Lai Xian-Rong, Zhou Bang-Hua, Du Ming-Sheng, Zheng Hai-Jie, Geng Zhi-Peng, Li Jia-Chuan, Meng Xian-Li, Zhang Yi, Zhang Jing
Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.
Zhongguo Zhong Yao Za Zhi. 2016 Dec;41(24):4579-4586. doi: 10.4268/cjcmm20162416.
To establish a method for determining the contents of six alkaloids (jatrorrhizine hydrochloride, columbamine hydrochloride, epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride) in six types of Coptidis Rhizoma pieces (crude pieces, ginger juice stir-fried pieces, vinegar stir-fried pieces, wine steamed pieces, wine stir-fried pieces, evodiae juice stir-fried pieces) by RP-HPLC, and explore the relationship with the curative effect of traditional Chinese medicine (TCM) and pharmacodynamics results. The chromatographic column was Welch XtimateTM C₁₈ (4.6 mm×250 mm, 5 μm), with 0.1% triethylamine solution (adjust pH at 10 with ammonium bicarbonate and ammonia) as mobile phase A and acetonitrile as mobile phase B for gradient elution (0-15 min, 10%-25%B; 15-25 min, 25%-30%B; 25-40 min, 30%-45%B) at a rate of 1.0 mL•min⁻¹. The column temperature was set at 30 ℃, and the wavelength was set at 270 nm. The six alkaloids showed a good linear relationship within the range of 0.85-16.96 mg•L⁻¹ (r=0.999 7), 1.25-24.96 mg•L⁻¹ (r=0.999 9), 2.05-40.96 mg•L⁻¹ (r=0.999 9), 3.65-72.96 mg•L⁻¹ (r=0.999 9), 2.88-57.60 mg•L⁻¹ (r=0.999 8), and 13.25-264.96 mg•L⁻¹ (r=0.999 6) respectively. The average recoveries (n=9) of the six alkaloids were 102.4% (RSD 1.2%), 101.8% (RSD 1.3%), 100.3% (RSD 1.8%), 100.7%(RSD 1.8%), 101.2% (RSD 1.5%) and 97.90% (RSD 2.0%) respectively, and their average contents were 3.55, 4.49, 9.12, 19.17, 15.69, 62.56 mg•g⁻¹, respectively. This determination method was accurate and repeatable, which could be used for the content determination in six types of Coptidis Rhizoma pieces. Data analysis on contents determination and preliminary pharmacodynamics results was conducted by using principal component analysis (PCA) and hierarchical clustering analysis (HCA). The analysis results showed that three types of Coptidis Rhizoma pieces (wine steamed pieces, wine stir-fried pieces, and evodiae juice stir-fried pieces) had significant differences with crude pieces, and the wine steamed Coptidis Rhizoma pieces showed most difference with crude pieces especially, mainly related to triglyceride (TG) and fasting blood glucose levels (FBG) in serum. In addition, columbamine hydrochloride was most affected among the six alkaloids. Those three types of Coptidis Rhizoma pieces (wine steamed pieces, wine stir-fried pieces, and evodiae juice stir-fried pieces), had more advantages for "anti-diabetes" in TCM clinical application, especially in the treatment of diabetic hyperlipidemia.
建立一种采用反相高效液相色谱法（RP-HPLC）测定6种黄连饮片（生片、姜汁炒片、醋炒片、酒蒸片、酒炒片、吴茱萸汁炒片）中6种生物碱（盐酸小檗碱、盐酸药根碱、盐酸表小檗碱、盐酸黄连碱、盐酸巴马汀、盐酸黄柏碱）含量的方法，并探讨其与中药疗效及药效学结果的关系。色谱柱为Welch XtimateTM C₁₈（4.6 mm×250 mm，5 μm），以0.1%三乙胺溶液（用碳酸氢铵和氨水调pH至10）为流动相A，乙腈为流动相B进行梯度洗脱（0 - 15 min，10% - 25%B；15 - 25 min，25% - 30%B；25 - 40 min，30% - 45%B），流速为1.0 mL•min⁻¹。柱温设定为30℃，检测波长设定为270 nm。6种生物碱在0.85 - 16.96 mg•L⁻¹（r = 0.999 7）、1.25 - 24.96 mg•L⁻¹（r = 0.999 9）、2.05 - 40.96 mg•L⁻¹（r = 0.999 9）、3.65 - 72.96 mg•L⁻¹（r = 0.999 9）、2.88 - 57.60 mg•L⁻¹（r = 0.999 8）、13.25 - 264.96 mg•L⁻¹（r = 0.999 6）范围内线性关系良好。6种生物碱的平均回收率（n = 9）分别为102.4%（RSD 1.2%）、101.8%（RSD 1.3%）、100.3%（RSD 1.8%）、100.7%（RSD 1.8%）、101.2%（RSD 1.5%）、97.90%（RSD 2.0%），其平均含量分别为3.55、4.49、9.12、19.17、15.69、62.56 mg•g⁻¹。该测定方法准确、重复性好，可用于6种黄连饮片的含量测定。采用主成分分析（PCA）和层次聚类分析（HCA）对含量测定数据和初步药效学结果进行分析。分析结果表明，3种黄连饮片（酒蒸片、酒炒片、吴茱萸汁炒片）与生片有显著差异，其中酒蒸黄连片与生片差异尤为明显，主要与血清中甘油三酯（TG）和空腹血糖水平（FBG）有关。此外，6种生物碱中盐酸药根碱受影响最大。这3种黄连饮片（酒蒸片、酒炒片、吴茱萸汁炒片）在中医临床应用中“抗糖尿病”方面具有更多优势，尤其在治疗糖尿病高脂血症方面。
