Abstract: Background and Objective: Human Herpes Virus-3 (Varicella Zoster Virus) causes Chickenpox in childhood and reactivates after decades from its latency to cause Herpes Zoster. The present study is an in vitro antiviral investigation on the leaves of Punica granatum L., against the clinical isolates of Human Herpes Virus-3 in comparison with acyclovir. Materials and Methods: Aqueous, ethanolic and aqueous ethanolic extracts were prepared by lyophilization process and were subjected to in vitro cytotoxicity assay in HEp-2 cells to estimate the maximum non-toxic concentration for the in vitro antiviral evaluation against the clinical isolates of HHV-3 in HEp-2 cells using the post-incubation assay. The structural features of leaf chemicals were docked with the HHV-3 protease through discovery studio. Results: Among the efficacious aqueous and ethanolic extracts tested, aqueous extract was superior than ethanolic extract in inhibiting the HHV-3 induced CPE at its MIC 15.625 μg mL–1 for the HHV-3 isolated from Chickenpox and 31.25 μg mL–1 for Zoster isolate. The efficacy of in vitro antiviral results of aqueous extract was comparable with that of the standard drug acyclovir. The leaf phytochemicals were found to interact with the protease of HHV-3 and the results are interpreted. Conclusion: The lyophilized aqueous extract from the leaves of Punica granatum L., had promising activity against HHV-3 and the leaf compound Apigenin was found to interact with the protease of HHV-3 with an interaction energy of -318.299 kcal moL–1.
INTRODUCTION
Human Herpes Virus-3 (HHV-3) usually called as Varicella Zoster Virus (VZV) is an alpha DNA virus of the Herpesviridae family. HHV-3 spreads through aerosolized droplets and undergoes latency in the dorsal root ganglions. The HHV-3 primarily causes Chickenpox in children and reactivates from its latency to cause Herpes Zoster in adults. However, recent epidemiologic studies conducted across different geographic regions have pointed out that Chickenpox was predominantly seen among children in temperate countries1-4 whereas the adults and adolescents in tropical regions are at higher risk of acquiring infection5-7. In spite of this Herpes Zoster has become a viral infection of all age groups among the Indian population8-9.
Nucleoside analogues namely the acyclovir and its derivatives are widely/being/used to treat HHV-3 infections. These drugs require viral encoded thymidine kinase (TK) for its phosphorylation and inactivate the viral DNA polymerase by competing with guanosine nucleosides that eventually results in the inhibition of viral DNA replication. However, these anti-virals are associated with side effects such as headache, vomiting and neurotoxic psychological effects10, renal dysfunction11 and nephropathy12. There is a possibility of eliminating unabsorbed drug in the urine due to inadequate oral bio-availability.
Furthermore, HHV-3 can cause severe and chronic infections in immuno-compromised patients making the lesions refractory to the acyclovir treatment. In such clinical conditions, repeated dosage of acyclovir renders resistance mechanism and hence there is a probability of patients to develop renal dysfunction and renal failure13-15. Cross resistance of HHV-3 to other antiviral drugs namely the Valacyclovir and penciclovir that require the viral thymidine kinase for its phosphorylation is also reported in the literature16,17. The pyrophosphate inhibitor Foscarnet (Phosphano Formic Acid, PFA) is currently employed as an alternate antiviral drug to treat acyclovir resistant HHV-3 infections. However, resistance was reported to foscarnet in HIV patients as well18-20.
Besides all these issues, the FDA has approved two live attenuated vaccines for HHV-3 infections namely the varivax for Chickenpox and zostavax for Herpes Zoster. Though these vaccines have reduced the disease incidence, the immunity generated is short lived for 1 year. Besides, the live attenuated Oka vaccine strain can cause vaccine associated break through varicella and zoster21,22.
