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Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India

Background Antimicrobial activity of 18 ethnomedicinal plant extracts were evaluated against nine bacterial strains (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Ervinia sp, Proteus vulgaris) and one fungal strain (Candida albicans). The collected ethnomedicinal plants were used in folk medicine in the treatment of skin diseases, venereal diseases, respiratory problems and nervous disorders. Methods Plants were collected from Palni hills of Southern Western Ghats and the ethnobotanical data were gathered from traditional healers who inhabit the study area. The hexane and methanol extracts were obtained by cold percolation method and the antimicrobial activity was found using paper disc diffusion method. All microorganisms were obtained from Christian Medical College, Vellore, Tamil Nadu, India. Results The results indicated that out of 18 plants, 10 plants exhibited antimicrobial activity against one or more of the tested microorganisms at three different concentrations of 1.25, 2.5 and 5 mg/disc. Among the plants tested, Acalypha fruticosa, Peltophorum pterocarpum, Toddalia asiatica,Cassia auriculata, Punica granatum and Syzygium lineare were most active. The highest antifungal activity was exhibited by methanol extract of Peltophorum pterocarpum and Punica granatum against Candida albicans. Conclusion This study evaluated the antimicrobial activity of the some ethnomedicinal plants used in folkloric medicine. Compared to hexane extract, methanol extract showed significant activity against tested organisms. This study also showed that Toddalia asiatica, Syzygium lineare, Acalypha fruticosa and Peltophorum pterocarpum could be potential sources of new antimicrobial agents.


Background
According to World Health Organization (WHO) more than 80% of the world's population relies on traditional medicine for their primary healthcare needs. Use of herbal medicines in Asia represents a long history of human interactions with the environment. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases.
A vast knowledge of how to use the plants against different illnesses may be expected to have accumulated in areas where the use of plants is still of great importance [1]. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive compounds of plants are alkaloids, flavanoids, tannins and phenolic compounds [2].
Rural communities, in particular paliyar tribes, depend on plant resources mainly for herbal medicines, food, forage, construction of dwellings, making household implements, sleeping mats, and for fire and shade. The use of medicinal plants as traditional medicines is well known in rural areas of many developing countries [3,4]. Traditional healers claim that their medicine is cheaper and more effective than modern medicine. In developing countries, low-income people such as farmers, people of small isolate villages and native communities use folk medicine for the treatment of common infections [5].
We chose eighteen plant species used in folk medicine to determine their antimicrobial activity (Table 1). In general, these plants are used in folk medicine in the treatment of skin diseases, venereal diseases, respiratory problems and nervous disorders. Properties of the collected plants are also provided in same table. Evidently, there are not many scientific studies that confirm the antimicrobial properties for most of the plants collected for this study. The phytochemical research based on ethnopharmacological informations is generally considered an effective approach in the discovery of new anti-infective agents from higher plants [6].
There are a few reports on the use of plants in traditional healing by either tribal people or indigenous communities of Tamil Nadu [7][8][9][10][11]. The development of drug resistance in human pathogens against commonly used antibiotics has necessitated a search for new antimicrobial substances from other sources including plants [12]. Screening of medicinal plants for antimicrobial activities and phytochemicals is important for finding potential new compounds for therapeutic use. This paper reports the results of a survey that was done based on folk uses by traditional practitioners in Palni hills of Tamil Nadu along with bioassay test for antimicrobial activity.

Ethnobotanical survey
Plants were selected for this study based on their medicinal use. Fresh plant parts were collected from the tribal villages (Paliyar tribe) in Palni hills of Tamil Nadu, India in Jan -April 2005. The tribal villages were approximately lie between 10° 12' -10° 15' N longitudes and 77° 26' -77°3 3' latitude. The ethnobotanical data (local name, mode of preparation, medicinal uses) were collected through questionnaire, interviews and discussions among the tribal practitioners in their local language (Tamil). The voucher specimens in duplicate were deposited in the herbarium of Entomology Research Institute, Loyola College, Chennai (India).

Preparation of hexane and methanol extract
Plant extracts were prepared by cold percolation method. The plant materials were dried under shade and ground into fine powder using electric blender. 50 g of dried powder was soaked in 300 ml hexane for 48 hours with intermittent shaking. The plant extracts were filtered through Whatman No. 1 filter paper into pill vials. The filtrates were dried until a constant dry weight of each extract was obtained. The residues were stored at 4°C for further use. The remaining plant residue was dried and soaked in 300 ml of methanol as above and the extract was collected as described earlier.

