Typhaneoside regulates the PI3K/Akt/mTOR autophagy transduction pathway. [1]

In a hypoxia/reoxygenation (A/R) model using primary neonatal rat cardiomyocytes, pretreatment with Typhaneoside-containing serum (40 mg/kg) significantly reduced the number of autophagosomes observed by transmission electron microscopy compared to the model (A/R) group. [1]
Immunofluorescence detection of LC3 showed that compared to the control group, the model group had increased red fluorescent spots (indicating abundant autophagosomes), while the Typhaneoside-containing serum group significantly reduced the number of red spots, suggesting decreased autophagosome formation. [1]
Western blot analysis revealed that compared to the model group, the Typhaneoside-containing serum group significantly decreased the LC3‑II/LC3‑I ratio and increased p62 protein expression. In addition, the ratios of p‑Akt/Akt and p‑mTOR/mTOR were significantly increased. [1]
In experiments with rapamycin (RA, an mTOR inhibitor) pretreatment: compared to the RA + A/R group, the RA + drug + A/R group showed no significant change in LC3‑II/LC3‑I ratio or p62 expression, indicating that RA blocked the autophagy inhibition by Typhaneoside-containing serum, and that the drug inhibits autophagy through the mTOR pathway. [1]
In experiments with API‑2 (an Akt inhibitor) pretreatment: compared to the drug + A/R group, the API‑2 + drug + A/R group showed significantly decreased p‑Akt/Akt ratio, and API‑2 blocked the autophagy inhibition by Typhaneoside-containing serum, indicating that the drug also inhibits autophagy via the Akt signaling pathway. [1]

In a rat model of heart failure after myocardial infarction (left anterior descending coronary artery ligation), Typhaneoside was administered at doses of 10, 20, and 40 mg/kg once daily for 4 weeks. Compared to the model group, the drug treatment significantly improved cardiac function: left ventricular end‑diastolic diameter (LVEDD) and left ventricular end‑systolic diameter (LVESD) decreased, while left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (LVFS), stroke volume (SV), and cardiac output (CO) increased. For example, at 40 mg/kg: LVEDD 7.45±1.02 mm (vs. model 8.82±0.43), LVESD 4.83±0.14 mm (vs. model 6.66±0.56), LVEF 59.88±3.90% (vs. model 41.66±4.44), LVFS 29.92±4.55% (vs. model 20.82±3.29), SV 0.25±0.07 ml (vs. model 0.18±0.03), CO 153.28±5.92 l/min (vs. model 53.89±7.83). [1]
Hemodynamic parameters: compared to the model group, Typhaneoside treatment (10, 20, 40 mg/kg) significantly increased mean arterial pressure (MAP), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP), left ventricular maximum ascending rate (+dp/dtmax), and maximum rate of decrease in left ventricular pressure (−dp/dtmax), while decreasing left ventricular end‑diastolic pressure (LVEDP). For example, at 40 mg/kg: MAP 209.11±12.03 mmHg (vs. model 179.40±10.72), LVDP 213.84±4.82 mmHg (vs. model 177.93±9.82), LVSP 221.30±7.65 mmHg (vs. model 180.63±10.45), LVEDP 101.76±4.44 mmHg (vs. model 121.94±8.72), +dp/dtmax 3334.76±145.09 mmHg/s (vs. model 2494.23±104.56), −dp/dtmax 3000.62±98.89 mmHg/s (vs. model 2480.78±134.89). [1]
ELISA results: compared to the model group, Typhaneoside treatment significantly reduced the elevated serum levels of N‑terminal pro‑brain natriuretic peptide (NT‑proBNP), matrix lysin 2 (ST2), interleukin‑6 (IL‑6), tumor necrosis factor alpha (TNF‑α), matrix metalloproteinase‑2 (MMP‑2), and MMP‑9. [1]

Primary neonatal rat cardiomyocytes were obtained from suckling rats. The ventricular muscles were cut into 1‑3 mm tissue pieces and digested with an equal volume of 0.08% type I collagenase and 0.08% trypsin at 37 °C for 5 min in a magnetic stirrer (200 rpm). The digestion was repeated until tissue blocks disappeared. The filtrate was centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in complete medium containing 20% fetal bovine serum and 0.1 mol/l Brdu, and cells were inoculated at a density of 5×10⁵/ml. After 48 h, cells were adherent and used for experiments. [1]
Drug‑containing serum (DS) was prepared from male Sprague‑Dawley rats. The drug‑administered group received 40 mg/kg Typhaneoside by gavage twice on the fifth day (1‑h gap between administrations), while the control group received saline. One hour after the last gavage, blood was collected from the abdominal aorta, centrifuged at 3000 rpm for 15 min at room temperature, and the serum was heat‑inactivated at 56 °C for 30 min, then sterilized by filtration through a 0.22 μm microporous membrane. [1]
Hypoxia/reoxygenation (A/R) model: primary cardiomyocytes were pretreated with DS, then the medium was removed, PBS was added, and cells were placed in an oxygen‑deficient box with nitrogen replacement for 12 h hypoxia at 37 °C. Afterwards, PBS was replaced with complete medium and cells were cultured for 4 h reoxygenation at 37 °C. Experimental groups included control, model (A/R), drug + A/R (40 mg/kg DS pretreatment), rapamycin (RA) + A/R, drug + RA + A/R, and drug + API‑2 + A/R. [1]
Electron microscopy: cells were fixed with 2.5% glutaraldehyde, then post‑fixed with 2% citrate fixative for 1 h, dehydrated in graded ethanol and acetone, embedded, sectioned (50‑70 mm), and double‑stained with 3% uranyl acetate‑lead citrate for observation under transmission electron microscopy. [1]
Immunofluorescence detection of LC3: paraffin sections were dewaxed in xylene, rehydrated, rinsed with PBS, incubated with primary antibody (LC3B) at 37 °C for 60 min, then with secondary antibody at room temperature for 60 min, stained with DAPI for 2 min, and observed under a fluorescence microscope. [1]
Western blot: cells were lysed in cell lysate with PMSF, centrifuged at 12,000 g for 15 min, and the supernatant was mixed with protein loading buffer, boiled for 5 min. Proteins were separated by 12% SDS‑PAGE, transferred to PVDF membrane, blocked, incubated with primary antibodies (LC3‑II/I, p62, Akt, mTOR, p‑Akt, p‑mTOR; 1:1000) overnight at 4 °C, then with secondary antibody (1:1000) for 1 h, and detected by chemiluminescence. [1]

