Inhibitory Effects of Twenty-Nine Compounds From Potentilla longifolia on Lipid Accumulation and Their Mechanisms in 3T3-L1 Cells

Potentilla longifolia Willd. ex D.F.K.Schltdl., which is a kind of traditional Chinese herb, is often referred to as “Ganyancao” in China, which means “the herb is effective in the treatment of liver inflammation”. Three new (ganyearmcaoosides A and B and ganyearmcaoic acid A; 1–3) and 26 known compounds (4–29) were isolated from the 95% ethanol extract of the dried aerial parts of this plant, of which 21 were isolated for the first time from this plant. The chemical structures of these compounds were elucidated using NMR and HR-ESI-MS analysis. The inhibitory effects of the 29 compounds with safe concentrations on the lipid accumulation in 3T3-L1 cells were evaluated using photographic and quantitative assessments of lipid contents by Oil Red O staining, and measurement of the triglyceride levels. Comprehensive analysis showed that compound 12 (3,8-dimethoxy-5,7,4′- trihydroxyflavone) showed the best inhibitory effect on lipid accumulation such as reducing the accumulation of oil droplets and triglyceride level, and was superior to the reference in positive control. Western blot analysis and RT-PCR results showed that compound 12 enhanced the phosphorylations of AMPK and ACC, and inhibited the expressions of adipogenesis-related proteins or genes including SREBP1c, FAS, SCD1, GPAT, PPARγ and C/EBPα, and thereby significantly inhibited lipid accumulation in a concentration-dependent manner. P. longifolia and its bioactive compounds could be promising as potential therapeutic agents for diseases related to lipid accumulation in the future.


INTRODUCTION
Excessive accumulation of lipids in the human body may lead to many problems, including hypertension, obesity, fatty liver disease, hyperlipidemia, and diabetes. These problems not only disturb the physical and mental health of human beings, but also seriously delay the development of human society and economy (Wada et al., 2007;Ideta et al., 2015;Ma et al., 2018). Solutions to these health problems are often focused on the prevention or suppression of fat accumulation. Human beings have been painstakingly trying to find non-toxic and effective lipid-lowering drugs, including drugs developed from traditional medicine sources. Natural products have been used in traditional medicines since ancient times. Some traditional medicines have been developed and standardized, and continue to contribute to human health. For example, many clinically used drugs of plant origin were derived from traditional medicines (Alves and Rosa, 2007;Li-Weber, 2009;Yuan et al., 2016).
"Chaoyao medicine" is a part of traditional Chinese medicine, mainly used by the people of Chaoxianzu nationality (a minority in China). Chaoyao medicine has formed its own particular details and styles in terms of the methods of use and species of medicinal materials after long-term use and development. A large amount of clinical trial data has been obtained from the long-term practice of Chaoyao medicine. Based on these clinical data, many aspects of Chaoyao medicine including the species and methods of use, have been continuously improved, and thus ensuring its curative effects. The aerial parts, or the whole plant, of Potentilla longifolia Wild. ex D.F.K.Schltdl., which is a kind of Chaoyao medicine, is usually used to treat jaundice and other liver injury diseases. Because of its remarkable curative effect in treating hepatitis and other liver inflammation, it is often referred to as "Ganyearmcao", or "Ganyancao" in China, which means "the herb is effective in the treatment of liver inflammation" (Piao et al., 2012). Potentilla species have been used for a long time as traditional herbs. This genus has been known and studied since ancient times for its possible therapeutic properties. In in vivo and vitro biological and pharmacological studies of Potentilla species are focused on antiviral, antimicrobial, antiinflammatory, hepatoprotective and antioxidative activities, etc. (Tomczyk and Latte, 2009). For example, one study demonstrated that asiatic acid from P. chinensis significantly ameliorated nonalcoholic fatty liver disease by inhibiting the Endoplasmic reticulum stress (ERS) pathway (Wang et al., 2018). The antioxidant experiments showed that the water extracts of both Potentilla species (P. argentea and P. recta) were the potent 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) [ABTS] scavenger. The methanol extracts of these two Potentilla species were active inhibitors of α-glucosidase (Sut et al., 2019). Other study also showed that methanol and water extracts of two Potentilla species (P. speciosa L. and P. reptans Willd.), and biflavanols and quercetin-3-O-α-L-rhamnopyranoside-2''-gallate isolated from P. anserina L. displayed good antioxidant activity (Uysal et al., 2017;Yang et al., 2020). Several tannins and flavonoids have been reported to be present in P. longifolia which was also known as P. viscosa. A few pharmaceutical research such as anti-oxidant activity of P. longifolia was also conducted (Wollenweber and Dorr, 2008;Tomczyk and Latte, 2009). Considering the traditional effect of P. longifolia on hepatitis, and the close relationship among hepatitis, fatty liver and lipid accumulation, we conducted the experiment of compounds isolated from P. longifolia on inhibitory effects on lipid accumulation in our previous study, and obtained good results. Therefore, the inhibitory effects of the herb on lipid accumulation was further studied.
AMP-activated protein kinase (AMPK) regulates fat and carbohydrates, adjusts the energy balance in cells, and is closely related to elements of lipid metabolism, such as adipogenesis and lipogenesis; thus, AMPK affects lipid accumulation in conditions such as obesity (Ha et al., 2016;Ma et al., 2018). After activation, AMPK inhibits the expression of Sterol regulatory element-binding protein 1c (SREBP1c), CCAAT/enhancer-binding protein α (C/EBPα), and Peroxisome proliferators-activated receptor-gamma (PPARγ) and their downstream genes, thereby inhibiting adipogenesis and lipogenesis, and regulating lipid metabolism (Gan et al., 2017). In addition, AMPK can inhibit lipid synthesis by inhibiting glycerol-3-phosphate acyltransferase (GPAT), the first key enzyme involved in catalyzing triglyceride (TG) synthesis, which results in lipid regulation (Lindeń et al., 2004;Henriksen et al., 2013). SREBP1c is a transcription factor involved in adipogenesis; SREBP1c activates genes and enzymes, including fatty acid synthase (FAS) and Stearoyl-Coenzyme A desaturase 1 (SCD1). PPARγ and C/EBPα are considered to be the main regulators of adipogenesis. PPARγ increases the expression of C/EBPα, which activates many adipocyte-specific genes, including SCD1. SCD1 is closely related to adipocyte maturation and the levels of TGs and fatty acids, which preferentially regulate adipogenesis (Ha et al., 2016;Ma et al., 2018). In addition, PPARγ combined with C/EBPα increased the expression of adipocyte-related genes and induced the formation of mature adipocytes in the late stage of adipogenesis (Moseti et al., 2016).
In a herbal medicine, there must be active chemicals acting alone or in concert to achieve the desired effects. Therefore, it is very important to study the chemical constituents in medicinal plants (Yuan et al., 2016). In this study, the chemical constituents of P. longifolia and their anti-lipid accumulation activity and mechanisms of action were investigated.

