12,23-Dione dammarane triterpenes from Gynostemma longipes and their muscle cell proliferation activities via activation of the AMPK pathway

The aging population is growing rapidly around the world and there is also an increase in sarcopenia, which is characterized by decreased muscle mass, strength and function in the elderly population. AMP-activated protein kinase (AMPK) is an essential sensor and regulator of glucose, lipid and energy metabolism throughout the body. Previous studies have shown that AMPK pathway activation by regular exercise and appropriate dietary control have beneficial effects on skeletal muscle. In the process of searching for new AMPK activators from medicinal plants, we isolated and characterized eight new 12,23-dione dammarane triterpenoids (1–3 and 5–9), as well as one known gypentonoside A from Gynostemma longipes. When all isolates were tested for their AMPK activation activities, seven compounds (1 and 3–8) were significantly activated AMPK phosphorylation in mouse C2C12 skeletal muscle cell lines. Since G. longipes contained a significant amount of active compound 1 (over 2.08% per dried raw plant), it suggested the potential of this plant to be developed as a functional food or botanical drug that enhances muscle proliferation by activating AMPK signaling pathways.

Dammarane triterpenes enhanced muscle proliferation through activating AMPK. Regeneration of damaged skeletal muscles depends on satellite cells (known as quiescent muscle precursor cells), which play an important role in the proliferation and differentiation of myoblasts to form or repair muscle fibers 18,19 . Recently, several studies have demonstrated that decreased proliferation of myoblasts and cytotoxicity can reduce the number of muscle fibers [20][21][22] . Therefore, mouse C2C12 cells, also known as myoblastic cells, were chosen in this research because they are a valid model to study muscle cell proliferation 7,20 . In a search for new bioactive natural products with promising activity for muscle proliferation, we found that the 95% EtOH eluted fraction on SP70 resin of the total extract could potentially be a 'hit' using an in vitro screening method. This fraction stimulated an increase in proliferation of C2C12 myoblast cell about 20-30% compared to the negative control during 24 and 48 hours of treatment using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) methods (Fig. 4A,B). The proliferation also expressed dose-dependently using a cell counting method ( Supplementary  Fig. S47). As AMPKα is an important regulator during the process of muscle proliferation 7,23 , we tested the effect   Bioassay-guided isolation from G. longipes resulted in the isolation of nine compounds (1-9) and all of the isolates were evaluated for their enhancing effect on muscle proliferation in C2C12 myoblast cells. After 48 hours of treatment, cell viability was assessed using the MTT and WST-1 methods. Seven compounds (1 and 3-8) increased cell viability by about 20-30% (Fig. 5A). Notably, compounds 6 and 7 exhibited the highest induction of cell proliferation at a value from 131.95 ± 2.54% to 136.19 ± 2.73%. Since compound 1 was identified as a major constituent of this plant (over 2.08% of the material), it was selected to examine the effects on the proliferation of two cancer cell lines, MCF-7 and MDA-MB-231 breast cancer cells. As shown in Supplementary Fig. S50, cell viability was not changed when the cells were incubated with a fraction of G. longipes (10 μg/mL) or compound 1 (10 μM) for 2 days. These results suggested that the active fraction and major compound of G. longipes stimulate muscle cell proliferation selectively and do not induce the outgrowth of harmful cancer cell lines.
A number of studies demonstrated that dammarane triterpenoids are potent classes of AMPK-activating agents 16,17,24 . In order to evaluate the potential activities of compounds from G. longipes, nine isolates (1-9) were measured the stimulatory effects on the p-AMPK and p-ACC expressions using mouse C2C12 myotubes. Results in Supplementary Figs S51 and S52 indicated that compounds 5, 6, and 7 increased the phosphorylation of AMPK and ACC proteins strongly, while compounds 1, 3, 4, and 8 exhibited less efficient. Therefore, potential candidates (1, 5, 6, and 7) were further confirmed the activation effect on p-AMPK in C2C12 myoblasts. These compounds clearly increased the phosphorylation of the target protein compared to Aicar as a positive control ( Supplementary Fig. S53).
Because compound 1 is the major constituent of the active fraction, it was selected for further evaluating the activation of p-AMPK in a time-dependent manner using the mouse C2C12 myoblast model. Remarkably, compound 1 up-regulated the phosphorylation of AMPK from 30 to 90 minutes ( Fig. 5B and Supplementary Fig. S54). Besides, compound 1 also induced the expression of AMPK slightly at 24 hours after treatment ( Supplementary  Figs S55 and S56). These results suggested that triterpenoids from G. longipes could significantly stimulate the p-AMPK expression in both mouse C2C12 myotubes and myoblasts.
