*Corresponding Author:

Shakir Ali
Department of Biochemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
E-mail: sali@jamiahamdard.ac.in

Abstract

This study reports the beneficial effect of aqueous extract of dried flower buds of Syzygium aromaticum (clove) in acute and chronic inflammation. Inflammation was induced in rats by injecting carrageenan in hind paw or implanting cotton pellet in the axilla. Administration of the extract (1 g/kg body weight) inhibited the formation of oedema induced by carrageenan and decreased granuloma in cotton pellet granuloma model. The extract, when compared with the disease control, is reported to decrease the elevated levels of succinate dehydrogenase (p<0.001), xanthine oxidase (p<0.05) and lipid peroxidation, and increase the activity of catalase (p<0.001) and glutathione peroxidase (p<0.01) in the two animal models. Potential role of xanthine oxidase in inflammation and the ability of the extract to alleviate oxidative stress and inflammation is discussed. The study advocates the use of aqueous extract, rather than the isolated bioactive principle for various reasons.

Keywords

Syzygium aromaticum; clove; oxidative stress; inflammation; xanthine oxidase.

Introduction

Dried fl ower buds of Syzygium aromaticum (Family Myrtaceae), an evergreen indigenous tree of India, Indonesia, Zanzibar, Mauritius and Sri Lanka, are esteemed as fl avoring agent, and have been traditionally used as carminative and antispasmodic [1]. Extract prepared from the bud has been found to inhibit platelet aggregation, alter arachidonic acid metabolism [2], used in Herpes simplex infection [3], and has anticarcinogenic potential [4]. Essential oil extracted from the bud is used topically to relieve pain and promote healing [5]. Free eugenol, eugenol acetate, caryophyllene, sesquetrepene ester, phenyl propanoid, and β-caryophyllene are the pharmacologically active compounds isolated from the bud [3,6]. Th is study reports the anti-infl ammatory activity of dried fl ower bud of S. aromaticum in acute and chronic infl ammation. Injection of carrageenan in the hind paw of rat and implantation of cotton pellet in its dorsal skin has been used earlier to study acute and chronic infl ammation, respectively [7,8]. Reduction in carrageenan-induced paw oedema (CIO) and cotton pellet granuloma (CPG) has signifi cant predictive value for antiinfl ammatory agents, and is commonly used to test the anti-infl ammatory activity of natural and synthetic compounds.

Methods

Materials and Preparation of Extract

Pathogen free adult female rats (Wistar strain) weighing 160-170 g were used in the study. Animals were used in accordance to the CPCSEA guidelines for the care and use of laboratory animals, as described earlier [9]. Chemicals and consumables for this study were obtained from standard commercial sources in India. Clove was purchased from local market and the extract was prepared as follows: 10 g crude powder was mixed well in 50 ml of warm distilled water, and the mixture was left overnight in a conical fl ask. The mixture was extracted again the following day, fi ltered through muslin cloth, and stored in pre-sterilized glass container at 4°C till further use. The amount of extract obtained from 100 g of crushed plant material (extractive value or percent yield) was calculated and found to be 40%. Extract was used within two days of preparation.

Experimental Protocol

Rats were divided randomly into the following groups: NC (normal control, the group that did not receive any treatment), DC (disease control, injected with carrageenan or implanted the cotton pellet; the groups were called CIO or CPG, respectively), AE (aqueous extract treated, treated with the extract prior to the induction of infl ammation, and DF (diclofenac treated, the group treated with diclofenac 1 h prior to the induction of infl ammation). Extract was administered orally at a dose level of 1 g/kg body weight. Diclofenac was administered to rats at a dose of 5 mg/ kg, orally. In CIO model, carrageenan was injected into rat hind paw to induce acute infl ammation [10], and in CPG, cotton pellet was implanted in the axilla of rat instead of carrageenan injection. Cotton pellet implantation leads to the formation of granuloma and infl ammatory response.

Evaluation of Anti-inflammatory Activity of Aqueous Extract

The anti-infl ammatory activity of the extract was determined in vivo in acute and chronic infl ammation. In CIO, carrageenan (0.1 ml of 1% carrageenan suspended in normal saline) was injected into rat hind paw one hour after the drug administration, as described by Winter et al [7]. Oedema (swelling) was measured for three consecutive h by plathysmom-eter (Ugo Basile, Italy). Mean increase in paw volume was measured, and percent inhibition was calculated as follows: V_c-V_t/V_c × 100; where V_c represents the oedema volume in control group, and V_t, the volume of oedema in treated group. In CPG, accurately weighed (10 ± 1 mg) pellet of sterile cotton, soaked in 0.1 ml distilled water containing 0.1 mg penicillin and 0.1 mg streptomycin, was implanted subcutaneously in the incision made in axilla [8]. Pellet was implanted under ether anesthesia and incision was stitched after implantation. The test drug was administered orally twice a day at 12 h interval for 10 days after implanting the cotton pellet. First dose of the drug was given an hour prior to cotton pellet implantation. Animals were sacrifi ced 12 h after administering the last dose, which was on day 10. Subsequently, the pellet was dissected out carefully, dried at 60°C and weighed. Diff erence in the weight of cotton pellet before and after implantation was determined, and % inhibition of granuloma formation in treated rats was calculated as follows: W_P-W_P-t/ W_P × 100; where, W_P is the weight of the implanted cotton pellet, and W_P-t, the weight of pellet in treated group.

