Screening of Korean Natural Products for Anti-Adipogenesis Properties and Isolation of Kaempferol-3-O-rutinoside as a Potent Anti-Adipogenetic Compound from Solidago virgaurea

In this study, the anti-adipogenetic activity of 300 plant extracts was investigated using an Oil Red O staining assay in a 3T3-L1 cell line. Our results indicate that three plants, including the stem and leaf of Physalis angulata, the whole grass of Solidago virgaurea, and the root of Dioscorea nipponica, produced over 90% inhibition of adipogenesis. Kaempferol-3-O-rutinoside, which demonstrated a 48.2% inhibitory effect on adipogenesis without cytotoxicity, was isolated from the butanol layer of a water extract of S. virgaurea guided by the anti-adipogenesis assay in 3T3-L1. PPAR-γ and C/EBPα expression levels were determined using western blot, and our results indicate that kaempferol-3-O-rutinoside has a strong anti-adipogenic effect in 3T3-L1 cells through the suppression of increases in PPAR-γ and C/EBPα expression.


Introduction
Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure. It is defined as abnormal or excessive fat accumulation that poses a health risk [1]. Many scientific communities have become increasingly interested in the molecular regulation of triglyceride synthesis and in phytochemical-based pharmaceutical approaches for reducing fat absorption and storage. Phytochemicals present an exciting opportunity for the discovery of new anti-obesity agents [2]. The regulation of fatty acid and triglyceride availability depends on the activity of the lipolytic enzymes of fatty acid metabolism in adipose tissue [3].
The characterization and identification of several genes involved in lipid metabolism have yielded a rich pool of potential targets for drugs to treat obesity and other metabolic syndromes [4]. One of the screening strategies used is to search for adipogenic inhibitors from plant extracts. Plants have traditionally been used as natural medicines for many diseases [5]. In particular, many oriental medicinal plants have been reported to have biological activity. Among the plant extracts screened, the extract of Solidago virgaurea var. gigantea (SV) was observed to significantly inhibit adipogenesis in 3T3-L1 adipocyte cells.
Molecules 2016, 21, 226; doi:10.3390/molecules21020226 www.mdpi.com/journal/molecules SV, a subspecies of Solidago virgaurea Nakai, is a perennial herb that grows on Ulreung Island in Korea. The whole plant (root and leaf) has been used as a stomachic and diuretic in Korean folk medicine, while the young aerial parts have been used as food [6]. A variety of plants belonging to the Solidago species has been reported to possess antibacterial, anti-oxidant [7], anti-inflammatory [8], and bone metabolic [9] activities. However, to our knowledge, no study has yet reported any anti-obesity effects of SV. In this study, we screened crude extracts from natural sources for potential anti-obesity effects on lipid accumulation in cultured 3T3-L1 adipocytes. Oil Red O staining and triglyceride contents served as indicators of lipid accumulation.

List of Natural Extracts That Showed 30% or More Inhibition of Adipogenesis of 3T3-L1 Cells
Three-hundred crude extracts were prepared from natural plant species found in Korea or Asia, and their anti-adipogenic activity by inhibition of adipogenesis was investigated at a concentration of 10 µg/mL. The results are reported in Table 1. Among the 300 plant extracts examined, 31 crude extracts from natural plant species showed a relatively high anti-adipogenic activity (more than 30%). A significant inhibition of adipogenesis of up to 92.5% was observed with SV. SV, a subspecies of Solidago virgaurea Nakai, is a perennial herb that grows on Ulreung Island in Korea. The whole plant (root and leaf) has been used as a stomachic and diuretic in Korean folk medicine, while the young aerial parts have been used as food [6]. A variety of plants belonging to the Solidago species has been reported to possess antibacterial, anti-oxidant [7], anti-inflammatory [8], and bone metabolic [9] activities. However, to our knowledge, no study has yet reported any anti-obesity effects of SV. In this study, we screened crude extracts from natural sources for potential anti-obesity effects on lipid accumulation in cultured 3T3-L1 adipocytes. Oil Red O staining and triglyceride contents served as indicators of lipid accumulation.

List of Natural Extracts That Showed 30% or More Inhibition of Adipogenesis of 3T3-L1 Cells
Three-hundred crude extracts were prepared from natural plant species found in Korea or Asia, and their anti-adipogenic activity by inhibition of adipogenesis was investigated at a concentration of 10 μg/mL. The results are reported in Table 1. Among the 300 plant extracts examined, 31 crude extracts from natural plant species showed a relatively high anti-adipogenic activity (more than 30%). A significant inhibition of adipogenesis of up to 92.5% was observed with SV.

