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

Abstract

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.

1. 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 of the gastrointestinal tract). However, these treatments have drawbacks and obstacles, including low efficacy, adverse effects, high cost, limited accessibility, low adherence, and a high recurrence rate. Therefore, more effective and individualized approaches are required to treat obesity and its comorbidities [2,6]. Generally, both hypertrophy (enhancing the size of existing adipocyte cells) and hyperplasia (boosting the quantity by differentiation of new adipocytes) of fat-storing cells are considered therapeutic targets for obesity treatment because they contribute to the abnormal expansion of adipocytes that is a characteristic of obesity [7,8,9]. Adipose tissue masses are controllable by adipogenesis inhibition, a process that turns fibroblastic preadipocytes into mature fat cells [10]. In vitro, 3T3-L1 preadipocytes, which are mouse embryonic fibroblasts generated cell lines, differentiate into mature adipocyte-like cells, and intracellular buildup of lipid droplets is seen during cell differentiation [11]. Thus, this cell line is a suitable screening model for obesity-related adipose tissue biology. Some natural products and plant extracts, such as Abelmoschus manihot [12], Albizia julibrissin [13], Amomum tsao-ko [14], indole derivatives [15], (−)-loliolide [16], kaempferol [17], Salix pseudolasiogyne [18], and shrimp oil [19], are known to prevent the lipid accumulation or the differentiation of adipocytes in 3T3-L1 cells.

Plants of the Smilax (Smilacaceae) genus, commonly called sarsaparilla, consist of approximately 300–350 species. The Smilax species, which are widely dispersed throughout the world’s tropical regions and the temperate zones of North America and East Asia, were commonly used as food and traditional medicine to cure inflammatory illnesses [20,21]. Smilax sieboldii Miq. is a climbing plant with prickly stems that grows in Korea, Japan, China, and Taiwan. Young leaves are harvested from the wild for local use as food. In addition, the subterranean parts have been employed in traditional folk remedies for arthritis, tumors, leprosy, psoriasis, and lumbago [22]. S. sieboldii exhibits various pharmacological activities, including antihyperlipidemic effects and cAMP phosphodiesterase inhibition [23,24]. Phytochemical studies revealed that steroids (spirostane and furostane skeleton) and their corresponding glycosides were present in the rhizome of this plant [22,24] and had several biological activities, such as antifungal, cytotoxic, anti-inflammatory, and anti-bacterial [20,21,25,26]. However, only a few studies have been conducted on the chemical composition of S. sieboldii. In our present research, we conducted investigations to identify potential bioactive phytochemicals in the ethyl acetate (EtOAc) and methylene chloride (CH₂Cl₂) fraction of the S. sieboldii EtOH extract. We included chromatographic purification and structural identification of one undescribed α-hydroxy acid derivative (16) and two new lanostane-type triterpenoids (17 and 18), which were isolated along with additional known constituents (1–15 and 19). The structure of the novel constituents was characterized using various spectral experiments (1D/2D NMR and HR-MS) and specific optical rotation analyses. Furthermore, we reported the isolation and structural elucidation of compounds 1–19 and evaluated the inhibitory effects of these isolated components on adipocyte differentiation using 3T3-L1 cells.

2. Results and Discussion

2.1. Isolation and Purification of Compounds 1–19

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, CH₂Cl₂, 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 CH₂Cl₂⁻ and EtOAc-layers showed a decrease in lipid accumulation in low-concentration (Figure 1). Chromatographic purification of the CH₂Cl₂⁻ 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).

2.2. Chemical Identification of Compounds 1–19

Compound 16 was isolated as a white amorphous powder with optical rotation of ${\left[\alpha \right]}_{\mathrm{D}}^{21}$ −4.8° (c = 0.14, MeOH). The molecular formula of C₇H₁₂O₅ was established from its high-resolution ESI-MS analysis (m/z 199.0575 [M + Na]⁺, calculated for C₇H₁₂O₅Na: 199.0577), indicating two degrees of unsaturation. ¹H NMR and distortionless enhancement by polarization transfer (DEPT) spectra in combination with the heteronuclear single quantum coherence (HSQC) spectrum revealed characteristic signals of the malic acid moiety [δ_H 4.16 (1H, dd, J = 8.4, 4.2 Hz), 2.71 (1H, d, J = 16.1, 4.2 Hz), and 2.60 (1H, d, J = 16.1, 8.4 Hz); δ_C 174.7 (s), 173.5 (s), 78.6 (d), and 39.4 (t)] [27,28]. In addition, the ¹H and ¹³C NMR data of 16 showed the presence of a methoxyl group [δ_H 3.41 (3H, s); δ_C 59.0 (q)], one oxygenated methylene signals [δ_H 4.22 (1H, dd, J = 7.0, 2.8 Hz) and 4.20 (1H, dd, J = 7.0, 3.5 Hz); δ_C 62.4 (t)], and a methyl group [δ_H 1.28 (3H, t, J = 7.0 Hz); δ_C 14.6 (q)] (

Table 1. ¹H and ¹³C NMR spectroscopic data for compound 16 in CD₃OD (δ in ppm) ^a.

Position	16
	δH (J in Hz)	δC (mult.)
1		173.5 s
2	4.16 dd (8.4, 4.2)	78.6 d
3	2.71 dd (16.1, 4.2); 2.60 dd (16.1, 8.4)	39.4 t
4		174.7 s
1′	4.22 dd (7.0, 2.8); 4.20 dd (7.0, 3.5)	62.4 t
2′	1.28 t (7.0)	14.6 q
OCH3	3.41 s	59.0 q

^a Assignments confirmed by ¹H-¹H COSY, HSQC, and HMBC experiments.

