key: cord-0307638-bozl1erw
authors: Damle, Latha; Damle, Hrishikesh; Ganju, Shiban; Chandrashekar, C; Bharath, BR
title: In silico, In vitro Screening of Plant Extracts for Anti-SARS-CoV-2 Activity and Evaluation of Their Acute and Sub-Acute Toxicity
date: 2021-09-08
journal: bioRxiv
DOI: 10.1101/2021.09.07.459230
sha: 9ff6d78f2efea5de42d299c0fbe6623d6d4204b2
doc_id: 307638
cord_uid: bozl1erw

Background In the absence of a specific drug for COVID 19, treatment with plant extracts could be an option worthy of further investigation. Purpose To screen the phytochemicals for Anti-SARS-CoV-2 in silico and evaluate their safety and efficacy in vitro and in vivo. Method The phytochemicals for Anti-SARS-CoV-2 were screened in silico using molecular docking. The hits generated from in silico screening were subjected for extraction, isolation and purification. The anti-SARS-CoV-2 activity of plant extracts of Z. piperitum (ATRI-CoV-E1), W. somnifera (ATRI-CoV-E2), C. inophyllum (ATRI-CoV-E3), A. paniculata (ATRI-CoV-E4), and C. Asiatica (ATRI-CoV-E5). The in vitro safety and anti-SARS-CoV-2 activity of plant extracts were performed in VeroE6 cells using Remdesivir as positive control. The acute and sub-acute toxicity study was performed in Wistar male and female rats. Results The percentage of cell viability for ATRI-COV-E4, ATRI-COV-E5 and ATRI-COV-E2 treated VeroE6 cells were remarkably good on the 24th and 48th hour of treatment. The in vitro anti-SARS-CoV-2 activity of ATRI-COV-E4, ATRI-COV-E5 and ATRI-COV-E2 were significant for both E gene and N gene. The percentage of SARS-CoV-2 inhibition for ATRI-COV-E4 was better than Remdesivir. For E gene and N gene, Remdesivir showed IC50 of 0.15 µM and 0.11 µM respectively, For E gene and N gene, ATRI-CoV-E4 showed IC50 of 1.18 µg and 1.16 µg respectively. Taking the clue from in vitro findings, the plant extracts A. paniculata (ATRI-COV-E4), W. somnifera extract (ATRI-COV-E5) and C. asiatica extract (ATRI-COV-E2) were combined (ATRICOV 452) and evaluated for acute and sub-acute toxicity in Wistar male and female rats. No statistically significant difference in haematological, biochemical and histopathological parameters were noticed. Conclusion The study demonstrated the Anti-SARS-CoV-2 activity in vitro and safety of plant extracts in both in vitro and in vivo experimental conditions.

Currently, there is no specific drug for SARS-CoV-2 infection. Efforts are being made globally to identify agents for preventive, supportive and therapeutic care. Many plants used in CA has been used in the Ayurvedic tradition of India and listed in an ancient Indian medical text called 'Sushruta Samhita' (Chopra et al. 1986 ; Diwan et al. 1991) . CA is also used in Indonesian islands. The primary active constituents of CA are triterpenoids, which include asiaticosides, madecassoside and madasiatic acid (Singh & Rastogi 1961) . CA in combination with Madura cochinchinensis (Lour.) Corner, and Mangifera indica was evaluated for anti-herpes simplex virus (HSV) activity and treatment for mucocutaneous HSV infection (Yoosook et al. 2000) .

AP is a common medicinal plant used in asian countries to treat various diseases like pharyngitis, tonsillitis, upper respiratory tract infection and acute monocytic leukemia (Thamlikitkul et al. 1991 The compounds inophyllum B, inophyllum P isolated from CI were found to have anti-HIV-1 reverse transcriptase activity (Laure et al, 2008) . The compound calanolide A from CI has also been reported to inhibit HIV-1 replication (Kashman et al. 1992 ).

