| Rhodiola rosea is the dry root and rhizome of Rhodiola crenulata(Hook.f.et Thoms.)H.Ohba and is included in the Chinese Pharmacopoeia 2015.Rhodiola rosea's main effect is to replenishing Qi,activating blood circulation,Tongmai asthma and curing cardiovascular disease.Traditional Chinese medicine broken pieces are broken through the modern smashing technology to break down the cell wall of plant herbs to a size of 45 ?m or less and a mesh size of 300 mesh or more.They are new Chinese medicinal powdered herbs used for direct oral administration.They retain Chinese herbal medicines and Chinese Herbal Pieces the characteristics of all ingredients of have the features and advantages of constant material basis,improved utilization rate,drastically reduced dosage,uniform and controllable quality,and ease of use.At present,Rhodiola rosea mainly exists in the form of traditional decoction pieces,which has the defects of unequal quality,uncontrollable quality,and tedious decoction.Compared with the traditional decoction pieces,duing to the disruption of cell walls,wether bring about changes in their chemical composition and content of active ingredients or not.Rhodiola rosea itself is more potent,and after processing into broken-walled decoction pieces,the active ingredients contained are rapidly and massively released,and at the same time,the insoluble ingredients may also increase with cell wall fragmentation,and the potential safety problems and effective dose exploration problems arise.Therefore,we compared the difference in fingerprints between Rhodiola rosea broken pieces and traditional decoction pieces,and the protective effect of Rhodiola rosea broken pieces and traditional decoction pieces on ischemic and hypoxic H9c2 cardiomyocytes.The standard aims to provide research basis for the exploration of the safety risks and effective doses in the later-staged,and it is of great significance for ensuring the quality of Rhodiola rosea broken pieces andsaving of medicinal resources.Object:(1)To establish and compare HPLC fingerprints before and after disruption of Rhodiola rosea,and to analyze the differences in chemical composition before and after crushed with Rhodiola rosea.(2)To investigate the difference of the protective effect of Rhodiola rosea broken pieces and traditional decoction pieces on hypoxia-ischemia H9c2 cardiomyocytes.Method:(1)Using HPLC method,the column is Agilent C18 column(4.6 mm × 250 mm,5 mobile phase:A acetonitrile-B0.02%phosphoric acid,gradient elution:6%A?6%A(0 to 18 min),6%A?20%A(18 to 45 min),20%A?to 20%A(45 to 55 min),20%A?22%A(55 to 65 min),22%A?22%A(65 to 90 min),DAD detection wavelength 278 nm,flow rate 0.8 mL·min-1,column temperature 30?,and injection volume 10 ?L.(2)CCK-8 method was used to determine the effect of different ischemic and hypoxic time on the proliferation of H9c2 cardiomyocytes and the activity of LDH in cell culture fluid to determine the optimal time for cell hypoxia and ischemia;CCK-8 method was used to determine the effects of different doses of Rhodiola rosea broken pieces and traditional decoction pieces on the proliferation rate of H9c2 cardiomyocytes,and to determine the best low,medium,and high-dose drugs;the experimental groups were normal group,model group,and Rhodiola rosea traditional decoction pieces low-dose group(0.002 mg·mL-1,Al),Rhodiola rosea traditional decoction pieces medium-dose group(0.020 mg·mL-1,A2),Rhodiola rosea traditional decoction pieces high-dose group(0.200 mg·mL-1,A3),Rhodiola rosea broken pieces low-dose group(0.002 mg·mL-1,B1),Rhodiola rosea broken pieces medium-dose group(0.020 mg·mL-1,B2),and Rhodiola rosea broken pieces high-dose group(0.200 mg·mL-1,B3);CCK-8 method was used to determine the effect of Rhodiola rosea broken pieces and traditional decoction pieces of low,middle and high dose groups on the survival rate of hypoxic-ischemic H9c2 cardiomyocytes;colorimetric method was used to detect Rhodiola rosea Low,medium,and high-dose groups of broken pieces and traditional decoction pieces on LDH activity and intracellularity in culture medium of hypoxic-ischemic H9c2 cardiomyocytes effects of MDA,ATP,T-SOD,CK content and activity;RT-PCR method was used to determine the effect of Rhodiola rosea Low,medium,and high-dose groups of broken pieces and traditional decoction pieces on the relative mRNA expression of Bax,Bcl-2 and Caspase3 in ischemic and hypoxic H9c2 cardiomyocytes;Western Blot assay was used to detect the difference between low dose,medium dose and high dose doses of Rhodiola rosea broken pieces and traditional decoction pieces on the effects of relative expression of Bax,Bcl-2,and Caspase3 proteins in hypoxic H9c2 cardiomyocytes.Result:(1)After the disruption of Rhodiola rosea,the number of chromatographic peaks did not increase or decrease,but the peak areas of chromatographic peaks of No.3,No.5,No.11,No.21,and No.22 increased significantly,and the remaining chromatographic peaks and peak areas were also different degrees of increase.(2)Compared with the model group,the survival rate of H9c2 cells could be significantly increased(P<0.05),and H9c2 could be significantly reduced at the same time.The activity of LDH in cardiomyocyte culture medium,the content of intracellular MDA and CK(P<0.05),the increase of intracellular T-SOD and ATP content(P<0.05),and the survival of H9c2 cardiomyocytes survived with the same concentration of Rhodiola rosea broken pieces,the rate of improvement is better than that of Rhodiola rosea traditional pieces in different degrees.(3)The results of RT-PCR and Western Blot showed that compared with the model group,Rhodiola rosea broken pieces and traditional decoction pieces could significantly reduce the mRNA and protein expression of Bax and Caspase3 in H9c2 cardiomyocytes after hypoxia-ischemia(P<0.05).Increased expression of Bcl-2 mRNA and protein in hypoxia-ischemia H9c2 cardiomyocytes(P<0.05),and the anti-apoptosis effect of Rhodiola rosea broken pieces was better than that of traditional Rhodiola rosea traditional decoction pieces.Conclusion:(1)This study can more comprehensively reflect the changes in the chemical composition before and after the disruption of Rhodiola rosea broke,which can provide a certain basis for the evaluation of the safety and effectiveness of Rhodiola rosea broken pieces.(2)The protective effect of Rhodiola rosea broken pieces on hypoxia-ischemia H9c2 cardiomyocytes is better than that of traditional decoction pieces.Its mechanism may be related to anti-oxidation and inhibition of cell apoptosis. |
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