Oyinlola O. Olaokun

1. The value of extracts of Ficus lutea (Moraceae) in the management of Type II diabetes in a mouse obesity model Oyinlola O. Olaokun

2. Introduction • Metabolic disorder characterized by chronic hyperglycemia • Defect in insulin secretion, impaired insulin action (insulin resistance) or both • Fourth/fifth leading cause of death globally • Chronic hyperglycaemia enhances glucose toxicity via oxidative stress which is responsible for β-cell dysfunction and diabetic complications • Type II diabetes accounts for about 90% of cases globally • Genetic predisposition, excessive caloric intake and inactivity

3. Introduction Fig 1 Pancreatic β-cell dysfunction in Type II diabetes (Prentki and Nolan, 2006). • Treatment: healthy diet, weight loss through physical exercise and/or with weight loss therapeutics, and oral hypoglycaemic drug

4. Introduction • Limitation of drugs: adverse side effects & failure to halt disease progression • Need for new drugs: herbal/medicinal plants may be a way of overcoming side effects • Plant polyphenols have been reported to have hypoglycaemic activity and weight reducing property • Many Ficus species are rich sources of polyphenols • Many Ficus species have been demonstrated in vivo to have hypoglycaemic activity but mechanism is speculative.

5. Aim To evaluate the effectiveness of selected South African Ficus species in the management of Type II diabetes using in vitroand in vivo models of efficacy.

6. Objectives • To evaluate the antioxidant activity and relation to the inherent total polyphenolic concentration. • To evaluate the in vitro α- amylase and α- glucosidase inhibitory activity • To evaluate the stimulation of glucose uptake in established muscle (C2C12) and liver (H-4-II-E) cells. • To evaluate the in vitrocytotoxicity using Vero kidney and C3A liver cell lines. • To evaluate the stimulating insulin release in established insulin producing pancreatic cell line (RIN-m5F) • To evaluate the most active extract from the above mentioned assays in the same assays through fractionation and the isolation of active compounds. • To evaluate the most active and non-toxic fraction from the above mentioned assays for stimulation of weight loss in a model of mouse obesity.

7. Materials • Leaves of ten Ficus species: Ficus capreifolia, Ficus cordata, Ficus craterostoma, Ficus glumosa, Ficus lutea, Ficus natalensis, Ficus polita, Ficus religiosa, Ficus sycomorus and Ficus thonningii were collected from University of Pretoria botanical garden. • Cell lines: C2C12 mouse muscle myoblast (CRL-1772), H411E rat hepatoma (CRL-1548) and C3A human liver cells (CRL-10741) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. • Vero African green monkey kidney cells were obtained from the Department of Veterinary Tropical Diseases (Faculty of Veterinary Sciences, University of Pretoria) • Ethics approval received for in vivo study(AUCC, University of Pretoria. Approval number: V060).

8. Methods Fig 2 Flow chart of methods

9. Methods • Total polyphenolic content (Djeridane et al., 2006) • Antioxidant activity: Trolox equivalent antioxidant capacity (TEAC) (Re et al.,1999) • α-Amylase inhibitory activity assay (Bernfeld 1955, Ali et al., 2006) • α-Glucosidase inhibitory activity assay (Bhandari et al., 2008) • Glucose uptake assay (Deutschlander et al., 2009) • Insulin release (DRG diagnostic Insulin (Rat) ELISA kit according to the manufacturer’s instructions) • Cytotoxicity activity - MTT colorimetric assay (Mosmann, 1983) • Structural elucidation was by 1H and 13C nuclear magnetic resonance (NMR)

10. Methods In vivo assay: 40 CD1 male mice Fed high calorie food prepared thrice weekly Food intake, body weight & faecal weight measured thrice weekly Once obesity attained (> 5 g weight/age), animals fasted for 6 h Fasting blood glucose and glucose tolerance test (GTT) conducted. Animals assigned to treatment group for 7 weeks. Week 6 - 2nd fasting blood glucose and GTT conducted Fig 3 Some activities of in vivo assay

11. Methods • All animals terminally bled • Blood samples: haematological and serum chemistry evaluation for each animal • Gross pathological changes of organs recorded for each animal • Statistical analysis • All data presented as the mean ± standard error of mean (S.E.M.) • Data evaluated by one-way analysis of variance (ANOVA) and considered significantly different at p˂0.05, followed by post hoctests • Non normal data log-transformed prior to statistical testing. • Glucose tolerance test: differences before and after treatment were ascertained using a paired t-test

