Plant material and preparation of decoction
A February collection of the unripe fruits of A. marmelos collected from Pangare village in the Parinche valley, about 53 km south east of Pune city in the state of Maharashtra, India, was used for the present study. February was chosen as the month of collection since the fruits achieve full size at this time but are still unripe. The plant material was authenticated by Dr. P. Tetali, Head, Research and Development, Naoroji Godrej Centre for Plant Research, Shirwal, Maharashtra, India. A voucher specimen has been deposited at the Botanical Survey of India (Western Circle), Pune, India, under herbarium number 124675. The fruits were cut into small pieces, shade dried and stored at 4°C.
A crude aqueous extract (decoction) was used for the study since it represents the nearest form to traditional preparations. The decoction was prepared as described in Ayurvedic text [30]. 1 g of the powdered dried fruit pulp was boiled in 16 ml double distilled water till the volume reduced to 4 ml. It was centrifuged and filtered through a 0.22 μm membrane before use. To replicate field conditions, each assay was performed with freshly prepared decoction.
The dry weight of the decoction thus obtained was 51.1 mg/ml ± 0.5 mg/ml and 20.4% ± 2.11% (w/w) with respect to the starting dried plant material. The decoction was diluted 1:100, 1:20 and 1:10 in appropriate media for each experiment and has been referred to as 1%, 5%, and 10% respectively throughout the text. The dry weight contents of these dilutions of the decoction were 0.51 mg/ml ± 0.005 mg/ml, 2.55 mg/ml ± 0.025 mg/ml and 5.11 mg/ml ± 0.05 mg/ml respectively.
Media, reagents, plastic ware and instrumentation
The bacterial media and the Minimal Essential Medium (MEM) were purchased from HiMedia laboratory, Mumbai, India. Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum (FCS) were procured from GibcoBRL, UK. The constituents of the Diamond's TYI-SS medium were procured from local Indian manufacturers, as were the antibiotics. Trypan blue, neutral red, ganglioside monosialic acid (GM1), anti-cholera toxin, ortho-phenylene diamine, bovine serum and bovine serum albumin were purchased from Sigma, USA. Peroxidase labeled swine anti-rabbit immunoglobulin was purchased from Dako, Denmark. All chemicals were from SD Fine Chemicals, Mumbai. Standard marmelosin was purchased from Natural Remedies, Bangalore, India. Gallic acid was kindly provided by Dr. KS Laddha, University Institute of Chemical Technology, Mumbai, India. Lactulose was a product of Intas Pharmaceuticals, Ahmedabad, India. The 24- and 96-well tissue culture plates and the 96-well ELISA plates were purchased from Nunclon, Denmark, the 55 mm diameter tissue culture plates were obtained from Tarsons, Kolkata, India, and the ELISA plate reader was purchased from Labsystems, Finland.
Cell culture
The human laryngeal epithelial cell line, HEp-2, and the embryonic monkey kidney derived cell line, MA-104, were obtained from National Centre for Cell Sciences, Pune, India. The cell lines were maintained in DMEM and MEM respectively, supplemented with 10% FCS, at 37°C in a 5% CO2 atmosphere. The cells were maintained in logarithmic growth by passage every 3-4 days.
Microorganisms used
Six bacteria, viz., enteropathogenic Escherichia coli (EPEC) strain B170, serotype 0111:NH, enterotoxigenic E. coli (ETEC) strains B831-2, serotype unknown (heat labile toxin producer) and strain TX1, serotype 078:H12 (heat stable toxin producer) (all strains obtained from Centre for Disease Control, Atlanta, USA), enteroinvasive E. coli (EIEC) strain E134, serotype 0136:H- (kindly provided by Dr. J. Nataro, Veterans Affairs Medical Centre, Maryland, USA), Vibrio cholerae C6709 El Tor Inaba, serotype 01 (kindly provided by Dr. S. Calderwood, Massachusetts General Hospital, Boston, USA) and Shigella flexneri M9OT, serotype 5 (kindly provided by Dr. P. Sansonetti, Institut Pasteur, France) were used for the present study. In addition, Giardia lamblia P1 trophozoites (kindly provided by Dr. P. Das, National Institute for Cholera and Enteric Diseases, Kolkatta, India) and simian rotavirus SA-11 (kindly provided by Dr. S. Kelkar, National Institute of Virology, Pune, India) were also included.
Phytochemical analysis
Qualitative phytochemical analysis of the decoction was carried out for assaying presence of carbohydrates, glycosides, proteins, amino acids, phytosterols, saponins, flavonoids, alkaloids and tannins [31]. High performance thin layer chromatography (HPTLC) fingerprinting of the methanol soluble fraction of the decoction was carried out with the solvent system n-Hexane:Ethyl acetate:Acetic acid (40:60:0.5). Marmelosin was used as a phytochemical reference standard for fingerprinting.
Antimicrobial activity
Antibacterial activity
The antibacterial activity was determined by a microtitre plate-based assay [32]. The bacterial strains were incubated in the absence (control) and presence of different dilutions of the decoction in nutrient broth and the optical density was measured after 24 h as a measure of growth. Three independent experiments were carried out. In each experiment, triplicate wells were set up for control as well as each dilution of the decoction.
Antigiardial activity
A 24 h culture of G. lamblia P1 trophozoites was incubated in the absence (control) and presence of different dilutions of the decoction in Diamond's TYI-SS medium. The number of viable trophozoites after 24 h was counted in a haemocytometer using trypan blue [33]. Three independent experiments were carried out. In each experiment, duplicate tubes were set up for control as well as each dilution of the decoction.