Considering the availability and sustainability of medicinal plants, 521 Indian medicinal plants were identified and the structure of phytochemicals from each plant were manually curated from a reported research article of high quality. The biological active conformations of 13,105 curated plant molecules were obtained through ligand preparation process. The structure of plant molecules were sketched using a 2D sketcher tool available in Schrodinger maestro and subjected for ligand minimisation using Ligprep (LigPrep, version 2.3, Schrödinger, LLC, New York, 2009). During the minimisation, force field OPLS_2005 were assigned and stereoisomers were calculated after retaining specific chiralities.

The proteins involved in, entry, replication and assembly of SARS-CoV-2 in human host such as Table 1 . The three-dimensional structure of target proteins were prepared using the protein preparation wizard workflow available in the Schrödinger 2019-2 glide module. During the protein preparation, the crystallographic water molecules with less than three H-bonds were deleted and hydrogen atoms corresponding to neutral pH were added in consideration of ionisation states of amino acids. Following this, coordinates for any missing side-chain atoms were added using Prime v4.0, Schrödinger 2019-2 and energy minimisation was performed using the OPLS_2005 force field.

The active site on the prepared receptor was confined with a 10Å radius by centering around selected residues for S-Protein and centering co-crystal molecules for other three target proteins. This generated a grid box measuring 20X20X20Å. The docking of phytochemicals over target proteins was performed using Glide v7.8. Schrödinger 2019-2 in different modes sequentially with defined and incremental precision, and computational time differences. The phytochemicals with best docked conformer and minimum glide energy were shortlisted for the evaluation of in vitro Anti-SARS-CoV-2 activity.

The aerial parts of ZP, CI, AP and CA, and root part of WS were procured from Herbo Ayurvedics Calicut Kerala and authenticated by using herbarium, maintained in our laboratory. Organic solvents such as Hexane, EtOAc, DCM, MeOH, and Silica gel (60-120 mesh) for sample preparation were procured by Avra Synthesis Pvt Ltd. Silica high performance Gold columns were purchased from Redi sepRf, Teledyne ISCO.

Solvents for HPLC, HPLC-grade Acetonitrile, Water and MeOH were procured from Sigma-Aldrich (Merck).

The plant materials were subjected for extraction using conventional bio-separation techniques.The 500g of plant material was packed in Soxhlet extractor, defatted by using hexane for about four hours. The dried defatted plant material was again subjected for extraction using 100% Methanol. The extraction was performed until the solvent in the thimble became colourless. The total filtrate was then concentrated by rotary evaporation under vacuum to obtain the ethanol crude extracts and the yield of the crude extracts was measured ( Table 2 ). The 0.1g of crude extracts were dissolved in 10ml of methanol and subjected for thin layer chromatography using silica gel as stationary phase and different sets of solvent system as mobile phase ( Table 3) .

The crude extracts were subjected for flash chromatography (Teledyne ISCO, CombiFlash NEXTGEN 300 system) using Redisep C-18 86G column for semi purification. Acetonitrile, water, and methanol were the solvents used at different concentrations volume by volume. The semi purified fractions were further subjected to HPLC for next level purification. The HPLC system used was WatersTM e2695 consisting of a quaternary pump, an automatic degasser, and an auto-sampler, having PDA and UV detector, column used was Shimpack C-18 (ODS) (250mm*4.6,5µm) column. The molecular weight of purified fractions were identified using the Shimadzu2020 LC-MS system. The temperature of the column was set to 40 °C and a 5 µL aliquot of the sample solution was injected at specific flow rate vis., 0.3ml/min,1ml/min, 1ml/min, 1ml/min, and 0.3ml/min for ZP, WS, CI, AP and CA fractions respectively. The gradient system followed for each sample was 70:30 for Water: Acetonitrile, 100% for Acetonitrile, and 70:30 for 0.1% formic acid in Water: 0.1% formic acid in MeOH. The positive ionization mode was used for compound ionization. The quantification was obtained in multiple reaction monitoring (MRM) mode with the precursor-product ion transition. High-purity nitrogen (N2) was used as the nebulizing gas, and nitrogen (N2) was used as the drying gas at a flow rate of 15 L/min. The mass spectrometer was operated at a capillary voltage of 4000 V, source temperature of 100 °C and desolvation temperature of 350 °C.

