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

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..Thesis (PhD)--University of Pretoria, 2012.Paraclinical Sciencesunrestricte

The potential role of GLUT4 transporters and insulin receptors in the hypoglycaemic activity of Ficus lutea acetone leaf extract

BACKGROUND: Some Ficus species have been used in traditional African medicine in the treatment of diabetes. The antidiabetic potential of certain species has been confirmed in vivo but the mechanism of activity remains uncertain. The aim was to investigate the hypoglycaemic potential of ten Ficus species focussing on glucose uptake, insulin secretion and the possible mechanism of hypoglycaemic activity. METHODS: The dried and ground leaves of ten Ficus species were extracted with acetone. The dried acetone extract was reconstituted with DMSO to a concentration of 100 mg/ml which was then serially diluted and used to assay for glucose uptake in muscle, fat and liver cells, and insulin secretion in pancreatic cells. RESULTS: Only the F. lutea extract was able to modulate glucose metabolism. In comparison to insulin in the primary muscle cells, the glucose uptake ability of the extract was 33% as effective. In the hepatoma cell line, the extract was as effective as metformin in decreasing extracellular glucose concentration by approximately 20%. In the pancreatic insulin secretory assay, the extract was 4 times greater in its secretory activity than commercial glibenclamide. With F. lutea extract significantly increasing glucose uptake in the primary muscle cells, primary fat cells, C2C12 muscle and H-4-II-E liver cells, the extract may act by increasing the activity of cell surface glucose transporters. When the 3T3-L1 pre-adipocytes were compared to the primary muscle, primary fat and C2C12 cells, the differences in the former’s ability to transport glucose into the cell may be due to the absence of the GLUT4 transporter, which on activation via the insulin receptor decreases extracellular glucose concentrations. Because the pre-adipocytes failed to show any active increase in glucose uptake, the present effect has to be linked to the absence of the GLUT4 transporter. CONCLUSION: Only F. lutea possessed substantial in vitro activity related to glucose metabolism. Based on the effect produced in the various cell types, F. lutea also appears to be a partial agonist/antagonist of the insulin cell membrane receptor. While the clinical effectiveness of F. lutea is not known, this plant species does possess the ability to modify glucose metabolism

Evaluation of the inhibition of carbohydrate hydrolysing enzymes, antioxidant activity and polyphenolic content of extracts of ten African Ficus species (Moraceae) used traditionally to treat diabetes

BACKGROUND: Some Ficus species have been used in traditional African medicine in the treatment of diabetes. The antidiabetic potential of certain species has been confirmed in vivo but the mechanism of activity remains uncertain. The aim of this study was to determine the activity and to investigate the mechanism of antidiabetic activity of ten selected Ficus species through inhibition of α-amylase and α-glucosidase activity, and the possible relationship between these activities, the total polyphenolic content and the antioxidant activity. METHODS: Dried acetone leaf extracts were reconstituted with appropriate solvents and used to determine total polyphenolic content antioxidant activity, α-amylase and α-glucosidase inhibitory activity. RESULTS: The crude acetone extract of F. lutea had the highest polyphenolic content (56.85 ± 1.82 mg GAE/g of dry material) and the strongest antioxidant activity with a TEAC value of 4.80 ± 0.90. The antioxidant activity of the acetone extracts of the Ficus species may not be ascribed to total polyphenolic content alone. The crude extract at a concentration of 0.5 mg/ml of F. lutea (64.3 ± 3.6%) had the best α-glucosidase (sucrase) inhibitory activity. The EC50 of F. lutea (290 ± 111 μg/ml) was not significantly different from that of F. sycomorus (217 ± 69 μg/ml). The α-amylase inhibitory activity of F. lutea (95.4 ± 1.2%) at a concentration of 1 mg/ml was the highest among the Ficus species screened. The EC50 for F. lutea (9.42 ± 2.01 μ g/ml), though the highest, was not significantly different (p < 0.05) from that of F. craterostoma and F. natalensis. It was apparent that the crude acetone extract of F. lutea is a partially non-competitive inhibitor of α-amylase and α-glucosidase. Based on correlation coefficients polyphenolics may be responsible for α-glucosidase activity but probably not for α-amylase activity. CONCLUSION: Antidiabetic activity potential via inhibition of α-amylase and α-glucosidase was discovered in Ficus lutea which has not been previously reported. The acetone extract of the leaves was high in total polyphenolic content and antioxidant activity, and was a potent inhibitor of α-amylase activity. Research is underway to isolate the active compound(s) responsible for the antidiabetic activity and to confirm the in vitro antidiabetic activity and to investigate in vitro toxicity.The National Research Foundation (NRF) of South Africahttp://www.biomedcentral.com/1472-6882/13/94am2014mn201

