Pharmacological Assessment Of Lupinus Arboreus Sims (Fabaceae) Methanol Extract And Three Active Constituents For Antinociceptive And Anti-Inflammatory Effects

ABSTRACT

The methanol extract and chemical constituents of Lupinus arboreus leaf were investigated

for antinociceptive and anti-inflammatory activities. The study was by experimental design.

the extract was partitioned to yield hexane, ethylacetate, and methanol fractions.

Phytochemical tests were done on the extract and fractions. Acute toxicity test (LD50) was

carried out on crude methanol leaf extract (CME). Extract hexane fraction (HEF),

ethylacetate fraction (EAF) and methanol fraction (MEF) were subjected to bioactivity

guided fractionation using mice tail immersion, hot plate, acetic acid- induced tests and

formaldehyde- and, egg albumin-induced rat paw oedema, as activity guide for

antinociceptive and anti-inflammatory studies respectively. The active constituents were

isolated by bioactivity-guided silica gel column chromatography eluted with gradient

mixtures. The isolated active compounds were characterized using a combination of

phytochemical analysis, m.p. determination, UV, IR, NMR and GC/MS spectral analyses.

The intraperitoneal (i.p) LD50 of the crude methanol extract was 84.85 mg/kg. Phytochemical

analysis of the methanol extract indicated the presence of steroids, flavonoids, glycosides,

terpenes and saponins. Tannin, resin, reducing sugar and protein were moderately present.

The hexane fraction contained steroids and terpenes while ethylacetate fraction contained

flavonoids and glycosides. Two active compounds AHF1 and AHF2 were obtained from the

hexane fraction while AEF1 was obtained from the ethyl acetate fraction. The AHF1

contained steroids. while AHF2 contained terpenes; AEF1 contained flavonoids. The crude

methanol extract (CME) (30 and 60 mg/kg,) i.p produced dose-related resistance against

thermal pain and significant (p< 0.01) inhibition of pain. On acetic-induced writhing test

CME exhibited a dose- related antinociceptive activity with 71.13 and 47.80 % at 60 and 30

mg/kg respectively. Fractions HEF, and EAF exhibited significant (p < 0.05) pain inhibition

of 73 and 64 % respectively while MEF produced 24 percent pain inhibition. AHF1 and

AHF2 fractionated from HEF significantly (p< 0.05) exhibited pain inhibition of 75 and 71 %

respectively at 30 mg/kg. AEF1 (30 mg/kg) also significantly (p< 0.05) inhibited pain reflex

by 71 %. In egg albumin-induced (acute) oedema in rats, CME (30 and 60 mg/kg) produced

a dose-related oedema inhibition of 81.10 and 91.50 % respectively at the 4th hour.

Similarly, the hexane fraction (HEF) and ethylacetate (EAF) at 60 mg/kg produced a

significant (p< 0.05) oedema inhibition of 79 and 40 % respectively at 4th hour. The effect of

methanol fraction (MEF) (60 mg/kg) was not significant (p> 0.05). The oedema inhibition

recorded by HEF and EAF were higher than the inhibition by aspirin (100 mg/kg). The CME

(30 and 60 mg/kg) significantly inhibited formaldehyde- induced arthritis, in a dose-related,

manner over a period of 4 hours (p< 0.05) (68 and 69 % inhibition respectively). Both HEF

and EAF at 60 mg/kg i.p, significantly (p< 0.05) inhibited the oedematous response to

formaldehyde-induced arthritis, causing 85.7 and 64.2 % inhibition respectively. The

inhibitory effects of the isolates AHF1, AHF2 and AEF1 on egg albumin-induced (acute)

oedema in rats (78; 72, and 66 % respectively) were significant and better than that of aspirin

(100 mg/kg) (46 %). The effect of AHF1, AHF2 and AEF1, (30 mg/kg i.p) on formaldehydeinduced

(chronic) oedema in rats were 79 %, 72 % and 65 % respectively. The isolated active

compounds were identified as stigmastene 3, 6-dione (AHF1), ursolic acid (AHF 2),

tetrahydroxyflavone-3a- rhamnoside (AEF1), and ellagic acid (AEF 2). In this study, the

extract and fractions of L. arboreus leaves exhibited antinociceptive effect in different

models of pain; and anti-inflammatory effects against both acute and chronic models of

inflammation. The isolated compounds AHF1, AHF2 and AEF 1 appear to be responsible for

the antinociceptive and anti-inflammatory effects. The compound AEF2 identified as ellagic

acid, known for its antimicrobial activity, was concomitantly isolated. These compounds

were isolated and characterized for the first time from L. arboreus.