Effect of the S. virgaurea var. gigantea Extracts on Inhibition of 3T3-L1 Adipocyte Differentiation
Each extract of SV and its anti-adipogenic activity was studied at a concentration of 10 μg/mL. The results represent lipid droplet accumulation, as triglycerides in adipocytes stain with Oil Red O staining solution separate from free fatty acids and phospholipids. As shown in Figure 1, in response to the administration of SVW (water extract of SV) at 10 μg/mL, the lipid content in 3T3-L1 adipocytes decreased significantly, showing a 79.39% inhibition.

Effect of the S. virgaurea var. gigantea Extracts on Inhibition of 3T3-L1 Adipocyte Differentiation
Each extract of SV and its anti-adipogenic activity was studied at a concentration of 10 µg/mL. The results represent lipid droplet accumulation, as triglycerides in adipocytes stain with Oil Red O staining solution separate from free fatty acids and phospholipids. As shown in Figure 1, in response to the administration of SVW (water extract of SV) at 10 µg/mL, the lipid content in 3T3-L1 adipocytes decreased significantly, showing a 79.39% inhibition.

Effect of Solvent Fractions of SVW on Inhibition of 3T3-L1 Adipocyte Differentiation
Solvent fractions of SVW were studied at a concentration of 10 μg/mL for inhibition of adipogenesis. As shown in Figure 2, lipid content in 3T3-L1 adipocytes decreased significantly in response to the SVW-Bf (n-butanol fraction of SVW) at 10 μg/mL, which showed a 78.25% inhibitory effect.

Effect of Solvent Fractions of SVW on Inhibition of 3T3-L1 Adipocyte Differentiation
Solvent fractions of SVW were studied at a concentration of 10 µg/mL for inhibition of adipogenesis. As shown in Figure 2, lipid content in 3T3-L1 adipocytes decreased significantly in response to the SVW-Bf (n-butanol fraction of SVW) at 10 µg/mL, which showed a 78.25% inhibitory effect.

Effect of Solvent Fractions of SVW on Inhibition of 3T3-L1 Adipocyte Differentiation
Solvent fractions of SVW were studied at a concentration of 10 μg/mL for inhibition of adipogenesis. As shown in Figure 2, lipid content in 3T3-L1 adipocytes decreased significantly in response to the SVW-Bf (n-butanol fraction of SVW) at 10 μg/mL, which showed a 78.25% inhibitory effect.  Confluent 3T3-L1 preadipocyte were differentiated into adipocytes in medium with or without 10 µg/mL of SVW fractions for 8 day. Con is control group; DM is differentiation media cells; SVW-Ef is ethyl acetate fraction from S. virgaurea var. gigantea water extract; SVW-Bf is n-butanol fraction from S. virgaurea var. gigante water extract; SVW-Wf is water fraction from S. virgaurea var. gigantea water extract. Three independent experiments have been carried out; * p < 0.05 vs. DM; *** p < 0.005 vs. DM.

Effect of SVW and SVW-Bf on Preadipocyte Viability
An MTS assay was performed to assess the effect of the SVW and SVW-Bf on 3T3-L1 cell viability. As shown in Figure 3, the SVW and SVW-Bf at 10, 50, and 100 µg/mL had no significant effect on viability after 72 h of treatment. The cells did not become toxic, even when the SVW and SVW-Bf were added at the highest concentration (100 µg/mL) for 72 h.

Effect of SVW and SVW-Bf on Preadipocyte Viability
An MTS assay was performed to assess the effect of the SVW and SVW-Bf on 3T3-L1 cell viability. As shown in Figure 3, the SVW and SVW-Bf at 10, 50, and 100 μg/mL had no significant effect on viability after 72 h of treatment. The cells did not become toxic, even when the SVW and SVW-Bf were added at the highest concentration (100 μg/mL) for 72 h.

Effect of SVW and SVW-Bf on Preadipocyte Viability
An MTS assay was performed to assess the effect of the SVW and SVW-Bf on 3T3-L1 cell viability. As shown in Figure 3, the SVW and SVW-Bf at 10, 50, and 100 μg/mL had no significant effect on viability after 72 h of treatment. The cells did not become toxic, even when the SVW and SVW-Bf were added at the highest concentration (100 μg/mL) for 72 h.

