Chinese Traditional and Herbal Drugs
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1 752 Chinese Traditional and Herbal Drugs * HPLC Hypersil GOLD aq C mm 4.6 mm 5 μm 1 ml/min % nm A -0.1% nm D -0.1% nm -0.1% nm 5-5-HMF-0.1% nm Accurasil NH mm 4.6 mm 5 μm ml/min 25 ELSD Alltech2000ES L/min SPSS t- A D 5-HMF A D5- R283.1 A (2016) DOI: /j.issn Content changes of chemical components and their effect of adjuvants during the process of Rehmanniae Radix Praeparata (steamed for nine times and shined for nine times) MENG Xiang-long, MA Jun-nan, ZHANG Shuo-sheng, LI Kun, XUE Fei-fei, ZHANG Jie, YUAN Zhi-cui, TIAN Ye-fei, WANG Ming-fang Institute of Pharmaceutical & Food Engineering, Shanxi University of Traditional Chinese Medicine, Jinzhong , China Abstract: Objective By HPLC, to explore the differences of adjuvants for the changes of chemical components of Rehmanniae Radix Praeparata during process by steamed for nine times and shined for nine times. Methods HPLC was conducted on a Hypersil GOLD aq C 18 column (250 mm 4.6 mm, 5 μm), flow rate was 1 ml/min, and the column temperature was 25. With the mobile phase of acetonitrile and 0.1% phosphoric acid water (1 99), the detection wavelength was 210 nm (catalpol), 203 nm (rehmannioside A, rehmannioside D, and leonuride), acetonitrile and 0.1% phosphoric acid water (3 97 and 4 96); 334 nm (actecosode), acetonitrile and 0.1% phosphoric acid water (16 84), and 284 nm (5-HMF), acetonitrile and 0.1% phosphoric acid water (11 89). Sugars was conducted on an Accurasil NH 2 column (250 mm 4.6 mm, 5 μm) with the mobile phase of acetonitrile and 0.1% phosphoric acid water (72 28), flow rate was 1 ml/min, the column temperature was 25. The detector was ELSD, Alltech2000ES, drift tube temperature was 93, and the gas flow rate was 2.6 L/min. The statistic analysis was carried out by SPSS 20.0 and the paired sample t-test was carried out for 12 kinds of chemical composition in Rehmanniae Radix Praeparata obtained by steamed for nine times and shined for nine times and wine-steamed for nine times and shined for nine times. Results With the increase of steamed times, the ZD sszywzh@126.com zhangshuosheng@aliyun.com
2 Chinese Traditional and Herbal Drugs contents of catalpol, leonuride, actecosode, stachyose, sucrose, and raffinose in Rehmanniae Radix Praeparata obtained by two ways are all decreased; While the contents of rehmannioside A and rehmannioside D were slightly increased; the contents of 5-HMF, fructose, glucose, and mannose trisaccharide were increased. Conclusion The established method conforms to the validation requirement of the methodology. In the process of Rehmanniae Radix Praeparata with steamed for nine times and shined for nine times, processing adjuvant has a marked impact on the quality of the processed product. And the related substances of Rehmanniae Radix Praeparata obtained by the two ways have a larger fluctuation in times 3, 4, and 6 of steamed and shined processes, and the reason for this still needs to be further studied. Key words: Rehmanniae Radix Praeparata; Chinese materia medica process; steamed for nine times and shined for nine times; adjuvants; catalpolreh; mannioside A; rehmannioside D; leonuride actecosode; 5-HMF; sugars Rehmanniae Radix [1] Scrophulariaceae Rehmannia Libosch. ex Fisch. et Mey. Rehmannia glutinosa Libosch [2] 4 [3] [4] [5] [6] 1 Thermo Fisher Scientific U3000 GZX-9076MBE HHS DV215CD Rehmannia glutinosa Libosch. - [7] 6 h60 24 h 9 60 S 1 S 9 - [5,8] SW 1 SW 9 A % D % % %5-5-HMF % % % % MST % MST % MST % % % S 1 S 9 SW 1 SW g 50 ml 1.5 h 25 ml 15% 5 ml 0.45 μm
