In Vitro
Chiisanogenin (10-40 μM; 24 h) causes no cytotoxicity when tested on differentiated L6-GLUT4myc cells[1]. A 15-min exposure to Chiisanogenin (40 μM) triggers calcium influx into L6-GLUT4myc cells, with an amplitude of 2.4-fold. The extracellular space is the main source of this influx, which can go on to promote AS160 phosphorylation and glucose uptake through calcium signaling[1]. Chiisanogenin (10 μM) crosses Caco-2 cell monolayers readily, with an efflux ratio of 1.31 and no significant active efflux[1]. For the human insulin receptor (PDB ID: 8VJB), Chiisanogenin shows strong binding, the predicted binding energy being -6.96 kcal/mol and the Kd being 0.318 μM. Key residues of the receptor take part in the binding mode through hydrophobic interactions and hydrogen bonding[1]. Glucose uptake in differentiated L6-GLUT4myc cells rises dose-dependently after Chiisanogenin (20-40 μM; 15 min): 1.8-fold at 20 μM and 2.6-fold at 40 μM[1]. After Chiisanogenin (20-40 μM; 15 min), GLUT4 translocates to the plasma membrane of differentiated L6-GLUT4myc cells, and GLUT4 levels are significantly higher both in the plasma membrane and at the cell surface. Plasma membrane GLUT4 is up 2.1-fold at 20 μM and 2.3-fold at 40 μM[1]. Differentiated L6-GLUT4myc cells treated with Chiisanogenin (20-40 μM; 15 min) show activation of the IRS1/PI3K/Akt/AS160 insulin signaling pathway; PI3K activity is required for this activation and for the GLUT4 translocation that follows[1]. Autophagic flux returns to normal in H/R-treated HK-2 cells given Chiisanogenin (12.5-50 μM), through stronger autophagosome synthesis and autophagosome-lysosome fusion[3]. Chiisanogenin (12.5-50 μM) drives TFEB into the nucleus of H/R-treated HK-2 cells, boosts autophagosome-lysosome fusion, and returns autophagy-lysosome homeostasis to normal[3]. Lysosomal membranes stay intact in H/R-treated HK-2 cells under Chiisanogenin (12.5-50 μM; 2 h), CTSB leakage is prevented, and the NLRP3 inflammasome activation and pyroptosis that would follow are inhibited[3]. Pyroptosis induced by H/R in HK-2 cells is inhibited by Chiisanogenin (12.5-50 μM; 2 h), which suppresses NLRP3 inflammasome activation together with downstream GSDMD cleavage, cytokine release and membrane damage[3]. In H/R-treated HK-2 cells, direct high-affinity binding to MLKL is seen with Chiisanogenin (12.5-50 μM; 2 h for Co-IP; 12 h for CETSA); MLKL phosphorylation and oligomerization are inhibited, and the MLKL-LAMP1 interaction is disrupted, so lysosomal membrane damage is prevented[3]. Through negative regulation of the MLKL signaling pathway, Chiisanogenin (12.5-50 μM; 48 h) blocks the fibrotic transdifferentiation and inflammatory response that TGF-β1 induces in HK-2 cells[3]. No anti-rotavirus activity is found for Chiisanogenin (up to 100 μM) in MA-104 cells[4]. Tumor cell lines P-388, L-1210, A549 and SNU C4 are cytotoxic targets in vitro for Chiisanogenin (0.2-0.5 mg/mL); ED50 values span 0.2 to 0.5 mg/mL, and SNU C4 cells respond most strongly[4]. Normal L02 hepatocytes show low cytotoxicity from Chiisanogenin (6.25-200 μM), with just a slight drop in viability at 200 μM, whereas growth of HepG2 hepatocellular carcinoma cells is inhibited dose-dependently[8]. Antibacterial activity of Chiisanogenin (overnight) is broad-spectrum: Bacillus subtilis ATCC 6633 is inhibited at an MIC of 50 μg/mL, and Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 65389 and Proteus vulgaris ATCC 3851, together with Salmonella typhimurium ATCC 14028 are inhibited at an MIC of 100 μg/mL (all). No inhibition is observed for *Escherichia coli* ATCC 35218 (>200 μg/mL)[5]. Chiisanogenin (12.5-100 μM; 2 h) offers potent protection to HK-2 cells from H/R-induced injury, and the optimal concentration for cytoprotection is 25 μM[3]. In cell-free molecular docking assays, specific and stable interactions form between Chiisanogenin and key active site residues of MLKL, with high-affinity binding[3]. At 0.5 mg/mL, Chiisanogenin is a weak inhibitor of β-glucuronidase and a potent inhibitor of partially purified rat gastric H+/K+-ATPase (IC50 of 0.5 mg/mL); HP urease is not inhibited at concentrations up to 0.5 mg/mL[4]. This UPLC-MS/MS method quantifies Chiisanogenin (5.00-500 ng/mL) reliably in spiked rat plasma. Linearity holds over 5.00-500 ng/mL, the LLOQ is 5.00 ng/mL, precision is below 11%, accuracy is within 8%, and stability is good under sample processing and storage conditions[6]. For H9c2 rat cardiomyocytes, Chiisanogenin (5-800 μM; 24 h) leaves viability unaffected at up to 100 μM, promotes proliferation at 50 μM, and inhibits viability from 200 μM upward[7]. Survival of aconitine-damaged H9c2 rat cardiomyocytes is significantly increased by Chiisanogenin pretreatment (2.5-50 μM; 2 h pretreatment followed by 24 h aconitine exposure) throughout the 2.5 μM to 50 μM range, although divaroside protects more strongly[7]. TNF-α-induced proliferation of HFLS-RA cells is significantly inhibited by Chiisanogenin (20 μmol/L; 24 h), and no cytotoxicity is observed within this concentration range[9]. Low cytotoxicity toward H9c2 rat cardiomyocytes is displayed by Chiisanogenin (0-1000 μM for 24 h): no obvious toxicity up to 50 μM, but significantly inhibited viability at 400 μM and above[10]. Against cytotoxic damage from 50 μM BaCl2, Chiisanogenin (50 μM; 2 h pre-incubation, then 24 h co-incubation with 50 μM BaCl2) gives H9c2 rat cardiomyocytes significant protection[10]. For PKA, Chiisanogenin has a predicted binding energy of -7.8 kcal/mol, reflecting high binding affinity and the superiority of its ligand-receptor interaction[10].
Cell Viability Assay[1]
| Cell Line | differentiated L6-GLUT4myc rat skeletal muscle cells |
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| Concentration | 10, 20 and 40 μM |
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| Incubation Time | 24 h |
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| Result | Maintained >95% cell viability relative to control at all tested concentrations. |
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Western Blot Analysis[1]
| Cell Line | differentiated L6-GLUT4myc rat skeletal muscle cells |
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| Concentration | 20 and 40 μM |
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| Incubation Time | 15 min |
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| Result | Increased total GLUT4 in the plasma membrane fraction by 2.1-fold at 20 μM relative to control. Increased total GLUT4 in the plasma membrane fraction by 2.3-fold at 40 μM relative to control. |
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Western Blot Analysis[1]
| Cell Line | differentiated L6-GLUT4myc rat skeletal muscle cells |
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| Concentration | 20 and 40 μM |
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| Incubation Time | 15 min |
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| Result | Increased phosphorylation of IRS1 (Tyr612), PI3K (Tyr458), Akt (Thr308), and AS160 (Thr642). Inhibited chiisanogenin-induced phosphorylation of PI3K, Akt, and AS160, and reduced GLUT4 levels in the plasma membrane when pretreated with wortmannin. |
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Western Blot Analysis[1]
| Cell Line | differentiated L6-GLUT4myc rat skeletal muscle cells |
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| Concentration | 40 μM |
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| Incubation Time | 15 min |
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| Result | Increased AS160 phosphorylation. Reduced chiisanogenin-induced AS160 phosphorylation when pretreated with BAPTA-AM. |
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Cell Viability Assay[3]
| Cell Line | human proximal tubular epithelial HK-2 cells (exposed to hypoxia/reoxygenation (H/R) injury) |
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| Concentration | 12.5, 25, 50 and 100 μM |
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| Incubation Time | 2 h (pretreatment before H/R injury) |
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| Result | Exhibited the most significant cytoprotective effect against H/R-induced injury compared to other candidate compounds. Provided optimal protection at 25 μM with stable, consistent effects and no observable toxicity or morphological abnormalities. Showed reduced efficacy at higher doses (e.g., 50 μM) due to mild compound-induced cytotoxicity. |
