Mumbai

16

MATERIALS

Plant Materials

Glycyrrhiza glabra Linn. NKCA Pharmacy, Mysore

Terminalia chebula NKCA Pharmacy, Mysore

Terminalia belerica NKCA Pharmacy, Mysore

Emblica officinalis NKCA Pharmacy,Mysore

Turbinella rapa Ashwini Oushadalaya,Mysore

Herbal Extracts Himalaya Drug Company, Bangalore

CHEMICALS

Acetone Thomas Baker Chemicals Ltd., Mumbai.

Anisaldehyde Loba Chemie Pvt.Ltd., Mumbai

Acetic Acid Glacial Ranbaxy Fine Chemicals Ltd.,New Delhi.

Ammonium Oxalate Reachem Lab. Chemicals Pvt. Ltd., Chennai

Calcium Carbonate Thomas Baker Chemicals Ltd.,Mumbai

Chloroform Thomas Baker Chemicals Ltd.,Mumbai

Carmoisine Creative Aromatics Pvt. Ltd., Chennai

17

Dimethylpolysiloxane Sigma Chemicals Ltd., Bangalore

Ethyl Acetate Thomas Baker Chemicals Ltd.,Mumbai

Ethyl Alcohol Reachem Lab.Chemicals Pvt. Ltd., Chennai

Formic Acid Thomas Baker Chemicals Ltd.,Mumbai

Folin Dennis Reagent S.D.Fine chemicals Pvt.Ltd.

Gallic Acid Thomas Baker Chemicals Ltd.,Mumbai

Glycyrrhetic Acid Sami Labs. Ltd., Mysore

Iodine Thomas Baker Chemicals Ltd.,Mumbai

Menthol Crystals Loba Chemie Pvt. Ltd., Mumbai.

Methanol Reachem Lab. Chemicals Pvt. Ltd., Chennai.

Methyl Paraben Loba Chemie Pvt.Ltd., Mumbai

Potassium Permagnate Loba Chemie Pvt.Ltd., Mumbai

Silica Gel GF254 Thomas Baker Pvt.Ltd., Mumbai

Sodium Carbonate Reachem Lab.Chemicals Pvt.Ltd.,Chennai

Sodium hydroxide Reachem Lab.Chemicals Pvt.Ltd.,Chennai

Sorbitol 70% liquid Loba chemie Pvt.Ltd., Mumbai.

Sulphuric Acid Ranbaxy Fine Chemicals, New Delhi.

Tannic Acid Thomas Baker Pvt.Ltd., Mumbai

Toulene Ranbaxy Fine Chemicals, New Delhi.

Xanthan Gum Loba Chemie Pvt. Ltd.,Mumbai

18

Reagents

Chloroform Water

Shaken 2.5 ml of Chloroform water with about 700 ml of distilled water until dissolved

and made the volume to 1000 ml with distilled water.

Anisaldehyde Sulphuric Acid

0.5 ml of Anisaldehyde is mixed with 10 ml of glacial acetic acid, followed by 85 ml of

methanol and 5 ml of concentrated Sulphuric acid. The TLC Plate with about 10 ml heated

at 100 ºC for about 5-10 min and then evaluated in the normal light.

19

METHODS

Qualitative and Quantitative chemical tests for Shankha Bhasma.20, 21

Qualitative tests

Test for the presence of calcium

a) Solution of Bhasma in dilute hydrochloric was treated with ammonium oxalate.

b) Solution of Bhasma in dilute hydrochloric was treated with potassium ferrocyanide.

Test for the presence of carbonate

a) Bhasma was treated with dilute hydrochloric acid and the liberated gas was passed

through limewater.

b) To about 5ml of the Bhasma solution in dilute hydrochloric acid, added 2ml of

10%w/v solution of magnesium sulphate was added.

Test for the presence of magnesium

The precipitate obtained from ammonium oxalate was filtered and the filtrate was

concentrated highly. To this concentrate solution sodium hydroxide pellets were added

and dissolve and then heated to remove ammonia.

Test for the presence of Manganese

The filtrate obtained after separation of calcium oxalate was treated with ammonium

sulphide, well stirred and left overnight.