, Figures S31–S36). Spin–spin coupling was observed as a cross-peak between the oxygenated methylene signals (δ_H 4.22 and 4.20) and the methyl signal (δ_H 1.28) in the ¹H-¹H correlation spectroscopy (COSY) spectrum, representing the ethoxy group (Figure 3). In the heteronuclear multiple bond correlation (HMBC) spectrum, the correlations between the methoxy group proton at δ_H 3.41 and C-2 (δ_C 78.6) confirmed that the methoxy group was linked to C-2, and the HMBC correlation of the oxygenated methylene protons (δ_H 4.22 and 4.20) and C-1 (δ_C 173.5) indicated that the ethoxy group was connected at C-1. Based on the above results, the structure of 16 was identified as 1-ethyl-2-methoxy malate and was named sieboldic acid.

Compound 17 was isolated as white amorphous powder and displayed a positive Liebermann–Burchard reaction. Its molecular formula was determined to be C₃₂H₅₄O from the molecular ion at m/z 455.4275 [M + H]⁺ based on HR-ESIMS, indicating six indices of hydrogen deficiency. The IR spectrum showed absorption bands at hydroxyl (3382 cm⁻¹) and terminal double bond (3043, 1371, and 890 cm⁻¹) functionalities [29]. The ¹H-NMR spectrum of 17 (

Table 2. ¹H and ¹³C NMR spectroscopic data for compounds 17 and 18 in CDCl₃ (δ in ppm) ^a.

Position	17		18
	δH (J in Hz)	δC (mult.)	δH (J in Hz)	δC (mult.)
1α	1.42 m	36.3 t	2.10 m	36.7 t
1β	1.77 td (13.3, 3.5)		1.81 dt (13.3, 4.9)
2α	1.74 m	28.0 t	2.40 m	34.9 t
2β	1.60 m		2.72 m
3α	3.20 dd (11.2, 4.2)	79.1 d		217.2 s
4		39.3 s		47.7 s
5α	0.86 m	52.7 d	1.37 m	53.4 d
6α	1.68 m	21.6 t	1.64 m	22.6 t
6β	1.45 dd (13.3, 3.5)
7α	1.28 m	28.3 t	1.36 m	27.7 t
7β	1.65 m		1.70 m
8β	2.16 m	42.0 d	2.22 m	41.9 d
9		148.7 s		147.1 s
10		39.6 s		39.1 s
11	5.21 d (5.6)	115.2 d	5.29 d (6.3)	116.3 d
12α	2.06 m	37.4 t	2.09 m	37.2 t
12β	1.88 m		1.93 m
13		44.5 s		44.3 s
14		47.2 s		47.0 s
15α	1.30 m	34.1 t	1.33 m	33.9 t
15β	1.37 m		1.40 m
16α	1.90 m	28.2 t	1.95 m	28.0 t
16β	1.27 m		1.31 m
17α	1.61 m	51.2 d	1.65 m	50.9 d
18	0.64 s	14.6 q	0.68 s	14.4 q
19	1.03 s	22.5 q	1.23 s	21.8 q
20β	1.39 m	36.71 d	1.43 m	36.5 d
21	0.90 d (6.3)	18.73 q	0.93 d (6.3)	18.5 q
22α	1.14 m	36.69 t	1.17 m	36.4 t
22β	1.56 m		1.59 m
23α	2.13 m	28.4 t	2.15 m	28.2 t
23β	1.85 m		1.89 m
24		159.2 s		159.0 s
25		36.5 s		36.3 s
26	1.04 s	29.6 q	1.06 s	29.3 q
27	1.04 s	29.6 q	1.06 s	29.3 q
28	0.97 s	28.5 q	1.08 s	25.6 q
29	0.80 s	15.9 q	1.07 s	22.0 q
30	0.73 s	18.71 q	0.75 s	18.4 q
31	4.82 s, 4.65 s	105.9 t	4.84 s, 4.67 s	105.8 t
32	1.04 s	29.6 q	1.06 s	29.3 q

^a Measured at 700 and 175 MH. The assignments were based on ¹H-¹H COSY, HSQC, HMBC, and ROESY experiments.

