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Annona muricata L. (AML), which is from the family of dicotyledon, has been widely used as ethnobotanical remedy in treating sedative, insecticidal, antiparasitic, antirheumatic and ametic (Santos, 2001). The possible anti-inflammatory, antinociceptive and anti-ulcer effects of ethanol extract of AML were investigated. The present results showed that AML exerted its effect in all the animal model tests.
5.1 Toxicity Study
The AML exerted its acute LD50 when given orally was found to be 890 mg/kg. Therefore, the largest dose administrated (300 mg/kg) in this study is the lowest dose that applied for the determination of LD50 without causing any death in mice.
5.2 Anti-inflammatory Activity
The efficacies of herbal medicines have been studied in many previous cases, including acute and chronic in¬‚ammatory as well as arthritic illnesses (Ahmed et al., 2005). In this study, experimental results show that the ethanolic extract of AML performs as an anti-inflammatory agent in mice and rats in both acute and chronic inflammation models.
Xylene-induced ear oedema test substitutes a skin inflammation model appropriate for the experimental of topical anti-inflammatory agent. Ear oedema model allows the evaluation of anti-inflammatory steroids and is less sensitive to non-steroidal anti-inflammatory agents (Zaninir et al., 1992). Xylene-induced mouse ear oedema also reflects the oedematization during the early stages of acute inflammation, which was probably related with the release and inhibition of the inflammation factors (Lin et al., 2007). Histopathologically, severe vasodilation, oedematous changes of the skin and infiltration of inflammatory cells are detected as signs of acute inflammation after topical application of xylene (Kou et al., 2003). In the present study, the increases in ear weight were inhibited significantly by a dose-related manner in the higher doses of the extract; which indicates possible anti-phlogistic but not anti-proliferative effects of the extract (Atta and Alkofahi, 1998). The effect of AML extract in this model may also suggest inhibition of phospholispase A2 (PLA2) (Akindele and Adeyemi, 2007).
Rheumatoid arthritis (RA), defined as a symmetric polyarticular arthritis that basically affects small diarthroidal joints of the hands and feet, is the most common inflammatory arthritis as well as a major cause of disability (Firestein, 2003). The model of adjuvant induced arthritis in rats has been extensively used in the study of in¬‚ammatory processes (Jones and Ward, 1966) and validated as a model of chronic pain (Colpaert et al., 1982). Rats were selected to induce arthritis because they develop a chronic swelling in multiple joints due to accumulation of inflammatory cells, erosion of joint cartilage and bone destruction. Furthermore, it also has close similarities to human rheumatoid diseases (Singh and Majumdar, 1996). The determination of paw swelling is apparently simple, sensitive and quick procedure for evaluating the degree of inflammation and the therapeutic effects of drugs (Tripathy et al., 2009).
CFA-induced polyarthritis is associated with an immune-mediated inflammatory reaction and the rat is unique in developing polyarthritis after CFA treatment (Cai et al., 2006). The initial reaction of edema and soft-tissue thickening at the depot site in this model is caused by the irritant effect of the adjuvant, whereas the late-phase arthritis and flare in the injected foot are presumed to be immunologic events (Ward and Cloud, 1965). The appearance of secondary lesions, i.e. non-injected paw swelling is a manifestation of cell-mediated immunity. The suppression of such secondary lesions by a drug shows its immunosuppressive activity (Singh et al., 2003; Bani et al., 2007). The arthritis-like symptoms in adjuvant induced rats share several histopathological features with human RA, such as mononuclear cell infiltration and synoviocyte hyperplasia that results in pannus formation followed by bone and cartilage destruction (Bendele et al., 1999).