Before sending the pure fractions for In-vitro studies, they were allotted code names: Z. piperitum as ATRI-CoV-E1, W. somnifera as ATRI-CoV-E2, C. inophyllum as ATRI-CoV-E3), A. paniculata as ATRI-CoV-E4), C. Asiatica as ATRI-CoV-E5. Then they were sent to Regional Centre for Biotechnology, Faridabad (RCBF) for in vitro studies.

In vitro cytotoxicity and Anti-SARS-CoV-2 activity was carried out at RCBF, using the methods described below:

The cytotoxicity assay was done in a 96-well plate, with 3 wells for each sample. 1x10e4 VeroE6 cells were plated per well and incubated at 37-degree C overnight for the monolayer formation. Next day, cells were incubated with the test substance (TS) at the indicated concentration (Table 4) in 0.5% Dimethyl sulfoxide (DMSO). The control cells were incubated without TS.

The treated incubated cells were collected at 24 and 48 hours and stained with Hoechst 33342 and Sytox orange dye. 16 images per well were taken at 10X, which covered 90% of well area using ImageXpress Microconfocal (Molecular Devices). The percentage of cell viability was measured by counting the number of dead cells in the Sytox image as described by Tan et al. 2004 .

The prepared cells were infected with SARS-CoV2 at a multiplicity of infection (MOI) of 0.01. Experimental animals were housed in groups of three for acute toxicity study and a group of six animals for sub-acute toxicity study as per standard laboratory conditions. Detail of animals is given in Table 5 .

The experimental female rats were bred and reared at the animal care facility in NUCARE, Nitte University, Mangalore and experiment was carried out in the same facility. Adult albino Wistar non-pregnant female rats weighing 200−220 g and between 8-10 weeks of age were used.

The test substance was prepared by standard procedure. Extracts weighing 833.33mg ATRI-CoV-E2, ATRI-CoV-E4 and ATRI-CoV-E5 each were placed in mortar. Small quantity of water was added, and the sample was triturated using a pestle. Once all samples were wet, remaining quantity of the water was added slowly to make up the total volume to 5ml, while trituration continued to get simple suspension and the dose formula was labeled as ATRICOV 452.

Three male rats and three female mice were selected and fasted overnight (12 h). Animals were not given food but had free access to water. The single dose of ATRICOV 452 at 2000 mg/kg was administered using an oral gavage needle. Animals were observed every 30 minutes for the first 4 hours, and daily thereafter, for a total of 14 days for toxic manifestations by observing clinical signs (salivation, excitability, draping, tremors, twitching, rising fur) and death. The weight and food consumption of the experimental animals were recorded on the first day and 14th day. After 14 days, they were sacrificed.

As per OECD (2001) recommendations, the dose at which the extract is not expected to produce mortality or severe acute toxicity is called the starting dose of the sub-acute toxicity (Tilahun et al. 2020 ).

The 2000 mg/kg body weight of the ATRICOV 452 was the highest dose determined. A total of 72 Wistar male and female rats were divided into 6 groups as shown in Table 6 ; each group consisted of 6 male and 6 female rats. The groups five and six were called satellite groups. Each group was given 1 mL/100 g of body weight of ATRICOV 452 at concentrations of 0.00 mg/kg (control), 200 mg/kg (low dose), 400 mg/kg (medium dose), 800mg/kg (high dose), 0.00 mg/kg (control reversal) and 800mg/kg (high dose reversal). All the six groups received the treatment for 28 days. The weight and food consumption of animals were measured weekly till the 28th day. On day 29, control, low, medium and high dose groups were sacrificed.

The satellite groups were maintained for another 14 days without any treatment to observe the reversible untoward effect of treatment if any. On day 43, satellite groups were sacrificed for hematological, biochemical and histopathological examinations.

