I= ANTI-INFLAMMATORY ACTIVITY OF AEGICERAS CORNICULATUM AND ITS MECHANISM OF ACTION USING IN VIVO AND IN VITRO SYSTEMS
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Title of Thesis
ANTI-INFLAMMATORY ACTIVITY OF AEGICERAS CORNICULATUM AND ITS MECHANISM OF ACTION USING IN VIVO AND IN VITRO SYSTEMS

Author(s)
TALAT ROOME
Institute/University/Department Details
H.E.J. Research Institute of Chemistry/ University of Karachi 
Session
2007
Subject
Chemistry
Number of Pages
355
Keywords (Extracted from title, table of contents and abstract of thesis)
aegiceras corniculatum, aegicerataceae, oedemogens, anti-inflammatory agents, inflammation, oedema, eedmogens, corniculatum extracts

Abstract
Aegiceras corniculatum L. Blanco (Aegicerataceae) from Indus Delta, Sindh, Pakistan, was evaluated for anti-inflammatory activity and its mechanism using various in vivo and in vitro model systems. Different oedemogens such as carrageenan, dextran, histamine, serotonin, bradykinin, prostaglandin E2, leukotriene B4, arachidonic acid, phospholipase A2 and hyaluronidase were injected into the hind paw of the rat. The hexane and ethyl acetate extracts from leaves and stem caused significant dose dependent inhibition (50-80%) of rat paw oedema in initial and late phases of inflammation (1-4 h). On the other hand, methanol was the only extract from leaves and stem that exhibited significant concentration dependent inhibitory response in the late phase of inflammation (3rd h) with an IC50 of 8.0 ± 0.35 mg/kg and 7.2 ± 0.48 mg/kg, respectively. This effect was significantly better than commonly prescribed nonsteroidal anti-inflammatory agent aspirin (IC50 = 48 ± 0.96 mg/kg) and was equipotent to naproxen sodium (IC50 = 8.7 ± 0.23 mg/kg). It appears that extracts from A. corniculatum particularly methanol extract possess appreciable anti-inflammatory property.

Dextran-induced paw oedema causes the release of histamine and serotonin that are responsible for the onset of initial phase of inflammation. Using this model, oedema was significantly reduced (~60%) by hexane and ethyl acetate extracts (leaves/stem). Whereas, histamineinduced paw oedema was suppressed in the presence of ethyl acetate extract of both leaves (62 ± 3%) and stem (49 ± 4%). Both hexane and ethyl acetate extracts from leaves and stem failed to suppress the serotonin, bradykinin and hyaluronidase induced paw oedema suggesting that these extracts neither antagonize the action of serotonin or bradykinin at their respective receptor sites nor interfere with the vascular permeability changes directly.

Arachidonic acid metabolites prostaglandin E2 (POE2) and leukotriene B4 (L TB4) are involved in late phase of inflammation. POE2-induced paw oedema was diminished significantly by methanol and ethyl acetate extracts (60-90%). This suppressive effect is mainly attributed to inhibition of cycIooxygenase enzyme and the production of POE2. However, hexane extracts were devoid of such effect. Noticeable reduction in L TB4-induced paw oedema was evident at 100 mg/kg of hexane (~ 60%) and ethyl acetate (~ 80%) extracts similar to selective 5-lipoxygenase inhibitors, caffeic acid (65%) and nordihydroguaiaretic acid (80%). Thus implying that these extracts interfere with the synthesis of 5-LOX metabolites (L TB4 and 5HETE). Methanol extract (stem) failed to produce any detectable effect against L TB4-induced paw oedema.

It is well established that arachidonic acid-induced paw oedema in rats is highly sensitive to dual inhibitors of cyclooxygenase and 5-lipoxygenase (phenidone), selective 5-LOX inhibitors (caffeic acid and nordihydroguaiaretic acid) and in current investigation hexane and ethyl acetate extracts (100 mg/kg) from leaves and stem inhibited AA-induced paw oedema by about 60% and 90%, respectively. This clearly demonstrates that these extracts acting as a selective 5-LOX inhibitor (hexane extract) and dual inhibitor of both the dioxygenases (ethyl acetate extract), which strengthens its previous results. Like aspirin and indomethacin, methanol extract failed to suppress arachidonic acid-induced paw oedema as this model is insensitive to COX inhibitors, thereby hinting that methanol extract acting as aspirin like drugs. Moreover, failure of extracts from A. corniclilatlim to suppress PLA2-induced paw oedema is an indicative that extracts do not interfere with the release of AA and supportive to that extracts inhibit arachidonic acid metabolism. It is well established that free radicals such as superoxide anions (O€™2), hydroxyl radical ('OH) and lipid peroxy radicals (LOO') are generated during inflammation and causes cellular and tissue damage leading to apoptosis. In the present study, inflammation was induced in mice using glucose oxidase and paw weight was noted. At 100 mg/kg, ethyl acetate and methanol extracts displayed 60%, whereas hexane extract produced only 30% reduction in paw oedema. From these results it could be inferred that the anti-inflammatory activity could probably be related to free radical ('OH) scavenging potential residing in this plant.

To explore the anti-inflammatory effect at cellular level rat peritoneal neutrophils were employed. These cells were stimulated with calcium ionophore A23187 and 5-LOX products leukotriene B4 (L TB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) were quantified using HPLC. The results highlighted that hexane and ethyl acetate extracts from leaves and stem and methanol extract from leaves significantly attenuated the production of both 5-LOX metabolites similar to nordihydroguaiaretic acid and phenidone. This phenomenon was potently suppressed by hexane (leaves) and ethyl acetate (stem) extracts with the IC50 of about 4.0 and 0.86 µg/ml, respectively and found to be equipotent to well known 5-LOX inhibitors quercetin (0.85 ± 0.18 µg/ml) and revealing that these extracts have potential to inhibit 5-LOX pathway. However, methanol extract (stem) was inactive in this model.

