Annona muricata L. ( AML ) , which is from the household of magnoliopsid, has been widely used as ethnobotanical redress in handling ataractic, insecticidal, antiparasitic, antirheumatic and ametic ( Santos, 2001 ) . The possible anti-inflammatory, antinociceptive and anti-ulcer effects of ethanol infusion of AML were investigated.
The present consequences showed that AML exerted its consequence in all the carnal theoretical account trials.
5.1 Toxicity Study
The AML exerted its ague LD50 when given orally was found to be 890 mg/kg.
Therefore, the largest dosage administrated ( 300 mg/kg ) in this survey is the lowest dosage that applied for the finding of LD50 without doing any decease in mice.
5.2 Anti-inflammatory Activity
The efficaciousnesss of herbal medical specialties have been studied in many old instances, including ague and chronic ini¬‚ammatory every bit good as creaky unwellnesss ( Ahmed et al.
, 2005 ) . In this survey, experimental consequences show that the ethanolic infusion of AML performs as an anti-inflammatory agent in mice and rats in both ague and chronic redness theoretical accounts.Xylene-induced ear oedema trial substitutes a skin redness theoretical account appropriate for the experimental of topical anti-inflammatory agent. Ear oedema theoretical account allows the rating of anti-inflammatory steroids and is less sensitive to non-steroidal anti-inflammatory agents ( Zaninir et al. , 1992 ) . A Xylene-induced mouse ear hydrops besides reflects the oedematization during the early phases of acute redness, which was likely related with the release and suppression of the redness factors ( Lin et al.
, 2007 ) . Histopathologically, terrible vasodilation, oedematous alterations of the tegument and infiltration of inflammatory cells are detected as marks of acute redness after topical application of xylene ( Kou et al. , 2003 ) . In the present survey, the additions in ear weight were inhibited significantly by a dose-related mode in the higher doses of the infusion ; which indicates possible anti-phlogistic but non anti-proliferative effects of the infusion ( Atta and Alkofahi, 1998 ) . The consequence of AML infusion in this theoretical account may besides propose suppression of phospholispase A2 ( PLA2 ) ( Akindele and Adeyemi, 2007 ) .Rheumatoid arthritis ( RA ) , defined as a symmetric polyarticular arthritis that fundamentally affects little diarthroidal articulations of the custodies and pess, is the most common inflammatory arthritis every bit good as a major cause of disablement ( Firestein, 2003 ) .
The theoretical account of adjuvant induced arthritis in rats has been extensively used in the survey of ini¬‚ammatory procedures ( Jones and Ward, 1966 ) and validated as a theoretical account of chronic hurting ( Colpaert et al. , 1982 ) . Rats were selected to bring on arthritis because they develop a chronic puffiness in multiple articulations due to accretion of inflammatory cells, eroding of joint gristle and bone devastation. Furthermore, it besides has close similarities to human rheumatoid diseases ( Singh and Majumdar, 1996 ) . The finding of paw puffiness is seemingly simple, sensitive and speedy process for measuring the grade of redness and the curative effects of drugs ( Tripathy et al. , 2009 ) .
CFA-induced polyarthritis is associated with an immune-mediated inflammatory reaction and the rat is alone in developing polyarthritis after CFA intervention ( Cai et al. , 2006 ) . The initial reaction of hydrops and soft-tissue thickener at the terminal site in this theoretical account is caused by the irritant consequence of the adjuvant, whereas the late-phase arthritis and flair in the injected pes are presumed to be immunologic events ( Ward and Cloud, 1965 ) . The visual aspect of secondary lesions, i.e. non-injected paw puffiness is a manifestation of cell-mediated unsusceptibility. 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 portion several histopathological characteristics with human RA, such as mononucleate cell infiltration and synoviocyte hyperplasia that consequences in pannus formation followed by bone and gristle devastation ( Bendele et al. , 1999 ) .
Paw puffiness is one of the major factors in measuring the grade of redness and curative efficaciousness of the drugs ( Begum & A ; Sadique, 1988 ) . The initial inflammatory response is developed within hours, but more critical clinical marks observed from the tenth post-inoculation twenty-four hours and thenceforth and the alterations remain noticeable for several hebdomads ( Clopaert et al. , 1982 ) . The present survey demonstrated that AML infusion is able to stamp down the puffiness of the paws in both ague and chronic stages possibly due to the suppression of inflammatory go-between released due to the initiation of CFA ( Tripathy et al. , 2009 )Further, we found that at higher dose i.e.
