生物测定法用于中药质量评价的探索研究——以夏枯草抗氧化活性与总酚酸含量相关性的研究为例

研究不同来源及不同部位夏枯草的抗氧化活性,并对其所含原儿茶酸、原儿茶醛、咖啡酸、异迷迭香酸苷和迷迭香酸5种酚酸类成分含量进行测定,探讨夏枯草抗氧化活性与总酚酸含量间的相关性,为制定科学合理的质量标准服务。以夏枯草50%甲醇提取液为研究对象,分别采用DPPH和HPLC测定夏枯草的抗氧化活性及原儿茶酸、原儿茶醛、咖啡酸、异迷迭香酸苷和迷迭香酸5种酚酸类成分的含量。DPPH采用夏枯草50%甲醇提取液0.5 m L与0.1 mmol·L~(-1)DPPH·乙醇溶液反应60 min,于517 nm波长处测定吸光度,用清除率及IC50进行抗氧化活性评价。HPLC采用Epic C18色谱柱,乙腈-0.1%甲酸水溶液为流动相,梯度洗脱,280 nm进行检测。采用偏最小二乘法对多批不同产地和不同部位夏枯草的抗氧化能力与其中5种酚酸类成分的总含量间的相关性进行分析。夏枯草50%甲醇提取液与0.1 mmol·L~(-1)DPPH·乙醇溶液反应的量效范围为0.300~1.65 g·L~(-1)(药材),原儿茶酸、原儿茶醛、咖啡酸、异迷迭香酸苷和迷迭香酸的进样量分别在0.007 84~0.980,0.011 5~1.44,0.008 64~1.08,0.080 0~1.00,0.079 8~0.998μg与各自峰面积积分值成良好的线性关系。5种成分的平均加样回收率分别为97.76%,96.88%,100.3%,102.1%,104.5%,RSD分别为1.8%,1.6%,1.7%,0.62%,0.75%。夏枯草各部位的抗氧化能力与总酚酸含量和在一定的药材质量范围内具有良好的线性相关性。因此,在确定的药材质量范围内,采用DPPH生物测定法结合HPLC含量测定法共同用于夏枯草药材的质量控制,可考虑作为一种新的尝试用于夏枯草质量标准的修订。     

分步醇沉对玛咖多糖单糖组成及抗氧化活性的影响

目的:分析分步醇沉所得玛咖多糖的单糖组成,并对各部分玛咖多糖的抗氧化能力进行研究。方法:采用水提醇沉法得到玛咖粗多糖,通过分步醇沉对玛咖粗多糖进行分离、纯化;采用气相色谱分析法对各玛咖多糖的单糖组成进行分析;最后采用三氯乙酸法和清除DPPH自由基的方法考察各部分玛咖多糖的抗氧化能力。结果:分步醇沉随乙醇浓度增加依次得到0~40%乙醇多糖部分,40%~50%乙醇多糖部分,50%~60%乙醇多糖部分,60%~70%乙醇多糖部分及70%~80%乙醇多糖部分,依次命名为MP1,MP2,MP3,MP4和MP5;其中收率最大的为MP1,纯度最高的为MP4,分别为0.856%和68.5%;经气相色谱技术分析发现不同体积分数乙醇所得玛咖多糖的单糖组成种类及比例不同;抗氧化能力试验结果表明各部分玛咖多糖的抗氧化能力存在显著性差异,其中抗氧化能力最强的为MP5,MP4抗氧化能力次之,抗氧化能力最弱的为MP1。结论:分步醇沉所得各部分玛咖多糖的抗氧化能力有较大差异,玛咖多糖的抗氧化活性与其单糖组成的种类、比例及其空间构象密切相关,分步醇沉对分离纯化玛咖多糖具有重要意义,为玛咖多糖进一步分离纯化、结构组成和药理研究提供可靠的参考依据。     

Antioxidant and hepatoprotective activity of aqueous extract of Solanum fastigiatum (false "Jurubeba") against paracetamol-induced liver damage in mice

Aim of the study

Solanum fastigiatum is a medicinal plant widely distributed in the south of Brazil and has been used mainly to treat hepatitis, spleen disorders, uterine tumors, irritable bowel syndrome and chronic gastritis. The present research was aimed to evaluate the potential antioxidant and hepatoprotective activity of aqueous extracts of leaves using in vitro and in vivo models to validate the folkloric use of the plant.

Materials and methods

Antioxidant activity was evaluated by different assays, including thiobarbituric acid reactive species (TBARS), total antioxidant, 2,2-diphenlyl-1-picrylhydrazyl (DPPH) radical and metal ion-chelating activities. The hepatoprotective activity of the aqueous extracts was studied on mice liver damage induced by paracetamol (250 mg/kg) by monitoring biochemical parameters.

Results

The extract showed inhibition against TBARS, induced by 10 μM FeSO₄ and 5 μM sodium nitroprusside in rat liver, brain and phospholipid homogenates from egg yolk. The plant exhibited strong antioxidant activity in the DPPH (IC₅₀, 68.96 ± 1.25 μg/ml) assay. The aqueous extract also showed significant hepatoprotective activity that was evident by enzymatic examination and brought back the altered levels of TBARS, non-protein thiol and ascorbic acid to near the normal levels in a dose dependent manner. Acute toxicity studies revealed that the LD₅₀ value of the extract is more than the dose 4 g/kg body weight of mice.

Conclusions

The results indicate that this plant possesses potential antioxidant and hepatoprotective properties and has therapeutic potential for the treatment of liver diseases.     

Glycation inhibitory activity and the identification of an active compound in Plantago asiatica extract

The glycation reaction involves a series of non-enzymatic reactions between the carbonyl group on reducing sugars and the amino group on proteins leading to the formation of advanced glycation end-products (AGEs), which are acknowledged to be involved in the pathogenesis of diabetic and aging-related complications. Consequently, the development of AGE inhibitors is considered to have therapeutic potential in patients with diabetes or age-related diseases. The preliminary results showed that a methanol extract (PAE) of Plantago asiatica, which is traditionally used as a folk medicine in Asian countries to treat fever, cough, wound etc., had strong glycation inhibitory activity. The effects of the extract on AGE fluorescence were dose-dependent, reaching 41% inhibition at 0.1 microg/mL of extract. The purified principle from PAE was identified as plantamajoside. As well as antioxidant activities, in vitro glycation inhibitory activities with 10 and 25 mm plantamajoside were higher than those with 10 and 25 mm aminoguanidine. The results demonstrate that PAE and plantamajoside had significant effects on in vitro AGE formation, and the glycation inhibitory activity and antioxidant activity of plantamajoside were comparable to those obtained using millimolar concentrations of the standard antiglycation agent aminoguanidine, and the antioxidant ascorbate, respectively.     

苯妥英钠代谢酶CYP2C9、CYP2C19基因多态性的分析

目的研究药物代谢酶基因多态性对苯妥英钠代谢的影响,并建立分析苯妥英钠代谢酶中CYP2C9、CYP2C19基因多态性的方法。方法从人全血中抽取DNA,设计引物扩增CYP2C9及CYP2C19,并运用限制性片段长度多态性（RFLP）技术的方法进行CYP2C9及CYP2C19的基因分型。结果成功设计了引物用来扩增CYP2C9及CYP2C19的基因片段,并测序验证。结论建立了简单可靠的CYP2C9及CYP2C19的RFLP基因分型方法。