Discussion
Obesity is quickly becoming one of the most serious health problems leading to death in modern society, owing to the consequent increased risk of hypertension, hyperlipidemia, cardiovascular diseases, diabetes, cancers, and non-alcoholic fatty liver disease [12]. It is generally associated with an excessive growth of adipose tissue mass caused by an increase in the number and size of fat cells, so the amount of adipose tissue mass can be regulated by the suppression of adipogenesis [13][14][15]. Thus, obesity could be prevented by lose weight through diet, physical activities, treatment with anti-adipogenesis agents, etc. [16,17]. To develop anti-adipogenesis agents, 3T3-L1 cells have served as a well-documented model system [18]. A promising source of such agents appears to come from Nature [1].
Using the 3T3-L1 model of adipogenesis, the anti-obesity activity of 300 crude extracts was screened at a concentration level of 10 μg/mL. This revealed 33 of 300 extracts that were active at 30% inhibition and greater. Among them, nine belonged to the Compositae family, five to the Liliaceae family, four to the Convolvulaceae family, three to the Eucommiaceae family, and three to the Solanaceae family. Their activities are hypothesized to be family-dependent due to their similar metabolic compositions. Out of these 31 plants, three, including the stem and leaf of Physalis angulata, the whole grass of Solidago virgaurea, and the root of Dioscorea nipponica, presented strong (over 90%) inhibition of adipogenesis in 3T3-L1 cells. The anti-obesity effect of Physalis angulata and Dioscorea nipponica have been reported in previous studies [19,20]. However, there have been no previous investigations of SV and its anti-adipogenesis activity. Thus, this plant could represent a new potential source of anti-adipogenesis activity and was selected for further study, including active component screening. SV (European goldenrod or woundwort) is an herbaceous perennial plant that has been traditionally used to treat urinary tract, nephrolithiasis, and prostate pathologies [21]. Its antioxidant activities and anti-cardiotoxicity effects have also been previously reported [22]. In the present study, we attempted to identify and isolate the active components of SV guided by an anti-adipogenic assay in 3T3-L1 cells, and K-3-O-R, which demonstrated a 48.2% inhibitory effects at 60 μg/mL without cytotoxicity within 72 h in accordance with previous results [23], was obtained from the SVW-Bf5. Interestingly, the inhibitory activity of K-3-O-R is no more than those of crude extract and fractions. Hence, synergistic effects and/or other efficient anti-adipogenesis components would be presented. In order to recognize whether synergistic effect it is, another two major components were isolated from SVW-Bf5, which are CA and PA also showed lower inhibitory than

Discussion
Obesity is quickly becoming one of the most serious health problems leading to death in modern society, owing to the consequent increased risk of hypertension, hyperlipidemia, cardiovascular diseases, diabetes, cancers, and non-alcoholic fatty liver disease [12]. It is generally associated with an excessive growth of adipose tissue mass caused by an increase in the number and size of fat cells, so the amount of adipose tissue mass can be regulated by the suppression of adipogenesis [13][14][15]. Thus, obesity could be prevented by lose weight through diet, physical activities, treatment with anti-adipogenesis agents, etc. [16,17]. To develop anti-adipogenesis agents, 3T3-L1 cells have served as a well-documented model system [18]. A promising source of such agents appears to come from Nature [1].
Using the 3T3-L1 model of adipogenesis, the anti-obesity activity of 300 crude extracts was screened at a concentration level of 10 µg/mL. This revealed 33 of 300 extracts that were active at 30% inhibition and greater. Among them, nine belonged to the Compositae family, five to the Liliaceae family, four to the Convolvulaceae family, three to the Eucommiaceae family, and three to the Solanaceae family. Their activities are hypothesized to be family-dependent due to their similar metabolic compositions. Out of these 31 plants, three, including the stem and leaf of Physalis angulata, the whole grass of Solidago virgaurea, and the root of Dioscorea nipponica, presented strong (over 90%) inhibition of adipogenesis in 3T3-L1 cells. The anti-obesity effect of Physalis angulata and Dioscorea nipponica have been reported in previous studies [19,20]. However, there have been no previous investigations of SV and its anti-adipogenesis activity. Thus, this plant could represent a new potential source of anti-adipogenesis activity and was selected for further study, including active component screening. SV (European goldenrod or woundwort) is an herbaceous perennial plant that has been traditionally used to treat urinary tract, nephrolithiasis, and prostate pathologies [21]. Its antioxidant activities and anti-cardiotoxicity effects have also been previously reported [22]. In the present study, we attempted to identify and isolate the active components of SV guided by an anti-adipogenic assay in 3T3-L1 cells, and K-3-O-R, which demonstrated a 48.2% inhibitory effects at 60 µg/mL without cytotoxicity within 72 h in accordance with previous results [23], was obtained from the SVW-Bf5. Interestingly, the inhibitory activity of K-3-O-R is no more than those of crude extract and fractions. Hence, synergistic effects and/or other efficient anti-adipogenesis components would be presented. In order to recognize whether synergistic effect it is, another two major components were isolated from SVW-Bf5, which are CA and PA also showed lower inhibitory than crude extracts. This seems to suggest that these results would be the result of a synergistic effect, which is a widespread phenomenon in natural products [24]. Scazzocchio et al. have reported a similar effect in the same pathway [25].
K-3-O-R is known as potent α-glucosidase inhibitor that exists in many plants [26]. As a flavonol glucoside, K-3-O-R would be ingested as a glycoside and adsorbed in the systemic circulation as K-3-O-R, kaempferol, kaempferol conjugated forms and other phenolic acids, and then arrive at target tissues [27]. A previous study has demonstrated that kaempferol inhibits lipid accumulation in adipocytes and zebrafish, and attenuates the late adipogenic factors such as PPAR-γ and C/EBPα [28]. In order to understand the mechanism of anti-adipogenesis action of K-3-O-R in 3T3-L1 cells, its effect on PPAR-γ and C/EBPα was studied in the present work. The biochemical pathways of adipogenesis in the 3T3-L1 cell line have been well characterized [29]. The transcription factors PPAR-γ and C/EBPα play key roles in the complex transcriptional cascade of adipocyte differentiation that will eventually activate and express adipocyte-specific genes such as fatty acid synthetase, fatty acid binding protein, leptin, adiponectin and etc., which closely related to obesity disease such as diabetes and non-alcoholic fatty liver disease [30]. PPAR-γ [10] and C/EBPα [11] are known to increase in 3T3-L1 cells as part of transcriptional regulation of differentiation; however, their levels were decreased after treatment with K-3-O-R in 3T3-L1 cells. Our results indicate that K-3-O-R isolated from SV has a strong anti-adipogenic effect in 3T3-L1 cells by suppressing the expression of PPAR-γ and C/EBPα.