) indicated the presence of eight tertiary methyls [δ_H 1.03, 0.97, 0.80, 0.73, 0.64 (3H each, s), and 1.04 (3H × 3, s)], one secondary methyl [δ_H 0.90 (3H, d, J = 6.3 Hz)], one axial methine proton bearing a hydroxy [δ_H 3.20 (1H, dd, J = 11.2, 4.2 Hz)], two germinal olefinic protons [δ_H 4.82, 4.65 (1H each, s)], and a typical H-11 proton of lanosta-9(11)-ene [δ_H 5.21 (1H, d, J = 5.6 Hz)]. The ¹³C-NMR spectrum of 17 revealed 32 carbon signals, which were identified with the aid of a DEPT experiment as four methines (δ_C 52.7, 51.2, 42.0, and 36.71), one oxygenated methine (δ_C 79.1), nine methylenes (δ_C 37.4, 36.69, 36.3, 34.1, 28.4, 28.3, 28.2, 28.0, and 21.6), nine methyls (δ_C 29.6 × 3, 28.5, 22.5, 18.73, 18.71, 15.9, and 14.6), five quaternary carbons (δ_C 47.2, 44.5, 39.6, 39.3, and 36.5), and four olefinic carbons (δ_C 159.2, 148.7, 115.2, and 105.9). Based on these NMR features and a comparison of ¹H and ¹³C-NMR data for 17 and agrostophyllinol, isolated from Agrostophyllum brevipes [30], it was confirmed that these two compounds have a same plane skeleton [lanost-9(11)-ene skeleton, observed in C-3, C-5, C-9, C-10, C-11, C-13, C-14, C-17, C-18, C-21, and C-30 (∆δ_C +0.2, +0.2, +0.2, +0.6, +0.3, +0.2, +0.2, +0.3, +0.3, +0.3, +0.4 (∆δ_C = δ₁₇ − δ_{lanost-9(11)-ene}))]. Moreover, the HMBC correlations of the five individual tertiary methyl signals on rings A-D [between CH₃-18 (δ_H 0.64) and C-12 (δ_C 37.4), C-13 (δ_C 44.5), C-14 (δ_C 47.2), and C-17 (δ_C 51.2); between CH₃-19 (δ_H 1.03) and C-1 (δ_C 36.3), C-5 (δ_C 52.7), C-9 (δ_C 148.7), and C-10 (δ_C 39.6); between CH₃-28 (δ_H 0.97) and C-4 (δ_C 39.3), C-3 (δ_C 79.1), C-5, and C-29 (δ_C 15.9); between CH₃-29 (δ_H 0.80) and C-3, C-4, C-5, and C-28 (δ_C 28.5); between CH₃-30 (δ_H 0.73) and C-8 (δ_C 42.0), C-13, C-14, and C-15 (δ_C 34.1)] and HMBC cross-peaks between H-11 (δ_H 5.21) and C-8, C-9, C-10, C-12, and C-13 firmly established the linkages of these partial structural units (Figure 4). The only distinction between them was the presence of an additional methyl group at C-25 of the side chain in compound 17. This was supported by the singlet signal of three chemically equivalent methyl groups [δ_H 1.04 (3H × 3, s, H-26, H-27, H-32)] observed in the ¹H-NMR spectrum of 17, which revealed the HMBC correlations with C-24 (δ_C 159.2) and ROESY correlations with H-31 (δ_H 4.82), elucidating the structure of the side chain as CH(CH₃)CH₂CH₂C(CH₂)C(CH₃)₃ (Figure 4). The relative configuration for 17 was determined as follows: first, the large coupling constant of H-3 (J_2–3 = 11.2, 4.2 Hz) indicated that the hydroxyl group (3-OH) was oriented equatorially (β-position) at C-3 [29,31]. Next, the ROESY correlations between H-18 (δ_H 0.64) and H-20 (δ_H 1.39); and H-17 (δ_H 1.61) and H-21 (δ_H 0.90) were determined as R configuration for the stereochemistry of C-20. Thus, compound 17 was accordingly determined as 25-methyl-24-methylenelanost-9(11)-en-3β-ol and was given the trivial name smilaxsiebolane A.

Compound 18 was obtained as white amorphous powder and exhibited a deprotonated molecular ion peak at m/z 453.4070 [M + H]⁺ (calcd for C₃₂H₅₃O, m/z 453.4091) on the HRESI-MS analysis, indicating its elemental formula as C₃₂H₅₂O. The molecular formula revealed seven indices of hydrogen deficiency. The following ¹H-NMR and HSQC analysis revealed one olefin proton [δ_H 5.29 (1H, d, J = 6.3 Hz)], eight tertiary methyls [δ_H 1.23, 1.08, 1.07, 0.75, 0.68 (3H each, s), and 1.06 (3H × 3, s)], one secondary methyl [δ_H 0.93 (3H, d, J = 6.3 Hz)], two germinal olefinic protons [δ_H 4.84, 4.67 (1H each, s)], and four methines (δ_H 2.22, 1.65, 1.43, and 1.37) (Table 2). The ¹³C-NMR data, in accordance with HRESI-MS data, showed 32 carbon signals, including one ketone carbon (δ_C 217.2), nine methyls (δ_C 29.3 × 3, 25.6, 22.0, 21.8, 18.5, 18.4, and 14.4), four olefinic carbons (δ_C 159.0, 147.1, 116.3, and 105.8), four methine carbons (δ_C 53.4, 50.9, 41.9, and 36.5), nine methylenes (δ_C 37.2, 36.7, 36.4, 34.9, 33.9, 28.2, 28.0, 27.7, and 22.6), and five quaternary carbons (δ_C 47.7, 47.0, 44.3, 39.1, and 36.3) (Table 2). The ¹H- and ¹³C-NMR spectra of 18 were comparable to those of 17, with the exception of the presence of a ketone functional group at δ_C 217.2 (C-3) and, meanwhile, the disappearance of the H-3 proton signal. As anticipated, the ¹H NMR spectrum of 18 is absent of the signal at δ_H 3.20 (hydroxy methine proton of compound 17) and instead displays two multiplet protons at δ_H 2.72 and 2.40, indicating a ketomethylene group. This assumption was validated by HMBC correlation of H-1 (δ_H 2.10 and 1.81), H-2 (δ_H 2.72 and 2.40), H-28 (δ_H 1.08), and H-29 (δ_H 1.07) with C-3 (δ_C 217.2) (Figure 5). Compound 18 has the same relative configuration as 17, confirmed by ROESY data (Figure 5) and positive specific rotation value ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +26.4° (c = 0.36, CH₂Cl₂). In conclusion, compound 18 was determined as 25-methyl-24-methylenelanost-9(11)-en-3-one and has been named smilaxsiebolane B.

The 16 known compounds were identified as trans-3,3′,5,5′-tetrahydroxy-4-methoxystilbene (1) [32], resveratrol (2) [33], 4-hydroxybenzoic acid (3) [34], 2-O-caffeoylglycerol (4) [35], 1-O-caffeoylglycerol (5) [36], juncusyl ester B (6) [37], acertannin (7) [38], 2-O-(E)-feruloyl glyceride (8) [39], maplexin D (9) [40], trans-caffeic acid (10) [41], syringic acid (11) [42], 1-syringoyl-1,2-dihydroxyethane (12) [43], 3,5-dihydroxy-4-methoxybenzoic acid (13) [44], 1-(3,4,5-trimethoxyphenyl)-1,2,3-propanetriol (14) [45], 1-O-trans-ρ-coumaroylglycerol (15) [37], and trans-ρ-ethyl coumarate (19) [46] by comparing their spectroscopic data with reference values from the previously published literature. To the best of our knowledge, this is the first study to isolate all the compounds from S. sieboldii. Seven compounds (1, 4, 7–9, 12, and 13) were identified in the Smilacaceae family for the first time.

