Plant material, preparation of 50% ethanolic extract, and compounds
The fruits of Terminalia chebula were collected from the medicinal plants garden of Ayurveda Research Institute, Poojappura, Kerala and the identity was confirmed at Ayurveda Research Institute, Trivandrum, Kerala. The voucher specimen (HLL/04/2013) has been kept at Natural Products Division of HLL Lifecare Limited, Trivandrum, Kerala, India. Air and shade dried fruits were grinded and strained through a 30 mesh (0.5 mm). The finely grinded powder (24 g) was subjected to pressurized sequential extraction using Accelerated Solvent Extractor (ASE 150, Dionex Inc., Sunnyvale, CA, USA) essentially as described previously [22, 23]. In brief, the material was mixed with diatomaceous earth at 4:1 ratio. The mixture was placed into a sample cell (100 ml) and loaded onto the ASE 150 system. Extraction was performed using 50% ethanol under pressure (1500 psi) at 60 °C with a flush volume of 60% using 2 static cycles. The solvent was then evaporated in a rotary evaporator (Büchi Labortechnik AG, 9230 Flawil, Switzerland). The dried extract was weighed and used for further studies.
Highly pure commercial chebulagic acid and chebulinic acid, both purified from T. chebula, were obtained from MP Biomedicals, Ohio, Solon, USA. These compounds were used as reference standard to identify the corresponding peaks in 50% ethanolic extract prepared from fruits of T. chebula as well as to determine their anti-HSV-2 activity in various assays as described below. Both the compounds were dissolved in absolute ethanol (10 mg/ml) and further dilutions were made in the culture medium before use. Acyclovir (commercial name acycloguanosin) was purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. It was dissolved in dimethyl sulphoxide (DMSO) to make stock solution of 7 mg/ml and further diluted in culture medium to prepare working concentration (s) prior to use. The final concentration of DMSO was < 0.1% in various assays where acyclovir was used.
High performance liquid chromatography analysis
The Reverse Phase High Performance Liquid Chromatography (HPLC; LC Agilent, Agilent Technologies, Boblingen, Germany) of the plant extract was performed using a Reverse phase XTerra RP 18 column (4.6 × 250 mm, 5 μm; Waters Corporation, Milford, USA) and pure compounds (chebulagic and chebulinic acids) were used as reference standards. HPLC grade solvents were purchased from Merck, Mumbai, India. The solvent system used was 0.01% orthophosphoric acid in water : acetonitrile (80 : 20) with a flow rate of 1 ml/min. The peaks were detected at 272 nm. The extract was prepared at a concentration of 5 mg/ml and the standards at 1 mg/ml in the mobile phase solvent system and 20 μl of each was injected for analysis. The data was processed using open lab software (Agilent Technologies).
Cells and viruses
African green monkey kidney cells (Vero cells) were obtained from National Centre of Cell Science, Pune, India and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Inc.) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz beit HaemeK, Israel) and an antibiotic-antimycotic cocktail [Penicillin (100 units/ml), Streptomycin (100 μg/ml) and Amphotericin B (250 ng/ml); Pen-Strep-Ampho sol, Biological Industries]. For HSV-2 G strain (VR-734; ATCC, Rockville, USA) production, the Vero cell culture was maintained at 37 °C in a humidified atmosphere of 5% CO2. The HSV-2 strain was propagated in the 25-cm2 tissue culture flask for 72 h at 37 °C in CO2 incubator at multiplicity of infection (MOI) of 0.01 PFU/cell . After three cycles of freezing/thawing, the supernatant was titrated on the basis of Plaque Forming Unit (PFU) as previously described  and stored in aliquots at −80 °C until use.
The cytotoxicity of the 50% ethanolic T. chebula extract, chebulagic acid, chebulinic acid and acyclovir was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich Inc.] assay . In brief, Vero cells (1.25 × 104/well) were seeded in 96-well cell culture plates (Greiner Bio-One, GmbH, Frickenhausen, Germany) and grown overnight at 37 °C in humidified atmosphere of 5% CO2. After 24 h, cells were treated with varying concentrations of the T. chebula extract, chebulagic acid, chebulinic acid and acyclovir in duplicate along with vehicle [dimethyl sulfoxide (DMSO) and/or ethanol] control for 48 h. Negative control included cells with medium only. After incubation, 20 μl of MTT (5 mg/ml in 50 mM PBS) was added per well and incubated for 4 h at 37 °C, followed by addition of MTT solvent (100 μl/well; 0.04 N HCl in absolute isopropanol). The absorbance (OD) was read at 540 nm with reference filter at 630 nm by using microplate spectrophotometer (ELX 800MS; BioTek Instrument Inc., Vermont, USA). Percent viability was calculated by dividing the OD obtained in treatment group by OD in the respective vehicle control multiplied by 100.