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Cell Viability Assay[7]
| Cell Line | H9c2 rat cardiomyocytes (non-injury baseline) |
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| Concentration | 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM and 800 μM |
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| Incubation Time | 24 h |
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| Result | Showed no significant effect on cell viability at concentrations up to 100 μM compared to the control group. Significantly promoted cell proliferation at 50 μM (p < 0.01). Significantly inhibited cell viability at 200 μM, 400 μM, and 800 μM (p < 0.01 or p < 0.001). |
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Cell Viability Assay[7]
| Cell Line | aconitine-injured H9c2 rat cardiomyocytes |
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| Concentration | 2.5 μM, 5 μM, 10 μM, 25 μM and 50 μM |
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| Incubation Time | 2 h (pretreatment); 24 h (aconitine exposure) |
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| Result | Significantly increased the viability of aconitine-injured H9c2 cells at all tested concentrations (p < 0.01 or p < 0.001). Exhibited a relatively weak protective effect compared to divaroside (DVS). |
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Cell Viability Assay[9]
| Cell Line | TNF-α-induced human fibroblast-like synoviocyte rheumatoid arthritis (HFLS-RA) cells |
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| Concentration | 20 μmol/L |
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| Incubation Time | 24 h |
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| Result | Significantly inhibited the proliferation of TNF-α-induced HFLS-RA cells, with cell proliferation activity percentage within the range of 53.73 to 94.4% compared to the TNF-α induction group. Showed no toxicity to HFLS-RA cells. |
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Cell Cytotoxicity Assay[10]
| Cell Line | H9c2 rat cardiomyocytes |
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| Concentration | 0, 2.5, 5, 10, 25, 50, 100, 200, 400, 800 and 1000 μM |
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| Incubation Time | 24 h |
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| Result | Exhibited no inhibitory effect on H9c2 cell viability and did not affect cell proliferation at concentrations up to 50 μM. Significantly inhibited H9c2 cell viability at concentrations of 400 μM and above. |
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Cell Cytotoxicity Assay[10]
| Cell Line | BaCl2-injured H9c2 rat cardiomyocytes |
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| Concentration | 50 μM |
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| Incubation Time | 2 h pre-incubation; 24 h co-incubation with 50 μM BaCl2 |
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| Result | Significantly protected H9c2 cells against cytotoxicity induced by 50 μM BaCl2. |
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In Vivo
In mice, Chiisanogenin (250-500 mg/kg; p.o.; single dose) lowers postprandial blood glucose in a dose-dependent way: peak postprandial glucose falls by 32% at 250 mg/kg and by 46% at 500 mg/kg[1]. In carrageenan-induced rat edema, Chiisanogenin (10-30 mg/kg; p.o.; daily; 7 days) shows dose-dependent antiinflammatory effects; at 30 mg/kg, paw edema is reduced by 30.4% at 3 hours, dye leakage by 50.1%, and trypsin activity is inhibited by 37.1%, while oxidative stress markers are attenuated[2]. In male C57BL/6 mice, Chiisanogenin (50-200 mg/kg; i.g.; daily; 7 days) limits the AKI-to-CKD transition and chronic renal fibrosis through regulation of MLKL-mediated inflammatory responses, with efficacy comparable to that of NAC[3]. In rats with BaCl2-induced ventricular arrhythmia, Chiisanogenin (25 mg/kg; i.p.; single dose; 30 minutes before arrhythmia induction) shows potent anti-arrhythmic activity, cutting the overall arrhythmia time to 59.33 seconds and restoring oxidative stress markers as well as myocardial ion pump activities to normal[10].
