In the present research work in vitro antibacterial activity of methanolic extract of aerial parts of Thymus serphyllum L. growing wild in Kashmir Himalaya was evaluated by agar well diffusion method and broth dilution assay against nine human pathogenic bacterial strains, known to cause serious infections. The extract was also screened for the presence of various bioactive phytoconstituents present in the plant. The extract in the present study possess appreciable potential of inhibiting the growth of all the bacterial strains at all tested concentrations (30, 60 and 90 mg/ml). The highest sensitivity was exhibited against Staphylococcus epidermidis MTCC- 435 and Staphylococcus aureus with mean zones of inhibition 20.66 and 20 mm respectively at the concentration of 90 mg/ml. Salmonella typhi showed the least activity with mean zone of inhibition of 10.00 mm at the concentration of 30 mg/ml. The MIC value ranged between 1.56 to 12.56 mg/ml. The phytochemical analysis of the crude extract revealed the presence of alkaloids, flavonoids, phenolics, saponins, tannins, cardiac glycosides, terpenes, steroids and carbohydrates. Anthraquinone glycosides were absent. The present study clearly indicate that the crude methanolic extract of Thymus serphyllum from high altitude of Kashmir Himalaya (2350 m) shows significant antibacterial activity in concentration dependent manner.
Thymus serphyllum was collected at flowering stage from Tangmarag area of Kashmir Himalaya at an altitude of 2350 m (a.s.l) by conducting field trips.
Preparation of extract:The aerial parts of the plant were properly cleaned and dried under shade for one week. After drying, the material was chopped and then grinded to powder. Dried plant powder was then packed in Soxhlet apparatus and extracted with methanol at 50-65 oC. The extract was then filtered through Whatmann filter paper No. 1. The pellet was discarded and the supernatant was collected and concentrated under reduced pressure at 35-45°C using Buchirotavapor (R-215). The extract was then dried, labelled and stored at 4oC in storage vials for experimental use16.
Antibacterial Activity:Microbial cultures of nine different species of both Gram positive and Gram negative bacteria were used for determination of antibacterial activity. Four bacterial strains viz. Proteus vulgaris MTCC- 321, Staphylococcus epidermidis MTCC- 435, Pseudomonas aeruginosa MTCC- 1688 and Bacillus subtilus MTCC-441 were standard laboratory isolates obtained from Microbial Type Culture Colletion, Chandigarh (India). The rest five bacterial strains were clinical isolates obtained from Department of Microbiology, Sheri Kashmir Institute of Medical Sciences- Srinagar (India). All the bacterial strains were sub-cultured at 37°C on Mueller- Hinton agar (Himedia) slants every fifteen days and stored at 4°C.
Antibacterial activity assay:In the present research work, the antibacterial activity of methanolic extract of Thymus serphylum was determined by agar well diffusion method as adopted by Perez et al17. Each microorganisms were grown overnight at 37°C in Mueller-Hinton Broth. Ten microlitres (10μL) of standardized inoculum (0.5 Mac- Farland) of each test bacterium was inoculated on molten Mueller-Hinton agar, homogenized and poured into sterile Petri dishes. The Petri dishes were allowed to solidify inside the laminar hood. A standard cork borer of 5mm in diameter was used to make uniform wells into which was added 30μl essential oil diluted in DMSO. Standard antibiotic kanamycin (30μg/disc) was used as positive control and DMSO as negative control. The plates were then incubated at 37 ± 1°C for 24h. The zone of inhibition was measured to the nearest size in mm with the help of standard scale18. The experiments were carried in strict aseptic conditions so as to achieve consistency. The experiments were carried out in triplicates and results were calculated as mean ± SD.
The methanolic extract of Thymus serphylum exhibited varying degree of antibacterial activity against the tested bacterial strains. The bacterial strains used were clinical and laboratory isolates. All these bacterial species are known to cause serious human infections. From clinical point of view, Klebsiella pneumonia causes neonatal nosocomial infection22. Escherichia coli cause’s septicemias and can infect the gall bladder, meninges, surgical wounds, skin lesions and the lungs23. Salmonella typhi causes serious public health problem in developing countries and represents a constant concern for the food industry24. Shigelladyssenteriae cause shigellosis. Staphylococcus aureus causes dermatitis and sialadenitis. Proteus vulgaris causes bacteremia, sepsis and urinary tract infections25-26. The most antibacterial sensitivity was shown by Staphylococcus epidermidis MTCC- 435 with mean zone of inhibition of 20.66 mm at the concentration of 90 mg/ml, while as Salmonella typhi showed the least activity with mean zone of inhibition of 10.00 mm at the concentration of 30 mg/ml. The minimum inhibitory concentration of methanolic extract of Thymus serphylum ranged between 1.56 to 12.56 mg/ml. The extract in the present study exhibited broad spectrum antibacterial activity which was comparable to the standard antibiotic drug (kanamycin). The Gram positive bacterial strains were found to be slightly more sensitive than Gram negative bacterial strains. It may be due to the absence of lipo-polysachride layer in Gram positive bacteria that might function as a barrier to the phytocemical substances that are responsible for antibacterial activity27-28. The plant extract being active against both clinical and laboratory isolates is also an indication that it can be a source of very potent antibiotic substances that can be used against multidrug resistant microorganisms. The phytochemical screening of methanolic extract of aerial parts of Thymus serphylum raveled the presence of alkaloids, flavonoids, phenolics, saponins, tannins, cardiac glycosides, terpenes, steroids and carbohydrates. Anthraquinone glycosides were found to be absent. These phytochemicals (Secondary plant metabolites) are responsible for the biological activities and are known to have antimicrobial, antioxidant activities29-30. The present study reports the presence of diverse phytochemicals in the plant than earlier reports by Kavita et al
14. The present research work supports the resourcefulness of the plant in terms of presence of
The results of the present study showed that the methanol extract can be an accessible source of promising therapeutic agents that can be used in combating infectious diseases caused by drug- resistant microorganisms. Further study is needed to isolate, structurally characterize the pure compounds and evaluate their antimicrobial activity against multidrug resistant microbial strains. The lead molecules will be further subjected to mechanistic studies and will be tested for the possibility of synergism.