2.3. Anti-Adipogenic Effects of Isolated Compounds on 3T3-L1 Cells

As a first step, to evaluate whether the separated phytochemicals had any influence on cell viability, 3T3-L1 preadipocytes were treated for 24 h with various concentrations (25–100 μM) of the compounds. No cytotoxicity was observed up to 100 μM for all compounds (Table S1, Supplementary Materials); thus, we treated the cells with phytochemicals at this concentration for further study. The anti-adipogenic effects of 19 compounds isolated from the active CH₂Cl₂ and EtOAc layer were screened by assessing fat accumulation in 3T3-L1 cells treated with 100 μM of phytochemicals (Figure 6). Different classes of compounds, such as stilbenoids, phenylpropanoid glycerides, simple phenolics, gallotannins (having a 1,5-anhydro-D-glucitol core), and lanostane-type triterpenoids were tested. Oil Red O staining was used to see how effectively the separated compounds inhibited intracellular lipid accumulation on day eight. Out of the 19 compounds, acertannin (7), maplexin D (9), and trans-ρ-ethyl coumarate (19) were the most active, with lipid accumulation percentages of 8.60 ± 0.41, 15.82 ± 1.24, and 17.73 ± 1.28%, respectively, at 100 μM treatment (** p < 0.01). In addition, 2-O-caffeoylglycerol (4), resveratrol (2), 1-O-caffeoylglycerol (5), juncusyl ester B (6), 2-O-(E)-feruloyl glyceride (8), 1-O-trans-ρ-coumaroylglycerol (15), and trans-3,3′,5,5′-tetrahydroxy-4-methoxystilbene (1) were moderately active with lipid accumulation % of 37.05 ± 0.96, 39.21 ± 3.98, 46.35 ± 3.56, 54.94 ± 7.40, 55.96 ± 10.52, 66.64 ± 4.78, and 67.93 ± 5.80%, respectively, at 100 μM (* p < 0.05, ** p < 0.01).

The structural class had a specific correlation with the inhibition of adipocyte differentiation. In the case of phenylpropanoid derivatives, the activity varied significantly with the replacement for glycerol. The trans-caffeic acid (10) has a free glycerol group and is negligibly active in preventing fat accumulation in adipocytes (lipid content % of 85.77 ± 17.18% at 100 μM). However, the glycerol substitution (2-O-caffeoylglycerol and 1-O-caffeoylglycerol) on the free carboxylic acid group of the trans-caffeic acid exhibited active fat accumulation inhibition from S. sieboldii, with 37.05 ± 0.96 and 46.35 ± 3.56% lipid accumulation at 100 μM, respectively. Among the isolates, gallotannins (acertannin and maplexin D) exhibited the most potent anti-adipogenesis effects, with an activity of 8.60 ± 0.41 and 15.82 ± 1.24% lipid content at 100 μM, respectively. Recently, the anti-adipogenic activity of a gallotannins mixture from Mangifera indica (mango) was demonstrated by the reduced number of lipid droplets and signaling pathway stimulation of the AMP-activated protein kinase (AMPK) [47]. The stilbenoid active compound from S. sieboldii, resveratrol (2), exhibits dose-dependent anti-adipogenic activity [48,49,50]. Moreover, resveratrol has been used as a positive control in the anti-adipogenic effect research literature [51,52]. Resveratrol stimulates deacetylating enzyme sirtuin-1 (SIRT-1) deacetylase, inhibiting adipogenesis, and adipocyte differentiation. Additionally, it increases adenosine monophosphate-activated protein kinase (AMPK) activity, which inhibits adipogenesis [48]. Based on these previous studies, the anti-obesity characteristics of trans-3,3′,5,5′-tetrahydroxy-4-methoxystilbene (1) are likely. The most active compounds, 2-O-caffeoylglycerol (4), acertannin (7), and maplexin D (9), were studied for dose dependency at concentrations of 25, 50, and 100 μM (Figure 7). According to these results, the stilbenoids (1 and 2), phenylpropanoid glycerides (4–6, 8, and 15), and gallotannins (7 and 9) isolated from S. sieboldii possessed potential anti-obesity effects because of their dose-dependent inhibitory activities against adipocyte differentiation and lipid formation in 3T3-L1 cells.

3. Materials and Methods

3.1. General Experimental Procedure

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

3.2. 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.