Paw swelling is one of the major factors in evaluating the degree of inflammation and therapeutic efficacy of the drugs (Begum & Sadique, 1988). The initial inflammatory response is developed within hours, but more critical clinical signs observed from the 10th post-inoculation day and thereafter and the changes remain detectable for several weeks (Clopaert et al., 1982). The present study demonstrated that AML extract is able to suppress the swelling of the paws in both acute and chronic phases maybe due to the suppression of inflammatory mediator released due to the induction of CFA (Tripathy et al., 2009)
Further, we found that at higher dosage i.e. 30 and 100 mg/kg, AML extract significantly decreased the concentration of the pro-inflammatory cytokines TNF-Î± and IL-1Î² at the local inflammation site in the AA. It was reported that increased expression of inflammatory cytokines, including TNF-Î± and IL-1Î² was observed in the bone region of the knee joint or serum samples from human osteoarthritis or rheumatoid arthritis patients (Kaneko et al., 2001). TNF-Î± and IL-1Î² enhance the proliferation of fibroblasts, stimulate the production of PGE2 (Arend and Dayer, 1995), and increase the expression of other cytokines and synthesis of collagen by synovial cells, contributing to cartilage and bone destruction (Dayer and Fenner, 1992). Thus, various strategies to block their activity are now being clinically applied and have been shown to be effective in the treatment of experimental arthritis (Moreland et al., 1999).In the present study, the anti-inflammatory action of AML extract is associated with significantly reduced TNF-Î± and IL-1Î² levels in the sera of AA.
5.3 Antinociceptive Activity
The antinociceptive activity of orally administered ethanol extract of AML was demonstrated in mice and rats in this study by two different chemical nociceptive test models as acetic acid-induced abdominal writhing and formalin test as well as by the thermal nociceptive test model as the hot plate test. The most prominent result of this study was the oral administrations of AML possessed pronounced antinociceptive activity as evaluated in all the test models.
Acetic acid that injected by intraperitoneal can lead to the peritoneal inflammation (acute peritonitis) which illustrated by contraction of the abdominal muscle in conjunction with an extension of the forelimbs and elongation of the body (Koster et al., 1959). Thus, acetic acid-induced writhing test are used as a visceral inflammation pain model for the screening of antinociceptive or anti-inflammatory activity of new analgesic agents (De Souza et al., 2009 M.M. De Souza, M.A. Pereira, J.V. Ardenghi, T.C. Mora, L.F. Bresciani, R.A. Yunes, F. Delle Monache and V. Cechinel-Filho, Filicene obtained from Adiantum cuneatum interacts with the cholinergic, dopaminergic, glutamatergic, GABAergic, and tachykinergic systems to exert antinociceptive effect in mice, Pharmacology Biochemistry and Behavior 93 (2009), pp. 40-46. Article | PDF (368 K) | View Record in Scopus | Cited By in Scopus (1)De Souza et al., 2009) and was permitted to evaluate the antinociceptive activity caused by both neurogenic and/or inflammatory pain (Benth et al., 2010). Besides that, acetic acid writhing test is used to evaluate the compounds for peripheral antinociceptive activities in general. Therefore, the writhing test is useful for distinguishing between central and peripheral nociception (Le Bars et al., 2001 D. Le Bars, M. Gozariu and S. Cadden, Animal models of nociception, Pharmacological Reviews 53 (2001), pp. 628-651.Le Bars et al., 2001).
Acetic acid injected into peritoneal cavity is believed can leads to an increased of cyclooxygenase (COX) and lipooxygenase (LOX) products in peritoneal fluids as well as promoting the release of other inflammatory mediators such as bradykinin, substance P, TNF-Î±, IL-1Î², IL-8, which finally stimulate the primary afferent nociceptors entering dorsal horn of the central nervous system (Ikeda et al., 2001). The results reported herein demonstrated that the administration of the AML significantly reduced the number of abdominal writhing induced by acetic acid in a dose-dependent manner. Thus, the present result suggests that the mechanism of AML may be partly mediated by the inhibition of COX and/or LOX and other inflammatory mediators in peripheral tissues. Also the antinociceptive activity could be suggested by the interruption of signal transduction in primary afferent nociceptors.
Although the acetic acid-induced abdominal writhing test exhibits good sensitivity in detecting the antinociceptive effects induced by NSAIDs, narcotics and other centrally-acting drugs (Bentley et al. 1981), but in some cases this method may demonstrates poor specificity due to the suppression of muscle relaxants and other types of drugs, eventually leads to misinterpretation of the results (Le Bars et al., 2001). Hence, in order to avoid misinterpretation, the formalin-induced paw licking as well as hot plate test models were employed in the present study.
