51

Journal of Health Sciences. 2013;3(11):051-062

The journal has had 5 points in Ministry of Science and Higher Education of Poland parametric evaluation.

Part B item 1107. (17.12.2013)

© TheAuthor (s) 2013;

This article is published with open access at Licensee Open Journal Systems of Radom University in Radom, Poland

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits

any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are

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This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License

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Conflict of interest: None. Received: 15.10.2013. Revised: 24.12.2013. Accepted: 24.12.2013.

Pharmacological, Phytochemical and Physicochemical

properties of methanol extracts of Erioglossum rubiginosum

barks

Santanu Barua1, S. M. Masud Rana

1*, Md. Mustahsan Billah

1,

Zannatul Naim2, Golam Sarwar

1

1Department of Pharmacy, Noakhali Science and Technology University, Sonapur,

Noakhali - 3814, Bangladesh 2Department of Pharmacy, Atish Dipankar University of Science and Technology, Banani,

Dhaka, Bangladesh

*Corresponding author

S. M. Masud Rana

Department of Pharmacy

Noakhali Science and Technology University, Sonapur, Noakhali- 3814, Bangladesh

Cell: +8801683788782

E- mail: kaktarua.r@gmail.com

Abstracts

Objectives: To evaluate the Pharmacological (cytotoxic and antimicrobial activities),

phytochemical properties and physicochemical properties of Erioglossum rubiginossum barks,

an evergreen plant, belonging to the family Sapindaceae.

Methods: Powdered barks of the plant were treated with methanol using hot extraction method

and the extract has been investigated for its biological activities. Crude methanol extracts of the

bark of E. rubiginosum were used for brine shrimp lethality bioassay. Vincristine sulphate was

used as standard. The crude methanol extract of E. rubiginosum was screened for their

antibacterial activity against a wide range of bacteria (both gram-positive and gram-negative)

by disc diffusion method, antifungal activity by modified poisoned food technique.

52

Phytochemical evaluation was by qualitative analysis and Physicochemical properties was

determined according to method (Anonymous 1968) described in British Pharmacopeia.

Results: The LC50 values of methanol extract were found to be 29.47µg/ml compared to

standard Vincristine sulphate (0.451µg/ml). From these results, it can be well predicted that the

barks of Erioglossum rubiginossum possess very low cytotoxic property. The crude methanol

extract showed significant antibacterial activity against gram-positive Staphylococcus aureus

(17.02±0.11mm), gram negative Salmonella typhi (7.05±0.03mm) and Shigella dysenteriae

(5.03±0.04mm) compared to Cephradine. Interestingly, gram- positive Bacillus cereus was

found resistant towards this extract. Again this methanol extract exhibited mild antifungal

activity against Aspergillus niger and Candida albicans. The phytochemical evaluation showed

the presence of alkaloids, flavonoids, phenols, saponins and carbohydrate. Various

physicochemical parameters like moisture content, total cash value, acid soluble ash value,

water soluble ash value, alcohol soluble extractive value and water soluble extractive value

were to be found 8.07%, 18.00%, 21.00%, 9.50%, 1.00% and 0.50% respectively.

Conclusion: Further investigations are required for isolating active compound that are

responsible for antimicrobial activity and low cytotoxicity and finding out the mechanism

behind these effects.

Keywords Erioglossum rubiginosum, Brine shrimp lethality, Antibacterial, Antifungal, Zone

of inhibition, Phytochemicals.

1. Introduction The plant under investigation Erioglossum rubiginosum belongs to the family Sapindaceae. The

common and local name of the plant is Kalayo and Boro Harina respectively. Erioglossum

rubiginosum is distributed throughout the Philippines, also occurs from northern India to Indo-

China and Thailand, through Malaya to tropical Australia1. Commonly using part of the plant is

seeds, roots, barks and leaves. Different study had shown that major components of flower

essential oil were nerolidol (34.8%), palmitic acid (13.2%), and farnesol (10.0%). Fruit

essential oil yielded palmitic acid (66.1%), myristic acid (10.0%), and linolenic acid (5.5%)2.