| Animal Model | ICR mice (female, 5 weeks old, 18–22 g) were orally administered D-(+)-glucose (2 g/kg in PBS) to induce postprandial hyperglycemia[1] |
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| Dosage | 250 mg/kg; 500 mg/kg |
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| Administration | p.o.; single dose |
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| Result | Reduced postprandial blood glucose levels by 32% at 30 minutes after glucose challenge (250 mg/kg dose) compared to the control group. Reduced postprandial blood glucose levels by 46% at 30 minutes after glucose challenge (500 mg/kg dose) compared to the control group. Confirmed significant reductions in overall blood glucose levels via area under the curve (AUC) analysis for both doses relative to the control group. |
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| Animal Model | Sprague-Dawley (4-week-old male, 250–300 g, carrageenan-induced hind paw edema)[2] |
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| Dosage | 10 mg/kg; 30 mg/kg |
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| Administration | p.o.; daily; 7 days |
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| Result | Reduced paw edema by 30.4% at 3 hours post-carrageenan injection at 30 mg/kg. Reduced pontamine sky blue dye leakage into the peritoneal cavity by 37.5% and inhibited trypsin activity by 27.4% at 10 mg/kg. Reduced pontamine sky blue dye leakage into the peritoneal cavity by 50.1% and inhibited trypsin activity by 37.1% at 30 mg/kg. Reduced serum lipid peroxide levels to 50.0 nmole/mL MDA, hydroxy radical levels to 5.51 nmole/mg protein, and increased serum SOD activity to 2.97 units/mg protein at 10 mg/kg. Reduced serum lipid peroxide levels to 41.8 nmole/mL MDA, hydroxy radical levels to 5.08 nmole/mg protein, and increased serum SOD activity to 3.21 units/mg protein at 30 mg/kg. Reduced RA factor from 10 to 6 and CRP factor from 10 to 7 at 10 mg/kg. Reduced RA factor to 5 and CRP factor to 5 at 30 mg/kg. Reduced hepatic XO activity to 11.7 nmoles uric acid/mg protein/min, increased SOD activity to 4.98 units/mg protein, GPX activity to 1.69 nmoles NADPH oxidized/mg protein/min, and catalase activity to 170.7 nmoles H2O2 dissipated/mg protein at 10 mg/kg. Reduced hepatic XO activity to 10.8 nmoles uric acid/mg protein/min, increased SOD activity to 6.17 units/mg protein, GPX activity to 1.90 nmoles NADPH oxidized/mg protein/min, and catalase activity to 183.5 nmoles H2O2 dissipated/mg protein at 30 mg/kg. |
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| Animal Model | C57BL/6 (male, 20–22 g, SPF conditions) underwent left nephrectomy followed by 40 min of right renal pedicle clamping to induce renal ischemia, then the clamp was released for reperfusion.[3] |
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| Dosage | 50 mg/kg; 100 mg/kg; 200 mg/kg |
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| Administration | i.g.; daily; 7 days |
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| Result | Significantly reversed IRI-induced elevations in serum creatinine and blood urea nitrogen levels at 100 mg/kg, with efficacy equivalent to MLKL siRNA knockdown and comparable to positive control NAC. Significantly reduced acute renal injury pathological scores and suppressed expression of renal injury markers KIM-1 and NGAL. Reversed IRI-induced increases in p-MLKL levels and downregulation of LAMP1, promoted nuclear translocation of TFEB, alleviated accumulation of LC3-II and p62, and restored autophagic flux. Inhibited IRI-induced pyroptosis, reducing extracellular LDH release, lowering IL-1β and IL-18 levels, suppressing cytosolic CTSB leakage, and blocking activation of the NLRP3/GSDMD-N axis. Significantly reduced IRI-induced elevations in serum creatinine and blood urea nitrogen levels at 14 days postreperfusion. Significantly decreased interstitial collagen deposition (measured via Masson's trichrome and Sirius red staining) and downregulated expression of fibrotic markers α-SMA, fibronectin, and Collagen I, with efficacy comparable to positive control NAC. |
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| Animal Model | Wistar rats (male, SPF grade, 220-250 g, ventricular arrhythmia induced by BaCl2)[10] |
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| Dosage | 25 mg/kg |
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| Administration | i.p.; single dose; 30 minutes before arrhythmia induction |
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| Result | Shortened the total duration of ventricular arrhythmia to 59.33 seconds, the duration of ventricular premature contractions to 5 seconds, and the duration of ventricular tachycardia to 54.33 seconds (all p < 0.001). Increased serum SOD levels and decreased serum MDA levels (p < 0.001). Increased myocardial Na+-K+-ATPase (p < 0.01) and Ca2+-Mg2+-ATPase (p < 0.01) activities. Reduced myocardial cell disorder, interstitial edema, and focal vacuolar degeneration compared with the model group. |
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Data provided by the manufacturer. Numbered citations refer to the Literature Cited list in the product description.
