Anti-Inflammatory Drug Tests

Anti-Inflammatory Drug TestsThe method described by Lorke with slight modification was utilise to determine the safety of the MEA. Briefly, normal profound male mice were divided into groups of five mice in each cage. MEA (100 and 1000 mg/kg) or fomite were intraperitone all in ally administered. Access to food and water, toxic symptoms and the general sort of mice were detect continuously for 1 h after the treatment, intermittently for 4 h, and thereafter over a period of 24 h. The mice were further observed for up to 14 eld following treatment for whatsoever signs of perniciousness and mortality.ResultOver the consume duration of 14 days, there were no deaths recorded in the groups of mice given 100 or 1000 mg/kg IP of MEA. During the observation period, MEA administration did not induce any variations in the general appearance or toxic signs in the animals.The sinuous attempt has long been used as a screening light beam for the assessment of painkiller or anti- infla mmatory properties of unsanded substances (Collier et al., 1968). This method testifys a good sensitivity, although it has poor specificity. To avoid misinterpretation of the results, in the present study the antinociceptive contract of MEA were confirmed in the formalin block out, a model of inflammatory discommode which has two distinctive variants which may indicate incompatible types of torment (Hunskaar and Hole, 1987). The early and late phases of formalin test view obvious first derivative properties, and whence this test is useful not exactly for assessing the analgesic substances, plainly overly for elucidating the mechanism of analgesia (Shibata et al., 1989). The early phase,named non-inflammatory pain, is a result of point arousal of nociceptors and reflects centrally-mediated pain the late phase,named inflammatory pain, is caused by local firing with a twist of inflammatory and hyperalgesic mediators (Hunskaarand Hole, 1987).The thermal model of the t ail-flick test is considered to be a spinal reflex, but could in addition involve senior high schooler neural structures, and thereof this method identifies mainly central analgesics (Jensen and Yaksh, 1986 Le Bars et al., 2001).Due to their tax deduction in virtually all human and animal diseases, inflammation and pain have become the focus of global scientific research. Adverse do of non-steroidal anti-inflammatory drug medicates (NSAIDs) and opioids have necessitated the search for new drugs with minimal side personnels (Dharmasiri et al.,2003 Vittalrao et al., 2011). The current trend of research is the investigation of medicines of plant origin because of their affordability and accessibility with minimal side effects.The thermal model of the tail-flick test is considered to be a spinal reflex, but could also involve higher neural structures, and therefore this method identifies mainly central analgesics (Jensen and Yaksh, 1986 Le Bars et al., 2001).The analgesic act of Cyathula prostrata in this study was investigated utilize the hot plate and mouse writhing tests. The hot plate test is useful for the evaluation of centrally acting analgesics which are known to elevate the pain threshold of mice towardsheat (Hiruma-Lima et al., 2000). It also indicates narcotic involvement with opioid receptor (Turner, 1965).The writhing model is a elegant method for screening skirting(prenominal) analgesic efficacy agents and it is more light-sensitive to non-steroidal analgesics (Collier et al., 1963). The analgesic effect of acetic acid is due to the liberation and increase level of some(prenominal) mediators such(prenominal) as histamine and serotonin which act by stimulation of peripheral nociceptive neurons (Cui et al., 2010).Over the centuries, phytopharmaceuticals have been utilized by different communities of the world 1.Acetic acid- bring forth writhing is a puff up recommended protocol in evaluating medicinal agents for their analgesic property . The pain induction caused by liberating endogenous substances as well as some some other pain mediators such as arachidonic acid via cyclooxygenase, and prostaglandin biosynthesis 10,23. This pain picture is widely used for the assessment of peripheral analgesic activity due to its sensitivity and solution to the compounds at a pane of glass which is not rough-and-ready in other methods. The local peritoneal receptor could be the cause of abdominal writhings 24. inconvenience sensation in acetic acid induced writhing paradigm is elicited by producing localized inflammatory response due to red of free arachidonic acid from wind phospholipids via cyclo-oxygenase (COX), and producing prostaglandin specifically PGE2 and PGF2, the level of lipoxygenase products may also increases in peritoneal fluids 10,23. These prostaglandin and lipoxygenase products cause inflammation and pain by increase capillary permeability. The substance inhibiting the writhings will have analgesic e ffect quite by ban of prostaglandin synthesis, a peripheral mechanism of pain inhibition 23.Thermal nociception models such as hot plat and the tail assiduousness tests were used to try central analgesic activity.The management of pain and inflammation related problems is a real challenge that people face daily. Although several drugs are available for these conditions, medicinal plants are believed to be an important denotation of new chemical substances with potential therapeutic effects (Gupta et al., 2006). formol testThe formalin test was carried out as described by Santos and Calixto, (1997). Groups