3 754 Chinese Traditional and Herbal Drugs D A 5 ml μg/ml 543 μg/ml D 78.2 μg/ml A 83 μg/ml 5 ml Hypersil GOLD aq C mm 4.6 mm 5 μm 1 ml/min % nm A -0.1% nm D -0.1% nm 1 SW 1 A A D D t/min 1 SW 1 HPLC Fig. 1 HPLC for reference substance of iridoid and sample SW μg/ml μg/ml A μg/ml D μg/ml 10 μl r Y X r μg/mly X r μg/ml A Y X r μg/ml D Y X r μg/ml RSD 0.74%
4 Chinese Traditional and Herbal Drugs RSD 0.60% A RSD 0.51% D RSD 1.55% h SW 1 RSD 1.55% RSD 2.07% A RSD 2.08% D RSD 2.04% SW 1 6 RSD 1.69% RSD 1.06% A RSD 0.30% D RSD 0.16% % RSD 1.59% S 1 S 9 SW 1 SW (n = 3) Table 1 Changes of iridoid contents affected by steamed times in process of Rehmanniae Radix Praeparata (n = 3) /(μg g 1 ) A/(μg g 1 ) D/(μg g 1 ) /(μg g 1 ) S SW S SW S SW S SW S SW S stands for steamed product; SW stands for wine-steamed product, same as below 10 ml 200 μg/ml 10 ml Hypersil GOLD aqc mm 4.6 mm 5 μm 1 ml/min % nm μg/ml 10 μl Y X r RSD 0.84% h SW t/min 2 SW 1 HPLC Fig. 2 HPLC for reference substance of acteoside and sample SW 1
5 756 Chinese Traditional and Herbal Drugs SW 1 RSD 1.03%SW 1 6 RSD 1.47% 98.91% RSD 0.86% S 1 S 9 SW 1 SW HMF Table 2 Changes of acteoside and 5-HMF contents affected by steamed times in process of Rehmanniae Radix Praeparata /(μg g 1 ) 5-HMF/(μg g 1 ) S SW S SW HMF HMF 10 ml 5-HMF μg/ml 5 ml Hypersil GOLD aq C mm 4.6 mm 5 μm 1 ml/min % nm μg/ml 10 μl 5-HMF Y X r HMF RSD 1.34% h SW 1 5-HMF RSD 2.02% SW HMF RSD 0.61% 5-HMF SW 1 5-HMF t/min 3 5-HMF SW 1 HPLC Fig. 3 HPLC for reference substance of 5-HMF and sample SW S 1 S 9 SW 1 SW S 1 S 9 SW 1 SW 9 1 g 20 ml 2 h PE 1 ml 10 ml 0.45 μm % 5 ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml
6 Chinese Traditional and Herbal Drugs % 5 ml Accurasil NH mm 4.6 mm 5 μm ml/min 25 ELSD Alltech2000ES L/min 4 SW t/min 4 SW 1 HPLC Fig. 4 HPLC for mixed reference substance and sample SW μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml 10 μl Y X r Y X r Y X r Y X r Y X r Y X r RSD 2.39% RSD 1.93% RSD 0.80% RSD 0.73% RSD 1.23% RSD 0.96% h SW 1 RSD 6.58% RSD 0.65% RSD 2.35% RSD 1.37% RSD 1.84% RSD 1.47%SW 1 6 RSD 6.58% RSD 0.65% RSD 2.35% RSD 1.37% RSD 1.84% RSD 1.47% % RSD 0.82% % RSD 1.39% S 1 S 9 SW 1 SW SPSS t 4 6 A D A D A 3 A 1 4 D 5 D 1 3
7 758 Chinese Traditional and Herbal Drugs Table 3 Changes of suger contents affected by steamed times in process of Rehmanniae Radix Praeparata /(μg g 1 ) /(μg g 1 ) /(μg g 1 ) /(μg g 1 ) /(μg g 1 ) /(μg g 1 ) S SW S SW S SW S SW S SW S SW ( x± s) Table 4 Comparison on iridoids in process of Rehmanniae Radix Praeparata ( x± s) /(μg g 1 ) A/(μg g 1 ) D/(μg g 1 ) /(μg g 1 ) x ± s t P x ± s t P x ± s t P x ± s t P * ** ** ** * ** ** * * ** * ** ** ** ** * ** ** ** * ** ** * P 0.05 ** P 0.01 * P <0.05 ** P <0.01, same as below 5 5-HMF Table 5 Comparison on acteoside and 5-HMF in process of Rehmanniae Radix Praeparata /(μg g 1 ) 5-HMF/(μg g 1 ) x ± s t P x ± s t P ** ** ** * ** * ** ** ** * ** ** ** ** HMF
8 Chinese Traditional and Herbal Drugs Table 6 Comparison on sugers in process of Rehmanniae Radix Praeparata /(μg g 1 ) /(μg g 1 ) /(μg g 1 ) x ± s t P x ± s t P x ± s t P ** ** * ** * * ** * ** ** ** * /(μg g 1 ) /(μg g 1 ) /(μg g 1 ) x ± s t P x ± s t P x ± s t P * ** * * * ** * * * ** * * 5-HMF HMF 6 5-HMF [1]. [J]., 1983(2): [2] [S] [3]. [J]., 1987, 3: 8-9. [4],,,. [J]., 2013, 44(11): [5]. [D]. :, [6]. [D]. :, [7],,,. [J]., 2005, 20(4): [8],,,. [J]., 2009, 44(9):
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