XANTHONES FROM GARCINIA MALACCENCIS IMPROVE GLUT4
AS WELL AS DECREASED PPARγ ACTIVATION ON ADIPOCYTES
P193
Muhammad Taher1*, Mohamed Zaffar Ali Mohamed Amiroudine1, Deny Susanti2,
Solachuddin JA Ichwan3
1Kulliyyah
of Pharmacy, 2Kulliyyah of Science, 3Kulliyyah of Dentistry,
International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan, Pahang
Phone: 60-95716400, Fax: 60-95716781, *E-mail: mtaher@iium.edu.my
Since most studies reported that the common link between
diabetes and obesity lies in the adipocyte, which stores excess
The Garcinia species are well-known for its phytochemical contents such as
energy in the form of triglyceride, therefore this research was
flavonoids, phenolic acids and xanthones. People in Malaysia often use Garcinia for
carried out to describe the xanthone compounds from Garcinia
traditional medicines including the treatment of abdominal pain, dysentery,
malaccencis species and evaluate its activity on adipocyte.
diarrhoea, suppuration, infected wound, leucorrhoea and chronic ulcer and
gonorrhea[1].
OBJECTIVES
Recently, it was reported that the stembark of G. malaccensis showed the presence
of xanthone, α-mangostin as major compound and together with other xanthone, β- 1. To measure the effect of G. malaccencis compound on
triglyceride accumulation and insulin-induced glucose uptake.
mangostin[2]. This species was reported to have a lot of medical properties
including antimicrobial, antioxidant and apoptosis but there was no reported data 2. To show the effect of G. malaccencis compound on the RNA gene
expression (mainly PPARγ and GLUT4) by qRT-PCR.
for its activity on adipocytes especially on obesity management.
INTRODUCTION
MATERIALS AND METHODOLOGY
1. Dried and powdered stembark of G. malaccensis was
collected from Taman Pertanian, Kuantan and extracted by
soxhlet with n-hexane, dichloromethane (DCM) and
methanol (MeOH). Two xanthone compounds were
successfully isolated: α-mangostin, β-mangostin.
2. For suitable dosage study (MTT), mature adipocytes were
seeded in 96-well plates. Cells were treated with mangostin
compounds for 48 hours at 37°C in humidified 5% CO2
atmosphere. Cells were then washed two times with PBS,
incubated with 20 µL MTT for 4 hours and 100 µL DMSO for
1 hour. Absorbance were measured at 570: 630 nm by ELISA
plate reader.
3. 3T3-L1 preadipocytes were culture in DMEM (+10% foetal
bovine serum, 1 % penicillin, 1 % streptomycin) at 37°C in
a humidify atmosphere of 5% CO2. Cells were grown until
reach confluent and were subculture every 2 days by
trypsinization.
4. Adipocyte differentiation: Two days post confluence (day
0), cells were stimulated to differentiate with differentiation
medium containing DMEM with 10% FBS, MDI [0.5 mM
IBMX, 0.25 µM dexamethasone, 1 µg/mL insulin] for 2 days.
On day 0 also, 3T3-L1 preadipocytes were tested with the
compounds to evaluate its differentiation-inhibitory effects.
After 8 days, cells were stained with Oil Red O and decreased
lipid accumulation were observed in these cells. For
quantitative analysis, Oil Red O staining was dissolved with
isopropanol and optical density was measured at 520 nm.
5. Glucose uptake assay: >90% fully differentiated adipocytes
were grown in 12-well plates. Glucose uptake was initiated by
the addition of 0.1 mL Krebs-Ringer HEPES (KRPH) buffer
containing 2-deoxy-D-[3H]glucose (0.037 MBq;Perkin elmer)
and glucose (0.001 mM). Later, glucose uptake was terminated
by washing the cells three times with ice-cold PBS. Cells were
lysed using 1% Triton X-100 and the levels of radioactivity
were assessed.
