Ethnobotanical Leaflets 12: 1269-82. 2008.

Antimicrobial Screening of some Selected Tribal Medicinal Plants

from Sriharikota Island, Andhra Pradesh, India

¹R. Bharath Kumar and ²B. Suryanarayana

¹Associate Professor, Dept. of Biotechnology,

Vignan's Engg. College, Vadlamudi, Guntar-522213

²Associate Professor (Retd), #8-307, Karnakamma Street,

Venkatagiri Town-524132

Issued 15 December 2008

Abstract

Sriharikota Island is important because of its rich vegetational diversity and for the fact that it is the site of a Rocket Launching Station built by the Indian Space Research Organization (ISRO). An aboriginal tribe called ‘Yanadi’ dwells in a few tribal pockets on Sriharikota Island. However, a few aged tribal men are still able to furnish information regarding their traditional practices of medicines and recipes. There is an urgent need to conserve their herbal wealth and preserve their traditional knowledge for the benefit of modern society.

It is with this background, along with the need to give a primary scientific basis to the tribal medicinal lore of Sriharikota Island, that a preliminary screening of antimicrobial (antibacterial and antifungal) activity screening was conducted for 81 tribal medicinal samples based on their medicinal importance and endemism/rare occurrence on the island.

Of these 81 samples, 28 have shown positive response for antimicrobial tests. Among these 8 species, namely Alangium salvifolium, Aristolochia indica, Ctrullus colocynthis, Datura stramonium, Ficus religiosa, Holoptelia integrifolia, Lawsonia innermis and Ventilago madaraspatana have shown congizable immunity zone of inhibition (i.e. above 20 mm). All the data is recorded in this paper. The results are mostly in conformity of the medicinal uses and they are discussed in detail in this article.

Introduction

Yanadies are an aboriginal tribe living on Sriharikota Island in Andhra Pradesh, even after the establishment of the Rocket Launching Station. They are said to have been originally from the Malaya Peninsula, Africa or Australia.

Until the establishment of the SHAR Centre at Sriharikota, Yanadies lived in forests and near the sea coasts in traditional ways and were in co-existence with the environment. In spite of the community having drifted from the natural way of life due to the introduction of agro-rural developmental activities, a few aged tribal men are still able to furnish ‘ethno-medicinal’ information pertaining to their traditional practices and healings. With the help of local tribal men, 300 ethno-medicinal plants were collected together with ample information relating to healing practices. The pharmaceutical uses of these plants are considered here under 16 categories (Table 1).

In order to give a primary scientific basis to the tribal medicinal lore of Sriharikota Island, a preliminary screening of Antimicrobial (antibacterial and antifungal) activity was conducted for 81 plant samples (such as root, root bark, stem bark, leaf, fruit, seed and whole plant etc.) chosen from the above mentioned 300 plant species. Twenty-eight samples showed positive responses (producing of an inhibition zone) for Antimicrobial screening tests.

Among the 28 samples, 8 species, namely Alangium salvifolium, Aristolochia indica, Citrullus colocynthis,Datura stramonium, Ficus religiosa, Holoptelia integrefolia, Lawsonia innermis and Ventilago madaraspatana, have shown a congizable immunity zone of inhibition (i.e. above 20 mm.). The results are discussed in this article.

Topography

Sriharikota Island is located at 80⁰ 21’ E and 13⁰ 22’ to 14⁰ N. It is a spindle shaped land mass sandwiched between the Bay of Bengal on the east and Pulicatlake on the west. It is 18 km east of Sullurpet, the nearest Railway Station connecting Chennai-Kolkata trunk line. Chennai is 98 km away from Sriharikota.

Materials and Methods

An intensive three year medico-ethnobotanical survey was conducted on Sriharikota Island from 1996-1999. Field tours at regular intervals were conducted covering all the seasons so as not to miss seasonal elements having pharmaceutical value. Plant species selected for Antimicrobial screening are used in ethnomedicine by the tribals for curing their general ailments such as infected wounds, itches, scabies, dandruff, blood motions, jaundice, fungal oral thrush and skin diseases etc. (Table 1).

