In-silico Molecular Docking Studies of some Isolated Phytochemicals from Biophytum veldkampii against Cyclooxygenase-II Enzyme and in vivo Anti-inflammatory Activity

Background


INTRODUCTION
Medicinal plants are a source of valuable therapeutic assistance for the alleviation of human diseases.According to the World Health Organization (WHO), more than 80 per cent of the world's population, mainly in developing countries, relies on conventional plantbased medicines for their primary health needs. [1]cientific confirmation concerns the screening of bioactive compounds from plants and has contributed to the creation of new medicines with successful roles in the defence and treatment of various diseases.Inflammation is the local reaction of living mammalian tissue to injury.It is a body's immune reaction to eliminate or limit the spread of the injurious agent.There are various components of an inflammatory reaction which may lead to the associated symptoms and tissue injury.Edema, leukocyte infiltration, and granuloma formation are all components of inflammation.It's a defence mechanism, though.Complex events and mediators involved in an inflammatory reaction can cause or aggravate a variety of reactions. [2]Nonsteroidal anti-inflammatory drugs (NSAIDs) are potent antiinflammatory agents acting by inhibition of the enzyme Cyclooxygenase (COX) and subsequent inhibition of prostaglandins at the site of inflammation.
Unfortunately, inhibition of gastrointestinal or renal prostaglandins is associated with mechanism-based sensitivity,that decreases the usefulness of these otherwise powerful and efficient drugs. [3]Two forms of COX enzyme have recently been identified: COX-1, which is constitutively expressed in many cells and tissues, and COX-2, which is selectively induced by proinflammatory cytokines at the site of inflammation. [4]The discovery of a second COX enzyme related to the hypothesis that the toxicity associated with clinically valuable NSAIDs was caused by inhibition of COX-1, while COX-1 was inhibited.In support of this assumption, the expression of the inducible COX-2 enzyme is selectively inhibited by the potent anti-inflammatory drug dexamethasone.Selective inhibition of COX-2 can produce superior anti-inflammatory drugs with considerable safety over existing NSAIDs. [5]iophytum veldkampii looks like a miniature palm and is native to India.The plants are usually found in wet lands (mostly plains) of tropical Africa, Asia and India, normally in the shades of trees and shrubs, in grasslands, open thickets, at low and medium altitudes. [6]he molecular docking process predicts ligand confirmation and orientation within the targeted binding site which holds great promise in the field of computer-based drug design. [7]n present study we focus on the in silico docking studies of phytochemicals present in Biophytum veldkampii against cyclooxygenase-2 enzyme

Plant material
Biophytum veldkampii whole plants were collected from the forest area of Rampachowdavaram, East Godavari and authenticated by Dr. P. Prasanna Kumari, Botany department, Dantuluri Narayana Raju College, Bhimavaram.A voucher specimen was kept at Pharmacology department, Shri Vishnu College of Pharmacy.

Preparation of plant extract
The whole plant was dried at room temperature.For removal of waste or fatty materials, the powder is extracted with petroleum ether for 7 days.Now the defatted marc is extracted with methanol by using soxhlet apparatus to get methanolic extract.This extract was dried under reduced pressure; brownish color of extract was obtained. [8]This extract was stored in desecrator for further use.

Phytochemical analysis
The methanolic extract was subjected to estimation of different phytochemicals as per literature. [9] vitro COX II inhibition assay The assay was performed by using Colorimetric COX (human ovine) inhibitor Screening assay kit.Briefly, the reaction mixture contains, 150 μl of assay buffer, 10 μl of heme, 10 μl of enzyme (either COX-1 or COX-2), and 10 μl of plant sample (1 mg/ml).The assay utilizes the peroxidase component of the COX catalytic domain.The peroxidase activity was assayed colorimetrically by monitoring the appearance of oxidized N, N, N, N'-tetramethyl-p-phenylenediamine (TMPD) at 590 nm.Diclofenac sodium 1mM was used as a standard drug.The percent COX inhibition was calculated using following equation: [10] COX II Inhibition activity (%) = 1-T/G*100 Where T = Absorbance of the inhibitor well at 590 nm.C = Absorbance of the 100 % initial activity without inhibitor well at 590 nm.

Animals
In this study male Wistar rats were used, animals were kept in animals house and maintained under standard quality conditions (RH 44-56%, temperature 27±2°C) and feed with standard diet and drinking water ad libitum for 1 week before and during study.Protocol of this study was approved by IAEC of Shri Vishnu college of Pharmacy and with the permission from CPCSEA Reg No. 439/PO/01/a/CPCSEA.

Assessment of anti-inflammatory activity Carageenan-induced paw edema
The anti-inflammatory activity of MEBV was assessed in rats using carrageen-induced paw edema model as per the previously described method. [11]Carageenan suspension (0.1 ml of 1% w/v in normal saline) was injected into the sub-plantar region of right hind paw.Total 25 rats were randomly divided into 5 groups each containing 5 rats.Rats were pretreated with vehicle or MEBV (400 and 200 mg/kg, orally) or reference standard Diclofenac (70/mg/kg) at 1 h earlier to carageenan injection.

