Abstract
The current study was conducted to investigate antibacterial activity of Alseodaphne andersonii. (King ex Hook. f.) Kosterm. (Lauraceae) leaf extracts against 15 strains of pathogenic bacteria. Inhibition of bacterial growth was investigated using disk diffusion methods. Methanol leaf extract of Alseodaphne andersonii. was active against all assayed bacteria. The minimal inhibitory concentration (MIC) values and minimal bactericidal concentration (MBC) values were also determined. Results indicated that Alseodaphne andersonii. extracts must possess major antibacterial components against infectious microorganisms. The MIC and MBC of methanol extract of leaves against Staphylococcus aureus. were found to be 0.414 and 6.62 mg/mL, and for Staphylococcus epidermidis. 0.207 and 3.31 mg/mL, respectively. Alseodaphne andersonii. might provide promising therapeutic agents against infections with bacterial organisms. This is the first report about the antimicrobial activity of Alseodaphne andersonii.. The plant is reported to be used as timber only.
Introduction
Even though the pharmaceutical industry has produced a number of new antibiotics in the past three decades, resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs that are used as therapeutic agents (Cohen, Citation1992). Such a fact is cause for concern, because of the number of patients in hospitals who have suppressed immunity, and because of new bacterial strains that are multiresistant, the problem of microbial resistance is growing and the outlook for the use of antimicrobial drugs in the future is still uncertain. Therefore, actions must be taken to reduce this problem and to continue studies to develop new drugs, either synthetic or natural. For a long period of time, plants have been a valuable source of natural products for maintaining human health, especially in the past decade, with more intensive studies for natural therapies. According to the World Health Organization, medicinal plants would be the best source to obtain a variety of drugs (Santos et al., Citation1995). About 80% of individuals from developed countries use traditional medicine, which have compounds derived from medicinal plants. Therefore, such plants were selected to better understand their properties, safety, and efficacy (Ellof, Citation1998). The use of plant extracts with known antimicrobial properties can be of great significance in therapeutic treatments. In view of this, Alseodaphne andersonii. (King ex Hook. f.) Kosterm. (Lauraceae), a plant growing in the Himalayan region, has been selected for the current study. There is very little reported about this plant. The plant is reported to be used as timber in Yuan-Nan (Anon., Citation1983). Only five gamma-lactones were isolated from the bark and roots of A. andersonii. (Lee et al., Citation2001). However, there were no reports about the antimicrobial activity of this plant. Therefore, an attempt has been made to investigate antibacterial activity of A. andersonii. leaf extracts against 15 strains of pathogenic bacteria.
Materials and Methods
Microbial cultures
The bacterial cultures were collected from the Department of Microbiology, SBSPGI, Balawala, Dehradun, India. The bacterial cultures were Staphylococcus aureus., Staphylococcus epidermidis., Shigella dysenteriae., Escherichia coli., Salmonella typhi., Klebsiella pneumoniae., Streptococcus pyogenes., Bacillus cereus., Mycobacterium tuberculosis., Enterococcus faecalis., Pseudomonas aeruginosa., Morexlla (Branhamella) catarrhalis., Pasturella multocida. subsp. multocida., Listeria monocytogenes., and Vibrio parahaemolyticus.. All the organisms were maintained on specified media slants at 4°C and revived prior to use.
Culture media and antibiotics
Lowstein-Jensen medium, corn meal agar, brain heart infusion and Mueller-Hinton agar procured from Hi-Media (Mumbai, India) were used. Gentamicin antibiotic disks (Hi-Media) were used as a standard.
Collection of plant material
The leaves of A. andersonii. (King ex Hook. f.) Kosterm (Lauraceae) were collected from the Forest Research Institute (F.R.I.), Dehradun, and authenticated by Dr. H.C. Pandey, Botanical Survey of India, Forest Research Institute. A voucher specimen (A-29) was deposited at the Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, S. Bhagwan Singh Post Graduate Institute of Biomedical Sciences & Research, Balawala, Dehradun, India.
Preparation of the extracts
Fresh plant leaves were shade-dried at room temperature, ground into a fine powder, and extracted with the solvents, viz., petroleum ether, chloroform, acetone, and methanol, by a Soxhlet apparatus. The solvents were removed under reduced pressure using a rotary evaporator to constant weight. The yields obtained for the petroleum ether, chloroform, acetone, and methanol extract were 6.25%, 3.0%, 3.5%, and 4.0% (w/w), respectively. The extracts were stored in clean glass bottles for 1 week at 4°C further used for antibacterial activity.