Human platelets are rich source of cyclooxygenase-I (COX-I) and 12-lipoxygenase (I2-LOX) and were used to examine the change in the production of their metabolites 12hydroxyheptadecatrienoic acid (12-HHT) and 12-hydroxyeicosatetraenoic acid (12-HETE),

respectively in the presence of plant extracts upon calcium ionophore A23187 stimulation. Ethyl acetate extract (leaves/stem) suppressed the production of both the metabolites with greater affinity for 12-HHT production (IC50 = 0.45 ± 0.01 μg/ml, leaves and 0.08 ± 0.002 μg/ml, stem). In case of 12-HETE the plant extract also reduced its production with 40x and 100x higher IC50 value. Interestingly, methanol extract of stem attenuated 12-HHT (IC50 = 41.1 ± 1.5 μg/ml) with a concomitant increase in 12-HETE production similar to selective cyclooxygenase inhibitors like indomethacin and aspirin thereby, reflecting that methanol extract of stem behaves like classical NSAIDs via selective inhibition of COX-l enzyme. Conversely, hexane extract (stem) reduced 12-HETE with a concomitant increase in 12-HHT. The IC50 for 12-HETE was calculated as 0.36 ± 0.12 μg/ml, which is about 18.8x more potent than baicalein, the only reported selective 12-LOX inhibitor.

The plant extracts was also analyzed in neutrophils infiltration model induced by carrageenan into the rat peritoneum, in which ethyl acetate extracts of leaves (IC50 = 38.6 ± 3.3 mg/kg) and stem (IC50 = 9.2 ± 0.44 mg/kg) markedly reduced the cell infiltration with about 2x and 8x lower IC50 values than that of phenidone, a dual acting anti-inflammatory agent. On the other hand, methanol extract (stem) failed to demonstrate appreciable effect likely to selective cyclooxygenase inhibitors (indomethacin and naproxen). These results provide supportive evidence that anti-inflammatory activity in ethyl acetate extract is due to dual inhibition of both COX and 5-LOX, whereas in methanol it is via selective inhibition of cyclooxygenase activity.

Free radical scavenging potential of plant extract was also analyzed in various biochemical reactions and cellular system. Both synthetic (DPPH) and biological (€˜OH, 0'2) radicals were scavenged by hexane, ethyl acetate and methanol extracts with varying degree of potency, along with Fe+3 ions chelating property which was found to be equivalent to classical antioxidants nordihydroguaiaretic acid, phenidone, quercetin and rutin. Anti-lipid peroxidative effect was observed in both Fe+3 / ascorbate dependent non-enzymatic lipid peroxidation and Fe+3 / ADP/ NADPH dependent enzymatic lipid peroxidation with an IC50 between 5.0-7.0 μg/ml, suggesting that extracts are acting as a free radical scavengers and chain breaking antioxidants.

Superoxide anion (O'2) generation from human neutrophils was also analyzed in the presesnce of plant extracts. Opsonized zymosan and phorbol-12-myristate-13-acetate (PMA) were used as stimulant to the cells that activate NADPH oxidase for O'2 generation. Hexane and ethyl acetate extracts (leaves/stem) suppressed the O'2 generation with an IC50 between 3.0-9.0 µg/ml. As already mentioned that extracts possess O'2 scavenging property but this free radical scavenging potency found to be significantly lower than that of O€™2 generation thereby, implying that extracts possess inhibitory effect on NADPH oxidase dependent O€™2 generation.

In conclusion, from the present study it could be interpreted that A. corniculatum extracts possess anti-inflammatory activity which . is governed by multiple mechanism such as inhibition of 1) histamine, 2) cyclooxygenase and 5-lipoxygenase products (PGE2 and L TB4) and 3) scavenging of various free radicals ('OH, O€™2) and their generation. Thus this plant has a promising potential to be included specifically in anti-inflammatory drug discovery program.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
229.14 KB
2 1 General Introduction 1
1209.98 KB
  1.1 Inflammation 1
  1.2 Inflammatory Mediators 3
  1.3 Anti-Inflammatory Agents 16
  1.4 Role Of Natural Products In Modem Medicine And Inflammation 28
  1.5 Role Of Reactive Oxygen Species In Inflammation 30
  1.6 Role Of Antioxidants In Inflammation 35
  1.7 Mangroves 37
  1.8 Collection Of Plant 52
  1.9 Extraction Procedure 52
  1.10 Objectives 53
3 2 Evaluation Of Anti-Inflammatory Activity Of A. Corniculatum Extracts In Rat Paw Oedema Model Using Various Eedmogens
837.7 KB
  2.1 Introduction 54
  2.2 Materials And Methods 57
  2.3 Results 61
  2.4 Discussion 104
4 3 Evaluation Of Anti-Inflammatory Activity Of A. Corniculatum Extracts At Cellular Level
1132.98 KB
  3.1 Introduction 111
  3.2 Materials And Methods 113
  3.3 Results 125
  3.4 Discussion 202
5 4 Evaluation Of Antioxidant And Free Radical Scavenging Potential Of A. Corniculatum Extracts Using In Vitro And In Vivo System
737.09 KB
  4.1 Introduction 213
  4.2 Materials And Methods 216
  4.3 Results 226
  4.4 Discussion 266
6 5 General Discussion 277
826.11 KB
  5.1 References 285