The Antiviral Efficacy of Withania somnifera (Ashwagandha) against Hepatitis C Virus Activity: In Vitro and in Silico Study

Objective: Evaluation antiviral effects of Withania somnifera (Ashwagandha) leaf extract against HCV. Methods: cell proliferation was assessed using MTT assay after isolation of lymphocyte cells and treated with Ashwagandha water extract (ASH-WX) (6.25 mg/ml 100 mg/ml). Assessment of quantitative Real-time PCR, Colony forming assay, TNF-α and molecular docking studies after infection of normal lymphocyte cells with 1 ml (1.5 × 106 HCV) serum then incubated with ASH-WX at concentration 25 mg/ml & 50 mg/ml. Results: MTT assay revealed a significant increase (p < 0.001) in normal lymphocyte proliferation at all concentration’s particularity at 25 mg/ml with SI (6.06) and at 50 mg/ml with (5.8). While TNF-α significantly decreased following ASH-WX treatment compared with control untreated infected cells (p < 0.05). PCR results showed a marked viral load reduction after treatment by ASH-WX at concentration 25 mg/ml to 6.241 × 103 IU/mL. Colony formation assay test revealed colony formation reduction compared to positive untreated control. Molecular docking analysis revealed good prediction of binding between Ashwagandha and NS5B and PKN2 compared to Sovaldi. Conclusion: ASH-WX may be a powerful antiviral against HCV infection.


Introduction
Hepatitis C is an infectious disease caused by hepatitis C virus (HCV) that essentially influences the liver [1] [2]. The global prevalence of HCV infected adults is estimated at 2.5% (177.5 million) ranging from 2.9% in Africa to 1.3% in Americas, with a global viraemic positive cases of 67% (118.9 million), varying from 64.4% in Asia to 74.8% in Australia [3] [4]. Most of the cases are caused by HCV genotypes 1 (70%) and 4 and less frequently by genotypes 2 and 3 [5].
HCV is epidemic in Egypt that has the highest prevalence in the world (15%) [6]. Genotype type 4 is the most prevalent in Egypt of about 73% followed by genotype 1 (26%), whereas 15.7% of HCV infection in Egypt were mixed genotypes [7] [8]. There is no protective vaccine available for HCV treatment but there are several recent drugs that could be used as a treatment for HCV including pegylated interferon (PEG IFN), boceprevir, ribavirin, Sofosbuvir (Sovaldi) and telaprevir [9]. Every drug has its mechanism against HCV, for example, Pegylated IFN is used due to its increased stability in vivo that activates cellular antiviral responses. Approximately 50% of responders relapse will appear upon withdrawal of treatment. Ribavirin had a broad-spectrum activity against several RNA and DNA viruses, however the treatment of chronic HCV using ribavirin alone had no significant effect on HCV RNA levels, so it has been used in combination with IFN-alfa [10] [11] [12]. Sofosbuvir (Sovaldi) can mimic the physiological nucleotide and competitively blocks the NS5B polymerase which is one of the non-structural proteins essential for viral RNA replication and inhibits the HCV-RNA synthesis by RNA chain termination. Due to the high-cost and severe side effects of current HCV treatments such as fatigue, hematologic toxicity, ophthalmologic disorders, cardiac diseases, myocardial infarction and the probability of virus recurrence [12], scientists are in great need to find new agents that are less expensive and less non-toxic and highly effective in combating HCV. Natural products have been used as traditional medicines in many parts of the world like Egypt, China, Greece, and India since ancient times [13]. Ayurvedic medicine eliminates many symptoms of different human diseases, including infectious diseases, and has been used for thousands of years [14]. One of the important natural products is Withania somnifera. Withania somnifera belongs to family Solanaceae and is commonly known as Ashwagandha or Indian ginseng and considered as a valuable medicinal herb in the Ayurvedic and indigenous medical systems [15]. Ashwagandha and its pharmaceutical derivatives; Withaferin A (WA) have vital role as antiviral agents against different types of viruses like; Infectious Bursal Disease Virus (IBDV) [16], HIV-1 [17], HPV [18], HSV [16] and the only one study investigated the effect of WA against HCV where, Sen et al showed that WA inhibits phosphorylation of PKC substrate peptide HCV [19] and suppresses HCV replication. Therefore, the present study was conducted to check antioxidant and antiviral activity of Ashwagandha against HCV replication.

Proliferation Analysis by MTT Assay
Lymphocyte proliferation assay was used as an indicator of cellular immune

Assessment of Anti-Oxidants' Activities Using Colorimetric Analysis
Antioxidants are synthesized or natural compounds that may avoid or delay some kinds of cell damage [26]. Total antioxidant, Glutathione S transferase, and Glutathione reductase were measured in normal lymphocytes after isolation and treated with ASH-WX at different concentrations (25 mg/ml & 50 mg/ml) and incubation for 48 h at 37˚C in a 5% CO 2 incubator using colorimetric assay kits (Biodiagnostic, Giza, Egypt) following the manufacturer's instructions.

