Iraqi J Pharm Sci, Vol.21(1) 2012 Anti-inflammatory effect of Ammi majus alcoholic extract 82 Dose Dependent Anti-inflammatory Effect of Ammi majus Alcoholic Extract in Rat: Chronic Study Shihab H. Mutlag *,1 * Department of Pharmacology and Toxicology,College of Pharmacy,University of Baghdad,Baghdad,Iraq. Abstract During treatment of inflammatory diseases, many conventional therapies (non-steroidal anti- inflammatory drugs) used to relief pain and inflammation. Chronic use of the intended drugs is frequently associated with serious side effect, which may lead to discontinuation of treatment . The efficacy and dose- response effect of ammi majus extraxt (2 , 4, 8 , 16, and 32 mg/rat) were assessed using formalin to induce paw edema in rats as a model of chronic inflammation respectively. In this study, 42 rats were used and allocated into 7 groups each containing 6 rats, representing control (Distilled water) , standard (piroxicam ) and test extract ( 2 , 4 , 8 , 16 and 32 mg/rat of Ammi majus alcoholic extract ). The test extract and control were given orally before induction of inflammation Paw edema was measured by using vernier caliper after 7 days for chronic inflammation. The result indicated that Ammi majus alcoholic extract significantly lower paw edema (p<0.05) compared to standard and control, while the dose 16mg/rat also lower the paw edema compared with other test groups but less compared with the dose 32mg/rat. In conclusion, Ammi majus alcoholic extract possess anti-inflammatory activity in animals model of chronic inflammation and the effect increased with increasing the dose. Key words: Ammi majus alcoholic extract, Chronic inflammation , Dose-Dependent . العالقة بيه الجرعة والتأثير للفعالية المضادة لاللتهاب للمستخلص الكحىلي للخلة الشيطاوية في الجرذان : دراسة مسمىة شهاب حطاب مطلك* ،1 ح تغذاد، تغذاد ، انعشاق.ظايع ،كهٛح انصٛذنح فشع األدٔٚح ٔانسًٕو ،* الخالصة انًسرًش ذمهم االنرٓاب نكٍ االسرعًال أٔ األنىانرٙ ذسكٍ انًسرعًهحُْانك انكصٛش يٍ االدّٔٚ االنرٓاتٛح األيشاضفٙ عالض ادٖ انٗ انثحس لطع انعالض انسثة انز٘ إنٗظاَثّٛ لذ ذؤد٘ تآشاسٚكٌٕ يصحٕتا انًضيُحاالدّٔٚ خصٕصا عُذ يشضٗ االنرٓاتاخ ِنٓز سخص شًُٓا . إنٗ إضافحانرٙ نٓا يفعٕل انعالظاخ انًصُعح ٔأشاس ظاَثّٛ الم انُثاذٛح انطثٛحعٍ عالظاخ تذٚهّ يرًصهّ تانًسرخهصاخ نك يعشفح انٓذف يٍ ْزِ انذساسح ْٕ ذمٛٛى فعانّٛ انخهّ انشٛطاَّٛ فٙ انًُارض انرعشٚثّٛ نالنرٓاتاخ انًضيُح فٙ انحٕٛاَاخ انًخرثشّٚ ٔكز ظشر يخرثش٘ ٔلسًد انٗ سثعح يعايٛع فٙ كم 24 انذساسحال.اسرعًم فٙ ْزِ أٔ انعشعحفًٛا ارا كاَد فعانٛح انعالض ذضداد تضٚادج يعًٕعح سد ظشراٌ نرمٛٛى ذؤشٛش انخهّ انشٛطاَّٛ فٙ كم ًَٕر ض يٍ انًُارض حٛس ذًد دساسح انرؤشٛش انًضاد نالنرٓاب نعشع يخرهفّ ٔكزنك ذى اسرخذاو يادج انثاٚشٔكسٛكاو ٔانًاء انًمطش عٍ طشٚك انفى نهعشر يهغى24يهغى 61ٔيهغى ،8يهغى ،2يهغى ،4نهًسرخهص % فٕسيانٍٛ ذحد ظهذ كم ظشر عهٗ 4. ذى اسرحذاز االنرٓاب فٙ ًَارض االنرٓاب انًضيُح تحمٍ كًُارض لٛاط ٔسٛطشِ اشُاء انرعشتّ لٛاط انسًك.فٙ ْزِ أداجاٚاو يٍ اسرحذاز االنرٓاب ٔذى لٛاط َسثح ذؤشٛش انعالض تطشٚمح 7يُح تعذ انرٕانٙ . ذى ذمٛٛى االنرٓاب انًض يهغى نهخهّ انشٛطاَّٛ فعانّٛ راخ فشق يعُٕ٘ فٙ ذمهٛم انٕريّ انُاذعّ يٍ حمٍ انفٕسيانٍٛ فٙ ًَٕرض 24انعشعّ أظٓشخ انذساسح يٕاص٘ نرؤشٛش االدٔاخ انمٛاسّٛ انًسرعًهح ٔانًرًصهّ تًضاداخ االنرٓاتاخ غٛش االنرٓاتاخ انًضيُّ عهٗ طٕل فرشج انرمٛٛى ٔترؤشٛش 24يهغى فٙ ذصثٛظ االنرٓاتاخ انًضيُّ ترؤشٛش الم يماسَح تانعشعح 61انسرشٔٚذّٚ )تاٚشٔكسٛكاو( ٔاضٓشخ انُرائط اٚضا ذؤشٛش نهعشعّ ائط انذساسّ تؤٌ ذؤشٛش انخهّ انشٛطاَّٛ اضٓش َرائط ظٛذِ فٙ ًَارض ًٔٚكٍ االسرُراض يٍ َر يهغى حٛس اصداد انرؤشٛش تضٚادج انعشعح. فعٕل انعالض ٚضداد تضٚادج انعشعح.ٔاٌ ييهغى 24االنرٓاب انًضيُّ ٔخصٕصا فٙ انعشعّ .