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目的建立隔山香药材中异芹菜脑(Isoapide,Iso)的高效液相色谱法(HPLC),测定方法并初步探讨异芹菜脑对体外培养乳鼠心肌细胞缺氧/复氧损伤的保护作用。方法采用高效液相色谱法,色谱柱:Kromasil C18柱(4.6 mm伊250 mm,5滋m);流动相:以甲醇-水(70颐30)洗脱,流速为1.0 mL·min-1,检测波长为280 nm,柱温室温,进样量:10滋L。并用原代培养的乳鼠心肌细胞建立缺氧复/氧损伤模型,异芹菜脑在建模前进行预干预,心肌细胞随机分为5组,空白对照组(Control,CON),缺氧/复氧组(hypoxia/reoxygenation,H/R),异芹菜脑低、中、高剂量干预组(IsoL、IsoM、IsoH)。缺氧4 h/复氧4 h后,采用CCK-8法检测心肌细胞存活率。结果异芹菜脑在0.02096~0.2096 mg·mL-1线性关系良好(r=0.9999),平均回收率为98.09%,RSD=1.25%。与空白对照组比较,缺氧/复氧组心肌细胞存活率显著降低(P约0.01);与缺氧/复氧组比较,IsoL、IsoM、IsoH剂量干预组心肌细胞存活率均显著升高。结论异芹菜脑对体外培养乳鼠心肌细胞缺氧/复氧损伤具有一定的保护作用,隔山香药材中异芹菜脑的HPLC测定方法准确、简便、重现性好,有助于完善隔山香药材的质量控制。
OBJECTIVE To establish a high performance liquid chromatography (HPLC) method for determination of isoapide (Iso) in Acorus gramineus and study its protective effect on hypoxia / reoxygenation injury in cultured neonatal rat cardiomyocytes. Methods The mobile phase was eluted with methanol-water (70:30) at a flow rate of 1.0 mL · min-1 on a Kromasil C18 column (4.6 mm × 250 mm, 5 μm) Detection wavelength was 280 nm, column temperature room temperature, injection volume: 10 Zizi. Hypoxia / reoxygenation injury model was established by primary cultured neonatal rat cardiomyocytes. Aneurysm of different celery was pre-treated before modeling. Cardiomyocytes were randomly divided into 5 groups: control group, control group, hypoxia / Hypoxia / reoxygenation (H / R) group, low, medium and high dose intervention group (IsoL, IsoM, IsoH). After hypoxia 4 h / reoxygenation for 4 h, the viability of cardiomyocytes was detected by CCK-8 assay. The results showed that there was a good linear relationship (0.09996 ~ 0.2096 mg · mL-1) in the brain of different celery (r = 0.9999), with an average recovery of 98.09% and a RSD of 1.25%. Compared with the blank control group, the survival rate of cardiomyocytes in hypoxia / reoxygenation group was significantly decreased (P = 0.01). Compared with hypoxia / reoxygenation group, the survival rate of myocardial cells in IsoL, IsoM and IsoH groups were significantly increased. Conclusion Different celery brain may have some protective effects on hypoxia / reoxygenation injury in neonatal rat cardiomyocytes cultured in vitro. The HPLC method of isolating celery in Chushan Shannon herb is accurate, simple and reproducible, Quality control.