20

Determination of calcium carbonate content in Shankha Bhasma

This was determined by redox titration method. The oxalate ion from calcium oxalate was

titrated against standard potassium permanganate. Potassium permanganate is a self –

indicator. The reactions are mentioned below: -

5CaC2O4 + 2KMn O4 + 8H2 SO4 2MnSO4 + K2 SO4 + 5CaSO4 + 8H2 O+ 10CO2

Procedure

Accurately weighed about 0.25 g of Bhasma and dissolved in 5ml of Hydrochloric acid

(1:1) and then added 50ml of 0.5N of ammonium oxalate and two drops of methyl orange

indicator. Then 10% of aqueous ammonia was added to it drop wise till pink color of the

solution turned to yellow, it was then heated to about 70-80 ºC & left for 1 hr. Then filtered

through whattman filter paper no.41 and washed thoroughly with excess of dilute (0.1N)

Ammonium Oxalate solution. The precipitate obtained from Bhasma was dissolved in 1:1

Hydrochloric acid again and then reprecipitated and washed as above. The precipitate

obtained from the Bhasma was washed thoroughly with distilled water till filtrate showed

negative test for oxalate (no discoloration of acidic KMnO4). The precipitate was then

dissolved quantitatively in 70ml of 10%Sulphuric acid and volume was made upto 100ml

with the 10% Sulphuric acid. The solution was then titrated against standard potassium

permanganate till a drop gave slight pink color persistent for 1min.

21

Determination of the antacid activity22

This was determined by Acid neutralization capacity test as mentioned in the USP. As

per the USP, an antacid substance has to raise the gastric pH above 3.5 within 15 min.

In this test, the number of milliequivalents of acid neutralized by 1 gm of Bhasma was

determined. Based upon the Acid neutralizing capacity, the dose of the Shankha Bhasma

was calculated and incorporated .

Procedure

Accurately weighed about 500mg of the shankha Bhasma and to it 30 ml of the

standardized 1N hydrochloric acid was added. Then it was stirred for 15 min. accurately

timed after the addition of the acid. Then it was titrated immediately (in a period not to

exceed an additional 5min.) with 0.5N sodium hydroxide to attain a stable pH of 3.5.

Calculated the number of milliequivalents of acid consumed and the result was expressed

in terms of milliequivalents of acid consumed per gm of Bhasma tested. The ANC in mEq

was calculated as shown below:

Each ml of IN Hcl consumed = 1mEq. Of acid consumed.

The same was converted into ANC (mEq) / gm of the Bhasma.

22

Monographic Studies on the herbal drugs23

Foreign Organic Matter.

Weighed 100 g of the sample and spread out as a thin layer and separated the foreign

organic matter. It was again weighed and percentage was calculated and values were

compared with the Pharmacopeial limits.

Total Ash

Incinerated 3 g of the drug accurately weighed in a silica crucible at a temperature not

exceeding 450 ºC until free of carbon. It was then cooled and weighed. Then calculated the

percentage ash with reference to the air-dried drug.

Acid Insoluble ash

Boiled the ash as obtained above for 5 min. with about 25ml of dilute hydrochloric acid.

The insoluble matter was collected on an ashless filter paper, washed with hot water and

then ignited to a constant weight. Calculated the percentage of the acid insoluble ash with

reference to the air-dried drug.

Alcohol Soluble extractive value

Macerated 5 g of coarsely powdered air-dried drug with about 100ml of ethanol in a closed

flask for 24 hrs, frequently shaken for 6 hrs and allowed to stand for 18 hrs. Then it was

filtered and 25 ml of the filtrate was evaporated to dryness in a tared flat-bottomed shallow

dish. Calculated the percentage of the alcohol soluble extractive with reference to the air-

dried drug.

Water Soluble extractive value

The same procedure was carried out as described under alcohol soluble extractive value

But here the extracting solvent used was chloroform water. The percentage of water-

soluble extractive value was calculated with reference to the air-dried drug.

23

Preparation of the Triphala powder24

Accurately weighed about 100 gms of the herbs Terminalia Chebula,Terminalia belerica

and Emblica officinalis. These were then passed individually through 120# mesh

separately. Then equal quantities of these herbs were taken in the mortar and then mixed

with the help of the pestle to give triphala powder. This powder was then used for

subsequent procedures.

Preparation of the Triphala extract 25

100 g of each of the herbs Terminalia chebula, Terminalia belerica and Emblica officinalis

(obtained after passing through 120#) were separately macreated with 95% of ethyl alcohol

for 4 days. Then it was filtered and the filtrate was evaporated to dryness (at a temperature

not to exceed 50ºC) when gummy mass was obtained. It was further dried at a temperature

not to exceed 50ºC in a vacuum drier .The powder obtained was passed through 120#.

