Vitex trifolia as Cyclooxygenase-2 Inhibitors in Anti-Inflammatory Drug Discovery

ABSTRACT: Inflammation is involved in several stages of diseases such as cardiovascular diseases, asthma, diabetes, neurodegenerative diseases, or cancer. Cyclooxygenase-2 (COX-2) is an important enzyme that catalyzes the production of prostaglandins from arachidonic acid and has an important role in various inflammatory-related pathologies. The selective suppression of COX-2 over COX-1 is thought to be a useful approach for treating inflammation. Vitex trifolia is traditionally used for various inflammatory ailments, thus giving scope for anti-inflammatory studies. Since the action of Ethanol extract of Vitex trifolia (EEVT) on COX-2 has not been previously described. In this study, we further investigated the effect of Cyclooxygenase-2 inhibitory effects spectrophotometrically in ELISA readers and evaluated anti-inflammatory activity in the carrageenan-induced rat paw edema model. The edema volume was measured using a plethysmometer. The results showed that the EEVt (200, 400, and 800 mg/kg BW) had significant anti-inflammatory activity and the EEVt demonstrated a COX-2 Inhibitory effect. The percentage of COX-2 inhibition was 8,04%, 20,74%, 21,92%, and the percentage of inhibition of paw edema was 39,56%, 45,60%, and 40,56%, respectively. These findings suggest that Vitex trifolia leaves extracts to possess promising and remarkable anti-inflammatory activity, which is directed against the enzymatic activity of COX-2.


Preparation of The Ethanol Extract of Vitex trifolia (EEVt)
The Vitex trifolia leaves was sun-dried. The dried Vitex trifolia was powdered using a conventional grinder. The powdered materials were then soaked in Ethanol (95%) for 24 hours by stirring at room temperature. The materials were filtered after 24 hours. The procedure was repeated three times. The filtrates were mixed and concentrated under vacuum using a rotary until a brownish semisolid extract was obtained, free of solvent. The extract was kept cold for further pharmacological testing.
Experimental Animal 18 adult male Wistar rats with body weights of 200-250 g and aged 2-3 months were obtained from West Sumatera animal houses were used for this study. Animals were housed and cared for in standard conditions with 12 h light/dark circle and were fed with a standard pellet diet and water ad libitum. All the animals were acclimatized for a minimum period of 1 week prior to the experiment. After 1 week, animals were randomly selected for different experimental groups (3 animal/ group) and used for the in vivo determination of anti-inflammatory activity. The rats were deprived of food, but not water, for 18-20 hours before an experiment.

Evaluation of Anti-Inflammatory activity
The anti-inflammatory activity was further examined by the carrageenan-induced rat paw edema method according to the method of Winter et al 28  By injecting 0.1 ml of 1% carrageenan in 0.9% saline into the right hind paw of the rat, edema was caused. After 1 h, 0.1 ml 1% carrageenan was injected subcutaneously into the subplantar area of the right hind paw of each rat except those in Group I. Edema volume was determined every 1 hour for up to 6 hours after carrageenan administration. The paw volumes were measured by a plethysmometer. The volume difference between before and after the right paw injection was measured to obtain the results. The inflammation degree of the paw and the rate of edema inhibition were calculated as follows: % edema inhibition = (Vc −Vt) × 100/ Vc; Vc and Vt are the average edema volumes of the control and test groups, respectively. The animal blood was collected in a heparin-coated tube at the end of the third hour. Groups I-VI was used to determine the activities of COX-2.

Evaluation of COX -2 Inhibitory Activity
Rat serum was prepared at the 3rd hour after induction of Carrageenan 1%. EEVt (200, 400, and 800 mg/kg BW) was used for inhibition studies.The ability of the test compound to inhibit COX-2 was determined by using the Enzyme-Linked Immunosorbent Assay (ELISA) kit according to the manufacturer's instructions. The product of this enzymatic reaction produced a distinct yellow color, determined by spectrophotometrically (Microplate reader) at 450 nm.

Statistical Analysis
The statistical software SPSS version 25 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Data were analyzed using one-way ANOVA followed by Duncan's multiple range test. p <0.05 was considered significant.

Anti-Inflammatory activity
In the present study, the edema was measured for 6h after carrageenan injection. EEVt given at dose 200, 400, and 800 mg/kg BW were effective in inhibiting the induced paw edema (Table 1) 40,56* a Data are expressed as the mean of Three observations (n = 3), bUsed as comparative group * Significant difference compared to the positive control (P < 0.05) Table 1 shows the effect of EEVt and standard drug as compared to carrageenan control at different hours in the carrageenan-induced paw edema model. From Table 1, a significant anti-inflammatory activity of EEVt could be confirmed through paw edema inhibition by 45,60%, 52,05% and 40,56% after 6 h, at 200, 400, and 800 mg/kg dosage, respectively, while celecoxib diminished paw edema by 40,56% at the same time. These results demonstrate that the EEVt (200, 400, and 800 mg/kg) significantly inhibited the inflammatory processes induced by the injection of carrageenan (p<0,005). These findings indicate that the EEVt posses potent anti-inflammatory properties.