The formalin test is not only a very well-described method in assessing antinociceptive drugs but also useful in the elucidation of the action mechanism. The advantage of using the formalin model of nociception is that it can distinguish between central and peripheral pain components (Tjolsen et al., 1992). This test is a model of nociceptive response iwhich can be characterized by two distinct phases involving different mechanisms. The first phase or the neurogenic pain is an intensely painful stage caused by the direct formalin action through the activation of nociceptive neurons in the periphery and the second phase correspond to the inflammatory pain occurs at the spinal cord level through the activation of the ventral horn neurons (Shibata et al., 1989).
Accordingly, it was reported that substance P and bradykinin participate in the appearance of the first-phase, while the late phase is due to a release of serotonin, histamine, bradykinin and prostaglandins (Le Bars et al., 2001; Parada et al., 2001; Verma et al., 2005). It is well known that drugs that act primarily on the central nervous system can inhibit in both phases equally such as morphine (Shibata et al., 1989), while peripherally acting drugs only can show pronounce inhibition in the late phase (Tjolsen et al., 1992). In the present study, our results produced antinociception against both neurogenic and inflammatory phase of formalin test. The time spent in licking the injured paw was significantly reduced by oral administration of the AML in both phases. Besides that, morphine, the centrally acting drugs, inhibited both phases equally in this test, while ASA, peripherally acting drugs, only significantly inhibited the second phase of the formalin. However, AML administered at 300 mg/kg, p.o. possessed stronger antinociceptive effect than ASA (100 mg/kg, p.o.) but less than morphine (5 mg/kg, i.p.) in the formalin. Due to the inhibitory possessions by AML on both neurogenic and inflammatory phase of the formalin-induced paw licking test, therefore suggests that the extract contains active properties acting both centrally and peripherally. In line with the present result, it also shows that the AML not only possesses antinociceptive effect but also anti-inflammatory activity.
The central antinociceptive action of the AML was supported by the result obtained in the hot plate test as it is a sensitive and specific method used to demonstrate the involvement of central mechanism ([Nemirovsky et al., 2001; Sulaiman et al., 2009). In the present study postulated that oral administration of AML shows pronounce dose-related prolongation in the latency time to the thermal nociceptive stimulus as compared to the control group with the dose 300 mg/kg provoking the longest latency, in consequence confirming the central activity of this extract.
The increased of nociceptive threshold of mice in the hot plate test together with the reduction of nociception in the first phase of formalin test have further strengthened the evidence of centrally mediated antinociceptive activity of the AML. In addition, this test was considered to be sensitive to drugs acting at the supraspinal modulation level (Yaksh and Rudy, 1977), suggesting the central antinociceptive effect of the AML which may also predominantly a spinal reflex (Nemirovsky et al., 2001).
Besides that, , it is well described that endogenous opioid system is largely involved in the central regulation of pain, as well as in the action of opioid-derived analgesic drugs (Sakurada et al., 2005 T. Sakurada, T. Komatsu and S. Sakurada, Mechanisms of nociception evoked by intrathecal high-dose morphine, Neurotoxicology 26 (2005), pp. 801-809. Article | PDF (168 K) | View Record in Scopus | Cited By in Scopus (17)Sakurada et al., 2005). In the present results exerted that the antinociception elicited by AML seems to be dependent of the activation of opioid system. This was observed base on the result showed that pre-treatment with a non-selective opioid receptor antagonist, naloxone, significantly antagonized the antinociceptive effect of AML but significantly reversed the antinociceptive effect of morphine in the hot plate test. These findings clearly suggest that AML possessed its antinociceptive activity which was mediated through opioid mechanism. The antinociceptive effect may cause of the activation of opioid receptors or the modulation of the effect of endogenous opioid peptides which may participate in the antinociceptive activity at both peripheral and central levels (Hui Ming et al., 2010).
