Anti-Adipogenic Polyacetylene Glycosides from the Florets of Safflower (Carthamus tinctorius)

Safflower (Carthamus tinctorius) is an annual herb belonging to the Compositae family; it has a history of use as a food colorant, dye, and medicine in oriental countries. LC-MS-UV-based chemical analysis of extract of the florets of C. tinctorius led to the isolation of two new C10-polyacetylene glycosides, (8Z)-decaene-4,6-diyne-1,10-diol-1-O-β-d-glucopyranoside (1) and (8S)-deca-4,6-diyne-1,8-diol-1-O-β-d-glucopyranoside (2), together with five known analogs (3–7). The structures of the new compounds were determined by using 1D and 2D NMR spectroscopic data and HR-MS data, as well as chemical transformations. Of compounds 1–7, compounds 2, 3, and 4 inhibited the adipogenesis of 3T3-L1 preadipocytes, whereas compounds 1 and 6 promoted adipogenesis. Compounds 2, 3, and 4 also prevented lipid accumulation through the suppression of the expression of lipogenic genes and the increase of the expression of lipolytic genes. Moreover, compounds 3 and 4 activated AMPK, which is known to facilitate lipid metabolism. Our findings provide a mechanistic rationale for the use of safflower-derived polyacetylene glycosides as potential therapeutic agents against obesity.


Introduction
Obesity is caused by energy imbalance, which leads to an excessive accumulation of body fat in adipose tissues [1]. The expansion of adipose tissue accompanies the differentiation of preadipocytes residing in adipose tissues to mature adipocytes and the generation and accumulation of lipid droplets in adipocytes [2]. Thus, the identification of compounds preventing adipogenesis and lipogenesis has been considered as an effective strategy for the alleviation of obesity and metabolic diseases.
Carthamus tinctorius L., also known as safflower, is a highly branched and thistle-like annual plant belonging to the Compositae family. C. tinctorius has a long history of use as a food colorant, dye, and traditional medicine for the treatment of cardiovascular diseases and gynecological symptoms [3]. The pistil of C. tinctorius is well-known as an edible dye agent in various European dishes, including paella, risotto, and pasta [4]. In previous studies, phytochemical investigation of this plant reported the presence of quinochalcones, such as carthamin, safflower yellow, and safflomin, flavonoids, alkaloids, and polyacetylenes [5]. Further pharmacological studies demonstrated that the polyacetylenes from C. tinctorius inhibited LPS-induced NO (nitric oxide) release, showing its potential as an agent for treating inflammatory diseases [6]. Recent studies have shown that extracts of safflower inhibited the adipogenesis of 3T3-L1 preadipocytes and alleviated high-fat diet-induced obesity in mice [7][8][9][10]. In addition, treatment with safflower extracts improved metabolic parameters, such as glucose metabolism and lipid profiles, in mice with diet-induced obesity [9,10]. Despite numerous evidences showing the beneficial effects of safflower on metabolism, the exact compounds responsible for the action and the mechanisms underlying their action remain undiscovered.
Biomedicines 2021, 9, x FOR PEER REVIEW 2 of 12 as an agent for treating inflammatory diseases [6]. Recent studies have shown that extracts of safflower inhibited the adipogenesis of 3T3-L1 preadipocytes and alleviated high-fat diet-induced obesity in mice [7][8][9][10]. In addition, treatment with safflower extracts improved metabolic parameters, such as glucose metabolism and lipid profiles, in mice with diet-induced obesity [9,10]. Despite numerous evidences showing the beneficial effects of safflower on metabolism, the exact compounds responsible for the action and the mechanisms underlying their action remain undiscovered. As a part of our continued search for natural products with novel structural and/or biological properties [11][12][13][14][15], seven polyacetylene glycosides (1-7), including two new C10-polyacetylene glycosides, (8Z)-decaene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (1) and (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside (2), were isolated from the extract of the florets of C. tinctorius by using LC-MS-UV-based chemical analysis. The structures of the new compounds were established by 1D and 2D NMR spectroscopic and high-resolution MS data analysis, and the absolute configurations of the sugar moiety were elucidated by chemical transformations followed by enzymatic hydrolysis. Herein, we have described the isolation and structural characterization of the compounds (1-7) ( Figure 1) and the evaluation of their effects on de novo adipogenesis and lipid metabolism in adipocytes.

