key: cord-1004726-uqm35x4b authors: Upadhyay, Saurabh; Tripathi, Praveen K.; Singh, Manju; Raghavendhar, Siva; Bhardwaj, Mohit; Patel, Ashok K. title: Evaluation of medicinal herbs as a potential therapeutic option against SARS‐CoV‐2 targeting its main protease date: 2020-08-04 journal: Phytother Res DOI: 10.1002/ptr.6802 sha: bc105f2e89f8d1fbca901385b9374b3d41204410 doc_id: 1004726 cord_uid: uqm35x4b The COVID‐19 disease caused by the SARS‐CoV‐2 has emerged as a worldwide pandemic and caused huge damage to the lives and economy of more than hundred countries. As on May 10, 2020, more than 4,153,300 people stand infected from the virus due to an unprecedented rate of transmission and 282,700 have lost their lives because of the disease. In this context, medicinal plants may provide a way to treat the disease by targeting specific essential proteins of the virus. We screened about 51 medicinal plants and found that Tea (Camellia sinensis) and Haritaki (Terminalia chebula) has potential against SARS‐COV‐2 3CL(pro), with an IC(50) for Green Tea as 8.9 ± 0.5 μg/ml and Haritaki 8.8 ± 0.5 μg/ml. The in‐silico studies suggested that Tea component Thearubigins binds to the cysteine 145 of protease active site and could be a pharmacoactive molecule. We predict that the inhibition in protease activity may be able to halt the SARS‐CoV‐2 replication cycle and therefore, we propose Green Tea, Black Tea, and Haritaki plant extracts as potential therapeutic candidates for SARS‐CoV‐2 infection. Further investigation on role of bioactive constituents of extracts is needed to establish the molecular basis of inhibition and towards expedited drug discovery. the development of drugs targeting the virus. The 3CL pro is very important for virus to replicate and propagate and its inhibitors may halt the disease at an early stage of replication. It recognises and cleaves the virus non-structural polyprotein at 12 sites. One of the site on the polyprotein includes Leu-Gln*(Ser, Ala, Gly) (* denotes the cleavage site). To impede virus replication, multiple strategies are being employed. Medicinal plants could be harnessed as one of the safest means of medication and have been used traditionally for various manifestations. The anti-viral activities of several plants have been elucidated so far (Newman & Cragg, 2007) . The role of plant lectins as anti-SARS-CoV-2 has been proposed (Capell et al., 2020) . We have shortlisted the medicinal plants which are reported to possess the anti-viral, anti-oxidant, and anti-inflammatory properties. Further, the aqueous extracts were screened for 3CL pro inhibition, invitro. We found that tea (Camellia sinensis) and Haritaki (Terminalia chebula) as potential inhibitors of 3CL pro activity of SARS-CoV-2. Additonally, we compared the activity inhibition by different types of Teas and found that Green Tea and Black Tea displayed comparable potencies to inhibit the 3CL pro activity of SARS-CoV-2. The pharmacoactive molecules like catechins, present in Green Tea, have been shown to possess multiple health-boosting activities like hypoglycemic, hypotensive, anti-tumor, anti-oxidative, hypolipidemic, anti-bacterial, and anti-viral effects (Cabrera, Artacho, & Giménez, 2006; Cooper, Morré, & Morré, 2005a , 2005b Mahmood et al., 2016) . Tea has a virucidal effect on the Zika virus (Carneiro, Batista, Braga, Nogueira, & Rahal, 2016) . Similarly, the Prodelphinidin B-2 3 0 -O-Gallate molecule has shown an anti-viral effect against the Herpes Simplex Virus type 2 (HSV-2; Cheng, Lin, & Lin, 2002) . Further, the Green Tea extract has also been demonstrated as a safe personal hygiene option against viral infections like human influenza A/H1N1 virus (Lee, Jang, Kim, Kim, & Seong, 2018) . Interestingly, Theaflavin-3, 3 0 -digallate (TF3) which is produced from polymerization and oxidization of the epicatechin gallate and EGCG during fermentation of fresh Tea leaves, displayed 3CL pro inhibition (Chen et al., 2005) . Similarly, Haritaki is also believed to possess anti-bacterial and antiviral activities (Lee et al., 2011) . Furthermore, it has been reported beneficial against viruses like HSV-2 (Kesharwani et al., 2017) and Influenza A virus (Li et al., 2020) . Epigallocathechin-3-gallate present in the C. sinensis inhibited the replication of hepatitis-B virus (Karamese, Aydogdu, Karamese, Altoparlak, & Gundogdu, 2015) . We assessed the proteolytic activity of the 3CL pro protein and found that the extracts of Tea (Green Tea and Black Tea) and Haritaki displayed a significant reduction in proteolytic activity. The 3CL pro gene (Wuhan-Hu-1, NC_045512.2, region 10, 972) which is 918 base pair synthesized in pEXA2 