Identification of Antimastits Componenets in Boerhavia diffusa as an Inhibitor of Staphalococus aureus Monofunctional Glycosyltransferase , Causing Bovine mastitis ( An Insilico Approach )

Bovine mastitis is an infection of cattle leading to a huge reduction in milk production that causes severe economic loss in dairy industry across the world. Causative of the disease includes bacteria, virus and non-bacterial pathogens. Among these, Staphalococus aureus is the common cause of mastitis and is highly resistant to the most routinely used antibiotics. So current antibiotic therapy has shown limited efficacy. The crude extract of locally available medicinal plant Boerhavia diffusa is used traditionally against mastitis and is found to be highly effective. The objective of the study is the identification of the phytochemicals in Boeravia diffusa responsible for the antimastitis activity by insilico docking analysis using Schrodinger Suit v 9.2. 20 phytochemicals in Boerhavia diffusa were selected for docking studies based on ADMET properties. The high resolution crystal structure of the target receptor protein of Staphalococus aureus was retrieved from PDB. Structure of the phytochemicals and the most commonly used antibiotic against Bovine mastitis were selected from PUB CHEM NCBI. The phytochemicals, Boeravinone A, B, C, D, E and F from Boehravia diffusa showed good docking scores and better interaction with the active sites of the target proteins used for the evaluation than the most commonly used commercially available drug. Results of this study are important for the designing of novel drugs for the treatment of mastitis.


INTRODUCTION
Bovine mastitis is a kind of inflammation of the udder of cattle due to wounds or scars.It is associated with the presence of infectious agents as bacteria, Bovine Herpes virus, non bacterial pathogens like mycoplasmas, fungi, yeast and chalmydia [1][2] .Among these, bacterial infections are most common.Bovine mastitis reduces the quantity of casein, lactoferrin and potassium in milk.As the major protein, casein in milk deteriorates, the calcium level in milk also decreases.During processing and storage also the milk protein undergo deterioration 3 .Milk from affected animals shows very high somatic cell count which lowers the quality of milk 4 .Hence, Bovine mastitis causes severe economic losses in dairy industry. 5rrent methods used in the control of Bovine mastitis are based on antibiotic therapy.The commonly used antibiotics are pencillins and semisynthetic pencillin derivatives targeting the pencillin binding proteins of Staphalococus aureus.Pencillin binding proteins or DD-transpeptidase form bonds between the oligopeptide crosslinks in peptidoglycan 6 .Inhibition of peptidoglycan synthesis leads to bacterial cell lysis 7 .Commonly used antibiotics target the DD-transpeptidase and inhibit peptidoglycan synthesis there by leading to bacterial cell lysis.But mutations in genes coding for transpeptidases leads to reduced interaction with antibiotic results in the generation of antibiotic resistance in Staphalococus aureus 8 .This is the main reason for the decreased antibiotic efficacy in the treatment of Bovine mastitis.Monofunctional glycosyl transferase 1 is an alternate potent target of Staphalococus aureus, the inhibition of which also leads to bacterial cell lysis.
The immunomodulator compounds derived from medicinal plants has potent application in controlling mastitis.Boerhavia diffusa is one of the medicinal plants in the whole or its peculiar parts have enormous medicinal properties 9 .Farmers of the Southern region of Kerala uses the crude extract of the plant as a whole as a medicine for mastitis from their traditional knowledge.

Methodology Bioinformatics analysis Preparation of protein
Crystal structure of the target proteins of Staphalococus aureus Monofunctional Glycosyltransferase was obtained from RCSB Protein Data Bank (PDB ID: 3HZS) 10 .1][12] Optimized the protein's hydrogen bond network by means of a systematic, cluster-based approach, which greatly decreased preparation times and then performed a restrained minimization that allows hydrogen atoms to be freely minimized.Here the crystal structure of the target protein was complexed with a ligand.So sitemap creation was exempted.

Ligand preparation
Twenty phytochemicals present in Boerahavia diffusa 13 were selected to find out the inhibitory activity towards the target protein.

Docking studies
The compounds were screened by Schrodinger docking software to study inhibitors of the target protein.Grid generation was done using the centroid of workspace ligand R0 48-8071.The rigid receptor docking using the Glide program was carried out against the target protein with the set of ligands.The mode of docking was selected as XP (Extra precision) for a high docking accuracy.The glide docking was carried out for the minimised protein 11,12,14 .

Ligand interaction study
The ligand molecules fit in to the active binding sites of the protein molecules by means of some interactions.The interactions include hydrogen bonding, pi-pi stalking interactions and pi-pi cation interactions.A detailed information of various interactions of the ligands with aminoacid residues pointing the type of bonds, bond length and various angles were studied using this option.

ADMET properties prediction
5] QikProp helped in analyzing the pharmacokinetics and pharmacodynamics of the ligand by accessing the drug likeness.

RESULTS AND DISCUSSION
Boeravia diffusa is a renowned medicinal plant due to its therapeutic potential.Aqueos leaf extract of Boeravia diffusa had hypoglycaemic effect 16 and nutritive properties 17 .Ethanolic extract of Boeravia diffusa leaves had antistress, adoptogenic, immunopotentiating 18 and anti-inflammatory activity 19 .Antibacterial activity of methanolic and ethanolic extracts of Boeravia diffusa leaves against both gram positive and Gram-negative bacteria were reported. 20Also aqueous and ethanolic extracts of Boeravia diffusa had antibacterial activity on E.Coli, Staphalococus aureus and P. aeruginosa. 21esent study revealed the phytochemicals in Boerhavia diffusa which are responsible for the antimastitis activity by inhibiting the most potent drug target in Staphalococus aureus.Table 1 shows the docking scores, number of hydrogen bonds, interacting amino acid residues in the active site of protein and hydrogen bond length.Table 2 explains the drug likeness of phytochemicals predicted by QikProp simulation.Fig. 1 and Fig. 2 shows the 2D and 3D interactions of the ligands with the target protein.
From the results obtained , it has been understood that there exist excellent binding interactions between phytochemicals in Boerhavia diffusa and target proteins (PDB ID:4HZS) compared to the commercial drug (cloxacillin) with good binding scores.In the protein monofunctional glycosyltransferase with PDB ID: 3HZS, the 110 th PHE,136 th GLN and130 th GLN aminoacid residues forms the binding pocket.The phytochemical Boeravinone E shows a binding score -6.151 kCal/mol and forms three hydrogen bonds with the aminoacid residues in the binding pocket.The hydroxyl group in the 11 th

CONCLUSION
The phytochemicals Boeravinone A, Boeravinone B, Boeravinone C, Boeravinone D, Boeavinone E and Boeravinon F has potent inhibitory activity towards the staphalococus aureus monofunctional glycosyltransferase compared to the commercial drug Cloxacillin.These phytochemicals are responsible for the antimastitis activity of Boerhavia diffusa.This study leads to the development of novel drugs for mastitis.