Antimicrobial screening
The hexane and methanol extracts of 18 plants were screened against a total of 9 bacterial strains and one fungal strain.

Antimicrobial susceptibility test
The disc diffusion method [13] was used to screen the antimicrobial activity. In vitro antimicrobial activity was screened by using Mueller Hinton Agar (MHA) obtained from Himedia (Mumbai). The MHA plates were prepared by pouring 15 ml of molten media into sterile petriplates. The plates were allowed to solidify for 5 minutes and 0.1 % inoculum suspension was swabbed uniformly and the inoculum was allowed to dry for 5 minutes. The different

Anthelmintic and narcotic
Toddalia asiatica Pers. Solanaceae Milagaranai Leaf paste prepared in water is taken internally to treat stomachache. Powder of bark from stem is used as tooth powder and also used to treat toothache.
Aromatic-tonic, stimulant, antiperiodic, pungent, stomachic concentrations of extracts (1.25, 2.5 and 5 mg/disc) were loaded on 6 mm sterile disc. The loaded disc was placed on the surface of medium and the compound was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37°C for 24 hrs. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter. The same procedure was followed for the fungus also. These studies were performed in triplicate.  Table 2.
Methanol extracts exhibited a higher degree of antimicrobial activity as compared with hexane extracts. Methanol extracts of Albizia procera,Cassia auriculata (leaves and flowers), Peltophorum pterocarpum, Punica granatum and Syzygium cumini showed activity. Punica granatum possesed 25% of tannin [20] and the antibacterial activity may be indicative of the presence of some metabolic toxins or broad-spectrum antibiotic compounds. Also ethanolic extract of Punica granatum was most active against E. coli. Prasanth et al [21] reported that, different extracts of Punica granatum fruit showed some antibacterial activity against P.vulgaris and B.subtilis. Rajakaruna et al [22] reported that Syzygium cumini showed good activity against Staphylococcus aureus and Bacillus subtilis.
Both hexane and methanol extracts of Syzygium lineare and Toddalia asiatica showed antimicrobial activity. The essential oils from the leaves of Toddalia asiatica were most active against E. coli, K. pneumoniae, P. aeruginosa and S. aureus [23]. Peltophorum pterocarpum and Syzigium lineare had the highest inhibitory activity against both gram positive and gram negative bacteria. On the other hand, Cassia alata showed only slight activity against bacteria such as S. aureus and B. subtilis. In the previous findings leaves, flowers, root and stem barks of Cassia alata showed a range of activity against several bacteria and protozoa [24]. In this study methanol extract of leaves of Cassia alata showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Somchit et al [25] also tested the whole plant parts of Cassia alata and showed activity in the leaves against Staphylococcus aureus.
Syzygium lineare, Punica granatum, Syzygium cumini and Toddalia asiatica produced the largest zones of inhibition against Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis. Voravuthikunchai et al [20] reported good antibacterial activity in P. pterocarpum and P. granatum against Escherichia coli using aqueous and methanol extracts. Methanol extracts of Peltophorum pterocarpum and Punica granatum showed activity against Candida albicans.
In general, among the tested microbial strains, bacteria were found to be more sensitive to many of the test agents than fungi.
The most sensitive bacterium was Bacillus subtilis, which was inhibited by methanol or hexane of 10 plants. On the other hand, no inhibition was observed in the Eruvinia sps. Some organisms exhibited only slight susceptibility. E. coli was inhibited by methanol extract of flowers of Cassia auriculata and hexane extract of Punica granatum. Proteus vulgaris was inhibited by methanol extract of Peltophorum pterocarpum and Syzigium lineare. Klebsiella pneumonia was inhibited by hexane extract of Olax scandens, methanol extracts of Peltophorum pterocarpum and Syzigium cumini.

Conclusion
The processing of the plants performed in this study was not comparable to the traditional approach when the Paliyar tribe used water for extracts whereas we have used hexane and methanol for extraction. In this sense it is not an exact replication of the traditional knowledge. All the same, given that methanol extracts were more effective then hexane extract, it is likely that water extracts were will be effective as well and possibly mort so.   5  10  11  11  10  ------5  12  13  13 13 2. Syzygium cumini ( S e e d )