148 5-HMF 5-hydroxymethylfurfural 1995 Kitagawa 16 GC TLC open column 5-HMF 1996 Kubo 5 HPCE SEWON PHARMACY 17 ODS column 5-HMF HMF catalpol

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1 J Chin Med 18(3,4): , ,2 1, Hypersil Hypercarb porous graphitic carbon UV ELSD 0.1% 60 catalpol (9) 5-Hydroxymethylfurfural (5-HMF) (8) glucose (1) sucrose (2) melibiose (3) manninotriose (4) raffinose (5) stachyose (6) verbascose (7) 9 ELSD 1~7 RSD 1.01~3.38% 1.24~3.33% UV 8 9 RSD % % stachyose (6) manninotroiose (4) A 9*[4/(4+6)] B log(4/6) A 0~1.57 B -2.42~-0.34 A 8.09~9 B 1.04~3.28 HPLC ELSD Rehmanniae. glutionsa (G AERTN. ) Libosch. R. glutionsa (G AERTN. ) Libosch. var. hueichingensis Chao et Schih 1-4 catapol 5 - H M F defructosylation raffinose series oligosaccharide defructose oligosaccharide 5 catalpol 6-10 catalpol 6,11-15 isocratic catalpol 6,11-15 catalpol Tel: chingche@sunten.com.tw

2 148 5-HMF 5-hydroxymethylfurfural 1995 Kitagawa 16 GC TLC open column 5-HMF 1996 Kubo 5 HPCE SEWON PHARMACY 17 ODS column 5-HMF HMF catalpol 5-HMF catalpol 5-HMF raffinose series oligosaccharide defructosylation 16 HPLC UV C18 NH 2 Fig. 1 Chemical structures of Maker substances in Rehmannia

3 149 catalpol 5-HMF Porous graphitic carbon Hypersil Hypercarb C 18 NH 2 ELSD HPLC Hypersil Hypercarb porous graphitic carbon, 100x2.1mm, 5 µ m ELSD catalpol 5-HMF glucose 1 sucrose 2 melibiose 3 isomaltotriose 4' raffinose 5 stachyose 6 verbascose 7 5-hydroxymethylfurfural 5-HMF 8 catalpol 9 HPLC-UV- ELSD 0.1% A B 0.2 ml/min manninotriose 4 stachyose 6 ELSD catalpol 5-HMF glucose (1), sucrose (2), melibiose (3), trehalose IS Arcos New Jersey, USA Raffinose (5) 5-HMF (5-hydroxymethylfuroic acid)(8) Aldrich Milwaukee, WIS, USA Isomaltotriose (4' ) and stachyose (6) Sigma St. Louis, MO, USA Verbascose (7) Fluka Steinheim, Germany formic acid Shimakyu's Pure Chemical Osaka, Japan Methanol Merck Darmatadt, Germany Milli-Q System Millipore, Bedford, MA, USA Shimadzu LC-10AD Rheodyne 7125, 20 µl loop Shimadzu SPD M10A photodiode Array Shimadzu CBM-M10A Shimadzu ELSD-LT - Finnigan LCQ advantage TSP LC quaternary pump P-4000 TSP UV-2000 TSP SN-4000 Finnigan ESI interface Finnigan Xcalibur Suntex SP-2200 Elma Transsonic Sigital Hettic Universal 10 ml 15mL 12 Nichiryo µl ml BIOHIT Germany µ L Guard-Pak µ-bondapak C18 (Millipore, Milford, MA, USA) Hypersil Hypercarb

4 150 porous graphite carbon 5 µm, 100x2.1 mm 0.2 ml/min (A) 0.1% HCOOH ph 2.75 (B) CH 3 OH (A) (B) 100: ~35 62: : :100 60~65 0: kpa Gain 8 ELSD HPLC UV-2000 LCQ ESI Sheath gas flow rate 40 arb Aux gas 20 arb I spray voltage 4.5 kv capillary temp 200 arb=arbitrary unit LCQ-tune tune sucrose 1 mg/ml syringe pump 5 µl/min 200 µl/min MeOH LC-MS (1) A A H 2 O H C O O H HOAc TFA H 2 O 0.1% B MeOH (2) B methanol acetonitrile B A 0.1% HCOOH trehalose 50 mg 10 ml 5 mg/ml IS glucose 1 sucrose 2 melibiose 3 isomaltotriose 4' raffinose 5 stachyose 6 verbascose 7 10 mg/ml 1 mg/ml 1.0 ml 200 µl IS (1) (Reproducibility) 0.1g 8 ml µm 25 ml 1 ml 200 µl IS 20 µl intraday interday (2) (Recovery) 0.1g 8 ml µm 25 ml 0.5 ml 0.5 ml glucose 0.5 mg/ml sucrose 0.3 mg/ml melibiose 0.2 mg/ml isomaltotriose 0.5 mg/ml raffinose 0.2 mg/ml stachyose 0.8 mg/ml verbascose 0.05 mg/ml 200 µl IS