30 and 100 mg/kg, AML extract significantly decreased the concentration of the proinflammatory cytokines TNF-I± and IL-1I? at the local redness site in the AA. It was reported that increased look of inflammatory cytokines, including TNF-I± and IL-1I? was observed in the bone part of the articulatio genus articulation or serum samples from human degenerative arthritis or rheumatoid arthritis patients ( Kaneko et al. , 2001 ) . TNF-I± and IL-1I? enhance the proliferation of fibroblasts, stimulate the production of PGE2 ( Arend and Dayer, 1995 ) , and increase the look of other cytokines and synthesis of collagen by synovial cells, lending to cartilage and cram devastation ( Dayer and Fenner, 1992 ) . Therefore, assorted schemes to barricade their activity are now being clinically applied and have been shown to be effectual in the intervention of experimental arthritis ( Moreland et al. , 1999 ) .
In the present survey, the anti-inflammatory action of AML infusion is associated with significantly reduced TNF-I± and IL-1I? degrees in the sera of AA.
5.3 Antinociceptive Activity
The antinociceptive activity of orally administered ethanol infusion of AML was demonstrated in mice and rats in this survey by two different chemical nociceptive trial theoretical accounts as acetic acid-induced abdominal writhing and formalin trial every bit good as by the thermic nociceptive trial theoretical account as the hot home base trial. The most outstanding consequence of this survey was the unwritten disposals of AML possessed marked antinociceptive activity as evaluated in all the trial theoretical accounts.Acetic acid that injected by intraperitoneal can take to the peritoneal redness ( acute peritoneal inflammation ) which illustrated by contraction of the abdominal musculus in concurrence with an extension of the forelimbs and elongation of the organic structure ( Koster et al. , 1959 ) . Therefore, acetic acid-induced writhing trial are used as a splanchnic redness hurting theoretical account for the showing of antinociceptive or anti-inflammatory activity of new analgetic 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 exercise antinociceptive consequence 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 measure the antinociceptive activity caused by both neurogenic and/or inflammatory hurting ( Benth et al. , 2010 ) . Besides that, acetic acid wrestling trial is used to measure the compounds for peripheral antinociceptive activities in general. Therefore, the wrestling trial is utile for separating between cardinal and peripheral nociception ( Le Bars et al. , 2001 D. Le Bars, M.
Gozariu and S. Cadden, Animal theoretical accounts of nociception, Pharmacological Reviews 53 ( 2001 ) , pp. 628-651.Le Bars et al. , 2001 ) .
Acetic acid injected into peritoneal pit is believed can take to an increased of Cox ( COX ) and lipooxygenase ( LOX ) merchandises in peritoneal fluids every bit good as advancing the release of other inflammatory go-betweens such as bradykinin, substance P, TNF-I± , IL-1I? , IL-8, which eventually stimulate the primary sensory nerve nociceptors come ining dorsal horn of the cardinal nervous system ( Ikeda et al. , 2001 ) . The consequences reported herein demonstrated that the disposal of the AML significantly reduced the figure of abdominal writhing induced by acetic acid in a dose-dependent mode. Therefore, the present consequence suggests that the mechanism of AML may be partially mediated by the suppression of COX and/or LOX and other inflammatory go-betweens in peripheral tissues. Besides the antinociceptive activity could be suggested by the break of signal transduction in primary afferent nociceptors.Although the acetic acid-induced abdominal wrestling trial exhibits good sensitiveness in observing the antinociceptive effects induced by NSAIDs, narcotics and other centrally-acting drugs ( Bentley et al. 1981 ) , but in some instances this method may show hapless specificity due to the suppression of musculus relaxants and other types of drugs, finally leads to misunderstanding of the consequences ( Le Bars et al.
, 2001 ) . Hence, in order to avoid misunderstanding, the formalin-induced paw defeat every bit good as hot home base trial theoretical accounts were employed in the present survey.The formalin trial is non merely a really well-described method in measuring antinociceptive drugs but besides utile in the elucidation of the action mechanism. The advantage of utilizing the formalin theoretical account of nociception is that it can separate between cardinal and peripheral hurting constituents ( Tjolsen et al.