Then the equal quantities of the extracts obtained from these herbs were mixed to get the

triphala extract. This was used for subsequent procedures.

Spectrophotometric estimation of tannins in the triphala powder and triphala extract

by Folin-Dennis Method 26,27

Principle

The method is based upon the oxidation of the molecules containing a phenolic hydroxyl

group. The tannins reduce phosphotungustomolybdic acid in alkaline medium to produce a

highly colored blue solution, the intensity of which is proportional to the concentration and

hence to the amount of the tannins and can be estimated against the standard tannic acid

solution at a wavelength of 700nm.

24

Fig1. Determination of the λmax. Of the tannic acid in distilled water.

Preparation of the standard solution of tannic acid in distilled water

Accurately weighed 100mg of tannic acid and dissolved in 100ml of distilled water in a

100 ml of volumetric flask. Then 10ml of the resulting solution was taken in another 100

ml volumetric flask and diluted upto the mark with the distilled water to give a

concentration of 100μg/ml solution. From this solution was pipetted out

0.2,0.4,0.6,0.8,1.0,1.2 and 1.4 ml into a series of 10 ml volumetric flask. To these solutions

were added 1ml of (1:10) folin’s reagent and 0.8 ml of 7.5% sodium carbonate solution.

Allowed to stand for the development of the color and then volume was made upto 10 ml

with distilled water. Then recorded the absorbance of the solution and plotted the

calibration graph.

25

Preparation of the sample solution

a) For Triphala powder

100 mg of the triphala powder was taken in 100ml volumetric flask and to this was added

95% of ethanol upto the mark, shaken well and then filtered. Then taken 1ml of the filtrate

in a 10ml volumetric flask, added 1ml of (1:10) Folin’s reagent and 0.8ml of 7.5% sodium

carbonate solution. Allowed to stand for the development of color and recorded the

absorbance at 703nm.The concentration corresponding to the absorbance was recorded

from the calibration curve of tannic acid and expressed as tannic acid equivalents.

b) For Triphala extract

Accurately weighed quantity of 100mg of the extract was taken in 100ml volumetric flask

and volume made upto the mark with 95% ethanol. Then 1ml of the filtrate was taken in a

10ml volumetric flask, added 1ml of (1:10) Folin’s reagent and 0.8ml of 7.5% of sodium

carbonate solution. Allowed to stand for development of color (about 30min.).Then

recorded the absorbance at 703nm.The concentration corresponding to the absorbance was

read from the calibration curve of tannic acid and expressed as tannic acid equivalents.

26

Thin layer chromatography 28,29,30,31

TLC provided for the entire drug in the monographs includes the identification of the drug

based on its major chemical constituents as markers. Silica gel GF254 plates of uniform

thickness were prepared and activated at 105ºC for 30min. and the plates were spotted with

the drug extract using capillary tube. The spotted plates were dried at R.T. The solvents

used were of analytical grade. Solvent system was prepared and the chromatographic

chamber was saturated for 30 min. The plates were placed in the chamber and left for the

development. Using different reagents did visualization of the spots and Rf values were

calculated.

Rf = Distance traveled by the spot

Distance travelled by the solvent front

Terminalia Chebula - gallic acid

Mobile Phase : -Toulene: Ethylacetate: Glacial acetic acid: Formic acid (20:45:20:5)

Sample Preparation :- Powdered sample was extracted with methanol.The methanol extract

was concentrated and residue dissolved in methanol.

Detection :- Anisaldehyde Sulphuric Acid.

Terminalia belerica - gallic acid





27

Emblica officinalis - gallic acid





Glycyrrhiza glabra - glycyrrhetic acid

Mobile Phase : -Toulene: Ethylacetate: Glacial acetic acid (9:1:0.5)

Sample Preparation :- Shaken 1 gm of the drug with about 20ml of chloroform for 15

min..Filtered and discarded the filtrate. Then refluxed the marc for

1hr with 30m 0.5M Sulphuric acid. Cooled and shaken the unfiltered

mixture with chloroform(2x20ml) and concentrated the combined

chloroform extract.Dissolved the residue in 1.0ml of chloroform :

methanol(1:1)mixture.