Plant Material
The florets of C. tinctorius were collected in Pocheon, Gyeonggi-do, Korea, and purchased from Dongyangpharm in September 2018. The plant was identified by one of the authors (K. H. Kim). A voucher specimen (HH-  was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Enzymatic Hydrolysis and Absolute Configuration Determination of the Sugar Moiety of 1 and 2
The absolute configuration of the sugar moiety was determined by using an LC-MS-UV-based method [18]. Compounds 1 and 2 (each 0.5 mg) were hydrolyzed with crude hesperidinase (10 mg, from Aspergillus niger; Sigma-Aldrich, Saint Louis, MO, USA) at 37 • C for 72 h, individually, and then, EtOAc was used for the extraction. Each aqueous layer was evaporated by using a vacuum evaporator and dissolved in anhydrous pyridine (0.5 mL) with the addition of L-cysteine methyl ester hydrochloride (1.0 mg). After the reaction mixture was heated at 60 • C for 1 h, O-tolylisothiocyanate (50 µL) was added, and the mixture was incubated at 60 • C for 1 h. The reaction product was evaporated by using a vacuum evaporator and dissolved in MeOH. Subsequently, the dissolved reaction product was directly analyzed by LC-MS [MeOH/H 2 O, 1:9→7:3 gradient system (0-30 min), 100% MeOH (31-41 min), 0% MeOH (42-52 min); 0.3 mL/min] using an analytical Kinetex C18 100 Å column (100 mm × 2.1 mm i.d., 5 µm). The sugar moiety from 1 and 2 was identified as D-glucopyranose based on the comparison of the retention time with an authentic sample (t R : D-glucopyranose 19.3 min).

Oil Red O Staining
To observe the accumulated lipid droplets in adipocytes, Oil Red O staining was performed after the differentiation [20]. After the adipocytes were fixed in 10% formaldehyde for 1 h and washed with 60% isopropanol, mature adipocytes were incubated with the Oil Red O working solution for 1 h. Then, the cells were washed with distilled water twice, and images of the stained lipid droplets were captured with Cytation™ 5.

Cell Counting
First, 3T3-L1 cells were treated with compounds 1-7 at concentrations of 10, 20, and 40 µM for 24 h; then, they were incubated with EDTA for 5 min for detachment. The detached cells were diluted with PBS, and the numbers of cells were counted by using a LUNA-II™ Automated Cell Counter (Logos Biosystems, Annandale, VA, USA).

Western Blotting
Proteins were extracted with Pro-Prep (Intron Biotechnology, Seoul, Korea) for 20 min on ice and then centrifuged at 13,000 rpm at 4 • C for 20 min. For Western blotting, 15 µg of each protein in the supernatant was separated by SDS-polyacrylamide gel (10%) electrophoresis. The proteins were transferred to polyvinylidene difluoride (PVDF, Millipore, Darmstadt, Germany) membranes using a semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the primary antibodies (dilution 1:2000) overnight at 4 • C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) for 1 h at room temperature. HRP signals reacting with chemiluminescence reagents (Abclon) were detected using AGFA X-ray films and quantified using the ImageJ software. Anti-phospho (T172) AMPKα (Abcam, ab2535) and anti-AMPKα (Abcam, ab2532S) antibodies were used for Western blotting.

Reverse Transcription and Quantitative Real-Time PCR (RT-qPCR)
Total RNA was extracted from adipocytes using Easy-Blue reagent (Intron Biotechnology) in accordance with the manufacturer's instructions. For reverse transcription (RT), cDNA was synthesized from 1 µg of total RNA using the Maxim RT-PreMix Kit (Intron Biotechnology). For quantitative real-time PCR (qPCR), cDNA from RNA was incubated with KAPA SYBR ® FAST qPCR Master Mix (Kapa Biosystems) and primers for each gene. The qPCR reaction was detected using a CFX96 Touch TM real-time PCR detector (Bio-Rad). The relative mRNA expression for each gene was normalized to the expression of β-actin. The sequences of qPCR primers used in this study are listed in Table 2. Table 2. Sequences of primers used for RT-qPCR.

Gene
Forward Reverse

Statistical Analysis
Statistical significance was analyzed by using a two-tailed Student's t-test with Excel and evaluated by using a p-value. The data represent the mean ± SEM for n = 3 replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the control group.

Isolation of the Compounds
The crude extract of the florets of C. tinctorius was solvent-partitioned using water and five organic solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, n-butanol, and acetone). The obtained fractions were primarily monitored and analyzed by LC-MS, which allowed us to identify the presence of components with a distinctive UV spectrum in the EtOAc-soluble fraction, which is characteristic of an enediyne chromophore [16,17]. To identify the compounds of interest, the LC-MS-based phytochemical analysis of the EtOAc fraction and preparative and semi-preparative HPLC were performed; the processes resulted in the isolation of two new C 10 -polyacetylene glycosides (1 and 2) and five known analogs (3-7).

