Insilico identification and invitro analysis of the antimastitis components present in Marsh Barbel.

Introduction

Almost any microbe that can opportunistically invade the udder tissue and cause inflammation results in mastitis. However, most infections are caused by various species of streptococci, staphylococci, and gram-negative rods, especially lactose-fermenting organisms of enteric origin, commonly termed coliforms. Among these Staphalococus aureus bacterial infections are most common. In most cases the strain of Staphalococus aureus is transmitted as nosocomial infection (hospital acquired/ human transmitted)¹ The antibiotics conventionally used in the medical treatment of mastitis in cows has not met much success. Many antibiotics kill the micro-organisms during replication where as non replicating pathogens remain unaffected and some others develop resistance.²

Plant based drugs plays a major role in treating infectious diseases. Marsh Barbel (Hygrophila auricualata) is a herbaceous medicinal plant in the acanthus family grows in marshy places. The plant is used for the treatment of arthritis, Oedema, ascites and bladder stone. It is a blood purifier and good aphrodiasic. Leaves and roots are taken for leucorrhoea. The plant as a whole is antihepatotoxic. Crude extract of the plant is found to be highly effective against Bovine mastitis. Present study focuss on the insilico identification and invitro analysis of the components which are responsible for the antimastitis activity.

Drug targets of Staphalococus aureus

Seperation ring formation regulator, EzrA (PDB ID:4YU3): Cytoskeletal proteins directed the bacterial cell division.³FtsZ⁴ and FtsA⁵ recruits other cell division proteins including separation ring formation regulator, EzrA which was identified as an inhibitor of FtsZ polymerization and its absence results in unwanted formation of multiple FtsZ rings not only at mid cell but also at cell pols.^6,7 EzrA is necessary to maintain the dynamics of Z ring during cell division⁷. It plays a major role in recruiting penciling binding protein⁸. Inhibition of EzrA varies the cellular patterns of both FtsZ and PBP 1 results defects and alteration in cell morphology, cell division and elongation^8,9. EzrA contributes to the regulation of cell size in Staphalococus aureus. EzrA knock out develop larger spherical cells in Staphalococus aureus and delayed cell division which could be lethal to Staphalococus aurus¹⁰.

 Extra cellular matrix binding protein, ebh (PDB ID:2DGJ): Genome analysis of several strains of Staphalococus aureus revealed the presence of a giant gene which encodes a major adhesion protein that binds to the extra cellular matrix of the host cell¹¹. Inhibition of ebh reduces its adhesion capacity which has no longer been bind to the host cell. Protein translocation sub unit (PDB ID:5TZI): Staphalococus aureus cell wall teichoic acid beta glycosyl transferase is an enzyme responsible for the methicillin resistance in methicillin resistant staphalococus aureus. TarS is therefore an excellent drug target whose inhibition results in Methicillin resistant Staphalococus aureus resensitation to beta lactam antibiotics¹².

Monofunctional glycosyl transferase(PDB ID:3VMR): For cell wall synthesis, bacterial transpeptidase and transglycosylase are essential. Most of the antibiotics target the transpeptidase. But the antibiotic resistance developed become a major threat in bacterial infection. Inhibiton of glycosyltransferase also results in cell lysis. There fore glycosyl transferase has also been considered as another excellent drug target in Staphalococus aureus¹³.

Capsular p1olysaccharide assembling protein, CapF(PDB ID:3VHR): Capsular polysaccharides are associated with the cell surface and is involved in mediating direct interactions between the bacteria and its environment¹⁴. The interaction between the bacteria and host cell can be removed by inhibiting CapF target.

Methodology

Bioinformatics analysis

Preparation of Protein

Crystal structure of the target proteins, Separation ring formation regulator(PDB ID:4YU3),Protein translocation subunit(PDB ID:5TZI),Extracellular matrix bindibg protein(PDB ID:2DGJ) and Monofunctional glycosyltransferase(PDB ID:3VMR)

 were obtained from RCSB Protein Data Bank¹⁵. The structure preparation efficiency was increased by protein preparation wizard. PDB files were imported automatically from the RCSB PDB website to the Schrodinger working interface.^16-17Missing hydrogen atoms were added. Ensured proper formal charge and force field treatment. Then removed the co-crystallized water molecules. By means of a systematic, cluster-based approach, optimized the protein’s hydrogen bond network which decreased the preparation times and then performed a restrained minimization.