Chemicals and Reagents
The 3T3-L1 cells were purchased from ATCC (Manassas, VA, United States). Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), fetal bovine serum (FBS), and penicillin−streptomycin were purchased from Gibco by Life Technologies (Grand Island, NY, United States). The TG assay kit was purchased from Nanjing Jiancheng Bioengineering Institute, China. Protein extraction, EASY BLUE total RNA extraction, and ECL-reagent kits were from Intron Biotechnology Inc. (Beverly, MA, United States). The Bio-Rad protein assay kit was from BioRad Laboratories (Hercules, CA, United States). ODS (50 μm) was obtained from YMC (Japan). All other chemicals and solvents were analytical grade.

Plant Material
The aerial parts of Potentilla longifolia Wild. Ex Schlecht. was acquired in Changbai Mountain, Jilin Province, China, in October 2014. The sample was authenticated by Prof. HZ Lv of College of Pharmacy, Yanbian University (voucher specimen: ID-2014106, stored in Chaoyao Herbarium of Yanbian University).

Cell Culture and Cytotoxicity Assay
The 3T3-L1 cells were cultured in DMEM containing 10% FCS, 100 unit/ml penicillin and 100 μg/ml streptomycin at 37°C in an atmosphere of 5% CO 2 . For the cytotoxicity assay, 3 × 10 4 3T3-L1 cells per well were cultured in 96-well plates and treated with the thirty compounds at the concentrations of 0, 10, 20, 40, or 80 μM for 96 h, respectively. Three parallel wells were set at per concentration. The cytotoxicities of these compounds were determined by the MTT assay. Absorbance was measured at 540 nm to determine viable cell numbers in wells.

Oil-Red O Staining
After discarding the medium in the 6-well plate, the differentiated cells were washed twice with phophate-buffered saline (PBS) and fixed with 10% formalin for 1 h. The cells then were stained with 1 ml Oil-Red O solution for 2 h to observe the differentiation of 3T3-L1 cells. The pictures were taken by an Olympus microscope. After that, 6-well plates were treated with isopropanol, and the absorbances were determined at 540 nm to evaluate the lipid accumulation.