Standard Preparation :-Refluxed 5mg of glycyrrhizin with 20ml of 0.5 M Sulphuric

acid.Cooled and extracted with chloroform(2x10 ml) . Evaporated

the combined chloroform extract and dissolved the residue in 1.0

ml of Chloroform: methanol (1:1) mixture.

Detection: - Anisaldehyde Sulphuric Acid.

28

PREPARATION OF THE SUSPENSION 32,33

1) In a 100 ml beaker about 40 ml of the distilled water was taken and to

this was added 35.0 gms of Sorbitol solution 70% and mixed well.

2) To the above mixture was added, Xanthan gum (in varying

concentration) and started stirring using a homogeniser.

3) In another beaker, methyl paraben was dissolved in a very little

quantity of alcohol and added to the above mixture.

4) Drugs and /or their extracts were passed through 120# sieve and then

was added in small quantity to the above mixture and stirred for 30

min.

5) Then dissolved the color in water and added to the above mixture.

Lastly flavor was added and the volume was made upto the mark.

29

FORMULATION CHART OF THE SUSPENSION

Ingredients

Quantity in g/ 100 ml

Formulation code

F I-1 FI-2 FI-3 FI-4 FII-1 FII-2 FII-3 FII-4

Powders

Glycyrrhiza glabra extract 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Triphala Churna 2.0 2.0 2.0 2.0 - - - -

Turbinella rapa 16 16 16 16 16 16 16 16

Triphala Churna Extract - - - - 2.0 2.0 2.0 2.0

Xanthan Gum 0.20 0.25 0.30 0.35 0.20 0.25 0.30 0.35

Sorbitol 70% liquid 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0

Distilled water to make 100ml 100ml 100ml 100ml 100ml 100ml 100ml 100ml

Preservative (methyl paraben) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Color q.s q,s q.s q.s q.s q.s q.s q.s

Flavor (Menthol) 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003

30

Evaluation of the suspension34

1) Particle size distribution

This was accomplished by using an optical microscope, a calibrated eyepiece that was

calibrated by using a stage micrometer.

Calibration of the eyepiece micrometer

The smallest division of the stage micrometer represents 0.01 mm in length.

Kept the eyepiece and the stage micrometer.

Focused under high power

Seen the superimposing

The stage micrometer was replaced with the slide of the object, which was suspended in

the paraffin oil. Sample was uniformly spread on a clean glass slide. Then placed under

microscope and the size was measured. The size obtained was multiplied by calibration

factor to get the actual dimension. The average value was taken. The details are mentioned

in page no.

Physico-chemical characterization of the suspension

pH measurement

pH of the prepared formulation was tested by using the calibrated systronic pH meter. The

prepared suspension formulations were taken separately in different glass beakers. The

electrodes were dipped in the formulations and pH was noted.

31

Viscosity

The viscosity of the prepared formulations were tested by using Brookfield’s viscometer,

attached with spindle no.21.The prepared formulations were placed in separate 100ml

beakers. The spindle of the viscometer was introduced into the formulation and the reading

was set to zero. The viscosity measured at 200 r.p.m was noted.

Ease of redispersibility

The conventional formulation was allowed to settle completely in a measuring cylinder.

The cylinder was inverted through 180 degrees and the number of inversions necessary to

restore a homogenous suspension was determined.

Sedimentation Volume (VS)

Sedimentation volume considers the ratio of the ultimate height (HU) of the sediment to the

initial height (HS) of the total suspension as the suspension settles in a cylinder under

standard conditions. The larger the fraction the better is the suspendibility. It is calculated

as follows: VS = HU/HS

Acid Neutralization Capacity

This was performed as per USP method by taking 5 ml of the suspension. The procedure is

same as described for Shankha Bhasma.

32

Stability studies 35,36,37

Stability studies were carried out on the selected formulations taking gallic acid as the

marker compound for 90 days at two temperatures i.e. 30 ºC /65% RH and 40 ºC / 75%

RH. The samples were withdrawn at regular intervals for 30 days for a total period of 90

days. HPTLC studies were performed to ascertain the stability data. The HPTLC graphs

were taken and discussed under results and discussion.

Anti-ulcer activity studies38

Antiulcer activity studies on the formulations were carried out on albino rats of either sex

weighing 200-250 g. The Histopathological studies were performed to ascertain the ulcer

prevention. The procedure is briefly described below: -

The rats were randomly divided into four groups, each group containing six animals.

Group 1: - control group i.e. no drug formulation was given.

Group 2: - ulcer was induced.