 Ligand Preparation

Twenty phytochemicals present in Marsh Barbel were selected to find out the inhibitory activity towards target proteins. The structure of  phytochemicals and commonly used antibiotic Pirlimycin Hydrochloride were downloaded from pub chem. in the (.sdf) format. These ligands were subjected to ligand preparation using the ligand preparation wizard (ligprep)^17-18.One low energy conformation was generated.  The Ligprep ligands were used for the Docking analysis.

Docking Studies

 The inhibitors of target proteins were studied by screening the 20 selected compounds using Schrodinger docking software. Grid generation was done using the centroid of workspace ligand R0 48-8071. The rigid receptor docking using the Glide program was carried out with the set of ligands. Extra precision (XP) mode of docking was selected for a high docking accuracy ^17-19.

Ligand Interaction study

A detailed information of various interactions of the ligands with aminoacid residues pointing the type of bonds, bond length and various angles can be studiesd using this option.

ADME properties prediction

^19-20QikProp module helps in analyzing the pharmacokinetics and pharmacodynamics of the ligands by accessing the drug likeness. Predicted significant ADME properties are Molecular weight (MW), hydrogen bond donor, hydrogen bond acceptor and logP(_O/W)

In Vitro Antibacterial Assay

Antibacterial assay was performed as per CLSI ( Clinical Laboratory Standards Institute)  Guidelines²¹. Filter paper disc diffusion technique was applied²². Test organisms were collected from Institute of Microbial Technology, Microbial Type Culture Collection Centre,( IMTECH), Chandigarh. The bacterial strains were maintained on their respective medium in slants at 2-8˚ C.  Preparation of Muller Hinton Agar ( MHA  )

Muller Hinton Agar (MHA)  medium was used for bacterial  culture. MHA was prepared and sterilized at  121˚ C for 15 minutes. After sterilization , required volume of the medium (25 ml) was poured in the sterile  petri dishes and allowed to solidified.

Inoculam preparation

Used pure culture as inoculam. Selected 3-4 similar colonies and transferred  them in to about 5 ml of suitable broth such as Tryptone Soya Broth (TSB). Incubate at 37 °C for 4-  8 hours till light to moderate turbidity develops.

Method of Inoculation

Dipped  a sterile non-toxic swab on a wooden applicator into the standardized inoculam and rotated  the soaked swab firmly against the upper side wall of the tube to express the excess fluid. Streaked the entire agar surface of the plate with the swab three times, turning the plates at 60 angle between each streaking . Allowed the inoculam to dry for 5-15 minutes with lid in place.Applied the discs ( Himedia -sterile 6mm disc -SD067) impregnated with the samples, luteoline,apigenine-7-o-glucuronide and apigenine-7-o-glucoside (Sigma Aldrich, London) with different concentrations (100%,75%,50% and 25% each) using aseptic technique. Then placed the discs with centers at  24mm apart.Incubated  immediately at  37° C and examined after 18 hours. Measured the zone showing complete inhibition and recorded the diameters of the zones to the nearest millimeter.Antibiotics discs (Himedia–HX022) were used as control. (Chloramphenicol-2µg and Streptomycin -10µg.)

Results and Discussion

Table-1: Phytochemicals having good docking scoreswith target proteins

Protein(PDB ID)	Ligand	Docking Score(Kcal/mol)
4YU3	Luteoline	-7.2
	Apigenine-7-o-glucuronide	-6.8
	Apigenine-7-o-glucoside	-6.3
	Lupeol	-5.6
	Linoleic acid	-4.9
	Betasitosterol	-4.5
	Pirlimycin Hydrchloride	-2.3
2DGJ	Luteoline	-6.8
	Apigenine-7-o-glucuronide	-7.5
	Apigenine-7-o-glucoside	-6.5
	Lupeol	-5.9
	Linoleic acid	-5.2
	Betasitosterol	-4.8
	Pirlimycin Hydrchloride	-2.9
5TZI	Luteoline	-6.7
	Apigenine-7-o-glucuronide	-7.1
	Apigenine-7-o-glucoside	-7.6
	Lupeol	-6.1
	Linoleic acid	-5.9
	Betasitosterol	-5.7
	Pirlimycin Hydrchloride	-4.5
3VMS	Luteoline	-7.6
	Apigenine-7-o-glucuronide	-8.1
	Apigenine-7-o-glucoside	-6.8
	Lupeol	-5.3
	Linoleic acid	-5.1
	Betasitosterol	-4.9
	Pirlimycin Hydrchloride	-3.6
3VHR	Luteoline	-5.9
	Apigenine-7-o-glucuronide	-6.1
	Apigenine-7-o-glucoside	-7.3
	Lupeol	-4.7
	Linoleic acid	-3.8
	Betasitosterol	-3.5
	Pirlimycin Hydrchloride	-2.3