Considering the structures and activities of all nine isolates, the core structure of the dammarane skeleton might contribute mainly to the AMPK activation effect. Further examination of structure-activity relationships (SAR) revealed that compounds with a longer saccharide side chain at C-3 showed lower AMPK inhibition effects: 4 (4 sugar units) < 1 (3 sugar units) < 5 (2 sugar units) < 6 and 7 (no sugar). It was also noteworthy that with the same number of sugar units, the attachment of a sugar unit to C-20 remarkably reduced the activity (2 < 4). Furthermore, the hydration of olefinic bond at C-20 and C-24 slightly increased the effect in the following order: 9 (two double bonds) < 1 (one double bond) < 8 (no double bond). The change of hydroxy (6) and ketone substitutions (7) at C-3 did not cause any difference in activity. Finally, to assess the effects of compound C during muscle proliferation, the cell growth and expression of p-AMPK were analyzed using the mouse C2C12 myoblast model. Interestingly, co-treatment of compound C with the active fraction or compound 1 decreased the percentage of cell viability after 48 hours of incubation, compared to the number of C2C12 cells exposed to the fraction or compound 1 alone (Fig. 5C). The decline of the p-AMPK expression was also observed after 1 hour of incubation. Similarly, the stimulation effect of compound 1 on p-AMPK was down-regulated by co-treatment with compound C (Fig. 5D and Supplementary Fig. S49). Consequently, the activation of the AMPK signaling pathway by G. longipes's fraction and compound 1 contributes relatively to muscle proliferation by inducing the growth of C2C12 myoblasts.
Effects of dammarane triterpenes on DNA synthesis during cell proliferation. It has been previously reported that 5-bromo-2′-deoxyuridine (BrdU) can be used to label the DNA of cells in the S-phase. This method is useful for quantifying the degree of DNA synthesis during cell proliferation 25 . Therefore, in order to confirm whether compound 1 from G. longipes induces cell proliferation, immunochemical staining analysis with a BrdU antibody was performed. In the first experiment, C2C12 myoblast cells were treated with compound 1 (20 μM) or the SP70-EtOH 95% fraction (20 μg/mL) and incubated for 8 hours. Next, the cells were incubated for 2 hours with or without BrdU. The BrdU incorporation into the nucleus was assessed using a fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody and DAPI staining. As shown in Fig. 6A and Supplementary Fig. S57, the number of BrdU-positive cells was significantly increased in the group treated with the active fraction of G. longipes or compound 1 compared to DMSO treatment alone. We also evaluated the BrdU-labeled cells using a flow cytometer for BrdU and PI staining. When C2C12 cells were incubated C2C12 cells were incubated with tested compounds for 48 hours and cell viability was assessed using the MTT or WST method. Each value is expressed as the mean ± SD (n = 3); *p < 0.05 and **p < 0.01, compared to negative control group using the WST method; while # p < 0.05 and ## p < 0.01, compared to the vehicle group using the MTT method. (B) C2C12 myoblast cells were exposed to compound 1 (20 μM) and incubated for 15 to 120 minutes. Phosphorylation of AMPK protein in the cells was assessed by Western blot analysis. Values are expressed as the mean ± SD (n = 3), *p < 0.05 and **p < 0.01, compared to the negative control group. (C) The effect of cotreatment compound C with the active fraction and compound 1 on the cell viability. C2C12 myoblasts were re-treated with compound C (20 μM) for 15 minutes and then the cells were continuously incubated with test samples for 2 days. After that, the percentage of cell viability was evaluated using MTT method. Results are presented as the mean ± SD (n = 3); *p < 0.05 and **p < 0.01, compared to negative control group (without compound C treatment); while # p < 0.05 and ## p < 0.01 compared to co-treatment with compound C and test samples group, respectively. (D) Down-regulatory effect of compound C on p-AMPK (Thr 172 ) when cotreatment with the active fraction or compound 1. After 15 minutes re-incubated with compound C (20 μM), mouse C2C12 myoblasts were then exposed to test samples for 1 hour. Phosphorylation of AMPK protein was measured by Western blotting. Data were calculated as the mean ± SD (n = 3); **p < 0.01 compared to the negative control, while ## p < 0.01 compared to co-treatment with compound C and test samples group, respectively. with or without BrdU treatment, the fluorescence intensity was significantly increased from 33.46 ± 0.58% to 55.41 ± 0.88%. In order to confirm the concentration dependence in the cells, compound 1 was treated for 8 hours at different concentrations (20, 10, and 5 μM) and then incubated with BrdU for 2 hours. The BrdU positive cell population increased in a concentration-dependent manner to 65.22 ± 3.43%, 59.90 ± 0.84% and 56.00 ± 0.81%, respectively (Fig. 6B). Moreover, to better understand the effects of co-treatment compound C with test samples on DNA synthesis during cell proliferation, immunofluorescence detection and flow cytometry analysis for BrdU staining were carried out. For qualitative analysis, the number of red stained cells notably reduced in the groups co-treated with compound C compared to the active fraction or compound 1 alone (Fig. 6A and Supplementary Fig. S57). To further examine the fluorescence intensity of each group, the BrdU-labeled cells were determined using flow cytometry for BrdU staining only (Supplementary Fig. S58). This results indicated that co-treatment of compound C with the active fraction or compound 1 failed to increase the number of BrdU-positive cells.
