Anti-Nociceptive Properties of the Ethanolic Extract Of Fruits of Xylopia Aethiopica (Dunal) A. Rich (Annonaceae) and its Major Constituent, Xylopic Acid

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The fruits of Xylopia aethiopica are traditionally used to treat malaria, fibroid (uterine), fungal infection, rheumatism, arthritis, amenorrhea, boil, haemorrhoids and flatulence whilst the crushed seeds are applied topically on the forehead in the treatment of headache and neuralgia. TLC and HPLC analyses of the extract revealed the presence of several compounds. The isolated xylopic acid produced a single spot in a number of solvent systems including petroleum ether: ethyl acetate (1:9) and hexane: ethyl acetate (1:9) and a single peak in HPLC analysis certifying the level of purity of the compound The extract and xylopic acid exhibited antinociception in all the pain models used. In the acetic acid-induced writhing test, the extract (30 – 300 mg kg-1) (F3,28=14.37, P<0.0001) and xylopic acid (10 – 100 mg kg-1) (F3,28=20.56, P<0.0001) significantly reduced abdominal writhes induced with acetic acid in mice with the highest dose of the extract inhibiting visceral nociception by 98.8 ± 0.8%. The highest dose of xylopic acid also inhibited visceral nociception by 93.8 ± 1.4%. Morphine (1 – 10 mg kg-1) (F3,28=9.77, P=0.00016) and diclofenac (1 - 10 mg kg-1) (F3,28=4.04, P=0.0165) used as controls in this model similarly exhibited significant antinociceptive activities in this test. The extract (30 – 300 mg kg-1) (F3,28=6.93, P=0.0012) significantly attenuated mechanical hyperalgesia in the Randall-Selitto test with maximum possible effect of 110 ± 16.17% at the highest dose used. Similarly xylopic acid (10 – 100 mg kg-1) significantly (F3,28=4.86, P=0.0076) attenuated mechanical hyperalgesia with a maximum possible effect of 94.58 ± 21.6% at the highest dose in the same test. The extract and xylopic acid were both relatively more effective in the Hargreaves thermal paw withdrawal test, (extract: F3,64= 8.10, P=0.0338, xylopic acid: F3,64= 7.11, P=0.03) compared to the tail flick test (extract: F3,64= 6.47, P=0.045, xylopic acid: F3,64= 19.5, P<0.0001). For the acute and chronic musculoskeletal pain tests morphine was most efficacious. The extract was also more efficacious than xylopic acid. Xylopic acid was however more potent in reducing both the chronic muscle and knee pain whereas the extract was more efficacious in the chronic skeletal pain model. The extract, xylopic acid and pregabalin ameliorated vincristine-induced neuropathic pain. Common symptoms experienced by patients with neuropathic pain such as mechanical hyperalgesia, tactile and cold allodynia were measured using Von Frey filaments and cold water. The extract (F3,28=5.12, P=0.006), xylopic acid (F3,28=3.72, P=0.0229) and pregabalin (F3,28=5.92, P=0.0029) produced tactile anti-allodynia. Similar effects were observed in the Von Frey intermediate and mechanical hyperalgesia as well as cold allodynia tests. In the formalin test the extract and xylopic acid inhibited both neurogenic and inflammatory phases of pain. The antinociception of the extract, xylopic acid and morphine involved the inhibition of opioid, NO-cGMP, 5-HT3, adenosine and muscarinic pathways Further determination of the mechanism of antinociception of xylopic acid carried using binding assay revealed the binding of xylopic acid to μ-opioid receptors with an enhancement of endogenous opioid binding. Capsaicin-sensitive C-fibres-glutamatergic-nociceptive pathway was found to participate in the antinociception of extract and xylopic acid. Tolerance to morphine antinociception on the opioid receptors developed after chronic treatment for eight days but failed to develop to the extract and xylopic acid. Also morphine tolerance did not cross-generalize to the extract and xylopic acid. In order to elucidate the drug—drug interaction between xylopic acid/morphine and xylopic acid/diclofenac in combination administrations, isobolographic analysis was performed. The experimental ED50’s (Zmix) of xylopic/morphine combination were smaller than their corresponding theoretical ED50’s (Zadd) in both phases of the formalin test indicating synergism. Isobolographic analysis of xylopic acid/diclofenac combination carried out also indicated potentiation of the combination as the experimental ED50 lay below the line of additivity. The degree of potentiation calculated as interaction index indicated that the combination synergized to produce antinociception. In summary these findings provide scientific data for the use of the fruit of X. aethiopica in the treatment of painful conditions and co- administration of xylopic acid/morphine and xylopic acid/diclofenac may be said to be beneficial as their various side effects may be reduced due to lower doses used with potentiation of their therapeutic effects.
A Thesis submitted to the School of Graduate Studies, Kwame Nkrumah University of Science and Technology, Kumasi in partial fulfillment of the requirements for the degree of Doctor of Philosophy