Figure 1: 3D docking and 2D interaction of ligands with the target proteins

Inhibition zone diameter for	Luteoline	Apigenine-7-o-glucuronide	Apigenine-7-o-glucoside
100%	32mm	36mm	40mm
75%	30mm	32mm	35mm
50%	27mm	30mm	32mm
25%	25mm	28mm	30mm

Table:2 Inhibition zone diameter of samples at different dilutions

Fig 2.a): Inhibition zone of Luteoline(100%,75%,50%,25% and control) Fig2.b): Inhibition zone of Apigenine-7-o-glucuronide(100%,75%,50%,25 and control)                            

Figure 3.c): Inhibition zone of  Apigenine-7-o-glucoside (100%,75%,50%,25% and control)

The phytochemicals, Luteoline, Apigenine-7-o-glucuronide and Apigenine-7-o-glucoside show better docking score and binding interactions compared to the most common commercially available drug towards all the target proteins. Three hydroxyl groups in Luteoline forms side-to-side hydrogen bonds with 171 GLU, 153 LYS and 178 GLU amino acid residues of separation ring formation regulator(PDB ID: 4UY3).(Bond length-2.20232,1.93319 and 2.00973; Bond angle-106.141, 108.81 and 143.97). With protein translocation subunit (PDB ID:5TZI) Luteoline forms five hydrogen bonds and one pi-pi stalking interaction. The hydroxyl groups in Luteoline forms hydrogen bonds with 316 GLU, 254 PHE, 196 LYS, 349 ASP and 349 ASP (Bond length- 2.67469, 2.19438, 2.69856, 2.05452 and 1.9085; Bond angle- 121.977, 122.478, 121.811, 139.442 and 129.917). Luteoline forms one pi-pi stalking interaction with 8 HIS amino acid residue and five hydrogen bonding interactions with 5 GLN, 5 GLN, 8HIS, 8 HIS and 69 ASP aminoacid residues(Bond lengths- 2.05515, 1.93819, 1.95829, 2.6519 and 1.77445; Bond angles- 130.599, 125.976, 113.336, 93.2074 and 139.563) of extra cellular matrix binding protein.(PDB ID: 2DGJ). With FMT Protein(PDB ID: 3VHR), Luteoline forms four hydrogen bonding interactions, one pi-pi stalking interaction(288 HIS amino acid residue) and one pi cation interaction(293 LYS amino acid residue) Four hydroxyl groups in the Luteoline forms hydrogen bond with 264GLU, 264 GLU, 277 ASN, and 288 HIS aminoacid residues.(Bond length-1.98351, 1.91817, 2.15452, 1.67927: Bond angles- 124.137, 151.814, 108.6 and 101.008). Luteoline forms one pi-pi stalking interaction (196 TYR amino acid rsidue) and two hydrogen bonding interactions with monofunctional glucosyltransferase.(PDB ID:3VMR). Two hydroxyl groups in the ligand forms hydrogen bonds with 231 ILE, and 215 GLN aminoacid residues.(Bond length-2.2819, 1.88984 and Bond angle-130.7, 150.179)