5.4 Anti-ulcerogenic Activity
Peptic ulcer is a benign gastric or duodenal mucosa lesion, which developed when the delicate balance between some gastroprotective and aggressive factor is lost. Major aggressive factors include acid, pepsin, Helicobacter pylori and bile salts (Jainu and Devi, 2006). According to Bighetti et al. (2005), the treatment of peptic ulcer is generally based on the inhibition of gastric acid secretion by H2-antagonists, such as omeprazole and antimuscarinics, as well as acid-independent treatment by sucralfate and bismuth. However, there is a major problem in the treatment of gastroduodenal ulcer which even though a healing rate of 80-100% after 4-8 weeks of therapy with H2-antagonists and proton pump inhibitors, the rate of ulcer recurrence within 1 year after suspending treatment is between 40 to 80 % (Miller and Faragher, 1986). In addition, most of these drugs produce several adverse reactions (Ariyphisi et al., 1986).
Gastric ulcer normally occurs in people who take non-steroid anti-inflammatory drugs (NSAIDs), which known to possess antinociceptive and anti-inflammatory effects depending on the particular drug at different doses. All of the NSAIDs appear to share a common mechanism, namely inhibition of cyclo-oxygenase (COX) enzyme(s), which leads to a decrease in the synthesis of various prostaglandins and thromboxanes. These will lead to an increase of 3- to 10- fold of ulcer complications in the stomach and duodenum and the affected patients treated with NSAIDs may be hospitalized and may also die from the ulcer (Seager and Hawkey, 2001). It has been demonstrated that extracts of AML posses an anti-inflammatory effect (Roslida et al., 2008; Souza et al., 2010). A search for new compounds or plant extracts with anti-inflammatory activity could represent a promising and practical approach for the management of inflammatory diseases, especially if these compounds do not harm the gastrointestinal tract is therefore essential.
Absolute ethanol can leads to severe damage of the gastric mucosa and it provokes multiple hemorrhagic red bands (patches) of different sizes along the long axis of the glandular stomach (Mincis et al., 1995). Even though the pathogenesis of ethanol-induced gastric mucosal damage is still unknown, but it may be due to stasis in gastric blood flow, which contributes to the development of the hemorrhage and necrotic aspects of tissue injury (Guth et al., 1984).
Exposure to ethanol helps in augment of the cellular damage extension in a dose-dependent way (Mutoh et al.. 1990). Ethanol is metabolized in the body and releases superoxide anion and hydroperoxy free radicals and it has been found that oxygen-derived free radicals are involved in the mechanism of acute and chronic ulceration in the gastric mucosa (Pihan et al., 1987). Furthermore, disturbances of mucosal microcirculation, ischaemia and appearance of free radicals, endothelin release, degranulation of mast cells, inhibition of prostaglandins and reduction of mucus production are observed after the administration of ethanol (Samonina et al., 2004).
Ethanol also induces solubilization of the mucus constituents in the stomach, a concomitant fall in the transmucosal potential difference and increase the flow of Na+ and K+ into the lumen, pepsin secretion, the loss of H+ ions and the histamine content in the lumen (Guth et al., 1984; Szabo and Vattay, 1990). Furthermore, the acute treatment with ethanol causes oxidative stress, DNA damage, enhanced xanthine oxidase activity and malonyldialdehyde levels and reduction in total glutathione content in gastric mucosal cells (Marotta et al., 1999).
Gastric mucus acts as an important protective factor for the gastric mucosa. It is not only capable of acting as an antioxidant agent but also reducing mucosal damage mediated by oxygen free radicals. However, the protective properties of the mucus barrier depend on the gel structure and also on the amount or thickness of the layer covering the mucosal surface (Penisi and Piezzi, 1999). Therefore, antiulcer agents should provoke mucosa-strengthening effect and cicatrisation action with low occurrence of side effects. These data suggest that antioxidant compounds could be active in this experiment model, producing antiulecrogenic effects. This effect is known as cytoprotection.
