Anti-inflammatory and Antioxidant Activities of Cordia dichotoma Forst

1Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India. 2Department of Pharmaceutical Sciences, North East Frontier Technical University, Along, Arunachal Pradesh, India. 3Department of Pharmacology, Himalayan Pharmacy Institute, Majhitar, Sikkim, India. 4Department of Pharmacy, Govt. Girls Polytechnic, Raipur, Chhattisgarh, India. *Corresponding Author E-mail: nhussain116@gmail.com

Chronic inflammatory diseases remain to be one of the major medical problems around the world. According to the World Health Organization (WHO), chronic inflammatory diseases are considered as the significant threat to public health. The prevalence of chronic inflammatory disorders is increasing with17.6 million clinical cases, particularly due to rheumatoid arthritis every year globally 1 . Approximately 3 of 5 people die and 50% of all deaths occur have been attributable to chronic inflammation-related diseases such as cardiovascular diseases (stroke, heart disorders), chronic respiratory diseases (allergic asthma, COPD), rheumatoid arthritis and joint disorders, systemic lupus erythematosus, inflammatory bowel disease (IBD), Crohn's disease, Alzheimer's disease, cancer, obesity, diabetes, chronic kidney disease and many others 2 . Some common FDA approved medications that include Non-steroidal anti-inflammatory drugs (NSAIDs), steroidal drugs and immuno suppressants are commonly used for the treatment of inflammatory disorders. These drug therapies are required to be administered for a long time and their use often induce some serious adverse effects such as gastrointestinal upset, heart burn, peptic ulcers, headache and so on. Because of this reason, there is an increasing need to develop some new and alternative anti-inflammatory drugs that would not only be efficacious against inflammatory illness and diseases, but also be safe and produce fewer side effects.
Plant-based traditional remedies have been used for the treatment of human diseases for thousands of years. About 80% of population depends on traditional herbal remedies for primary health care across the globe 3 . Traditional or folklore herbal medicines play a significant role in the management of a variety of human disorders including cancer, neurological disorders, diabetes and pain and inflammatory disorders, just to name a few 4 . WHO has recommended the evaluation of traditional plant-based remedies or herbal preparations for antidiabetic activity because they are more effective and safe as compared to synthetic antidiabetic drugs 5 . In addition, herbal medicines have the property of synergistic action due to the presence of a variety of active constituents in a single drug/ medicinal preparation. Numerous indigenous medicinal plants of India have been found to be useful in the management of pain and inflammations. Ayurveda, Unani and Siddha, are the notable systems of medication documented in ancient practice basically utilizing plants/ plant-based preparations as medicines for curing human ailments/ diseases like diabetes 6 . In view of their traditional and ethnopharmacological importance, herbal medicine may have potential role in the management of pain and inflammations.
Moreover, medicinal plants provide valuable source of new chemical moieties with potential therapeutic properties. Plants that have been found to have anti-inflammatory potential can be screened in search for novel bioactive secondary metabolites or phyto constituents as new and effective, but safe anti-inflammatory agents.
Oxidative stress (OS) induced by the reactive oxygen species (ROS) produced from the action of free radicals in the biological matrix may be increased abnormally during diabetes, causing an imbalance between the cellular metabolism and the antioxidant system of the body. The oxidative stress produces several inflammatory cascades that damage the cellular components 7 . Further, oxidative stress is undoubtedly claimed to have significant role in the progression of chronic inflammation mediated diseases. The cellular oxidative stress can be suppressed to an appreciable extent by potentiating the action of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSH) and glutathione peroxidase (GPx) by alternative herbal therapies 8 .
Cordia dichotoma Forst. (also known as Indian cherry) belonging to the family Boraginaceae is an average-sized tree of tropical and subtropical origin. It is widely found in Sri Lanka, India, and other tropical regions of the world. The use of this plant has been on ancient practice for the management of a variety of human disorders. It is also an important plant species found in various traditional Indian systems of medicine including Ayurveda, Unani and Siddha 9 . Seeds of C. dichotoma are used for the management of various inflammatory disorders 10 . Fruits are used as expectorant, astringent, laxative and anthelmintic 9 . Some common ethnomedinal uses of C. dichotoma includes antidiabetic, immunomodulator, diuretic, anthelmintic, wound healing, antiulcer, gastroprotective, anti-inflammatory, antileprotic, antidiabetic, and hepatoprotective and antioxidant activities 10 . The bark of C. dichotoma has been reported to a variety of phyto constituents such as betulin, á-amyrin, octacosanol, â-sitosterol, lupeol-3-rhamnoside, â-sitosterol-3-glucoside, hentricontanol, taxifolin-3,5-dirhmnoside, and hesperitin-7-rhamnoside 9, 11 .
There has been no scientific study on the anti-inflammatory activity of the C. dichotoma bark previously reported in literature. The present study was, therefore, aimed at investigating the antiinflammatory activity of the methanolic extract of C. dichotoma bark (MECD) with a view to justify the traditional use of this particular plant species in the treatment of edema. The antioxidant activity was evaluated to justify the anti-inflammatory action of MECD on the basis of its radical scavenging effect.