Apigenin-7-o-glucuronide exhibit good binding interactions with all the five target proteins. There exist one pi cation interaction(with 153LYS amino acid residue) and three hydrogen bonding interactions with(153LYS, 178 GLU and 178 GLU) separation ring formation regulator(PDB ID:4UY3) (Bond length- 1.96331, 1.76908, 1.93861: Bond angles- 103.985, 162.753, 123.26). With protein translocation subunit (PDB ID:5TZI), Apigenine-7-o-glucuronide forms three hydrogen bonding interactions. Hydroxyl groups in the molecule forms side-to-side hydrogen bonds with 260 ASN, 320 ARG and 265 LYS aminoacid residues.(Bond length- 1.76436, 1.99256, 2.40928; Bond angle- 150.118, 152.092, 142.185) 8 HIS, 5 GLN, 68 LYS and 58 ASP aminoacid residues of the extracellular marix binding protein (PDB ID: 2DGJ)forms the binding pocket for Apigenine-7-o-glucuronide. The molecule binds to the protein through four strong hydrogen bonds. With FMT Protein(PDB ID: 3VHR), Apigenine-7-o-glucuronide forms two pi-pi interactions (with 262 PHE, 288 HIS) and two hydrogen bonding interactions(with 264 GLU and 293 LYS; Bond length- 2.17424, 2.67643; Bond angle- 125.076 and 142.806). Hydroxyl group in Apigenine-7-o-glucuronide forms one strong hydrogen bond with monofunctional glycosyltransferase(PDB ID: 3VMR).

Apigennine-7-o-glucoside is the next better binding phytochemical to all the target proteins. It shows a pi cation interaction with 153 LYS aminoacid residue in the Seaparation ring formation regulator.  Better binding is observed with protein translocation sub unit. The hydroxyl groups in the molecule forms five hydrogen bonds with 260 ASN, 260 ASN, 320 ARG, 260 ASN and 316 GLU aminacid residues.(Bond lengths-2.34635, 2.15509, 2.27555, 1.968 and 1.82668; Bond angles-111.19, 91.2809, 116.739, 132.626 and 124.805). Hydroxyl group in the molecule forms one hydrogen bond with 12 ASP of the extracellular matrix binding protein. With FMT protein, the molecule forms one pi-pi interaction(with 357 PHE) and four hydrogen bonds (with 257 ASP, 285 LYS, 295 GLU and 295 GLU; Bond lengths- 1.79566, 1.95087, 1.68729, 1.81636; Bond angles- 111.521, 137.487, 156.243 and 121.299). With monofunctional glycosyltransferase, the molecule forms two hydrogen bonds(with 196 TYR and 232 ASN; Bond lengths-1.89569, 2.02513; Bond angles- 153.125 and 107.875).

Pirlimycin hydrochloride shows least interactions compared to the above phytochemicals in Marsh Barbel. It forms three hydrogen bonds with separation ring formation regulator, four hydrogen bonds with protein translocation sub unit, four hydrogen bonds with extra cellular matrix binding protein, four hydrogen bonds with FMT protein and two hydrogen bonds with monofunctional glycosyltransferase. From the insilico docking analysis, it is evident that the phytochemicals Luteoline, Apigenine-7-o-glucuronide and Apigenine-7-o-glucoside are responsible for the antimastitis activity of the plant Marsh Barbel.

Anti bacterial activity of the above three components were tested by Disc diffusion method using Staphalococus aureus strain. Three different concentrations of the active components were chosen for the antibacterial analysis. From the results obtained, it has been observed that Luteoline, Apigenine-7-o-glucuronide and Apigenine-7-o-glucoside exhibit excellent antibacterial activity. The result of antibacterial activity study of the active phytochemicals against Staphalococus aureus is shown in figures 2.a,b and c. The results suggests the inhibition zone of the three components in different concentrations. (Table 2). In earlier studies the antibacterial activity of Luteoline isolated from Scutellaria barbata was reported^23, And also the antibacterial activity of Apigenine-7-o-glucoside was reported.²⁴

Conclusion

In the present study, the phytochemicals in marsh Barbel, Luteoline, Apigenine-7-o-glucuronide and Apigenine-7-o-glucoside are identified as the antimastitis components by insilico docking analysis since these phytochemicals showed better inhibitory activity towards all the potent drug targets of Staphalococus aureus bacteria. From the invitro antibacterial analysis it is confirmed that the Staphalocoucs aures is susceptable to Luteoline, Apigenine-7-o-glucuronide and Apigenine-7-o-glucoside.

