Ten New Dammarane-Type Saponins with Hypolipidemia Activity from a Functional Herbal Tea—Gynostemma pentaphyllum

Gynostemma pentaphyllum (thumb.) Makino is a functional herbal tea commonly used in Asian countries and regions to reduce blood lipid levels. G. pentaphyllum saponin is the main component, but there are still a large number of components with lipid-lowering activity that have not been found. In this study, 10 novel dammarane-type saponins, (1–10) and a known one (11) were isolated from G. pentaphyllum. Ten new compounds were identified and named as yunnangypenosides A-J (1–10), and another known one (11) was also obtained. Their chemical structures were determined by MS, NMR spectroscopic analyses. Moreover, the cytotoxicities on human HepG-2 hepatocellular carcinoma cells of these isolates were evaluated, and the results showed that compounds 1–11 had no obvious cytotoxicity. Finally, all these compounds were evaluated for their lipid-lowering effect by means of the oil red O staining method. Ten compounds could significantly reduce lipid levels except of 2, especially 8 exhibite the strongest hypolipidemia activity.


Introduction
Nowadays, much attention has been paid to people's physical health, and healthy eating and physical exercise are ways of maintaining general good health. Meanwhile, high-sugar and high-fat diet habits affect people's health and have been associated with obesity, hypertension, diabetes, dyslipidemia, and other cardiovascular risk factors. Amongst these factors, dyslipidemia has a great impact on human health. It is the main pathogenicity of atherosclerotic cardiovascular disease and an important risk factor for ischemic stroke. So, how to prevent hyperlipidemia through an effective lifestyle has become a topic of increasing concern [1][2][3]. Tea, with leaves or buds from the plant, constitutes one of the beverages popularly consumed in different parts of the world, such as green tea, oolong tea, or black tea. Recent reports demonstrated that some functional tea drinks may exert a positive effect on lowering blood lipid and cholesterol. For example, green tea such as Longjing tea in China has antioxidant and blood lipid lowering effects [4].
that some functional tea drinks may exert a positive effect on lowering blood lipid and cholesterol. For example, green tea such as Longjing tea in China has antioxidant and blood lipid lowering effects [4]. The constituents of these tea, including polyphenols, flavonoids, and volatile oil, etc., have several good bioactivities, such as the ability to lowering blood lipid levels and blood pressure, in addition to anti-oxidant protectives and inhibition of inflammation [5,6].
Gynostemma pentaphyllum is a folk medicine and functional herbal tea, which has a good reputation for lowering blood lipid and blood pressure [7][8][9][10][11]. It consists of numerous chemical components, such as saponins, vitamins, polysaccharides, flavonoids, and amino acids [12,13]. Previous studies have shown that Gypenoside is a marker component in this plant, and its main chemical components have excellent anti-hyperlipidemia, anti-oxidative, anti-inflammatory, antitumor, and other biological activities [11,[14][15][16][17]. In view of its good pharmacological activity and edible value, the chemical composition aroused our research group's interest. Consequently, chemical constituents of G. pentaphyllum, especially triterpene saponins, were systematically studied in this paper. The saponins of this plant were isolated and identified by column chromatography (CC) and preparative HPLC methods. As a result, based on the physicochemical properties and spectral data, 11 compounds ( Figure 1) were obtained and their structures were determined. The 10 novel compounds were identified and named as yunnangypenosides A-J (1-10), followed by a known one, 3β, 20S-dihydroxydammar-24-ene- 21 (11). Moreover, the toxicities of compounds 1-11 were detected using CCK-8 assay (Cell Counting Kit-8), and 11 compounds' activities in lowering lipid by oil red O staining method in HepG-2 cells were also estimated. The results showed that compounds 1-11 had no obvious cytotoxicity, ten compounds (1, 3-11) were significant lipid lowering activity, with the exception of 2, and compound 8 showed the best hypolipidemia activity.

