Computational screening of phytocompounds from Moringa oleifera leaf as potential inhibitors of SARS-CoV-2 Mpro


 Background: Coronavirus Disease (COVID-19), caused by novel SARS CoV-2 is rapidly spreading all over the World creating a global public health emergency at unprecedented levels. Till today, no effective treatments or vaccines against this global pandemic is reported and hence to identify lead compounds having potential action in controlling the spread the pandemic is a global concern. This study aimed at in silico screening of phytocompounds from M.oleiera leaf against novel SARS CoV-2 main protease (Mpro) through molecular docking. M.oleiera is an Indian medicinal plant as well as a vegetable, all parts of the plant is medicinally useful and is being used in many of the traditional and Ayurvedic medicinal preparations. Result: When the 19 compounds identified from M.oleifera leaf methanolic extract by Liquid Chromatography Mass Spectrometry (LCMS/MS) analysis and 5 FDA approved anti-viral drugs were screened in silico with SARS CoV-2 main protease (Mpro), the following compounds showed top interaction; apigenin-7-O-rutinoside (-8.8 kcal/mol), Mudanpioside (-8.3 kcal/mol), isoquercetin (-8 kcal/mol), isoquercitrin (-8 kcal/mol), quercetin (-7.8 kcal/mol) and dihydroquercetin (-7.8 kcal/mol). Anti-viral drugs: Raltegravir (-7.2 kcal/mol), Lopinavir-Ritonavir (-7.7 kcal/mol), maraviroc (-8.2 kcal/mol), Nelfinavir (-8.3 kcal/mol) and Tipranavir (-9.2 kcal/mol) also showed active interaction with Mpro. Preliminary phytochemical screening of methanol extract showed the presence of flavonoids, cardiac glycosides, phenols, coumarins, saponins, steroids and phytosteroids. In vitro antioxidant activity of methanolic extract of M.oleifera also showed greater activity, which would ameliorate the post-COVID secondary infection. Conclusion: Hence these compounds from M.oleifera, which are our diet based components, which can interact with the Mpro and curtail COVID-19 virus multiplication in the host cell.


Background
Corona viruses (SARS-CoV-2) are enveloped viruses belonging to the family Coronaviridae, and subfamily Coronavirinae. The virion of SARS-CoV-2 consists of crown shaped peplomers and possess positive stranded genome of ∼30kb size consisting of a 5' cap and 3' poly A tail. A helical capsid is also present within the viral membrane consisting of genomic RNA complexed with nucleocapsid. There are four genera of corona viruses; alpha, beta, gamma and delta. Alpha and beta causes infections in mammals while the other two affects birds and mammals commonly [1]. HCoV-229E, HKU1, HCoV-NL63 and HCoV-OC43 are the human corona viruses which cause mild upper respiratory infection to highly contagious Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS CoV) [2].
As per the recent report on 29 th July 2020 of COVID-19 by WHO total 16 558 289 were infected people and a total of 656 093 deaths globally (https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200729-covid- 19). The causative agent of ongoing COVID-19 pandemic is highly contagious novel Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2). No vaccines or effective treatments are available till today against this globally affected pandemic and scientists all over the World are investigating the possibility to develop vaccines and/or medicines. All the prevailing prophylaxis against SARS-CoV-2 is symptomatic to handle mild to severe forms of symptoms associated with the infection [3]. Hence the situation warrants discovery of potential medications to control SARS-CoV-2 pandemic from spreading.
The corona virus uses the heteromeric spike protein present on its surface to interact with the ACE2 (angiotensin-converting enzyme-2) abundantly present in many human cell types [4]. After entering the cell, two polyproteins encoding several crucial non-structural proteins from their genomic RNA is translated including Chymotrypsin-like protease (3CLpro) or main protease (M pro ) and papain like protease (P pro ) [5,6]. Open reading frames, ORFs, ORF1a and ORF1b of SARS CoV-2 and MERS CoV-2 encodes two cysteine proteases, viz; a papain-like protease (PL pro ) and a 3C like protease (main protease (M pro ) are encoded by ORF1a. PL pro is involved in the cleavage of rst three cutting sites of its polyprotein and M pro is involved in cleaving other eleven positions leads to the release of sixteen non-structured proteins (nsp). Since this autocleavage process is necessary for viral maturation and replication, M pro remains as the important drug target against COVID-19 infection [7]. In this study, we aimed to identify bioactive compounds from Moringa oleifera leaves, which bind with M pro of SARS CoV-2.