6. Total RNA was extracted from 3T3-L1 adipocytes cultured
using Trizol® reagent. Later, total RNA was reverse transcribed
into cDNA using QuantiTect® Reverse Transcription and gene
expression was evaluated by qRT-PCR.
Collection of plant materials
Plant compounds preparation
Suitable dosage study (MTT)
Cell culture maintenance
Adipocyte
differentiation
Oil red O
staining
Fig. 1: The fruit of Garcinia malaccensis
Absorbance
reading at
520 nm
Fig. 2: Structure of
α-mangostin
Fig. 3: Structure of
β-mangostin
Glucose uptake
activity assay
Level of radioactivity
were determined by
using a Tri-Carb
2700TR liquid
scintillation counter
Trizol reagent
Total RNA
Extraction
and cDNA
Analysis
qRT-PCR
Fig. 4: Flowchart of the study
Oil Red O
image
RESULTS AND DISCUSSION
α-mangostin
Untreated
(DMSO control)
Fig. 6: Lipid accumulation (OD:520nm)
Fig. 8: Pparγ binding activity
Insulin
β-mangostin
Fig. 7: 2-Deoxyglucose uptake analysis
Fig. 9: Glut4 expression
• Since obesity is a side effect of some anti-diabetic drugs, we first evaluated the
effects of the compounds on 3T3-L1 adipogenesis. Our results show that the
compounds lacks pro-adipogenic or adipocyte differentiation effects since its
addition to the adipocyte reduced triglyceride accumulation at any of the tested
concentrations.
• Figure 5 and 6: Effects of the compounds on adipocyte differentiation. The Oil
Red O stained adipocytes were photographed at 100x magnification. All values are
presented as means ± SD of three independent experiments. As shown by the Oil
red O elution, treatment with α-mangostin and β-mangostin reduced intracellular
fat accumulation by up to 50% relative to MDI-treated control cells at dose 50 µM.
• Figure 7: Glucose uptake activity. Adipocytes in 12-well plates were incubated for
60 minutes with α-mangostin and β-mangostin (10, 20 and 50 µM) or with
metformin (1mM) and sodium orthovanadate (5mM) as a positive control, or
DMSO treated as a negative control. Levels of radioactivity in the cell lysates were
determined using a liquid scintillation counter. Data are means ± SD, (n = 3). *p <
0.05 vs. untreated group (DMSO control), a = significant.
• Figure 8: Effect of the compounds on pparγ binding activity. Pparγ expression
was measured after 48 hours of treatment. B-actin was used as the control.
Results are expressed as means ± SD.
• Figure 9: Glut4 expression. Glut4 is the major insulin-dependent transporter
responsible for the uptake of glucose from blood stream into muscle and fat.
CONCLUSION
It was suggested that effects of α and β-mangostins were mediated in inhibition of
adipocyte differentiation and stimulation of glucose uptake. These dual findings might
elucidate as one of the potential candidate for obesity management and may
potentially benefit for preventing metabolic disorders such as diabetes and obesity.
REFERENCES
[1] Yu, Zhao, M., Yang, Zhao, Q. and Jiang (2007). Phenolics from hull of Garcinia mangostana fruit and their
antioxidant activities. Food chemistry. 104(1): 176-181.
[2] Taher, Susanti, Rezali, Zohri, Ichwan, Alkhamaiseh (2012). Apoptosis, antimicrobial and antioxidant activities
of phytochemicals from Garcinia malaccensis Hk.f. Asian Pacific Journal of Tropical Medicine 5(2): 136-141.
FUTURE WORK / PROJECT POTENTIAL
The research will be expanded to evaluate the effects of the
xanthone compounds by using animals model (in vivo study) and
examined the protein expression by using the western blot
techniques. This can further support our claims for its obesity
management. Later, a pattern of drugs will develop and analyzed for
its bioactivity using in vitro and in vivo model.
ACKNOWLEDGEMENT
This work is supported by e-science fund through 02-01-08-SF0110.