Antimicrobial activity of ethanolic extracts of Justicia neesii

Materials and Methods

Collection and identification of plant: Plant material free from infection was collected from different areas of East Godavari district, Andhra Pradesh during the month of February 2014 on day time. The plant was taxonomically identified by the experts of Botanical Survey of India, Hyderabad (BSI/DRC/2013-14/Tech./915-A).

Extraction of plant material: Whole plant parts including leaves, stem, twigs, flowers, seeds, roots were separated and made free from soil matter. They were dried and powdered by using hand pulveriser to a course powder. Then the powder was extracted with ethanol by using sohxlet apparatus at a temperature of 50-55ºC for 8 hours. The extracts were concentrated using vacuum evaporator and the semisolid mass was dried in vacuum desiccators. The yield of plant extract was found to be 10.58 % (w/w).

Preparation of nutrient agar: The weighed amount of NaCl (5 g), peptone (10 g), beef extract (10 g) are dissolved in 1,000 mL of the water, then agar (20 g) is added slowly on heating with continues stirring until agar is completely dissolved and pH is adjusted to 7.2 to 7.4. This nutrient agar medium is then sterilized by moist heat sterilization method using autoclave at temperature of 120°C at 15 lb pressure maintained for 15 min.

Preparation of potato dextrose agar (PDA): A potato was peeled and 100 g was measured, finely chopped and boiled to a mash in distilled water. The dextrose was measured (12.5 g) and placed in a 1L measuring cylinder. Agar was measured (12.5 g) and added to the measuring cylinder (with the dextrose). The potato mash was stirred and strained into the cylinder. Hot distilled water was added to make up 500 mL. The contents was continuously poured and stirred until consistency was achieved. The content was then poured into a conical flask, plugged with cotton wool, over which aluminum foil was tightly wrapped. The flask was then autoclaved at 121°C for 24 hours (Murray, 1995).

Microbial cultures: Bacterial cultures of Gram positive bacteria- Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 3160), S. faecalis (MTCC 459), Gram negative bacteria- Escherichia coli (MTCC 46), Pseudomonas aeruginosa (MTCC 1688), Klebsiella pneumonia (MTCC 4032) and fungal cultures of Aspergillus flavus (MTCC 277), Aspergillus niger (MTCC 2723), Candida albicans (MTCC 183), Fusarium axisporum (MTCC 1755), Saccharomyces cerevisiae (MTCC 4742) were procured from Institute of Microbial Technology, Chandigarh, Punjab, India. All the test organisms were maintained on nutrient/potato dextrose agar slopes and sub-cultured once in every two weeks.

Preparation of inoculums: The test inoculums are prepared as per the McFarland method. McFarland standard was prepared by adding 0.5 mL of 1.175%w/v barium chloride to 85 mL of 1%v/v sulfuric acid and mixed. Then the volume was made up to 100 mL with 1% sulfuric acid. The optical density of the prepared solution was checked at 625 nm range gives an absorbance ranging from 0.08 to 0.10. The bacterial inoculums are prepared from 24 hour old cultures by taking 3-5 morphologically similar colonies of respective micro organisms and transferred into 5 mL sterile saline solution and adjusted to 0.5 McFarland turbidity standards equivalent to the cell density of 1-5 x 10⁸ CFU/mL. The fungal inoculums are prepared from seven days old culture plates by taking 3-5 morphologically similar colonies of respective micro organisms and transferred into 5 mL sterile saline solution and adjusted to 0.5 McFarland turbidity standards. From this solution 1:10 dilution are preformed three times with growth medium to get inoculums density of 1-5 x 10³ CFU/mL (Mcfarland, 1907).

Antimicrobial assay: The antimicrobial assay was carried out by agar well diffusion assay (Perez, 1990). The sterilized microbial medium was cooled to 50°C. 20 mL of the microbial media was taken into sterile universal bottles and seeded with 0.2 mL of respective cultures of standard inoculums size aseptically. Then the seeded media was transferred to the sterile petri dishes under aseptic conditions. The wells are created by using sterile cork borer of 6 mm diameter at equidistance points. The respective wells are supplied with 200, 400 and 800 µg of test drug in 25 µL volume. The petri plates were then kept in refrigerator for diffusion and then transferred to biological incubator and bacterial culture plates are incubated at 37°C for 24 hours and fungal culture plates at 27°C for 48 hours. Ciprofloxacin and nystatin are used as positive controls at 10 µg/well concentration to compare the antibacterial and antifungal affects respectively. Each experiment was performed in triplicate and the zone of inhibition values are noted after incubation period by using Antibiotic zone reader. Activity index for each extract was calculated by using following formula.

Activity index (AI) = Inhibition Zone of the sample/ Inhibition Zone of the standard.

Minimal inhibitory concentration (MIC): The minimum inhibitory concentration (MIC) is the concentration required to inhibit growth of a specific isolate in vitro under standardized conditions. The MIC of test extract on each organism was determined by using serial dilution method. For this purpose 10 mg/mL concentration of test solution was prepared and two fold serial dilutions are made to get 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, 0.019 mg/mL concentrations of solutions. 1 mL of test solution is then taken into each test tube and diluted with 1 mL of sterile nutrient agar media (for bacteria) or potato dextrose agar media (for fungi). The test tubes were then inoculated with 0.1 mL of microbial suspension of standard size. The tubes were incubated at 37°C for 24 hours for bacteria and 28°C for 48 hours for fungi in a biological incubator and observed for change in turbidity and compared with the growth in control tubes which contains 75% ethanol in water (Pelczer et al., 2004).

Minimum bactericidal/ fungicidal concentration (MBC/MFC): The MBC/MFC is the lowest concentration at which the culture has been completely sterilized. 50 µL of the media from each test tube showing no visible growth was taken and inoculated into 2 mL of sterile broth media and incubated as done in MIC determination.

Total activity (TA) determination: Total activity is the volume at which the test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated and is expressed in mL/g (Eloff, 2004).

Total activity = Extract per gram dried plant part/MIC of extract