Determination of six alkaloids in six types of Coptidis Rhizoma pieces by RP-HPLC and spectrum-effect relationships with anti-diabetes pharmacodynamics data
【Abstract】To establish a method for determining the content of six alkaloids (jatrorrhizine hydrochloride, columbamine hydrochloride, epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and 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 and 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), 2.88–57.06 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 content 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 the most difference with crude pieces especially, mainly related to triglyceride (TG) and fasting blood glucose levels (FBG) in serum. In addition, columbamine hydrochloride was the 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.
【Keywords】 Coptidis Rhizoma pieces; HPLC; jatrorrhizine hydrochloride; columbamine hydrochloride; epiberberine hydrochloride; coptisine hydrochloride; palmatine hydrochloride; berberine hydrochloride; content determination; pharmacodynamics data; principal components analysis (PCA) ; hierarchical clustering analysis (HCA) ;
(Translated by XIONG Y)
 Chinese Pharmacopoeia. Volume 1 [S]. 2015: 303 (in Chinese).
 Ye Y L, Zhao P P, Luo Z L. 黄连及其炮制品中5种生物碱含量测定 [J]. Chinese Traditional Patent Medicine, 1996, 18 (3): 16 (in Chinese).
 Liu H R, Tang J F, Wu X L. 酒对黄连炮制品质量影响的研究 [J]. China Journal of Chinese Materia Medica, 2000, 25 (10): 638 (in Chinese).
 Wu J F, Li H Y, Yang A P, et al. 高效液相色谱法分析黄连炮制品中盐酸小檗碱的含量变化 [J]. Chinese Traditional Patent Medicine, 1999, 21 (8): 408 (in Chinese).
 Liao Q W, Fan D L, Xiao X H, et al. Fingerprints of different samples of processed Rhizoma Coptidi by HPLC [J]. China Journal of Chinese Materia Medica, 2007, 32 (3): 210 (in Chinese).
 Song P S, Wei Y L, Zhao J B, et al. 黄连不同炮制品中3种生物碱含量的高效液相色谱法测定 [J]. Lishizhen Medicine and Materia Medica Research, 2008, 19 (10): 2396 (in Chinese).
 Geng Z P, Zheng H J, Zhang Y, et al. Simultaneous determination of six alkaloids in Coptis chinensis of different regions by RP-HPLC [J]. China Journal of Chinese Materia Medica, 2010, 35 (19): 2576 (in Chinese).
 Zhao L, Li J H, Zhu D Z, et al. Principal component analysis of nutrients in five varieties of velvet antler (Cornu Cervi Pantotrichum) [J]. Spectroscopy and Spectral Analysis, 2010, 30 (9): 2571 (in Chinese).
 Li J C, Meng X L, Cui R, et al. Comparison in pharmacodynamic effects of different types of processed Rhizoma Coptis on mouse diabetes [J]. Chinese Traditional Patent Medicine, 2010, 32 (11): 1922 (in Chinese).
Kong W J, Zhao Y L, Xiao X H, et al. Spectrum-effect relationships between ultra performance liquid chromatography fingerprints and anti-bacterial activities of Rhizoma Coptidis [J]. Anal Chim Acta, 2009, 634 (2) : 279.
 Wan X H, Tan Y L. 主成分分析原始数据的预处理问题 [J]. Chinese Journal of Health Statistics, 2005, 22 (5): 327 (in Chinese).
 Dai X L, Ma X Q, Wang T T. The improved principal component analysis of arabidopsis thaliana gene method [J]. Microcomputer & Its Applications, 2015, 34 (14): 51 (in Chinese).
 Wang Z M, Qian Z Z, Zhang Q W, et al. 一测多评法建立的技术指南 [J]. China Journal of Chinese Materia Medica, 2011, 36 (6): 657 (in Chinese).
 Liu J L, Meng X L, Liu Y S. 古代中医用黄连治疗消渴的方药探讨 [J]. Journal of Sichuan of Traditional Chinese Medicine, 2010, 28 (4), 41 (in Chinese).
 Zhao Q P, Cui Q B, Zhang Y. 黄连用于治疗糖尿病的研究进展 [J]. Pharmacy and Clinics of Chinese Materia Medica, 2010, 1 (1): 55 (in Chinese).
 Lai X R, Meng X L, Zhang Y, et al. 酒炙黄连炮制品的新用途: 中国, ZL 201010174854.3 [P]. 2016-05-25 (in Chinese).
 Lai X R, Meng X L, Zhang Y, et al. 萸炙黄连炮制品的新用途: 中国, ZL 201010174835.0 [P]. 2015-05-20 (in Chinese).
 Lai X R, Meng X L, Zhang Y, et al. 酒蒸黄连炮制品及其制备方法和用途: 中国, ZL 201010174851.X [P]. 2012-06-27 (in Chinese).
 Li J C, Meng X L, Lai X R, et al. 基于3T3-L1 细胞的不同黄连炮制品“止消渴”药效比较研究 [J]. Pharmacology and Clinics of Chinese Materia Medica, 2010, 26 (2): 52 (in Chinese).