Anti-HSV-2 activity of the extract of T. chebula, chebulagic acid and chebulinic acid
Direct anti-viral activity assay
To evaluate direct inactivation of HSV-2 by 50% ethanolic T. chebula extract, chebulagic acid and chebulinic acid, HSV-2 virus (100 PFU) was pre-incubated with sub-toxic serial concentrations of the T. chebula extract, chebulagic acid, chebulinic acid and acyclovir at 37 °C for 1 h. Subsequently, the contents were added to the confluent Vero cells monolayer growing in a 24-well cell culture plate (Corning Incorporated Costar, NY, USA) followed by incubation at 37 °C for 1 h under the humidified 5% CO2 atmosphere. After virus adsorption, the supernatant was aspirated and infected cells were washed twice with serum free DMEM and overlaid with 1% low melting point (LMP) agarose overlay medium. After 48 h incubation, cells were fixed with 10% formaldehyde (in 50 mM PBS), stained with 0.2% crystal violet and number of plaques counted. The percent plaque reduction was calculated as 100 – [(P
C) × 100], where P
T and P
C refer to the number of plaques in the presence and absence of the compound, respectively. The direct anti-viral activity was measured by plaque reduction as compared to the vehicle treated virus control. The minimal concentration of the compound required to inhibit 50% of plaque numbers (IC50) was calculated by regression analysis of the dose–response curves of the extract/compounds.
HSV-2 attachment and penetration assay
The attachment and penetration assays were performed essentially as described previously [26, 27] with slight modifications. In brief, for attachment assay, confluent Vero cells grown in 24-well cell culture plates were pre-chilled at 4 °C for 1 h. The medium was aspirated and virus (100 PFU/well) was added in the presence or absence of the varying concentrations of 50% ethanolic T. chebula extract, chebulagic acid, chebulinic acid and acyclovir to the Vero cells and incubated for 3 h at 4 °C. Subsequently, cells were washed twice with plain DMEM medium, and processed as described in direct anti-viral activity assay to determine IC50. In the penetration assay, HSV-2 virus (100 PFU/well) was added to the pre-chilled confluent monolayer of Vero cells growing in 24-well culture plate for 3 h at 4 °C to allow attachment. The medium was replaced with pre-warmed fresh medium containing varying concentrations of test extract/compounds and incubated at 37 °C to maximize virus penetration. After 30 min, the infected monolayer cultures were treated with warm PBS (pH 3.0) for 1 min to inactivate the non-penetrated viruses. After washing three times with serum-free medium, cells were overlaid with 1% LMP agarose and processed to quantitate the number of plaques after 48 h incubation as described above.
Post-infection virus plaque reduction assay
The anti-HSV-2 activity of 50% ethanolic T. chebula extract, chebulagic acid, chebulinic acid and acyclovir has been evaluated by using post-infection plaque reduction assay . In brief, Vero cells (8 × 104/well) were seeded in 24-well cell culture plate and incubated at 37 °C in a humidified atmosphere of 5% CO2 to form complete monolayer. After incubation, medium was removed and cells were infected with HSV-2 virus (100 PFU/well) for 1 h at 37 °C under humidified atmosphere of 5% CO2. After virus adsorption, viral inocula was subsequently removed by washing the cells twice with fresh serum free DMEM and cells were overlaid with 1% LMP agarose, containing varying concentrations (less than calculated CC50 values) of the 50% ethanolic extract from T. chebula, chebulagic acid and chebulinic acid. Acyclovir was used as positive control and agar overlay containing appropriate solvents (DMSO/ethanol), used to prepare test compounds comprises the vehicle control. After 48 h incubation, plates were processed and number of plaques counted.
The values are expressed as mean ± standard error mean of three/four independent experiments performed in duplicate. For determination of the CC50 and IC50 values, nonlinear regression of concentration-response curves were prepared using GraphPad Prism 4 (GraphPad Software Inc., CA, USA). The statistical significance in reduction of plaques in presence of T. chebula extract, chebulagic acid, chebulinic acid and acyclovir versus respective vehicle treated virus control was determined by one-way ANOVA and p < 0.05 was considered statistically significant. Significance among various treatment groups was further analysed by using Tukey post-hoc test.