DISCUSSION
The paw edema caused by carrageenan is a useful phlogistic tool for studying systemic anti-inflammatory drugs. This test is divided into two phases and is sensitive to the majority of clinically effective anti-inflammatory drugs. The first phase, which occurs within 1-2 hours of carrageenan injection, is caused by serotonin release as well as an increase in bradykinin, histamine, and prostaglandins in the inflammatory location. The second phase occurs 3-5 hours after carrageenan injection and is associated with the production and release of kinin and prostaglandins in the inflamed area 29,30 . Throughout the second phase, The macrophages are known to produce more interleukin-1 (IL-1) which caused an increase in the accumulation of polymorphic nuclear cells (PMNs) in the inflammatory area. Activated PMNs then begin to release lysosomal enzymes and active oxygen species, resulting in connective tissue destruction and paw swelling 31 . This study reported the anti-inflammatory activity of the ethanolic extract of Vitex trifolia leaves and the possible anti-inflammatory mechanism in the experimental model. In the present study, the EEVt (200, 400, and 800 mg/kg) significantly inhibited the inflammatory processes. This is indicated by the ability of EEVt to significantly decrease rat paw swelling as shown in Tables 1. These results further corroborate previous studies that the hydroalcoholic extract Vitex trifolia leaves at doses 100 and 200 mg/kg BW showed anti-inflammatory activity in both acute and subacute stages of inflammation 25 . Other studies reported that Aqueous extract of Vitex trifolia leaves showed significant inhibitory activity on interleukin (IL)-1β, IL-6 and iNOS mRNA synthesis, all of which are involved in the inflammatory response. Moreover, the plant extract seemed to induce the LPS-dependant IL-10 anti-inflammatory cytokine 32 . Extract of Vitex trifolia leaves at doses 25-100 mg/kg BW showed analgesic activity. Pain receptors can be found all over the body and are sensitive to mechanical, thermal, toxic chemical, or inflammatory stimuli 33 . In most cases, regardless of etiology, the genesis of pain is inflammatory; thus, targeting inflammation and pain together is an important concept in pain management 34 . Because inflammation and pain are linked, a number of medications have both analgesic and anti-inflammatory properties. COX and 5-LOX are two important enzymes that catalyze the formation of inflammatory mediators. Cox inhibitors are the main strays of current therapy aimed at modulating pain, inflammation, and fever control 35 . Many COX-2 or 5-LOX inhibitors have been developed as anti-inflammatory drugs; however, some have been withdrawn from the market, suggesting the need for inhibitors with minimal side effects 36 . Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their analgesic effects by inhibiting cyclooxygenase activity, antipyretic, anti-inflammatory, and antithrombotic effects 37,38 . The anti-inflammatory potential of EEVt may be due to the presence of active phytoconstituents such as flavonoids. The previous report describes flavonoids can reduce the risk of atherosclerosis and atherothrombotic disease and several other inflammatory diseases 39,40 . The flavanol quercetin was found to suppress the expression of COX 2 mRNA in rat paw pouch exudates cells, indicating that quercetin's antiinflammatory action may be due in part to suppressing COX-2 up-regulation 41 . Flavonoids can interfere with the oxidative synthesis of (Arachidonic Acid) AA from phospholipids and decrease the downstream production of inflammatory metabolites from AA metabolism, oxidative damage, and initiation of inducible pathways of Inflammation due to their powerful antioxidant capacity 42,43 . Other studies have demonstrated that flavonoids with antioxidant properties can reduce the cellular conversion of Corresponding Author: Ifora Ifora AA to MDA (Malondialdehyde) in patients with chronic inflammation 44 . Radical scavenging activities of phenolic and polyphenolic compounds have been shown in previous studies 45,46 . There are also many studies on antiinflammatory activities of plant extracts that contain flavonoids 47,48 . Furthermore, Vitex trifolia possessed enormous potential as a medicinal drug, particularly in cancer treatment and antioxidant 49,50 . The significant antioxidant capacity and flavonoid content of the extract used may have contributed to the anti-inflammatory effect. This is the first report on the potent COX-2 inhibitory properties of ethanol extract of Vitex trifolia leaves. The obtained results suggest that the biological effects of this natural compound may be due to inhibition of prostaglandin synthesis via the arachidonic acid pathway.

CONCLUSION
From the results of the present investigation, it can be concluded that the ethanol extract of Vitex trifolia (EEVt) leaves possesses significant anti-inflammatory activity. The mechanism of anti-inflammatory action is thought to be mediated by COX-2 inhibition. The results presented also suggest the need for further research on the COX-1 inhibitory effect.

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.