3.3. Extraction and Separation/Compound Isolation

The shade-dried whole plant of S. sieboldii (7 kg) was percolated with 70% aqueous EtOH at room temperature. Following evaporation of the solvent under reduced pressure, the residue (395 g) was suspended in water, successively partitioned with CH₂Cl₂, EtOAc, and water-saturated n-BuOH to afford 86 g, 25.5 g, and 39 g, respectively. Part of the EtOAc-soluble fraction was chromatographed over a Diaion HP-20 resin and eluted with water−MeOH stepwise gradient solvent system (1:0 to 0:1) to yield five fractions (G99A₁ to G99A₅). Of these, fraction G99A₂ (2.5 g) was subjected to MPLC over ODS eluting with a gradient of 10–30% MeOH in H₂O to give 11 subfractions (G99B₁ to B₁₁). Fraction G99B₁ (72.7 mg) was separated by preparative high-pressure liquid chromatography (HPLC) (Kromasil 100-5-C18 column; 250 × 21.2 mm; flow rate 10 mL/min; solvent A–0.05% trifluoroacetic acid in the water, solvent B–MeCN; gradient elution, 0 min 10% B to 50 min 15% B, detection at 254 and 350 nm). HPLC separation led to the purification of compounds 12 (2.3 mg, t_R = 18.3 min), 13 (4.5 mg, t_R = 19.8 min), and 14 (15.8 mg, t_R = 21.4 min). Further purification of subfraction G99B₂ (86.9 mg) using the above HPLC system (gradient elution, 0 min 15% B to 50 min 27% B) resulted in the isolation of compound 3 (15.3 mg, t_R = 16.9 min). Fractions G99B₄ (125 mg), G99B₈ (95 mg), and G99B₁₀ (86.7 mg) were separated by preparative HPLC (same conditions as for fraction G99B₂) to obtain pure compounds 4 (25.8 mg, t_R = 14.3 min), 5 (31.3 mg, t_R = 16.9 min), 16 (3.5 mg, t_R = 22.0 min), 6 (35.9 mg, t_R = 21.4 min), 15 (4.5 mg, t_R = 25.5 min), 7 (20.1 mg, t_R = 16.5 min), and 8 (13.4 mg, t_R = 22.2 min). Subfraction G99B₆ (69.7 mg) was isolated using preparative HPLC (gradient elution, 0 min 15% B, 50 min 23% B) to obtain three compounds 9 (1.5 mg, t_R = 12.8 min), 10 (3.1 mg, t_R = 18.8 min), and 11 (8.2 mg, t_R = 19.5 min). G99A₃ (3.5 mg) was separated using RP-MPLC with MeCN−H₂O (elution 0 min 10:90 to 110 min 40:60) to obtain eight subfractions (G99C₁ to C₈). Subfraction G99C₁ (125 mg) was purified using preparative HPLC (gradient elution, 0 min 17% B, 30 min 30% B) to purify compound 1 (24.8 mg, t_R = 32.5 min). Subfraction G99C₇ (150 mg) and subjected to preparative HPLC (detection 280 and 325 nm, gradient elution, 0 min 25% B, 50 min 33% B) to obtain compound 2 (36.7 mg, t_R = 27.7 min). The CH₂Cl₂-soluble fraction was subjected to silica gel column chromatography (CC) and eluted using a CH₂Cl₂-MeOH gradient system (1:0 to 1:1) to yield 12 fractions (G99D₁ to G99D₁₂). Compound 17 (125.1 mg) was isolated from fraction G99D₃ (1.2 g) by recrystallization in CH₂Cl₂-MeOH. G99D₅ (931 mg) was further chromatographed by silica gel CC with an n-hexane-CH₂Cl₂ (10:1 to 0:1) mixture to obtain nine fractions (G99E₁ to G99E₉). The fraction G99E₈ (68.9 mg) was isolated by Sephadex LH-20 CC (CH₂Cl₂-MeOH, 1:1, isocratic elution) to obtain compounds 18 (15.4 mg) and 19 (7.5 mg). The isolation part of the present research is summarized in Figure 8.

3.4. 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 ¹H and ¹³C, respectively). The ¹H and ¹³C NMR spectra are shown in Figures S2–S61 (Supplementary Materials).