Antimicrobial screening was done by the standard procedures described by Bhakuni et.al (1969,1971,1988,1990), Ikram & Inamul (1980), Aldesanmi (1988),Mbah (1993) and Ahmad et.al (1994), etc.

Preparation of Plant Extracts

The plant parts (Leaf, Rt, Rt.b, St., St.b., Wp. etc.) were washed with water, chopped into small fragments and shade dried. The dried samples were ground to powder (i.e. 20 gr. each); they were separately extracted in 200 ml methanol and acetone solvents with soxhlet apparatus. They were carefully collected into conical flasks and properly evaporated by placing them on hot water bath. Later on this, these powdered samples were carefully packed in small bottles and stored at room temperature.

Extracts of plant samples were prepared by taking 2g of dried powdered plant material in 5 ml methanol and acetone concentrations in small bottles. Filter paper discs were prepared with Whatmann No.1 filter paper discs of 6 mm diameter and they were autoclaved by keeping in a clean and dried Petri plate. The filter paper discs soaked in plant extracts were taken as test material and dried. The filter paper discs were carefully transferred to spread on cultured Petri plates. Filter paper discs were immersed in methanol and acetone also prepared and used as a control.

Media & Microorganisms

The suitable culture media was prepared by dissolving the below mentioned ingredients for the respective microorganisms. The contents were autoclaved at 15 lbs for 15 min. Microorganisms are taken as test organisms both Gram +ve, Gram-ve bacterial species and fungal sp. for antibacterial and antifungal activities of plant extracts.

Medium for bacterial sp.: Nutrient Broth/Nutrient Agar Medium (NBM/NAM) ingredients:

Peptone -- 10 gr

Beaf extract -- 10 gr

Nacl -- 5 gr

Dist. H₂O -- 1000 ml

Agar -- 15 gr

P^H -- 7

Medium for fungal sp.: Sabaroud Broth/ Sabaroud Agar Medium (SBM/SAM) ingredients:

Glucose -- 40 gr

Peptone -- 10 gr

Agar -- 10 gr

Dist. H ₂O --1000 ml

P^H -- 6

Microorganisms:

Bacteria:

Gram +ve:

Staphylococcus aureus - This major pathogenic sp. cause boils, wound infections and food

poisoning in humans.

Micrococcus sp. – Harmless saprophyte occurs in soil and fresh water and also found on

the skin of humans and animals.

Gram –ve:

Salmonella tiphymurium- Pathogenic for humans causing enteric fevers (such as Typhoid
and Paratyphoid fevers).

Escherichia coli – Occurs in the lower portion of intestine of humans and warm blooded

animals –causes gastroenteritis, urinary tract infection.

Pseudomonas aeruginosa – Pathogenic for humans and animals. Isolated from wounds,
burns and urinary tract infections.

Fungi:

Pathogenic fungi:

Candida albicans – Pathogenic, causing cadidiasis -a disease of the mucus, vagina and
alimentary tract infections.

The above mentioned microbial cultures were maintained on their respective media in slants at 4⁰C. (as per standard procedures).

Preparation of Test Plates & Antimicrobial Testing:

The agar test plates (Petri dishes) were prepared by pouring about 15 ml of the medium. These test plates were placed under aseptic conditions at 4⁰C for 24 hours to control sterility. After solidifying the media (NAM and SAB), the inoculums (bacteria 24 hrs and fungi 48 hrs.) homogenous suspension was uniformly spread on their respective test plates. The filter paper discs were prepared in methanol (M) and acetone (A) extracts as taken for control.

The filter paper discs were carefully placed the spreaded culture test plates and incubated at appropriate temperature for bacteria at 37⁰C for 24 hrs. and fungi 30⁰C for 48 hrs. After the incubation period, the test plates were examined for inhibitory zones. The diameters of inhibitory zones were recorded (Table 2). All determinants were made at least in triplicate for each of the test organisms in different extracts, and recorded.

Results & Discussion:

Sixteen field tours of 5-7 days duration for each tour were conducted for three years during 1996-1999, with the assistance of local tribal men. Three hundred species having medicinal uses plus ample field notes were collected, including flowering, fruiting season, flower colour and other phenological data.