Lymphocyte Cell Infection with HCV Serum
After isolation of Lymphocyte from normal cells as described above, cells were

RNA Extraction
Total RNA from cultured lymphocyte cells was extracted using QIAamp® RNA Blood Mini Kit according to the manufacturer's instructions (QIAGEN, Hilden, Germany).

Quantitative Real Time RT-PCR
The RNA copy number of HCV in the supernatant of infected normal lympho-

Colony Forming Assay for HCV Replication
Colony-forming assays could be used as a pre-clinical tool to assess HCV colony formation as a reflection of the antiviral drugs effectiveness as previously described [27]. Briefly, lymphocyte normal cells (1 × 10 6 cell/ml) were isolated and infected with 1 ml (1.5 × 10 6 HCV) serum as described above, then treated in 24 well plate with ASH-WX at concentrations (25 mg/ml and 50 mg/ml), the cells were centrifuged and washed with 1.0 M PBS, pH 7.4. Coomassie blue stain (Coomassie® Brilliant blue G 250, Sigma-Aldrich, St Louis, MO, US) was added as follows: fixing solution: 50% methanol and 10% glacial acetic acid, staining solution: 0.1% Coomassie® Brilliant blue G 250, 50% methanol and 10% glacial acetic acid, storage solution (5% glacial acetic acid) on the cells. The cells were then incubated at 37˚C in a 5% CO 2 incubator with fixing solution for 1 h to overnight with gentle agitation, then staining solution was added for 20 minutes with gentle agitation and finally destaining solution was added, the solution was replenished several time until background of the gel was being fully destained and the cells (1 × 10 6 cell/ ml) were divided in 24 well plates. Finally, the cells in each panel were examined under an inverted microscope (ZeissAxio Vert.A1; Zeiss; Gottingen, Germany) at 40× magnification, morphological changes were observed, and cells were photographed using the digital camera of an inverted microscope (Color Digital Imaging-SPOT Idea 3MP).

Assessment of Protein Concentration of TNF-α, Using ELISA
Tumor necrosis factor (also known as TNF-α or cachectin) is one of the most vital cytokines which regulate the cell signaling. The TNF-α system is enhanced in patients with HCV chronic infection with high levels of circulating TNF-α and a parallel increase in the level of the soluble TNF receptors [28]. TNF-α levels were measured using an ELISA Kit (K0331131P; KomaBiotech, Seoul, South Korea) was measured in lymphocytes normal cells after isolation and infection with1ml (1.5 × 10 6 UI HCV). HCV serum as described above and treated with ASH-WX (25 mg/ml & 50 mg/ml) following the manufacturer's instructions.

Molecular Modelling (Docking Study)
The purpose of this study is to analyze the inhibitory action of variables between 2 study groups was done using Mann-Whitney U-test for independent samples while analysis of variance (ANOVA) with Bonferroni correction was done for more than 2 groups' comparison Two-tailed P-values < 0.001 were considered statistically significant.

Lymphocyte Proliferation Using MTT Assay
Proliferation of lymphocyte cells increased following treated with gradual concentrations (6.25 mg/ml -100 mg/ml) of ASH-WX, particularity at concentration 25 mg/ml ( Table 2). The mean of cell proliferation concentration was 0. 75 ± 0.01 with SI of 6.06 and at concentration 50 mg/ml of ASH-WX, the mean of cell proliferation concentration was 0.72 ± 0.05 with SI of 5.8. While the untreated cell proliferation concentration was 0.13 ± 0.04 with the stimulation index of 1. Also, increase the number of cells after incubation with ASH-WX at gradual concentration (6.25 mg/ml -100 mg/ml) for 24 h and 48 h compared to untreated cells was observed, particularity at concentration 25 mg/ml and 50 mg/ml of ASH-WX (10 g/100ml distillated water).

Assessment of Anti-Oxidant Activities
Normal lymphocyte cells treated with ASH-WX at different concentrations (25 mg/ml & 50 mg/ml) for 48 h revealed a significant increase in anti-oxidant activities compared with the untreated control cells (P < 0.001). The highest activity of total antioxidant (331.8 ± 9.6) was observed when lymphocyte normal cells were treated with 25 mg/ml of ASH-WX, while the highest activity of Glutathione reductase (946.3 ± 26.1) was observed when lymphocyte normal cells were treated with 50 mg/ml of ASH-WX. Glutathione-S-transfers revealed the highest activity (1534.6 ± 9.7) when treated with 25 mg/ml of ASH-WX ( Table 2, Table 3).

