Antimicrobial activity of Leucas clarkei

Materials and Methods

Materials

Solvents such as petroleum ether, benzene, chloroform and ethanol used were of analytical grade and obtained commercially from Merck-Limited, India, Mumbai.

Collection and identification of plant material

The plant L. clarkei were collected in the month of October from Bolangir,Odisha, India. The plant material was taxonomically identified and authenticated by Dr. Uma Devi, Head, Department of Botany, Govt. Women’s College, Sambalpur, Odisha. A voucher specimen (GWC/B-315/09) has been deposited in the Herbarium of the Department of School of Pharmaceutical Education & Research, Berhampur University,  India for future reference.

Preparation of plant material

The whole plant was first sun dried for several weeks, crushed by hands and dried again. Then the crushed parts of the plant were ground into coarse powder with the help of a mechanical grinder. By using the concept of the nature of solubility and distribution of the active ingredients, powdered material (1,500 g) was packed in soxhlet apparatus (Reuben et al., 2008; Harborne, 1998) and extracted successively with petroleum ether (60-80) (43 g) to deffat the drug, petroleum ether was removed from the powdered defatted drug which was then extracted with benzene (39 g), chloroform (41 g) and 95% of ethanol (43 g) as increasing polarity. The whole each mixture then underwent filtration through what man filter paper. The filtrates (petroleum ether, benzene, chloroform and ethanol filtrate) obtained were evaporated by rotary evaporator at 5 to 6 rpm and at 40ºC temperature. It rendered a gummy concentrates. The gummy concentrate was designated as crude extract which was then freeze dried and preserved at 4ºC.

Phytochemical screening

Qualitative phytochemical tests for the identification of alkaloids, flavonoids, steroids, glycosides, saponins, tannins and terpenoids were carried out for all the extracts by appropriate method (Sazada et al., 2009; Sofowora, 1993; Harborne, 1993; Kulkarni, 2006; Ndukwe et al., 2006).

The freshly prepared extracts of L. clarkei were qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extracts was performed using the following reagents and chemicals: Alkaloids with Wagner reagent, flavonoids with the use of concentrated hydrochloric acid, tannins with 5% ferric chloride, saponins with ability to produce suds, gum with Molish reagents and concentrated sulfuric acid, steroids with sulfuric acid, reducing sugar with the use α-napthol and sulfuric acid and terpenoids with chloroform and concentrated hydrochloric acid.

Microbial cultures

The following microorganisms were used to test the activity of the extracts. Pure isolates of two Gram positive bacteria such as Escherichia coli (MTCC-443), Salmonella typhi and two Gram negative bacteria such as Staphylococcus aureus (MTCC-1430), Bacillus subtilis (MTCC-441) and fungi Candida albicans (MTCC-183), Candida krusei were collected from the Department of Life Science, Sambalpur University, Odisha, India.

Culture media

Nutrient broth, nutrient agar and Sabouraud dextrose agar (SDA) were purchased from the Himedia.

Reference antibiotics

Amoxycillin and Miconazole were used as reference antibiotics (RA) against bacteria and yeast, respectively. This was collected as standard sample from Medico Remedies Pvt. Ltd, Maharashtra, India.

Inoculum preparation

Nutrient broth and SDA were used for growing and diluting the microorganism suspensions. Inoculums from bacterial cultures were prepared by picking colonies from 24 hours old cultures. Colonies were suspended in 5 mL of a solution containing 0.145  mol  of saline per liter. The density was adjusted by spectrophotometer to that of a 0.5 McFarland standard at a wavelength of 530 nm to yield a stock suspension of 1 x 10⁶ to 5 x 10⁶ cells per mL. Two different inoculums sizes were evaluated: 1 x 10⁶ to 5 x 10⁶ cells per ml and a 1:100 dilution containing 1 x 10⁴ to 5 x 10⁴ cells per mL. Plates were swabbed in three directions.

