Ginsenoside monomers increase mitochondrial function in cardiomyocytes by activating the SIRT1 pathway
As we showed, GS are a complex mixture of ginsenoside monomers of different proportions (Supplementary Fig. 1-2) . To further gain insight into which ginsenoside in GS promotes mitochondrial function through increased glucose oxidation and SIRT1 activation, we performed a series of experiments to investigate the effects of different ginsenosides on basal OCR, ATP content, and key protein levels in H9c2 cells. Similar to that of GS, different ginsenoside monomers, such as Rg1, Re, Rf, Rb1, Rc, Rh1, Rb2, Rb3, Rd, S-Rg3, R-Rg3, and Rk1, at individual concentrations of GS increased basal OCR in H9c2 cells after 48 h treatment compared with the control group (Fig. 8A) . Moreover, ATP content was higher in Re-, Rf-, Rb1-, Rc-, Rh1-, Rb2-, or Rb3-treated H9c2 cells than the control or GS group (Fig. 8B) . These results confirm that these ginsenosides can promote mitochondrial energy metabolism. Additionally, key proteins of the glucose oxidation process, HK-II and MPC1, were increased by Rg1, Re, Rf, and Rc, which was similar to GS (Fig. 8C-8D) . For SIRT1 expression, we found that the level of SIRT1 was upregulated by most of the ginsenoside monomers, with the exception of Rk3 (Fig. 8C-8D) . Especially, ginsenosides Rg1, Re, Rc, Rh1, Rb2, and Rb3 had a similar role in increasing SIRT1 expression as GS (Fig. 8C-8D) . Taken together, these results identified the major ginsenosides in GS, including Rg1, Re, Rf, Rb1, Rc, Rh1, Rb2, and Rb3, which promote aerobic cellular respiration and SIRT1-mediated mitochondrial biosynthesis to protect cardiomyocytes.