of mice (n=5) were treat with HAAE (150 and 200 mg/kg), HAME (150 and 200 mg/kg), Aspirin (100 mg/kg), morphine (10 mg/kg) and distilled water. Formalin (1% v/v) was injected into the sub-plantar realm of the right hind glove of the animals, one hour post treatment. The duration of paw licking was metrical for 0-5 proceedings (neurogenic phase) and 15-30 minutes (inflamm atory phase) after formalin administration.ResultThe formalin test exhibited the characteristic biphasic response. Phase 1 response which was recorded from the judgment of conviction of formalin injection and 5 minutes post-injection was not affected by some(prenominal) extract at either dose level. Morphine however, demoed significant (pminutes post formalin injection. The extracts of HAAE (150 mg/kg and 200 mg/kg) and HAME (150 mg/kg and 200 mg/kg) as well as acetylsalicylic acid and morphine showed significant (pAcetic acid induces pain by the electrical outlet of endogenous mediators of pain such as prostaglandin through the activity of cyclooxygenase (COX) (Satyanarayana et al., 2004 Ballou et al., 2000). Therefore this model of pain should be tick by peripheral analgesics through the inhibition of COX activity. Our results therefore show that the higher doses of HAAE and HAME have peripheral analgesic properties similar to aspirin by inhibition of the tumble of endoge nous pain mediatorsThe formalin test is said to be a model of pain which closely resembles clinical pain compared to the other nociceptive models (Tjolsen and Hole, 1997). This test has two distinct phases the first phase (neurogenic pain) due to deal chemical stimulation of nociceptors, results from the stimulation of myelinated and unmyelinated nociceptive sensory nerve fibers, mainly C fibers, which can be suppressed by opioid analgesic drugs like morphine (Sayyah et al., 2004). The second or late phase seems to be an inflammatory response which elicits inflammatory pain and can be inhibited by anti-inflammatory drugs (Young at al., 2005). The second phase is caused by the release of inflammatory mediators such as prostaglandins and histamine in the peripheral tissues, as well as functional changes in the neurons, of the spinal cord which may allay transmission in the spinal cord (Franca et al., 2001 Garcia et al., 2004) like dead reckoning induced granuloma tissue formation FPEO, BPEO and diclofenac sodium were orally administered for 16 full-strength days in Groups III-VII. On eighth day, the animals (Groups II-VII) were mildly anaesthetised with ether, cardinal sterile cotton plant plant fiber wool dead reckonings (50 mg) were subcutaneously implanted in the dorsal region of the rats and two at the axilla and two at the groin regions. On 16th day, all the rats were killed using anaesthetic ether and the cotton pellets were dissected out without poignant the surrounding granuloma tissues (Winter and Porter 1957). Chronic inflamed tissues (from axilla and groin regions) were excised and stored in 0.9% saline at -20_C for biochemical summary. The moist pellets were weighed and then dried at 60_C for 48 h and then again reweighed. The percentage decrement in cotton pellets weight of the test samples was observed and compared with that of respective cotton pellet and diclofenac sodium treated groups. This provides a measure to assess the anti -inflammatory effect of the test samples.Experimental designSeven groups were employed in the present anti-inflammatory study. Each group consists of vi rats and experimental protocol include 16 days study. Each group of animals was employed with sterile cotton pellets (50 mg each) implantation in the dorsal region of rats at eighth day. Group I (vehicle control group) 1% of carboxy methyl cellulose (1 mL, p.o.) was administered to the rats for 16 consecutive days. Group II (negative control group) four sterile cotton pellets, 50 mg each were implanted in the dorsal region of rats at eighth day. Group III (positive control group) reference standard drug and diclofenac sodium (12.5 mg/kg, p.o.) were administered to the rats for 16 consecutive days. Groups IV-VII (test groups) rats were pretreated with free and bound phenoplast compounds of E. officinalis (20 and 40 mg/kg, p.o.) for 16 consecutive days.ResultEffect of E. officinalis on granulomatous tissue formation Table 1 shows the effect of FPEO and BPEO on granulomatous tissue changes due to cotton pellet induced inveterate inflammation. Changes in the cotton pellets weight (wet weight- wry weight) of the test samples were compared with the cotton pellet and diclofenac sodium (12.5 mg/kg) treated groups. Pretreatment (i.e. on days 1-8) of diclofenac and the phenolic fractions of E. officinalis did not show any behavioral changes. Both the fractions have shown lessening in granulomatous tissue mass as compared to cotton pellet treated group. However, whole high doses (40 mg/kg) of each fraction have shownsignificant (p.05) reduction which was comparable to that of diclofenac sodium pretreated group.The hot plate method is very effective for evaluating drugs possessing analgesic property, which act centrally (Vale et al., 1999 Haque et al., 2001 Silva et al., 2003 Al-Naggar et al., 2003). Prolongation of reaction time in hot plate test inferred possible central analgesic effects of the oil. The oil increase d the reaction time significantly at the dose levels used compared to control group. Acetic Acid-induced writhing has been used to evaluate drugs possessing peripheral analgesic effects (Koster et al., 1959 Viana et al., 