Muhammad Taher1*, Mohamed Zaffar Ali Mohamed Amiroudine1, Deny Susanti2,

1
P193 XANTHONES FROM GARCINIA MALACCENCIS IMPROVE GLUT4 AS WELL AS DECREASED PPAR? ACTIVATION ON ADIPOCYTES
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 [phone], Fax
60-95716781, E-mail mtaher_at_iium.edu.my
Since most studies reported that the common link
between diabetes and obesity lies in the
adipocyte, which stores excess energy in the form
of triglyceride, therefore this research was
carried out to describe the xanthone compounds
from Garcinia malaccencis species and evaluate
its activity on adipocyte.
INTRODUCTION
The Garcinia species are well-known for its
phytochemical contents such as flavonoids,
phenolic acids and xanthones. People in Malaysia
often use Garcinia for traditional medicines
including the treatment of abdominal pain,
dysentery, diarrhoea, suppuration, infected
wound, leucorrhoea and chronic ulcer and
gonorrhea1. Recently, it was reported that the
stembark of G. malaccensis showed the presence of
xanthone, a-mangostin as major compound and
together with other xanthone, ß-mangostin2.
This species was reported to have a lot of
medical properties including antimicrobial,
antioxidant and apoptosis but there was no
reported data for its activity on adipocytes
especially on obesity management.
OBJECTIVES

• To measure the effect of G. malaccencis compound
  on triglyceride accumulation and insulin-induced
  glucose uptake.
• 2. To show the effect of G. malaccencis compound
  on the RNA gene expression (mainly PPAR? and
  GLUT4) by qRT-PCR.

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
a-mangostin, ß-mangostin.
5. Glucose uptake assay gt90 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-3Hglucose (0.037
MBqPerkin 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.

• 2. For suitable dosage study (MTT), mature
  adipocytes were seeded in 96-well plates. Cells
  were treated with mangostin compounds for 48
  hours at 37C 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.

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.
3. 3T3-L1 preadipocytes were culture in DMEM
(10 foetal bovine serum, 1 penicillin, 1
streptomycin) at 37C 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.
Fig. 1 The fruit of Garcinia malaccensis
Fig. 4 Flowchart of the study
Fig. 2 Structure of a-mangostin
Fig. 3 Structure of ß-mangostin
RESULTS AND DISCUSSION

• 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.

Oil Red O image
Untreated (DMSO control)
Insulin
a-mangostin
ß-mangostin

• 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
  a-mangostin and ß-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
  a-mangostin and ß-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 lt 0.05 vs. untreated group (DMSO control),
  a significant.

Fig. 6 Lipid accumulation (OD520nm)
Fig. 7 2-Deoxyglucose uptake analysis

• Figure 8 Effect of the compounds on ppar?
  binding activity. Ppar? 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.

Fig. 8 Ppar? binding activity
Fig. 9 Glut4 expression
FUTURE WORK / PROJECT POTENTIAL
CONCLUSION
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.
It was suggested that effects of a and
ß-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.
ACKNOWLEDGEMENT
This work is supported by e-science fund through
02-01-08-SF0110.