A total of 81 methanol and acetone soxhlet extracts from 77 plant species popularly used by the tribals as herbal remedies were screened in, in-vitro studies to evaluate their antibacterial and antifungal activity. Twenty eight samples exhibited positive activity (Table 2). Of these 28 samples, 8 species exhibited a cognizable inhibitory zone (> 20 mm.), i.e. 10 % of the total test samples (81 plant extracts), Table 4.

The inhibitory activity of methanol extracts is comparatively more than those of acetone extracts. The methanol and acetone extracts had high inhibitory activity in Gram +ve bacteria and comparatively less in both Gram –ve bacteria and in the pathogenic fungus, Candida albicans.

Popular medicinal plants useful to tribals selected based on therapeutic values and screened in vitro for antimicrobial activity of acetone and methanol plant extracts was observed on five strains of two gram-positive, three gram-negative bacteria and one pathogenic fungi. The plant species were selected based on their tribal medicinal importance, and corresponding pharmaceutical categories are given in Table 1.

Table 1. Classification of Selected Tribal Medicinal Plants of Antimicrobial Analysis.

CATEGORY PHARMACEUTICAL CATEGORY No.of Sps.
No.
1. Respiratory and bronchial problems 4
2. Cordiac 1
3. Urinary and kidney ¾
4. Piles and constipation ¾
5. Arthritis and rheumatism 22
6. Anodyne elements 1
7. Sexual problems 3
8. Skin and dermatitis 9
9. Opthalmic and E.N.T 7
10. Pscychoactive and nervous disorders 1
11. Jaundice and liver disorders 1
12. Digestive and stomachic 4
13. Cooling and allterative 1
14. Antipyretic, anthelmintic and

antiperiodic 2
15. Antidotes, antivenom ( scorpion sting

and Snake bite) 2
16. Diabetes 3

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Total = 61
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The samples that were selected based on their therapeutic uses, fall more or less under all 16 pharmaceutical categories. Out of these 28 samples which have shown positive samples selected based on their therapeutic uses, among these 16 pharmaceuical categories, antimicrobial results were exhibited in the form of immunity zone, fall under 11 pharmaceutical categories only. Further 7 samples of antifungal 22 samples of antibacterial in the 3 samples have exhibited both antifungal and antibacterial inhibition.

Further immunity response for PC - 5, was observed in 7 species and for PC - 8 is observed in 8 species, and immunity response against other PC. categories are only seen 1 or 2 species. (Table 3).

Table-3. Summary Of Antimicrobial Screening.

--------------------------------------------------------------

S.No. Plant extract sample plant Microbial test elements.

PC.No. part (G+ve) (G+ve) F.

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

--------------------------------------------------------------

Respiratory and

bronchial : PC - 1.

1. Centella asiatica Lf. + - -

2. Datura stramonium Sd. + - -

3. Hybanthus enneaspermus Lf. + - -

4. Sarcostemma acidum St. - - +

Arthritis & rheumatism:

PC - 5.

5. Alangium salivifolium Lf. - - +

6. Caesalpinia bonduc Sd. - + -

7. Citrullus colocynthis Lf. - - +

8. Ficus religiosa St.b + - -

9. Garcinia spicata Rt.b + - -

10. Holoptelia integrifolia St.b + - +

11. Toddalia asiatica Rt. + - -

Anodyne elements :

PC - 6.

12. Ficus religiosa St.b. + - -

--------------------------------------------------------------

---------------------------------------------------------------

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

---------------------------------------------------------------

Skin and dermatitis :

PC - 8.:

13. Crataeva magna Lf. + - -

14. Cissus vitiginea Rt. + - -

15. Datura stranonium Lf. + - -

16. Eugenia bracteata Lf. + - -

17. Ficus religiosa Lf. - - +

18. Gmelina asiatica Fr. + - -

19. Lawsonia inermis Lf. + - -

20. Ventilago madraspatana Rt.b + - -

Opthalmic and ENT :

PC - 9.:

21. Eugenia bracteata Rt. + - -

22. Lawsonia inermis Lf. + - -

23. Sarcostemma acidum St. - - +

Pscychoactive and

nervous disorders :

PC - 10:

24. Centella asiatica Lf. + - -

25. Datura stranonium Sd. + - -

Jaundice and Liver

disorders: PC – 11.