12. Results Table 1 Total polyphenolic contents and antioxidant activity of extracts of the ten Ficus species aMeans of values, bAntioxidant activity (Trolox equivalent antioxidant capacity), c,d,e,fNosignificant difference between extracts with same value, significant difference p˂0.05 between different values

13. Table 2 The inhibition (EC50) of α-amylase and α-glucosidase activity by extracts of the ten Ficus species a,b,Nosignificant difference between fractions with same value, but significant difference p˂0.05 between different values

14. α-Amylase and α-glucosidase (sucrase) enzymes are therapeutic targets for modulation of postprandial hyperglycaemia • F. lutea extractpotently inhibited α-amylase and α-glucosidase (sucrase) activity • Correlation between polyphenolic content of the Ficus species and α-amylase inhibition (0.80) and α-glucosidase (sucrase) inhibition (0.84), suggest polyphenols may in part responsible for the evident activity • Correlation between antioxidant activity of Ficus species and their polyphenolic content, suggest that the metabolites responsible for the observed activities may be part of the natural constituent of the plant

15. Table 3. Cytotoxicity activity of extracts of the ten Ficusspecies (LC50µg/ml)

16. Fig 4 The effect of extracts of the ten Ficusspecies and insulin on glucose uptake in C2C12 muscle cells (expressed as percentage of untreated control cells).

17. Fig 5 The effect of extracts of the ten Ficus species, metformin and insulinon glucose uptake in H-4-11-E rat liver cells (expressed as percentage of untreated control cells).

18. Fig 6 The effect of extract of F. luteaand glibenclamide on insulin secretion in RIN-m5F pancreatic cell (expressed as percentage of untreated control cells) in glucose free medium.

19. Enhancing glucose uptake and insulin secretion are therapeutic targets for impaired insulin action and deficient insulin secretion • F. lutea extract enhanced superior glucose uptake into muscle and liver cells.Mechanism probably insulin-mimetic property, probably due to increase translocation of GLUT4 transporter • F. lutea extract enhanced 4.58 fold insulin secretion suggesting insulin secreting properties • The Ficus species contained compounds that were generally relatively more nephrotoxic than hepatotoxic. • Those in F. lutea extract were relatively more hepatotoxic • F. lutea extract being the most active was fractionated into six fractions

20. Table 4 Total polyphenolic content of fractions of F. lutea extract aMeans of values, bTotalpolyphenolic contents (mg gallic equivalent/g dry weight of extract) of crude acetone extract of F. lutea. c,d,eNosignificant difference between fractions with same value, but significant difference p ˂ 0.05 between different values

21. Table 5 The inhibition (EC50) of α-amylase and α-glucosidase activity by fractions of F. lutea extract a,b,cNo significant difference between fractions with same value, but significant difference p˂0.05 between different values

22. Table 6 Cytotoxicity activity of fractions of F. luteaextract (LC50in µg/ml ± SE) ND: not determined

23. Fig 7 The effect of the fractions of F. lutea extract on glucose uptake in C2C12 muscle cells (expressed as percentage of untreated control cells)

24. Fig 8 The effect of the fractions of F. lutea extract on glucose uptake in H-4-11-E rat liver cells (expressed as percentage of untreated control cells).

25. Fig 9 The effect of ethyl acetate fraction of F. lutea extract on insulin secretion in RIN-m5F pancreatic cells (expressed as percentage of untreated control cells).

26. Activities of F. lutea extract are within the intermediate polar solvents with the ethyl acetate fraction being most active • Fractionation potentiated inhibition of α-glucosidase (sucrase) activity but attenuated α-amylase inhibitory activity supporting the presence of synergism of the extract • Fractionation also potentiated glucose uptake into cells but reduced insulin secretory activity (3.49 fold) • Correlation between polyphenolic content, inhibition of sucraseactivity and glucose uptake suggest polyphenols may in part responsible for the evident activities • The hepatotoxic compounds in F. lutea extract reside in the n-butanol fraction • The ethyl acetate fraction was subjected to column chromatographic isolation of compounds.

27. Fig 10 Compounds isolated from ethyl acetate fraction

28. Table 7 The inhibition (EC50) of α-glucosidase (sucrase) activity by compounds isolated from ethyl acetate fraction of F. lutea a,bNo significant difference between compounds with same value, but significant difference p˂ 0.05 between different values.

