Novel medical use of 2 alpha, 23- dihydroxyl ursolic acid
A technology of dihydroxyursulic acid and application, applied in the field of medicine, can solve the problems of no hepatocyte protection effect, no clinical application, etc.
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Embodiment 1
[0018] Measurement of mitochondrial swelling
[0019] Preparation of mitochondria and measurement of mitochondrial swelling. According to the Appril method, the liver was separated in the pre-cooled separation solution (including 0.225mol·L -1 Mannitol, 0.075mol L -1 Sucrose, 0.05mmol·L -1 EDTA, 10mmol·L -1 Tris-HCl, pH 7.4), cut into pieces, washed, homogenized, centrifuged at 500×g for 5 minutes, took the supernatant, centrifuged at 8800×g for 10 minutes, discarded the supernatant, the precipitate was the mitochondria, and used the separation solution Wash 3 times and finally resuspend in separation medium. Protein concentration was determined by the Coomassie brilliant blue method.
[0020] Mitochondria were resuspended in the assay solution (125mmol·L -1 Sucrose, 50mmol·L -1 KCl, 2mmol L -1 K H 2 PO 4 , 5mmol·L -1 Succinic acid, 50μmol·L -1 CaCl 2 , 5 μmol L -1 Rotenone, 10mmol·L -1 Hepes, pH7.4). Normal mouse liver mitochondria were divided into 6 gr...
Embodiment 2
[0023] Determination of Superoxide Anion Scavenging Rate
[0024] The scavenging effect of 2,23-hydroxyursolic acid on superoxide anion was detected by xanthine / xanthine oxidase oxygen radical generation system. In 3mL 10mmol·L -1 K H 2 Add 50μmol·L to PO4-KOH buffer (pH7.4) -1 , 150μmol·L -1 , 500μmol·L -1 2α, 23-Dihydroxyursolic acid, 100 μmol L -1 Xanthine and 0.02 U / mL xanthine oxidase, add nitroblue tetrazolium (NBT) 100 μmol L -1 , incubated at 25°C for 5min, and measured the absorbance at 550nm. With 500μmol·L -1 Oleanolic acid was used as a positive control. The formula for calculating superoxide anion scavenging rate is as follows: (ΔA control -ΔA drug ) / ΔA control ×100%, ΔA=A 0min -A 5min .
[0025] Table 1 Scavenging effect of 2,23-dihydroxyursolic acid on superoxide anion produced by xanthine / xanthine oxidase system.
[0026] Group Absorbance decrease (ΔA) Clearance
[0027] Control 242.00±16.86
[0028] Oleanolic acid 500μmol·L -1 147.00...
Embodiment 3
[0035] Determination of hydroxyl radical scavenging rate
[0036] Using CuSO 4 / Cytochrome C free radical generation system detects the scavenging effect of DHUA on hydroxyl free radicals. In 3mL 0.15mmol·L -1 Phosphate buffer (containing 100 μmol L -1 VitC, 100μmol·L -1 CuSO 4 and 100μmol·L -1 Cytochrome C, pH 7.4) was added with 50 μmol L -1 , 150μmol·L -1 , 500μmol·L -1 Ursolic acid or DHUA, the reaction system was incubated at 25°C for 90 minutes, and the absorbance value was measured at 550nm, with 500μmol L -1 Oleanolic acid was used as a positive control. The calculation formula of hydroxyl radical scavenging rate is the same as above, where, ΔA=A 0min -A 90min .
[0037] Table 2 Effect of 2,23-dihydroxyursolic acid on vitamin C / CuSO 4 The scavenging effect of hydroxyl radicals generated by the system.
[0038] Group Absorbance decrease (ΔA) Clearance
[0039] Control 442.83±23.42
[0040] Oleanolic acid 500μmol·L -1 241.33±27.21 45.6%
[0041]...
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