Effects of dammarane triterpenes on glucose uptake and ATP levels. Recent studies suggested the importance of AMPKα enhances glucose uptake and ATP production during the process of muscle proliferation 7 . Considering that the glucose uptake level was evaluated using a fluorescent-tagged glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), to monitor intracellular glucose uptake. As illustrated in Fig. 6C, the active fraction and compound 1 as well as insulin (as a positive control) increased glucose uptake in mouse C2C12 myoblasts.
The effects of dammarane triterpenes from G. longipes on the mitochondrial ATP synthesis was examined by treatment mouse C2C12 myoblasts with the active fraction and compound 1 in approximately 70 minutes. The ATP levels of the active fraction and compound 1 increased drastically from 30 to 50 minutes comparison with the vehicle group (Fig. 6D). However, the rate of ATP production reduced rapidly after 1 hour of incubation with these samples. During the proliferation of myoblasts, the production of ATP is an essential downstream target of glycolysis, and cells used these ATP (approximately 30%) which is supplied by oxidative phosphorylation (OXPHOS) as the major source of metabolic energy 26 . Taken together, the role of triterpenoids from G. longipes, which regulate muscle proliferation by increasing glucose uptake and ATP production has been suggested.

Enhancement of cell proliferation by dammarane triterpenes through cell cycle regulation.
Several previous studies have proposed that ginseng root extract promotes glucose uptake in muscle cells and also induces the proliferation of β-cells 27 , and ginsenoside Rg 1 from Panax ginseng enhances myoblast differentiation and myotube growth 28 . Thus, the effect of dammarane triterpenoid from G. longipes on cell proliferation through regulation of cell cycle was also evaluated using propidium iodide (PI) staining method 29 . C2C12 myoblasts were exposed to compound 1 (20 μM) for 12 and 24 hours. The cell cycle distributions at different phases were significantly changed, compared to the vehicle group. After 12 hours of incubation with compound 1, the cells with 2 N DNA content (G 0 /G 1 phases) decreased remarkably from 72.07 ± 0.63% to 65.09 ± 2.49%, while the cells with >2 N DNA content (S or G 2 /M phases) increased about 8% compared with negative control ( Supplementary  Fig. S59). Moreover, the proportion of G 2 /M phases increased significantly from 13.64 ± 1.47% to 22.41 ± 0.81% when the cells were incubated with compound 1 for 24 hours in comparison with the group treated with DMSO. In our study, we have described novel natural product compounds that enhance myoblast proliferation. It can be noted that previous research indicates that compounds promoting skeletal muscle stem cell proliferation also have the potential to enhance muscle regeneration in vivo 30 . As a further study, we believe that assessment of the novel compounds described in this manuscript as promoters of muscle regeneration is warranted.

Conclusion
In this study, we reported the findings of eight new 12,23-dione dammarane triterpenes from G. longipes, a traditional Vietnamese medicinal plant. Seven of the compounds (1 and 3-8) activated significantly AMPK phosphorylation in mouse C2C12 cell lines. The result of AMPK activation by compound 1 could stimulate glucose uptake into the cells and increase mitochondrial ATP synthesis. The effect of dammarane triterpenes on DNA synthesis during cell proliferation also showed that compound 1 increased the BrdU positive cell population in a dose-dependent manner. Analysis of cell cycle using flow cytometry displayed clearly that compound 1 reduced the 2 N DNA content (G 0 /G 1 phases) while the ratio of G 2 /M phases was significantly increased. These results suggested strongly that compound 1 enhances significantly the proliferation of C2C12 myoblast cells (Fig. 7). Furthermore, a considerable amount of active compound l (over 2.08% per dried raw plant) in G. longipes suggested that it may be a promising candidate for development of functional food or botanical drug. These results also indicated that new dammarane-type compounds are promising candidates for muscle proliferation via activation of AMPK signaling pathways and could be further studied and developed as therapeutics for geriatric diseases.

