Mangosteen Peel Extract (Garcinia mangostana L.) and its Constituents to Lower Lipid Content on Adipogenesis Cells Model (3T3-L1)

Obesity is one of the risk factor for dyslipidemia incident, cancer, diabetes mellitus, and cardiovascular disease. Treatment of obesity using common commercial drugs shows low rate of success, hence natural products may provide better or more efficient therapeutic approach. Mangosteen (Garcinia mangostana L.) (Clusiaceae) peel extract contains xanthones and it is potentially used as alternative medicine for obesity. This research was done to determine the anti-obesity characteristics of Mangosteen Peel Extract (MPE) and its xanthones α-Mangostin (AM) and γ-Mangostin (GM) on 3T3L1 cell line. Anti-obesity effects of MPE and xanthones were investigated using differentiated-3T3L1 preadipocyte cells. Inhibitory activity of the extract and compounds on the production of Triglyceride (TG), Cholesterol (CHOL), Glucose-6-Phosphate Dehydrogenase (G6PDH) activity, and lipid droplets were examined. MPE and its compound were capable to inhibit the production of TG, CHOL, G6PDH, lipid droplets. MPE 50 μg/mL and GM 75 μM were the most active to lower TG 57.95% and 59.72%, CHOL 33.33% and 31.68%, G6PDH 52.90%, 41.95%, lipid droplets 72.99% and 70.07% respectively. In conclusion mangosteen peel extract and γ-Mangostin are the most active antiobesity compared to α-Mangostin.


Introduction
Obesity is a complex disorder that has effects on the normal functions of the body. Obesity is a worldwide threat for public health, as it is involved in various diseases, such as hypertension, coronary heart disease, aosteoarthritis, cancer, type 2 diabetes, and many more 1 . In the first half of 21 st century, obesity became one of a great challenge in public health 2 . Many studies to prevent and treat obesity have been conducted 3 . There are many strategies known for effective obesity therapy, some of them are inhibiton of adipocyte differentiation, stimulation of energy expediture, suppression of food intake, regulation on lipid metabolism, and lipase inhibition 4 .

Mangosteen Peel Extraction
The mangosteen (G. mangostana L.) fruit were obtained from farms in Cisalak-Subang, West Java, Indonesia. The plant was identified by a herbarium staff from Department of Biology in School of Life Science and Technology, Bandung Institute of Technology, Bandung, Indonesia. The peel was collected from mangosteen fruit, then dried and then extracted by maceration with distilled ethanol (70%). The filtrate was collected after 24 hours this procedure was repeated until the filtrate was colorless. The filtrate was then evaporated with a rotatory evaporator at 40°C to yield Mangosteen Peel Extract (MPE). It was stored in -20 o C 11,14,15 until further used.

Cell Cultures and Adipogenesis Induction
The

Triglyceride (TG) Assay
The 3T3-L1 adipocytes were harvested 5 days after the initiation of differentiation. The cells were washed twice with cold PBS, collected, and lysed in lysis buffer (1% Triton X-100 in PBS). The total TG content in the cells was determined with a colorimetric enzymatic test using Glycerol-3-Phospate-Oxidase (GPO) (DiaSys 1 5760 99 10 023). 500 μl mixed reaction contained 450 μl reagent with five microlitre sample (cell lysate) was incubated in 37 ºC for 5 minutes. The absorbance was measured at 500 nm 15,22 .

Cholesterol (CHOL) Assay
The cholesterol level of lysed differentiated cells was measured with enzymatic photometric test (DiaSys 1 1300 99 10 021). Briefly, 5 μl sample (cell lysate after MPE, AM, GM treatment) was introduced into the sample well which contained 450 μl reagent, while 5 μl of ddH 2 O was used as blank sample, incubated at temperature 37 ºC for 10 minutes. The absorbance was measured at 500 nm 15,22 .

