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

model

Oyinlola O. Olaokun

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

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

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.

Aim

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

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 vitro cytotoxicity 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.

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).

Methods

Assays conducted with standard methods

Dried ground leaves of plants of Ficus species prepared for extraction Extracted with acetone

(1:10 w/v) to yield crude extract

Total polyphenolic content

Antioxidant activity

Alpha- Amylase inhibition assay

Alpha-glucosidase inhibition assay

Glucose uptake assay

Insulin secretion assay

Cytotoxicity activity

Most active plant extract selected for solvent/solvent fractionation Successively and exhaustively

partitioned with solvents of increasing polarities

Hexane fraction

Chloroform fraction

Ethyl acetate fraction

n-Butanol fraction

Water fraction

Dichloromethane fraction

All the above assays conducted with the fractions

The most active fraction selected

Compounds isolated from most active fraction

In vivo assay-weight reducing activity of most active fraction in diet induced obese CD1 mouse model

All the above assays conducted with isolated compounds Fig 2 Flow chart of methods

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)

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

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 hoc tests

Non normal data log-transformed prior to statistical testing.

Glucose tolerance test: differences before and after treatment were ascertained using a paired t-test

Results

Plants extract aTotal polyphenol(mg GAE/g dry weight)

abAntioxidant activityTEAC

Ficus capreifolia 4.73 ± 0.26c 0.34 ± 0.05c

Ficus cordata 8.23 ± 1.00d 0.27 ± 0.03c

Ficus craterostoma 9.80 ± 0.93d 0.66 ± 0.06d

Ficus glumosa 19.24 ± 0.79e 1.29 ± 0.30e

Ficus lutea 56.85 ± 1.82f 4.80 ± 0.90f

Ficus natalensis 4.75 ± 0.92c 0.69 ± 0.08d

Ficus polita 8.04 ± 0.52d 0.31 ± 0.06c

Ficus religiosa 5.40 ± 0.35c 0.59 ± 0.18c

Ficus sycomorus 12.33 ± 0.26e 1.91 ± 0.19e

Ficus thonningii 4.64 ± 0.48c 0.77 ± 0.06d

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,fNo significant difference between extracts with same value, significant difference p˂0.05 between different values

Table 2 The inhibition (EC50) of α-amylase and α-glucosidase activity by extracts of the ten Ficus speciesPlant extract α-Amylase inhibition

(EC50) µg/mlα-Glucosidase

inhibition (EC50) µg/mlFicus capreifolia ˃100 ˃ 1000 Ficus cordata ˃ 100 ˃ 1000Ficus craterostoma 11.41 ± 4.68a ˃ 1000Ficus glumosa ˃ 100 ˃ 1000Ficus lutea 9.42 ± 2.01b 290 ± 111a

Ficus natalensis 17.85 ± 4.42a ˃ 1000Ficus polita ˃ 100 ˃ 1000Ficus religiosa ˃ 100 ˃ 1000Ficus sycomorus ˃ 100 217 ± 69a

Ficus thonningii ˃ 100 ˃ 1000Acarbose 0.04 ± 0.03 3.4 ± 0.5

a,b,No significant difference between fractions with same value, but significant difference p˂0.05 between different values

• α-Amylase and α-glucosidase (sucrase) enzymes are therapeutic targets for modulation of postprandial hyperglycaemia

• F. lutea extract potently 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

Table 3. Cytotoxicity activity of extracts of the ten Ficus species (LC50 µg/ml)

Plant Extract Vero cells (µg/ml) C3A cells (µg/ml)

Ficus capreifolia 85.3 ± 2.0 108.4 ± 0.8Ficus cordata 76.7 ± 1.4 166.3 ± 1.9Ficus craterostoma 356.2 ± 9.6 ˃1000Ficus glumosa 72.7 ± 9.2 127.6 ± 2.6Ficus lutea 214.8 ± 5.0 126.0 ± 6.8Ficus natalensis 69.2 ± 8.0 113.8 ± 7.4Ficus polita 90.9 ± 1.4 44.8 ± 1.8Ficus religiosa 110.9 ± 8.2 922.9 ± 4.7Ficus sycomorus 101.8 ± 1.1 151.6 ± 4.3Ficus thonningii 68.0 ± 1.0 491.4 ± 9.9Doxorubicin 17.0 ± 0.1 6.7 ± 0.6