Therefore, the need for the development of a potential antiviral drug with equal efficacy or amplified potential than the antiviral drug acyclovir is in demand. Phyto kingdom remains the source of potential anti-microbials for various infections and its usage has been in practice since the antique civilization. Globally substantial investments have been made on the traditional herbal medicinal research and the annual market for these herbal medicinal products approaches US dollar 60 billion. Antiviral research has been focused on various medicinal plants and its phytochemicals as an alternate source of antiviral for other groups of herpes viruses namely the HHV-1, HHV-2, HHV-4 and HHV-5. However, very limited antiviral research publications are available on the usefulness of drugs from plant origin as an effective antiviral drug23-30 for Human Herpes Virus-3. Also the antiviral potency in the leaves of Punica granatum L., is yet to be investigated for any other viruses especially against the HHV-3. Hence this study was undertaken to explore the antiviral efficacy of Punica plant leaves on the clinical isolates of HHV-3.
MATERIALS AND METHODS
CELLS and viruses: Human Epithelial diploid cells (HEp-2) were procured from National Centre for Cell Sciences (NCCS) Pune and were successfully propagated in MEM supplemented with FBS, antibiotics and antifungal agents under 5% CO2 atmosphere at 37°C. The clinical isolates of HHV-3 from Chickenpox and Herpes Zoster lesions were isolated in HEp-2 cells at 33°C. The isolated strains of HHV-3 were confirmed by conventional PCR technique targeting the IE62 gene. The amplified IE62 gene of the HHV-3 isolates were then run in 2% agarose gel and the gel was documented using Bio-Rad Gel documentation system. The tissue culture infectious dose fifty (TCID50) was estimated by Reed and Muench31 method. The viral stocks were stored at -80°C till use.
Collection and processing of plant leaves: The leaves of Punica granatum L., (semi wild) were collected from a small village called Nathanankudi near to Chidambaram (Fig. 1). The leaves were identified and authenticated by the Department of Plant Biology and Plant Biotechnology at Presidency College, Chennai. The collected leaves were initially washed in sterile double distilled water and shade dried. The dried leaves were then finely grounded in a mixer and sieved to fine powder. The leaf powder was then stored in an air tight container till its use.
Preparation of lyophilized extracts: The lyophilized form of aqueous, ethanol and aqueous ethanol extracts from the leaves of Punica granatum L., were prepared by adopting the procedure described by Kothandan and Swaminathan32.
| Fig. 1: | Leaves of Punica granatum L. |
| Fig. 2(a-c): | Lyophilized extracts from the leaves of Punica granatum L. |
Briefly 20 g of leaf powder was soaked in 100 mL of sterile double distilled water (aqueous 100%) and kept for overnight at 4°C. The same procedure was adopted for ethanolic (100%) and aqueous ethanolic extracts in which the ethanol and water were added in the ratio of 1: 1 (V/V). The infusions in the aqueous, ethanolic and aqueous ethanolic extracts were squeezed and filtered separately using a gauze cloth. The crude extract was then centrifuged and the clarified supernatant was filtered using a membrane filter apparatus with 0.22 μm Millipore filter. The filtered extracts were loaded into sterile flasks and lyophilized (Fig. 2).
In vitro cytotoxicity assay: The in vitro cytotoxicity of the lyophilized extracts in comparison with the standard drug acyclovir was done by adopting the procedure followed by Serkedjieva and Ivancheva33.
In brief a 96 well microtitre plate was seeded with 100 μL of HEp-2 cell suspension and replenished with 100 μL of 10% growth medium. The plate was then allowed to reach overnight confluence at 37°C under 5% CO2 humidified atmosphere. The stock solution of the extracts and the standard drug acyclovir were prepared and each of the extract was serially diluted through double dilution technique. Hence, the drug concentrations 500, 250, 125, 62.5, 31.25, 15.625, 7.8 and 3.9 μg mL–1 were achieved. The growth medium from the confluent titre plate was emptied with careful pipetting without disturbing the confluent layer and 100 μL of the respective drug dilutions were added into the corresponding wells. About 100 μL of 2% maintenance medium was then added and the titre plate was incubated at 37°C under 5% CO2 atmosphere for 120 h. The results were observed for every 24 h to record the in vitro cytotoxic effects such as unusual morphological variations or changes in cellular shape and size if any. The maximum drug concentration in which there was no absolute in vitro cellular toxicity was considered as maximum non-toxic concentration (MNTC) of the lyophilized extracts and the standard drug acyclovir.