vector was procured from Eurofins Genomics, Japan and was used for sub cloning in protein expression vector since it has been designed with BamHI and XhoI restriction enzyme sites. The digested desired gene insert was purified and ligated to a modified pET-28a vector between BamHI and XhoI sites. This modified vector contains the sequences coding for PreScission, flag tag, and hexahistidine. The cloned gene in the pET-28a vector was subjected to protein expression. After analyzing the soluble expressions from various expressing strains of Escherichia coli, we chose BL-21DE3 Rosetta RIL cells for expressing the protein. The 3CL pro protein tagged with 6-histidines at N-terminal end was expressed using an auto-induction method of protein production with appropriate antibiotics kanamycin (50 μg/ml) and chloramphenicol (34 μg/ml; Studier, 2005) . The cells were first cultured for 2 hr at +37 C, further grown at +18 C for 18 hr. The cells were harvested by centrifugation, and the pellet was re-suspended in buffer A (50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol, 10 mM Imidazole) containing protease inhibitors 1 mM DTT, 0.5 mM PMSF and DNAseI 0.001 mg/ml with 5 mM MgCl 2 . The cells were lysed by adding lysozyme having final concentration 1 mg/ml and disrupted by sonication on ice with 30 s ON and 30 s OFF for 10 min, and the lysate was clarified by centrifugation at 10,000g for 30 min. The clear lysate was applied onto a Ni-NTA column pre-equilibrated with buffer A on ÄKTA™ start FPLC system (GE Healthcare). The column was washed with 100 mL of Buffer A, and 3CL pro protein was eluted with gradient elution 0-500 mM imidazole in buffer A. The fractions were analyzed on SDS-PAGE for purity. The eluted fractions were pooled, dialyzed, and purified through QFF anion exchange chromatography with a linear gradient of NaCl (0-500 mM). The purified protein was homogeneous with about >95% purity and confirmed with western blot analysis. Fractions containing 3CL pro protein were pooled and cleaved with recombinant Precision protease at +4 C overnight. The overnight cleaved protein was reapplied on the Ni-NTA column and the unbound sample has been collected. It was concentrated, buffer exchanged (20 mM Tris HCl pH 7.5, 150 mM NaCl, 10% Glycerol, and 1 mM DTT), flash frozen and stored at −80 C for all biochemical and biophysical studies. The protease activity of the purified 3CL pro was tested using a modified protocol from previously reported protocol using casein as a substrate (Banik, Biswas, & Karmakar, 2018; Cupp-Enyard, 2008) . The 3CL pro enzyme is non-specific as it cleaves at 12 different locations in SARS-CoV-2. Casein substrate contains peptide sequences that can be cleaved by 3CL pro of SARS-CoV-2. Hence, we have used the casein substrate for activity measurements. The substrate stock of 0.65% The plants aqueous extracts were evaluated for their potential to inhibit the enzyme activity. The 10 μM of 3CL pro enzyme and 137 μM of casein substrate were used to study the inhibition effects of extracts. Different concentrations (0-450 μg/ml) of extracts were screened to assess their inhibitory potential. Two sets of reactions were prepared, one for reaction with extracts having 3CL pro and another without 3CL pro , as control. The rate of inhibition and potential efficacy was calculated by standard kinetics curve. Fluorescence quenching was performed to analyze the effect of plants extracts on the 3CL pro structural dynamics. The tertiary structure perturbations post-interaction to ligands present in the extracts were assessed using intrinsic fluorescence of 3CL pro . Ten μM of 3CL pro protein was incubated with or without extracts at different concentrations (0--80 μg/ml), fluorescence intensity was monitored on Cary Eclipse fluorescence spectrophotometer (Agilent technologies). The parameters used for excitation wavelength is 295 nm and emission scan between 310 and 400 nm with the slit width for excitation (5 nm) and emission (10 nm). The experiments were performed at controlled temperature 25 C. The change in intensity or wavelength was used for the protein ligand interaction analysis. To decipher the difference in efficacies to inhibit the viral protease by universally used Tea, we compared the enzymatic inhibition for Green Tea as wells as Black Tea mentioned above using the 10 μM of 3CL pro at varying concentrations of Tea. Moreover, we have analyzed the effective protease inhibition by both tea with Haritaki. All the parameters like substrate, enzyme, and inhibitor concentrations were kept constant during the reactions. The crystal structure PDB 6LU7 of the 3CL pro was taken from the RCSB