Formalin induced Inflammation
MEBV was assessed for evaluation of anti-inflammatory activity in formalin induced inflammation rat model. [12]Total 25 rats were randomly divided into 5 groups each containing 5 rats.After 1 h orally pretreated rats with vehicle (4% gum acacia) or MEBV (400 and 200 mg/kg) or Diclofenac (70 mg/kg) intraperitoneal administration of 0.05 ml of 2.5% commercially available 37% formalin in dorsal surface of left hind paw in rats.The paw volume at 0h,3h,5h and 24 hr.After carageenan administration was measured in ml using Plethysmometer (Ugo Basile, Italy).

Motility Test
The motility pattern of the rats was observed for a period of 5 min.If the rat was avoided touching the floor and walked difficulty, score is 0, if the rat walked with little difficulty but with toe touching the floor, score is 1 and if the rat walked easily score is 2. [13]

Sample Collection
After completion of experimental study, according to CPSCEA guidelines all animals were anesthetize by using ketamine and blood was collected by retriorbital puncture.Now blood samples were centrifuged for 15 m at 10000 rpm at 37°C collect serum and used for estimation of cytokines.

Cytokines Assay
Cytokines like IL-6, TNF -α and IL-10 (pg/ml) were estimated by using cytokines ELISA kits and assays were conducted as per ELISA kits manufacturer's guidelines. [14]

ADMET Analysis
By using of admetSAR, ADMET properties of ligands were studied it is very important to know the pharmacokinetic properties of ligands to establish their function inside of the body. [18]

Statistical analysis
All the data were expressed as mean ± S.D and performed by using one-way analysis of variance followed by Tukey test.p<0.05 is considered as statistically significant.

RESULTS
Phytochemical studies of methanolic extract revealed the presence of various phytochemicals shown in Table 1.

Effect of MEBV on COX II inhibition
The result of COX II inhibition by using methanolic extract of Biophytum veldkampii are summarized in Table 2

Effect of Biophytum veldkampii on paw thickness of rats (cm) on Carageenan induced inflammation
Injection of carageenan into the hind paws a progressive edema reaching it maximum at 5 hr.In case of Group I animals had shown (Normal control) paw thickness was found 3.028 ± 0.12 at 0 hr and this remains constant up to 24 hr.Group II animals (Carageenan induced group) had showed an increase in a paw thickness for each hr, at 0 hr.028 ± 0.12cm, at 5 hr 3.59±0.56cm, and at 24 hr 4.01 ±0.18 cm.The paw thickness of Group III (Diclofenac) animals was 3.35±0.41cm which showed a mild increase at end of 3 hr, after end of 3 hr it decreased the paw thickness to 3.04 ±0.58 cm and 2.99 ±0.74 cm at the end of 5 and 24 hr respectively.Group IV (MEBV 400 mg/kg) shows decreases the paw thickness to 3.014 ±0 cm at the end of 24 hr.Group V (MEBV 400 mg/kg) show decreases the thickness of paw to 3.42±0.75cmat the end of 24 hr. Figure 1 Effect of Biophytum veldkampii on paw thickness of rats (cm) on Formalin induced inflammation Injection of formalin into the hind paws a progressive edema reaching it maximum at 5 hr.In case of Group I animals had shown (Normal control) paw thickness was found 3.028 ± 0.12 at 0 hr and this remains constant up to 24 hr.Group II animals (Formalin induced group) had showed an increase in a paw thickness for each hour, at 0 hr 3.028 ± 0.12cm, at 5 hr 3.12 ±0.08 cm, and 3.89 ±0.58** cm at 24 hr.The paw thickness of Group III (Diclofenac) animals was 3.66 ±0.89 cm which showed a mild increase at end of 3 hr, after end of 3 hr it decreased the paw thickness to 3.28±0.85cm and 2.98 ±0.78cm at the end of 5 and 24 hr respectively.Group IV (MEBV 400 mg/kg) shows decreases the paw thickness to 3.014 ±0.79 cm at the end of 24 hr.Group V (MEBV 200 mg/kg) show decreases the thickness of paw to 3.22±0.87cm at the end of 24 hr. Figure 2 Effect of Biophytum veldkampii on motility of rats Walking ability of the rats to climb the staircase at the time of peak inflammation was checked by score of motility.Group I (normal control) animals showing the motility score is 2. Group II (carageenan) animals show 0.16 ± 0.35 motility score.Whereas Group IV (MEBV 400mg/kg) and V (MEBV 200mg/kg) animals exhibits motility score like 1.58 ±0.78 and 1.22 ±0.48 respectively.This indicates MEBV decrease the inflammation and increase the walking ability of animals.

