Introduction

The screening of plant extracts and plant products for antimicrobial activity has shown that higher plants represent a potential source of novel antibiotic prototypes. Numerous studies have identified compounds within herbal plants that are effective antibiotics. Traditional healing systems around the world that utilize herbal remedies are an important source for the discovery of new antibiotics. some traditional remedies have already produced compounds that are effective against antibiotic-resistant strains of bacteria .The results of this indicate the need for further research into traditional health systems. It also facilitates pharmacological studies leading to synthesis of a more potent drug with reduced toxicity. The need of the hour is to screen a number of medicinal plants for promising biological activity.

Ayurveda, the traditional Indian herbal medicinal system practiced for over thousands of years have reports of antidiabetic plant with no known side effects. Such plants and their products have been widely prescribed for diabetic treatment all around the world with less known mechanistic basis of their functioning. Here these natural products need to be evaluated significantly and methodically in order to check for their properties^{1, 2}.

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 plants against different illness may be expected to have accumulated in areas were the use of plants is still of great importance³. 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 are alkaloids, flavanoids, tannins and phenolic compounds⁴. 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.

Screening of medicinal plants for antimicrobial activities and phytochemicals is important for finding potential new compounds of therapeutic use.  Syzygium cumini has been introduced to many different places where it has been utilized as a fruit producer, as an ornamental and also for its timber.  Bark, leaves and fruits are used in medicine. The leaves are antibacterial and are used for strengthening the teeth and gums. The tender leaves are used for vomiting. The fruits and seeds are sweet, acrid, sour, tonic and cooling, and are used in diabetes, diarrhoea, pharyngitis, splenopathy, urethrorrhea.

Materials and Methods

Collection of plant materials and preparation of extracts

Plants were collected in and around Tirupati of Chittoor dist. of A.P. The leaves were washed and air dried at room temperature. The chloroform, ethanol and aqueous extracts were prepared sequentially in soxhlet extractor using 30 gms of dried plant tissue mixed with 150 ml of respective solvents (100% v/v) for 24 hours^{5, 6}. Chloroform and ethanol extracts were evaporated to dryness in rotary evaporator, where as aqueous extracts are lyophilized⁷. 25 mg of dry weight of each crude extract was further reconstituted in 2.5ml of distilled water and 1:15 dilutions of all these extracts were used for further studies.

 Antimicrobial screening

The chloroform and ethanolic extracts of three plants were screened against six bacterial strains. The test organisms were Bacillus subtilis (ATCC 441), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 15380), Pseudomonas aeruginosa (ATCC 27853), and Proteus vulgaris (MTCC 1771) obtained from IMTECH, Chandigarh.

Preparation of inoculum

Stock cultures were maintained at 4⁰C on slants of nutrient agar. Active cultures for experiments were prepared by transferring a loop full of culture from the stock cultures to test tubes of Mueller-Hinton broth (MHB) for bacteria that were incubated with out agitation for 24 hours for 37⁰C. The cultures were diluted with fresh Mueller-Hinton broth to achieve optical densities corresponding to 2.0 x 10⁶ Colony Forming Units (CFU/ml) for bacteria.

Antimicrobial susceptibility test

The disc diffusion method Bauer et al⁸ was used to screen the antimicrobial activity. Invitro antimicrobial activity was screened by Mueller-Hinton Agar (MHA) obtained from Himedia (Mumbai). The MHA plates were prepared by pouring 20 ml of molten media in to sterile Petri plates. The plates were allowed to solidify for 10 minutes and 0.1% inoculum suspension was swabbed uniformly and the inoculum was allowed to dry for 5 minutes. The different concentrations of extracts were loaded on 6mm sterile discs. The loaded discs were placed on the surface of the medium and the compounds were allowed to diffuse for 5 minutes and the plates were kept for incubation at 37⁰C for 24 hours. At the end of incubation, inhibition zones were formed around the discs were measured with transparent ruler in millimeters. These studies were performed in triplicate.