F. cap

reifolia

F. co

rdata

F. cat

erosto

ma

F. glu

mosa

F. lutea

F. nata

lensis

F. polita

F. rel

igiosa

F. syc

omorus

F. thonningii

DMSOInsulin

0.00

5.00

10.00

15.00

20.00

25.00Extract (500 µg/ml), Insulin (100 µM) Extract (250 µg/ml), Insulin (10 µM)Extract (125 µg/ml), Insulin (1 µM) Extract (63 µg/ml), Insulin (0.1 µM)Extract (31 µg/ml) Extract (15 µg/ml)

Crude acetone plant extracts, solvent control (DMSO) and insulin

Gluc

ose

upta

ke (p

erce

ntag

e of

unt

reat

ed c

ells)

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

F. cap

reifolia

F. co

rdata

F. cat

erosto

ma

F. glu

mosa

F. lutea

F. nata

lensis

F. polita

F. rel

igiosa

F. syc

omorus

F. thonningii

DMSO

Metform

inInsulin

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00 Extract (500 µg/ml), Metformin & Insulin (100 µM) Extract (250 µg/ml), Metformin & Insulin (10 µM)Extract (125 µg/ml), Metformin & Insulin (1 µM) Extract (63 µg/ml), Metformin & Insulin (0.1 µM)Extract (31 µg/ml) Extract (15 µg/ml)

Crude acetone plant extracts, solvent control (DMSO), metformin and insulin

Gluc

ose

upta

ke (p

erce

ntag

e of

unt

reat

ed c

ells)

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

62.5 125 250 500 1 10 1000

20

40

60

80

100

120

140

160

Extract of Ficus lutea (μg/ml) Glibenclamide (μM)

Insu

lin se

creti

on (p

erce

ntag

e of

unt

reat

ed c

ells)

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

• 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

Table 4 Total polyphenolic content of fractions of F. lutea extract

FractionsabTotal polyphenol (mg GEA/g dry

weight extract)

Hexane 14.86 ± 1.43c

Chloroform 10.32 ± 0.82c

Dichloromethane 11.83 ± 2.32c

Ethyl acetate 100.51 ± 1.60d

n-Butanol 79.58 ± 0.50e

Water 13.34 ± 0.85c

aMeans of values, bTotal polyphenolic contents (mg gallic equivalent/g dry weight of extract) of crude acetone extract of F. lutea. c,d,eNo significant difference between fractions with same value, but significant difference p ˂ 0.05 between different values

Table 5 The inhibition (EC50) of α-amylase and α-glucosidase activity by fractions of F. lutea extract

Fractions α-Amylase inhibition (EC50) µg/ml

α-Glucosidase inhibition (EC50) µg/ml

Hexane ˃1000 ˃1000

Chloroform ˃1000 ˃1000

Dichloromethane ˃1000 854.51 ± 56.92a

Ethyl acetate 39.53 ± 7.10a 126.78 ± 30.62b

n-Butanol 26.50 ± 1.22b 195.17 ± 63.60c

Water ˃1000 558.40 ± 51.67a

a,b,cNo significant difference between fractions with same value, but significant difference p˂0.05 between different values

Table 6 Cytotoxicity activity of fractions of F. lutea extract (LC50 in µg/ml ± SE)Fractions Vero kidney cells C3A liver cells

Hexane ˃1000 ˃1000

chloroform 389.6 ± 1.8 615.7 ± 3.9

Dichloromethane 302.3 ± 2.1 ˃1000

Ethyl acetate 126.9 ± 1.5 ˃1000

n-Butanol 216.1 ± 2.9 76.8 ± 0.4

Water ND ND

ND: not determined

hexa

ne

chlor

oform

dichlo

rometh

ane

ethyla

cetate

butan

olwate

r0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50500 µg/ml250 µg/ml125 µg/ml63 µg/ml31 µg/ml15 µg/ml

Fractions of Ficus lutea

Glu

cose

upt

ake

(Per

cent

age

of u

ntre

ated

cel

ls)

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)

hexa

ne

chlor

oform

dichlo

rometh

ane

ethyla

cetate

butan

olwate

r0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

500 µg/ml250 µg/ml125 µg/ml63 µg/ml31 µg/ml15 µg/ml

Fractions of Ficus lutea

Glu

cose

upt

ake

(Per

cent

age

of u

ntre

ated

cel

ls)

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).