Evaluation of Biochemical Changes in Inflammation in Extract Treated Rats

All biochemical procedures were carried out on the hepatic tissue obtained from rats sacrifi ced four hours after administering carrageenan or 10 days after implanting cotton pellet. Samples were processed as described earlier [9] and kept at 0 to 4°C till fi nal analysis, which was done on the same day. Succinate dehydrogenase, catalase, glutathione peroxidase, and xanthine oxidase were measured by the spectrophotomeric procedure and enzyme activity was calculated as described earlier [11-13,9]. Lipid peroxidation and glutathione were measured according to the method described by Wright et al [14] and Jollow et al [15], respectively. Protein was measured by Lowry’s method [16].

Statistical analysis

Statistical analysis was carried out using Graph Pad Prism 4.0, and results were expressed as Mean ± S.E.M. (n = 6). Statistical signifi cance was calculated by Student’s t-test. The group of animals treated with the extract was compared with the respective disease control groups, either CIO or CPG; p<0.05 was considered as signifi cant.

Results and Discussion

Infl ammation is a protective biological response of vascular tissue to harmful stimuli including pathogens, damaged cells, or irritants to remove the injurious stimuli and initiate the healing process. It is also considered as a mechanism of innate immunity. Persistent infl ammation, however, may lead to a host of diseases including diabetes, rheumatoid arthritis and even cancer. Th is study reports inhibition of infl ammation by the aqueous extract of clove. The extract could inhibit both acute and chronic infl ammation in rat, and modulated a cascade of biochemical reactions that propagate and mature the infl ammatory response. We observed inhibition in the formation of oedema and granuloma, and found the extract to decrease elevated levels of several biochemical parameters involved in infl ammation and oxidative stress. The increase in average paw volume of rats injected with carrageenan was signifi cantly less in the extract treated group. In the treated rats, paw volume, measured over a period of 3 h, was 84%, when compared to the untreated group. In the untreated rats, paw volume increased to 174% in comparison to normal control. Extract also caused a signifi cant decrease in the weight of cotton pellet implanted in the animal skin. The weight of granuloma at the end of the experiment was 20.5 mg in the treatment group vs 33 mg in untreated control rats (Table 1). In both animal models of acute and chronic infl ammation, pharmacological response of the extract was comparable to diclofenac, which was used as a standard drug.

Table 1: Effect of clove extract on carrageenan-induced oedema and cotton pellet granuloma.

One of the objectives of the study was to investigate the eff ect of extract on the levels of succinate dehydrogenase (SDH), which has been used as a biochemical marker in infl ammation [17,18]. Th is study reports a considerable increase in SDH activity in untreated rats in both acute and chronic infl ammation (Table 2). SDH is a key enzyme linked with the energy yielding citric acid cycle in mitochondria. An increase in SDH would mean increased supply of ATP to the liver and possibly other tissues including the infl amed tissue.

Table 2: Effect of clove extract on the marker of inflammation and oxidative stress in rats.

Infl ammatory process comprises of the nonphagocytic/phagocytic infl ammatory response, infl ammation mediators including neutrophilderived reactive oxygen species or ROS, nitric oxide, prostaglandins, and cytokines. In infl ammation, the infl ammatory processes induce oxidative/ nitrosative stress and lipid peroxidation (LPO), thereby producing excess ROS, reactive nitrogen species (RNS), and DNA-reactive aldehydes, which aff ect the tissue and organ. Persistent oxidative and/or nitrosative stress and increased LPO induced by infl ammatory processes deregulate cell homeostasis. Neutrophils migrate to the site of infl ammation and upon activation by a stimulus, generate ROS in abundance [19-21]. Prevention of infl ammatory and nociceptive responses by preventing the generation of ROS has been reported earlier in acute and chronic infl ammation. In animals, xanthine oxidase (XOD) is one of the en- zymes involved in the generation of ROS and cause injury [9]. Th is study reports an increase in XOD activity in infl ammation, and found that the extract could attenuate the elevated levels of enzyme in acute and chronic infl ammation (Table 2). The fi nding assumes signifi cance and opens up avenues to further investigate the role of inhibitors of xanthine oxidase in infl ammation. Clove extract could almost normalize the enzyme activity. Diclofenac, however, did not decrease the enzyme activity in CIO rat, but reduced it in CPG animals. Th is study further correlates the level of XOD-derived ROS with lipid peroxidation (LPO), which has been reported to increase in infl ammation. LPO indicates changes in membrane fl uidity and permeability. Th is study reports increased LPO in both CIO and CPG, and the level reduced signifi cantly in extract treated rats (Table 2). The extract was also found to normalize total leukocyte count, neutrophil, lymphocyte, and platelet count (data not shown), which are aff ected in infl ammation. Recruitment and activation of leukocytes and neutrophils, which produce ROS upon activation, has been reported by others to contribute to tissue damage in infl ammation [19].