Results and Discussion

The results of the antibacterial properties of extracts on the microorganisms are tabulated as follows.

 Table 1. Sensitivity Pattern of Syzygium cumini on test organisms.

Table 2. Sensitivity Pattern of Azadirachta indica on test organisms

Table 3. Sensitivity Pattern  of Bougainvillea spectabilis on test organisms.

Isolation of chemical compounds from plant material is largely dependent on the type of solvent used in extraction procedure. In this assay the extracts were prepared using sterile distilled water and solvents like chloroform and Ethanol. We found in this study that none of the aqueous extracts produced any zones of inhibition. The ethanolic and chloroform extracts were found to show consistent antimicrobial activity in comparison to the aqueous extracts. This might have resulted from the lack of solubility of active constituent in aqueous solutions while the extracts showed increased solubility in solvents like chloroform and ethanol.

The tested plant extracts were most active against gram positive microorganisms than gram negative microorganisms. This is in agreement with previous reports by several workers^{9, 10, 11, 12, 13, 14}. The antimicrobial assay was carried out on gram positive bacteria i.e., Bacillus subtilis, Staphylococcus  aureus and gram negative bacteria i.e., Escherichia coli, Proteus vulgaris, Klebsiella pneumonia, Pseudomonas aeruginosa.

Bacillus subtilis and Staphylococcus aureus were found to be susceptible to ethanolic extracts of Syzygium cumini.  Klebsiella pneumonia and Proteus vulgaris were found to be more sensitive to the extracts of Azadirachta indica and Bougainvillea spectabilis respectively. It was found that methanolic extracts of Syzygium cumini, Azadirachta indica and Bougainvillea spectabilis inhibited the growth.

Prasanth et al.¹⁵ reported that, different extracts of Azadirachta indica showed some antibacterial activity against some gram positive and negative bacteria such as P.vulgaris and B.subtilis.  Rajakaruna et al.¹⁶ reported that Syzygium cumini showed good activity against Bacillus subtilis and Staphylococcus aureus. The essential oils from the leaves of Syzygium cumini were most active against Escherichia coli, Proteus vulgaris, Klebsiella pneumonia, Pseudomonas aeruginosa and Staphylococcus aureus¹⁷.

These differences in the bacterial susceptibility to different extracts may be attributed to the fact that cell wall in gram positive bacteria are of single layer whereas gram negative cell wall is multilayered. Alternatively the passage of the active compound through the gram negative cell wall may be inhibited. It is thought that observed differences may result from the doses used in this study. In addition microorganisms show variable sensitivity to chemical substances related to different resistance levels between strains. The microorganism susceptibility to different extracts did not correlate with the susceptibility or resistance to a particular antibiotic within same species. Further the results obtained by this method may vary as many factors such as microbial growth, exposure of microorganisms to different chemical substances and the quantity of the substance. The factors responsible for this high susceptibility of gram positive and negative organisms to the extracts are not exactly known but may be attributed to the presence of secondary metabolites¹⁸. It is worthy to note that the antimicrobial activities of these plants extracts were dependent on the concentration of the extracts as reported by Ekwenye et al. ¹⁹ also, if the extract has high molecular weight, the rate of diffusion is always slow, reduced and also takes longer time, whereas an extract of low molecular weight diffuses faster and at a quicker rate.

Conclusion

In conclusion the negative result does not mean the absence of bioactive compounds nor is the plant inactive. Active compounds may be present in insufficient quantities in the crude extracts to show activity with the dose levels employed. Lack of Activity can thus only be proven using higher doses. Alternatively, if the active principle is present in high enough quantities there could be other constituents exerting antagonistic effects or negating the positive effects of bioactive agents.

