INTRODUCTION:
Diabetic mellitus is a chronic endocrine
disorder characterized by hyperglycemia. The prevalent type 2 DM occurs in more
than 90% of diabetics and is caused by combination of peripheral insulin
resistance and impaired insulin secretion. This metabolic disorder includes
alterations in carbohydrate, lipid and protein metabolism and is grouped
together under metabolic syndrome with other lifestyle related diseases.
Chronic hyperglycemia of diabetes often causes long term damage and dysfunction
to various organs, especially eyes and kidneys.
Lifestyle changes such as exercise is
suggested to be helpful in alleviating the disease[1-3] but
worldwide prevalence of type 2 DM is increasing. This is evident by the
estimate of number of adults with diabetes in 1995 of 135 million and which was
projected to rise to 300 million in 2025[4]. The projected increase
is also seen where in 2010, diabetic prevalence was calculated to affect 285
million and estimated to increase to 439 million by 2030[5].
The morbidity of type 2 DM is associated
with increased glucose concentrations and this is often due to postprandial
glucose concentrations. The increase after a meal is caused by hydrolysis of
starch by pancreatic α-amylase as well as uptake of glucose by intestinal
α-glucosidase and therefore, the strategy for type 2 DM management would
be strong inhibition of pancreatic α-amylase and intestinal
α-glucosidase[6].
Plant polyphenols have been reported to
possess inhibitory effect on α-amylase and α-glucosidase as well as
increasing glucose uptake into skeletal muscle and adipocytes[7].
Compounds from plants have also shown beneficiary effect on glucose uptake in
the liver and this is vital as the organ is an important regulator of plasma
glucose level and plays a key role in glucose metabolism and regulation.
Natural products have always been a source
for development of new drugs even in the present era of combinatorial chemistry
and drugs of plant or microbial origin account for more than 30% worldwide
sales of natural products. This coupled with the fact that pharmacological
approach using synthetic oral hypoglycemic cause serious side effects[8-9],
makes the search for alternatives with enhanced therapeutic but reduced side
effects an ever going process.
Morinda citrifolia L. (Rubiaceae) is a small
tropical evergreen tree that is indigenous to Pacific Islands, South East Asia
and other tropical as well as semitropical regions. It has been used
traditionally in folk medicine as a treatment for diabetes as well as other
related diseases[10-11]. Its fruit contains a variety of natural
products. Other than flavanoids such as rutin, quercetin and kaempferol, other
key markers such as asperulosidic acid, dimethyl morindol and scopoletin were
also detected[12-15].
One of the authors have reported
antihyperglycemic[16] and antihyperlipidemic[17] effect
of MCF extract in streptozotocin-induced diabetic rats. However, the
antidiabetic mechanism responsible for the fruits effect has not been studied.
Therefore, this study would investigate the effect of MCF extract and
scopoletin on glucose uptake in HepG2 cells.
In addition, the in vitro inhibitory effect of the extract and
scopoletin on α-amylase and α-glucosidase will be studied.
MATERIALS AND METHODS:
Chemicals
Anthrone reagent (C14H10O),
Bromo Phenol Blue (BPB), DMSO and scopoletinat scopoletin were purchased from
Nacalai tesque (Kyoto, Japan). Insulin and metformin were purchased from Tocris
(Bioscience, Bristol, UK). Triple E/Trypsin
and fetal bovine serum (FBS) were from
GIBCO (UK). Dulbecco’s Modified Eagle’s Medium (DMEM), D-glucose
anhydrous, sodium bicarbonate (NaHCO3) and thiazolyl blue tetrazolium
bromide were from SIGMA (UK), whereas phosphate buffered saline (PBS) tablet
was from Calbiochem®(Darmstadt, Germany). Sodium hydroxide (NaOH)
and Hydrochloric acid (HCI) from Dulchefa (Netherland). Chloroform, ethanol, ethyl acetate, methanol
and H2SO4were from R&M (Essex, U.K).