Evaluation of Biological Activity of the Isolated Compounds
Prior to the assessment of the anti-adipogenic properties of the isolated compounds, we first evaluated the cytotoxic effects of compounds 1-7 in 3T3-L1 preadipocytes. No compounds exhibited cytotoxicity at concentrations of 10 and 20 µM, but compounds 1, 5, and 6 decreased cell viability at a concentration of 40 µM (Figure 4). Hence, we treated 3T3-L1 cells with compounds 1-7 at a concentration of 20 µM during the entire process of adipogenesis for the evaluation of the anti-adipogenic activities of these compounds. Oil red O staining data showed that compounds 2-4 prevented the de novo generation of adipocytes and lipid accumulation within adipocytes ( Figure 5A). The transcription levels of mature adipocyte marker genes (Adipsin and Fabp4) were significantly reduced by treatment with compounds 2-4 ( Figure 5B). These data indicated that the new C 10polyacetylene glycoside (2) and two known analogs (3 and 4) inhibited the adipogenesis of 3T3-L1 preadipocytes.
As the failure of lipid fusion was observed in the groups treated with compounds 2-4 ( Figure 5A), we then investigated whether compounds 2-4 affected lipid metabolism. Upon exposure to compounds 2-4 during adipogenesis, the expression of the lipogenic gene SREBP1 was suppressed ( Figure 6A), whereas the mRNA expression of the lipolytic gene ATGL was increased ( Figure 6B). In the case of compound 4, the expression of another lipolytic gene, HSL, was also markedly enhanced ( Figure 6C). These results suggested that compounds 2-4 can enhance lipid metabolism through the inhibition of lipogenesis and the facilitation of lipolysis.

Evaluation of Biological Activity of the Isolated Compounds
Prior to the assessment of the anti-adipogenic properties of the isolated compounds, we first evaluated the cytotoxic effects of compounds 1-7 in 3T3-L1 preadipocytes. No compounds exhibited cytotoxicity at concentrations of 10 and 20 µM, but compounds 1, 5, and 6 decreased cell viability at a concentration of 40 µM ( Figure 4). Hence, we treated 3T3-L1 cells with compounds 1-7 at a concentration of 20 µM during the entire process of adipogenesis for the evaluation of the anti-adipogenic activities of these compounds. Oil red O staining data showed that compounds 2-4 prevented the de novo generation of adipocytes and lipid accumulation within adipocytes ( Figure 5A). The transcription levels of mature adipocyte marker genes (Adipsin and Fabp4) were significantly reduced by treatment with compounds 2-4 ( Figure 5B). These data indicated that the new C10-polyacetylene glycoside (2) and two known analogs (3 and 4) inhibited the adipogenesis of 3T3-L1 preadipocytes. As the failure of lipid fusion was observed in the groups treated with compounds 2-4 ( Figure 5A), we then investigated whether compounds 2-4 affected lipid metabolism. Upon exposure to compounds 2-4 during adipogenesis, the expression of the lipogenic gene SREBP1 was suppressed ( Figure 6A), whereas the mRNA expression of the lipolytic gene ATGL was increased ( Figure 6B). In the case of compound 4, the expression of another lipolytic gene, HSL, was also markedly enhanced ( Figure 6C). These results suggested that compounds 2-4 can enhance lipid metabolism through the inhibition of lipogenesis and the facilitation of lipolysis.
AMP-activated protein kinase (AMPK) is known to be a key controller of energy metabolism through the inhibition of adipogenesis and the stimulation of lipid metabolism upon its activation [27]. It has been reported that treatment of brain cells with safflower yellow AMPK. Western blotting data showed that compounds 3 and 4 significantly increased the phosphorylation of AMPK compared with the total amounts of AMPK ( Figure 6D).   AMPK. Western blotting data showed that compounds 3 and 4 significantly increased the phosphorylation of AMPK compared with the total amounts of AMPK ( Figure 6D).

Conclusions
In this study, we identified two novel C 10 -polyacetylene glycosides (1 and 2) and five known compounds (3-7) from the MeOH extract of the florets of C. tinctorius. The effects of the seven identified compounds on adipogenesis were evaluated; compounds 2-4 efficiently inhibited adipocyte differentiation from 3T3-L1 preadipocytes, reducing the mRNA expression levels of Adipsin and Fabp4. Furthermore, compounds 2-4 promoted the expression of lipolytic genes while downregulating the expression of lipogenic genes. Compounds 3 and 4 induced AMPK phosphorylation, which is known to improve energy metabolism ( Figure 7); in contrast, compound 2 appeared to regulate lipid metabolism through other pathways not involved in AMPK signaling. Our findings provide experimental evidence to support the metabolic role of safflower-derived polyacetylene glycosides in the prevention of excessive lipid accumulation in obesity.

Conclusions
In this study, we identified two novel C10-polyacetylene glycosides (1 and 2) and five known compounds (3-7) from the MeOH extract of the florets of C. tinctorius. The effects of the seven identified compounds on adipogenesis were evaluated; compounds 2-4 efficiently inhibited adipocyte differentiation from 3T3-L1 preadipocytes, reducing the mRNA expression levels of Adipsin and Fabp4. Furthermore, compounds 2-4 promoted the expression of lipolytic genes while downregulating the expression of lipogenic genes. Compounds 3 and 4 induced AMPK phosphorylation, which is known to improve energy metabolism ( Figure 7); in contrast, compound 2 appeared to regulate lipid metabolism through other pathways not involved in AMPK signaling. Our findings provide experimental evidence to support the metabolic role of safflower-derived polyacetylene glycosides in the prevention of excessive lipid accumulation in obesity.

Conflicts of Interest:
The authors declare no conflict of interest.