In vitro antiviral assay: The in vitro antiviral assay of the lyophilized extracts in comparison with the standard drug acyclovir was done by following the procedure suggested by Yarmolinsky et al.26 in triplicates.
The antiviral assay was performed individually for the clinical isolates of HHV-3 isolated from Chickenpox and Herpes Zoster through post incubation assay. In short, a 96 well titer plate was seeded accordingly to the above said procedure and was then incubated at 37°C under 5% CO2 atmosphere in order to reach the confluence. The monolayered wells were infected with TCID50 dose with the exclusion of cell control wells in which 100 μL of 2% maintenance medium was added. The virus control well was also set up as positive control. The monolayer in wells was allowed for 1 h viral adsorption at 33°C in 5% CO2 humidified atmosphere. After adsorption, the unadsorbed viral particles were removed and 100 μL of the respective maximum nontoxic concentration of the serially diluted lyophilized extracts and the standard drug acyclovir were added into the corresponding wells. The entire set up was incubated at 33°C under 5% CO2 atmosphere in a humidified condition for 120 h (5 days). The plates were observed under an inverted phase contrast microscope (Nikon Ti-eclipse 100) for every 24 h from the first day of the antiviral evaluation till the end of the experiment to check for the presence or absence of virus induced CPE and the results were recorded with appropriate interpretation. The least drug concentration at which there was no observable viral induced CPE comparatively with cell control and virus control was estimated as the Minimum Inhibitory Concentration (MIC) of the respective drug. The experiment was done in triplicates and the values are represented in mean±SD.
In silico docking study: The protein structure of HHV-3 protease was retrieved from PDB (ID: 1VZV). The ligand and crystallographic water molecules were removed and loop refinement was carried out to screen the violations and determination of disallowed amino-acids in the protein. Ligand molecules were prepared and energy was minimized using CHARMm force field in Discovery Studio. The active site was predicted in Discovery Studio version 4.0 and the binding site was defined by current selection method that creates a sphere around the centroid of selected binding site points with the radius adjusted by increasing the sphere radius by 1 A°. Molecular docking was performed by flexible docking analysis. The docked poses were analyzed for docking score, binding energy and hydrogen bond interactions.
RESULTS
In vitro isolation of HHV-3: The inoculated flasks produced CPE consisting of alteration in the cellular morphology, followed by degeneration and disintegration of cells at the end of 120 h. The CPE produced by the clinical isolates of HHV-3 after 120 h in comparison with control cells was shown in Fig. 3a-c.
The amplified IE62 gene through Polymerase Chain Reaction has produced a PCR product size of >100 bp and the gel image was shown in Fig. 4.
In vitro cytotoxicity assay: The cytotoxic and non-cytotoxic concentrations of the lyophilized leaf extracts are tabulated the following Table 1 and the corresponding images of its cytotoxicity are given in Fig. 5.
| Fig. 3(a-c): | HEp-2 cell control in comparison with inoculated cells after 120 h (Magnification: 40X) (a) HEp-2 cell control, (b) CPE caused by HHV-3 isolate from chickenpox and (c) CPE caused by HHV-3 isolated from zoster |
The arrow in b and c indicate the typical CPE of HHV-3 in HEp-2 cells consisting of alteration in cell morphology and degeneration of cells at the end of 120 h |
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| Table 1: | In vitro cytotoxicity of Punica leaf extracts and acyclovir |
| T: Presence of toxicity, NT: Presence of non-toxicity, PT: Presence of partial toxicity | |
| Table 2: | Antiviral activity of the lyophilized extracts of Punica leaves to HHV-3 isolated chickenpox and zoster |
| VZV: Varicella zoster virus | |
| Table 3: | Best 5 lead molecules and their important properties |
| Fig. 4: | Gel image of amplified IE62 gene of HHV-3 isolated from chickenpox and herpes zoster 2% agarose gel electrophoresis image with the amplified HHV-3 IE62 gene of size ~100 bp isolated from Chickenpox and Herpes Zoster |
| Lane a: Positive control, Lane b: Amplified IE62 gene of HHV-3 isolated from Chickenpox, Lane c: Amplified IE62 gene of HHV-3 isolated from Zoster and Lane d: 100 basepair ladder | |
In vitro anti-viral assay: Among the lyophilized leaf extracts from Punica granatum L., the aqueous extract exhibited a predominant antiviral activity to the HHV-3 clinical isolates at its MIC 15.625 and 31.25μg mL–1, respectively. The ethanolic extract inhibited the HHV-3 isolates at 62.5 μg mL–1 while the aqueous ethanolic extract inhibited the HHV-3 isolated from chickenpox at 125 μg mL–1 and partially at the HHV-3 isolated from zoster at 125 μg mL–1. The standard drug acyclovir inhibited the HHV-3 isolates at 15.625 and 31.25 μg mL–1.