Plant materials
and preparation of extracts
The leaf and fruits of M. citrifolia were obtained from Kampung Tok Dor, Besut, Terengganu
in September 2012. Herbarium samples are deposited at Universiti Sultan Zainal
Abidin (UniSZA) Herbarium, Faculty of Agriculture, Biotechnology and Food
Sciences, voucher no: 0.00218. The fruits were cut into small pieces, dried in
an oven at temperature of 45oC and ground into powder. The
dried fruit powder was macerated for 3 days with methanol at ratio of 1 g of
sample to 10 ml of methanol. The extract was then filtered and evaporated using
rotary evaporator.
Cell culture and
treatment
The human liver hepatocellular carcinoma
cell line (HepG2 cells) was provided by the Animal cell culture laboratory of
Faculty of Agriculture, Biotechnology and Food Sciences, UniSZA. The cells were
grown in a humidified atmosphere containing 5% CO2 at 37°C[18].
The cells were routinely maintained in growth medium which consisted of DMEM
supplemented with 10% heat-inactivated FBS and 0.5% pens/strep (antibiotic).
Proliferating cells were subcultured into fresh growth medium every 2-3 days.
For routine maintenance, trypsin in phosphate-buffered saline (PBS) was used as
the treatment to detach the cells from the t-flask and after 5-10 minute
exposure at 37°C, the cells were seeded in growth medium.
Cell viability
(MTT assay)
Viability
of HepG2 cells after treatment with the plant extract and standard drug was
determined by assaying for the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to formazan. HepG2 cells were seeded in 96 well plates at density
of 2 ×106 cells per well. Cells were inoculated in a volume of 100
μl per well and 100 μl aliquot of growth media was added to the free
cells. Fresh media containing MCF extract or scopoletin at indicated
concentrations was added 24 hours after seeding. Control cells were incubated
without test sample and with maintenance medium. The microplates were incubated
at 37°C for a period of 24 hours. The cell medium was replaced with 100 μl
fresh medium per well containing 0.5 mg/ml MTT and incubated for another 4
hours in the dark. Lastly, 100 μl of isopropanol was added to solubilize
the formazan and its absorption was measured at 570nm (620 nm as reference)
using a micro-plate reader (Infinite 200 PRO NanoQuant, TECAN).
Glucose uptake
assay
Glucose
uptake was measured as previously described by Tandrasasmita et al. (2011)[19]. Briefly,
HepG2 were grown to about 1 × 104 cells/ml in 6 cm diameter plates.
When the cells reached 80% confluency, the cells were washed twice with
phosphate-buffered saline and incubated with3 ml glucose solution (10 mg/ml)
with or without administration of MCF extract, metformin, scopoletin and
insulin at 37°C for 10 min. During incubation the cells took up glucose,
enabling free glucose to be measured in the media, from which the concentration
of glucose uptake could then be determined. Measurements of glucose
concentrations were performed by the reaction of glucose with anthrone in
presence of sulphuric acid.
The
concentration of glucose taken up by the cells is described by the equation:
Initial glucose-free glucose within media
%Glucose
uptake=-------------------------------------X 100%
Initial glucose
α-Amylase
inhibition assay
The assessment of inhibitory effect of MCF
extract on α-amylase activity was measured based on modified method of
Apostolidis et al. (2007)[20].
Sample solutions of 1 mg/ml of MCF extract or 0.2 μM scopoletinat volume
of 500 μl and 0.2 M phosphate buffer pH 6.9 (500 μl) containing
α-amylase solution (0.5 mg/ml) were incubated at 25°C for 10 min. After
preincubation, 500 μl of 1% starch solution in 0.02 M sodium phosphate was
added and the reaction mixture was incubated at 25°C for 10 min. The reaction
was stopped with 1.0 ml of dinitrosalicylic acid (DNS). The reaction mixture
was then incubated in a boiling water bath for 5 min and allowed to cool to
room temperature. The reaction mixture was then diluted with 10 ml distilled
water and absorbance was read at 540 nm (Infinite 200 PRO NanoQuant, TECAN).
The inhibitory effect of the extract was compared to standard inhibitor,
acarbose.
