In Vitro Propagation and Evaluation of Antimicrobial Activity of Selected Medicinal Plants against Human Disease Causing Fungi and Bacteria

Abstract

The present investigation was conducted with a view to establish a standard method for rapid micropropagation, antibacterial activity and antifungal activity of Achyranthes aspera L. and Centella asiatica L. Urban. In this investigation, shoot tip and nodal segment explants from field grown plants were used as experimental materials for micropropagation. For surface sterilization of both explants, treatment of HgCl2 (0.1 %) was found to be most effective for 6 minutes. It was found that MS medium containing 2.0 mg/I SAP gives the best result for culture initiation as well as in axillary shoot proliferation in both explants. Medium supplemented with BAP and GA3 produced a smaller number of shoots than single use of BAP, but enhance shoot length for both plants. Nodal segment was found more responsive than shoot tip explant for rapid micropropagation. For rooting, half strength of MS supplemented with 0.5 mg/I NAA was found more effective than others. Propagated plantlets were successfully transferred into the soil. More than 90% of the transplanted clones were survived. For antibacterial activity test leaf and stem of both plants were used for collecting extracts using three solvent named petroleum ether, chloroform and ethanol. These three crude extracts of both plants were tested by agar disc diffusion method against some Gram positive (Staphylococcus aureus, Bacillus cereus, Streptococcus haemolytica, Bacillus subtilis, Bacillus megaterium, Sarcina lutea) and Gram negative ( Shigella sonnei, Escherichia coli, Shigella shiga, Klebsiella pneumoniae, Salmonella typhi, Klebsiella sp, Pseudomonas sp, Shigella dysenteries, Pseudomonas aeruginosa) bacteria that caused human disease. The antibacterial activity was compared with commercial antibiotics such as tetracycline. Leaf and stem extracts (ethanol, petroleum ether and chloroform) of both plants were effective against both Gram-negative and Gram-positive bacteria. In case of Shigella Sunnie, Shigella Shiga, Salmonella typhi, Pseudomonas aeruginosa and Shigel/a dysenteries, the antibiotic tetracycline did not show inhibition activity but the leaf-stem extracts (petroleum ether and chloroform) exhibit the zone of inhibition against them. In case of Achyranthes aspera, among the three solvents, ethanol and petroleum ether extracts showed higher activity against pathogen than chloroform extract, where the petroleum ether extracts showed minimum inhibitory concentration (MIC) ranged from 156.25 to 625 µgmr1 concentrations. Chloroform extracts showed MIC of 312.5 to 625 µgmr1 concentrations. The lowest MIC value was calculated in the ethanol extract against Staphylococcus aureus and it was 156.25 µgmr1 concentrations. On the other hand, in Gentelia asiatica, among the three solvents, petroleum ether and chloroform extracts showed higher activity than ethanol extract, where the petroleum ether extracts showed minimum inhibitory concentration (MIC) ranged from 156.25 to 625 µgmr1 concentrations. Chloroform extracts showed MIC of 312.5 to 625 µgmr1 concentrations. The lowest MIC value was calculated in the petroleum ether extract against Klebsiella pneumoniae and it was 156.25 µgmr1 concentrations. The antifungal activity of the three organic solvent extracts of Achyranthes aspera and Centella asiatica against Aspergillus niger, Aspergillus fumigatus, Aspergil/us flavus, Penicillum sp, Microsporum sp Trichophyton sp and Candida sp, were determined at different concentrations. For the both plants extract among the three solvents, ethanol and petroleum ether extracts showed higher activity than chloroform extract, whereas, petroleum ether extracts showed minimum inhibitory concentration (MIC) for Achyranthes aspera and ethanol extract for Centella asiatica ranged from 625 to 1250 µgmr1 concentrations. The antifungal activity was compared with commercial antifungal drug itraconazole. In case of Achyranthes aspera, standard drug itraconazole showed higher antifungal activity than the all extracts but not in case of Ethanol and Petroleum ether extracts of Centella asiatica. In the present investigation, petroleum ether and ethanol extracts showed the higher antifungal activity than the chloroform extracts. For the phytochemical analysis the ethanolic leaf and stem extract of Achyranthes aspera was considered for assessment of alkaloids and other secondary metabolites. Alkaloid tests were performed using different alkaloid detecting reagents like Dragendroff's (D), Wagner's (W), Mayer's (M), Hager's (H) and Tannie acid (T). The ethanolic leaf and stem extracts of Achyranthes aspera gave positive result for Saponin, Tannin, Flavonoids, Cardiac Glycoside and Terpenoids. On the other hand, ethanolic extract of Gentelia asiatica gave positive test for Tannin, flavonoid, glycosides, saponin and terpenoids but negative test for alkaloid. From the results it can be concluded that in future, there is a possibility to develop new drugs from the studied plant against the tested bacteria and fungi.