Antirotaviral activity
The entry and subsequent survival of rotavirus SA-11 in MA-104 cells was assayed by the neutral red uptake assay [34]. Briefly, MA-104 cells were grown in 96-well tissue culture plates for 72 h after which they were infected with rotavirus for 90 min in absence (control) and presence of different dilutions of the decoction. Subsequently, the extracellular virus and the decoction were washed off and the culture was further incubated for 72 h. Thereafter, the cells were incubated with 0.03% neutral red dye for 30 min. The intracellular dye was released with 1:1 solution of 100 mM acetic acid and ethanol and the intensity measured at 540 nm (reference 630 nm) in an ELISA plate reader. Three independent experiments were carried out. In each experiment, triplicate wells were set up for control as well as each dilution of the decoction.
Effect on bacterial colonization
Effect on adherence
The effect on the adherence of E. coli strain B170 to epithelial cells was assayed by the method described by Cravioto et al. [35]. Briefly, a 48 h culture of HEp-2 cells on glass coverslips was infected with a log phase culture of the bacterium (5 × 107/ml) and incubated for 3 h. Non-adherent bacteria were washed off, the coverslips were fixed in 10% formaldehyde and stained with toluidine blue stain (0.1% w/v). HEp-2 cells having typical EPEC micro-colonies [36] were counted under light microscope. Three independent experiments were carried out. In each experiment, duplicate cover-slips were set up for control as well as each dilution of the decoction.
Effect on invasion
The effect on invasion of E. coli E134 and S. flexneri into epithelial cells was studied by the method described by Vesikari et al. [37]. Briefly, a 48 h culture of HEp-2 cells grown in a 24-well tissue culture plate was infected with log phase culture of the bacteria (108/ml) and incubated for 2 h. The culture was further incubated with gentamycin (100 μg/ml) for 3 h. The epithelial cells were then lysed by cold shock with chilled distilled water and the released bacteria were counted by plating on nutrient agar. Three independent experiments were carried out. In each experiment, duplicate wells were set up for control as well as each dilution of the decoction.
Two different protocols were performed for both the adherence and the invasion assays to understand whether the bacterial adherence and invasion respectively were affected by the effect of the decoction on the epithelial cells or through competitive inhibition. The HEp-2 cells were incubated in absence (control) and presence of different dilutions of the decoction either for 18-20 h prior to infection (pre-incubation) or simultaneously with infection (competitive inhibition) respectively.
Effect on bacterial enterotoxins
Effect on E. coli heat labile toxin (LT) and cholera toxin (CT)
LT, which is localized in the bacterial cell membrane, was obtained by lysing E. coli B831-2 with polymyxin B sulphate (1 mg/ml) whereas CT, which is released extracellularly, was obtained as a culture supernatant of V. cholerae. LT and CT were assayed by the GM1-enzyme linked immunosorbent assay (GM1-ELISA) [38]. Briefly, the toxins were added to ELISA plates pre-coated with 1.5 μmol/ml of GM1. Anti-cholera toxin and peroxidase labeled swine anti-rabbit immunoglobulin used at dilutions of 1:300 and 1:200 were used as primary and secondary antibodies respectively. Ortho-phenylene diamine (6 mg) with hydrogen peroxide (4 μl) in 10 ml citrate buffer (pH 5.5) was used as the substrate. The intensity of the color thus developed was read at 492 nm in an ELISA plate reader.
To study the effect on production of the toxins, the respective bacteria were grown in Casein Hydrolysate Yeast Extract (CAYE) in absence (control) and presence of different dilutions of the decoction and the toxins produced were assayed by GM1-ELISA. To study the effect on the binding of the toxins to GM1, the toxins obtained by growing the respective bacteria in CAYE were added to the assay system in absence (control) and presence of the decoction. Three independent experiments were carried out. In each experiment, triplicate wells were set up for control as well as each dilution of the decoction.
Effect on E. coli heat stable toxin (ST)
ST was assayed by the method originally described by Gianella [39]. Briefly, ST, which is released extracellularly, was obtained as the culture supernatant of E. coli TX1. The toxin was inoculated intra-gastrically in 2-3 days old Swiss White suckling mice. Following an incubation of 3 h at room temperature, the pups were sacrificed and the ratio of gut weight to that of the remaining carcass weight was calculated. Ratio of ≥ 0.083 was considered as positive.
To study the effect on production of ST, the bacterium was grown in CAYE in absence (control) and presence of different dilutions of the decoction and the toxin produced was assayed. To study the effect on the action of ST, the toxin obtained by growing the bacterium in CAYE was intra-gastrically injected in absence (control) and presence of different dilutions of the decoction. CT was used as a negative control. Three independent experiments were carried out. In each experiment, three animals were inoculated for control as well as each dilution of the decoction.
The Institutional Ethics Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) cleared the use of animals in the study. The Foundation for Medical Research (FMR) is registered with CPCSEA (registration No. 424/01/a/CPCSEA, June 20th, 2001).
Statistical analysis and presentation of data
The results for each assay have been expressed as the mean ± standard error of the percentage values from three independent experiments. The percentage in each experiment was calculated using the formula {(C or T)/C} × 100, where C is the mean value of the duplicate/triplicate readings of the control group and T is mean value of the duplicate/triplicate readings of the test (dilutions of the decoction) groups. Hence, the value of control is 100% and the values of the test groups have been represented as percentages relative to control.
Data were analyzed by analysis of variance (ANOVA) and Dunnett's post-test. Statistical analyses were performed using the software Prism 4.0 (GraphPad, Inc.). P ≤ 0.05 was considered to be statistically significant.