Glucose-6-Phosphate Dehydrogenase Activity Assay
The differentiated cells were added in 96 well plates (5 x 10 3 cells/well) in 100 μl medium (DMEM containing 10% FBS and 1% ABAM) for 24 hours at 37 o C and 5% CO 2 , then assayed using G6PDH kit (Abcam, AB102529). Briefly 50 μl reaction mix were added into positive control and sample wells (MPE, AM, GM treatment), while background mix was added into background control wells. Samples were incubated at 37ºC in dark room. The absorbance of samples was read at 450 nm after 30 minutes 22,24 .

Statistical Data Analysis
Statistical analysis of the data was evaluated using Statistical Package for the Social Sciences statistics version 17.0 software. Statistical analysis was evaluated by One-way Analysis of Variance (ANOVA). And then analysis was followed by Duncan post-hoc test and was considered to be significant (p<0.05). Data are presented as mean±Standard Deviation.

Effect of MPE, AM, GM on Lipid Accumulation in 3T3-L1 Adipocytes
Obesity is a disorder of lipid metabolism 25 . To measure the lipid accumulation in obesity model in 3T3-L1, quantified by measuring the Optical Density (OD) at 490 nm 26 . Lipid accumulation is associated with the development and occurrence of obesity. Lipid accumulation in the adipocytes is a result of a hyperplasia and hypertrophy of adipocyte cells. Inhibition and prevention of accumulation of cytoplasmic lipid droplets and adipogenesis in 3T3-L1 cells that were treated at the differentiation and maintenance stages are shown to reduce lipid accumulation 24 ( Figure 1). As shown in the Figure 1, treatment of MPE, AM, and GM reduced the lipid accumulation as indicated by lipid droplet compared to differentiated cells (0.7968). MPE of 50 mg/ml showed highest decrease among treated groups. This was also supported with the results of quantitative lipid measurement in which treatment of MPE 50 mg/ml showed the lowest lipid level (0.2153) among treatments (Table 1). Negative control (without treatment) showed no differentiation as indicated by the lipid droplet formation ( Figure 1) and lowest of absorbance value (Table 1).

Effect of MPE, AM, GM on Cholesterol Level in 3T3-L1 Adipocytes
Consumption of long-chain saturated fatty acids increases cholesterol level 25 . High level of cholesterol is associated with both degree and distribution of obesity 27 . Induced expression of Lipo-Protein Lipase (LPL) and leptin also play roles in cholesterol reduction 28 . Effect of MPE and xanthones treatment on CHOL level of adipocytes (3T3-L1) is presented in Table 2. MPE of 50 mg/ml showed the highest inhibitory activity among treatments (33.33%) with cholesterol level of 177.42 mg/dl. The inhibition activity of MPE was comparable with negative control (29.43%). However, MPE 50 mg/ml and GM 75 μM, have antiobesity potency due to higher CHOL inhibitory activity (Table 2).  Obesity is correlated with high level of TG 29 . TG in adipose tissue acts as a major energy storage form.

Effect MPE, AM, GM on G6PDH Level in 3T3-L1 Adipocytes
G6PDH is responsible in adipogenesis by generating ligand Peroxisome Proliferator-Activated Receptor γ (PPARγ) activating adipocyte-specific gene expression and differentiation, as well as regulating adipose tissue mass which is associated with obesity development 31 . Effect MPE and xanthones treatment on G6PDH level of adipocytes (3T3-L1) can be seen Table 4. In G6PDH level, MPE 50 μg/ml showed the lowest level (0.37 nmol/min/ml), this result was comparable with negative control (0.33 nmol/min/mL). MPE 50 mg/ml also showed the highest inhibitory activity of G6PDH among treatments (52.90%), and this data was comparable with negative control (57.84%). However, MPE of 50 mg/ml has antiobesity potency due to higher G6PDH inhibitory activity (Table 4).

