Identification of C21 Steroidal Glycosides from Gymnema sylvestre (Retz.) and Evaluation of Their Glucose Uptake Activities

Gymnema sylvestre (Retz.) Schult is a multi-purpose traditional medicine that has long been used for the treatment of various diseases. To discover the potential bioactive composition of G. sylvestre, a chemical investigation was thus performed. In this research, four new C21 steroidal glycosides sylvepregosides A-D (1–4) were isolated along with four known compounds, gymnepregoside H (5), deacetylkidjoladinin (6), gymnepregoside G (7) and gymnepregoside I (8), from the ethyl acetate fraction of G. sylvestre. The structures of the new compounds were established by extensive 1D and 2D nuclear magnetic resonance (NMR) spectra with mass spectroscopy data. Compounds 1–6 promoted glucose uptake by the range of 1.10- to 2.37-fold, respectively. Compound 1 showed the most potent glucose uptake, with 1.37-fold enhancement. Further study showed that compounds 1 and 5 could promote GLUT-4 fusion with the plasma membrane in L6 cells. The result attained in this study indicated that the separation and characterization of these compounds play an important role in the research and development of new anti-diabetic drugs and pharmaceutical industry.


Introduction
Diabetes mellitus (DM) is a disease caused by lack of insulin secretion or insulin resistance, which cause the body to experience persistent hyperglycemia and long-term metabolic disorders. DM is classified into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), in which T2DM accounts for nearly 95% of individuals, and the number of patients is increasing year by year [1,2]. Insulin resistance (IR) is one of the essential conditions in the development of type 2 diabetes mellitus, and insulin reduces blood sugar levels and improves IR by promoting glucose absorption [3]. Traditional Chinese medicine has used natural products (NPs), including TCM formulations and their extracts, to treat human diseases for thousands of years. Many Chinese herbal medicines and their active ingredients have anti-diabetic properties with minimal side effects, and are widely used in the treatment of T2DM [4]. Therefore, it is of great significance to screen new effective hypoglycemic drugs from natural products through glucose uptake.
Gymnema sylvestre (Retz.) Schult is a genus of tropical plants belonging family Apocynaceae, and mainly distributed in Guangxi, Guangdong, Yunnan, Fujian and Zhejiang provinces of China [5]. G. sylvestre was used as a natural remedy for over two millennia. As a traditional Chinese herbal medicine, G. sylvestre has the effects of detumescence, fever removal, detoxification, promoting muscle growth, reducing swelling, dispelling wind and relieving pain, and is used to treat diseases such as vasculitis, snake bites, rheumatism, waist and knee pain, etc. [6,7]. Furthermore, G. sylvestre has also demonstrated other   Figure 1 were not correct due to oversight. The oxygen atoms in the a compounds 1-4 were missing. The authors would like to apologize for any i caused to readers by this correction. Replacing this figure will not affect conclusions of the paper. The manuscript will be updated and the origina online on the article webpage, with reference to this correction. olecules 2022, 27, x. https://doi.org/10.3390/xxxxx www.mdpi.com/ ure 1 were not correct due to oversight. The oxygen atoms in the acyl g pounds 1-4 were missing. The authors would like to apologize for any caused to readers by this correction. Replacing this figure will not affect conclusions of the paper. The manuscript will be updated and the origin online on the article webpage, with reference to this correction.
Compound 2, was isolated as a white powder. The molecular formula of 2 was determined as C 49 H 70 O 15 by HRESIMS data (m/z 921.4578 [M + Na] + , calcd for 921.4607) ( Figure S13). The nmR data (Table 1) Figure S19) correlations from H-20 (δ H 4.68) to C-1 (δ C 166.1) enabled location of the 2-methyl-2-butenoyl group at C-20. The aglycone of 2 was the same as stephanoside K isolated from Stephanotis lutchuensis [29]. Finally, the coupling constants of the anomeric proton δ H 4.81 (1H, dd, J = 9.6, 1.8 Hz, H cymI -1) and 4.67 (1H, dd, J = 10.0, 2.3 Hz, H cymII -1) illustrated that the sugars were β-linkage. By comparison of the 1 H and 13 C nmR data for 2 with those of 1, 2 was observed to have the similar aglycone and the same sugar sequence as those of 1, and the key HMBC correlations confirmed that the sugar moiety was attached to C-3 of the aglycone ( Figure 2). Thus, the structure of 2 was elucidated as 12  Figure S25). The IR spectrum of 3 ( Figure S36) showed the characteristic for hydroxy (3441 cm −1 ), ester (1701 cm −1 ) and olefinic (1681 cm −1 ) groups. By comparison of the nmR (Table 2) -19) to δ C 139.9 (C-5) explained that the double bond