Anti-inflammatory, anti-diabetic, anti-oxidant and cytotoxicity assays of South African herbal teas and bush tea blends

DATA AVAILABILITY STATEMENT : The data in this study are available in the article.South Africa is home to a variety of herbal teas, such as bush tea (Athrixia phylicoides DC.), honeybush tea (Cyclopia intermedia E. Mey and C. subternata Vogel), special tea (Monsonia burkeana Planch. ex Harv.), and rooibos tea (Aspalathus linearis (Burm.f.) R. Dahlgren) that are known to possess anti-oxidant, anti-inflammatory and anti-diabetic properties. The objective of this study was to determine the in vitro anti-oxidant activity of selected tea blends using 2,20-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays, as well as to assess their anti-inflammatory properties using the 15-lipoxygenase inhibitory assay. Furthermore, the study measured glucose utilisation in C2C12 myotubes. Lastly, 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to test the safety of the tea extracts on Vero cells (African green monkey kidney cell line). Special tea and its blend with bush tea exhibited potent anti-oxidant and anti-inflammatory activity. The blending of bush tea with special tea at different ratios resulted in increased anti-oxidant activity. Although special tea had a level of cell toxicity, its toxicity was lowered during blending. All of the tea samples showed anti-diabetic effects, although with less potency as compared to insulin. The current investigation supports the use of blended herbal teas, and the positive anti-inflammatory effect of special tea warrants further research.Research, Innovation and Commercialisation Department, University of South Africa.https://www.mdpi.com/journal/foodsam2023Paraclinical Science

Antidiabetic activity of the ethyl acetate fraction of Ficus lutea (Moraceae) leaf extract : comparison of an in vitro assay with an in vivo obese mouse model

BACKGROUND : Ficus lutea crude acetone leaf extracts were previously shown to stimulate glucose uptake and insulin secretion of established cells and, inhibit α-amylase and α-glucosidase activities. METHODS : For this study, F. lutea acetone extracts were subjected to solvent-solvent fractionation to yield fractions with differing polarities (hexane, chloroform, dichloromethane, ethyl acetate, n-butanol and water) in an attempt to obtain a more potent fraction with in vitro and probably in vivo activity. RESULTS : Among these fractions, the ethyl acetate fraction had the highest total polyphenol content (100.5 ± 1.6 mg GAE/g dried extract) and α-glucosidase inhibitory activity (126.8 ± 30.6 μg/ml). It also stimulated the highest glucose uptake of C2C12 muscle cells and decreased extracellular glucose concentration of H-4-II-E liver cells with low cytotoxic activity. The ethyl acetate fraction (10.88 ± 0.55 μg/L at 250 μg/ml) enhanced insulin secretion in RIN-m5F pancreatic β-cells to the same degree as the positive control glibenclamide (11.09 ± 0.07 μg/L at 1μM). While fractionation increased α-glucosidase inhibition and glucose uptake of cells, in the ethyl acetate fraction, the α-amylase inhibition and insulin secretion decreased. The weight reducing and glucose control potential of the ethyl acetate fraction in an obese mouse model, important factors in the amelioration of type II diabetes was determined. The extract had no statistical significant weight reducing activity. CONCLUSION : A major finding was the decrease in the area under the curve of the glucose concentration over time in animals that were treated with both a change in diet and with the plant extract. This is linked to increased glucose uptake within the cells, the most likely mechanism is either an increased insulin response or increased insulin secretion.The National Research Foundation (NRF) of South Africa and the University of Pretoria.http://www.biomedcentral.com/bmccom/plementalternmedam2016Paraclinical Science