Group 3: - Animals pretreated with formulation containing herbal powders

@0.2ml/animal.

Group 4: - Animals pretreated with formulation containing herbal extracts @0.2ml/animal

33

The overnight fasted rats belonging to all the groups were administered 1ml of absolute

alcohol. After 1 hr of administration of the ulcerogen all the rats were anaesthetized and

sacrificed by cervical dislocation. The stomachs were removed, and opened along the

greater curvature, washed with normal saline and observed under 100X for the presence of

the ulcers. The ulcers were scored as below:

0 Normal colored stomach. 0.5 Red coloration.

1 Spot ulcer 1.5 Hemorrhagic streaks

2 Ulcers equal to 3mm but less than

5mm

3 Ulcers greater than 5

Mean ulcer score for each group was determined and ulcer index (UI) was calculated as

under:

UI = (n. lesion 1) + (n. lesion 2) + (n. lesion 3) + …….

n. Animals

34

Qualitative and Quantitative Analysis of Shankha Bhasma:

Table No. 1 Qualitative tests

Test

Observations

Inference

Tests for calcium

White precipitate

Calcium Present

Tests for Carbonate

White Precipitate

Carbonate Present

Tests for Magnesium

White Precipitate

Magnesium Present

Tests for Manganese

Green precipitate in soluble in dil. HCL.

Manganese Present

Table No.2 Quantitative tests

Observations

Parameter

Trial 1

Trial 2

Trial 3

Mean ± S.D

1) Calcium carbonate Content (%)

94.1

93.6

95.3

94.33±0.873

2) Acid Neutralization Capacity (mEq/gm)

16.6

17.2

17.0

16.93±0.305

35

Monographic analysis of the herbs.

The monographic analysis of all the herbal ingredients was performed with

reference to the Herbal Pharmacopoeia of India and the Ayurvedic Pharmacopoeia of

India. Both these Pharmacopoeias suggest tests like foreign organic matter, total ash, acid

insoluble ash, water-soluble extractive and alcohol soluble extractive for the purity and

identification of the individual drugs. The values of the monographic analysis are reported

along with the standard values for comparison purposes. The results are within the range of

the pharmacopoeias.

Table No. 3: Foreign organic matter of herbal drug. Drug Obtained value

(In percentage) Pharmacopeial Standards

I II III Mean ±S.D

Terminalia Chebula 0.85 0.74 0.45 0.68±0.206 NMT 1%

Terminalia belerica 1.17 1.26 1.24 1.22±0.047 NMT 2%

Emblica officinalis 2.2 2.36 2.81 2.45±0.316 NMT 3%

Glycyrrhiza glabra 1.53 2.11 2.63 2.09±0.5503 *NS

*NS = not specified *S.D = Standard deviation, n=3

36

Table No. 4: Determination of total ash value.

Obtained value (In percentage)

Drug

I II III Mean ±S.D*

Pharmacopeial limits

Terminalia Chebula

3.41 3.241 3.042 3.23±0.184 NMT 5%

Terminalia belerica

5.824 5.833 5.714 5.79±0.066 NMT 7%

Emblica officinalis 4.933 4.582 4.621 4.712±0.192 NMT 5%

Glycyrrhiza glabra 6.51 7.23 7.81 7.183± 0.6513

NMT 10%

*S.D. = Standard deviation, n=3 Table No.5: Acid insoluble ash value.


Drug

I II III Mean ±S.D*

Pharmacopeial limit

Terminalia Chebula

1.8 1.782 1.923 1.835± 0.0767

NMT 5%

Terminalia belerica

0.866 0.814 0.823 0.834± 0.0277

NMT 1%

Emblica officinalis 0.933 0.925 0.907 0.9223± 0.010

NMT 2%


NMT 2.5%

S.D*. = Standard deviation, n=3

37

Table No.6: Determination of extractive values: Alcohol soluble extractive values.


Drug

I II III Mean ±S.D*.

Pharmacopeial limit

Terminalia Chebula

44.26 46.22 40.38 43.62±2.972 NLT 40%

Terminalia belerica

25.84 27.20 27.54 26.86±1.311 NLT 8%

Emblica officinalis 40.6 43.18 49.36 44.38±4.5016 NLT 40%

Glycyrrhiza glabra 12.96 13.92 15.12 14.0±1.082 NLT 10%

S.D*. = Standard deviation, n=3 Table No.7: Extractive values: Water-Soluble extractive values.