• trans-3,3′,5,5′-Tetrahydroxy-4-methoxystilbene (1): pale brown amorphous powder; UV (MeOH) λ_max (log ε) 224 (3.55), 314 (3.75) nm; ¹H-NMR (700 MHz, acetone-d₆) δ 6.91 (1H, d, J = 16.1 Hz, H-8), 6.87 (1H, d, J = 16.1 Hz, H-7), 6.64 (2H, s, H-2′, 6′), 6.55 (2H, d, J = 2.1 Hz, H-2, 6), 6.30 (1H, d, J = 2.1 Hz, H-4), 3.82 (3H, s, 4′-OCH₃); ¹³C-NMR (175 MHz, acetone-d₆) δ 159.6 (C-3, 5), 151.5 (C-3′, 5′), 140.6 (C-1), 136.3 (C-4′), 134.3 (C-1′), 129.3 (C-8), 128.8 (C-7), 106.8 (C-2′, 6′), 105.9 (C-2, 6), 103.0 (C-4), 60.8 (4′-OCH₃); ESI-MS (positive ion mode) m/z 275 [M + H]⁺.
• Resveratrol (2): pale brown amorphous powder; UV (MeOH) λ_max (log ε) 216 (3.56), 305 (3.93) nm; ¹H-NMR (700 MHz, CD₃OD) δ 6.95 (1H, d, J = 16.1 Hz, H-8), 6.80 (1H, d, J = 16.1 Hz, H-7), 7.35 (2H, d, J = 8.4 Hz, H-2′, 6′), 6.76 (2H, d, J = 8.4 Hz, H-3′, 5′), 6.44 (2H, d, J = 2.1 Hz, H-2, 6), 6.16 (1H, t, J = 2.1 Hz, H-4); ¹³C-NMR (175 MHz, CD₃OD) δ 159.8 (C-3, 5), 158.5 (C-4′), 141.5 (C-1), 130.6 (C-1′), 129.5 (C-8), 128.9 (C-2′, 6′), 127.2 (C-7), 116.2 (C-3′, 5′), 105.9 (C-2, 6), 102.8 (C-4); ESI-MS (positive ion mode) m/z 229 [M + H]⁺.
• 4-Hydroxybenzoic acid (3): white amorphous powder; UV (MeOH) λ_max (log ε) 199 (3.95), 210 (sh), 255 (3.70) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.88 (2H, d, J = 8.4 Hz, H-2, 6), 6.82 (2H, d, J = 8.4 Hz, H-3, 5); ¹³C-NMR (175 MHz, CD₃OD) δ 170.2 (C-7), 163.5 (C-4), 133.1 (C-2, 6), 122.8 (C-1), 116.2 (C-3, 5); ESI-MS (positive ion mode) m/z 139 [M + H]⁺.
• 2-O-Caffeoylglycerol (4): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (3.65), 240 (3.25), 295 (sh), 325 (3.95) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.60 (1H, d, J = 16.1 Hz, H-7), 7.05 (1H, d, J = 2.1 Hz, H-2), 6.95 (1H, dd, J = 8.4, 2.1 Hz, H-6), 6.78 (1H, d, J = 8.4 Hz, H-5), 6.31 (1H, d, J = 16.1 Hz, H-8), 4.98 (1H, p, J = 5.6 Hz, H-2′), 3.76 (2H, dd, J = 11.9, 4.9 Hz, H_a-1′, 3′), 3.73 (2H, dd, J = 11.9, 5.6 Hz, H_b-1′, 3′); ¹³C-NMR (175 MHz, CD₃OD) δ 169.1 (C-9), 149.7 (C-4), 147.2 (C-7), 147.0 (C-3), 127.9 (C-1), 123.1 (C-6), 116.6 (C-5), 115.5 (C-8), 115.3 (C-2), 76.7 (C-2′), 61.9 (C-1′, 3′); ESI-MS (positive ion mode) m/z 255 [M + H]⁺.
• 1-O-Caffeoylglycerol (5): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (3.68), 241 (3.29), 295 (sh), 325 (3.97) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.59 (1H, d, J = 15.4 Hz, H-7), 7.05 (1H, d, J = 2.1 Hz, H-2), 6.95 (1H, dd, J = 8.4, 2.1 Hz, H-6), 6.78 (1H, d, J = 2.1 Hz, H-5), 6.29 (1H, d, J = 15.4 Hz, H-8), 4.25 (1H, dd, J = 11.9, 4.2 Hz, H_a-1′), 4.16 (1H, dd, J = 11.9, 4.2 Hz, H_b-1′), 3.89 (1H, p, J = 6.3, 4.2 Hz, H-2′), 3.61 (1H, dd, J = 11.2, 4.9 Hz, H_a-3′), 3.59 (1H, dd, J = 11.2, 5.6 Hz, H_b-3′); ¹³C-NMR (175 MHz, CD₃OD) δ 169.4 (C-9), 149.7 (C-4), 147.3 (C-7), 146.9 (C-3), 127.9 (C-1), 123.1 (C-6), 116.6 (C-5), 115.3 (C-2), 115.0 (C-8), 71.4 (C-2′), 66.7 (C-1′), 64.2 (C-3′); ESI-MS (positive ion mode) m/z 255 [M + H]⁺.
• Juncusyl ester B (6): white amorphous powder; UV (MeOH) λ_max (log ε) 210 (3.45), 226 (3.55), 297 (sh), 309 (4.05) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.67 (1H, d, J = 16.1 Hz, H-7), 7.46 (2H, d, J = 8.4 Hz, H-2, 6), 6.81 (2H, d, J = 8.4 Hz, H-3, 5), 6.37 (1H, d, J = 16.1 Hz, H-8), 4.99 (1H, p, J = 4.2 Hz, H-2′), 3.76 (1H, dd, J = 11.9, 4.9 Hz, H_a-1′, 3′), 3.73 (1H, dd, J = 11.9, 5.6 Hz, H_b-1′, 3′); ¹³C-NMR (175 MHz, CD₃OD) δ 169.1 (C-9), 161.4 (C-4), 146.8 (C-7), 131.3 (C-2, 6), 127.4 (C-1), 117.0 (C-3, 5), 115.5 (C-8), 76.8 (C-2′), 61.9 (C-1′, 3′); ESI-MS (positive ion mode) m/z 239 [M + H]⁺.
• Acertannin (7): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (4.15), 275 (3.53) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.09 (2H, s, H-2″, 6″), 7.08 (2H, s, H-2′, 6′), 4.91 (1H, ddd, J = 10.5, 9.1, 5.6 Hz, H-2), 4.55 (1H, dd, J = 12.6, 2.1 Hz, H_a-6), 4.38 (1H, dd, J = 11.2, 4.9 Hz, H_b-6), 4.10 (1H, dd, J = 11.2, 5.6 Hz, H_a-1), 3.71 (1H, t, J = 9.1 Hz, H-3), 3.53 (1H, m, H-5), 3.52 (1H, m, H-4), 3.35 (1H, t-like, J = 10.5 Hz, H_b-1); ¹³C-NMR (175 MHz, CD₃OD) δ 168.5 (C-7″), 167.9 (C-7′), 146.7 (C-3″, 5″), 146.6 (C-3′, 5′), 140.1 (C-4′), 140.0 (C-4″), 121.5 (C-1″), 121.3 (C-1′), 110.4 (C-2′, 6′), 110.3 (C-2″, 6″), 80.3 (C-5), 77.1 (C-3), 73.3 (C-2), 72.1 (C-4), 68.1 (C-1), 65.0 (C-6); ESI-MS (positive ion mode) m/z 469 [M + H]⁺.
• 2-O-(E)-Feruloyl glyceride (8): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (3.60), 237 (2.50), 296 (sh), 325 (3.95) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.66 (1H, d, J = 16.1 Hz, H-7), 7.19 (1H, d, J = 2.1 Hz, H-2), 7.08 (1H, dd, J = 8.4, 2.1 Hz, H-6), 6.81 (1H, d, J = 8.4 Hz, H-5), 6.40 (1H, d, J = 16.1 Hz, H-8), 4.99 (1H, p, J = 4.9 Hz, H-2′), 3.89 (3H, s, 3-OCH₃), 3.76 (1H, dd, J = 11.9, 4.2 Hz, H_a-1′, 3′), 3.73 (1H, dd, J = 11.9, 5.6 Hz, H_b-1′, 3′); ¹³C-NMR (175 MHz, CD₃OD) δ 169.1 (C-9), 150.7 (C-4), 149.5 (C-3), 147.1 (C-7), 127.9 (C-1), 124.2 (C-6), 116.6 (C-5), 115.5 (C-8), 111.9 (C-2), 76.8 (C-2′), 61.9 (C-1′, 3′), 56.6 (3-OCH₃); ESI-MS (positive ion mode) m/z 269 [M + H]⁺.
