Bioguided fractionation of hypoglycaemic component in methanol extract of Vernonia amygdalina: an in vivo study

Abstract Nine components (C1–C9) were isolated from chloroform fraction of fractionated methanol extracts of Vernonia amygdalina leaves (FMEVA) by column chromatography. All the components C1 to C9 were purified and screened for hypoglycaemic activities in type-2 diabetic rats. The most potent hypoglycaemic component was elucidated on the basis of extensive spectroscopic (1D-, 2D-NMR, GC-MS, FTIR) data analysis. The Component C5 was found to be the most potent hypoglycaemic in reducing blood glucose by 12.55 ± 3.55% at 4 h post-oral administration, when compared to the positive (18.07 ± 1.20%) and negative (−1.99 ± 0.43%) controls. The spectroscopic data analysis reveals that the isolated compound has a structure consistent with 11β,13-dihydrovernolide. The isolated compound is part of the hypoglycaemic components present in V. amygdalina leaves that is responsible for the anti-diabetic activities. Further research is needed in the development of this compound or its derivatives for pharmaceutical use. Graphical Abstract


Introduction
Type-2 diabetes is the specific and common form of diabetes mellitus that is of major dilemma to everyone in the world today, particularly in the way it deteriorates the quality of human life. It accounts for about 90% of all reported cases of diabetes and affects everyone irrespective of age (Okoduwa, Umar, Ibrahim, Bello, Habila 2015; World Health Organisation (WHO) 2017), gender and/or socio-economic status (Okoduwa, Umar, Ibrahim, Bello, Ndidi 2015). Despite the current development of therapeutic agents, there is no effective treatment without side effects (Gheibi et al. 2017). Quite a lot of drugs (such as sulfonylurea, biguanide, pioglitazone and glucosidase inhibitors) currently exist for the treatment of diabetes mellitus. But, the use of these drugs are restricted by their pharmacokinetics properties, secondary failure rates and an accompanying side effects, such as lactic acidosis, diarrhoea and liver problem (Ezuruike and Prieto 2014;Tariq et al. 2016). This has necessitated the scientific search for a new class of compounds of relatively less toxic natural resources to overcome the diabetic problem.
Vernonia amygdalina Delile (VA) is commonly known as a bitter leaf in English language due to its bitter taste. African familiar names for VA include 'chusar-doki' (Hausa), 'onugbu' (Igbo), 'etidot' (Efik, Ijaw and Ibibio), 'ewuro' (Yoruba), 'oriwo' (Edo) and 'ndoleh' (Cameroon) (Toyang and Verpoorte 2013). V. amygdalina belong to the family Asteraceae (Compositae) and genus Vernonia. Traditional healers use VA leaf extract to treat diabetes mellitus (Ezuruike and Prieto 2014;Asase and Yohonu 2016) and as anti-malarial, anti-helminth, digestive tonic, appetizer and for treatment of wounds (Ijeh and Ejike 2011;Okpe et al. 2016). Several works have been done in the past on VA to verify its folkloric uses (Okolie et al. 2008;Toyang and Verpoorte 2013). Earlier study by the authors on the fractionated extracts of the plant on a unique rat model of T2D (fortified diet-fed streptozotocin-treated (FDF-STZ) rat model of T2D) have established some significant anti-diabetic properties (Okoduwa et al. 2017). Furthermore, the toxicity of the plant extract has been studied and reported in previous research by the authors (Okoduwa et al. 2017). Therefore, the present work is an effort geared towards the bioassay-guided chromatographic analysis of the most active fraction from methanol extracts of VA with a view to isolating and identifying the most effective constituents responsible for the hypoglycaemic properties observed in the leaves of the plant.

Experimental
This is provided in the supplementary material section.

Results and discussion
The Supplementary material Table S1 shows the change (%) in fasting blood glucose after oral administration of the purified components from VACF. It was observed that the purified components C5 was most active in reducing the blood glucose of diabetic rats by 12.55% when compared to the untreated diabetic (DC) and metformin-treated diabetic group (PC) with À1.99% and 18.07% respectively. The decrease (%) in fasting blood glucose after oral administration of the purified component C5 at three different doses (5, 10 and 20 mg/kg b.w.) respectively are presented in the Supplementary material Table S2.
The result of the spectroscopic analysis of the most active component of C5 was confirmed to be 11b,13-dihydrovernolide (see Supplementary materials Figures S1-S7  and Tables S3-S4). The compound (11b,13-dihydrovernolide) was shown in this investigation to have hypoglycaemic effect (Table S1). The compound has been isolated previously from related specie called V. colorata, where it was reported to exhibit little antibacterial action in respect to vernolide and vernodalin (Rabe et al. 2002). The FTIR spectra broad band at 3324 cm À1 was conspicuous for the hydroxyl group and 2832.8 cm À1 for ¼ C-H group. The saturation of the double bond at 11-13 positions may have reduced the activity as an antibacterial observed by Rabe et al. (2002). Although, the activity of a molecule may be related to its 3D-conformation, in the structure of 11b,13-dihydrovernolide there are one oxygen bridge between C-14 and C-15 which confer rigidity to the whole structure. Moreover, there is present also an epoxide (oxirane) function which is an extremely reactive group. This functionality may be responsible for some of its activity as observed in this study. Furthermore, a biologically active compound may have more than one specific activity. Only a few compounds show specific activity and this statement is valid also for drugs currently used in therapeutic purposes. For instance, the side effect of a drug is due to the drug itself which is administered to cure or treat a specific disease. The same is applicable for natural products which may be active towards several biological targets. A typical example is the case of daphnetin, a simple coumarin contained in Daphne spp. (Venditti et al. 2017). Dapnetin resulted to be an active anti-inflammatory (Li et al. 2017), anti-cancer (Kumar et al. 2016) and anti-diabetic and modulator of apoptosis pathways (Vinayagam and Xu 2017). Also, the polyphenols offer an example of the multi-target action of natural compounds. In fact, besides the wellknown antioxidant activity, they may exert in in vivo systems very different bioactivity, such as the antiobesity effect showed by the polyphenol-rich extract from Citrus sinensis (L.) Osbeck var. moro in a clinical study (Cardile et al. 2015). Another example is that of syringin which showed a dose-dependent effect on sleep induction in mice (Cui et al. 2015) but exert also an antifeedant properties against stored products insect pests (Cis et al. 2006). The possibility that natural compounds could be active towards more than one cellular target does not have to be a surprise because the secondary metabolites have developed and selected in a sort of evolutionary process together with the plant species that biosynthesize and use them to protect itself from predators.

Conclusions
The most potent bioactive compound responsible for the hypoglycaemic activities of V. amygdalina was identified. This is the very first of its kind in available published literatures. The compound 11b,13-dihydrovernolide isolated from V. amygdalina leaves resulted to exert hypoglycaemic activity.