Drugs and chemicals
Carrageenan and indomethacin were purchased from Merck Pvt. Ltd., Mumbai, India. Carboxy methyl cellulose (CMC) was procured from Sigma-Aldrich, Mumbai, India. All other chemicals and solvents used in this study were of analytical grade.

Plant material
The barks of C. dichotoma Forst. were collected during the month of April-May, 2012 from the Duhai forest of Ghaziabad, Uttar Pradesh, India. The plant material was identified from CSIR-National Institute of Science Communication and Information Resources (CSIR-NISCAIR), New Delhi. A voucher specimen (NISCAIR/ RHMD/ Consult/2012-13/2025/33) of the bark of C. dichotoma was submitted at the herbarium for future reference.

Extraction methodology
The shade-dried barks of C. dichotoma were pulverized to coarse powder, defatted using petroleum ether, and extracted by Soxhlation using methanol as solvent. The methanolic extract was subsequently evaporated to dryness and the concentrated extract so obtained was preserved in a refrigerator at 4 o C for further use. The percentage yield of the methanolic bark extract of C. dichotoma (MECD) was found to be 7.11% w/w on dry weight basis.

Phytochemical analysis
The MECD was analysedfor the presence of presence of phyto constituents as per the standard procedure previously described in literature 12 .

Experimental animals
Adult Wistar female albino rats weighing around 300-330 g were procured from the Institutional Animal House for the experimental study. Acclimatization of animals was done in accordance with standard laboratory conditions (temperature: 25 ± 2 o C, relative humidity: 50 ± 5 %) with a 12 h light/12 h dark cycle for a week prior to the beginning of experiments, and were provided with free access to the standard pellet diet and drinking water ad libitum. The experimental protocol was approved by the IAEC vide approval no. IAEC/DU/58 dated. 24/09/2013.

Acute toxicity study
The oral acute toxicity of MECD was done as per OECD guidelines. The rats were randomly distributed into seven groups of three animals each. The first three groups were administered with MECD at 10, 100, and 1000 mg/kg doses, respectively. The animals were observed for signs of mortality or morbidity or death for 24 hours. Further, doses of 2,000, 3,000, and 5,000 mg/kg were given to rest of the three groups and observed for 48 hours. One group administered with vehicle was treated as normal control 4 .

Evaluation of anti-Inflammatory activity
The anti-inflammatory activity was evaluated by carrageenan induced rat paw edema method according to the previously reported methods [13][14][15][16] with minor modifications. The rats were randomly distributed into five groups containing six rats in each group (one control, one toxic, standard and two test groups). The acute inflammation in rats was induced by the injecting carrageenan (1% w/v solution in 0.9 % w/v sodium chloride, 0.1 ml) in the planter region of rat's paw. The extract was formulated in two different doses (250 and 500 mg/kg body weight) in the form of a suspension using 0.3% CMC. The extract formulations were administered orally into experimental animals one hour prior to injecting carrageenan. Indomethacin (5 mg/kg body weight, oral route) was used as standard drugs.   The paw volume was measured using a Plethysmometerat 0, 1, 3 and 4 hours after the administration of carrageenan. The percentage inhibition of paw edema was estimated using the following formula. % Inhibition of edema = (V t -Vc/Vt) × 100 where, V t = Paw volume in test group, V c = Paw volume in control group