The detailed antiviral profile of Punica leaf extract and acyclovir were tabulated in Table 2 and illustrated in Fig. 6 and 7.
In silico docking study: Nine phytochemicals were chosen and docked with the target protein protease. All the 9 compounds interacted with the protease and only 3 compounds were scrutinized based on their energy interaction namely the apigenin followed by luteolin and granatin A. The binding energy of the phytochemicals with the VZV protease are given in Table 3 and in Fig. 8.
| Fig. 5(a-d): | In vitro cytotoxicity of lyophilized extracts from the leaves of Punica granatum L. (Magnification: 40X) HEp-2 cells showing cytotoxicity of Punica leaf extracts (a) Partial toxicity of aqueous extract at 500 μg mL–1, (b) Toxic concentration of ethanolic extract at 250 μg mL–1, (c) Toxic concentration of aqueous ethanolic extract at 500 μg mL–1 and (d) Non-toxicity of acyclovir at 500 μg mL–1 |
| Fig. 6(a-f): | MIC of lyophilized extracts from the leaves of Punica granatum L. and acyclovir to HHV-3 isolated from chickenpox in comparison with virus and cell control (Magnification:40X), In vitro antiviral results to Punica leaf extracts against HHV-3 isolated from chickenpox in HEp-2 cells (a, b and c shows MIC of aqueous, ethanolic and aqueous ethanolic extracts at (a) Aqueous15.625 μg mL–1, (b) Ethanol 62.5 μg mL–1, (c) Aqueous ethanol 125 μg mL–1, (d) MIC of acyclovir at 15.625 μg mL–1, (e) Cell control and (f) Virus control |
DISCUSSION
Anti-viral studies on Human Herpes Virus-3 were limited with special reference to plants namely the Pongamia pinnata24, Ficus benjamina25, Passiflora edulis28 and also the antiviral efficacy residing in the Punica plant leaves was annulled in the literature. Hence the research objective was framed with an in vitro antiviral screening of Punica leaves against the clinical isolates of HHV-3 isolated from Chickenpox and Herpes Zoster.
The HHV-3 was isolated from vesicular fluid lesions at 33°C in HEp-2 cells however the authors O’Neil et al.34 and Ozaki et al.35 propagated HHV-3 in human embryonic cells (MRC-5) at 37°C. We confirmed the HHV-3 isolates using conventional PCR technique targeting IE62 gene amplification which supported the results of Loparev et al.36 in which the VZV isolates were identified by IE62 gene amplification PCR technique.
The lyophilized Punica leaf extracts exhibited least in vitro cytotoxicity to the HEp-2 cells as it is evidenced from their maximum non-toxic concentration (MNTC).
| Fig. 7(a-f): | MIC of lyophilized extracts from the leaves of Punica granatum L. and acyclovir to HHV-3 isolated from Zoster in comparison with virus and cell control (Magnification:40X), in vitro antiviral results to Punica leaf extracts against HHV-3 isolated from zoster in HEp-2 cells, a, b and c shows MIC of aqueous, ethanolic and aqueous ethanolic extracts at (a) Aqueous 31.25 μg mL–1, (b) Ethanol 62.5 μg mL–1, (c) Aqueous ethanol 125 μg mL–1, (d) MIC of acyclovir at 31.25 μg mL–1, (e) Cell control and (f) Virus control |
| Fig. 8(a-c): | Docking of top lead phytochemicals with the active site residues of HHV-3 protease. Interaction between the lead phytochemicals of Punica with HHV-3 protease (a) Apigenin, (b) Luteolin and (c) Granatin A |
The green colour indicates the amino acids involved in the hydrogen bond formation, orange colour show the amino acids that forms pi pi interaction, thick wire frame structure depicts the ligand |
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The MNTC of acyclovir was 500 μg mL–1 which was a fold higher than the in vitro non toxic concentration of acyclovir reported by Shebl et al.27 which was 250 μg mL–1 in vero cells.