was located at H-5 and H-6. In addition, the 13 C nmR spectroscopic data of the skeleton of 3 matched well with those of the sarcostin skeleton isolated from Cynanchum botanicals [31,32]. By detailed analyses of its HSQC, 1 H-1 H COSY and HMBC spectra ( Figures S29-S31) to δ C 171.3 (C-1 ) revealed the placement of the two ester groups at C-20 and C-12, respectively. The aglycone of 3 was the same as marstomentoside J isolated from Marsdenia tomentosa [33]. The 1 H nmR spectrum of 3 showed two anomeric proton signals at δ H 4.84 (1H, dd, J = 9.6, 1.7 Hz, H cymI -1) and 4.67 (1H, dd, J = 9.5, 1.8 Hz, H cymII -1), and the coupling constants of the anomeric protons illustrated that the sugars were β-linkage. By comparison of the 1 H and 13 C nmR data for 3 with those of 1, 3 was observed to have the similar aglycone and the same sugar sequence as those of 1, and the key HMBC correlations confirmed that the sugar moiety was also attached to C-3 of the aglycone (Figure 3). Based on the above evidence, the absolute configuration of 3 was elucidated as 12-O-acetyl-20-O-(E)-2-methyl-2-butenoyl-sarcostin-(20S)-3-O-β-cymaropyranoside-(1→4)-β-cymaropyranoside.  Four known compounds were also isolated and identified as gymnepregoside H (5), [23] deacetylkidjoladinin (6), [34] gymnepregoside G (7) [23] and gymnepregoside I (8) [35], by comparison of spectroscopic data and physicochemical properties with those reported values in the literatures.
Researches showed that berberine has been shown to have antidiabetic properties. Berberine could downregulated the expression of genes involved in lipogenesis and upregulated those involved in energy expenditure in adipose tissue and muscle [36,37]. The abundant compounds 1-6 (>2 mg) were selected to evaluate their glucose uptake activity in L6 cells. Berberine (BBR, 30 µg/mL) was used as the positive control, and we used a cell-based fluorescently-labeled deoxyglucose analog kit to test the uptake activity. After incubating with samples, the 2-NBDG fluorescence intensity at the plasma membrane shows varying degrees of change. Compared to normal control (NC) group, three new compounds sylvepregosides B-D (2-4) and deacetylkidjoladinin (6) exerted weak activity, and showed glucose uptake activity with the enhancement by 0.2-, 0.16-, 0.13-and 0.1-fold at 30 µg/mL (Figure 4). Gymnepregoside H (5) possesses a moderate effect on promoting glucose uptake, which increased glucose uptake to 1.96 folds at 30 µg/mL. Sylvepregosides A (1) was the most active compound, and exhibited glucose uptake activity with 1.37-fold enhancement at 30 µg/mL. Comparing 1 and 5, both exhibited two groups of aromatic signals on C-12 and C-20, the only difference was the oligosaccharide chain in compound 1, whereby the polysaccharide may enhance the activities of glucose uptake.
For further evaluation of the efficacy of 1 and 5, a L6 cell line which stably expressed Myc-GLUT4-mOrange was used to test whether samples could also facilitate the fusion of GLUT4 with the plasma membrane. The red fluorescence of GLUT4-mOrange and the green fluorescence of FITC-myc were detected using laser confocal microscopy. As a result, we found that gymnepregoside H (5) possesses a moderate effect on promoting GLUT-4 fusion with the plasma membrane in L6 cells. Sylvepregosides A (1) was the most active compound promoting GLUT-4 fusion with the plasma membrane in L6 cells ( Figure 5). Quantification of this effect revealed that the percentage of FITC positive cells were 69% and 65%, respectively ( Figure 6).

Figure 4.
Effects of compounds 1-6 (30 µg/mL) on glucose uptake in L6 cells. Glucose uptake activities of compounds 1-6 in L6 cells using a fluorescent derivative of glucose, 2-NBDG. Berberine (30 µg/mL) was used as a positive control. After incubation for 24 h with or without 2-NBDG, the fluorescent signals were detected at Ex/Em = 485/535 nm. The results were calculated as the means ± standard error of mean (n = 5), with each performed five times; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to non-control group.  The results were calculated as the means ± standard error of mean (n = 3), with each performed three times; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to control group.

Chemicals and Reagents
Chromatography grade solvents were used for HPLC, and all other chemical reagents were analytical grade. HPLC grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Sephadex LH-20 dextran gel was purchased from Amersham Pharmacia Biotech Co. (Piscataway, NJ, USA).

Materials
The aerial part of G. sylvestre were collected from Nanning, Guangxi, China, in June 2019. The roots were dried at room temperature, macerated into a fine powder, and stored at room temperature. The plant was identified by Professor Songji Wei of School of Pharmaceutical Sciences, Guangxi University of Chinese medicine, Nanning, China.