2000).Acetic acid has been reported to cause hyperalgesia by liberating endogenous substances such as prostaglandins, leukotrieines, 5-HT, histamine, kinins, H+ and K+, etcetera which have been implicated in the mediation of pain perception (Forth et al., 1986 Rang et al., 1999).Yin et al (2003) reported that many studies have shown that the earlier phase (1st phase) of formalininduced pain reflects the top effect of formalin on nociceptors whereas the late phase (2nd phase) reflects inflammatory pain, which has been coupled to prostaglandin synthesis (Hong and Abbot, 1995 Yin, et al., 2003). Opioid analgesics have been reported to possess antinociceptive effects in both phases having more effect at the 2nd phase (Le Bars et al., 2001). Non-steroidal anti-inflamm atory drugs (NSAIDS) such as indomethacin is said to be effective only in the 1st phase especially if the formalin is injected at high concentration (Yashpal andCoderre, 1998). In this study, the oil dose-dependently inhibited nociception induced in the Formalin Test significantly compared to control group in the 1st phase (neurogenic) and 2nd phase (inflammatory). These results therefore further suggest that the oil contain constituents that exhibit anti-inflammatory propertiesCommonly used Non-Steroidal anti-inflammatory Drugs (NSAID) such as aspirin and indomethacin are widely used to reduce clump associated with pain and inflammation through inhibition of prostaglandin synthesis by direct effect on cyclo-oxygenase (COX) in the arachidonic acid (AA) metabolism (Amos et al., 2001 Nwafor and Okwuasaba, 2003)Inflammation is a disorder involving localized increases in the number of leukocytes and a variety of analyzable mediator molecules 4. Prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation. Their biosynthesis has also been implicated in the pathophysiology of cardiovascular diseases, cancer, colonic adenomas and Alzheimers disease 5,6.Medicinal plants are believed to be an important citation of new chemical substances with potential therapeutic effects 7,8. The research into plants with maintain folkloric use as pain relievers, antiinflammatory agents, should therefore be viewed as a fruitful and logical research strategy in the search for new analgesic and anti-inflammatory drugs 9.Acute toxicity testThe animals were divided into six groups containing eight animals in each group. MEPA was suspended in normal saline and administered orally as a single dose to groups of mice at different concentrations (500, 750, 1000, 1250, 1500 and 2000 mgkg-1 b.w). These animals were observed for a 72 h period. The number of deaths was expressed as a percentile and the LD50 was obdurate by probit a tes t using the death percentage versus the log dose 12.ResultAcute toxicity testIn the acute toxicity assay no deaths were observed during the 72 h period at the doses tested. At these doses, the animals showed no uninventive symptoms associated with toxicity, such as convulsion, ataxy, diarrhoea or increased diuresis. The median lethal dose (LD50) was determined to be higher than highest dose tested i.e., 2.0 gkg-1 b.w. cotton pellet-induced granulomaThe cotton pellets-induced granuloma in rats was studied according to the method DArcy et al., 1960 16. The animals were divided into four groups of six animals in each group. The rats were anaesthetized and sterile cotton pellets weighing 10 1 mg were implanted subcutaneously into both sides of the groin region of each rat. Group I served as control and received the vehicle (0.9% NaCl, 5 mlkg-1 b.w. The extract MEPA at the concentration of 250 and 500 mgkg-1 b.w was administered orally to groups II and III animals for seven consecutiv e days from the day of cotton pellet implantation. Group IV animals received indomethacin at a dose of 10 mgkg-1 b.w for the same period. On 8th day the animals were anaesthetized and the pellets together with the granuloma tissues were guardedly removed and made free from extraneous tissues. The wet pellets were weighed and then dried in an oven at 60C for 24 h to invariable weight, after that the dried pellets were weighed again. Increment in the dry weight of the pellets was interpreted as a measure of granuloma formation The antiproliferative effect of MEPA was compared withcontrol.Statistical analysisThe values were expressed as mean S.E.M. The statistical significance was determined by using the student t-test 17. Values of P ResultCotton pellets-induced granulomaThe effects of MEPA and indomethacin on the proliferative phase of inflammation are shown in table 1. A significant reduction in the weight of cotton pellets was observed with MEPA (250 and 500 mgkg-1 b.w) compared to the vehicle treated rats. However the degree of reduction was less than the effect caused by indomethacin.The cotton pellet method is widely used to evaluate the transudative and proliferative components of the chronic inflammation. The wet weight of the cotton pellets correlates with the transuda the dry weight of the pellets correlates with the amount of the granulomatous tissue 20,21. Administration of MEPA (250 and 500 mgkg-1 b.w) and indomethacin (10 mgkg-1 b.w) appear to be effective in inhibiting the wet weight of cotton pellet. On the other hand, the MEPA effect on dry weight of the cotton pellet was almost near to that of indomethacin. These data support the hypothesis of the greater effect of the MEPA on the inflammation in rats. This effect may be due to the cellular migration to injured sites and accumulation of collagen an mucopolysaccharides.