Methanolic fraction isolated a tetrasaccharide derivative of farnesol named rubiginoside along

with known triterpenoid saponins3.

The plant commonly used for the treatment of leprosy4.

This plant is extensively used as

folkloric medicine such as roots are used as astringent, leaves and fruits are used for the

treatment of fever and poultice5, 1

. Recent investigation had shown the leaves of the plant could

be used as a natural source of membrane stabilizers6.

Another recent investigation had proved

that the leaves of the plant possess antioxidant, thrombolytic and cytotoxic activity7. CNS

depressant activity was found significantly by the plant8.

According to literature survey, the study, investigation and information of barks of Erioglossum

rubiginosum is still insufficient. So, the present study was undertaken for crude methanol

extract of barks of Erioglossum rubiginisum for investigation of cytotoxic, antibacterial,

antifungal activities and phytochemical properties.

53

2. Materials and Methods

2.1 Plant material and extraction

The barks of Eriglossum rubiginosum was collected from National Botanical Garden, Mirpur,

Dhaka. The plant was identified by the taxonomist of Bangladesh National Herbarium, Mirpur,

Dhaka, Bangladesh and a voucher specimen has been deposited in the herbarium unit

(Accession no DACB 38566).

The barks were sun dried for several days and then oven dried for 24 hours at considerably low

temperature (not more than 400o C) for better grinding. The dried barks were then ground to a

coarse powder using high capacity grinding machine in the Phytochemical Research

Laboratory, Faculty of Pharmacy, University of Dhaka, Bangladesh. The current study used hot

extraction method. The powdered material (500gm) was taken in a cleaned, ambered color

reagent bottle (2.5 liters) and soaked in 1.5 L of 99% methanol. The container with its content

was sealed by bottle cap and kept for a period of 20 days accompanying occasional shaking and

stirring. The whole mixtures were then filtered through a fresh cotton plug and finally with a

Whatman No.1 filter paper separately in two separate beakers. The volume of the filtrate was

then allowed to evaporate at Rotary evaporator at 40 degree Celsius temp until approximately

80% solvent was evaporated. It rendered a blackish-brown gummy concentrated extract. The

blakish-brown extract concentrated was designated as crude extract of methanol.

2.2 Test Microorganisms

Authentic pure cultures of human pathogenic gram positive bacteria (Bacillus cereus ATCC

10876, Staphylococcus aureus ATCC-29740), gram negative bacteria (Salmonella typhi ATCC-

14028, Shigella dysenteriae ATCC-9027) and fungi (Aspergillus niger ATCC-16404, Candida

albicans ATCC-10231) were obtained from Poultry Research & Training Center (PRTC),

Chittagong Veterinary and Animal Sciences University, Chittagong, Bangladesh.

2.3 Brine shrimp lethality bioassay

Brine shrimp lethality bioassay was used for probable cytotoxic action according to

McLaughlin (1998)9 and Persoone (1980)

10.

According to this method, natural product extracts, the pure compounds and fractions can be

tested for their biological activity. In this method, in vivo lethality in a simple zoological

organism (Brine shrimp nauplii, Artemis salina) was used as a favorable monitor for screening

and fractionation in the discovery of new bioactive natural products. Vincristine sulphate was

used as a positive control group whereas DMSO was used as negative control group according

to Tyler and Brady (1988)11

; McKey (1994)12

and Lewis et al., (2005)13

.

2.4 Antibacterial screening

Antibacterial screening was determined by the disc diffusion method14

against gram positive

(Bacillus cereus, Staphylococcus aureus) and gram negative bacteria (Shigella dysenteriae,

Salmonella typhi). Cephradin was used as standard.

2.5 Antifungal Screening

Antifungal screening was determined against Aspergillus niger and Candida albicans by the

poisoned food technique15

with slight modification. Briefly, 1st Plate preparation: Preparing

potato dextrose agar > Placing it in Petridis > Mixing extract with media > Drying up for 30

54

minutes. 2nd

Test suspension preparation: Selection of a few colonies of fungus > Placing it

in in savored agar media > Incubation at room temperature for 3 days > Match with standard.