Antioxidant activity
The in vitro antioxidant activity was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay method 5,8 . A solution of 0.1 mM DPPH in methanol was prepared and 2.4 ml of this solution was mixed with 1.6 ml of the MEXD in methanol at different test concentrations (10-200 ìg/ml). The reaction mixture was vortexed thoroughly and kept at room temperature in the dark for 30 min. The absorbance of the mixture was determined spectrophotometrically at 517 nm. Ascorbic acid was used as standard drug. The experiment was carried out in triplicate observations. The percentage of radical scavenging activity was calculated using the following formula. Then % of inhibition of scavenging activity was plotted against concentration and from the graph IC 50 was calculated.
where, A o and A t are absorbance values of the control and the test compound (standard/ test), respectively

Statistical Analysis
Results are presented as mean ± standard error of mean (SEM). The one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test was used to analyse data. Statistical analysis was performed using the IBM SPSS 19.0 statistical software package, for Windows. Statistical differences at 1% (p < 0.01) level of probability between the groups were considered statistically significant.

Phytochemical analysis
Qualitative phytochemical analysis showed the presence of alkaloids, steroids, cardiac glycosides, flavonoids, tannins, terpenoids, saponins and carbohydrate in the MECD.

Acute oral toxicity
Results of oral acute toxicity study reveals that the LD 50 of the MECD was above 5,000 mg/kg body weight. No gross behavioural changes, other symptoms of toxicity and mortality were observed up to 48 hours of experimentation. The body weight and food consumption of treated rats were normal as compared to vehicle control.

Anti-inflammatory activity
The MECD showed anti-inflammatory activity against carrageenan-induced paw edema model in rats. The extract exhibited dose-dependent anti-inflammatory activity. Maximum inhibition of edema was observed after 4 hours of experimental period. At lower test dose (250 mg/kg b.w.), the percentage inhibition of paw edema was 29.7 %, while 48.6 % inhibition of edema was observed at higher dose (500 mg/kgb.w.). Results of antiinflammatory activity are presented in Figure 1. Both lower and higher test doses of the MECD demonstrated significant (p< 0.01) inhibition of paw edema as compared to control group. The standard indomethacin group also exhibited sufficiently high level of anti-inflammatory effect with 56% inhibition of paw edema at 5 mg/kg dose, relative to the control group. The anti-inflammatory effect of MECD is comparable to some extent with that of the standard drug, indomethacin.

Antioxidant activity
In the in vitro radical scavenging activity, the significant reduction in the DPPH radicals was seen due to the free radical scavenging property of MCED. The antioxidant activityrose with increasing the concentration of the test extract, reaching maximum level with the IC 50 vale of 62.46 µg/ml. On the other hand, the standard drug, ascorbic acid exhibited comparatively more antioxidant activity with IC 50 of 27.66 µg/ ml ( Figure 2). However, the MECD extract of C. dichotoma bark possesses antioxidant activity.
Results reveal the anti-inflammatory potential of the MECD of C. dichotoma bark along with antioxidant property. It is attributed that the anti-inflammatory effect might be due to the phytochemical content such as phenolic compounds, flavonoids, saponins or alkaloids of the extract. Many studies have investigated the antioxidant potential of plant polyphenols and flavonoids. Phenolic phytoconstituents and flavonoids have been attributed to exhibit pharmacological effects against heart diseases, cancer, neurological disorders, diabetes, inflammatory disorders and so on owing to their radical scavenging actions [17][18][19] . Literature reports suggest that the flavonoids content of plant extract may be responsible for the anti-inflammatory effect which might be because of their radical scavenging actions 20 . The antioxidant activity of MECD can help reduce the oxidative stress induced generation of inflammatory mediators and associated tissue damages. Restoring the levels of antioxidant enzymes, herbal drugs could act as free-radical scavengers and eventually prevent generation of ROS and thereby OS induced cellular damages 21, 22 . In this study, the antioxidant activity of MECD may be the underlying reason behind its anti-inflammatory action. Phenolic compounds and flavonoids of MECD could reduce cellular oxidative stress induced inflammatory damages.

CONCLUSION
It is concluded that the methanolic extract of C. dichotoma(MECD) bark possesses anti-inflammatory activity in carrageenaninduced paw edemamodel in rats. Our study scientifically validates the folkloric claim as well as traditional uses of C. dichotomaas antiinflammatory medication. It is suggested that the anti-inflammatory activity of C. dichotomamay be due to the presence of phenolic phytoconstituents or plant flavonoids in the methanolic bark extract. Further studies can be carried out in order to identify the specific phytochemical(s) responsible for the anti-inflammatory potential of C. dichotoma..