Antiviral potential of selected Indian medicinal (ayurvedic) plants against Herpes Simplex Virus 1 and 2

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) belong to diverse family of Herpesviridae, causing oral herpes lesions (HSV-1), genital lesions (HSV-2), meningitis, and encephalitis.[1] Primary infection within the genital tract, followed by an established latency phase give rise to life-long infection in humans.[2] Treatment of herpes infection is thus cause of major concern owing to the difficulty in eliminating it from the ganglion, high cost of treatment, increasing drug resistance, and association with HIV-1.[3–7] We investigated the plants selected on the basis of their traditional usage by comparing the disease symptomatology and recommended use from the texts of Ayurveda and used them in the forms as used traditionally. Primary screening of 24 extracts of seven plants was done by cytopathic effect inhibition (CPE) assay followed by dose response, antiviral, and cytotoxicity assay conducted at eight concentrations from 3.125 to 400 μg/ml. Five extracts, Punica granatum aqueous extract (PGAE), peals powder (PGPP), freeze-dried powder (PGFP), Ocimum sanctum methanol extract (OSME), and Azadirachta indica leaves powder (AZLP) demonstrated favorable activity in primary screening. PGFP showed antiviral activity at 50, 100, and 200 μg/ml concentrations followed by PGAE, which showed highest selectivity index (SI) (14 and 12.5) followed by PGFP (7.6 and 12.9). Thus, aqueous extract and freeze-dried powder of P. grantum have potential to be further explored for its possible anti-HSV activity. The plants selected, namely, A. indica, Curcuma longa, Punica granatum, O. sanctum, Nyctanthes arbortristis, Carica papaya, and Holarrhena antidysentrica were collected from Gujarat State of India. The plant species were taxonomically identified and confirmed using morphological and anatomical techniques. They were authenticated at the Botanical Survey of India, Pune and voucher specimens were deposited and standardized using High Performance Thin Layer Chromatography (HPTLC) method (data not provided here) [Figures ​[Figures11 and ​and22]. Figure 1 In-vitro screening of plant extracts against HSV-1 and HSV-2 Figure 2 Twenty-four extracts of seven plants were screened for their antiviral activity in-vitro. The figure depicts the plants and the methodology used for screening We screened 24 extracts of 7 selected plants, of which 7 were water extracts, 3 were methanol extracts, and 14 were powders of various plant parts [Table 1]. The herb powders were prepared by drying and grinding the plant materials. Water and methanol extracts were prepared from 500 g of fresh raw material of plant parts in Soxhlet extraction vessel under reflux with stirrer for 3 cycles with methanol or water. After each cycle, solvent was added to replenish the remaining volume and dried in a rotatory evaporator at temperature not exceeding 35°C. The stock solution of 20 mg/ml was prepared in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, USA) and subsequent serial dilutions were carried out in Dulbecco's modified eagle medium (DMEM) (GIBCO Cat. No. 12430) supplemented with 2% fetal bovine serum (FBS) (GIBCO Cat. No. 16000-044). The final concentration of DMSO was <0.2%.[8] Table 1 Details of the plant and their extracts screened against herpes virus All reagents and media for cell culture were purchased from GIBCO-BRL (Grand Island, NY, USA). Confluent monolayer of African Green Monkey kidney cell line (Vero) (ATCC CCL-81) was grown in T-25 flasks in a 5% carbon dioxide (CO2) incubator at 37°C for 48 hours, in DMEM supplemented with 10% FBS, penicillin G sodium 200 U/ml, streptomycin sulfate 200 mg/L, and amphotericin B 0.5 mg/L. HSV-1 clinical strain and HSV-2 ATCC G strain virus stocks were propagated in Vero cells and used at a titer of 1:10,000 for HSV-1 and 1:1000 for HSV-2 in all in-vitro experiments. Acyclovir (ACV) powder (Matrix Laboratories Limited, Hyderabad, India) was used as a standard antiviral drug at four different concentrations: 3.125, 6.25, 12.5, and 25 μg/ml for HSV-2 and 0.78, 1.56, 3.125, 6.25, and 12.5 μg/ml for HSV-1. It was dissolved with DMSO to give a stock concentration of 10 mg/ml and then diluted with DMEM-2% FBS (maintenance medium) before use. For all the assays, ACV control consisted of the cells, ACV at different concentrations: 3.125, 6.25, 12.5, and 25 μg/ml for HSV-2 and 0.78, 1.56, 3.125, 6.25, and 12.5 μg/ml for HSV-1 made by 1:2 serial dilutions and the virus suspension. Primary screening of these extracts was conducted by CPE assay at 100 μg/ml concentration against HSV-2 in triplicates. Confluent Vero cells were grown in 96-well microtiter plates (2 × 104 cells/100 μl) for 24 hours. Two-fold serial dilutions of the samples were prepared in DMEM with 2% FBS. The cell control consisted of 200 μl maintenance medium; the virus control consisted of 100 μl of maintenance medium and 100 μl of the virus suspension. After 1 hour incubation of the cells with different sample concentrations, the virus (HSV-2) was added to the plates and the plates were again incubated for 48 hours in 5% CO2 at 37°C. The plates were then washed with saline and stained with crystal violet and incubated for 30 min at room temperature after which the plates were gently washed under running tap water and left overnight for drying and visually analyzed (qualitatively).[9] Eight concentrations of extracts that exhibited activity in CPE assay were assayed from 3.125 μg/ml onwards higher in the ratio 1:2 till 400 μg/ml. After 1 hour incubation of test samples with the cells, virus was added and the plates were incubated for 48 hours before washing with saline and staining with crystal violet. After overnight drying, the plates were analyzed.[10,11] The extracts that exhibited activity in CPE assay were analyzed at eight concentrations for antiviral assay. The assay plates contained virus and cell controls and the samples. The plates were then incubated for 48 hours after which the plates were washed with plain DMEM followed by the addition of tetrazolium compound MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent diluted 1:10 in 2% FBS-DMEM. After incubation of 4 hours, detergent was added to the wells and left overnight for incubation in a 5% CO2 at 37°C. The absorbance was then read with the help of a plate reader at a wavelength of 570 nm. The data were analyzed by plotting cell number versus absorbance, allowing quantification of changes in cell proliferation.[12,13]