Plant material collection, Identi cation and preparation
The plant material was identi ed by DNA barcoding analysis and the voucher specimen and herbarium (No. 486) was deposited at St. Albert's College Herbarium (SAC), Ernakulam, Kerala, India. M.oleifera leaves were collected, shade dried and powdered. Soxhlet extraction was carried out by taking 20 g of the leaf powder in 1000 ml of methanol. The extract thus obtained was concentrated using rotary evaporator and lyophilized.

Identi cation of compounds fromoleifera methanolic extract by LCMS/MS Analysis
Bioactive compounds present in the extract was tentatively identi ed by LCMS/MS (Schimadzu) analysis. The column used was C18 and a gradient elution of 0.1% formic Acid in water and acetonitrile was employed. The ow rate was 0.3ml/min and the duration of the analysis was 30 minutes.

Molecular Docking Analysis
High-resolution three-dimensional X-ray crystal structure of SARS CoV M pro (PDB ID: 6Y2F) was downloaded from Protein Data Bank. Water molecules were removed and hydrogen atoms were added using PyMol software. The SDF structure of the selected ligands were downloaded from PubChem and they were converted to PDB format using Open babel software and then to PDBQT using AutoDock tools. The following compounds were identi ed tentatively by LCMS/MS analysis of M.oleifera methanolic extract, which were used for docking studies; Apigenin-7-O-rutinoside, Mudanpioside J, Isoquercetin, Isoquercitrin, Quercetin, Dihydroquercetin, Luteolin, Neochlorogenic acid, Vicenin 2, Catechin, Epicatechin, Rutin , Marumoside B, Scutellarin, Rhamnetin, Astragalin, Apiin, Marumoside A and Niazirin. The FDA approved drugs Nel navir, Raltegravir and Lopinavir-Ritonavir, maraviroc, Tipranavir were also docked against SARS CoV2 M Pro . Molecular docking analysis was done using AutoDock Vina [35].

Phytochemical Screening
Presence of avonoids, cardiac glycosides, phenols, coumarins, saponins, steroids and phytosteroids were detected from the methanolic extract of M.oleifera leaf.

Discussion Preliminary Phytochemical Screening
Potterat, 1997 [36] reported the presence of alkaloids, triterpenoids, avonoids, tannins, saponins, and glycosides in methanolic extract of M.oleifera. These are essential phytochemicals in the leaf that represent total phenolics, different enzymes (ascorbic acid oxidase, polyphenol oxidase, and catalase) and vitamins [37] , [38]. Phenolic compounds are also reported from moringa leaf extracts which act as natural antioxidants and serve as rst line defense against free radical induced cellular damage [39]. Flavonoids are the bioactive phytocomponents which are used for treatment of allergies, in ammation, ulcers, tumors and viral infections while saponins are reported to have antihypertensive and hypocholesteremic effects [10] , [40].

In vitro antioxidant activity
Most of the medicinal plants are a major source of phytochemicals with proven antioxidant property [41]. Moringa especially treated as a nutritive as well as a medicinal plant in the Indian traditional medicine. Results of the present study reveal that the antioxidant and anti-viral property of Moringa oleifera from a natural source. There was a 90% and 98.9% inhibition of DDPH free radical at a concentration of 20 μg/ml and 100μg/ml respectively, showing that even at a low dose of 20 μg/ml the moringa extract proved to be an effective antioxidant (table:2). Similar results were also reported by Fitriana et al., 2016 [42] revealing that the methanolic extract shows highest antioxidant activity. It is proven that avonoids and other phenolic compounds present in the plant have potent antioxidant and chelating property. According to Suphachai, 2014, [43] there exists a correlation between antioxidant activities with total phenolic compound. Methanolic extracts will have a total phenolic compound higher than the other solvents because methanol is the highest polar solvent which can pull out more polyphenol compounds. The presence of polyphenol compounds such as quercetin and kaempferol in the leaves may also account for greater antioxidant activity. The observed antioxidant property of the extract would ameliorate the post-COVID secondary infection. kcal/mol) but it was found to be a P-gp substrate, having low GI absorption and low bioavailability score. Whereas Mudanpioside J (-8.3 kcal/mol) is a monoterpene glycoside which also showed higher a nity with M pro than Raltegravir (-7.2 kcal/mol), Lopinavir-Ritonavir (-7.7 kcal/mol), Maraviroc (-8.2 kcal/mol) and showed same a nity as of nel navir (-8.3 kcal/mol). it was also found to have low GI absorption. Isoquercetin, hyperoside, quercetin and dihydroquercetin showed much higher binding a nity with M pro than the anti-viral drugs Raltegravir and Lopinavir-Ritonavir. Luteolin also exhibited binding a nity similar to the anti-viral drug Lopinavir-Ritonavir. Isoquercetin and hyperoside, showed low GI absorption where quercetin and dihydroquercetin had high GI absorption rate and none of them were P-gp substrate. All the anti-viral drugs except maraviroc were having low GI absorption. Also, all of them were P-gp substrate.