Fungal cultures were aseptically inoculated on petri dishes containing autoclaved, cooled, and settled SDA medium. The petri dishes were incubated at 31ºC for 48 hours to give white round colonies against a yellowish background. These were aseptically subcultured on SDA slants. The yeast colonies from SDA slants were suspended in sterilized 0.9% sodium chloride solution (normal saline), which was compared with McFarland solution. According to the manufacturer's directions, 1 mL of yeast suspension in normal saline was added to 74 mL of sterile medium and kept at 45°C to give a concentration of 2 x 10⁷ cells/mL.

Determination of MIC

Double strength nutrient agar was prepared in 500 mL of distilled water which was swirled and mixed thoroughly by heating to allow uniform dissolution, after which 5 mL of it was dispensed into universal bottles and sterilized in an autoclave at 121°C for 15 min. The agar was allowed to cool to 45ºC and each graded solution was then mixed gently with molten double strength nutrient agar in a petri dish and allowed to solidify for one hour. Extracts’ concentrations of 100, 90, 80, 70, 60, 50, 40, 30, 20, 10  and 5 μg/mL (Ndukwe et al., 2006) respectively were prepared by serial dilution. Each plate was divided into six equal sections and labeled accordingly to correspond to six test organisms. Two 6 mm diameter paper discs (Whatman No. 1) were placed aseptically into each labeled section of the plate using sterilized forceps. With an automatic micropipette, 20 μL of each bacterial suspension was taken and transferred aseptically and carefully into each appropriate pre-labeled paper disc on the agar plates. The plates were incubated for 24 hours at 37°C after which they were observed for growths or death of the test organisms. In contrast, fungal cultures were incubated  at 31°C for 48 hours in SDA medium. The lowest concentration inhibiting growth was taken as the minimum inhibitory concentration (MIC). The average of 3 values was calculated and that was the MIC for the test material.

Determination of MBC and MFC

This was carried out to know  if  the  organisms  could  be  killed  completely  or their growths could only be inhibited. Another set of plates of nutrient agar were prepared and sterilized in an autoclave as earlier described. The paper discs in all the plates from the MIC tests were reactivated. Emphasis was mostly paid to the MIC plates and the preceding plates. The reactivation was done in a mixture of 0.5% egg lecithin and 3% Tween 80 solution in a test tube. The reactivated organisms were sub-cultured into appropriately labeled quadrants of the sterilized nutrient agar plates using wire loop into each test tube and streaking uniformly on the labeled quadrants. This was then incubated for 24 hours at 37°C after which they were observed for growths (Ndukwe et al., 2006; Navarro et al., 1996). In contrast, fungal cultures were incubated at 31°C for 48 hours in SDA medium. The MBC was the quadrant with the lowest concentration of the extract without growth.

Disc diffusion assay (Navarro et al., 1998; Swain et al., 2008)

Stock solutions of different extracts of L. clarkei were prepared separately by initially dissolving 0.05 g of the extract in 0.5 mL of DMSO to obtain a stock solution of concentration 100 mg/mL. From this stock solution, concentrations of 1 mg/mL were prepared by serial dilution. From the above stock solution further dilutions were made to get 10, 20, 30, 40, 60 μg/mL working solutions. The cork and bore diffusion method of Bauer et al. (1966) and Barry and Thornsberry (1985) were used in the antimicrobial screening. Nutrient agar was inoculated with a microbial cell suspension (200 μL in 20 mL of medium) and poured into sterile petri dishes followed by cross-streaking with the same wire loop to achieve uniform spread on the plate. Sterile filter paper discs 6 mm in diameter were impregnated with 20 μL of each extract concentration (30 and 60 μg/ mL), which were prepared using the same solvents employed to dissolve the plant extracts, and placed on the inoculated agar surface. Standard 6 mm discs containing rifampicin 30 μg/disc was used as positive control. Negative controls were made using paper discs loaded with 20 μL of the solvents. After pre-incubation for 2 hours in a refrigerator the plates were incubated overnight at 37°C for 18-24 hours. In contrast, fungal cultures were incubated at 31°C for 48 hours in SDA medium using miconazole 10 μg/disc (Sur-Altiner et al., 1999). Negative controls were made using paper discs loaded with 20 μL of the solvents. At the end of the incubation period antimicrobial activity was evaluated by measuring the zones of inhibition.