, 1992 ) . This trial is a theoretical account of nociceptive response iwhich can be characterized by two distinguishable stages affecting different mechanisms. The first stage or the neurogenic hurting is an intensely painful phase caused by the direct formol action through the activation of nociceptive nerve cells in the fringe and the 2nd stage correspond to the inflammatory hurting occurs at the spinal cord degree through the activation of the ventral horn nerve cells ( Shibata et al.
, 1989 ) .Consequently, it was reported that substance P and bradykinin participate in the visual aspect of the first-phase, while the late stage is due to a release of 5-hydroxytryptamine, histamine, bradykinin and prostaglandins ( Le Bars et al. , 2001 ; Parada et al. , 2001 ; Verma et al. , 2005 ) . It is good known that drugs that act chiefly on the cardinal nervous system can suppress in both stages every bit such as morphia ( Shibata et al.
, 1989 ) , while peripherally moving drugs merely can demo pronounce suppression in the late stage ( Tjolsen et al. , 1992 ) . In the present survey, our consequences produced antinociception against both neurogenic and inflammatory stage of formalin trial. The clip spent in creaming the injured paw was significantly reduced by unwritten disposal of the AML in both stages. Besides that, morphia, the centrally moving drugs, inhibited both stages every bit in this trial, while ASA, peripherally moving drugs, merely significantly inhibited the 2nd stage of the formol. However, AML administered at 300A mg/kg, p.o. possessed stronger antinociceptive consequence than ASA ( 100A mg/kg, p.
o. ) but less than morphia ( 5A mg/kg, i.p. ) in the formol. Due to the repressive ownerships by AML on both neurogenic and inflammatory stage of the formalin-induced paw creaming trial, hence suggests that the infusion contains active belongingss moving both centrally and peripherally.
In line with the present consequence, it besides shows that the AML non merely possesses antinociceptive consequence but besides anti-inflammatory activity.The cardinal antinociceptive action of the AML was supported by the consequence obtained in the hot home base trial as it is a sensitive and specific method used to show the engagement of cardinal mechanism ( [ Nemirovsky et al. , 2001 ; Sulaiman et al. , 2009 ) . In the present survey postulated that unwritten disposal of AML shows pronounce dose-related protraction in the latency clip to the thermic nociceptive stimulation as compared to the control group with the dose 300A mg/kg arousing the longest latency, in effect corroborating the cardinal activity of this infusion.
The increased of nociceptive threshold of mice in the hot home base trial together with the decrease of nociception in the first stage of formalin trial have further strengthened the grounds of centrally mediated antinociceptive activity of the AML. In add-on, this trial was considered to be sensitive to drugs moving at the supraspinal transition degree ( Yaksh and Rudy, 1977 ) , proposing the cardinal antinociceptive consequence of the AML which may besides predominantly a spinal physiological reaction ( Nemirovsky et al. , 2001 ) .Besides that, , it is good described that endogenous opioid system is mostly involved in the cardinal ordinance of hurting, every bit good as in the action of opioid-derived analgetic drugs ( Sakurada et al. , 2005 T.
Sakurada, T. Komatsu and S. Sakurada, Mechanisms of nociception evoked by intrathecal high-dose morphia, 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 consequences exerted that the antinociception elicited by AML seems to be dependent of the activation of opioid system. This was ascertained base on the consequence showed that pre-treatment with a non-selective opioid receptor adversary, Narcan, significantly antagonized the antinociceptive consequence of AML but significantly reversed the antinociceptive consequence of morphia in the hot home base trial.
These findings clearly suggest that AML possessed its antinociceptive activity which was mediated through opioid mechanism. The antinociceptive consequence may do of the activation of opioid receptors or the transition of the consequence of endogenous opioid peptides which may take part in the antinociceptive activity at both peripheral and cardinal degrees ( Hui Ming et al. , 2010 ) .
5.4 Anti-ulcerogenic Activity
Peptic ulcer is a benign gastric or duodenal mucous membrane lesion, which developed when the delicate balance between some gastroprotective and aggressive factor is lost. Major aggressive factors include acerb, pepsin, Helicobacter pylori and gall salts ( Jainu and Devi, 2006 ) . Harmonizing to Bighetti et Al. ( 2005 ) , the intervention of peptic ulcer is by and large based on the suppression of stomachic acerb secernment by H2-antagonists, such as omeprazole and antimuscarinics, every bit good as acid-independent intervention by Carafate and Bi.