1 mL of blood samples were collected from each rat. The hematological examination was carried out to measure differences in Red Blood Cell (RBC), Packed cell volume (PCV), Haemoglobin, Neutrophils, Basophils, Lymphocytes and Monocytes counts.

The levels of glucose, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Total Protein (TP), Albumin, Total cholesterol (T.Chol), Triglyceride (TG), Blood urea nitrogen (BUN), creatinine, total bilirubin, calcium, phosphate, sodium and potassium were measured.

All rats were sacrificed using Isoflurane anesthesia at the end of the 14th day for acute toxicity and at the end of the 28th day for sub-acute toxicity studies. Macroscopic and histopathological examinations were done for liver, kidney and jejunum.

The data were analysed using Graph Pad Prism version 6 Software. For each group, one way ANOVA analysis was done followed by Dunnett's comparison test. The data were expressed as mean ± standard deviation (SD). A p-value < 0.05 was considered as statistically significant.

The virtual screening of 13,105 phytochemicals at different precision levels like high throughput virtual screening (HTVS), standard precision (SP) and extra precision (XP) has suggested the interaction of phytochemicals from ZP, WS, CI, AP and CA with SARS-CoV-2 drug targets.

The SARS-CoV-2 enters the host by interacting its S-Protein with a host receptor called ACE2. The S-protein consists of two subunits, S1 as the receptor-binding domain (RBD) and S2 subunit as the fusion RdRp through non-obligate RNA chain termination mechanism, and the crystal structure of the template-RTP RdRp complex provides an experimental model to rationalise how these drugs inhibit the SARS-CoV-2

RdRp activity. In the present study, the conformation of the Andrographidine C from AP has also shown nonobligate RNA chain termination type of interaction (Figure 2 ) and the interaction was similar to the cocrystal ( Figure 2D ) . The orientation of Andrographidine C was parallel to the base Uracil 20 (U 20) of RNA and the interaction was found stable due to pi-pi stacking between coumarin moiety and Uracil base.

Additionally, the H-Bonds formed by electron donating hydroxyl groups present in the Andrographidine C have strengthened the interaction with minimum docking energy ( Table 7) . are involved in the formation of the S1 subsite. The lactam at P1 of N3 inserts into the S1 subsite and forms a hydrogen bond with His163 of protomer A. Similarly, the Coagulin G from WS, Zanthoxyl flavone from ZP and 3,4,6,8-Tetrahydroxy-7-(3-hydroxy-3-methylbutyl)-9H-xanthen-9-one from CI showed interaction with His163 (Figure 3, 4 and 5) . The interactions were favoured by other residues in the cleft ( Table 7) .

The fractions of interest were purified using flash chromatography and confirmed for the presence of phytochemicals Zanthoxyl flavone, Coagulin G, 3,4,6,8-Tetrahydroxy-7-(3-hydroxy-3-methylbutyl)-9Hxanthen-9-one, Andrographidine C, and Kaempferol in ZP, WS, CI, AP and CA methanol extracts respectively.

The flash chromatographic fractionation of crude methanol extracts of ZP has led to four different polarity fractions (Figure 6A ) at different RT ( Table 8) The flash chromatographic fractionation of crude methanol extracts of AP has led to two different polarity fractions (Figure 9A ) at different RT ( Table 8 ). The HPLC chromatogram for the second fraction obtained from flash chromatography at Rt2 of 25min has shown a high resolution peak at the retention time of 6.5min (Figure 9B ) based on UV wavelength at 210 nm. The HPLC fraction obtained at the retention time of 6.5min has shown a peak corresponding 463 m/z in MS spectra ( Figure 9C ) has confirmed the presence of Andrographidine C in AP methanol extract. Hence, it is inferred that the Andrographidine C in the extract can interact with RdRp of SARS-CoV-2 and offer the Anti-SARS-CoV-2 activity.