References

1. Owens, W. E.; Nickerson, S.;  Boddle,  R.L.;  Ray, C.H.  Large Anim Practice. 2002,21, 26-28.
2. Sandholm, M. 1.; Kaartinen, L.; Pyörälä, S. J Vet Pharmacol. 1990, 13, 248-260.
3. Robert, M. Cleverley; Jeffrey, R.; Barrett Arnaud Baslé; Nhat Khai Bui; Lorraine Hewitt;  Alexandra Solovyova; Zhi-Qiang Xu; Richard A. Daniel; Nicholas E. Dixon;  Elizabeth J. Harry; Aaron J. Oakley; Waldemar Vollmer; Richard J. Lewis. Nature Communications. 2014, 5, 521-527.
4. Lowe, J.; Amos, L. Nature. 1998, 391, 203-206.
5. Szwedziak, P.; Wang, Q.; Freund, S.M.; Lowe, J. EMBO J. 2012, 31, 1-12.
6. Levin, P. A.; Kurtser, I. G.; Gross mann, A. D. Proc.Natl.Acad.Sci.USA. 1999, 96, 9642-9647.
7. Haeusser, D.; Schwartz, R.; Smith, A.; Oates, M.; Levin, P. Mol.Microbiol. 2004,52, 801-814.
8. Claessen, D. Mol.Microbiol. 2008, 68, 1029-1046.
9. Jorge, A. M.; Hoiczyk, E.; Gomes, J. P.; Pinho, M.G. Plos One 6. 2011, 522-527.
10. Jorge, A. Metal; Steele, V. R.; Bottomley,  A. L.; Garcia Lara, J.; Kasturiarachichi, J.; Foster, S. J. Mol.Microbiol. 2004, 80, 542-555.
11. Tanaka, Y.; Sakamoto, S.; Kuroda, M.; Goda, S.; Tsumoto, K.; Hiragi, Y.; Yao, M.; Watanabe, N.; Ohta, T.; Tanaka, I. J.str.2008, 16, 488-96.
12. Solmaz Sobhanifar; Lian, J. Worrall; Dustin, T. King.; Gregory, A. Wasney.; Lass Baumann; Robert,T.Gale; Michael Nosella; Eric, D. Brown; Stephen, G. Withers; Nathalic, C.J. PlosPathogens12. 2016, 12, 154-159.
13. Huang, C.Y.; Shish, H.W.; Lin,L, Y.; Jien, Y.W.; Cheng, T.J.; Cheng. W.c.; Wong, C.H.; Proc.Natl.Sci.USA. 2012, 17, 6496-501.
14. Miyafusa, T.; Caaveiro, J.M.; Tanaka,Y.; Tsumoto, K. Biochem J. 2012, 3, 619-621.
15. https://www.rcsb.org/
16.  Schrodingersuit 2009 Protein Preparation Wizard; Epik version 2.0; Impact version          5.5. Schrodinger, LLC, New York.2010
17.  Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Maniz,D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P.S. J.Med.Chem. 2004, 47, 1739-1749.
18. Babitha Agarwal; Sunanda Das; Archana Pandey. Asian journal of Applied Sciences. 2011, 3, 436-441.
19.  Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Maniz, D. T. J.Med.Chem. 2006, 49, 6177-6196.
20. Schrodinger suit , Protein Preparation Guide, Site Map 2.4; Glide Version 5.6,Lig Prep          2.4,Qik Propn3.3, Schrodinger, LLC, New York, NY,2010
21. Michael Hombach Guido, V.; Bloemberg Erik, C. Böttger. Journal of Antimicrobial Chemotherapy. 2012.  67,  622–632.
22. Lawrence Drew, W.; Barry, A. L.;  Richard O’Toole; John C. Sherris. Applied and Environmental Microbiology. 1972. 24, 240-247.
23. Yoichisato; Shihosuzaki; Takako; Nishikawa; Masaru Kihara; Hirofumi Shibata; Tomihiko Higuti. Journal of Ethnopharmacology. 2000. 72, 483-488.
24. Nilufar; Z. Mamadalieva; Florian Herrmann; Mahmoud;Z. Ei-Readi; Ahmad Tahrane; Razan Hamoud; Dilfuza;R. Egamberdieva; Shahnoz;S. Azimova; Michael Wink. Journal of Pharmacy and Pharmacology. 2011. 63, 1346-1357.