Extraction and Isolation
The dried aerial parts of the plant (15.0 kg) were milled and then extracted with 70% EtOH (3 × 20 L, 3 days each) at room temperature to yield 1752.1 g of crude extract. Subsequently, the crude extract was suspended in H 2 O and partitioned with petroleum ether (PE) (8 × 10 L), ethyl acetate (EtOAc) (8 × 10 L), and n-butyl alcohol (n-BuOH) (8 × 10 L) to give a PE fraction (73.1 g), EtOAc fraction (383.3 g), and n-BuOH fraction (758.0 g), respectively. The EtOAc fraction (360.0 g) was subjected to HP-20 column (6 × 61 cm) chromatography eluting with a gradient solvent system of EtOH /H 2 O (10%, was changed every 24 h and cultured for 7 days to promote the differentiation of L6 cells into myotube cells. α-MEM, FBS, penicillin and streptomycin were obtained from Hyclone (Logan, UT, USA). The stock cultures were grown in 25 cm 2 culture flasks, and the experiments were carried out in 60 mm petri dishes and 96-well microtiter plates. We used R-250 (Coomassie blue) staining to identify the difference between myotubes and normal L6 cells, the difference was the myotubes showed increasing cytoplasmic tonofilaments [38].

Glucose Uptake Assay
Differentiated L6 myotubes were seeded in a 96-well black plate with the density of 1 × 10 4 -5 × 10 4 cells/well and incubated in 100 µL α-MEM for 12 h until 100% confluence. Thereafter, L6 cells were treated with proper dosages of sample or berberine dispersed in 100 µL glucose-free α-MEM dissolved in 150 µg/mL 2-NBDG (Cayman Chemical, Ann Arbor, MI, USA). After 24 h incubation in the cell incubator, the 96-well black plate was centrifuged for 5 min at 400× g at room temperature. Discarding the supernatant, 200 µL cell-based assay buffer was added into each well, and then the 96-well black plate was centrifuged for 5 min at 400× g at room temperature. After aspirating the cell-based assay buffer, each well was added into 100 µL of assay buffer. Finally, we set the excitation/emission of the fluorescent microplate reader at 485/535 nm and analyzed fluorescence intensity of 2-NBDG in each well. Zen 2010 Software (Carl Zeiss, Jena, Germany) was used to analyze the fluorescence intensity of 2-NBDG.

GLUT-4 Fusion with the Plasma Membrane
Construction of the myc-GLUT4-mOrange plasmid and cell line was performed as described previously [39] GLUT-4 fusion with the plasma membrane Myc-GLUT4-mOrange cells were cultured in six-well plates and grown on coverslips. After 2 h of starvation treatment, the cells were incubated in the presence of sample or berberine for 30 min. Then, cells were fixed with 3% polyformaldehyde for 30 min. After blocking with 2% bovine serum albumin (BSA; Hyclone, Logan, UT, USA) in phosphate buffered saline (PBS; Hyclone, Logan, UT, USA) for 30 min at room temperature, the cells were incubated with anti-myc mouse monoclonal antibody (TransGen Biotech, Beijing, China) for 1 h at room temperature. Then, the cells were washed three times with 2% BSA in PBS and incubated with goat anti-mouse-FITC (TransGen Biotech, Beijing, China). After being washed three times with 2% BSA in PBS, the coverslips were turned over and placed on a glass slide. Finally, mOrange red fluorescence and FITC green fluorescence were observed using a laser confocal microscope. GLUT-4 externalization was quantitated by determination of the percentage of GLUT4-mOrange-positive cells that exhibited FITC fluorescence at the cell surface.

Statistical Analysis
Differences between groups were analyzed by one-way analysis of variance (ANOVA). Data are shown as means ± standard error (M ± SEM). Tukey's post hoc test of Graph-Pad Prism 5.0 software packages was used to perform statistical analyses. A probability (p) values < 0.05 were regarded as statistically significant.

Conclusions
In this study, four new C 21 steroidal glycosides sylvepregosides A-D (1-4), as well as four known compounds (5)(6)(7)(8) were obtained from Gymnema sylvestre. Compounds 1-6 presented the effects of glucose uptake in L6 cells at 1.10-to 2.37-fold, respectively. Specifically, compound 1 exerted the strongest activity for glucose uptake, with 1.37-fold enhancement, and compound 5 showed moderate uptake activity, by increasing glucose uptake by 0.96-fold. Further study shows that compounds 1 and 5 could promote GLUT4 fusion with the plasma membrane in L6 cells. Our research suggested that compounds 1 and 5 could offer promising lead structures with glucose uptake activity, which could be meaningful to the development of pharmaceutical products. Meanwhile, it also provided a clue for potentially active anti-diabetic constituents in the plants of genus Gymnema.