databank. The active site prediction was performed using the metaPocket2.0 server and the best hit near to catalytic site having His41 and Cys145 was fixed. This was used for site-specific docking The 3CL pro was expressed in E. coli expression system and purified to homogeneity with affinity and anion exchange chromatography ( Figure 1B) . The confirmation of the protein was done using western blot analysis using anti-His antibody ( Figure 1C ). The activity assay to validate the enzymatic property of recombinantly purified 3CL pro was established by using the casein substrate. The calculation of K m for casein was performed by the varying casein substrate and it was obtained as 137 μM (Figure 2 ). The plant extracts were screened for protease activity and inhibition potential is shown in Table 1 . Results shown a significant reduction in protease activity of 3CL pro by Green Tea (C. sinensis) and Haritaki (T. chebula). The IC 50 values of Green Tea and Haritaki was found to be 8.9 ± 0.5 and 8.8 ± 0.5 μg/ml, respectively (Figure 3) . The aqueous extracts of plants Mulethi (Glycyrrhiza glabra), Tulsi (Ocimum tenuiflorum), Giloy (Tinospora cordifolia), and Neem (Azadirachta indica) have less efficiency in inhibiting the protease activity of 3CL pro . These plants could not inhibit the 3CL pro protease activity to 50% even with 450 μg/ml of the plant extracts. The interactions of the active moieties present in the extracts interacted with the 3CL pro protein and resulted in the tertiary structure perturbation. The fluorescence quenching was observed as depicted by the decrease in the intensity of the fluorescence spectra ( Figure 4 ). The comparison in the 3CL pro protease inhibition efficacies of Green Tea, Black Tea, and Haritaki were studied. Both Tea (Green Tea and Black tea) and Haritaki displayed comparable inhibition to protease activity; 50% inhibition at 10 μg/ml and more than 90% inhibition beyond 40 μg/ml. The results suggest that either form of Tea and Haritaki can be a potential anti-viral agent against the SARS-CoV-2 ( Figure 5 ). The The COVID-19 disease has garnered immediate attention from scientists and medical experts worldwide. The hunt for an effective therapy and potential intervention against SARS-CoV-2 has become a primary goal. In this context, we report medicinal plant extracts from Tea (Green Tea and Black Tea) and Haritaki as potential therapeutic options for the management of infection. The Ab-Initio drug discovery programs require huge capital investment and some undesirous events like immunogenicity, toxicity, and efficacy remain a matter of concern in the clinical trials (Corsello et al., 2017 (Varga et al., 2020) . The inflammatory and oxidative damage to the cells conferred by the virus can be countered by the anti-inflammatory and anti-oxidative properties of these plants (Cabrera et al., 2006; Chan, Lim, & Chew, 2007; Karamese et al., 2015; Lee et al., 2018) . Also, as these plants are routinely used for treatment in different kinds of diseases and disorders, the risks of cytotoxic effects are also relatively low (Cheng et al., 2002; Karamese et al., 2015) . The anti-viral properties of Tea have been demonstrated against the influenza virus, zika virus, hepatitis-B virus, and SARS virus (Carneiro et al., 2016; Mahmood et al., 2016) . We found that components present in the aqueous extract of Tea and Haritaki inhibited the 3CL pro of SARS-CoV-2 ( Figure 3) . However, the anti-viral effect of these extracts and bioactive constituents needs to be validated in the in-vivo studies. The activity data ( Figure 5 ) suggests that both Tea and Haritaki might be good source for the design of potential inhibitors for the 3CL pro of SARS-CoV-2. We have tried to investigate the potential and probable molecules that are abundantly present in Tea. Among the virtually screened molecules, thearubigin, quercetin-3-O-rutinoside, and hesperidin dis- Coronavirus main proteinase (3CLpro) structure: Basis for design of anti-SARS drugs Extraction, purification, and activity of protease from the leaves of Moringa oleifera Beneficial effects of green tea-A review Potential applications of plant biotechnology against SARS-CoV-2 The green tea molecule EGCG inhibits Zika virus entry. Virology Antioxidant activity of Camellia sinensis leaves and tea from a lowland plantation in Malaysia. 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The Lancet Liver injury during highly pathogenic human coronavirus infections Evaluation of medicinal herbs as a potential therapeutic option against SARS-CoV-2 targeting its main protease Authors declare no conflict of interest. https://orcid.org/0000-0001-6134-7794