3rd

Streking the plates: Strek all plates with test organism > Dry up for 30 minutes >

Incubation at room temperature for 3 days > Recording zone of growth > Calculating

percentage of inhibition by following formula:

Here,

C= Diameter of control = 08 cm

T= Diameter of zone of growth

I= Percentage of inhibition

2.6 Phytochemical screening

The methanol extracts of barks of Erioglossum rubiginosum were subjected to qualitative tests

for the identification of various phytochemicalconstituents.16, 17, 18, 19, 20

.

2.7 Physicochemical Profile21-26

2.7.1 Ash value

Total ash, acid insoluble ash and water soluble ash were determined as reported in the

Anonymous (1968)21

and MHFW (1999)22

. Briefly, total ash was determined using 2 g of the

air-dried powdered sample. The total ash was boiled for 5 minutes with 25 ml of distilled water;

the insoluble matter was collected on an ashless filter paper, washed with hot distilled water,

and ignited for 15 minutes at a temperature not exceeding 450oC. The weight of the insoluble

matter was subtracted from the weight of the total ash; the difference in weight represents the

water-soluble ash. The percentage of the water-soluble ash was calculated with reference to the

air-dried powdered plant sample.

2.7.2 Extractive values and Moisture content

Extracts of the plant samples were prepared with different solvents for the study of extractive

values. For present study alcohol and water were used as solvent for the study of extractives

value

3. Result

3.1 Brine shrimp lethality bioassay

In case of brine shrimp lethality bioassay, the methanol extracts of bark of Erioglossum

rubiginosum demonstrated very low cytotoxic potentiality against Artemis salina with LC50

value of 29.47µg/ml as compared to Vincristine sulphate (0.451µg/ml) Table 1 and Table 2.

3.2 Antibacterial screening

The antibacterial activity of Erioglossum rubiginosum test samples (400μg/disc) was evaluated

against two gram positive and two gram negative bacteria and the results were found significant

compared with standard, Cephradin. The test samples of Erioglossum rubiginosum revealed

antibacterial activity with zone of inhibition ranging from 5.03±0.04 mm to 17.02±0.11 mm.

The highest zone of inhibition 17.02±0.11 mm and 7.05±0.03 mm was shown against gram

55

positive bacteria (Staphylococcus aureus) and gram negative bacteria (Salmonella typhi)

respectively. But, gram- positive Bacillus cereus was found resistant towards this extract

Table 3.

3.4 Antifungal screening

The antifungal activities of methanol extracts of Erioglossum rubiginosum (100 µl/disc) and

standard Griseofulvin (30 μl/disc) were determined against two pathogenic fungi (Aspergillus

niger, Candida albicans) where, 25% and 30.5% of inhibition was found against Aspergillus

niger and Candida albicans respectively in comparison to Griseufulvine (100% and 98.5%)

Table 4

3.5 Phytochemical screening

In preliminary phytochemical screening, the methanol extract of Erioglossum rubiginosum

confirmed the presence of alkaloids, saponins, phenolic compound and carbohydrate Table 5.

3.6 Physicochemical screening

3.6.1 Ash value

Results of total ash value, acid insoluble ash and water soluble ash value has shown in Table 6.

Total ash value was found to be 9.50 %), acid soluble ash value 1.00% and water soluble ash

value was found to be 0.50%.

3.6.2 Extractive value and Moisture content

Result of the extractive value has shown in Table 6. Alcohol soluble and water soluble

extractive value were found to be 18.00 % and 21.00% respectively. Moisture content to found

8.07%

4. Discussion Plants may offer a new source of cytotoxic, antibacterial, antifungal and antiviral agents with

significant activity27, 28

. Brine shrimp cytotoxicity assay has been considered as prescribing

assay for antibacterial, anti-fungal, insecticidal, anti-parasitological and various pharmacologic

activities29

. Several studies have shown that brine shrimp bioassay has been an excellent

method to screen the cytotoxic property of medicinal plants and for the isolation of a great

variety of biologically active compounds30

.

