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].

Table 1 Uses and properties of ethnomedicinal plants collected for antimicrobial screening
Full size table

There are a few reports on the use of plants in traditional healing by either tribal people or indigenous communities of Tamil Nadu [7–11]. 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.

Methods

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° 33' 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. The test organisms were Bacillus subtilis (ATCC 441), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (MTCC 3615), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 15380), Pseudomonas aeruginosa (ATCC 27853), Ervinia sp (MTCC 2760), Proteus vulgaris (MTCC 1771) and one fungal strain Candida albicans (MTCC 227) obtained from the Christian Medical College, Vellore, Tamil Nadu.

Preparation of inoculum

Stock cultures were maintained at 4°C on slopes of nutrient agar. Active cultures for experiments were prepared by transferring a loopful of cells from the stock cultures to test tubes of Mueller-Hinton broth (MHB) for bacteria and Sabouraud dextrose broth (SDB) for fungi that were incubated without agitation for 24 hrs at 37°C and 25°C respectively. The cultures were diluted with fresh Mueller-Hinton and Sabouraud dextrose broth to achieve optical densities corresponding to 2.0·106 colony forming units (CFU/ml) for bacteria and 2.0·105 spore/ml for fungal strains.

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 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.

Results and discussions

Table 1 provides the botanical name, family, local name, plant parts used together with their traditional therapeutic uses and properties for the 18 ethnomedicinal plants collected from Palni hills of Tamil Nadu. Out of the 18 plants tested for antimicrobial activity, 10 plant species showed antimicrobial activity by inhibiting one or more microorganisms. The results of the antimicrobial screening of the crude extracts of all species of plants are shown in Table 2. Among the plants screened, Acalypha fruticosa, Peltophorum pterocarpum, Toddalia asiatica,Cassia auriculata, Syzygium cumini and Syzygium lineare showed promising activity against tested microorganisms. The tested plant extracts were most active against gram-positive microorganisms than gram-negative microorganisms. This is in agreement with previous reports by the several workers [14–19].

Table 2 Antimicrobial activity of the hexane and methanol extracts of collected ethnomedicinal plants
Full size table

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.

The antibacterial activity of Syzigium lineare, Olax scandens, Albizia procera and Acalypha fruticosa are reported for the first time. No previous report on the antibacterial activity of these species could be found in the literature. Among the medicinal plants tested in this work, Peltophorum pterocarpum and Punica granatum showed the most promising antimicrobial properties indicating the potential for discovery of antibacterial principles. Further phytochemical studies are required to determine the types of compounds responsible for the antibacterial effects of these species. The results also indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness might warrant fruitful results. Several plants used by Paliyar tribe exhibit some degree of antibacterial activity towards gram-positive bacteria such as, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis. These plants could serve as useful sources for new antimicrobial agents.