The flash chromatographic fractionation of crude methanol extracts of CA has led to four different polarity fractions (Figure 10A ) at different RT ( Table 8) . The HPLC chromatogram for the second fraction obtained from flash chromatography at Rt2 of 30min has shown a high resolution peak at the retention time of 8.71min (Figure 10B ) based on UV wavelength at 370nm. The HPLC fraction obtained at the retention time of 8.71min has shown a peak corresponding 290 m/z in MS spectra ( Figure 10C ) has confirmed the presence of Kaempferol C in CA methanol extract. Hence, it is inferred that, the Kaempferol in the extract can interact with S-Protein of SARS-CoV-2 and offer the Anti-SARS-CoV-2 activity.

The extracts ATRI-CoV-E3, ATRI-CoV-E4 and ATRI-CoV-E5 showed promotion of VeroE6 cell growth with 110.31%, 103.31% and 106.59% cell viability at 24hr post treatment respectively. ATRI-CoV-E3, ATRI-CoV-E4 and ATRI-CoV-E5 showed 88.72%, 83.53% and 94.48% of cell viability at 48hr post treatment. The extracts ATRI-CoV-E1 and ATRI-CoV-E2 showed 88.75% and 77.05%, 85.35% and 88.11% of cell viability at 24hr and 48hr post treatment respectively. Remdesivir at 10µM concentration showed 99.23% and 94.37 % cell viability at 24 and 48 hr respectively as shown in Figure 10 . TS showed greater cell viability after 48 hr of treatment as compared to Remdesivir.

For E gene, at 24 hr, Remdesevir, ATRI-CoV-E1, ATRI-CoV-E2, ATRI-CoV-E3, ATRI-CoV-E4 and (Figure 12) .

ATRI-CoV-E4 and ATRI-CoV-E5 were subjected for seven-point IC50 estimation to identify the minimum dose required to inhibit the replication of SARS-CoV-2.

The dose-response curves were obtained using seven different concentrations of Remdesivir, ATRI-CoV-E2, ATRI-CoV-E4, and ATRI-CoV-E5. For E gene and N gene, Remdesivir showed IC50 of 0.15 µM and 0.11 µM respectively (Figure 13A and 13B) , For E gene and N gene, ATRI-CoV-E4 showed IC50 of 1.18 µg and 1.16 µg respectively (Figure 14A and 14B) . Remdesivir and ATRI-CoV-E4 showed significant delta CT for both E and N genes as shown in Table 9 . The Delta CT of ATRI-CoV-E2 and ATRI-CoV-E5 were moderate. In IC50 estimation, ATRI-CoV-E2 and ATRI-CoV-E5 did not show consistent CT values, 

No abnormalities were observed in clinical signs as shown in Table 10 . Body weights of each animal were recorded prior to the administration of ATRICOV 452 (Day 0) and at the end of the experiment (Day 15). The highest dose of ATRICOV 452 2000 mg/kg body weight did not induce acute toxicity in rats during the study and the ATRICOV 452 fed animals showed healthy growth during the observation period as shown in Table 11 .

During the study period, clinical signs of all treated animals were found normal as tabulated in Table   12 . The food intake and body weights increased as shown in Table 13 .

The administrations of ATRICOV 452 in high dose showed increase in RBC and reticulocyte count in both male (8.707 × 10 6 /mm3 Cells ) and female (7.278 × 10 6 /mm3) compared with the control (4.75 × 10 6 /mm3) however it was not statistically significant (Figure 15 and Figure 16) . showed no change (Figure 15 and Figure 16 ).

Biochemical analysis was conducted on day 29 and 43; the following observations were made by comparing with control groups.

The male animals receiving high dose treatment showed statistically significant (P<0.05) decrease in glucose. The low and medium dose groups showed no statistically significant change (Figure 17 ).

Male animals receiving low, medium and high dose treatment showed significant (P<0.0001, P<0.01, P<0.001) increase in AST (Figure 17) . However, such changes were not evident in female animals ( Figure 18 ).

Male animals receiving medium and high dose showed significant (P<0.001) decrease in ALP ( Figure 17 ).