Structural Elucidation
The 60% ethanol extract from the G. pentaphyllum was isolated by CC and eluted with different proportions of mobile phase systems to obtain compounds 1-10. Compound 11 was a known one, whose structure could be illustrated by referring the data to those in the literature [12].
Compound 7 was a white powder, and its MF of C 54 H 92 O 22 was inferred from the HR-ESI-MS data. The NMR spectroscopic properties of 7 and 4 were similar, with the differences being the sugar unit and the absence of the hydroxyl (C-12) in 7. Through analysis of the NMR data, 54 carbons were obtained, of which 30 were allocated to the triterpene skeleton. Acid hydrolysis of 7 gave four d-glucopyranosyl units according to analysis using the HPLC method. The β configuration was also determined based on the coupling constants (7.6, 7.6, 7.6, and 7.4 Hz). The HMBC cross-peaks from the anomeric protons δ H 4.96 (Glc-1') to δ C 89.4 (C-3), δ H 5.13 (Glc-1") to δ C 83.5 (C-20), δ H 5.38 (Glc-1"') to δ C 83.7 (C-2"), and δ H 5.36 (Glc-1"") to δ C 83.7 (C-3") suggested the locations of those sugar units at C-3, C-20, C-2", and C-3", respectively. Some characteristic carbon and hydrogen symbols were proposed from the 13 C-NMR (Table 3) and DEPT-135 spectra. The skeleton of compound 7 was suggested based on the NMR spectral data, and the basic mother nucleus of 7 was the same as that of vina-ginsenoside-R 3 [24]. This confirmed the above conjecture through key correlations based on the HBMC spectrum ( Figure 2). The HR-ESI-MS, 1 H-NMR, 13  Compound 8 was a light-yellow powder, with its MF of C 54 H 92 O 22 inferred from the adduct ion. Comparing the NMR spectra between 8 and 4 showed that the key difference was the sugar unit. Acid hydrolysis of 8 gave three β-d-glucopyranosyls and one α-l-rhamnopyranosyl unit based on analysis using the HPLC method and the NMR spectra. The HMBC correlations from δ H 4.96 (Glc-1') to δ C 89.1 (C-3), from δ H 5.17 (Glc-1") to δ C 84.4 (C-20), from δ H 5.64 (Glc-1"') to δ C 81.7 (C-2"), and from δ H 6.56 (Rha-1"") to δ C 78.3 (C-2"') determined that the three β-d-glucopyranosyl units were located at C-3, C-20, and C-2", while the α-L-rhamnopyranosyl unit was located at C-2"', respectively. The NMR data of 8 can be seen in Table 4. Comparison of the NMR spectrum of 8 with that of (3β, 12β,

Bioactivity Evaluation
The G. pentaphyllum is a functional herbal tea, which has a lot of biological effects. In our study, the lipid-lowering activity of 11 components on human HepG-2 hepatocellular carcinoma cells was determined.

Cytotoxic Activity Assay
The CCK-8 assay provides a convenient and robust way of determining cell viability, which uses water-soluble tetrazolium salt (WST-8) to produce an orange formazan dye upon bio-reduction in the presence of an electron carrier by dehydrogenases. Cytotoxicity screening on the isolated compounds 1-11 was measured using the CCK-8 assay in HepG-2 cells. The results (Figure 3) of activity experiments showed that these compounds had no significant cytotoxicity at concentrations from 6.25 µg/mL to 100 µg/mL. These cytotoxic results could tell us that, at the appropriate concentration, these compounds were almost non-cytotoxic; so, there was no toxic effect seen during our determination of hypolipidemic activity.
presence of an electron carrier by dehydrogenases. Cytotoxicity screening on the isolated compounds 1-11 was measured using the CCK-8 assay in HepG-2 cells. The results (Figure 3) of activity experiments showed that these compounds had no significant cytotoxicity at concentrations from 6.25 μg/mL to 100 μg/mL. These cytotoxic results could tell us that, at the appropriate concentration, these compounds were almost non-cytotoxic; so, there was no toxic effect seen during our determination of hypolipidemic activity.

Hypolipidemia Activity Assay
To evaluate the inhibitory effects of the isolates on oleic acid-induced lipid accumulation, HepG-2 cells were treated with compounds 1-11 (50 μg/mL) in the presence of oleic acid for 24 h. The results were displayed in Figure 4, and HepG-2 cells in the control group contained numerous red lipid droplets fused each other, indicating that the HepG-2 cell lipid accumulation model was set up successfully. The lipid drops in cells treated with compounds 1, 3-11 had light colors and smaller volumes, while compound 8 was the most obvious by comparison with the control group. Through

Hypolipidemia Activity Assay
To evaluate the inhibitory effects of the isolates on oleic acid-induced lipid accumulation, HepG-2 cells were treated with compounds 1-11 (50 µg/mL) in the presence of oleic acid for 24 h. The results were displayed in Figure 4, and HepG-2 cells in the control group contained numerous red lipid droplets fused each other, indicating that the HepG-2 cell lipid accumulation model was set up successfully. The lipid drops in cells treated with compounds 1, 3-11 had light colors and smaller volumes, while compound 8 was the most obvious by comparison with the control group. Through the above phenomena, we could conclude that compounds 1, 3-11 had strong antilipidemic activity, while compound 8 had the strongest antilipidemic activity.

Plant Material
We collected the plants of G. pentaphyllum from Yunnan province, China, in October 2016, which were identified by Chunhua Wang, Tianjin University of Traditional Chinese Medicine. One sample (No. TJTCMBH20161006CH) was kept at the College of Pharmaceutical Engineering of TCM, Poyanghu Road, Jinghai, Tianjin, China.