Real-Time PCR (Normal Infected Lymphocyte with HCV)
Real time PCR results revealed a reduction of the viral load from a very high viral titer which recorded 1.5 × 10 6 IU/mL to 3.71 × 10 5 IU/mL and after treatment by ASH-WX at concentration 25 mg/ml, viral load was reduced to 6.241 × 10 3 IU/mL and at concentration 50 mg/ml, viral load was reduced to 2.6878 × 10 4 IU/mL (Table 4).

Colony Formation Assay
Colony formation assay results revealed that ASH-WX enhanced reduction of HCV colony formation compared to untreated infected lymphocyte cells (positive control), where ASH-WX at 25 mg/ml had more significant effect on the reduction of colony formation than 50 mg/ml (Figure 1).

Human Tumor Necrosis Factor Alpha (TNF-α) Activity in Infected Lymphocytes with Hepatitis C Virus
ELISA analysis showed a significant decrease in the TNF-α concentration (P < 0.05) of infected lymphocytes cells with Hepatitis C virus treated with ASH-WX at concentrations (25 mg/ml and 50 mg/ml) for 48 h at 37˚C in a 5% CO 2 incubator compared with the control untreated cells (Figure 2).

Molecular Docking Study of Antiviral Activity
The binding mode of Sovaldi (1) and Ashwagandha (2) in the active sites of Human Protein Kinase N2, and NS5B, molecular docking was carried using the Glide software. Human Protein Kinase N2 can bind with Sovaldi (1) through two hydrogen bonds with Arg 917, Leu 918 ( Figure 3; Table 5). Ashwagandha (2) can form one hydrogen bond with Sep 755. The docking scores of Human protein kinase N2 (PKN2) with compounds Sovaldi (1) and Ashwagandha (2) were −2.002 and −3.474 kcal/mol, respectively. The binding mode of Sovaldi (1) with NS5B showed it can form four hydrogen bonds with Asn 142, Glu 398, Trp 397, Ser 39. It has a calculated docking score of −4.688 kcal/mol. The docking results also showed that Ashwagandha (2) having the highest docking score of −5.599 kcal/mol and maximum inhibitory activity with NS5B ( Figure 4, Table  5). It forms hydrogen bonds with Arg 394 and Asn 411.

Discussion
The present study investigated the anti-viral effects of Egyptian Ashwagandha leaves, a well-known herbal medicine that is full of anti-oxidants, against hepatitis C virus. The phytochemical analysis of Egyptian Ashwagandha leaves suggests that it belongs to chemotype III, which is different to the Indian Ashwagandha regarding the antioxidant activity [21] [30] [31]. To the best of our knowledge, this is the first investigation of this chemotype against HCV.
Our results showed increased proliferation of lymphocyte normal cells after These results agreed with previous studies which showed that Ashwagandha has powerful anti-oxidant action as it increased the levels of three natural anti-oxidants; superoxide dismutase, catalase, and glutathione peroxidase in the rat brains [34]. Moreover, agreed with Andallu and Radhika who reported that Ashwagandha is as an important medicinal plant that has good antioxidant potentials throughout its root [35].
The effect of ASH-WX on Hepatitis C virus revealed a reduction of the viral load in infected lymphocyte normal cells before treated with ASH-WX from a very high viral titer which recorded 1.5 × 10 6 IU/mL to 3.71 × 10 5 IU/mL then, colony forming assay was performed and the results showed that ASH-WX enhances reduction of colony formation compared to positive control. Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced in response to infectious pathogens. Previous studies demonstrated that the blood level of TNF-α is increased in HCV patients and that correlated with increase of HCV pathogenesis and the severity of liver diseases [36] [37]. Our results of effect of ASH-WX on TNF-α in infected lymphocyte normal cells with Hepatitis C revealed a significant decrease in the TNF-α activity in infected lymphocytes treated with ASH-WX compared to control with significant p value (<0.05) and that agreed with previous studies which demonstrated that ASH-WX has anti-inflammatory effects, specifically reducing gene expression of CCL2 and CCL5 in response to TNF-α stimulation [38]. Another study on effect of ASH-WX on TNF-α showed a significant decrease in the TNF-a concentration (P < 0.05) of HepG2 cells treated with ASH-WX at the IC50 concentration (5.0 mg/ml) for 48 h compared with the control untreated cells [21].
Based on the in-silico docking study between ASH-WX and Sovaldi, which an example of current drugs in HCV treatment, with Human protein kinase N2 (PKN2, PRKCL2) and NS5B revealed that Ashwagandha, has a better binding affinity and inhibitory activity against PKN2 and NS5B than Sovaldi. Our results on the effect of Ashwagandha on Hepatitis C replication agreed with previous studies that reported that Withaferin A has an effective role in suppression of HCV replication, where it inhibits phosphorylation of PKC substrate peptide HCV [19].

Conclusion
In conclusion, Ashwagandha (Withania somnifera) water extract is a powerful anti-oxidant and has antiviral properties in HCV infected lymphocyte cells. It might have potential as a promising anti-viral agent against HCV and these results should be confirmed in animal studies.