Verification of the folkloric and anecdotal antidiabetic effects of Hypoxis hemerocallidea (Fisch., CA Mey. & Ave-Lall) and isolated, beta-sitosterol using early-stage type II spontaneous diabetic mutant BKS-Lepr(db) mice

BACKGROUND : Previous studies in our laboratory in ex vivo assays have demonstrated H. hemerocallidea extract as potential antidiabetic agent through increased insulin release from pancreatic beta cells. Thus, for this study the early stage type II spontaneous diabetic mutant mice model was used to evaluate and determine the degree of the antidiabetic efficacy of H. hemerocallidea. METHODS : Eight-weeks-old type II spontaneous pre-diabetic mutant BKS-Leprdb mice were fed with feed supplemented with either H. hemerocallidea extract, isolated compound (β-sitosterol) or chlorpropamide (positive control) for 4 weeks. The haematological parameters, clinical chemistry, glucose tolerance, feed intake, faecal output and body weights were measured. RESULTS : The blood glucose concentrations of all the animals treated with plant extract, β-sitosterol compound and non-treated pre-diabetic animals did not return to baseline levels. Only the β-sitosterol treatment and positive control groups resulted in a respective small decrease of 5.8 and 5.2% in the mouse weights over the study period, with no significant changes (p > 0.05) in food intake. However, there was a general trend for decrease in faecal output for all the groups. Albumin, triglycerides, and total cholesterol levels in β-sitosterol and chlorpropamide-treated animals were lower, relative to untreated-animals. Animals fed with plant extract showed large amounts of internal fat. There were no significant changes (p > 0.05) in total serum protein, globulin, alanine aminotransferase, alkaline phosphatase, urea nitrogen and creatinine attributed to administration of treatments. In all groups, some animals showed lesions associated with cardiac puncture. Few animals except animals treated with plant extract, showed presence of a leftventricular hypertrophic cardiomyopathy. The liver and kidneys for all groups appeared macroscopically normal and the thymuses were small (±2 mg). There were pathological signs in some of the animals particularly in myocardial fibres, renal tubular, glomerular, hepatocyte granularity and pancreas islets. However, there was no significance trend between the groups. CONCLUSION : Based on the results, none of the treatments could be considered highly effective for the management of type II pre-diabetes as sole therapeutic intervention.The Department of Higher Education and training (DHET) Research Development Grant (RDG) and Faculty of Veterinary Science University of Pretoria.https://bmccomplementmedtherapies.biomedcentral.comam2023Paraclinical Science

Phytochemical analysis and in-vitro anti-African swine fever virus activity of extracts and fractions of Ancistrocladus uncinatus, Hutch and Dalziel (Ancistrocladaceae)

BACKGROUND: African swine fever (ASF), a highly contagious fatal acute haemorrhagic viral disease of pigs currently has no treatment or vaccination protocol and it threatens the pig industry worldwide. Recent outbreaks were managed by farmers with ethnoveterinary preparations with various claims of effectiveness. RESULTS: We identified 35 compounds using GC-MS protocol and ASF virus (NIG 99) was significantly reduced by some extracts and fractions of the plant. However, the plant was poorly extracted by water and cytotoxicity was found to be a major problem with the use of the plant since its extracts also reduced the primary cells used in the assay. CONCLUSION: It is confirmed that the plant has antiviral potentials against ASF virus and farmers’ claims seem to have certain degree of veracity, but finding the best means of exploring the potential of the plant while reducing its cytotoxic effect in-vitro and in-vivo will be necessary.The Executive Secretary, Agricultural Research Council of Nigeria, Abuja for part funding of this project under the Project Code 025060410100000 (Development of rapid and effective Diagnostic and Control tools for African Swine Fever)http://www.biomedcentral.com/1746-6148/9/120ab201