Drug

I II III Mean ±S.D*.

Pharmacopeial limit

Terminalia Chebula

62.02 65.92 62.34 63.42±2.165 NLT 35%

Terminalia belerica

39.2 37.76 41.08 39.34±1.664 NLT 60%

Emblica officinalis 52.4 54.8 63.2 56.8±5.570 NLT 50%


NLT 20%

S.D* = Standard deviation, n=3

38

Table No.8: TLC Identification of the Individual Herbs

HERBAL DRUGS RF Value

Obtained Literature

1) Glycyrrhiza glabra 0.45 0.46

2) Terminalia Chebula 0.66 0.68

3) Terminalia belerica 0.70 0.68

4) Emblica officinalis 0.69 0.68

The Thin Layer Chromatographs of all the herbal ingredients were taken and

are shown on next page.

39

TLC Photographs of the herbal drugs

STD SAMPLE STD SAMPLE Terminalia belerica Terminalia chebula

STD SAMPLE STD SAMPLE Emblica officinalis Glycyrrhiza glabra

39(a)

Estimation of tannin content by Folin-Dennis Method.

Table No.9: Standard graph of the tannic acid by Spectrophotometric method.

Sl.No. Concentration. (μg/ml)

Absorbance Mean±S.D*.

1 2 0.1278±0.012 2 4 0.2235±0.025 3 6 0.3307±0.023 4 8 0.4673±0.041 5 10 0.5769±0.044 6 12 0.6665±0.043 7 14 0.7823±0.051 8 Triphala Powder 0.1755±0.034 9 Triphala extract 0.3297±0.046

S.D*= Standard deviation, n=3 Fig. No.2: Calibration curve of tannic acid in distilled water at 703 nm.

Stan d ad r d g r ap h o f T an n ic Acidy = 0 .0 5 6 4 xR 2 = 0 .9 9 8 3

0

0 .2

0 .4

0 .6

0 .8

1

0 2 4 6 8 1 0 1 2 1 4

Conce ntra tion M cg/m l

Abs

orba

nce

Quantity of tannins present = Conc. from graph x Vol. taken x Dilution Factor (%W/w) 1000

40

Table No.10. Total tannin Content HERBAL DRUGS Tannin content (%w/w)

Triphala churna powder 29.1

Triphala churna extract 59.5

Standard deviation, n=3

Physicochemical Characterization of the suspension.

Table No. 10: Particle Size Distribution data

1 division of eyepiece micrometer = 13.1 μm

0

5

10

15

20

25

30

#19.

5

#32.

5

#45.

5

#58.

5

#71.

5

#84.

5

#97.

5

Mean particle size in microns

% fr

eque

ncy

From the particle size distribution data it can be seen that most of the particles lies between

50-75 micrometers.

41

TABLE NO.11: pH and Viscosity

PARAMETER pH

Viscosity (in cps)

Formulation code

Trial1 Trial 2 Trial 3 Mean±S.D Trial1 Trial 2 Trial 3 Mean±S.D

FI-1

7.88

8.02

8.16

8.02±0.14

30.0

30.3

29.7

29.967± 0.3512

FI-2

8.22

8.17

8.29

8.227± 0.06028

40.2

41.3

40.8

40.767± 0.5508

FI-3

8.09

8.11

8.32

8.173± 0.1274

50.7

50.3

51.2

50.73± 0.4509

FI-4

7.96

8.21

8.05

8.07± 0.1266

61.6

60.3

61.4

61.1± 0.70

FII-1

7.90

8.11

8.12

8.0467± 0.1321

38.5

39.7

40.3

39.5± 0.9165

FII-2

8.0

8.21

7.98

8.063± 0.1263

51.4

52.8

50.6

51.6± 1.114

FII-3

8.22

8.37

8.16

8.25± 0.1082

63.3

62.7

62.1

62.7±0.60

FII-4

8.15

7.95

8.26

8.12± 0.1572

76.6

77.9

80.3

78.267± 1.877

1) The results show that the pH of all the formulations lies around 8.0, which is desirable

for an antacid suspension.

