International Journal of Drug Development and Research

Key words

Antibaterial (•) Antifungal (•) disc diffusion method (•) Indian medicinal plants.

Introduction

Consequent to frequent and indiscriminate use of antimicrobial agents, the microbes have developed resistance to many antibiotics [1]. In addition to this, antibiotics are sometimes associated with deleterious effects on the host, which include allergic reactions, adverse effects by eliminating beneficial microorganisms, which are present in the gastro intestinal tract, besides immunosuppression.

In view of these facts there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases. Medicinal plants having a wide variety of chemicals from which novel antibacterial and antifungal chemotherapeutic agents could therefore be discovered. [2-10].

Leguminosae: This is the second largest family of flowering plants and contains 600 genera and about 12000 species. It is divided into subfamily papilionaceae, the minosoideae and caealpinoideae.

Papilionaceae: Herbs, shrubs (or) trees, leaves simple (or) compound, flowers zygomorphic and papilionaccous, stamens 10, monadelphous (or) diadelphous fruit.

Rutaceae-contain 150genera & 900 species, mainly shrubs and trees distributed in both temperate and tropical countries but abundant in S. Africa & Australia, oil glands are present in the leaves & other parts. The flowers are usually cymes, with 4-5 sepals, 4-5 petals, 8-10 stamens & a superior ovary, fruits are various types. Common constituents include alkaloids, volatile oils, rhamnoglucosides, coumarins, terpenoids. Alkaloids type includes alkaloidal amines, imidazole, indole, quinidine, pyridine, pyorolidine, quinazoline and quinoline. Many of its plant fruits contain rich in citric acid and vitamin C. Meliaceae: A family of 50 genera and about 1400 spp. Trees (or) shrubs some yield timber & other seed oil. Genera include Cedrela, Swietenia, Khaya, Carapa, Melia & Azadirachta. Main constituents of the family are triterpenoids and limonoides (Trease and Evans, 1996). The present study is designed to evaluate antimicrobial activities of 12 crude extracts from the authentic samples of 6 Indian medicinal plants Crotalaria retusa, Stylosanthes fruticosa, Aegle marmelos, Todddalia asiatica, Toona ciliata and Swietenia macrophylla belonging to the above 3 families.

Experimental

Plant material

Pharmacognostically identified individual plants were collected from Kancheepuram District and Munar Hills Kerala during September 2000 and the plants were identified by Prof. P. Jeyaraman, Plant Anatomy Research Centre, Tambaram, Chennai - 45. Toona ciliata Roxb. (Meliaceae) heartwood was collected from Western Ghats of South India and authenticated by Dr. Venkatasubramanian, Institute of Forest Genetics and Tree Breading (IFGTB), Coimbatore. Voucher specimens were deposited in the Asthagiri Herbal Research Foundation (AHRF 001- 006).

Preparation of extracts

Authenticated samples of plant materials were shade dried powdered (250 g) and subjected to subsequent extraction with hexane (250 ml) and 95% ethanol (250 ml). These extracts were concentrated under vacuum by using rotary vacuum evaporator.

Test organism

The bacterial culture was maintained in nutrient agar medium (M087) (Himedia), final pH at 25 ?C is 7.3 ± 0.2. Fungal cultures were maintained on Sabouraud dextrose-agar medium (M063) (Himedia), final pH at 25 ?C is 5.6 ± 0.2. The following microorganisms were selected for the study, Gram-positive organisms: Staphylococcus aureus (ATCC 9144), Staphylococcus epidermitis (ATCC 155), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 11778), Micrococcus luteus and Gram-negative organisms: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027). Fungi: Candida albicans (ATCC 1091) and Aspergillus niger (ATCC 6275). The organisms were procured from the Institute of Microbial Technology, Sector 39A, and Chandigarh, India.

Screening of antimicrobial activity of crude plant extract

Antimicrobial test was carried out by the disc diffusion method [15] using the inoculums containing seeded nutrient agar medium. The dried plant extracts were dissolved in dimethyl formamide (DMF), 50 mg/ml. 5 μl of the plant extracts (250 μg/disc) was applied to the sterile filter paper disc (diameter, 6 mm). The discs were placed on the inoculated nutrient agar / Sabouraud dextrose medium along with solvent control and an appropriate reference antibiotic disc was applied for bacteria and fungi. For bacteria ciprofloxacin 10 μg /disc and for fungi ketoconazole 10 μg /disc served as a positive control. The filter paper disc impregnated with DMF was used as solvent control. The bacterial Petri-dishes were incubated at 37.5 ºC for 24 h and fungi Petri-dishes were incubated at 37 ºC for 24-48 h. The antimicrobial activities were measured by the zone of inhibition expressed in millimeter around the disc. All experiments were carried out in duplicates.

Results and Discussion

The antimicrobial activity of C. retusa, S. fruticosa, A. marmelos, T. asiatica, T.ciliata and S. macrophylla was recorded as the zone of inhibition in mm (Table 1). The results indicated that out of the 6 plants tested S. fruticosa, T. asiatica and T. ciliata extracts showed activity against more number of organisms as compared to the other three plants C. retusa, A. marmelos and S. macrophylla. Antifungal activity was pronounced in T. asiatica especially against C. albicans. Alcohol extract of S. fruticosa, T. ciliata, and hexane extract of T. asiatica, T. ciliata showed little activity against A. niger. The S. macrophylla extract showed no antimicrobial activity at the concentration tested.

