Anti-Nociceptive And Anti-Inflammatory Effectsof An Ethanolic Extract Oftheaerial Parts Of Hilleria Latifolia (Lam.) H. Walt. (Phytolaccaceae)

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Pain and inflammation remain a real and current problem in clinical medicine and require new agents with improved efficacy for more effective therapy. The aerial parts of the perennial herb Hilleria latifolia (Lam.) H. Walt. (Family: Phytolaccaceae) are used in Ghanaian traditional medicine for the treatment of various painful and inflammatory conditions. The present study examined the anti-nociceptive, anti-inflammatory, some neurobehavioural properties as well as toxicity of an ethanolic extract of the aerial parts of Hilleria latifolia in animal models. Preliminary phytochemical screening of the Hilleria latifolia extract (HLE) revealed the presence of saponins, tannins, glycosides, steroids, terpenoids as well as small amounts of flavonoids and alkaloids. HLE (10–300 mg kg-1, p.o.), together with morphine and diclofenac (positive controls), showed significant anti-nociceptive activity in chemical (acetic acid-induced abdominal writhing, glutamate, formalin and capsaicin tests) and thermal (tail immersion test) behavioural pain models in rodents. The anti-nociceptive effect exhibited by HLE in the formalin test was partly or wholly reversed by the systemic administration of naloxone (a non-selective opioid receptor antagonist), theophylline (a non-selective adenosine A1/A2 receptor antagonist) and atropine (a non-selective muscarinic receptor antagonist). Cyproheptadine (a 5-HT2A receptor antagonist), ondansetron (a 5-HT3 receptor antagonist), yohimbine (a selective α2-adrenoceptor antagonist), nifedipine (L-type Voltage-gated calcium channel blocker), glibenclamide (an ATP-sensitive K+ channel inhibitor) and NG-L-nitro-arginine methyl ester /L-NAME (a NO synthase inhibitor), however, did not significantly block the anti-nociceptive effect of the extract. HLE, unlike morphine, did not induce tolerance to its anti-nociceptive effect in the formalin test after chronic administration; morphine tolerance did not also cross-generalize to the anti-nociceptive effects of HLE. Interestingly also, chronic concomitant administration of HLE and morphine significantly suppressed the development of morphine tolerance. Hexamethonium antagonised the neuronal nicotinic effects of HLE on isolated guinea pig ileum preparation. Together, these results indicate that HLE produces dose-related anti-nociception in several models of chemical and thermal pain—without tolerance induction—through mechanisms that may involve an interaction with adenosinergic, nicotinic cholinergic, muscarinic cholinergic and opioidergic pathways. iii Oral administration of HLE (10-300 mg kg-1, p.o.), either pre-emptively or curatively, significantly inhibited carrageenan-induced foot oedema in 7-day old chicks with maximal inhibitions of 38.11±5.55 % (pre-emptive) and 30.91±4.66 % (curative). Similarly, the NSAID diclofenac (10-100 mg kg-1, i.p.) dose-dependently reduced the oedema by 59.33±10.82 % and 42.87±7.46 % respectively for pre-emptive and curative treatments. Dexamethasone (0.3-3 mg kg-1, i.p.), a steroidal anti-inflammatory agent inhibited the oedema with maximal effect of 42.77±7.64 % (pre-emptive) and 36.60±6.76 % (curative). In the Freund‘s adjuvant induced-arthritis model in rats, HLE as well as the positive controls, dexamethasone and methotrexate, showed significant anti-arthritic properties when applied to established adjuvant arthritis. Oral administration of HLE (10-300 mg kg-1 p.o.) significantly reduced oedema in the ipsilateral paw of rats with a maximal inhibition of 32.64± 2.74 %. HLE (10-300 mg kg-1 p.o.), however, did not significantly affect joint oedema or systemic arthritic spread which is usually indicated by the inhibition of the spread of the oedema from the ipsilateral to the contralateral paw. The DMARD methotrexate (0.1-1 mg kg-1, i.p.) and the steroidal anti-inflammatory agent dexamethasone (0.3-3 mg kg-1, i.p ) reduced very significantly the total polyarthritic oedema as well as the spread of the arthritis from the ipsilateral to the contralateral paws of the treated animals. In all the in vitro antioxidant tests performed, with the exception of the total phenol assay and total antioxidant capacity, n-propyl gallate was used as the reference antioxidant. The extract (0.03-1 mg ml-1) exhibited Fe3+ reducing activity (EC50=2.071±0.782 mg ml-1), scavenged DPPH (EC50 =0.2269±0.037 mg ml-1) and prevented lipid peroxidation (IC50 =0.1122±0.010 mg ml-1). N-propyl gallate showed similar effects like the extract but was more potent. The total phenol content of HLE was estimated to be 29.40±1.09 mg tannic acid equivalent/g of HLE while the total antioxidant capacity was 55.16±13.60 mg ascorbic acid equivalent/g of HLE. These findings reveal that the extract has antioxidant properties which may partly account for its anti-inflammatory activity. Neurobehavioural properties of HLE were evaluated in various behavioural paradigms—elevated plus maze (EPM), the light/dark box, forced swimming test (FST), tail suspension test (TST) and pentobarbitone sleeping time test. HLE (10-300 mg kg-1, p.o.) displayed anxiolytic activity similar to diazepam in all the anxiety models used by significantly increasing the number of inter-compartment transitions and time spent in the lit area of the light/dark box as well iv as significantly increasing open arm entries, percentage open arm entries and percentage open arm time in the EPM. The extract (10-300 mg kg-1, p.o.) also exhibited antidepressant effects by reducing the duration of immobility in both the FST and TST. The extract (10-300 mg kg-1, p.o.) neither modified motor performance in the beam walk test nor caused CNS depression in the pentobarbitone sleeping time test. These results suggest that the extract has anxiolytic and antidepressant effects. Acute and sub-acute toxicity tests were conducted by the oral route in rats. During the experiment, no deaths were observed in any groups and there were no remarkable changes in general appearance, as well as in food and water consumption. The LD50 of HLE was estimated to be above 3000 mg kg-1. The no-observed-adverse-effect level (NOAEL) of H. latifolia was 300 mg kg-1. No significant changes were observed in haematological parameters, body weights and organ/body weight ratios. There were, however, significant changes in some serum biochemical parameters (plasma proteins and serum bilirubin) of extract-treated groups compared to control. No significant histopathological changes were noted in the kidneys, stomach, liver and spleen of rats at extract doses up to 1200 mg kg-1. Based on these findings, it can be inferred that HLE is relatively non-toxic in rats but has the potential to cause toxicity at high dose levels—demanding that caution be taken when using H. latifolia for medicinal purposes. Putting all together, this study has shown that the ethanolic extract of Hilleria latifolia aerial parts has anti-nociceptive, anti-inflammatory, antioxidant, anxiolytic and antidepressant activities. The extract also has low oral toxicity but should be used with caution.
Thesis Submitted In Fulfillment of The Requirement of the Degree of Doctor of Philosophy in the Department of Pharmacology.