• Maplexin D (9): white amorphous powder; UV (MeOH) λ_max (log ε) 216 (4.10), 276 (3.47) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.10 (2H, s, H-2″, 6″), 7.08 (2H, s, H-2′, 6′), 4.99 (1H, ddd, J = 10.5, 9.1, 5.6 Hz, H-2), 5.01 (1H, t, J = 9.1 Hz, H-4), 4.18 (1H, dd, J = 11.2, 5.6 Hz, H_a-1), 3.98 (1H, t, J = 9.1 Hz, H-3), 3.62 (1H, brd, J = 9.8 Hz, H_a-6), 3.52 (1H, m, H_b-6), 3.54 (1H, m, H-5), 3.40 (1H, t, J = 11.2 Hz, H_b-1); ¹³C-NMR (175 MHz, CD₃OD) δ 167.78 (C-7″), 167.82 (C-7′), 146.69 (C-3′, 5′), 146.65 (C-3″, 5″), 140.22 (C-4″), 140.18 (C-4′), 121.23 (C-1″), 121.2 (C-1′), 110.5 (C-2″, 6″), 110.4 (C-2′, 6′), 81.2 (C-5), 75.1 (C-3), 73.5 (C-2), 73.0 (C-4), 68.1 (C-1), 62.8 (C-6); ESI-MS (positive ion mode) m/z 469 [M + H]⁺.
• trans-Caffeic acid (10): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (3.62), 239 (3.20), 297 (sh), 324 (3.92) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.53 (1H, d, J = 16.1 Hz, H-7), 7.03 (1H, d, J = 2.1 Hz, H-2), 6.93 (1H, dd, J = 8.4, 2.1 Hz, H-6), 6.77 (1H, d, J = 8.4 Hz, H-5), 6.21 (1H, d, J = 16.1 Hz, H-8); ¹³C-NMR (175 MHz, CD₃OD) δ 171.2 (C-9), 149.6 (C-4), 147.2 (C-7), 147.0 (C-3), 127.9 (C-1), 123.0 (C-6), 116.6 (C-5), 115.2 (C-2), 115.7 (C-8); ESI-MS (positive ion mode) m/z 181 [M + H]⁺.
• Syringic acid (11): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (4.15), 275 (3.42) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.33 (2H, s, H-2, 6), 3.88 (6H, s, 3, 5-OCH₃); ¹³C-NMR (175 MHz, CD₃OD) δ 170.1 (C-7), 149.0 (C-3, 5), 141.9 (C-4), 122.0 (C-1), 108.4 (C-2, 6), 56.9 (3, 5-OCH₃); ESI-MS (positive ion mode) m/z 199 [M + H]⁺.
• 1-Syringoyl-1,2-dihydroxyethane (12): white amorphous powder; UV (MeOH) λ_max (log ε) 205 (3.87), 230 (sh), 304 (3.35) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.34 (2H, s, H-2, 6), 5.14 (1H, dd, J = 5.6, 4.2 Hz, H-8), 3.91 (6H, s, 3, 5-OCH₃), 3.89 (1H, dd, J = 11.2, 4.2 Hz, H_a-9), 3.75 (1H, dd, J = 11.2, 4.2 Hz, H_b-9); ¹³C-NMR (175 MHz, CD₃OD) δ 199.9 (C-7), 149.3 (C-3, 5), 143.2 (C-4), 126.9 (C-1), 107.9 (C-2, 6), 75.7 (C-8), 66.4 (C-9), 57.1 (3, 5-OCH₃); ESI-MS (positive ion mode) m/z 243 [M + H]⁺.
• 3,5-Dihydroxy-4-methoxybenzoic acid (13): white amorphous powder; UV (MeOH) λ_max (log ε) 211 (3.97), 258 (3.39), 294 (3.15) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.03 (2H, s, H-2, 6), 3.89 (3H, s, 4-OCH₃); ¹³C-NMR (175 MHz, CD₃OD) δ 170.0 (C-7), 151.8 (C-3, 5), 141.2 (C-4), 127.3 (C-1), 110.5 (C-2, 6), 60.9 (4-OCH₃); ESI-MS (positive ion mode) m/z 185 [M + H]⁺.
• 1-(3,4,5-Trimethoxyphenyl)-1,2,3-propanetriol (14): white amorphous powder; UV (MeOH) λ_max (log ε) 204 (3.85), 227 (sh), 269 (2.55) nm; ¹H-NMR (700 MHz, CD₃OD) δ 6.72 (2H, s, H-2, 6), 4.59 (1H, d, J = 5.6 Hz, H-7), 3.84 (6H, s, 3, 5-OCH₃), 3.74 (3H, s, 4-OCH₃), 3.67 (1H, m, H-8), 3.54 (1H, dd, J = 11.2, 4.2 Hz, H_a-9), 3.40 (1H, dd, J = 11.2, 5.6 Hz, H_b-9); ¹³C-NMR (175 MHz, CD₃OD) δ 154.5 (C-3, 5), 139.9 (C-1), 138.4 (C-4), 105.2 (C-2, 6), 77.5 (C-8), 75.4 (C-7), 64.4 (C-9), 61.2 (4-OCH3), 56.7 (3, 5-OCH₃); ESI-MS (positive ion mode) m/z 259 [M + H]⁺.
• 1-O-trans-ρ-Coumaroylglycerol (15): white amorphous powder; UV (MeOH) λ_max (log ε) 210 (3.35), 226 (3.45), 296 (sh), 310 (3.90) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.65 (1H, d, J = 16.1 Hz, H-7), 7.46 (1H, d, J = 9.1 Hz, H-2, 6), 6.80 (1H, d, J = 9.1 Hz, H-3, 5), 6.36 (1H, d, J = 16.1 Hz, H-8), 4.26 (1H, dd, J = 11.2, 4.2 Hz, H_a-1′), 4.16 (1H, dd, J = 11.2, 6.3 Hz, H_b-1′), 3.89 (1H, m, H-2′), 3.61 (1H, dd, J = 11.2, 5.6 Hz, H_a-3′), 3.59 (1H, dd, J = 11.2, 5.6 Hz, H_b-3′); ¹³C-NMR (175 MHz, CD₃OD) δ 169.3 (C-9), 161.5 (C-4), 146.9 (C-7), 131.3 (C-2, 6), 127.3 (C-1), 117.0 (C-3, 5), 115.1 (C-8), 71.4 (C-2′), 66.7 (C-1′), 64.2 (C-3′); ESI-MS (positive ion mode) m/z 239 [M + H]⁺.
• Sieboldic acid (16): white amorphous powder; ${\left[\alpha \right]}_{\mathrm{D}}^{21}$ −4.8° (c = 0.14, MeOH); ¹H-NMR (700 MHz, CD₃OD) and ¹³C-NMR (175 MHz, CD₃OD) (Table 1); HRESI-MS (positive ion mode) m/z 199.0575 [M + Na]⁺ (calcd. for C₇H₁₂O₅Na, 199.0577).
• Smilaxsiebolane A (17): white amorphous powder; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +46.4° (c = 0.11, CH₂Cl₂); IR (KBr) ν_max 3382, 3043, 2920, 2856, 1706, 1634, 1462, 1371, 1044, 979, 890 cm⁻¹; ¹H-NMR (700 MHz, CDC1₃) and ¹³C-NMR (175 MHz, CDC1₃) (Table 2); HRESI-MS (positive ion mode) m/z 455.4275 [M + H]⁺ (calcd. for C₃₂H₅₅O, 455.4247).
• Smilaxsiebolane B (18): white amorphous powder; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +26.4° (c = 0.36, CH₂Cl₂); IR (KBr) ν_max 2959, 2870, 1706, 1635, 1461, 1368, 1114, 986, 890 cm⁻¹; ¹H-NMR (700 MHz, CDC1₃) and ¹³C-NMR (175 MHz, CDC1₃) (Table 2); HRESI-MS (positive ion mode) m/z 453.4071 [M + H]⁺ (calcd. for C₃₂H₅₃O, 453.4091).
• trans-ρ-Ethyl coumarate (19): white amorphous powder; UV (MeOH) λ_max (log ε) 217 (3.44), 236 (3.53), 294 (sh), 325 (4.13) nm; ¹H-NMR (700 MHz, CD₃OD) δ 7.60 (1H, d, J = 16.1 Hz, H-7), 7.45 (1H, d, J = 8.4 Hz, H-2, 6), 6.80 (1H, d, J = 8.4 Hz, H-3, 5), 6.31 (1H, d, J = 16.1 Hz, H-8), 4.21 (2H, q, J = 7.0 Hz, H-1′), 1.31 (1H, t, J = 7.0 Hz, H-2′); ¹³C-NMR (175 MHz, CD₃OD) δ 169.5 (C-9), 161.4 (C-4), 146.5 (C-7), 131.3 (C-2, 6), 127.3 (C-1), 117.0 (C-3, 5), 115.5 (C-8), 61.6 (C-1′), 14.8 (C-2′); ESI-MS (positive ion mode) m/z 193 [M + H]⁺.