In present brine shrimp lethality bioassay, ten different concentrations (0, 0.78, 1.56, 3.13, 6.25,

12.5, 25, 50, 100, 200, 400 μg/ml) of E. rubiginosum extract were used to determine its

cytotoxicity by brine shrimp lethality bioassay (Table 2, Figure 1). The test samples showed

different mortality rates at different concentrations where the percentage of mortality increased

with an increase in concentration. The variation in results may be due to the difference in the

amount and kind of cytotoxic substances (e.g. tannins, flavonoids, triterpenoids, or coumarins

etc.) present in the crude extracts. The LC50 value and Chi- square was found 29.47μg/ml and

0.783 respectively (Table 1). From these results, it can be well predicted that the barks of

Erioglossum rubiginossum possess very low cytotoxic property which may be due to little

presence of cytotoxic substances.

In antibacterial screening, significant zone of inhibition was observed against Staphylococcus

aureus and both gram negative bacteria (Salmonella typhi, Shigella dysenteriae) as well.

Though Bacillus cereus is gram positive, but it revealed maximum resistance against the test

sample. It was also observed that the test samples showed most significant and best activity

against Shigella dysenteriae than the standard Cephradin.

56

In general, gram-negative bacteria are more resistant to antibiotics than gram-positive

bacteria31, 32

. The resistance is due to the differences in their cell wall composition. In gram-

negative bacteria the outer membrane acts as a great barrier to many environmental substances

including antibiotics33

. Presence of thick murine layer in the cell wall prevents the entry of the

entry of the inhibitors34

. The ability of the extracts to exhibit antibacterial activity against the

bacteria suggested the presence of hydrophilic and hydrophobic antibacterial compounds such

as luteolin, apigenin etc35

.

But the present study revealed a controversy report that gram-negative bacteria were more

susceptible to the crude extracts than gram-positive bacteria. It may be due to the presence of

broad spectrum of antibacterial compounds in the bark of E. rubiginosum.

The antifungal activities of methanol extracts of Erioglossum rubiginosum was expressed in

little amount against Aspergillus niger and Candida albicans due to the presence of antifungal

constituents in less quantity.

From several previous study we know that, e.g. presence of alkaloids, which may be used as

antimicrobial activity36

, phenolic compounds and flavonoids, which can be referred to as

nature’s biological response modifiers, have shown antimicrobial and anticancer (cytotoxic)

activities37

. Again flavonoids and polyphenolic compounds are considered as one of the major

chemical constituents responsible for cytotoxic and antimicrobial activity38

. Our present study

had shown the presence of these biological response modifiers such as phenolic compounds,

saponins, alkaloids and flavonoids. So we can say that the presence of these chemical

constituents may be the reasons for cytotoxic and antimicrobial activity.

Physicochemical parameters of the plants prove that it possess good quantitative properties. Its

dried form is expected to have a long shelf-life with reduced chance of microbial growth due to

its relatively low moisture content of 8.07%. Total ash value of 9.50% indicates low inorganic

components in the herbal plant. Acid insoluble ash value of 1.00% indicates high digestibility

when the plant is consumed. Water soluble ash value of 0.50% is indicative of negligible level

of water-soluble minerals absorption from the plant when it is consumed. The alcohol-soluble

extractive value of 18.00% and water-soluble extractive value of 21.00% shows that both

solvents would be good for extraction of this potential drug-plant.

5. Conclusion From the present study, we can say that the methanol extracts of bark of E. rubiginosum can be

used as significant antibacterial agent having mild anti-fungal and less cytotoxic potentiality.

So, further chemical and pharmacological investigations to isolate and identify chemical

constituents responsible for these potential bioactivities should be suggested.