ADME Prediction and Molecular docking Analysis
All the other phytocompounds showed binding a nity greater than -7 kcal/mol, but with lesser a nity with M pro than the anti-viral drugs used here. Neochlorogenic acid (-7.6kcal/mol) is a phenolic acid which showed low GI absorption and low bioavailability score. Vicenin 2 (-7.6 kcal/mol), catechin (-7.5 kcal/mol), Epicatechin (-7.4 kcal/mol) and Rutin (-7.4 kcal/mol) are avonoids showed potential interaction by forming more hydrogen bonds with M pro . Catechin and epicatechin had high GI absorption and high bioavailability score unlike vicenin2.
Maramoside B is a phenol, showed a binding a nity of -7.3kcal/mol but GI absorption was low. Scutellarin (-7.3 kcal/mol), is a avonoid exhibited strong interaction with M pro but without forming any conventional hydrogen bonds. It also showed high GI absorption and bioavailability score. Rhamnetin (-7.2 kcal/mol), astragalin (-7.2 kcal/mol) and apiin are avonoids and Marumoside A and niazirin (-7 kcal/mol) both are phenolic compounds also showed good binding a nity with M pro. Rhamnetin, marumoside A and niazirin had high GI absorption rate and bioavailability score whereas astragalin and apiin were having low GI absorption and bioavailability score.
Anti-viral drugs such as Raltegravir, Lopinavir-Ritonavir, Maraviroc, Nel navir and Tipranavir were also screened for tracing possible interaction with M pro . Raltegravir (-7.2 kcal/mol) is an anti-retroviral drug, used along with other drugs to relieve the HIV infection [44]. Lopinavir-Ritonavir (-7.7 kcal/mol) is a combination of two medications; lopinavir and ritonavir, used to control HIV/AIDS infection [45]. Maraviroc (-8.2 kcal/mol) is a FDA approved chemokine receptor type (CCR5) antagonist, which blocks the entry of HIV to cells [46]. Nel navir (-8.3 kcal/mol), is anti-retroviral drug and a protease inhibitor, used for treating Human Immunode ciency Virus (HIV) [47].Tipranavir (-9.2 kcal/mol) is also an anti-viral medication used to treat HIV infection in combination with ritonavir.
After the screening and molecular docking of bioactive components from M.oleifera leaf, it was found exhibiting strong binding a nity with the novel corona virus main protease (M pro ). The results were comparable to the interactions of the FDA approved antiviral drugs studied here. Further cell line studies and biochemical assays would establish its activity.
Since natural products are always serve as an excellent source of lead molecules for many diseases without much side effects unlike in the case of many synthetic drugs in use today. M. oleifera is a universal tropical plant easily available, easily extractable and economically viable source of these compounds.

Conclusion
The present study in an in silico-based approach which reveals the possibility of identi cation of potent SARS CoV2 M pro inhibitors from plant sources. Our study gives an idea of the action of phytoconstituents from M.oleifera leaf against the main protease of Corona Virus. Among the 19 compounds screened, apigenin-7-O-rutinoside showed highest activity against SARS CoV-2 M pro . Moreover, these compounds are also found to have antioxidant property which would ameliorate the post-COVID secondary infection. Availability of data and material

Abbreviations
All data and materials are available upon request.

Competing interests
The authors do not have con icts of interest.

Funding
The authors thank University Grants Commission, India, for providing the nancial assistance to carry out the work in the form of UGC-JRF to the corresponding author.
Authors' contributions AND carried out the analysis and drafted the manuscript. TJJ supervised the work and nalized the manuscript.