الكلمات المفتاحية : المستخلص الكحىلي للخلة الشيطاوية ، االلتهاب المسمه ، تأثير زيادة الجرعة Introduction Inflammation is an important physiological reaction which occurs in response to a wide variety of injurious (bacterial infection or physical trauma) ultimately aiming to perform the dual function of limiting damage and promoting tissue repair (1) . It requires the participation of various cell types expressing and reacting to diverse mediator along a very precise sequence (2) . The inflammatory response is often initiated by the activation of resident macrophage through pattern-recognition receptors: this triggers the sequential release of pro-inflammatory mediators such as eicosanoids, cytokines, chemokines and protease which derive leukocyte recruitment and activation (3) . 1 Corresponding author E- mail : shihab_hattab@yahoo.com Received : 22/10/2011 Accepted : 1/4/2012 mailto:shihab_hattab@yahoo.com Iraqi J Pharm Sci, Vol.21(1) 2012 Anti-inflammatory effect of Ammi majus alcoholic extract 83 Chronic inflammation is a process of prolonged duration (weeks or months to years) in which active inflammation, tissue injury and healing proceed simultaneously (4) . It is characterized by a- infiltration with mononuclear cells including macrophage, lymphocytes and plasma cells b-tissue destruction, largely induced by the product of inflammatory cells c- repair, involving new vessel proliferation (angiogenesis) and fibrosis (5) .Resolution of inflammation (anti- inflammatory response) is an active process controlled by endogenous mediators that suppress pro-inflammatory gene expression and cell trafficking, induce inflammatory cell apoptosis and phagocytosis. An optional balance between pro- and anti-inflammatory response is required to prevent the highly detrimental effect of extensive, prolonged or unregulated inflammation (3) .Ammi majus is ancient herbal remedy used in the treatment of various therapeutic conditions , asthma and angina (6) , an infusion is used to calm the digestive system, while the decoction of the seed, taken after intercourse appears able to prevent implantation of fertilized ovum in the uterus (7) and the seed contain furancoumarins which stimulate pigment production in skin expose to bright light (6,7) .Ammi majus contain Linear furancoumarins (xanthotoxin , bergabten , imperatorin and isioimpiellin) which inhibt human liver CYP450 (8,9) simple coumarins induce number of enzymes like aldehyde reductase , glutathione S- transferase(GST), and NAD(P)H quinone oxidoredctase in the liver that are possible for detoxification of alfatoxin B1 (10) and also contain flavoniods (quercetin and keampferol) which has antioxidant and anti-tumor activity (11,12) The present study was designed to evaluate the efficacy and dose response effect of Ammi majus alcoholic extract in experimental animal model of chronic inflammation. Materials and Methods The present study was carried out on 42 rats of both sexes weighing 250, selected from the Animal House of the College of Pharmacy, University of Baghdad. The animals were maintained on normal temperature, humidity and light/dark cycle. They fed standard rat pellet diet and had free access to water until the night of the day of investigation.The animals were allocated into seven groups 6 animals each as follows . The control group was administered with 2ml/kg distilled water orally by gastric gavage tube , the standard group was treated with 5mg/kg piroxicam intraperitoneally while the test groups was treated with either 2, 4, 8, 16 or 32 mg/rat orally respectively. Chronic inflammation was induced by injection of 0.1 ml of 2% formalin into sub planter area of the right hind paw of the rat (13) . All treatment were administered 30 minutes prior to formalin injection and continued for seven consecutive days. The increase in paw thickness was measured by vernier