The value of extracts of Ficus lutea (Moraceae) in the management of Type II diabetes in a mouse obesity model

Abstract

Diabetes mellitus is a chronic disease characterised by prolonged hyperglycaemia,especially postprandial, in association with the consumption of diets that promote obesity. While different types of the disease have been identified, Type II diabetes also known as insulin dependent diabetes is most prevalent. Treatment for patients with this disease is usually a combination of exercise, low caloric diet and specific medical intervention through the use of allopathic medicines or surgery. While the number of treatment option is large, unfortunately, treatment is usually associated with complication such as drug adverse reactions and failure to halt disease progression. As a result new therapies are required. Herbal medicines such as those derived from the Ficus species, which have been used traditionally in the treatment of diabetes, may serve as new source of drug therapies. The aim of this study was to evaluate the effectiveness of selected South African Ficus species for their potential ability to manage Type II diabetes using in vitro and in vivo screening models. Dried and ground leaves of ten Ficus species were extracted separately with acetone, chloroform and hexane for determination of its phytochemical constituents. Since acetone extracted more variety of compounds, the extracts was used for determination of total polyphenol content, antioxidant activity, α-amylase and α-glucosidase inhibitory activity, cytotoxicity, glucose uptake in primary cell cultures and established cell lines, and insulin release in pancreatic cell lines. The most active extract (F. lutea) was subjected to solvent-solvent fractionation and the six fraction subsequently evaluated by the same assays. The most active fraction (ethyl acetate) was hereafter subjected to fractionation for the isolation of bioactive compound(s) or direct evaluation in a mouse obesity model. The acetone extract of F. lutea had the highest polyphenolic content (56.85 ± 1.82 mg GAE/g dry weight), the strongest antioxidant activity (4.80 ± 0.90 TEAC) and the highest α-amylase inhibitory activity with an EC50 value of 9.42 ± 2.01 μg/ml. Although the extract of F. lutea had the highest sucrase (64.31 ± 3.57%) inhibitory activity at concentration of 0.5 mg/ml, the ECβ of F. sycomorus (217 ± 69 μg/ml) was the best followed by F. lutea (289 ± 111μg/ml). Based on the correlation coefficient between polyphenol and alpha amylase inhibition (0.80) and alpha glucosidase (sucrase) inhibition (0.84), and the partial non-competitive manner by which the acetone extract of F. lutea inhibited the α-amylase and α-glucosidase enzymes, the polyphenols appear to be in part responsible for the evident activity. All ten Ficus species were less toxic than doxorubicin (positive control) but contained compounds that are generally relatively more toxic to the Vero kidney cells than to the C3A liver cells. The extract of F. craterostoma was the least toxic to the C3A and Vero cells, while the LCβ for the extract of F. lutea extract were relatively non-toxic to the Vero cells (214.8 ± 5.0 μg/ml) and more toxic (126.0 ± 6.8 μg/ml) to the C3A cell line. In the glucose uptake assays using primary rat abdominal muscle or epididymal fat cells, F. lutea acetone extracts (200 μg/ml) induced greater glucose uptake of 10.8 ± 1.8% for muscle and of 32.0 ± 8.4% for fat respectively, in comparison to the DMSO control wells.A similar response was seen with the established C2C12 muscle and H-4-II-E liver cell lines, where F. lutea in a dose related manner increased glucose uptake and at the highest concentration (500 μg/ml) increase glucose uptake by 14.9 ± 2.3% and 19.3 ± 0.6% respectively. In contrast no result was quantifiable in the established 3T3-L1 pre-adipocytes cell line, most likely due to a flaw in the methodology. The concurrent insulin addition, (1 and 10 μM) also potentiated the glucose utilisation in the F. lutea treated C2C12 and H-4-II-E cells. On addition of extracts to the RIN-m5F pancreatic β-cells, the extract of F. lutea stimulated a dose related increase in insulin release with insulin secretion of 120.8 ± 11.1% at the highest concentration (500 μg/ml) and concurrent dose related decrease in cell viability in comparison to the untreated control. As a result it would appear that F. lutea acetone extracts have a dual mechanism behind its ability to reduce glucose concentrations. The extract of Ficus lutea, was further subjected to solvent-solvent fractionation in hexane, chloroform, dichloromethane, ethyl acetate, n-butanol and water due to its superior response. The ethyl acetate fraction had the highest polyphenolic content (100.5 ± 1.6 mg GEA/g dried extract) and the highest sucrase inhibitory activity (126.8 ±30.6 μg/ml), while the n-butanol fraction had the highest α-amylase inhibitory activity (26.5 ± 1.3 μg/ml). Nonetheless the inhibition of the α-amylase enzyme activity by the various fractions was in all cases lower than that for the crude extract. In the cytotoxic assay using Vero monkey kidney and C3A liver cell line, the hexane fraction was the least toxic while the ethyl acetate fraction was relatively non-toxic, it had the lowest LDβ against the Vero cells (LDβ = 126.9 ± 1.5 μg/ml). In the glucose uptake assays, the ethyl acetate fraction stimulated the greatest glucose uptake into the C2C12 muscle and H-4-II-E liver cells in dose responsive manner, with no added benefits being achieved through the concurrent addition of insulin. The ethyl acetate fraction also enhanced insulin secretion in RIN-m5F pancreatic β-cells, albeit to a lower extent than the crude extract with dose related decrease in cell viability. With the ethyl acetate fraction being the most active fraction with moderately toxicity, further isolation was attempted. Five compounds were isolated, namely lupeol, stigmasterol, α-amyrin acetate, epicatechin and epiafzelechin, with all of the compounds except epiafzelechin previously known to possess antidiabetic activity. The ethyl-acetate fraction was also evaluated for its weight reducing potential in obese mouse model. Unfortunately no in vivo activity was discernible. In conclusion, this study is the first to report on the in vitro antidiabetic activity of the extract of F. lutea..