62.5 125 250 5000

20

40

60

80

100

120

140

Concentration of the ethyl acetate fraction (µg/ml)

Insu

lin se

creti

on (p

erce

ntag

e of

unt

reat

ed c

ells)

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).

• 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 sucrase activity 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.

Fig 10 Compounds isolated from ethyl acetate fraction

Lupeol

Epicatechin

Epiafzelechin

Stigmasterol

Alpha-amyrin acetate

Table 7 The inhibition (EC50) of α-glucosidase (sucrase) activity by compounds isolated from ethyl acetate fraction of F. lutea

Compound EC50 (µg/ml)

Lupeol >1000

Stigmasterol 115.71 ± 11.6a

α-Amyrin acetate 335.82 ± 22.6a

Epicatechin 5.72 ± 2.6b

Epiafzelechin 7.64 ± 4.9b

a,bNo significant difference between compounds with same value, but significant difference p˂ 0.05 between different values.

Lupeo

l

Stigm

astero

l

α- Amyri

n aceta

te

Epica

techin

Epiaf

zelech

in0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

250 µg/ml125 µg/ml63 µg/ml31 µg/ml15 µg/ml

Compounds isolated from ethyl acetate fraction

Glu

cose

upt

ake

(per

cent

age

of u

ntre

ated

cel

ls)

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).

Lupeo

l

Stigmast

erol

α- Amyri

n acet

ate

Epicate

chin

Epiafze

lechin

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

250 µg/ml125 µg/ml63 µg/ml31 µg/ml15 µg/ml

Compounds isolated from ethyl acetate fraction

Glu

cose

upt

ake

(per

cent

age o

f unt

reat

ed c

ells

)

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 ).

62.5 125 250 5000.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

Concentration of the isolated compound (epiafzelechin) (µg/ml)

Insu

lin se

creti

on (p

erce

ntag

e of

unt

reat

ed c

ells)

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

• 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

0 5 10 15 20 2537.038.039.040.041.042.043.044.045.046.047.0

High calorie diet High calorie diet with treatmentNormal diet Normal diet with treatment

Period of treatment of CD1 mice

Mea

n bo

dy w

eigh

t (g)

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.

0 5 10 15 20 256.07.08.09.0

10.011.012.013.014.015.016.0

High calorie diet High calorie diet with treatmentNormal diet Normal diet with treatment

Period of treatment of CD1 mice

Mea

n fo

od in

take

(g)

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.

0 5 10 15 20 250.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

High calorie diet High calorie diet with treatmentNormal diet Normal diet with treatment

Period of treatment of CD1 mice

Mea

n fa

ecal

wei

ght (

g)

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.

0 10 20 30 40 50 60 70 80 906.00

11.00

16.00

21.00

26.00

High calorie diet High calorie diet with treatmentNormal diet Normal diet with treatment

Time (min)

Mea

n gl

ucos

e co

ncen

trati

on (m

M)

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.

0 10 20 30 40 50 60 70 80 905.00

10.00

15.00

20.00

25.00

30.00

High calorie diet High calorie diet with treatmentNormal diet Normal diet with treatment

Time (Min)

Mea

n gl

ucos

e co

ncen

trati

on (m

M)

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.

• 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 administrationBioavailability of active compound(s) and this was probably

becausePoorly absorbed from the intestine because of hydrophilicityHighly metabolised and rapidly eliminatedMetabolites in blood and target organs differ from native

compounds with biological activity

Conclusion

• F. lutea extract 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.

Future work

• The following questions may be addressed: To what extent will the complications of type II diabetes be reduced by

F. lutea extracts?

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?

Acknowledgment• Prof J.N. Eloff: Leader Phytomedicine Programme

• Prof Vinny Naidoo: 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 • Tharien DeWinnaar for administrative issues and purchase of materials/reagents• Ms. Magds Nel, Ms. Elsa van Wyke and Mr. Jason Sampson for assistance in the

collection, identification and authentication of the plants• Mrs. Ilse Janse van Rensburg and Mrs. Santa Meyer (UPBRC) for the in vivo

assay. Dr Tamsyn Pulker for treating sick animals

Thank you