References

1. Patwardhan, B., Vaidya A.D.B., Chorghade, M. “Ayurveda and natural product drug discovery”. Curr. Sci., 86: 789-799 (2004).
2. Said O., Fulder S., Khalil K., Azaizeh H., Kassis E., Saad B. Maintaining a physiological blood glucose level with ‘Glucolevel’, a combination of four anti-diabetes plants used in the traditional Arab herbal medicine. Evid. Based. Complement. Med., 5: 421-428 (2007).
3. Diallo, D., Hveem, B., Mahmoud, M.A., Betge, G., Paulsen, B.S., Maiga, A. “An ethanobotinical survey of herbal drugs of Gourma district, Mail”. Pharmaceutical Biology, 37: 80-91 (1999).
4. Edeoga, H.O., Okwu, D.E., Mbaebie, B.O. “Phytochemical constitutions of some Nigerian medicinal plants”. African. J. Biotech, 4:685-688 (2005).
5. Adesegun, S.A., Fajana, A., Orabueze, C.I., Coker, H.A.B. “Evaluation of antioxidant properties of Phaulopsis fascisepala C.B.Cl (Acanthaceae)”. Evid. Based. complement Med., 6:227-231 (2009).
6. Punitha, I.S.R., Rajendra, K., Shiwaikar, A. “Alcoholic stem extract of Coscinium fenestratum regulates carbohydrates metabolism and improves antioxidant status in streptozotocin nicotinamide induced diabetic rats”. Cam, 2: 375-381 (2005).
7. Tafesse, G., Mekonnen, Y., Makonnen, E. “Antifertility effect of aqueous and ethanol extracts of the leaves and roots of Asparagus africanus in rats”. Afr. Health. Sci., 6:81-85 (2006).
8. Bauer, R.W., Kirby, M.D.K, Sherris, J.C., Turck, M. “Antibiotic susceptibility testing by standard single disc diffusion method”. Am. J. Clin. Pathol, 45:493-496 (1966).
9. Buwa, L.V., Staden, J.V. “Antimicrobial and antifungal activity of traditional medicinal plants used against venereal diseases in South Africa”. J. Ethno. pharm, 103:139-142 (2006).
10. Valsaraj, R., Pushpangadan, P., Smitt, U.W., Adsersen, A., Nyman, U. “Antimicrobial screening of selected medicinal plants from India”. J. Ethno. Pharm., 58:75-83 (1997).
11. Srinivasan, D., Nathan, S., Suresh, T., Lakshmana perumalsamy, P. “Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine”. J. Ethno. Pharm., 74: 217-220 (2001).
12. Samy, R.P., Ignacimuthu, S. “Antibacterial activity of some folklore medicinal plants used by tribals in Westeran Ghats in India”. J. Ethno .pharm., 69 :63-71 (2000).
13. Perumalsamy, R., Ignacimuthu S., Raja, D.P. “Prelimenary screening of ethanomedical plants from India”. J. Ethno. Pharm., 66: 235-240 (1999).
14. Parekh, J., Jadeja, S., Chanda, S. “Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity”. Turkish J. Biol., 29:230-210 (2005).
15. Prashanth, D., Asha, M.K., Amit, A. “Antimicrobial activity of Punica granatum”. Fitoterapia, 72:171-173 (2001).
16. Rajakaruna, N., Harris, C.S., Towers, G.H.N. “Antimicrobial activity of plants from Serpentine outcrops in Sri Lanka”. Pharm. Biol., 40:235-244. (2002).
17. Saxena, V.K., Sharma, R.N. “Antimicrobial activity of the essential oil of Toddalia asiatica”. Fitoterapia, 40:235-244 (2002).
18. Nweze, E.I., Okafor, J.I., Njoku, O. “Antimicrobial Activities of Methanolic extracts of Trema guineesis (Schumm and Thorn) and Morinda lucida Benth used in Nigerian Herbal Medicinal Practice”. J. Biol. Res. Biotech., 2: 39-46 (2004).
19. Ekwenye, U.N., Elegalam, N.N. “Antibacterial activity of ginger (Zingiber officinale Roscoe) and garlic (Allium sativum L.) extracts on Escherichia coli and Salmonella typhi”. J. Mol. Med. Advan. Sci., 1: 411-416 (2005).