α-Glucosidase
inhibition assay
The α-glucosidase method was
determined according to the method of Apostolidis et al. (2007)[20]. In brief, 50 μl of the test
samples and 100 μl of 0.1 M
phosphate buffer (pH 6.9) containing yeast α-glucosidase solution (1.0
U/ml) were preincubated in 96 well plates at 25°C for 10 min. After incubation,
50 μl of 5 mM pNPG solution in 0.1 M phosphate buffer (pH 6.9) was added
to each well and the reaction mixtures were incubated at 25°C for 5 min. The
absorbance of the reaction mixtures was recorded with a micro-plate reader at
405 nm (Infinite 200 PRO NanoQuant, TECAN) before and after incubation with
pNPG solution and compared to that of the control which had 50 μl buffer
solutions instead of test samples. The experiments were performed in triplicate
and the α-glucosidase inhibitory activity was expressed as percentage
inhibition. Acarbose was prepared in distilled water at 1.0 mg/ml concentration
and used as positive control.
Statistical
analysis
All the data points are mean values ±
standard error. Where appropriate,
statistical analysis were performed using one-way analysis of variance
(ANOVA) to treat difference between mean while Tukey’s multiple comparison test
with P ≤ 0.05 was taken as significant. The software employed for
statistical analysis was SPSS.
RESULTS:
Cell viability by
MTT assay
Cell viability by MTT assay was used to
assess the safe dose of MCF extract and scopoletin for monitoring glucose
uptake with HepG2 cells. It is important to use
the safe dose as we do not want the extract or compound to be harmful to the
tested cells. The safe dose was identified at the concentration of which
the cells viability is about 80% and the concentration for safe dose of MCF extract was 1.0mg/ml while
scopoletin was 0.2 µM.
In vitro
α-amylase inhibition study
The ethanolic extract of MCFat
concentration of 1 mg/ml showed mild inhibition of amylase but its percentage
of inhibition of 43.5% was higher than that for 0.2 μM scopoletin at 23.9%
as well as that for the 1 mg/ml standard acarbose at 35.2%. The percentage
values for inhibition of amylase by ethanolic extract of MCF, acarbose and
scopoletin are shown in Fig. 1.
Figure 1 Percentage inhibition of ethanol extract of MCF,
scopoletin and acarbose on α-amylase in
vitro. Values represent mean ± SEM of triplicate tests. Bars with
different letters are significantly different (p<0.05).
In vitro
α-glucosidase inhibition study
The ethanolic extract of MCF at
concentration of 1 mg/ml showed strong inhibition of α-glucosidase and its
percentage of inhibition of 57% was comparable to that of the standard acarbose
at concentration of 1.0 mg/ml (Percentage of inhibition of 57.7%). However, the
percentage of inhibition for0.2 μM scopoletin at concentration was lower
than both of these and its value was only 35.7%. The values for percentage of
inhibition by α-glucosidase are shown in Fig. 2.
Figure 2 Percentage inhibition of ethanol extract of MCF,
scopoletin and acarbose on α-glucosidase in vitro. Values represent mean ± SEM of triplicate
tests. Bars with different letters are significantly different (p<0.05).
In vitro glucose uptake
study
The effect on glucose uptake by 1 mg/ml of
MCF extract, 0.2 μM scopoletin as well as the two positive controls of
metformin and insulin were studied in
vitro using HepG2 cells and the results show that insulin had the highest
activity. Its percentage of glucose uptake of 69.3% was higher than both of
metformin as well as for the MCF extract. The values of 59.5% for the fruit
extract and 58.2% for metformin were not statistically different from each
other but they were higher than that of scopoletin, which value was only
30.6%. The percentage values for glucose
uptake by 1 mg/ml of ethanolic extract of MCF, 0.2 μM scopoletin, 100
μg/ml metformin and 1 IU/ml insulin are shown in Fig. 3.
Figure 3 Percentage of glucose uptake by ethanol extract of MCF,
scopoletin, metformin and insulin in HepG2 cells. Values represent
mean ± SEM of triplicate tests. Bars with different letters are significantly different
(p<0.05).