General Information
Diaion HP-20 resin and sephadex LH-20 used for separation were purchased from Sigma-Aldrich (St. Louis, MO, USA) and GE Healthcare (Uppsala, Sweden), respectively. All organic solvents used for extraction and isolation were obtained from Samchun (Pyeongtaek-si, Gyeonggi-do, Korea). Ultrapure water used for extraction, isolation and all solutions was obtained using a Milli-Q laboratory water pufification system (Millipore, Bedford, MA, USA) with a resistivity over 18.2 MΩ¨cm. Fourier-transform-NMR spectrometer used for nuclear magnetic resonance were obtained from Bruker Korea (Seongnam, Korea). Signal processing and interpretation were performed using the Bruker DPX 400 MHz (9.4T) package. JMS-700 Mstation used for EI-MS were obtained from JEOL (Tokyo, Japan). Voyager-DE-STR-MALDI-TOF Mass Spectrometer used for MALDI-TOF MS was purchased from Applied Biosysterms (Foster City, CA, USA).

Preparation of Natural Extracts
The natural products were extracted in water or ethanol, by using evaporative solvent removal. The concentrated samples were stored at -20˝C for further study.

Preparation of S. virgaurea var. gigantea
SV (1.5 kg) was extracted twice with chloroform (15 L, room temperature) at for 48 h. After filtration, the dried SV was extracted with 70% ethanol (EtOH, 15 L, room temperature) twice for 48 h. Afterwards, the dried residue was extracted two times with water (15 L) for 2 h at 100˝C.

Cell Culture and Differentiation
3T3-L1 fibroblasts were obtained from ATCC (Manassas, VA, USA) and grown at 37 °C under a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DEME, Gibco, Waltham, MA, USA) containing 10% bovine calf serum (GenDEPOT, Katy, TX, USA) and 100 U/mL The MS and 1 H-NMR data for PA are identical to those reported previously [34].

Oil Red O Staining
SV extracts and its solvent fractions treated to 3T3-L1 cells at the concentration of 10 µg/mL on the day 4 after differentiation induction; Diaion HP-20 fractions of SVW-Bf treated to 3T3-L1 cells at the concentration of 10 and 50 µg/mL; each isolated compound treated to 3T3-L1 cells at the concentration of 10, 30, and 60 µg/mL. The Oil Red O staining was performed on the day 8 after differentiation induction as following. Briefly, the 3T3-L1 adipocyte cells were washed with phophate buffered saline (PBS) and fixed with 10% formalin. After the Oil Red O staining, cells were photographed using a phase-contrast microscope (Olympus CKX41, Tokyo, Japan) in combination with a digital camera (Canon Inc., Tokyo, Japan) at 200ˆmagnification. The lipid droplets were dissolved in isoprapanol and measure at 540 nm using a microplate reader (Sensident scan, Labsystems, Helsinki, Finland). The relative lipid content and adipogensis inhibitory percentage were calculated using the following equations:

Statistical Analysis
All values are mean˘S.E.M. For statistical analysis, the p value was calculated using a two-tailed unpaired Student's t-test with p < 0.05 considered statistically significant.