Extraction and Isolation
We dried the plants at 60 • C in the oven; next, the processed herbs of G. pentaphyllum (about 5000 g) were soaked in 60% C 2 H 5 OH/H 2 O solution (v/v) for three days, three times in total, and then filtered. The extracted solution was mixed and rotated to evaporate until no alcohol was present. We suspended the extract with water, and then extracted it with three different polar solvents (petroleum ether (PE) three times, ethyl acetate (EtOAc) five times, and five times with n-butanol). After this step, the EtOAc extract was analyzed using silica gel column chromatography (v/v, MeOH: CH 2 Cl 2 , 0-75%, gradient elution), giving 10 fractions (Fr

Acid Hydrolysis of Dammarane-Type Glycosides
The sugar parts of compounds 1-5, 8 were obtained using acid hydrolysis and HPLC analysis. This approach was based on methods published in the literature [28]. Each isolated compound (4 mg) was treated with 2 mol/L HCl (2 mL) under reflux conditions at 85 • C for 2 h. Each mixture was extracted with ethyl acetate to afford the aglycone portion, and the aqueous layer was desiccated under reduced pressure. Then, pyridine (1 mL), l-cysteine methyl ester (4 mg), and O-tolyl isothiocyanate (4 mg) were added to the evaporated filtrate in sequence, and the mixture was stirred at 60 • C for 1 h. Each derivative fraction was subjected to HPLC (LC, column, Symmetry Shield TM RP C18; column temperature, 35 • C; mobile phase, 25% acetonitrile contained 0.1% formic acid; flow rate, 0.8 mL/min; ultraviolet detection wavelength, 254 nm). Under these conditions, the sugars of each reactant were identified by comparison with authentic standard derivatives (d-glucose, l-glucose, and l-rhamnose).

Cell Culture and Reagents
HepG-2 cells were obtained from the Binhai Lab of Tianjin University of Traditional Chinese Medicine (Bio-Swamp, MD, USA). The culture process was derived from the literature [29,30].

Cell Viability Assays
The CCK-8 test was used to evaluate the cytotoxic activities of these isolated natural products. The measurements and statistical methods were carried in reference to the literature [28,31].

Hypolipidemia Activity Assay
We tested the lipid-lowering bioactivities of 11 compounds using the oil red O staining method. The cells were grown on six-well plates, where each well (500 µL) contained 2.5 × 10 5 cells, which were incubated for 24 h. After the culture medium was substituted by medium with the 11 compounds (50 µL/mL), oleic acid (0.5 mmol/L) was added [27]. Cells were washed three times with phosphate buffer saline (PBS) after 24 h. Then, the cells were fixed with 4% paraformaldehyde (1 mL) for 30 min. Then, they were rewashed with PBS, and the cells were infiltrated with 60% isopropyl alcohol (1 mL) for 10 s. Finally, the cells were stained using oil red O for 1 h in a dark environment. For optical microscopy observation, the cells were washed three times with PBS [32,33].

Statistics
The t-test was used to analyze the differences between groups of data, with the significance of difference among groups determined using SPSS version 17.0 (International Business Machines Corporation, Armonk, NY, USA) and statistical diagrams generated using GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). A p-value less than 0.05 (*p < 0.05) was considered statistically significant. A p-value less than 0.01 (**p < 0.01) denoted notable statistical significance.

Conclusions
G. pentaphyllum, as a kind of functional tea beverage commonly used by people, attracted the interest of our research group due to its lipid-lowering chemical activity. As far as we know, G. pentaphyllum is becoming more and more popular as a food and beverage. Therefore, much chemical analysis work was carried out for G. pentaphyllum, and researchers found a large number of Gynostemma saponins. However, no detailed chemical composition report was found on the plants we picked in Yunnan province, and some unknown antilipidemic active ingredients were not clarified. Based on the above reasons, we collected some samples from Yunnan Province, China, and carried out a systematic chemical separation with ethanol extract using the method of food chemical analysis. Interestingly, 10 previously undescribed dammaranne-type saponins (1-10) and one known compound (11) were obtained. To study the antilipidemic activity of these compounds, an oil red O staining assay was carried out to determine their bioactivities. Interestingly, these isolated compounds produced hypolipidemia activity in HepG-2 cells except for 2, while compound 8 exhibited the best hypolipidemia activity through the oil red O staining assay. This study provides some scientific evidence for people drinking G. pentaphyllum tea to reduce blood lipid levels, while it also provides new compounds which can be used to enrich the chemical composition of this functional herbal tea. We believe that our research will encourage further studies of the chemical composition and antilipidemic activity of G. pentaphyllum, leading to the development of a healthy tea food based on G. pentaphyllum.