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
Extracted with acetone
(1:10 w/v) to yield
crude extract
Dried ground leaves of plants of
Ficus species prepared for extraction
Assays conducted with standard methods
Total
polyphenolic
content
Antioxidant
activity
AlphaAmylase
inhibition
assay
Successively and exhaustively
partitioned with solvents of
increasing polarities
Hexane
fraction
Chloroform
fraction
Alphaglucosidase
inhibition
assay
Glucose
uptake
assay
Insulin
secretion
assay
Cytotoxicity
activity
Most active plant extract selected for
solvent/solvent fractionation
Dichloromethane
fraction
Ethyl
acetate
fraction
n-Butanol
fraction
Water
fraction
All the above assays conducted with the fractions
The most active fraction selected
Compounds isolated
from most active
fraction
All the above assays
conducted with isolated
compounds
In vivo assay-weight
reducing activity of most
active fraction in diet
induced obese CD1 mouse
model
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
resonance (NMR)
1H
and
13C
nuclear magnetic
• In vivo assay: 40 CD1
male mice
Methods
• 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
Table 1 Total polyphenolic contents and antioxidant activity of extracts of the
ten Ficus species
aTotal polyphenol
abAntioxidant activity
Plants extract
(mg GAE/g dry weight)
TEAC
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
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 species
Plant extract
Ficus capreifolia
α-Amylase inhibition
(EC50) µg/ml
˃100
α-Glucosidase
inhibition (EC50) µg/ml
˃ 1000
˃ 100
˃ 1000
11.41 ± 4.68a
˃ 1000
˃ 100
˃ 1000
9.42 ± 2.01b
290 ± 111a
17.85 ± 4.42a
˃ 1000
Ficus polita
˃ 100
˃ 1000
Ficus religiosa
˃ 100
˃ 1000
Ficus sycomorus
˃ 100
217 ± 69a
Ficus thonningii
˃ 100
˃ 1000
0.04 ± 0.03
3.4 ± 0.5
Ficus cordata
Ficus craterostoma
Ficus glumosa
Ficus lutea
Ficus natalensis
Acarbose
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 ± 0.8
Ficus cordata
76.7 ± 1.4
166.3 ± 1.9
356.2 ± 9.6
˃1000
72.7 ± 9.2
127.6 ± 2.6
214.8 ± 5.0
126.0 ± 6.8
Ficus natalensis
69.2 ± 8.0
113.8 ± 7.4
Ficus polita
90.9 ± 1.4
44.8 ± 1.8
Ficus religiosa
110.9 ± 8.2
922.9 ± 4.7
Ficus sycomorus
101.8 ± 1.1
151.6 ± 4.3
Ficus thonningii
68.0 ± 1.0
491.4 ± 9.9
Doxorubicin
17.0 ± 0.1
6.7 ± 0.6
Ficus craterostoma
Ficus glumosa
Ficus lutea
Glucose uptake (percentage of untreated cells)
25.00
20.00
Extract (500 µg/ml), Insulin (100 µM)
Extract (125 µg/ml), Insulin (1 µM)
Extract (31 µg/ml)
Extract (250 µg/ml), Insulin (10 µM)
Extract (63 µg/ml), Insulin (0.1 µM)
Extract (15 µg/ml)
15.00
10.00
5.00
0.00
Crude acetone plant extracts, solvent control (DMSO) and insulin
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).
Glucose uptake (percentage of untreated cells)
25.00
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)
20.00
15.00
10.00
5.00
0.00
Crude acetone plant extracts, solvent control (DMSO), metformin and insulin
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).
Insulin secretion (percentage of untreated cells)
180
160
140
120
100
80
60
40
20
0
62.5
125
250
Extract of Ficus lutea (μg/ml)
500
1
10
100
Glibenclamide (μM)
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
Fractions
abTotal
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
α-Glucosidase
(EC50) µg/ml
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
˃1000
558.40 ± 51.67a
Water
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
Glucose uptake (Percentage of untreated cells)
35.00
500 µg/ml
250 µg/ml
30.00
125 µg/ml
25.00
63 µg/ml
20.00
31 µg/ml
15 µg/ml
15.00
10.00
5.00
0.00
Fractions of Ficus lutea
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)
Glucose uptake (Percentage of untreated cells)
45.00
500 µg/ml
250 µg/ml
125 µg/ml
63 µg/ml
31 µg/ml
15 µg/ml
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
Fractions of Ficus lutea
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).
Insulin secretion (percentage of untreated cells)
160
140
120
100
80
60
40
20
0
62.5
125
250
500
Concentration of the ethyl acetate fraction (µg/ml)
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.
Lupeol
Stigmasterol
Epicatechin
Epiafzelechin
Alpha-amyrin acetate
Fig 10 Compounds isolated from ethyl acetate fraction
Table 7 The inhibition (EC50) of α-glucosidase (sucrase) activity by compounds
isolated from ethyl acetate fraction of F. lutea
Compound
Lupeol
EC50 (µg/ml)
>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.
Glucose uptake (percentage of untreated cells)
45.00
40.00
250 µg/ml
35.00
125 µg/ml
30.00
63 µg/ml
25.00
31 µg/ml
20.00
15 µg/ml
15.00
10.00
5.00
0.00
Compounds isolated from ethyl acetate fraction
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).
Glucose uptake (percentage of untreated cells)
60.00
250 µg/ml
50.00
125 µg/ml
40.00
63 µg/ml
31 µg/ml
30.00
15 µg/ml
20.00
10.00
0.00
Compounds isolated from ethyl acetate fraction
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 ).
Insulin secretion (percentage of untreated cells)
160.00
140.00
120.00
100.00
80.00
60.00
40.00
20.00
0.00
62.5
125
250
500
Concentration of the isolated compound (epiafzelechin) (µg/ml)
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
Mean body weight (g)
High calorie diet
Normal diet
High calorie diet with treatment
Normal diet with treatment
47.0
46.0
45.0
44.0
43.0
42.0
41.0
40.0
39.0
38.0
37.0
0
5
10
15
Period of treatment of CD1 mice
20
25
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.
Mean food intake (g)
16.0
15.0
14.0
13.0
12.0
11.0
10.0
9.0
8.0
7.0
6.0
0
High calorie diet
High calorie diet with treatment
Normal diet
Normal diet with treatment
5
10
15
20
25
Period of treatment of CD1 mice
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.
High calorie diet
Normal diet
Mean faecal weight (g)
7.0
High calorie diet with treatment
Normal diet with treatment
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0
5
10
15
20
25
Period of treatment of CD1 mice
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.
Mean glucose concentration (mM)
High calorie diet
High calorie diet with treatment
Normal diet
Normal diet with treatment
26.00
21.00
16.00
11.00
6.00
0
10
20
30
40
50
60
70
80
90
Time (min)
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.
Mean glucose concentration (mM)
High calorie diet
High calorie diet with treatment
Normal diet
Normal diet with treatment
30.00
25.00
20.00
15.00
10.00
5.00
0
10
20
30
40
50
60
70
80
90
Time (Min)
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 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
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