Qualitative analysis
All the extracts of the leaf of A. andersonii. were screened for their phytoconstituents. Extracts were evaporated to dryness in vacuo. at about 45°C further dried to a constant weight at the same temperature in a hot-air oven. A portion of the residue was used to test for qualitative analysis by the methods followed by Sofowora (Citation1993) and Wall et al. (Citation1952).
Screening for the antibacterial potential of the plant extracts
The disk diffusion method was used to determine in vitro. antibacterial activity of the extracts (Bauer et al., Citation1996). The cultures were subcultured in agar medium and incubated at 37°C 24–120 h, and from this the spore suspension was prepared containing 105 cell/mL. A suspension of 100 µL was seeded on agar medium. A sterile empty disk (Hi-Media) previously soaked in a known concentration of extract (100 mg/mL) was carefully placed on the labeled seeded plate (Olukoya et al., Citation1986). These disks were placed on the agar plates against the control (solvent) and standard drug (gentamicin). Plates were incubated at 37°C 24–120 h and observed for the zone of inhibition. Disk diameter (6 mm) was deducted while recording the zone size. The tests were conducted in triplicate.
Minimum inhibitory concentration
Minimum inhibitory concentration (MIC) was evaluated by tube dilution method against Staphylococcus aureus. and Staphylococcus epidermidis. only. The methanol extract MIC was determined by dilution of the extract to various concentrations (0.097–50 mg/mL) and compared with the standard (gentamicin). All the tubes were incubated at suitable temperature for 24–120 h. The tubes were observed for the appearance of any growth. The MIC was interpreted as the lowest concentration of the extract that did not permit any visible growth when compared with control tubes.
Minimum bactericidal concentration
Minimum bactericidal concentration (MBC) was determined by subculturing method (Alade & Irobi, Citation1993). Subcultures made from samples obtained from those test tubes that showed no visible turbidity or growth in MIC assays were made on freshly prepared nutrient agar plates. After 24 h incubation, the MBC was regarded as the lowest concentration of the extract that did not permit any growth on the agar plate surface used.
Results
The leaf extracts (petroleum ether, chloroform, acetone, and methanol) were subjected to qualitative analysis for the presence of various phytoconstituents. Acetone and methanol extracts showed the presence of alkaloids, glycosides, proteins, and amino acids, while petroleum ether and chloroform extracts showed the presence of steroids. The methanol extract revealed prominent effects on Staphylococcus aureus. (35 mm), Staphylococcus epidermidis. (37 mm), Salmonella typhi. (28 mm), Escherichia coli. (23 mm), and Bacillus cereus. (23 mm), respectively, in terms of zones of inhibition, while the acetone extract exhibited moderate effect and the other two extracts showed negligible effects (). The zone of inhibition being prominent in methanol extract, therefore the MIC and MBC studies of extract were carried out. The MIC and MBC of methanol extract of leaf against Staphylococcus aureus. was found to be 0.414 and 6.62 mg/mL, and for Staphylococcus epidermidis., 0.207 and 3.31 mg/mL, respectively (). This is the first report of antimicrobial studies for this plant.
Table 1.. Antibacterial activity of different Alseodaphne andersonii. leaf extracts.
Table 2.. Determination of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of methanol extract of A. andersonii. leaf.
Discussion
Medicinal plants constitute an effective source of both traditional and modern medicines. Herbal medicine has been shown to have genuine utility and about 80% of rural population depend on it for primary health care. Over the years, the World Health Organization (Citation1978) advocated that countries should interact with traditional medicine with a view to identifying and exploiting aspects that provide safe and effective remedies for ailments of both microbial and nonmicrobial origins. Our results revealed that the leaf extracts of Alseodaphne andersonii. showed remarkable antibacterial activity against a number of pathogenic bacteria responsible for severe infections. Further research is required for purification of the active principles and elucidation of their nature and structures. We can conclude that plant extracts have great potential as antimicrobial compounds against microorganisms. Thus, they can be used in the treatment of infectious diseases caused by microbes.
Acknowledgments
The authors are immensely thankful to Management, S.B.S. (P.G.) Institute of Biomedical Sciences and Research, Balawala, Dehradun (U.A.), for providing the requisite facilities and financial support, and offer special thanks to Dr. Ram Lal Thakur, Head, Department of Microbiology, S.B.S. (P.G.) Institute of Biomedical Sciences and Research, Balawala, Dehradun (U.A.), for valuable guidance.
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