Discussion
Obesity is a disorder which involves lipid metabolism and the enzymes that are involved in this process can be targeted to develop various antiobesity drugs. Xanthones the anti-inflammatory effect by reducing COX-2, IL-6, IL-1β, and NO production in LPS-induces RAW 264.7 cells 14 . Mangosteen peel and its compound have high antioxidant activities 33 . α-Mangostin can suppress intracellular lipid accumulation in differentiating adipocytes and stimulated lipolysis in mature adipocytes; inhibit Fatty Acid Synthase (FAS) 34 . Mangosteen peel extract and its constituents have antiinflammatory, antioxidant, antiadipogenesis and these mechanism can be useful in treating or preventing obesity 34 .
Lipid-lowering activity of MPE, and its constituents (Table 1), is caused by the inhibition of the transcriptional regulation of lipid synthesis and/or stimulation of lipolysis in 3T3-L1 adipocytes 35 . The differentiation of preadipocytes into adipocytes is regulated by a complex network of transcription factors. After differentiation, C/EBPβ was induced immediately, while C/EBPα and PPARγ are master regulators of adipogenesis; their maintenance is critical to the progression of the final stages of adipocyte differentiation 36,37 . Adipocyte differentiation and fat accumulation are associated with the occurrence and development of obesity 38 . Increase in the number of fat cells and adipose tissue mass further cause obesity.
Theoretically, the higher the lipid droplets formation the higher the optical density and therefore, the plants with more formation of lipid droplets results higher absorbance which would be effective in the induction of differentiation of pre-adipocyte to adipocyte. Control (without treatment) showed no differentiation as indicated by the lipid droplet formation and absorbance reading. There was significant difference for undifferentiated cells compared with the control (differentiated cells) 39 .
In this study, MPE and its constituents decreased CHOL level compared to positive control ( Table 2). The consumption of long-chain saturated fatty acids (C>10) has led to increase in TG and CHOL levels 25,40 . Furthermore, high TG level leads to increase in Very Low Density Lipoprotein (VLDL) and chylomicron levels, as transporters of TG. LDL is the last stage of VLDL catabolism, therefore raised VLDL levels also increase LDL levels. LDL is responsible for transporting the cholesterol to peripheral tissues for oxidation or to adipose tissues for storage 41 .
High plasma TG is associated with obesity 29 . In the current study, MPE reduced TG level in 3T3-L1.
Metabolism of TG is activated by the expression of adipocyte-specific fatty acid binding protein (aP2), Fatty Acid Synthase (FAS), and Acetyl-CoA Carboxylase (ACC) genes. The decrease of TG content may be resulted from decreasing lipid synthesis. MPE also decreased level of G6PDH which plays a role in adipogenesis through generating ligand Peroxisome Proliferator-Activated Receptor γ (PPARγ) which contributes to activation of adipocyte-specific gene expression and differentiation and there by controls energy accumulation in the form of adipose tissue mass which is associated with obesity development 31 . These results suggest that MPE has anti-adipogenesis effect. MPE caused the lowest body weight gain percentage as well as the lowest FAS concentration in adipose tissue and serum of experimental rats. Antiobesity potency of MPE might strongly relate to its α-Mangostin content (29.13%) based on HPLC assay 42 . Based on study of Adnyana et al., (2015), MPE has antiobesity activity higher than AM due to α-amylase and pancreatic lipase inhibitory activities 43 .
In this study, MPE showed good activity compared to marker compounds, AM and GM. Many studies have reported beneficial properties of MPE toward the lipid profile. A study carried by Adiputro et al., (2013) revealed that the ethanolic extract of mangosteen pericarp reduced total CHOL, TG, and LDL levels along with increased High-Density Lipoprotein (HDL) levels in rats fed high-lipid diet. There were several compounds of xanthones involved in the stimulation of adipolysis in differentiated 3T3-L1 and primary human adipocytes. Several studies reported that α-Mangostin plays a role in reducing lipid accumulation with decreased peroxisome proliferator activated PPARγ expression along with stimulation of the glucose uptake and free fatty acid release from 3T3-L1 adipocytes via GLUT4 and leptin expression 44 . Mangosteen and its xanthones have good potential to control and modify the metabolic syndrome and its related disorders such as obesity, disrupted lipid profile, diabetes and its complications 45 .

Conclusion
Mangosteen peel extract, and its constituents reduced the levels of lipid, cholesterol, triglyceride, and G6PDH which makes it as a promising antiobesity agent.