Antimicrobial activity of natural compounds from Kalanchoe crenata against pathogenic bacteria

Abstract

For routine susceptibility testing of non-fastidious bacteria Mueller-Hinton agar is considered to be the best. Medium formulations only that have been tested according to, and that meet the acceptance limits describe in National Committee for Clinical laboratory Standards (NCCLS) document protocols for evaluating dehydrate [24]. Mueller-Hinton agar was prepared according to the manufacturer's instructions from a commercially available dehydrated base. Evaluation of antimicrobial potential of Kalanchoe crenata natural compounds For in vitro investigation of antimicrobial activity of natural compounds of Kalanchoe crenata against selected isolates, disc diffusion method [25] and agar well diffusion method [26] were applied. The accuracy and reproducibility of this test are dependent on maintaining a set of standards recommended by the NCCLS [24]. All the identified isolates were subjected to various concentrations of natural compounds of Kalanchoe crenata for the evaluation of antibiotic activity pattern. Agar well diffusion method In agar well diffusion test, the natural compounds of Kalanchoe crenata were allowed to diffuse out into the medium. The antimicrobials present in the natural compounds of Kalanchoe crenata interact with the freshly seeded test organisms. This interaction results in a uniform circular zone of inhibition zone was measured in millimeters. From an agar plate of pure culture, 3-5 colonies of the same morphological type were selected. The top of each colony was touched with a loop and the growth was transferred into a tube containing 4-5 ml of Nutrient broth. The broth culture was then incubated at 37° C to attain the preferable turbidity. Sterile cotton swab was dipped into the suspension. The excess fluid was removed by pushing and rotating the swab firmly against the wall, just above the fluid level. The entire dried surface of Mueller-Hinton agar plate was streaked by the swab 2-3 times. That results an even distribution of the inoculums ever the entire surface [26]. Disc diffusion method Antimicrobial activities of the natural compounds of Kalanchoe crenata were determined by using the disc diffusion method [24]. The bacterial culture was streaked on Mueller Hinton Agar plate. Blank discs containing the natural compounds of Kalanchoe crenata were then placed on the inoculated plate surface and incubated at 37°C for 24 hours. Antimicrobial activity was determined by measuring the diameter of zones of inhibition produced after incubation [24]. All samples of dry residue were dissolved in autoclaved distilled water. The discs were impregnated with the natural compounds of Kalanchoe crenata and placed aseptically on the inoculated agar. Statistical analysis We used analysis of variance with an F-test, followed by a t-test. P values less than 0.05 were considered significant. The data are presented as mean ± standard deviation values of independent replicates. Results In the present study, five bacteria (gram-negative and gram-positive bacteria) were used. The antibacterial assays were performed by disc diffusion and agar-well diffusion methods. Therefore, it Sample preparation The plant part of Kalanchoe crenata (leaf) was separately shade dried, finely powdered using a blender, and subjected to extraction of flavonoids in following the method as described elsewhere with some modifications [20-23]. Briefly, two hundred grams of each finely powdered sample was Soxhlet extracted with 80% hot methanol (1000 ml) on a water bath for 24 h and filtered. Filtrate was re-extracted successively with petroleum ether, ethyl ether and ethyl acetate using separating funnel. Petroleum ether fractions were discarded as being rich in fatty substances, whereas ethyl ether and ethyl acetate fractions were analyzed for free and bound flavonoids, respectively. Ethyl acetate fraction of each of the samples was hydrolyzed by refluxing with 7% H 2 SO 4 for 2 h for removal of bounded sugars from the flavonoids. Resulting mixture was filtered and filtrate was extracted with ethyl acetate in separating funnel. Ethyl acetate extract thus obtained was washed with distilled water to neutrality. Ethyl ether and ethyl acetate fractions flavonoids were dried and weighed. The extracts were stored at 4°C for farther used and were re-suspended in their respective. Total flavonoids determination Total flavonoids content of each extract was determined by aluminum chloride as described elsewhere [with some modifications [20-23]. Briefly, plant extracts (0.5 ml of 1:10 g/ml) were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It remained at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm with a spectrophotometer, and quercetin was used as a standard for calibration curve. Total flavonoids values are expressed in terms of mg equal quercetin in 1 g powder. Collection of bacterial isolates Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-Negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli) were obtained from MAG Osmani Medical College, Sylhet, Bangladesh and identified at the microbiology laboratory, Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology. Test solution preparation For preparation of test solution of appropriate concentration, natural compounds of Kalanchoe crenata were solubilized to autoclaved distilled water and diluted to desired concentration. Precautions were taken during the operation for not to contaminate the culture with foreign contaminants. The test solution was poured into the well using a micropipette. The plates were kept in sterile condition for 30 minutes for diffusion of the test solution to the surrounding media. Addition of the test solution to the plates then inverted into incubator at 37°C and then after 24 hours, we examined of each plate. The circular inhibition zones were formed around the well on the surface. The diameters of the complete zones of inhibition were measured using a ruler [24].