Effect of Biophytum veldkampii on cytokines expression (pg/ml)
In Group II (Carageenan treated animals) shows highest levels of serum IL-6 (82.4±1.25 pg/ml), TNF-α (779 ±0.68 pg/ml) and lowest values of IL-10 (18.4 ±089 pg/ml) compared to normal group.On the contrary, Diclofenac sodium treated animals show statistically decreased the IL-6    2, as well as the protein-ligand interactions revealing hydrogen bonding and also binding settings are additionally published in Tables 3 (Figure 5-7).

ADME/T evaluation by using admetSAR
In this research by using admetSAR, ADMET properties of ligand were estimated.All ligands shows excellent human intestinal solubility, Blood brain barrier infiltration (BBB).All ligands were AMES negative.The and TNF-α and increases levels of IL-10.Whereas Biophytum veldkampii treated groups like Group IV(MEBV 400mg/kg) and Group V (MEBV 200mg/kg) exhibits low levels of IL-6 (64.8 ±0.69 pg/ml; 72.8 ±0.62 pg/ml) and TNF-α(646 ±2.75 pg/ml; 692±0.36pg/ml)and elevated levels of IL-10 (32.6 ±0.85 pg/ml; 24.9 ±0.63 pg/ml).Figure 3 Computational Study In silico studies of compounds present in Biophytum veldkampii, using Auto Dock 4.0 showed the following results.Of 11 compounds studied, 2, 6, 3' , 4'-Tetrahydroxy-2-benzylcoumaranone, 2(1H)-Quinazolinone satisfy the Lipinski's rule of five for drug-likeness.The other compounds which do not follow the Lipinski's properties were not considered for further docking studies.The structures of compounds from Biophytum veldkampii are shown in Figure 4.The binding energy for each chosen compound with the COX-2 enzyme is given in Table 3.

DISCUSSION
For screening of anti-inflammatory activity of drugs, carageenan induced rat paw edema model is suitable and commonly used method.
Carageenan is a polysaccharide it cause the inflammation by release of pro inflammatory mediators like prostaglandins, leuckotrienes, histamine and TNF alpha. [19]The inflammation occurs in two phase.In first phase started with release of histamine, 5HT and kinins after the injection of carageenan in the first few hours.In second phase related to release of prostaglandins in next 2-3 hr.Prostaglandins are main chemical agents responsible for inflammation.Second phase is the most target phase for steroidal and non-steroidal anti-inflammatory drugs. [20]n this research Biophytum veldkampii had very consistent anti-inflammatory action and exhibit significant decreases thickness of paw.Although, the Cyclooxygenase and lipoxygenases pathways play a vital role in the inflammatory process, the inhibition of Cyclooxygenase is more effective in inhibiting carageenan-induced inflammation than lipoxygenases inhibitors. [21]iophytum veldkampii might have inhibited the synthesis of PGs mediated by Cyclooxygenase.Oral administration of methanolic extract of Biophytum veldkampii significantly decreases the pro inflammatory cytokines and elevated the anti-inflammatory cytokines.COX enzyme increases the prostaglandins synthesis which causes the decreases the antiinflammatory cytokines like IL-10, IL-4 and IL-13.Our present findings Biophytum veldkampii shows elevated levels of IL-10 hence IL-10 has been found as a potent macrophage deactivator, which blocked TNF-α, IL-1, IL-6, IL-8, and GM-CSF by human monocytes. [22]n silico molecular docking studies is one of the significant method to development of new medications for various pathological conditions, advantage of this method is less time and required less budget compared to standard lab experiments.In this research, we used a docking method making use of open software programs as well as virtualized.Interactions of ligands 2, 6, 3' , 4'-Tet-rahydroxy-2-benzylcoumaranone and 2(1H)-Quinazolinone with anti-inflammatory protein COX-II enzyme.ΔG indicates informative of ligand docking in the active site of a protein, kind of molecular communications, such as hydrogen bond, hydrophobic, as well as likewise electrostatic interactions, with necessary amino acid, which is a step of ligand docking in favorable conformations.Hydrophobic synergy is the main aspect of the firmness of proteins.Hydrogen bonding furthermore maintains protein firmness, yet to a minimized degree than hydrophobic synergy.Our results disclose that electrostatic, hydrophobic and hydrophilic communications are regulated by numerous amino acid deposits in each ligand-protein communication.2,6,3' ,4'-Tetrahydroxy-2-benzylcoumaranone exhibits hydrogen bond, hydrophobic bond and electrostatic interactions whereas 2(1H)-Quinazolinone exhibits only hydrogen bond, and hydrophobic bond interactions.

CONCLUSION
To conclude with the compounds from the Biophytum veldkampii showed better binding features with the COX-2 enzyme.Thus, these compounds can be effectively used as drugs for treating inflammation which is predicted on the basis of docking scores.The insights gained in this work can be further used in experimental studies for designing anti-inflammatory drugs with novel targets and mechanism of action.
. The average COX II inhibition was calculated by taking the mean values.Diclofenac show 86.3 % of COX II inhibition, Whereas MEBV at dose of 200 mg/kg exhibit 60.34% and at dose of 400 mg/kg show 78.3 % inhibition of COX II.
of HIA, BBB and LD50 values of the ligands are shown in Table4 and 5.