However, there is a major job in the intervention of gastroduodenal ulcer which even though a mending rate of 80-100 % after 4-8 hebdomads of therapy with H2-antagonists and proton pump inhibitors, the rate of ulcer return within 1 twelvemonth after suspending intervention is between 40 to 80 % ( Miller and Faragher, 1986 ) . In add-on, most of these drugs produce several inauspicious reactions ( Ariyphisi et al. , 1986 ) .Gastric ulcer usually occurs in people who take non-steroid anti-inflammatory drugs ( NSAIDs ) , which known to possess antinociceptive and anti-inflammatory effects depending on the peculiar drug at different doses. All of the NSAIDs appear to portion a common mechanism, viz. suppression of cyclo-oxygenase ( COX ) enzyme ( s ) , which leads to a lessening in the synthesis of assorted prostaglandins and thromboxanes. These will take to an addition of 3- to 10- crease of ulcer complications in the tummy and duodenum and the affected patients treated with NSAIDs may be hospitalized and may besides decease from the ulcer ( Seager and Hawkey, 2001 ) . It has been demonstrated that infusions of AML posses an anti-inflammatory consequence ( Roslida et al.
, 2008 ; Souza et al. , 2010 ) . A hunt for new compounds or works infusions with anti-inflammatory activity could stand for a promising and practical attack for the direction of inflammatory diseases, particularly if these compounds do non harm the GI piece of land is hence indispensable.Absolute ethyl alcohol can take to terrible harm of the stomachic mucous membrane and it provokes multiple hemorrhagic ruddy sets ( spots ) of different sizes along the long axis of the glandular tummy ( Mincis et al. , 1995 ) .
Even though the pathogenesis of ethanol-induced stomachic mucosal harm is still unknown, but it may be due to stasis in stomachic blood flow, which contributes to the development of the bleeding and necrotic facets of tissue hurt ( Guth et al. , 1984 ) .Exposure to ethanol aids in augment of the cellular harm extension in a dose-dependent manner ( Mutoh et al.. 1990 ) . Ethanol is metabolized in the organic structure and releases superoxide anion and hydroperoxy free groups and it has been found that oxygen-derived free groups are involved in the mechanism of ague and chronic ulceration in the stomachic mucous membrane ( Pihan et al.
, 1987 ) . Furthermore, perturbations of mucosal microcirculation, ischemia and visual aspect of free groups, endothelin release, degranulation of mast cells, suppression of prostaglandins and decrease of mucous secretion production are observed after the disposal of ethyl alcohol ( Samonina et al. , 2004 ) .Ethanol besides induces solubilization of the mucous secretion components in the tummy, a attendant autumn in the transmucosal possible difference and increase the flow of Na+ and K+ into the lms, pepsin secernment, the loss of H+ ions and the histamine content in the lms ( Guth et al. , 1984 ; Szabo and Vattay, 1990 ) .
Furthermore, the acute intervention with ethanol causes oxidative emphasis, DNA harm, enhanced xanthine oxidase activity and malonyldialdehyde degrees and decrease in entire glutathione content in stomachic mucosal cells ( Marotta et al. , 1999 ) .Gastric mucous secretion acts as an of import protective factor for the stomachic mucous membrane. It is non merely capable of moving as an antioxidant agent but besides cut downing mucosal harm mediated by O free groups.
However, the protective belongingss of the mucous secretion barrier depend on the gel construction and besides on the sum or thickness of the bed covering the mucosal surface ( Penisi and Piezzi, 1999 ) . Therefore, antiulcer agents should arouse mucosa-strengthening consequence and cicatrisation action with low happening of side effects. These informations suggest that antioxidant compounds could be active in this experiment theoretical account, bring forthing antiulecrogenic effects. This consequence is known as cytoprotection.In the present survey, the control group treated orally with ethanol clearly produced the expected characteristic zone of necrotizing mucosal lesions. On the other manus, one hr before the initiation of the stomachic lesion, unwritten disposal of AML extract significantly decreased the entire lesion country and the per centum of lesion. The best repressive consequence on the ulcerative country was observed with unwritten intervention of AML demoing lessening of 51.
77 % ( 30 mg/kg ) , 75.43 % ( 100 mg/kg ) and 92.80 % ( 300 mg/kg ) , severally, when compared to the ulcerative country of 273.3 A± 11.13mm2 of the control group ( Table 4.8 ) .
