Dihydromyricetin is a potent inhibitor with an IC₅₀ of 48 μM on dihydropyrimidinase. Dihydromyricetin can activate autophagy through inhibiting mTOR signaling. Dihydromyricetin suppresses the formation of mTOR complexes (mTORC1/2). Dihydromyricetin is also a potent influenza RNA-dependent RNA polymerase inhibitor with an IC₅₀ of 22 μM.

Dihydromyricetin, a flavonol, significantly inhibits the catalytic activities of dihydropyrimidinase toward both the natural substrate dihydrouracil and xenobiotic substrate 5-propyl-hydantoin. Dihydromyricetin exhibits a significant inhibitory effect on the activities of dihydropyrimidinase for both substrates, even more than Myricetin does. The IC₅₀ values of Dihydromyricetin for dihydropyrimidinase determined from the titration curves using Dihydrouracil and 5-propyl-hydantoin are 48±2 and 40±2 μM, respectively^[1]. Dihydromyricetin (DHM) supplementation significantly reverses the increased phosphorylation of mTOR at Ser²⁴⁴⁸ (p-mTOR) during D-gal administration, which suggests that Dihydromyricetin can activate autophagy through inhibiting mTOR signaling^[2].

Shanghai Jinpan Biotech Co Ltd has not independently confirmed the accuracy of these methods. They are for reference only.

Changes in learning and memory capacity in rats administrated normal control group, D-gal group, D-gal+Dihydromyricetin (100 mg/kg) group, D-gal+Dihydromyricetin (200 mg/kg) group assessed by morris water maze (MWM) (n=10 per group). Dihydromyricetin (DHM) treatment significantly shortens the escape latency when compared with D-gal-induced model group^[2].

Shanghai Jinpan Biotech Co Ltd has not independently confirmed the accuracy of these methods. They are for reference only.

320.25

C₁₅H₁₂O₈

27200-12-0

二氢杨梅素；白蔹素

Room temperature in continental US; may vary elsewhere.

DMSO : ≥ 100 mg/mL (312.26 mM)

* “≥” means soluble, but saturation unknown.

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (7.81 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (7.81 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (7.81 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (7.81 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (7.81 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (7.81 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Huang CY. Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases. PLoS One. 2015 May 19;10(5):e0127634.

  [2]. Chang H, et al. Ampelopsin suppresses breast carcinogenesis by inhibiting the mTOR signalling pathway. Carcinogenesis. 2014 Aug;35(8):1847-54.

  [3]. Kou X, et al. Ampelopsin attenuates brain aging of D-gal-induced rats through miR-34a-mediated SIRT1/mTORsignal pathway. Oncotarget. 2016 Nov 15;7(46):74484-74495.

  [4]. Václav Zima, et al. Unraveling the Anti-Influenza Effect of Flavonoids: Experimental Validation of Luteolin and its Congeners as Potent Influenza Endonuclease Inhibitors. Eur J Med Chem. 22 August 2020, 112754.

A rapid spectrophotometric assay is used to determine the enzymatic activity for hydantoinase, allantoinase, dihydroorotase, and imidase. Dihydrouracil, 5-propyl-hydantoin, and phthalimide are used as substrates. Unless explicitly stated otherwise, Dihydrouracil (2 mM) is used as the substrate in the standard assay of dihydropyrimidinase. Briefly, the decrease in absorbancy at 230, 248, and 298 nm is measured upon hydrolysis of Dihydrouracil, 5-propyl-hydantoin, and Phthalimide as the substrate at 25°C, respectively. To start the reaction, the purified dihydropyrimidinase (10-70 μg) is added to a 2 mL solution containing the substrate and 100 mM Tris-HCl (pH 8.0). Substrate hydrolysis is monitored with a UV/vis spectrophotometer. The extinction coefficient of each substrate is determined experimentally by direct measurement with a spectrophotometer. The extinction coefficients of Dihydrouracil, 5-propyl-hydantoin, and Phthalimide are 0.683 mM^-1cm^-1 at 230 nm, 0.0538 mM^-1cm^-1 at 248 nm, and 3.12 mM^-1cm^-1 at 298 nm, respectively. The initial rates of change are a function of enzyme concentration within the absorbance range of 0.01-0.18 min^-1. A unit of activity is defined as the amount of enzyme catalyzing the hydrolysis of 1 μmol substrate/min, and the specific activity is expressed in terms of units of activity per milligram of enzyme. The kinetic parameters K_m and V_max are determined from a non-linear plot by fitting the hydrolyzing rate from individual experiments to the Michaelis-Menten equation^[1].

Shanghai Jinpan Biotech Co Ltd has not independently confirmed the accuracy of these methods. They are for reference only.

Hippocampus and cortex tissue samples are homogenized in lysis buffer containing 20 mM Tris (pH 7.5), 135 mM NaCl, 2 mM EDTA, 2 mM DTT, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 10 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM PMSF for 30 min on ice and centrifuged at 12000×g at 4°C for 30 min. The supernatant is collected and protein quantification is carried out using a BCA kit. The protein samples are boiled in the presence of sample buffer at 95°C for 5 min. The target protein is separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and then probed by corresponding primary and secondary antibodies. Finally, the target protein is visualized by enhanced chemiluminescence (ECL) reagent exposure to X-ray film^[2].

Shanghai Jinpan Biotech Co Ltd has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[2]
Totally 40 male Sprague-Dawley (SD) rats (age: 8 weeks old; body weight: 160±20 g) are used. The rats are randomly divided into four groups including normal control group, D-gal model group, and D-gal combined with DHM at the doses of 100 and 200 mg/kg-d groups with 10 rats in each group. All rats are housed at the environment with room temperature of 22±2°C and a dark-light cycle (12 h: 12h), and provided the accessibility to food and water ad libitum. After adapting to new environment for 1 week, the rats from DHM groups are administered with DHM dissolved in distilled water at the designated dosages by gavage once a day at 8:00am for 6 consecutive weeks. The rats from the normal control group are administrated with distilled water. Except from the normal control group, the rats from other groups are subjected to subcutaneous injection of D-gal at the dose of 150 mg/kg.d for 6 consecutive weeks. Each administration of DHM should be 2 h ahead of D-gal injection.

Shanghai Jinpan Biotech Co Ltd has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Huang CY. Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases. PLoS One. 2015 May 19;10(5):e0127634.

  [2]. Chang H, et al. Ampelopsin suppresses breast carcinogenesis by inhibiting the mTOR signalling pathway. Carcinogenesis. 2014 Aug;35(8):1847-54.

  [3]. Kou X, et al. Ampelopsin attenuates brain aging of D-gal-induced rats through miR-34a-mediated SIRT1/mTORsignal pathway. Oncotarget. 2016 Nov 15;7(46):74484-74495.

  [4]. Václav Zima, et al. Unraveling the Anti-Influenza Effect of Flavonoids: Experimental Validation of Luteolin and its Congeners as Potent Influenza Endonuclease Inhibitors. Eur J Med Chem. 22 August 2020, 112754.