Methods
General experimental procedures. Optical rotations were measured in a JASCO P-2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). IR data were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA). NMR data were analyzed using an AVANCE 500 MHz spectrometer (Bruker, Germany) or a JNM-ECA 600 MHz spectrometer (Jeol, Japan). HRESIMS values were analyzed using an Agilent Technologies 6130 Quadrupole LC/MS spectrometer equipped with an Agilent Technologies 1260 Infinity LC system (Agilent Technologies, Inc., Santa Clara, CA, USA) and an INNO C18 column (4.6 × 150 mm, 5 μm particle size, 12 nm, J.K.Shah & Company, Korea). Silica gel (particle size: 63−200 μm) and RP-C 18 (particle size: 40-63 μm) were purchased from Merck (Darmstadt, Germany) and Sephadex LH-20 from Sigma-Aldrich (St. Louis, MO, USA) were used for column chromatography (CC). Silica gel 60 F 254 and RP-18 F 254 TLC Figure 6. Increased DNA synthesis in C2C12 myoblasts cells by dammarane triterpenes from G. longipes. (A) C2C12 myoblast cells were re-treated with compound C (20 μM) for 15 minutes and the cells were then incubated with the active fraction (20 μg/mL) or compound 1 (20 μM) for 8 hours. Cells were then incubated for 2 hours in the presence or absence of BrdU. After fixation and permeabilization, the cells were incubated with antibodies and stained with the DAPI solution. Cells images were observed by fluorescence microscopy. (B) C2C12 myoblast cells were incubated with compound 1 at different concentrations for 8 hours. After incubation for 2 hours with or without BrdU, the cells were harvested and flow cytometric analysis for BrdU and PI staining was performed. The cell distribution was displayed using a histogram. Data presented as the mean ± SD (n = 2), *p < 0.05 and **p < 0.01, compared to the DMSO group. (C) Stimulation effects of the active fraction or compound 1 on glucose uptake in C2C12 myoblasts using the 2-NBDG probe. The cells were incubated with test samples or insulin (as a positive control) for 1 hour. The fluorescent intensity was observed at ex/em = 450/535 nm using a fluorescence microplate reader. Each value is represented as the mean ± SD (n = 3), *p < 0.05 compared to negative control. (D) Dammarane triterpenes from G. longipes increased intracellular ATP level of C2C12 myoblasts. The cells were treated with the active fraction (20 μg/mL) or compound 1 (20 μM) from 0 to 70 minutes. Mitochondrial ATP synthesis was measured using ATP bioluminescence determination assay. Data were calculated as the mean ± SD (n = 3); the p value for compound 1 (*p < 0.05, **p < 0.01) and total fraction ( # p < 0.05) compared to negative control. for partial maturase K gene, rbcL134-F (5′-GGACAACTGTGTGGACCGAT-3′), rbcL590-R (5′-AAACGGTCTC TCCAACGCAT-3′) for partial ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene and rp143-F (5′-ACCTTCCCGACCACGATTTC-3′), rp686-R (5′-CGGAACAACCCCGTTCACTA-3′) for rpl20-rps12 intergenic spacer region. All nucleotide sequence data were recorded in the GenBank sequence database, National Center for Biotechnology Information (NCBI), with the accession numbers KX619478, KX619479, KX619480 and KX619481 respectively. DNA sequencing data were analyzed using the Geneious DNA sequencing analysis software (version 8.1.8, Biomatters Ltd, New Zealand). DNA sequences were compared to published sequences available from Genbank using the Basic Local Alignment Search Tool (BLAST). All analysis sequences were matched with Gynostemma longipes with pairwise identity 99.9% (ITS1-5.8S-ITS2 region), 99.6% (rbcL gene), 99.8% (rpl20-rps12 intergenic spacer region) and 99.9% (matK gene). This additional genetic information supported morphological authentication of Gynostemma longipes (Fig. 1).