The present study primarily focused on identifying plants for use as potential antimicrobial agents in infectious diseases. The results demonstrated that S. fruticosa, T. asiatica and T. ciliata were effective against more number of organisms when compared to other plant extracts. Difference in antimicrobial activity of the plant may be related to the presence of varied active compounds. The most active plant extracts referred above possess alkaloids, flavonoids, triterpenoids and sterols, which are classified as active antimicrobial compounds [11]. Hexane and ethanol extract of the plants differ in their activity against the tested microorganism. The antimicrobial activity against Gram- positive bacteria was more pronounced than Gram- negative, which in accordance with the results [12-13].

The extracts of all the plants were effective against P. aeruginosa but not with E. coli. Though E. coli and P. aeruginosa are Gram-negative organisms the difference in activity of the extract against these organisms are not clearly understood. Test organisms differed significantly in relation to their susceptibility to the different plant extracts. S. aureus, B. subitilis, M.luteus and S. epidermitis are the most susceptible organisms (Gram-positive) studied. The cell wall in Gram-positive bacteria is single layered, where as in Gram-negative organism it is a multi-layered, structure bounded by an outer cell membrane [14]. Hence it can be hypothesized that the single layered cell wall may be more susceptible to the antibacterial activity of the plant extract possibly through enhanced penetration of the antibacterial components of the extracts into the cell wall as compared to the organism with multilayered cell wall.

The study yielded the first report of scientific documentation of antibacterial activity of S. fruticosa and T. ciliata. The activity of T. ciliata (heart wood) may be attributed to the presence of triterpenoids cedrelone, caffeic acid, gallic acid, vanillic acid, ferulic acid, protocatechuic acid, catechin and chlorogenic acid and in the case of S. fruticosa the activity may be due to the presence of flavanoids and tannins. T.asiatica the activity may be due to the presence of flavanoids and triterpenoids. Some plants previously screened by other investigators were included in this study because different methods were used and different microorganisms were used in the assay. It is evident from the present study that the above plant extracts may be useful as antimicrobial agents against the aforementioned pathogens. Further studies include isolation of active molecule, toxicity studies, safety, and absorption pattern in the body.

Tables at a glance

Table 1

References

1. Davis J, Inactivation of antibiotics and the dissemination of resistance genes. Science 1994; 264: 375-382.
2. Anonymous, Wealth of India, Raw materials. Publication and Information Directorate, CSIR, New Delhi,1982, pp IV: 35-36.
3. Chopra RN, Chopra IC, Handa KL, Kapur LD. Chopra ?s Indigenous Drugs of India.Calcutta, Academic Publication, 1982, pp 520.
4. Iyengar MA. Study of Crude Drugs. ed 2nd.Udupi, Navayuga Press, 1985, pp 13-78.
5. Kirtikar KR, Basu BD.Indian Medicinal Plants. Dehradun: International Book Distributors, 1985, Vol. 1-5.
6. Asima Chatterjee, Satyesh Chandra Pakrashi. The Treatise on Indian Medicinal Plants.New Delhi, Publication and Information Directorate, 1995, Vol.1-5.
7. Evans WC, Trease GE. Trease and Evans Pharmacognosy. ed 14th, New Delhi, WB Saunders company Limited, 1996, pp 28-55.
8. Rashid MA, Hossain MA, Hasan C M, Reza MS. Antimicrobial diterpenes from Polyalthia longifolia var. Pendulla (Annonaceae). Phytother Res 1996; 10: 79-81.
9. Rana BK, Singh UP, Taneja V. Antifungal activity and kinetics of inhibition by essential oil isolated from leaves of Aegle marmelos. J Ethnopharmacol 1997; 57: 29-34.
10. Chatterjee TK. Herbal Options.ed 3rd, Calcutta: Books and Allied (P) Ltd. 2000; 203-256.
11. Rojas A, Hernandes L, Pereda Miranda R, Mata R. Screening for antimicrobial activity of crude drug extracts and pure natural products from Mexicanmedicinal plants.J Ethnopharmacol 1992; 35: 275- 283.
12. Gonzalez AG, Moujir L, Bazzochi IL, Correa MD, Gupta MP. Screening of antimicrobial and cytotoxic activities of Panamanian plants. Phytomedicine, 1994; 1: 149-153.
13. Grosvenor PW, Supriono A, Gray DO.Medicinal plants from Riau Province, Sumatra,Indonesia, Part 2: antibacterial and antifungal activity. J Ethnopharmacol 1995; 45: 97-111.
14. Yao J, Moellering R. Antibacterial agents In: Mannual of Clinical Microbiology. Washington DC, ASM, 1995, pp 1281-1290.
15. Murray PR. Baron EJ, Pfaller MA, Tenover FC, Yolke RH. Manual of clinical Microbiology, ed 7th, Washington, ASM, 1999, pp 1527-39.