The use of plants in the traditional management of diabetes in Nigeria: Pharmacological and toxicological considerations

Ethnopharmacological relevance: The prevalence of diabetes is on a steady increase worldwide and it is now identified as one of the main threats to human health in the 21st century. In Nigeria, the use of herbal medicine alone or alongside prescription drugs for its management is quite common. We hereby carry out a review of medicinal plants traditionally used for diabetes management in Nigeria. Based on the available evidence on the species׳ pharmacology and safety, we highlight ways in which their therapeutic potential can be properly harnessed for possible integration into the country׳s healthcare system. Materials and methods: Ethnobotanical information was obtained from a literature search of electronic databases such as Google Scholar, Pubmed and Scopus up to 2013 for publications on medicinal plants used in diabetes management, in which the place of use and/or sample collection was identified as Nigeria. ‘Diabetes’ and ‘Nigeria’ were used as keywords for the primary searches; and then ‘Plant name – accepted or synonyms’, ‘Constituents’, ‘Drug interaction’ and/or ‘Toxicity’ for the secondary searches. Results: The hypoglycemic effect of over a hundred out of the 115 plants reviewed in this paper is backed by preclinical experimental evidence, either in vivo or in vitro. One-third of the plants have been studied for their mechanism of action, while isolation of the bioactive constituent(s) has been accomplished for twenty three plants. Some plants showed specific organ toxicity, mostly nephrotoxic or hepatotoxic, with direct effects on the levels of some liver function enzymes. Twenty eight plants have been identified as in vitro modulators of P-glycoprotein and/or one or more of the cytochrome P450 enzymes, while eleven plants altered the levels of phase 2 metabolic enzymes, chiefly glutathione, with the potential to alter the pharmacokinetics of co-administered drugs. Conclusion: This review, therefore, provides a useful resource to enable a thorough assessment of the profile of plants used in diabetes management so as to ensure a more rational use. By anticipating potential toxicities or possible herb–drug interactions, significant risks which would otherwise represent a burden on the country׳s healthcare system can be avoided

Antidiabetic Activity, Molecular Docking, and ADMET Properties of Compounds Isolated from Bioactive Ethyl Acetate Fraction of <i>Ficus lutea</i> Leaf Extract

Diabetes contributes to the rising global death rate. Despite scientific advancements in understanding and managing diabetes, no single therapeutic agent has been identified to effectively treat and prevent its progression. Consequently, the exploration for new antidiabetic therapeutics continues. This study aimed to investigate the antidiabetic bioactive ethyl acetate fraction of F. lutea at the molecular level to understand the molecular interactions and ligand-protein binding. To do this, the fraction underwent column chromatography fractionation to yield five compounds: lupeol, stigmasterol, α-amyrin acetate, epicatechin, and epiafzelechin. These compounds were evaluated in vitro through α-glucosidase inhibition and glucose utilization assays in C2C12 muscle and H-4-11-E liver cells using standard methods. In silico analysis was conducted using molecular docking and ADMET studies. Epicatechin exhibited the most potent α-glucosidase inhibition (IC50 = 5.72 ± 2.7 µg/mL), while epiafzelechin stimulated superior glucose utilization in C2C12 muscle cells (33.35 ± 1.8%) and H-4-11-E liver cells (46.7 ± 1.2%) at a concentration of 250 µg/mL. The binding energies of the isolated compounds for glycogen phosphorylase (1NOI) and α-amylase (1OSE) were stronger (<−8.1) than those of the positive controls. Overall, all tested compounds exhibited characteristics indicative of their potential as antidiabetic agents; however, toxicity profiling predicted epiafzelechin and epicatechin as better alternatives. The ethyl acetate fraction and its compounds, particularly epiafzelechin, showed promise as antidiabetic agents. However, further comprehensive studies are necessary to validate these findings