Male and animals receiving low, medium and high dose treatment showed statistically significant (P<0.05) increase in total protein value (Figure 17and Figure 18 ).

Male animals receiving low and medium dose treatment showed significant (P<0.05) increase in total cholesterol (Figure 17 ).

Female animals receiving low and high dose treatment showed significant (P<0.05) increase in triglyceride ( Figure 18 ).

Female animals receiving medium dose treatment showed significant ( P<0.01) decrease in BUN (Figure 18 ).

Male and female animals receiving low and medium dose treatment showed significant (P<0.0001) decrease in creatinine (Figure 17 and Figure 18 ).

Female animals receiving high dose treatment show significant (P<0.05) decrease in total bilirubin ( Figure 18 ).

Male animals receiving low dose treatment showed significant (P<0.01) decreases in phosphorus ( Figure 17 ).

Bone marrow analysis was conducted on day 29 and day 43. At low dose, treatment groups showed mild reactive plasmacytosis. At medium dose, treatment groups showed mild reactive myeloid hyperplasia.

At high dose, treatment groups showed mild to moderate myeloid hyperplasia and mild reactive plasmacytosis. The control groups showed no bone marrow change. (Table 14) .

Urine analysis showed no changes on day 29 and on day 43.

The organ weight comparison was done with control groups. Liver weight of male animals receiving high dose treatment showed significant (P<0.05) decrease. Spleen of male animals receiving low dose and female animals receiving medium dose showed significant (P<0.05) increase in weight. Lungs of female animals receiving medium dose showed significant (P<0.05) decrease (Table 15 ).

For the rest of the organs (Kidneys, Heart, Brain, Testes and Adrenal) no changes in the weight was observed.

No histopathological changes were observed in the liver, kidney, and jejunum of animals treated with low, medium and high dose (Figures 19, Figures 20 and Figures 21) .

We have demonstrated that the in vitro Anti-SARS-CoV-2 activity of plant extracts ATRI-CoV-E4, ATRI-CoV-E5, and ATRI-CoV-E2, showed no cytotoxicity in VeroE6 cells. The seven-point IC50 study of extracts showed consistent Anti-SARS-CoV-2 activity of ATRI-CoV-E4. The in vivo acute and sub-acute toxicity study of ATRICOV 452 did not show any significant toxicity. Further, in vivo Anti-SARS-CoV-2 activity needs to be demonstrated and worth investigating the test samples in clinical trials.

Competing interests: Latha Damle is the founder of Atrimed Biotech LLP and holds equity in Atrimed Pharmaceuticals. Shiban Ganju and Hrishikesh Damle hold equity shares in Atrimed Pharmaceuticals.

The study was sponsored by Atrimed Pharmaceuticals Pvt. Ltd and no external funding was received for the study. All the six bone marrow smears from the second control group 6CF1 to 6CF6 show features suggestive of normal haematopoiesis.

All the six bone marrow smears in the Low Dose group from 6LM1 to 6LM6 and 6LF1 to 6LF6

show features of MILD REACTIVE PLASMACYTOSIS.

No pathological changes seen.

All the six bone marrow smears in the medium dose group form 6MM1 to 6MM6 and 6MF1 to 6MF6 show features indicative of MILD REACTIVE MYELOID HYPERPLASIA.

No specific pathological changes seen.

All the six bone marrow smears in the high dose group from 6HM1 to 6HM6 and 6HF1 to 6HF6

show features of MILD TO MODERATE MYELOID HYPERPLASIA AND MILD REACTIVE

No specific pathological changes seen.

All the six bone marrow smears in the reversal control group 6CRM1 to 6CRM6 and 6CRF1 to 6CRF6 show features of NORMAL HAEMATOPOIESIS.

No pathological changes seen.

All the six bone marrow smears in the reversal high dose control group 6HRM1 to 6HRM6 and 6HRF1 to 6HRF6 show features indicative of NORMAL HAEMATOPOIESIS / MILD REACTIVE

No other haematopoitic abnormalities seen in any of these smears in the reversal groups. 

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