In the present study, the control group treated orally with ethanol clearly produced the expected characteristic zone of necrotizing mucosal lesions. On the other hand, one hour before the induction of the gastric lesion, oral administration of AML extract significantly decreased the total lesion area and the percentage of lesion. The best inhibitory effect on the ulcerative area was observed with oral treatment of AML showing decrease of 51.77% (30 mg/kg), 75.43% (100 mg/kg) and 92.80% (300 mg/kg), respectively, when compared to the ulcerative area of 273.3 ± 11.13mm2 of the control group (Table 4.8). These results indicate that AML extract displays an antiulcerogenic effect related to cytoprotective activity, since it significantly reduced the ethanol-induced ulcer. In addition, the inhibition of ulcer after treatment with AML leaf extract was similar to lansoprazole at dose of 30 mg/kg.
The next step of this work was to investigate the probable antiulcerogenic mechanism of action promoted by AML. The roles of nitric oxide (NO) and sulfhydyl (SH) compounds were both investigated because these endogenous substances are involved physiologically in gastric mucosa protection. Studies designate the involvement of NO, an endogen substance that enhances the mechanisms of cytoprotection of mucosa observed experimentally by promoting blood vessel dilatation and reducing lipoperoxidation while acting similarly to an anti-inflammatory drug (Ancha et al., 2003). It is also well recognized that NO was involved in the modulation of acid and alkaline secretion, mucus secretion and gastric mucosal blood flow (Chandranath et al., 2002). Besides that, nitric oxide also modulates the secretion of some cellular growth factors, primarily the epidermal growth factor (Elliot et al., 1995).
In order to investigate the role of endogenous NO in cytoprotection, we used the NOS inhibitor, L-NAME to investigate the protective effect of AML on ethanol-induced gastric haemorrhagic lesions (Fig 4.4). Pre-treatment with L-NAME did not alter the cytoprotection induced by AML. Oral administration of AML (300 mg/kg) to L-NAME (70 mg/kg) - pre-treated animals produced a reduction (93.88%) in the gastric haemorrhagic lesions when compared with the L-NAME-pre-treated control value of 291.17 ± 20.62. This gastroprotection effect of AML observed by pre-treatment with L-NAME was very similar to the protective activity observed by pre-treatment with saline (86.83%). This result showed that the previous administration of L-NAME, a NO-synthase inhibitor did not alter the AML-induced cytoprotection of ethanol-induced gastric lesions, thus it is possible to affirm that the main gastroprotective mechanism involving AML extract is not related to NO physiological action in ulcer locally induced by ethanol.
Besides the NO, the role of endogenous SH compounds in mucosal protection has been demonstrated in ethanol-induced gastric injury where the development of damage was accompanied by a lowering in production of mucosal SH compounds (Avila et al., 1996). The synthesis of mucus which strengthens the mucosa barrier against harmful agents also plays it vital role in gastric production. The continuous adherent mucus layer is also a barrier to luminal pepsin, thus protecting the underlying mucosa from proteolytic digestion (Allen and Flemstrom, 2005). The adherent mucus gel layer is a structural one to create a stable, undisturbed layer to support surface neutralization of acid and also acts as a protective physical barrier against luminal pepsin. Since the SH compounds bind to free radicals that form tissue injury by noxious agents and these agents may also protect mucus, since mucus subunits are joined by disulfide bridges that, if reduced, render mucus water-soluble (Avila et al., 1996).
When cells containing mucus are damaged by extracellular oxygen radicals, intracellular mucus may be released into the gastric tissue to prevent further damage by scavenging these radicals (Berenguer et al., 2007). Furthermore, the growth in damage is accompanied by a decrease of the concentration of mucosal SH compounds which is caused of the SH-groups bind the free radicals formed due to the action of noxious agents. For this reason, we investigated the possible involvement of endogenous SHs in the gastroprotective effect of AML extract by pre-treating animals with NEM (a SH-blocker) in gastric lesion induced by ethanol (Figure 4.5). Pre-treatment with NEM alters the cytoprotection induced by AML. Pre-treatment of animals with NEM noticeably increased the gastric lesions when compared to control groups. The animals treated with AML (300 mg/kg) reduce the gastric lesion almost by one third (82.02% with saline versus 58.21% with NEM). This significant (p<0.001) attenuation of gastric lesions indicated the strong involvement of endogenous SHs in the gastroprotection effects of the AML extract.