In our observation we found that the lyophilized aqueous extract from the leaves of Punica granatum L., exhibited appreciable and superior antiviral activity to both the clinical isolates of HHV-3 and was highly effective in preventing the HHV-3 induced cytopathic effect at its MIC 15.625 μg mL–1 (HHV-3 isolated from Chickenpox) and at 31.2 5μg mL–1 (HHV-3 isolated from Zoster), respectively, whose inhibitory activity was much superior to the anti-HHV-3 activity of the licorice powder extract containing 125 μg of glycyrrhizin observed by Shebl et al.27. Additionally, the aqueous leaf extract of Punica showed a comparable antiviral inhibition with the standard acyclovir whose MIC was 15.625 and 31.25 μg mL–1 for the HHV-3 isolates. Besides these the ethanol and the aqueous ethanol extracts of Punica granatum L., also inhibited the HHV-3 isolates slightly at higher drug concentrations.
The crude ethanolic extract prepared from the fruit rind of Punica granatum inhibited HHV-1 in the adsorption stage with an IC50 value of 37.7±6.7 μg mL–1 in vero cells37. Though this study supports the antiviral potency of Punica granatum, our finding is novel since it has revealed the anti HHV-3 activity in the leaves of Punica granatum L. The leaf extracts possibly would have caused an interruption in the replication of HHV-3 as the antiviral assay was done and evaluated post infection till the final day of observation.
Furthermore, we have attempted to screen the active phytochemicals which were reported to be present on the leaves of Punica granatum L., through in silico docking study which plays an important role in rational drug design38,39. This has inspired us to perform a docking study for the phytochemicals from the leaves of Punica granatum L., with the target protein, the HHV-3 protease, a major protein involved in the capsid assembly and DNA packaging of the virus.
This has resulted in the identification of Apigenin which had the highest binding affinity against the protease of VZV with a binding energy of -318.299 Kcal moL–1 forming hydrogen bond with GLY146, Leu212, Arg148 and Arg147 and the bond length falls between 1.9-3.4 Å followed by Luteolin and Granatin A with a binding energy of -219.607 Kcal mol–1 and -174.581 Lcal mol–1. This energy interaction score was effective than Erysenegalensein E with binding affinity of -114.4 using iGEMDOCK and it formed hydrogen bond40 with ILE 63 and ILE 64.
CONCLUSION
The plant Punica granatum L., holds potential antiviral activity against the HHV-3 and the phytochemicals present in the leaves interacted with the protease. Thus we conclude that the plant Punica granatum L. has to be further evaluated on the aspect of isolating the potential phytochemicals whose antiviral efficacy against the HHV-3 should be investigated with special reference to the drug should resistant Human Herpes Virus-3.
SIGNIFICANCE OF STATEMENT
This study is the first comprehensive study on evaluating the in vitro antiviral efficacy of Punica granatum L., to HHV-3 clinical isolates from Chickenpox and Herpes Zoster in HEp-2 cells that can be beneficial for the development of novel potential drug against the infections caused by human Herpes Virus-3. This study will help to uncover the areas of antiviral drug development that many researchers were not able to explore.
ACKNOWLEDGMENT
The authors are extremely thankful to the Department of Science and Technology, Govt of India for providing an excellent laboratory facility to the PG and Research Department of Microbiology and Biotechnology, Presidency College, Chennai-05 for carrying out the research activities. The authors are grateful to late Mr. N. Angamuthu. M.A., for his help rendered during the collection of plant leaves.