caliper method (14) before and seven days after induction of inflammation.. Statistical Analysis All data were expressed as mean ±SEM. Comparisons between treated groups were performed by ANOVA and students t-test to evaluate the statistical difference. The P value < 0.05 was considered significant. Results The anti-inflammatory effect of Ammi majus alcoholic extract on chronic inflammatory model was illustrated in( table 1 )and (figure 1). Treatment with piroxicam significantly reduce formalin induce paw thickness (p<0.05) compared to control . Ammi majus alcoholic extract 16mg /rat and 32mg/rat showed significant reduction in paw thickness (p<0.05) compared to control, while Ammi majus alcoholic extract 2mg, 4mg and 8mg/rat showed non-significant reduction in paw thickness( p>0.05) compared to control .Both piroxicam and Ammi majus alcoholic extract 16mg/rat produced comparable effect on formalin-induced chronic inflammation while Ammi majus alcoholic extract 32mg/rat showed significant difference to that produced by control , standard and all other treated groups (2mg, 4mg, 8mg and 16mg/rat ) which produced the greatest effect with increase the dose of Ammi majus alcoholic extract . Figure 1: Mean increase in paw thickness during experimentally-induced chronic inflammation of the study groups. Iraqi J Pharm Sci, Vol.21(1) 2012 Anti-inflammatory effect of Ammi majus alcoholic extract 84 Table 1: Effect of different doses of Ammi majus alcoholic extract on formalin-induce chronic inflammation in rats. % of inhibition Mean increase in paw thickness(mm) after 7 days Treatment Groups - 2.96 ± 0.37 Control ( 2ml/kg D.W) n=6 16.11 1.63± 0.28 *a Piroxicam 5mg/kg n=6 0 2.86 ± 0.12 b Extract 2mg/rat n=6 0.23 2.9 ± 0.14 b Extract 4 mg/rat n=6 0.94 2.85 ± 0.19 b Extract 8 mg/rat n=6 12.79 2.06 ± 0.25 *c Extract 16 mg/rat n=6 24.88 1.2 ±0.24 *d Extract 32 mg/rat n=6 Data are presented as mean ± SEM; n=number of animals; *P<0.05 with respect baseline value ; values with non-identical superscript (a,b,c and d) among different groups are considered significantly different (P<0.05). Discussion The inflammatory process is invariably characterized by a production of prostaglandins, leukotrienes , histamine , bradykinin , platelet-activating factor (PAF) and by a release of chemicals from tissues and migrating inflammatory cells (15) . The initial phase of inflammation (edema,0-1 hour) which is not inhibited by NSAID like indomethacin or aspirin , has been attributed to the release of histamine , 5-hydroxytryptamine and bradykinin (16) , followed by a late phase (1-6 hours) mainly sustained by prostaglandin release and more recently has been attributed to the induction of inducible cyclooxygenase (COX-2) in the tissue (17) . Inflammatory events are initiated, enhanced, or coordinate by the action of various chemical mediators such as mast cells, platelets, and leukocytes are responsible for the release of inflammatory mediators and play an important role in the development of inflammation. These mediators include cytokines and chemokines , which promote inflammation and further function to amplify the response (18) , low molecular weight lipids derived from arachidonic acid (AA) , gases like nitric oxide (NO) and carbon monoxide , reactive oxygen species (ROS) and nucleotides (19) , small peptides such as kinins, complement and clotting system and finally amines such as histamine and 5-HT.Chronic inflammation begins 2-4 days after the onset of the acute response and can last for weeks to months or years due to the persistence of the initiating stimulus, interference of the normal healing process, repeated bouts of acute inflammation or low-grade smoldering