Acknowledgment The authors are grateful to Bangladesh National Herbarium, Bangladesh to identify the plant

and Chittagong Veterinary and Animal Sciences University, Bangladesh to supply the

microorganism. Authors are also thankful to Department of Pharmacy, Noakhali Science and

Technology University; Poultry Research and Training Center, Bangladesh for providing the

laboratory facilities and technical support.

Conflict of interest statement We declare that we have no conflict of interest.

57

References 1. Philippine Medicinal Plants: Kalayo. http://www.stuartxchange.com/Kalayo.

2. Stephen G. Pyne, Boonsom Liawruangrath, Saisunee Liawruangrath, A Teerawutkulrag, A

comparative study of the essential oil from flowers and fruits of lepisanthes rubiginosa. J

Chuangbunyat. Acta Pharmaceutica Sciencia. 2011; 53 (4): 535-542.

3. Adesanya SA, Martin MT, Hill B, Dumontet V, Van Tri M, Sévenet T, Païs M. Rubiginoside, a

farnesyl glycoside from Lepisanthes rubiginosa. Phytochemistry. 1999; 51(8):1039-41.

4. Vinod M, Sharma M, Kesharwani A, Thakur R. Anti-leprotic Plants of Chhattisgarh: A Review

. Verma Rungta College of Pharmaceutical Science and Research, Kohka Road, kurud, Bhilai,

C.G, India.

5. Find Me a Cure: Kalayo. http://findmeacure.com/2011/01/04/kalayo. Accessed 07.02.13.

6. Pankaj Chandra Debnath, Abhijit Das, Amirul Islam, Md. Ariful Islam, Md. Mahadi Hassan,

Sultan Md. Gias Uddin Membrane stabilization – A possible mechanism of action for the anti-

inflammatory activity of a Bangladeshi medicinal plant: Erioglossum rubiginosum (Bara

Harina). Pharmacognosy Journal. 2003; 5(3): 104-107

7. Amirul Islam, S. M. Masud Rana, Abhijit Das, Monika Rani Saha, Sultan Md. Gias Uddin. In

vitro Antioxidant, Thrombolytic and Cytotoxic Activities of Methanolic Leaf Extract and Its

Fractionates of Erioglossum rubiginosum (Roxb.) Blume: Dhaka Univ. J. Pharm. Sci. 2013;

12(2): 105-110

8. Sattar M A, Gan EK, Loke SE, Mah KF, Wong WH. Effect of an extract of Erioglossum edule

on the central nervous system. J Ethnopharmacol. 1989; 25:217–220.

9. Maclaughlin JL, Anderson JE, Rogers and Lingling L. Drug Info Journal. 1998; (32): 513-524.

10. Persoone G. Proceeding of the international symposium on brine shrimp, Vol- 4 Universal

Press, Belgium. 1980.

11. Tyler VD., Brady LR. & Robbers JE. Pharmacognosy. (Alkaloids). 9th ed. Lea and Febiger

Publisher. Philadelphia.1988; 227

12. McKey D. Legumes and nitrogen: the evolutionary ecology of a nitrogen-demanding lifestyle in

Advances in Legume Systematics, part 5, the nitrogen factor, Royal Botanic Gardens, Kew,

UK. 1994; 211–228.

13. Lewis G., Schrire B., MacKinder B. & Lock M. Legumes of the world Royal Botanical

Gardens, Kew, UK. 2005.

14. Bayer AW, Kirby WMM, Sherris JC, Turck M. Antibioticsusceptibility testing by a

standardized single disc method. Am J Clin Pathol. 1966; 45: 493- 496.

15. Grover RK, Moore JD: Toximetric studies of fungicides against brown rot organism-

Sclerotinia fructicola and S. laxa. Phytopathology 1962; 52: 876–880.

16. Toshiya, Kondo; Takafumi, Yoshikawa; School of Pharmaceutical Sciences, Kitosato

University, Minato‐ KU, Tokyo. Journal of Natural Medicines. 2007; 61(2): 108 – 186.