3.5. Cell Culture and Adipocyte Differentiation

The mouse 3T3-L1 preadipocytes were procured from Zenbio (SP-L1-F, Zenbio, Durham, NC, USA), and cells were cultured in 3T3-L1 Preadipocyte Medium (PM-1-L1, Zenbio) in a humidified incubator with 95% air and 5% CO₂ at 37 °C. Two days after cells reached confluence, induction with initiation media [3T3-L1 Differentiation Medium (3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI)); DM-2-L1, Zenbio] was performed. The initiation medium was changed on day two with the 3T3-L1 Adipocyte Medium (AM-1-L1, Zenbio). On days four and six, the progression medium was replaced with the 3T3-L1 Adipocyte Medium (AM-1-L1, Zenbio). From days zero to eight, the cells were treated with nontoxic concentrations of the extracts and isolated constituents, which were identified via a cell cytotoxicity assay.

3.6. 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.

3.7. Cell Viability Assay

The cell viability was analyzed by MTT cytotoxicity assays [11]. The 3T3-L1 preadipocytes were seeded at a density of 5 × 10³ cells/well in 100 μL of culture medium. A time-zero control plate was prepared one day after plating. The isolated compounds (1–19) were applied directly, and the cells were incubated for 24 h in a humidified atmosphere with 5% CO₂ at 37 °C. The proliferation of the cells was then determined. MTT [5 mg/mL in phosphate-buffered saline (PBS)] solution was added to the wells, followed by incubation for 3 h. The medium was removed from the wells by aspiration, and buffered dimethyl sulfoxide (DMSO, 0.1 mL) was added to each well, after which the plates were shaken. Subsequently, absorbance was measured using a SpectraMax 190PC microtiter plate reader at 540 nm.

3.8. 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.

4. 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.