Animal model of heart failure after myocardial infarction: 8‑week‑old male Sprague‑Dawley rats (200±20 g) were anesthetized with 2% isoflurane inhalation (ventilation rate 1.5 l/min). The left anterior descending coronary artery was ligated with a 6/0 nonabsorbable suture at 2 cm below the left atrial appendage. The sham group underwent the same procedure without ligation. Serum troponin I level was measured 24 h after surgery to confirm successful model establishment. Postoperatively, penicillin (200,000 u/kg) was administered intraperitoneally for 7 days as anti‑inflammatory treatment. Rats were routinely fed for 4 weeks after surgery. [1]
After successful modeling, rats were divided into 5 groups (n = 16 for sham, n = 20 per model group): sham (normal saline), model (normal saline), Typhaneoside low‑dose (10 mg/kg), middle‑dose (20 mg/kg), and high‑dose (40 mg/kg). Typhaneoside infusion was administered once a day for 4 weeks. [1]
Ultrasound examination: after 4 weeks of treatment, left ventricular end‑diastolic diameter (LVEDD), left ventricular end‑systolic diameter (LVESD), left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (LVFS), stroke volume (SV), and cardiac output (CO) were measured. [1]
Hemodynamic testing: after 4 weeks of treatment, mean arterial pressure (MAP), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP), left ventricular end‑diastolic pressure (LVEDP), left ventricular maximum ascending rate (+dp/dtmax), and maximum rate of decrease in left ventricular pressure (−dp/dtmax) were determined. [1]
ELISA: serum levels of NT‑proBNP, ST2, IL‑6, TNF‑α, MMP‑2, and MMP‑9 were detected by enzyme‑linked immunosorbent assay. Standard and sample holes were filled with 50 μl samples, then 40 μl sample dilution and 50 μl horseradish peroxidase‑labeled detection antibody were added, incubated at 37 °C for 60 min, washed five times, then 50 μl substrate A and B were added and incubated in darkness at 37 °C, then 50 μl stop solution was added and OD450 measured within 15 min. [1]
Western blot (tissue): tissue samples were lysed, centrifuged, and processed as described in the Cell Assay section to measure LC3‑II/I, p62, p‑Akt/Akt, and p‑mTOR/mTOR expression. [1]

[1]. Effect of typhaneoside on ventricular remodeling and regulation of PI3K/Akt/mTOR pathway. Herz. 2019 Jun 14.

Autophagy plays a key role in the occurrence and development of heart failure (HF). Typhaneoside can improve cardiac morphological structure, enhance heart function, and improve myocardial remodeling in a rat model of HF after myocardial infarction. The beneficial effects may be related to reduction of inflammatory factors (IL‑6, TNF‑α) and myocardial matrix metalloproteinases (MMP‑2, MMP‑9). Typhaneoside mediates autophagy inhibition in cardiomyocyte hypoxia/reoxygenation through the PI3K/Akt/mTOR pathway by increasing the phosphorylation levels of Akt and mTOR, thereby inhibiting excessive autophagy and improving HF. [1]

C34H42O20

770.68

770.226

104472-68-6

5489389

Light yellow to yellow solid powder

1.7±0.1 g/cm3

1065.0±65.0 °C at 760 mmHg

332.6±27.8 °C

0.0±0.3 mmHg at 25°C

1.716

2.27

11

20

9

54

1320

14

C[C@H]1[C@@H]([C@H]([C@H]([C@@H](O1)OC[C@@H]2[C@H]([C@@H]([C@H](C(O2)OC3=C(OC4=CC(=CC(=C4C3=O)O)O)C5=CC(=C(C=C5)O)OC)O[C@H]6[C@@H]([C@@H]([C@H]([C@@H](O6)C)O)O)O)O)O)O)O)O

POMAQDQEVHXLGT-QQVXUORWSA-N

InChI=1S/C34H42O20/c1-10-20(38)24(42)27(45)32(49-10)48-9-18-22(40)26(44)31(54-33-28(46)25(43)21(39)11(2)50-33)34(52-18)53-30-23(41)19-15(37)7-13(35)8-17(19)51-29(30)12-4-5-14(36)16(6-12)47-3/h4-8,10-11,18,20-22,24-28,31-40,42-46H,9H2,1-3H3/t10-,11-,18+,20-,21-,22+,24+,25+,26-,27+,28+,31+,32+,33-,34?/m0/s1

3-[(3R,4S,5S,6R)-4,5-dihydroxy-3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxy-5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~324.38 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)