Anti-Adipogenic Activity of Secondary Metabolites Isolated from Smilax sieboldii Miq. on 3T3-L1 Adipocytes

Smilax sieboldii, a climbing tree belonging to Smilacaceae, has been used in traditional oriental medicine for treating arthritis, tumors, leprosy, psoriasis, and lumbago. To evaluate the anti-obesity effects of S. sieboldii (Smilacaceae), we screened methylene chloride (CH2Cl2), ethyl acetate (EtOAc), aqueous-saturated n-butanol, and ethanol (EtOH) extracts of the whole plant at various concentrations to inhibit adipogenesis in adipocytes. The 3T3-L1 cell line with Oil red O staining with the help of fluorometry was used as an indicator of anti-obesity activity. Bioactivity-guided fractionation of the EtOH extract and subsequent phytochemical investigation of the active CH2Cl2- and EtOAc-soluble fractions resulted in the isolation of 19 secondary metabolites (1–19), including a new α-hydroxy acid derivative (16) and two new lanostane-type triterpenoids (17 and 18). The structures of these compounds were characterized using various spectroscopic methods. All the isolated compounds were screened for adipogenesis inhibition at a concentration of 100 μM. Of these, compounds 1, 2, 4–9, 15, and 19 significantly reduced fat accumulation in 3T3-L1 adipocytes, especially compounds 4, 7, 9, and 19, showing 37.05 ± 0.95, 8.60 ± 0.41 15.82 ± 1.23, and 17.73 ± 1.28% lipid content, respectively, at a concentration of 100 μM. These findings provide experimental evidence that isolates from S. sieboldii extracts exert beneficial effects regarding the regulation of adipocyte differentiation.


Introduction
Obesity, the most prevalent chronic metabolic condition, is a state associated with excess body fat accumulation caused by a negative energy balance between caloric intake and expenditure, with more than 1.9 billion overweight adults and 650 million clinically obese people worldwide [1]. Obesity is a significant risk factor for a number of comorbid illnesses, including cardiovascular disease, certain types of cancer, cerebrovascular incidents, metabolic syndrome, nonalcoholic fatty liver disease, obstructive sleep apnea, osteoarthritis, psychiatric troubles, respiratory problems, and type 2 diabetes mellitus, amongst many others [2][3][4]. According to the guidelines for the treatment of obesity, the most effective method for weight management is a multidisciplinary approach that may include behavioral therapy, medication, lifestyle adjustments, and/or bariatric surgery [5]. These strategies include changes in lifestyle (such as diet, exercise, and behavioral therapy), drug therapies (such as anti-obesity drugs that target appetite, absorption, or metabolism), and surgical interventions (such as bariatric surgery that modifies the anatomy or function Dried whole S. sieboldii plant was crushed and extracted with 70% EtOH at room temperature to obtain the crude EtOH extract using rotary evaporation. The EtOH extract was sequentially employed in the solvent partition process with three solvents, CH 2 Cl 2 , EtOAc, and aqueous-saturated n-butanol (BuOH), which yielded three main solvent fractions with increasing polarity. To determine the cell viability, an MTT assay was performed by treating 3T3-L1 cells with crude extract (50,100, and 150 µg/mL). As shown in Figure S1 (Supplementary Materials), crude extract showed no significant adverse effect on viability after 24 h, indicating a noncytotoxic effect of crude extract on 3T3-L1 cells. The crude extracts and solvent layers of S. sieboldii whole plant were screened for inhibition of adipocyte differentiation at various concentrations (ranging from 3.125 to 150 µg/mL, Figure 1). The CH 2 Cl 2 − and EtOAc-layers showed a decrease in lipid accumulation in low-concentration ( Figure 1). Chromatographic purification of the CH 2 Cl 2 − and EtOAc-soluble fractions afforded one new α-hydroxy acid derivative (16) and two new lanostane-type triterpenoids (17 and 18), along with 16 known compounds ( Figure 2).

General Experimental Procedure
Detailed information on the general experimental procedures is provided in the Supplementary Materials.

General Experimental Procedure
Detailed information on the general experimental procedures is provided in the Supplementary Materials.

Source of Plant Material
The whole plant of S. sieboldii was collected at Yeoncheon-gun, Gyeonggi-do, Republic of Korea, in August 2021. Botanical identification was performed, and a voucher specimen (G99) was deposited at the Bio-Center, Gyeonggido Business and Science Accelerator (GBSA), Suwon, Republic of Korea.

Spectroscopic Data Analysis
NMR spectra were obtained in acetone-d6, CD3OD, and CDCl3. NMR data were acquired using a Bruker Ascend III 700 spectrometer (resonance frequency was 700.53 and 176.15 MHz for 1 H and 13 C, respectively). The 1 H and 13 C NMR spectra are shown in Fig-Figure 8. The isolation scheme of compounds 1-19.

Oil Red O Staining of Adipocytes Lipid Droplets
On day eight, following differentiation induction, differentiated 3T3-L1 cells were stained with a lipid (Oil Red O) staining kit (ST-R100, Zenbio). Briefly, the cells were washed with PBS, fixed with a fixation solution (Zenbio) for 30 min in the dark, and washed two times with PBS and 70% EtOH. The lipid droplets within the differentiated 3T3-L1 cells were then stained with an Oil Red O solution (Zenbio) for 30 min. The excess stain was removed by washing with 70% EtOH and PBS. The stained lipid droplets were dissolved in isopropanol containing 4% nonidet P-40 (Sigma-Aldrich, St. Louis, MO, USA), and quantitative analysis using an enzyme-linked immunosorbent assay (ELISA) reader (SPECTRAmax 190PC, Molecular Devices, San Jose, CA, USA) at 510 nm was performed.

Statistical Analysis
The presentation of all data is as means standard deviation (SD). All experiments were performed at least three times independently. Differences between the means of each experimental group were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (IBM SPSS statistics 29). Values were considered statistically significant differences, which means sharing the different superscript letters when p < 0.05. ANOVA and Student's t-test were used to determine the significance of the results. Values of * p < 0.05 and ** p < 0.01 were considered statistically significant.

Conclusions
In this study, as part of an ongoing research project to discover bioactive natural products, we identified anti-adipogenic secondary metabolites from the whole plant of the ethanolic extracts of S. sieboldii that inhibit adipocyte differentiation in 3T3-L1 cells. Our biological data for the first time revealed that stilbenoids, phenylpropanoid glycerides, and gallotanins might be responsible for the reported inhibitory activities on adipocyte differentiation in 3T3-L1 cells of the S. sieboldii extracts. Therefore, we conclude that the anti-obesity effects of S. sieboldii extracts are mediated via anti-adipogenesis. The bioactive constituents with anti-adipogenic activity, 2-O-caffeoylglycerol (4), acertannin (7), maplexin D (9), and trans-ρ-ethyl coumarate (19) can be further chemically optimized to obtain the additional active compounds for the inhibition of adipocyte differentiation. In addition, the active compounds can be further studied for their exact underlying mechanisms, and these can also be investigated in obesity models in vivo for establishing therapeutic potential. Taken together, our findings suggest that the regulation of adipocyte differentiation is possible, thereby making S. sieboldii a promising source for the development of therapeutic agents and health-promoting components to treat diseases associated with obesity.