2) The viscosity data show that the viscosity of the suspension containing herbal extracts is

more than those containing herbal powders. This may be attributed to the fact that the

herbal extracts being sticky in nature contribute some value to the viscosity of the

suspension. 42

Table no. 12: sedimentation volume and redispersibility PARAMETER

Formulation code

Sedimentation volume

Redispersibility

FI-1

0.88

2

FI-2

0.91

3

FI-3

0.93

3

FI-4

0.96

4

FII-1

0.90

3

FII-2

0.92

3

FII-3

0.95

4

FII-4

0.98

5


1) Hence from the above data, it can be concluded that as all the formulations are

characterized by high sedimentation volume, the suspension may be flocculated

suspension.

2) From the redispersibility data it can be seen that formulations FI-2, FI-3 FII-1 and FII-2

have good redispersibility with good consistency.

43

Table no. 12: Acid Neutralization Capacity PARAMETER Acid Neutralization Capacity (mEq/ml)

Formulation code

Trial1 Trial 2 Trial 3 Mean±S.D

FI-1

2.65

2.85

2.81

2.77±0.1058

FI-2

3.0

2.91

2.52

2.81±0.2551

FI-3

2.55

2.98

2.31

2.613± 0.3395

FI-4

2.86

3.15

2.99

3.0± 0.1453

FII-1

2.58

2.38

2.40

2.453±0.1102

FII-2

1.93

2.31

2.90

2.38±0.4888

FII-3

2.45

3.0

2.20

2.55±0.4093

FII-4

2.20

2.19

2.54

2.31± 0.1769

1) From the above data it is evident that the acid neutralization capacity of all the

formulations lies between 2-3 mEq/ml. The minimum acid that has to be neutralized by

an antacid should be 12 mEq-40 mEq and hence 5ml of the above formulations

satisfies the requirement.

44

ANTI-ULCER ACTIVITY

TABLE NO.13: Ulcer Index

GROUP ULCER INDEX

Control 1.01 ± 0.68

Ulcerated 8 .68 ± 1.51

Treated (FI-2) 3.57± 2.14*

Treated (FII-2) 2.11±1.07*


P < 0.05 as compared to the ulcerated group , * indicates statistically significant

From the above data is very obvious that the Suspension containing the herbal extracts

shows a decrease in the ulcer index as compared to the one containing the fine powders of

these drugs.

Histolopathological Studies

The tissue samples were fixed in 10% buffered formalin and processed with paraffin wax.

For this study 5mm sections were stained with haematoxylin and eosin. The extent and

depth of the ulceration and hemorrhage were evaluated.

Statistical Analysis

Statistical Analysis was carried out by using one-way ANOVA followed by Duncan’s

multiple comparison tests. All the results obtained in the study were compared with each

group. P values than 0.05 were considered stastically significant.

The Photos of Histopathological studies were taken and discussed on next page

45

Histopathological studies:

The fig.no.1 indicates the normal mucosa of the stomach with mucus and presence of

chief cells.

The fig. No. 2 indicates the ulcerated mucosa with prominent loss of mucus, chief

cells.

The fig no.3 indicates the pretreated mucosa with suspension containing herbal

powders that shows less loss of mucus and chief cells.

The fig no. 4 indicates the mucosa pretreated with suspension containing herbal

extracts with near normal appearance to that of normal mucosa.

ANTI-ULCER ACTIVITY STUDIES

NORMAL MUCOSA ULCERATED MUCOSA

TREATED MUCOSA FII-2 TREATED MUCOSA FI-3

45(a)

HPTLC method for estimation of gallic acid

Table no.14: Percentage of gallic acid by HPTLC Method

Formulation Temperature Days % Gallic acid

0th Day 11.54 30ºC/65%RH

90th day 11.52

0th Day 11.54

FI-3

40ºC/75%RH

90th day 11.49

0th Day 19.63 30ºC/65%RH

90th day 19.59

0th Day 19.63

FII-2 40ºC/75%RH

90th day 19.57

The HPTLC data shows that there is no significant change in the concentration of gallic

acid at the end of 90 days at two temperatures. The graphs are shown on next page.

Table No.15: TLC of the suspension at 0th and 90th Day

Rf value Formulation Temperature Days

Obt. Std.

0th Day 0.67 0.68 30ºC/65%RH

90th day 0.66 0.68

0th Day 0.67 0.68

FI-3

40ºC/75%RH

90th day 0.66 0.68

0th Day 0.67 0.68 30ºC/65%RH

90th day 0.69 0.68

0th Day 0.67 0.68

FII-2 40ºC/75%RH

90th day 0.69 0.68

The TLC Photographs are shown on next Page. TLC Photographs shows no secondary

spots, which shows that there is no degradation of the product during storage.