17. Wallis T.E.; Practical Pharmacognosy, VI Edn. 1953.

18. Kokate, C.K.; Practical Pharmacognosy, 1st Edn. Vallabh Prakashan, Delhi. 1986.

19. Brain, K.R.; Turner, T.D. The Practical Evaluation of Phytopharmaceuticals, Wright

Scientechnica, Bristol, 1975.

20. Chandrika, C.; Aruna, R., Practical Biochemistry, 1st Edn. Augustine Publishers, Madurai,

1988: 1 – 10.

21. Anonymous, British Pharmacopoeia, General Medical Council, Pharmaceutical Press, London,

1968.

58

22. Okhale, Samuel Ehiabhi, Amanabo, Mercy Omachonu, Jegede, Ibikunle Adeola, Egharevba,

Henry. MHFW. (190with slight modifications Omoregie 1, Muazzam, Ibrahim Wudil 2 , Kunle,

Oluyemisi Folashade) The Ayurvedic Pharmacopeia of India, part 1, Vol. II (first edition),

Published by Ministry of health and family welfare, Government of India, Department of Indian

system of medicine and Homeopathy, 1999.

23. Sumitra Singh, Vijay Naresh, Surendra Kr, Sharma. Pharmacognostic Parameters of, Salvadora

Oleoides Decne. Leaves. Asian. Journal of Pharmaceutical Research and Development. 2013; 1

(3): (in press).

24. Sumitra Singh, Vijay Naresh, Surendra Kr, Sharma. Pharmacognostical and physicochemical,

studies on the stem bark of Prosopis, cineraria (l.) druce.: A medicinal plant indigenous to

southwest asia. Universal Journal of Pharmacy. 2013; 02 (02): (in press).

25. R. K. Issar, The botanical identification of market sample of Brahmadandi. Jour. Res.

Ind.Med.1974; 91- 92.

26. D. A. Johansen. Plant Microtechnique. New York, McGraw-Hill; 1940; 126.

27. Mun˜oz-Mingarro, D., N. Acero, F. Llinares, J.M. Pozuelo, A. Gala´n de and J.A. Mera

Vicenten,. Biological activity of extracts from Catalpa bignonioides Walt. (Bignoniaceae). J.

Ethonopharmacol., 2003; 87: 163-167.

28. Coelho de Souza, G., A.P.S. Haas, G.L. Von Poser, E.E.S. Schapoval and E. Elisabetsky.

Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J.

Ethnopharmacol. 2004; 90: 135-43.

29. McLaughlin JL: in Assays for Bioactivity. In Methods in Plant Biochem. 6th

edition. Edited by

Hostettmann K. San Diego, USA: Academic Press; 1991; 1–32.

30. Quignard EL, Pohlit AM, Nunomura SM, Pinto AC, Santos EV, Morais SK, et al. Screening of

plants found in Amazonas state for lethality towards brine shrimp. Acta Amazon 2003; 33: 93-

104.

31. Paz EA, Lacy RN, Bakhtiar M. The betalactum antibiotics penicillin and Cephalosporin in

Prespective Hodder Stongton, London, 1995, 227.

32. Chowdhury AA, Islam MS. Antibacterial activity of Trema orientalis. Dhaka University J.

Pharamaceutical Sci. 2004, 3(1-2): 115-117.

33. Tortora GJ, Funke BR, Case CL. Microbiology: An Introduction. Benjamin Cummings, San

Francisco. 2001, 88.

34. Evans JS, Pattison E, Moris P., Antimicrobial agents from plant cell culture, in secondary

metabolites in plant cell culture (edited by Moris PA, Scraggs A, Stafford A, Flower M)

Cambridge University, London. 1986.

35. Z.A. Zakaria, H. Zaiton, E.F.P Henie, A.M Mat Jais and E.N.H. Engku Zainuddin. In vitro

Antibacterial Activity of Averrhoa bilimbi L. Leaf and Fruits Extracts, Int. J. Trop. Med., 2007;

2(3): 96-100.

