Ethnobotanical Leaflets 12: 337-342. 2008.
Preliminary Phytochemical Screening and Antibacterial Studies on the Ethanolic Leaf Extract of Nyctanthes arbortristis Linn.
Sathiya, M., Parimala, P., and Muthuchelian, K.
Department of Bioenergy, School of Energy, Environment and Natural Resources,Madurai Kamaraj University, Madurai - 625 021 email@example.com
Nyctanthes arbor-tristis Linn. is extensively used in Indian traditional and folklore medicines to cure various human ailments. The preliminary phytochemical screening of the leaves revealed the presence of alkaloids, tannins, flavonoids, cardiac glycosides and sugars. In vitro antibacterial studies on the ethanolic leaf extracts were carried out on ten medically important bacterial strains, including Salmonella typimurium, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli, Psudomonas sp. Staphylococcus epidermis, Micrococcus luteus, Staphylococcus aureus, Streptococccus sp. and Bacillus subtilis, which were procured from the Microbial Type Cultrue and Collection, Chandigarh, India, using agar disc diffusion method. The bacterial strains were exposed to the following four different concentrations of extracts: 50mg/ml, 100mg/ml, 200mg/ml and 300mg/ml solvent. The results of our antibacterial assay revealed that the extract showed good inhibitory activity against all the tested pathogens compared with standard antibiotics like streptomycin and penicillin. The inhibitory activity was found to be dose dependent.
Since antiquity, man has used plants to treat common infectious diseases, and some of the traditional medicines are still included as part of the habitual treatment of various maladies (Heinrich et al., 2004; Rios et al., 2005). Scientific interest in medicinal plant has burgeoned in recent times due to increased efficiency of new plant derived drugs and rising concerns about the side effects of modern medicine. The continuing emergence of drug resistant organisms and the increasing evolutionary adaptations by pathogenic organisms to commonly used antimicrobials have reduced the efficacy of antimicrobial agents currently in use. Therefore, the search for new drugs from novel sources such as plants continues to be necessary (Fransworth and Morris, 1976). To date, plants continue to be a major source of commercially consumed drugs. Even most synthetic drugs have their origin from natural plant products (Sofowara, 1982).
The leaves of Nyctanthes arbortristis are extensively used in tribal and Ayrvedic medicine for the treatment of various diseases such as sciatica, chronic fever, rheumatism, internal worm infections, asthma, inflammations, dyspepsia, dermatopathy, bronchitis, cough, constipation, hepatopathy, grayness of hair and baldness (Kuvaev and Blinova, 1960; Saxena et al., 1987). This study aimed at investigating the phytochemical and antibacterial properties of the ethanolic leaf extract from this ethnic Indian medicinal plant against ten bacterial isolates in order to validate or otherwise prove the claims of the herbalists who use it as an antimicrobial remedy. This study will also hopefully expose new frontiers by improving on the current applications of the plant extract. It has been suggested that aqueous and ethanolic extracts from plants used in allopathic medicine are potential sources of antiviral, antitumoural and antimicrobial agents (Chung et al. 1995; Vlietinck et al. 1995). The selection of crude plant extracts for screening programs has the potential of being more successful in initial steps than the screening of pure compounds isolated from natural products (Kusumoto et al. 1995).
Collection of plant materials
Mature leaves were collected from the healthy plant in
early morning at the Centre for Biodiversity and Forest Studies,
Preparation of plant extract
The leaves were washed in tap water, shade dried for 10 days and made into a fine powder of 40 mesh size using the laboratory mill. Following that, 100g of the powder was filled in the thimble and extracted using 500 ml of distilled ethanol in soxhlet apparatus for 8 – 10 hours. The extract was filtered through Whatman no.1 filter paper to remove all unextractable matter, including cellular materials and other constitutions that are insoluble in the extraction solvent. The entire extract was concentrated to dryness using rotary flash evaporator under reduced pressure. The dried extract was redissolved in ethanol to yield solutions containing 50, 100, 200 and 300mg of leaf extract per ml solvent.
The extract was tested on the following five Gram positive bacteria: Staphylococcus epidermis, Micrococcus luteus,
sp. and Bacillus subtilis. Five Gram negative bacteria
were also tested, including Salmonella typimurium, Pseudomonas
aeruginosa, Klebsiella pneumonia, Escherichia
coli, Psudomonas sp.
All the strains were procured from the Microbial Type Culture and collection,
Phytochemcial analysis of the extract was conducted following the procedure of Indian Pharmacopeia (1985). By this analysis, the presence of several phytochemicals like alkaloids, flavonoids, tannins, saponins, esters, resins, sugars, glycosides and acids were tested.