29. Fig 11 The effect of the compounds isolated from ethyl acetate fraction of F. lutea extract on glucose uptake in C2C12 muscle cells (expressed as percentage of untreated cells control cells).

30. Fig 12 The effect of the compounds isolated from ethyl acetate fraction of F. lutea extract on glucose uptake in H-4-11-E rat liver cells (as percentage of untreated control cells ).

31. Fig 13 The effect of the isolated compound (epiafzelechin) on insulin secretion in RIN-m5F pancreatic cells (expressed as percentage of untreated control cells).

32. Epicatechin and epiafzelechin - potent inhibitors of α-glucosidase (sucrase) activity than the crude extract and fraction • Epicatechin and epiafzelechin – enhanced superior glucose uptake into cells than crude extract and fraction. Mechanism - probably via the insulin-mimetic mode of action. • Epiafzelechin enhanced insulin secretion similar to crude extract but superior to fraction. • Some of the isolated compounds are speculated to have weight reducing property

33. Fig 14 The effect of high calorie and normal diet with and without treatment (the ethyl acetate fraction of F. lutea) on body weight of CD1 mice. The initial body weight at period 0 was when obesity state was attained by mice prior to commencement of treatment for about 7 weeks.

34. Fig 15 The effect of high calorie and normal diet with and without treatment (the ethyl acetate fraction of F. lutea) on food intake of CD1 mice. Food intake at period 0 was when obesity state was attained by mice prior to commencement of treatment for about 7 weeks.

35. Fig 16 The effect of high calorie and normal diet with and without treatment (the ethyl acetate fraction of F. lutea) on faecal output. Faecal output at period 0 was when obesity state was attained by mice prior to commencement of treatment for about 7 weeks.

36. Fig 17 The effect of high calorie diet on blood glucose concentrations of CD1 mice. Fasting blood glucose concentrations and glucose tolerance tests (GTT) at period 0 when obesity state was attained by CD1 mice prior to commencement of treatment.

37. Fig 18 The effect of different diets on blood glucose concentrations of CD 1 mice. Fasting blood glucose concentrations and glucose tolerance tests (GTT) of CD1 mice 6 weeks of treatment.

38. Haematological and serum chemistry evaluation: no significant difference between treated and untreated groups • Gross pathological evaluation: no significant changes • Weight reducing assay: no significant weight loss discerned • Failure may be due to many factors including: • The dose administered • The mode of administration • Bioavailability of active compound(s) and this was probably because • Poorly absorbed from the intestine because of hydrophilicity • Highly metabolised and rapidly eliminated • Metabolites in blood and target organs differ from native compounds with biological activity

39. Conclusion • F. luteaextract was the most active • The mechanisms underlying the anti-diabetic activity of F. lutea extract includes the inhibition of α-amylase and α-glucosidase activities, enhancing of glucose uptake in cells and stimulation of insulin secretion • The isolated compounds have reported anti-diabetic activity while epiafzelechin is reported to have anti-diabetic activity for the first time. • This study is the first study to report on the in vitro anti-diabetic activity of F. lutea extracts.

40. Future work • The following questions may be addressed: • To what extent will the complications of type II diabetes be reduced by F. luteaextracts? • Could the extracts of F. lutea reduce the extent of oxidative stress leading to these complications? • Does ingestion of polyphenolic compounds present in extracts of F. lutea influence endogenous antioxidant enzymes and non-enzymatic reactions? • Could the measurement of glycated haemoglobin (HbA1c) in the rodent blood be a better way of evaluating the management of type II diabetes in a mouse obesity model treated with the F. lutea extract?

41. Acknowledgment • Prof J.N. Eloff:Leader PhytomedicineProgramme • Prof VinnyNaidoo: Supervisor • Dr L.J. McGaw: Co-supervisor • National Research Foundation (NRF) South Africa • Faculty of Veterinary Science and the Department of Paraclinical Sciences for research funding • Federal Institute of Industrial Research Oshodi (FIIRO) • Ms Annette Venter for helping with cell culture • Drs Ahmed Aroke and Maurice D. Awouafack for isolation and elucidating the structure of compounds • TharienDeWinnaar for administrative issues and purchase of materials/reagents • Ms.MagdsNel, Ms. Elsa van Wyke and Mr. Jason Sampson for assistance in the collection, identification and authentication of the plants • Mrs.IlseJanse van Rensburg and Mrs. Santa Meyer (UPBRC) for the in vivo assay. DrTamsynPulkerfor treating sick animals

42. Thank you