SOURCE:

Faculty: Pharmaceutical Sciences
Department: Pharmacology And Toxicology

CONTRIBUTORS:

Ajaghaku, D. L.
Akah, P. E

ABSTRACT:

Millettia aboensis(Hook. F.) Baker is popular in ethno-medicine particularly for its acclaimed general healing properties.Validation of these claims and elucidation of possible mechanisms through which the active ingredients might be actinghave not been carried out. This study investigated the antioxidant and immune-enhancing potentials of the leaf of M. aboensis. Dried pulverized leaves of the plant were extracted with 70% ethanol by cold maceration for 72 h. Three solvent fractions of the extract were obtained through liquid-liquid fractionation.Phytochemical studies were carried out on the extract and fractions. In vitro antioxidant activities of the extract and fractions were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and hydrogen peroxide scavenging activity tests; while in vivo protection against oxidative damage was assessed using carbon tetrachloride (CCl4)-induced liver damage and streptozotocin (STZ) induced systemic oxidative stress models. In vivo immune enhancing properties were monitored using primary and secondary immune responses to tetanus toxoid. Active fractions (ethyl acetate and butanol) of the extract were subjected to vacuum liquid chromatography on silica gel, Sephadex LH-20 gel chromatography and semi-preparative reverse phase HPLC to isolate compounds 1 and 2 from ethyl acetate fraction and compound 3 from butanol fraction. The structures of these compounds were elucidated by a combination of 1D and 2D NMR and mass spectrometry. In vitro DPPH test and inhibition of liver microsome lipid peroxidation were used to evaluate the antioxidant activity of compounds 1 and 2 while stimulation of specific T-lymphocytes was used to evaluate immune enhancing activity of compound 3. Phytochemical studies showed that tannins and resins were abundant in the extract while phenolic compounds were concentrated in the ethyl acetate fraction (EAF). The extract exhibited both antioxidant and immune-enhancing properties. Antioxidant activity was more prominent in the EAF, while butanol fraction (BF) expressed more immune-enhancing activity. Against the DPPH radical, the extract gave EC50 value of 116.67 µg/mL while EAF and BF had EC50 of 35.33 and 79.17 µg/mL respectively. EAF had total phenolic content of 305.2 mgGAE/g compared to 26.1 and 247.6 mgGAE/g in n-hexane and butanol fractions respectively. EAF (200 mg/kg) produced significant (p<0.05) in vivo inhibition of lipid peroxidation (69.3%) similar to that of α-tocopherol (74.9%).Structural elucidation of the active compounds revealed compound 1 as epicathechin-(2β→O→7, 4β→8)-cathechin(Procyanidine A1), compound 2 as epicathechin-(2β→O→7, 4β→8)-epicathechin (Procyanidine A2) and compound 3 as isomeric mixtures of quercetin-3O-rutinoside and quercetin-3O-robinobioside. Compounds 1 and 2 demonstrated strong inhibition of liver microsome lipid peroxidation in vitro, with EC50 of 46 and 55 µg/mL respectively. Equal ratio combination of the two compounds produced synergistic inhibition of DPPH radical with EC50 of 7 µg/mL against 9 µg/mL produced by ascorbic acid. At 400 mg/kg, normalised mean secondary IgG1 and IgG2a antibodies response of the butanol fraction were 9.0 and 7.7 respectively compared to 13.2 and 16.5 produced by Noni capsule®. Findings from the nature of cytokine up-regulation by butanol fraction following secondary challenge with tetanus toxoid revealed that IL-12, IL-17A, IFN-γ and IL-4 were expressed by 48.14, 41.37, 38.22, and 31.03% respectively. Compound 3 isolated from butanol fraction exhibited in vitro up-regulation of specific CD4+ lymphocytes that were largely interferon gamma releasing. Up to 43.7% stimulatory effect of IFNγ was produced at 6.25 µg/mL compared to the baseline effect in DMSO control group. The extract, fractions and isolated compounds from M. aboensis expressed strong antioxidant and immune-enhancing properties, which may explain its ethnopharmacological use for general healing.