36. Joshi N, Bhatt S, Dhyani S, Nain J. Phytochemical screening of secondary metabolites of

Argemone mexicana linn. flowers. Int J Curr Pharm Res. 2013; 144-147

37. Rievere C, Van Nguyen JH, Pieters L, Dejaegher B, Heyden YV, et al. Polyphenols isolated

from antiradical extracts of Mallotus metcalfianus. Phytochem. 2009; 70: 86-94.

38. Paria S, Maity S, Mookerjee M. Phytochemial Investigation and Evaluation of Anthelmintic

activities of V. negundo leaf extract. Int J Res Pharm Biomed Sci. 2012; 3: 1143-1146.

59

Table 1: LC50 values of the methanol extracts E. rubiginosum barks

Test

samples Regression line R

2 LC50 (g/ml)

VS y = 30.8x + 60.64 0.972 0.451

ME y = 24.88x + 13.44 0.783 29.47

VS = Vincristine sulphate

ME = Methanol extract of E. rubiginosum bark

Table 2: Effect of crude methanol extract (ME) of barks of E. rubiginosum on shrimp

nauplii

Conc. (µg/ml) Log10 conc. % of mortality LC50 (µg/ml)

0 0 0

29.47

0.78 -1.1072 10

1.56 0.19382 10

3.13 0.49485 20

6.25 0.79588 20

12.5 1.09691 30

25 1.39794 40

50 1.69897 40

100 2 70

200 2.30103 80

400 2.60206 100

Table 3: Antibacterial activity of methanol extracts of Erioglossum rubiginosum barks

Test

microorganisms

Diameter of zone of inhibition (mm)

Test sample

(400μg/disc)

Cephradin

(30 μg/disc)

Gram positive bacteria

Bacillus cereus 00.00±0.00 14.01±0.03

Staphylococcus aureus 17.02±0.11 30.00±0.06

Gram negative bacteria

Salmonella typhi 7.05±0.03 10.01±0.07

Shigella dysenteriae 5.03±0.04 2.0±0.05

Values are the mean of three replicates ± standard error. P<0.05

60

Table 4: Antifungal activity of methanol extracts of Erioglossum rubiginosum barks

Test microorganisms Percentage of inhibition (

)

Test sample

(100 μl/disc)

Griseofulvin

(30 μl/disc)

Aspergillus niger 25% 100%

Candida albicans 30.5% 98.5%

Table 5: Phytochemical constituents identified in the methanol extracts of Erioglossum

rubiginosum barks

Plant constituents test / Reagent used Methanol extracts

Alkaloids

+

+

+

a) Mayer’s Test

b) Wagner’s Test

c) Hager’s Test

Phenolic compound

+

+

+

a) 5% FeCl3 Solution

b) Reaction with lead acetate

c) Drug + K3Fe (CN)6 + NH3

Saponin

+ a) Foam test

Carbohydrate

+

+

+

a) Molisch’s Test

b) Fehling’s Test

c) Benedicts’ Test

Phytosterols

_ a) Libermann’s Test

Flavonoids

+

+ a) Fluorescence Test

b) Reaction with lead acetate

61

Table 6: Physicochemical parameter of the plant of Erioglossum rubiginosum

Parameter Value (%)

Moisture content 8.07

Alcohol-soluble extractive

value

18.00

Water-soluble extractive value 21.00

Total ash 9.50

Acid-insoluble ash 1.00

Water-soluble ash 0.50

Figure 1: Plot of % mortality and predicted regression line of Vincristine sulphate

Effect of Vincristine sulphate (positive control) on

shrimp nauplii

y = 30.8x + 60.645

R2 = 0.9729

0

20

40

60

80

100

120

-2 -1 0 1 2

Log. concentration

% m

ort

ali

ty

62

Figure 2: Plot of % mortality and predicted regression line of methanol extract

Effect of Vincristine sulphate (positive control) on

shrimp nauplii

y = 24.883x + 13.449

R2 = 0.7835

-20

0

20

40

60

80

100

120

-2 -1 0 1 2 3

Log. concentration

% m

ort

ali

ty