Measurement of the TG Level
The 3T3-L1 cells were lysed in lysis buffer which included 25 mM sucrose, 20 mM Tris−HCl, 1 mM EGTA, and 1 mM EDTA, and the cells were collected and centrifugated at 13,000 rpm for 15 min. A TG assay kit was used to measured the levels of TG in accordance with the instructions of the manufacturer. A Bio-Rad protein assay reagent (Bio-Rad Laboratories) was used to determine the concentration of protein in accordance with the manufacturer's instructions. Furthermore, the experimental details in the Western blot analysis, RNA isolation and reverse transcription polymerase chain reaction (RT-PCR) and statistical analysis, including primers and sequences, etc., followed the relevant contents of our previous paper (Ma et al., 2018).

Identification of Compounds 1-29
Compound 1 Compound 1 was obtained as a white amorphous powder, the molecular formula was assigned as C 18 H 26 O 9 based on the [M + H] + peak at m/z 387.1643 (calcd for C 18 H 27 O 9 , 387.1650) in the HR-ESI-MS data (Supplementary Figure S5). The observed NMR data suggest that compound 1 is a phloroglucinol glucoside which was similar to 2-β-D-gluco-pyranosyloxy-4-methoxy-6hydroxyisovalero-phenone) (Wang et al., 2014a), except the positions of methoxy group and hydroxyl group were exchanged.

Compound 2
Compound 2 was obtained as a white amorphous powder, the molecular formula was assigned as  Figure S10). The 1 H and 13 C NMR data revealed that the framework of 2 was the same as 1, with an additional glucopyranosyl moiety at C-4 position, as shown in Figure 2.

The Effects of the 29 Compounds on Cell Viability of 3T3-L1 Cells
To examine the cellular toxicity, 3T3-L1 cells were treated with each of the 29 compounds for 96 h at various concentrations (0-80 μM). The MTT assay showed that concentrations from 0-40 μM of compounds 1, 2, 4, 5, 8, 10, 11, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, 24, 27, and 29; from 0-20 μM of compounds 3, 6,7,9,15,19,26 (reference compound in positive control), and 28; and from 0-10 μM of compound 25 showed no toxicity (see Figure 4). The compounds at the corresponding concentrations were employed in the subsequent experiments. Ursolic acid, which is a pentacyclic triterpene natural compound and can be isolated from the leaves, flowers and fruits of many medicinal herbs such as Rosmarinus officinalis, has aroused great interest for its inhibitory effects on lipid accumulation (Chu et al., 2015;Katashima et al., 2017). In this experiment, ursolic acid was isolated as one of the 30 compounds from P. longifolia and therefore it was selected as the reference compound in the positive control.
The results of Oil Red O staining showed that compared with the differentiation medium (DM) group, the accumulation of oil droplets in the 3T3-L1 cells treated with compounds 1, 2, 3,5,8,9,11,12,13,15,19, and 26 (reference compound in the positive control) were decreased and the differentiation of 3T3-L1 cells was inhibited to some extent. Among them, compounds 1, 9, 11 and 12 were similar to compound 26 (positive control) in terms of reducing the accumulation of oil droplets. (see Figure 5).
Triglycerides (TGs) are the most abundant lipid component in the human body, and are also used as an index of clinical lipid-related diseases. The amount of TGs in the 3T3-L1 cells were determined. The results showed that TG levels in cells treated with compounds 1, 2, 3, 4, 9, 11, 12, 13, and 26 were decreased compared with the DM group to 72,73,67,72,64,61,56,67, and 66%, respectively in which compounds 9, 11 and 12 were greater than compound 26 (reference compound in the positive control) in terms of reducing TG accumulation and compounds 3 and 13 were very close to positive control. (see Figure 6B).
All the above results indicated that compounds 1, 2, 3, 9, 11, 12 and 13, especially compound 12 could inhibit the differentiation of 3T3-L1 cells and lipid accumulation. Therefore it was necessary to study the chemical constituents and their mechanisms of action in a more in-depth way.