Tissue damage related to oxidative stress and consequent LPO can be reversed by the reduced form of glutathione (GSH). In states of oxidative stress, glutathione is depleted leading to further peroxidation of lipids, and a fall in glutathione is usually accompanied by a rise in lipid peroxidation [22]. GSH has pleiotropic roles including the maintenance of cells in a reduced state, serving as an electron donor for certain antioxidative enzymes, for example, glutathione peroxidase, and the formation of conjugates with some harmful endogenous and xenobiotic compounds by glutathione s-transferase. Th us, the eff ect of the extract on GSH may further benefi t its anti-infl ammatory action. We observed a slight (nonsignifi cant) decrease in GSH in CIO, while it increased slightly in CPG. GSH, however, increased considerably in the extract treated rats (Table 2), which could be due to its rapid turnover by glutathione reductase (GR). The activity of GR was found to increase signifi cantly in CPG rats, while it decreased in CIO rats (Table 3). The results correlate with the level of GSH, which showed a slight change in CIO rats. Increase in LPO in CIO group was also considerably less when compared to CPG group (Table 1). Higher level of GR in CPG could be due to increased GSH turnover that can be attributed to increased LPO. Oxidative stress seems to play a more prominent role in CPG. GSH is particularly important because it neutralizes the eff ect of peroxides generated in oxidative stress. The process involves glutathione peroxidase (GPx), which converts glutathione to its oxidized form (GSSG). We observed an increase in GPx in both CIO and CPG rats. Catalase, another enzyme involved in peroxide metabolism, also increased in the two animal models, and clove extract attenuated the activity of these enzymes. Diclofenac, the drug used in infl ammation, is also reported to increase the activity of catalase and GPx (Table 3). These results suggest the clove extract might exert its effect by ameliorating oxidative stress in infl ammation.

Table 3: Effect of clove extract on peroxide and glutathione metabolizing enzymes.

A large number of phytochemicals, for example, the phenylpropanoids (carvacrol, thymol, eugenol and cinnamaldehyde) [5,23,24], alkaloids, amino acids, fl avonoids, proteins, sterols, reducing sugars, tannins, phenols [25], ascorbic-acid, benzyl-acetate, β-carotene, β-caryophyllene, β-pinene, β-sitosterol-glucoside, carvone, furfural, kaempferol, mucilage, naphthalene, oleanolic acid, stigmasterol, aluminum, zinc, boron, calcium, and manganese [18] have been isolated from clove and several have been reported for various pharmacological actions [26]. Earlier, eugenol was reported to inhibit ROS generated by macrophages [27], inhibit prostaglandin biosynthesis, formation of thromboxane B2, and arachidonic acid-induced platelet aggregation in vitro [28]. β-Sitosterol present in clove is also reported for antiulcer, anticancer, anti-diabetic, and T-cell proliferative activity in human [29]. Th us, it is diffi cult to assign the reported eff ect of clove extract to any specifi c ingredient, and various bioactive principles present in the extract are proposed to act synergistically. It is proposed that the extract should be used in its crude form in order to achieve better results. The idea is not to negate the use of single purified compound, but highlight the relevance of using the extract in the form it has been used traditionally. The presence of more than one ingredient in the dose is well documented to potentiate the eff ect of purified active principle. Further, the presence of antioxidants in the herb is proposed to attenuate the oxidative stress and, thus, infl ammation. In addition to this, the use of purified active principle is often limited due to its toxicity and cost, which is not the case when the whole extract is used. Eugenol, which is present in the clove, has been reported to induce cell death (apoptosis), and might not account for all benefi cial eff ects of clove.

Conclusion

Clove extract is reported to inhibit acute and chronic infl ammation, possibly through a mechanism that ameliorates the oxidative stress possibly by inhibiting xanthine oxidase. The study further reveals signifi cant anti-oxidative eff ect of clove in vivo, and suggests the eff ect to be the consequence of multiple phytochemicals present in the extract.

Acknowledgments

The Department of Biochemistry acknowledges UGC for providing fi - nancial assistance under SAP (DRS-1). Authors contributed equally to the study.

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