46

TLC of the Formulations at 0th Day TLC of the formulations at 90th Day

G 1 2 3 4 5 6 G 1 2 3 4 5 6

std samples std samples

G : gallic acid G : gallic acid

46(h)

Table no.15: Viscosity, pH and acid neutralization capacity.

Formulation

Temperature/

Humidity

Days Viscosity pH Acid Neutralization

capacity

0th Day 50.73±0.4509 8.17±0.1274 2.61± 0.3395

30th day 50.64±0.4487 8.12±0.1258 2.60± 0.3346

60th day 50.56±0.4410 8.11±0.1192 2.59±0.3277

30ºC/65%RH

90th day 50.43±0.4361 8.10±0.1132 2.58±0.3201

0th Day 50.73±0.4509 8.17±0.1274 2.61± 0.3395

30th day 50.57±0.4438 8.16±0.1265 2.60± 0.3313

60th day 50.45±0.4397 8.16±0.1259 2.59± 0.3298

FI-3

40ºC/75%RH

90th day 50.23±0.4337 8.15±0.1247 2.58±0.3288

0th Day 51.63±1.114 8.06±0.1263 2.38±0.4888

30th day 51.32±1.111 8.05±0.1257 2.32±0.4873

60th day 50.81±1.109 8.04±0.1252 2.27±0.4864

30ºC/65%RH

90th day 49.45±1.085 8.0±0.1179 2.19±0.4856

0th Day 51.66±1.114 8.06±0.1263 2.38±0.4888

30th day 51.54±1.108 8.04±0.1253 2.29±0.4865

60th day 51.43±1.104 8.03±0.1247 2.23±0.4857

FII-2

40ºC/75%RH

90th day 51.39 ±1.080 7.9±0.1243 2.15±0.4790

47

Table no.16: Organoleptic properties.

Formulation

Temperature/

Humidity

Days Color Odour Taste

0th Day No Change No change No Change

30th day color change No Change No Change


30ºC/65%RH


0th Day No Change No Change No Change



FI-3

40ºC/75%RH





30ºC/65%RH





FII-2 40ºC/75%RH


48

From the Present study we can conclude the following: -

1) From the particle size distribution data it can be concluded that the suspension is a

coarse dispersion with particle size predominantly lying between 50-75 micrometers.

2) The suspensions containing the herbal extracts show greater viscosity than those

containing the herbal powders for the same concentration of the suspending agent. This

difference may be due to the fact that the extracts obtained are sticky in nature that may

contribute to the viscosity of the formulation.

3) The suspension containing herbal powders and 0.3% concentration of Xanthan gum

shows the optimum quality of the suspension with regards to the consistency and

redispersibility, while in case of suspension containing herbal extracts, 0.25%

concentration of the Xanthan gum shows the optimum quality of the suspension with

regards to the consistency and redispersibility. Hence these formulations were selected

for in vivo studies and stability studies.

4) All the formulations show an acid neutralization capacity between 2-3 mEq /ml .

5) All the formulations show the pH of around 8.0 that is the desirable pH for an antacid

suspension.

6) All the formulations show high sedimentation volume and good ease of redispersibility

except FII-4 which has poor ease of redispersibility.

7) The HPTLC graphs indicate that there are no interactions between the ingredients

during the stability studies.

8)The TLC Photographs taken during stability studies show no secondary spots

that indicates that there is no degradation-taking place during stability studies.

49

9) From the data on the viscosity, pH and acid neutralization capacity of the

suspension, which was evaluated during the stability studies, indicates that there is no

change in these parameters.

10) The TLC Photo graphs that were taken during stability studies show no secondary

spots that indicate that there is no degradation-taking place during stability studies.

11) The extracts obtained from the herbs show significantly greater Antiulcer Activity

than those of the powdered drugs. This may be due to the fact that the extracts may

contain more concentration of the active ingredients responsible for antiulcer activity.

12) Thus efforts to formulate an herbal antacid suspension were found to be

satisfactory.

Future Work that can be carried out: -

1) The Anti-ulcer activity can be evaluated by varying the concentrations of the herbal

Powders / extracts.

2) The extracts of other antiulcer herbs may also be evaluated to find out extent of

anti- ulcer activity.

50

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52

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APPENDICES

APPENDIX - I

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Sri Shivarathreeshwara Nagara Mysore

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