The Structure-Activity Relationships of the 29 Compounds
Among the 29 compounds isolated from P. longifolia, 12 flavonoids and nine phenolic compounds were identified. Among the 12 flavonoids, compounds 11, 12, and 13 exhibited significant inhibitory activity on lipid accumulation. Compared with the FIGURE 5 | Inhibitory effects of the 29 compounds on lipid accumulation from Oil-Red O staining in 3T3-L1 cells. Lipid accumulation was evaluated by Oil-Red O staining in 3T3-L1cells. 3T3-L1cells were divided into normal control (Con) group, differentiated control treated with differentiation medium (DM) group, differentiated positive control treated with differentiation medium plus pioglitazone (PIO) group, and thirty individual treatment groups treated with thirty compounds alone respectively.
Frontiers in Pharmacology | www.frontiersin.org November 2020 | Volume 11 | Article 555715 9 other nine compounds, each of these three compounds had a methoxy group at the C-3 position, rather than hydroxyl or sugar moieties. In addition, these compounds had oxygen-containing substituents at the C-4′ position, i.e. hydroxyl or methoxy groups, while there were no oxygen-containing substituents at the adjacent positions (C-3' or C-5'). Five of the nine phenolic compounds were phenolic glycosides. According to the results of the Oil Red O staining, isopropanol decolorization, TG content, and other aspects, stronger inhibition of lipid accumulation was observed for the five phenolic glycosides compared with the non-phenolic glycosides.
The Effects of Compound 12 on Lipid Accumulation in 3T3-L1 Cells From the above experimental results, compound 12 showed a considerable effect on inhibiting lipid accumulation, so we reconfirmed the cytotoxicity, photographic and quantitative assessments of lipid contents by Oil Red O staining, and TG content of compound 12 (see Figure 7), and the results were consistent with the previous experiments.

The Effects of Compound 12 on Adipogenesis-Related Gene Expressions in 3T3-L1 Cells
To investigate the authenticity of the results of the molecular docking study, and to explore the inhibitory mechanism of compound 12 on lipid accumulation in 3T3-L1 cells, the effects of compound 12 on lipid metabolism-related genes and protein expression were studied by RT-PCR and western blot analysis.
The Effects of Compound 12 on the Phosphorylations of AMPK and ACC, and Adipogenesis-Related Proteins in 3T3-L1 Cells As shown in Figure 9, compound 12 significantly inhibited the protein expression of SREBP1c, FAS, SCD1, PPARγ, and C/EBPα The triglyceride levels were measured by using TG assay Kit. Data represent the mean ± S.D. of three separate experiments. ## p < 0.01 as compared with CON group; ** p < 0.01compared with DM group. The significant difference was only shown to p < 0.01, even when the difference was p < 0.001.
Frontiers in Pharmacology | www.frontiersin.org November 2020 | Volume 11 | Article 555715  ## p < 0.01 as compared with CON group. ** p < 0.01 compared with DM group. The significant difference was only shown to p < 0.01, even when the difference was p < 0.001.
Frontiers in Pharmacology | www.frontiersin.org November 2020 | Volume 11 | Article 555715 11 in a concentration-dependent manner; all of these proteins are related to adipogenesis. These results were consistent with the RT-PCR results for genes related to lipid accumulation.
Compared with the DM group, the expression of pAMPK/ AMPK and pACC/ACC were significantly increased, suggesting that compound 12 significantly enhanced the phosphorylations of AMPK, and inhibited the expressions of adipogenesis-related proteins and genes such as SREBP1c, FAS, and SCD1, and thereby significantly inhibited lipid accumulation.
Although there have been many studies on Potentilla species, only a few studies on chemical constituents and pharmacological activities of P. longifolia were conducted. In this research, the inhibitory effects of P. longifolia on lipid accumulation was studied for the first time. In this study, 29 compounds were isolated from 95% ethanol extract of the plant and their chemical structures were identified. It was found that three of them were new compounds and 18 compounds were isolated from this plant for the first time. Then 29 compounds were then screened for their inhibitory effects on lipid accumulation, and the structureactivity relationship was analyzed. Among the 12 flavonoids, when there was a methoxy group at the C-3 position and an oxygen-containing substituent at the C-4′ position, while there were no oxygen-containing substituents at the adjacent positions (C-3' or C-5'), the compound seemingly showed good activity. Then, the mechanism of action of compound 12 with the best activity was studied. The results showed that adipogenesis-related proteins or genes including SREBP1c, FAS, SCD1, GPAT, PPARγ and C/EBPα, AMPK and ACC, were all more or less involved in the inhibitory effects of lipid accumulation by compound 12.

CONCLUSION
Three new and 26 known compounds were identified from the 95% ethanol extract of the dried aerial parts of P. longifolia. Among them, compounds 1, 2, 3, 9, 11, 12, and 13, significantly inhibited the differentiation and lipid accumulation in 3T3-L1 cells. Especially, compound 12 was superior to reference compound in positive control. Western blot analysis and RT-PCR results showed that compound 12 enhanced the phosphorylation of AMPK and inhibited the expressions of adipogenesis-related proteins and genes such as SREBP1c, FAS, and SCD1, and thereby significantly inhibited lipid accumulation. P. longifolia and its bioactive compounds will play an important role in the treatment of diseases related to lipid accumulation in the future. ## p < 0.01 as compared with CON group, ** p < 0.01 compared with DM group. The significant difference was only shown to p < 0.01, even though the difference was p < 0.001.