Anti ‐ Differentiation Effect of B , D ‐ Seco limonoids of Swietenia mahogani

© 2017 Pharmacognosy Magazine | Published by Wolters Kluwer ‐ Medknow 293 ABSTRACT Background: Obesity is a pathological state caused by abnormal or excessive accumulation of fat. Swietenia mahogani JACQ., known as West Indian mahogany, is a medium-sized semi-evergreen tree belonging to Meliaceae. Their seeds are used in Indonesian folk medicine as a treatment for hypertension, diabetes, malaria, and it also has anti-feedant activities. The major components of S. mahogani are B, D-seco limonoids, a type of irregular triterpenes are well known. Objective: We tried to find the bioactive components, which have the inhibitory activity on adipocyte differentiation from the seeds of S. mahogani. Material and Methods: 3T3-L1 cells, derived from mouse preadipocyte, are widely used in studying adipogenesis process. In this study, we used 3T3-L1 cells to find natural products with the inhibitory activity on adipocyte differentiation. S. mahogani seeds were dried and extracted with 100% MeOH. Results: The methanolic extract was fractionated by bioassay-guided method to give nine B, D-seco limonoids (1-9) with slight structural modifications. Among nine compounds, compounds 4, 6 and 8 exhibited significant inhibitory effects of cell differentiation on 3T3-L1 cells. Those compounds have tigloyl residue at C-3 in common. Besides, compounds with no tigloyl residue at C-3 showed insignificant effect. Nevertheless, not all compounds with tigloyl residue at C-3 exerted significant inhibitory effect. Conclusion: These results suggested that tigloyl residue at C-3 may play a role in the anti-proliferative activity on a dipogenesis and the refined extract of S. mahogani may have a potential to be developed as a therapeutic agent to treat obesity.


INTRODUCTION
Obesity is a major cause of the risk factors for various metabolic and cardiovascular diseases such as fatty liver, diabetes, hypertension, and hyperlipidemia. [1] Adipogenesis is a process where fibroblastic preadipocytes become mature adipocytes. [2] It brings the abnormal accumulation by an increase in the sizes or numbers of adipocytes from precursor cells. [3] Thus, the control of the differentiation of preadipocytesinto adipocytes is the crucial point for the prevention of obesity. However, many medicines available in the market for losing or controlling of weight are not successful in the long-term maintenance of over-weight patients. [4] To find the anti-adipogenetic agents from natural products, 3T3-L1 cells were selected as in vitro screening tool. These cells are preadipocytes derived from mouse and sub-clones of swiss mouse embryo 3T3, and differentiate into adipocyte-like phenotype under the exposure of adipogenic inducers, such as fetal bovine serum, dexamethasone, isobutylmethylxanthine, and insulin. [5] In the present study, we used 3T3-L1 cells to find natural products with the inhibitory activity on adipocyte differentiation, which are expected to lose or control body's over-weight. Swietenia mahogani JACQ., known as West Indian mahogany, is a medium-sized semi-evergreen tree belonging to Meliaceae. It is native to West Indies and cultivated in tropical countries. Its seeds are used as a treatment for hypertension, diabetes and malariain Indonesian folk medicine. [6] Decoction of its bark has been used as febrifuge or antimalarial drug. [7] Furthermore, some studies reported that it acts as a blood platelet aggregation inhibitor. [8] The well known constituents are mainly limonoids, especially B, D-seco limonoids, which are derivatives of limonoids oxidized rings B and D of limonoid. [9] To the best of our knowledge, the study of the extract or compounds of S. mahogani on anti-adipogenesis has not been reported. As a result, we isolated B, D-seco limonoids (1-9) from the seeds of S. mahogani and elucidated their anti-adipogenesis against 3T3-L1 cells.

Plant materials
The Seeds of S. mahogani were collected at Jakarta, Indonesia in August 2012 and air-dried. These seeds were identified by Prof. Sang Hyun Sung, one of the authors in the present study; (SNUPH-0830) has been stored in the Herbarium of the Medicinal Plant Garden, Seoul National University.