DISCUSSION:
The MCF has been used for treating diabetes
mellitus but its mechanism of action for alleviating blood glucose is not
established although leaf extract from another species from the same genus in
Africa has been reported to inhibit α-amylase and α-glucosidase[21].
Therefore, in our study we evaluated the effect of inhibition of both enzymes
in addition to the in vitro evaluation
of glucose uptake by the fruitextract as well as the biomarker, scopoletin.
Often glucose uptake for diabetic studies are carried out using adipocytes[22-24]
and differentiated skeletal muscle cells[25-26] but our experiment
utilized HepG2 cells as they have similar physiological function to normal
hepatocytes[27] and also they are stable during many passages.
Considerable previous studies have used HepG2 cell to monitor glucose uptake[28-29].
Glucose uptake in our study was determined by measuring the differences in
concentration of glucose in media before and after incubation with test
compounds.
Prior to evaluating the effect of plant
extracts and compounds, viability studies were carried out using the MTT assay.
The MTT assay which measures the activity of the mitochondrial reductase enzyme[30]
is an estimate of the number of viable cells and these studies help in
eliminating the cytotoxic doses of plant extracts and for determining the
precise range of concentrations of test samples for further study. The safety
dose is often ascertained as the dose which gives viability of about 80%[31-32]
and the value of this for MCF extract was 1.2 mg/kg whereas for scopoletin was
0.2μM.
The therapeutic approach in using M. citrifolia extract to treat diabetes
is suggested to decrease post-prandial hyperglycemia. Firstly, this is achieved
by increased glucose uptake into the liver. The result of our study shows that
MCF extract increases glucose uptake in HepG2 cells. This is the first study
that reports this mechanism for extract of this species as earlier study by
Nguyen and co-workers[33] reported increased glucose uptake in
adipocytes by isolated compounds from M.
citrifolia. The compounds were episesamin 2,6-dicatechol, lirioresinol B,
lirioresinol B dimethyl ether, and ursolic acid, and did not include the
predominant biomarker, scopoletin.
Secondly, the decrease in postprandial
hyperglycemia is achieved by hindered absorption of glucose by inhibition of
the carbohydrate hydrolyzing enzymes in the digestive organs. The enzymes that
are affected are α-amylase, that catalyses the breakdown of starch to maltose
and finally to glucose, as well as α-glucosidase, present in the small
intestine and catalyzing the breakdown and absorption of complex sugars. Examples
of such inhibitors in clinical use are acarbose, miglitol and voglibose but
they do have side effects[34].
The present study indicates that ethanolic
extract of MCF possesses inhibitory effect on glucosidase and amylase. However,
scopoletin which is the major biomarker in ethanolic extract of M. citrifolia possesses only mild
activity on inhibition of both these enzymes. Various studies have shown the
presence of flavonoids such as rutin, kaempferol and quercetin in fruits of M. citrifolia. These compounds are
suggested to be responsible for this activity as they have been shown to
possess inhibitory effect on amylase[35-36] and glucosidase[37-40]
in in vitro studies.
CONCLUSION:
This
study investigated the potential antidiabetic effect of M. citrifolia with focus on increased glucose uptake as well as
inhibition of α-amylase and α-glucosidase, which would be beneficial
by reducing hyperglycemia. The ethanolic extract of the fruit showed these
three activities and at the concentration of the safe dose, they were higher
for the fruit than the biomarker scopoletin. In conclusion, the results from
this study give scientific support to the use of M. citrifolia in traditional medicine for the treatment of diabetes
and show, for the first time, the potential role of α-glucosidase and
α-amylase inhibition as well as increased glucose uptake by hepatocytes in
its activity.
CONFLICT OF INTEREST:
The authors declare no conflict of
interest.
ACKNOWLEDGEMENTS:
The authors graciously acknowledge the financial backing of
Ministry of Higher Education, Malaysia for granting Dr. Mahadeva Rao the research grant for the
execution of this work (FRGS/1/2012/SKK03/UNISZA/02/01).
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