General experimental Procedures
Mature leaves of S. villosum were collected from the outskirts of Burdwan (23°16' N, 87°54' E), WB, India from March 2006 to February 2007. Plant samples were identified by a plant taxonomist, Dr. G. G. Maity, Department of Botany, Kalyani University, Kalyani, West Bengal, India.
All chemical reagents used in this study were of analytical grade. Reagents for SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Protein standard kit for PAGE was purchased from GENEI, Bangalore, India.
Preparation and preservation of protein from decoction
5 g of dried decoction of mature leaves were soaked in phosphate buffer (pH 7.2) for over night and filtered through Whatman filter paper (No. 40). Leaf protein was extensively dialyzed for 48 hrs at 10°C against deionised distilled water and lyophilized. Dried protein was then kept in a freeze and stored at 5°C for Bioassay. Protein was bio assayed against laboratory-reared 3rd instar larvae of An. stephensi, Cx. quinquefasciatus and St. aegypti mosquitoes. They were treated with different percent concentrations of protein solutions of the leaves, following the standard WHO larval susceptibility test method [8]. The tests were conducted at room temperature (27–30°C). Concentrations (0.03, 0.05% and 0.1%) of the extract in water were prepared fresh and used for the tests during mid-May to mid-June. Solutions of the extract were prepared in distilled water. At each of the given concentrations, three replicates comprising 10 larvae each were exposed. Results were scored after 24 h of continuous exposure to the test solution and expressed as per cent mortality. A control set was also prepared having the same larval density of each species in distilled water without the application of the protein sample.
Antibacterial assay
Four bacterial strains were used for the study. Gram positive bacteria include S. aureus MTCC 2940 and B. subtilis MTCC 441 and Gram negative bacteria include E. coli MTCC 739 and P. aeruginosa MTCC 2453. All the tested strains were reference strains and were collected from the Microbiology Laboratory of Burdwan Medical College, Burdwan, India. The bacterial cultures were maintained in nutrient broth (Himedia, M002) at 37°C and maintained on nutrient agar (Himedia, MM012) slants at 4°C.
Disc diffusion method
Antibiogram was done by disc diffusion method [9, 10] using protein and commonly used antibiotics. The test quantity of protein was dissolved in sterile water. The surfaces of media were inoculated with bacteria from a broth culture. High potency bio-discs (Himedia) were placed on the agar. After 18 h of incubation at a specific temperature [(30 ± 1) °C for B. subtilis and 37°C for S. aureus, E. coli and P. aeruginosa], the plates were examined and the diameters of the inhibition zones were measured to the nearest millimeter and compared against standard antibiotic amoxicillin. A control set was prepared with the DMSO in which no isolated protein/antibiotics were added.
MIC value determination
The MIC was determined by macrobroth dilution [11] and agar well diffusion [12]. 100 μl volume of two-fold serial dilutions of extracts reconstituted in 5% DMSO was introduced into triplicate wells in Muller Hinton Agar plates (MHA) pre inoculated with test bacterial strains. The protein fraction was allowed to diffuse into the MHA at room temperature before incubation at 37°C for 18 h. The reconstituted extract was serially diluted two-fold in Muller Hinton Broth (MHB, Oxoid). Duplicate tubes of each dilution were inoculated with 5 × 105 cells (cfu) of the test bacterial strain and cultures incubated in a water bath at 37°C for 18 h. Two-fold serial dilution amoxycillin (μg/ml concentration) were included in each experiment as controls. The MIC was taken as the lowest concentration of protein fraction or drugs showing clear zone of inhibition in the agar well diffusion technique and as the highest dilution (least concentration) of protein fraction showing no detectable growth in macrobroth assay.
Amino acid analysis
Amino acid analysis of the isolated protein was performed using PICO.TAG amino acid system according to PICO.TAG operation manual (Waters, USA). Dialysed and dried leaf protein (20 μg) of S. villosum was hydrolyzed by 6 N HCl containing 5% thioglycollic acid [13] for 24 h at 105°C in the PICO.TAG workstation. Hydrolysed sample and standard amino acid mixture, standard A (0.005 ml) was taken in respective tubes (vials) and was dried completely. These were then derivatized [14] by phenyl isothiocyanate (PITC) solution (ethanol: triethyl acetate: water: PITC: 7:1:1:1 by volume) for 20 min at 25°C in a Nitrogen atmosphere. The vials were then dried and the samples were reconstituted in a diluent solution (Na2HPO4, 0.071% w/v in distilled water, pH 7.4; pH was adjusted by 10% H3PO4 containing 5% v/v acetonitrile. The samples were analyzed by HPLC at 38°C as per the PICO.TAG manual using a PICO.TAG C18 hydrophobic column (5 μm, 3.9 × 150 mm, waters and detection at 254 nm. Amino acids present in the unknown sample were determined quantitatively by comparing the peak areas (745 B data module print out) of amino acids present in standard A.
SDS-PAGE electroporesis
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli, 1970 [15]. It was carried out on Bio-Rad gels composed of stacking gel (5% w/v) using 1.0 M Tris-glycine buffer containing 0.4% SDS at pH 6.8 and resolving gel (12%, w/v) using 1.5 M Tris-glycine buffer containing 0.4% SDS at pH 8.8. Protein sample was dissolved in phosphate buffer (5 mg/ml) and mixed with a solubilization buffer Tris – HCl 6.22 mμ (pH 6.8) which contains 2% (w/v) SDS, 50% glycerol, a pinch of bromophenol blue and reduced with 0.9 mμ 2-mercaptoethanol in boiling water for 3 min. Protein sample was loaded onto each well and electrophoresis (Bio-Rad electrophoresis apparatus, Bio-Rad Laboratories, Hercules, CA) was conducted at constant current of 60 volts by a Bio-Rad electrophoresis constant power supply unit (Model 200/2, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA). After electrophoresis, gels were stained with 0.2% (w/v) AgNO3 solution after being treated with fixing solution (methanol-acetic acid-H2O-p-formaldehyde) and sodium thiosulphate solution. It was then treated with developer (Na2CO3-sodium thiosulphate-37% p-formaldehyde) until the bands came out. The gels were soaked with stop solution and stored in 30% methanol (v/v) at 4°C. Molecular masses were determined using the molecular weight standard kit from GENEI, Bangalore, India. The result was placed in Table 6.
Statistical Analysis
Mortality rates were corrected with Abbott's correction formula [16]. The data obtained were subjected to Probit analysis, to calculate the median lethal concentration LC50 and LC90 value [17]. Since the readings of control (distilled water) experiments in vitro, antibacterial studies against those bacteria were zero, the data were analyzed by simple arithmetic means of the different extracts and the standard errors were compared with the control.