26. Phyllanthus amarus W.pl. + - +

---------------------------------------------------------------

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

---------------------------------------------------------------

Digestive and stomachic :

PC - 12:

27. Aristolochia indica Rt. + - -

28. Mangifera indica St. + - -

29. Mimusops elengi St.b. + - -

Cooling and alterative :

PC - 13:

30. Colubrina asiatica Lf. - + +

Antidotes and antivenom:

PC – 15 :

31. Aristolochia indica Rt. - + +

32. Caesalpinia bonduc Sd. - + -

Diabetes : PC – 16.

33. Casearia esculenta Rt. - + -

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Eighty-one samples when subjected to inhibition activity test 28 samples, are positive for inhibition; rest of the 54 samples are passive. Details are as follows:
Immunity response of species against microbial activity as per pharmaceutical categories is as follows:

PC 8 : 8 sps ; PC 5 : 7 sps ; PC 1 : 4 sps ; PC 9 & 12 : 3 sps ;

PC 10 & 15 : 2 sps ; PC 6, 11, 13 & 16 : 1 sps.

Upon critical study and to summarize the antimicrobial activity test, it is noticed that out of 28 samples with positive response the congizable inhibition could be observed i.e. above 20 mm in 8 species only 6 species have exhibited immunity zone against G+ve bacteria. Expression for inhibition is either for both test elements vig. bacteria and fungus or one element ie. bacteria or fungus the solvent extracts in A/M (Acetone/ Methanol ) [ Table 4].

Table 4. Above 20 mm Inhibition.

-----------------------------------------------------------
S.No. Plant name part G- G+ F.

used

(1) (2) (3) (4) (5) (6)
-----------------------------------------------------------
1. Alangium salvifolium Lf. - - 15

mm/M
2. Aristolochia indica Rt. - 22 50 -
mm/A mm/M
3. Citrullus colocynthis Lf. - - 20

mm/M

4. Datura stramonium Lf. - 22 -

mm/M

5. Ficus religiosa St.b - 20 -

mm/M

6. Holoptelia integrifolia St.b - 10 25

mm/M mm/M

7. Lawsonia innermis Lf. - 25 22 -

mm/A mm/M

8. Ventilago madraspatana Rt.b - 20 -

mm/M

-------------------------------------------------------------------------

A: Acetone pl. extract.

M: Methanol pl. extract.

A/M: Acetone and Methanol pl. extract.

In all, eight species only, out of 81 plant extract samples, exhibited a congizable inhibitory zone ( 20 mm ) i.e. 10% of the total test samples.

Antifungal inhibition could be noticed in three species Alangium salvifolium is positive in both A/M, solvent extracts but distinct exhibition is at 15 mm diam. in methanol Presumably it must be antifungal and it may be preferred for further studies for conformation.

Antibacterial and antifungal inhibitory expression is observed in Holoptelia integrifolia St.b. extracts of both in M/A solvents respectively.

Although antibacterial expression showing 10 mm diam. inhibition only. Its antifungal expression is 25 mm diam. in methanol solvent similar to Citrullus colosynthis.

Regarding antibacterial inhibition, 6 species are positive with cognizable expression. Thus only 10% of the total samples are positive for antimicrobial immunity and correlating with the tribal medicinal uses specific for dermal (PC. 8), Arithritis and rheumatism, (PC. 5) and digestive disorders (PC. 12), etc.

Acknowledgements

The authors are thankful to the Principal, Visvodaya Government College, Venkatagiri, for providing facilities and encouragement to the Director SHAR Centre, Sriharikota, for providing necessary facilities to carry field work and to the Head, C&LD Division, SHAR Centre, Sriharikota, for help during fieldwork. The authors are also thankful to ISRO Bangalore, for extending financial assistance.

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