These consequences indicate that AML infusion displays an antiulcerogenic consequence related to cytoprotective activity, since it significantly reduced the ethanol-induced ulcer. In add-on, the suppression of ulcer after intervention with AML leaf infusion was similar to lansoprazole at dosage of 30 mg/kg.The following measure of this work was to look into the likely antiulcerogenic mechanism of action promoted by AML. The functions of azotic oxide ( NO ) and sulfhydyl ( SH ) compounds were both investigated because these endogenous substances are involved physiologically in stomachic mucous membrane protection.
Surveies designate the engagement of NO, an monocot substance that enhances the mechanisms of cytoprotection of mucous membrane observed by experimentation by advancing blood vas distension and cut downing lipoperoxidation while moving likewise to an anti-inflammatory drug ( Ancha et al. , 2003 ) . It is besides good recognized that NO was involved in the transition of acid and alkalic secernment, mucus secernment and stomachic mucosal blood flow ( Chandranath et al. , 2002 ) . Besides that, azotic oxide besides modulates the secernment of some cellular growing factors, chiefly the cuticular growing factor ( Elliot et al. , 1995 ) .
In order to look into the function of endogenous NO in cytoprotection, we used the NOS inhibitor, L-NAME to look into the protective consequence of AML on ethanol-induced stomachic haemorrhagic lesions ( Fig 4.4 ) . Pre-treatment with L-NAME did non change the cytoprotection induced by AML. Oral disposal of AML ( 300 mg/kg ) to L-NAME ( 70 mg/kg ) – pre-treated animate beings produced a decrease ( 93.
88 % ) in the stomachic haemorrhagic lesions when compared with the L-NAME-pre-treated control value of 291.17 A± 20.62. This gastroprotection consequence of AML observed by pre-treatment with L-NAME was really similar to the protective activity observed by pre-treatment with saline ( 86.83 % ) . This consequence showed that the old disposal of L-NAME, a NO-synthase inhibitor did non change the AML-induced cytoprotection of ethanol-induced stomachic lesions, therefore it is possible to confirm that the chief gastroprotective mechanism affecting AML infusion is non related to NO physiological action in ulcer locally induced by ethyl alcohol.Besides the NO, the function of endogenous SH compounds in mucosal protection has been demonstrated in ethanol-induced stomachic hurt where the development of harm was accompanied by a lowering in production of mucosal SH compounds ( Avila et al.
, 1996 ) . The synthesis of mucous secretion which strengthens the mucous membrane barrier against harmful agents besides plays it critical function in stomachic production. The uninterrupted disciple mucous secretion bed is besides a barrier to luminal pepsin, therefore protecting the underlying mucous membrane from proteolytic digestion ( Allen and Flemstrom, 2005 ) . The adherent mucous secretion gel bed is a structural one to make a stable, undisturbed bed to back up surface neutralisation of acid and besides acts as a protective physical barrier against luminal pepsin. Since the SH compounds bind to free groups that form tissue hurt by noxious agents and these agents may besides protect mucous secretion, since mucous secretion fractional monetary units are joined by disulfide Bridgess that, if reduced, render mucous secretion water-soluble ( Avila et al. , 1996 ) .
When cells incorporating mucous secretion are damaged by extracellular O groups, intracellular mucous secretion may be released into the stomachic tissue to forestall farther harm by scavenging these groups ( Berenguer et al. , 2007 ) . Furthermore, the growing in harm is accompanied by a lessening of the concentration of mucosal SH compounds which is caused of the SH-groups bind the free groups formed due to the action of noxious agents. For this ground, we investigated the possible engagement of endogenous SHs in the gastroprotective consequence of AML infusion by pre-treating animate beings with NEM ( a SH-blocker ) in stomachic lesion induced by ethyl alcohol ( Figure 4.5 ) .
Pre-treatment with NEM alters the cytoprotection induced by AML. Pre-treatment of animate beings with NEM perceptibly increased the stomachic lesions when compared to command groups. The animate beings treated with AML ( 300 mg/kg ) cut down the gastric lesion about by one tierce ( 82.02 % with saline versus 58.
21 % with NEM ) . This important ( p & lt ; 0.001 ) fading of stomachic lesions indicated the strong engagement of endogenous SHs in the gastroprotection effects of the AML infusion.