Anti bacterial assay
The four different concentrations of the leaf extracts were tested for antibacterial activity using agar disc diffusion assay according to the method of Bauer et al., 1966. The strains of microorganisms obtained were inoculated in conical flask containing 100 ml of nutrient broth. These conical flasks were incubated at 370 C for 24 h and were referred to as seeded broth. Media were prepared using Muller Hinton Agar (Himedia), poured on Petri dishes and inoculated with the test organisms from the seeded broth using cotton swabs. Sterile discs of six millimeter width had been impregnated with 20 µl of test extract and introduced onto the upper layer of the seeded agar plate. The plates were incubated overnight at 370 C. Antibacterial activity was assigned by measuring the inhibition zone formed around the discs. The experiment was done three times and the mean values were presented. Streptomycin (10µg/disc) and penicillin (10µg/disc) were used as standards.
The preliminary phytochemical analysis of the leaf extract revealed the presence of alkaloids, tannins, glycosides, esters, sugars acids and resins as presented in Table 1. The results obtained from the disc diffusion assay showed that there has been an increasing effect on bacterial growth inhibition with increasing concentration of the extract. And the extract showed good inhibitory activity on almost all the bacteria tested. It has been found that among all the tested organisms, the Gram negative bacterial strain, Pseudomonas aeroginosa, was found to be more susceptible to the plant extract by showing inhibition zone ranging from 7.2 – 17.1 mm and the gram positive strain Staphylococcus epidermis was least susceptible with the inhibition zone ranging from 7.1 – 11. 95 mm. The antimicrobial activity in terms of zone of inhibition was presented in Table 2. The observed activity may be due to the presence of potent phytoconstituents in the leaf extracts.
Table 1. The phytochemical profile of the leaf extract.
Table 2. Antibacterial activity of ethanolic leaf extract of Nyctanthes arbortristis.
*All the values are mean ± standard deviation of three determinations.
DISCUSSION & CONCLUSION
Antibiotics provide the main basis for the therapy of bacterial infections. However, the high genetic variability of bacteria enables them to rapidly evade the action of antibiotics by developing antibiotic resistance. Thus, there has been a continuing search for new and more potent antibiotics (Heisig, 2001). According to World Health Report of Infectious diseases 2000, overcoming antibiotic resistance is the major issue of the WHO for the next millennium. Hence the last decade witnessed an increase in the investigation of plants as a source of human disease management (Prashanth et al. 2001). Nyctanthes arbortristis showed maximum antibacterial activity and so this plant can be used to discover bioactive natural products that may serve as leads for the development of new pharmaceuticals that address hither unmet therapeutic needs. Such screening of various natural organic compounds and identifying active agents is the need of the hour, because successful prediction of lead molecule and drug like properties at the onset of drug discovery will pay off later in drug development.
The authors are thankful to the University Grant Commission under the project University with Potential for Excellence for providing funding facilities.
Anon. (1996). Pharmacopiea of
Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M. (1966). Antibiotic
susceptibility testing by a standardized single disk method. American journal of clinical pathology. 45: 493-496.
Chung, T.H., Kim, J.C. and Kim, M.K. (1995) Investigation of Korean plant
extracts for potential phytotherapeutic agents against Hepatitis B- Virus. Phytotherapy research. 9: 429-434.
Fransworth, N.R., Morris, R.N. (1976). Higher plants: The sleeping giants of drug development. American journal of Pharmaocology. 17(2): 46-56.
Heinrich, M., Barnes, J., Gibbons, S., Williamson, E.M. (2004). Fundamental of pharmacognosy and phytotherapy. Curchill Livingstone, Edinburgh.
Heisig, P. (2001). Planta medica. 67: 4-12.
Kusumoto, I.T., Nakabayashi, T. and Kida, H. (1995). Screening of various plant extracts used in Ayurvedic medicine for inhibitory effects on human immunodeficiency virus type I (HIV-1) protease. Phytotherapy Research. 9: 180-184.
Kuvaev, V.B., Blinnova,
K.F. (1960). Preliminary chemical evolution of medicinal plants used in
Prashanth, D., Asha, M. K. and Amit, A. (2001). Fitoterapia. 72: 171-173.
Rios, J.l., Recio, M.C. (2005). Medicinal plants and antimicrobial activity. Journal of Ethnopharmacology. 100: 80-84.
Saxena, R.S., Gupta, B., Saxena, K.K., Singh, R. And Prasad, D.N., Study of anti inflammatory activity of the leaves of Nyctanthes arbortristis Linn. – An Indian medicinal plant. Journal of Ethnopharmacology. 11: 319-30, 1987.
Sofowara, E.A. (1982). Medicinal plants and
traditional Medicines in
Vlietinck, A.J., Van Hoof, L. and Totte, J. (1995). Screening of hundred Rawandese medicinal plants for antimicrobial and antiviral properties. Journal of Ethnopharmocology. 46: 31-47, 1995.
Warrier, P.K. (1995). Indian Medicinal
Plants, Vol. IV, (Orient Longman Pvt. Ltd.,