due to continued production of immune response mediators (4) . The histological characteristics of chronic inflammation are the increase presence of macrophage and increased numbers of fibroblasts and other tissue matrix cells, such as osteoclasts and chondrocytes, via cytokine and growth factor-induced proliferation. Fibroblast is associated with secretion of both collagen and collagenase leading to fibrosis and reactive tissue remodeling. Phagocytic cell can also contribute directly to tissue injury through the release of proteolytic enzymes and free radicals (20) .The effect of the of Ammi majus alcoholic extract on formalin-induced paw edema, as chronic inflammatory models was assessed by vernier caliper method. Ammi majus alcoholic (32mg/rat) significantly reduced paw thickness (p<0.05) and the level of inhibition was found to be higher than standard drug utilized in the study as shown in (figure 1) and (table 1) while Ammi majus alcoholic extract (16mg/rat) show less effect in paw thickness compared with piroxicam. Ammi majus alcoholic extract (2mg , 4 mg and 8mg/rat) showed no effect on paw thickness compared to other test groups .The anti- inflammatory effect of Ammi majus alcoholic extract may be explained by the many active constituents of the extract : 1(quercetin), which is the most common flavoniod that scavenger both reactive oxygen species (ROS) and reactive nitrogen species (RNS). Consequently the flavonoid might be used to reduce both oxidative stress i.e an imbalance between the production of and protection against reactive species, and the inflammation. Moreover , querectin can also inhibit TF-kB activation, thereby directly reducing the cytokine production via this transcription factor (21) . Both these capacities of the intended flavonoid may contribute to the counteracting effect of quercetin on the lipopolysacchride(LPS) -induced tumor necrosis factor alpha(TNFα) . 2(kaempferol) suppressed nuclear factor –kappaB(NF- kappaB) activating and expression of its target genes cyclooxygenase-2 inducible nitric oxide synthase , monocyte chemoattractant protein-1 , and regulate upon activation , and normal T- cell expressed and secreted in aged rat kidney. Furthermore , kaempferol suppressed the increase of the pro-inflammatory NF-kappaB Iraqi J Pharm Sci, Vol.21(1) 2012 Anti-inflammatory effect of Ammi majus alcoholic extract 85 cascade through modulation of nuclear factor- inducing kinase (NK)/kappaB kinase(IKK) and mitogen-activated protein kinases (MAPKs) in aged rat kidney (22) . 3(coumarines) like bergabten showed significant anti- inflammatory and analgesic activity; however xanthotoxin only have anti-inflammatory activity and isoimperatorin only analgesic effect . The anti-inflammatory and analgesic constituents seem to be related to the peripheral inhibition of inflammatory substance and to their effect on the central nervous system (23) . Imperatorin and isoimperatorin showed dual inhibitory activity due to their significant effect on 5- lipoxygenase and showed comparable inhibition on cycloxygenase1( COX1) and cycloxygenase2( COX2), when compared to indomethacin and nimesulide . Only imperatorin caused a significant reduction of nitric oxide (NO)generation (24) . Also psoralen , xanthotoxin have shown COX-2/5-LO dual inhibitory activity (25) . oxypeucedanin , imperatorin and isoimpertorin these compound have been reported to exhibit pharmacological effect such as inhibition of lipopolysaccharide- induced prostaglandin E2 (26) , inhibition of IL- 1B-induced cyclooxygenase-2 (cox2) (27) and inhibitory effects on the GABA degradative enzyme , GABA transaminase (28) . 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