Cell culture and adipocyte differentiation
Mouse embryo fibroblasts 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.) and incubated in DMEM supplemented with 10% bovine calf serum until confluence. Two days after confluence (designated day 0), preadipocytes were stimulated to differentiate with differentiation medium (DM, DMEM with 10% FBS, 0.5 mM 3-isobutyl-1-methyl-xanthine, 10 µg/mL insulin and 1 µM dexamethasone) for 2 days (day 2). Cells were then maintained in DM (DMEM 10% FBS and 10 µg/mL insulin) for other 2 days (day 4), followed by culturing with DM (DMEM with 10% FBS) for additional 4 days (day 8). All media contained 100 IU/mL penicillin and 100 mg/ mL streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 95% air -5% CO 2 . Test compounds were dissolved in DMSO (final concentration of 0.1% in media). The cultures were treated with test samples for the whole culture period (day 0-8).

Oil red o staining
Lipid droplets in cells were stained with Oil Red O (ORO) on day 8. Briefly, culture dishes were washed three times with PBS and fixed with 10% formalin for 1 hour at room temperature. After fixation, cells were washed once with PBS and stained with a filtered ORO solution (6 parts of saturated 0.6% ORO in isopropyl alcohol and 4 parts of water) for 15 minutes at room temperature. Cells were washed twice with water for 15 minutes and visualized. To quantify the intracellular lipids, spectrophotometrical quantification of the stain was performed by dissolving the stained lipid droplets with 4% Nonidet P-40 in isopropyl alcohol for 5 minutes. The absorbance was measured at 544 nm. Lipid contents (% of control) =A 540nm (sample-undifferentiated control)/A 540nm (differentiated control-undifferentiated control) × 100.

Statistical analysis
Statistical analysis was performed using Student's t test. Data were expressed as the means ± standard deviation (SD). Statistical significance was represented with an asterisk for p values < 0.05, two asterisks for p values < 0.01 and three asterisks for p values < 0.001.
Anti-differentiation activities of total extract, fractions and the compounds from the seeds of S. mahogani against 3T3-L1 cells Inhibitory effect on adipocyte differentiation in 3T3-L1 cells was investigated with total extract, CHCl 3 and BuOH fraction, and aqueous residue [ Figure 3A]. After inducing of the differentiation of 3T3-L1 cells, total extract and CHCl 3 fraction showed significant inhibitory activities on 3T3-L1 cell differentiation compared to the other fractions, BuOH fraction and aqueous residue. Compounds 1-9 isolated from CHCl 3 fraction were treated on 3T3-L1 cells [ Figure 3B]. Among them, compounds 4, 6 and 8 showed significant anti-differentiation activities in dose-dependent manner at the concentrations of 50 and 100 μM against 3T3-L1 cells.

DISCUSSION
S. mahogani produces active secondary metabolites, limonoids, which have been known to possess a wide range of biological activities. We isolated nine B, D-seco limonoids derivatives with slight modifications, such as hydroxylation, acetylation and tigloylation, from the seeds of S. mahogani and evaluated their anti-differentiation activities against 3T3-L1 cells. Blocking adipogenesis (adipocyte differentiation) has been considered as an effective prevention strategy for obesity. 3T3-L1 cells are being used as a useful tool to investigate biological factors affecting adipogenesis. We performed in vitro study for the evaluation of the inhibitory effect on adipocyte differentiation in 3T3-L1 cells by total extract, sub-fractions, and B, D-seco limonoids (1-9) isolated from CHCl 3 fraction. Compounds 1-9 were highly substituted with hydroxyl, acetyl or tigloyl moieties and showed different inhibitory activities against 3T3-L1 cells. Compounds 4, 6 and 8 showing the significant anti-differentiation activities had a tigloyl moiety at C-3 in common. But it is in appropriate to make an assumption that it is essential for leading to inhibitory effect on adipocyte differentiation because compounds 5, 7 and 9 having a tigloyl moiety showed weak inhibitory activities. It was estimated that a tigloyl residue may not be an essential factor for anti-differentiation of 3T3-L1 cells, but give an effect on anti-differentiation.

CONCLUSION
Limonoids are known to have a wide range of activities including antimalarial, anti-cancer, anti-HIV, and anti-feedant on insects. We reported B, D-seco limonoids have an anti-differentiation against 3T3-L1 cells. It might need the structure and activity relationship study against the 3T3-L1 cells differentiation according to the types of backbone or functional groups in the further study.