5 µl (3) 0.1 mg/ml signal /noise=3/1 (4) isomaltotriose manninotriose isomaltotriose manninotriose isomaltotriose ELSD 0.1g 8 ml ml 0.45 µm 1 ml 200 µl IS 20 µl A1~A8 B1~B3 C1~C3 D1~D8 E1~E3 F1 F2 200 g g (1) Hypercarb porous graphitic carbon Hypersil silicas catalpol 5-HMF NH 2 column C 18 column porous graphitic carbon (2) catalpol iridoid 甙

6 152 UV 206nm 11-13,15 206nm 5-HMF UV λ max 283nm 280nm (3) A ELSD A ELSD HCOOH HOAc TFA 0.1% A ELSD A ELSD 0.1mg/mL MS 0.01mg/mL 1, glucose 2, sucrose 5, raffinose 6, stachyose 7, verbascose A ELSD TFA HCOOH HOAc LC-MS A LC-MS LCQ advantage ESI+ TIC TFA HCOOH HOAc HCOOH HCOOH A A (4) B porous graphite carbon B Hypersil methanol acetonitrile A 0.1% HCOOH ELSD ELSD B ELSD methanol 5-HMF methanol acetonitrile methanol B A 0.1% HCOOH ph 2.75 B MeOH 2 LC-MS (1) LC-MS 1 dimer trimer HPLC-ELSD 1 1~7 manninotriose 4 [M+18] +, 18=H 2 O [M+23] +, 23=Na, [M+H] + MS m/z pattern

7 153 Fig. 2 HPLC chromatogram of Rehmanniae Radix. (a) HPLC-ELSD chromatogram of standards 1-7. (b) HPLC-UV chromatogram of Rehmanniae Radix, 206nm. (c) HPLC-ELSD chromatogram of Rehmanniae Radix. (d) HPLC-UV chromatogram of Rehmanniae Preparata Radix, 280nm. (e) HPLC-ELSD chromatogram of Rehmanniae Preparata Radix.

8 154 pattern (2) LCQ ion extraction m/z SIM [M+H 2 O] + [M+Na] m/z ~7 2 1~7 Hypercarb doublet LC-MS melibiose 2 manninotriose 4 doublet LC- MS doublet 5-HMF 8 catalpol HMF 8 m/z=127 catalpol 9 m/z=380 ion extraction 5-HMF verbascotetraose catalpol manninotriose UV 5-HMF catalpol manninotriose 4 ELSD catalpol doublet catalpol doublet catalpol manninotriose Table 1 Mass data of Maker substances Sugar compounds m.w. MS (m/z, %) Fragment of base peak [M+H 2 O] + [M+Na] + m/z, ion 1 glucose (100) 203 (5) 180 M + 2 sucrose (100) 365 (12) 325 [M+H] + 3 melibiose (100) 365 (8) 325 [M+H] + 4 manninotriose (100) 527 (11) 487 [M+H-H 2 O] + 5 raffinose (100) 527 (73) 343 [M+H-162] + 6 stachyose (100) 689 (61) 505 [M+H-162] + 7 verbascose (100) 851 (70) - - Organic compounds m.w. MS (m/z, %) Fragment of base peak [M+H] + [M+ H 2 O] + [2M+H-Glc*] HMF (100) 144 (24) - 9 catalpol (72) 563 (100) * Glc=glucosyl group 162u

9 155 ELSD A = a C b A a b C ln A = k + b ln C k ln A ln C b 1~2 ELSD 23 Shimadzu ELSD-LT ELSD melibiose 0.02~0.6 mg/ml y= x sucrose 2 isomaltotriose 4' raffinose 5 stachyose 6 verbascose 7 2 glucose RSD manninotriose doublet catalpol HPLC doublet doublet Table 2 Calibration curves of standards LC-ELSD calibration curve Standard Range (mg/ml) Function R 2 Type 1 glucose 0.1~ 3.0 y = x (Peak area / IS) Power* 2 sucrose 0.01~1.3 y = x Power 3 melibiose 0.01~0.5 y = x Power 4 isomaltotriose 0.03~1.8 y = x Power 5 raffinose 0.01~0.5 y = x Power 6 stachyose 0.01~1.7 y = x Power 7 verbascose 0.001~0.16 y = x Power LC-UV calibration curve Standard Range (mg/ml) Function R 2 Type 8 5-HMF 0.2~500 y = x Power * calibration curve: y = a x b log y = a' + b log X

10 156 manninotriose doublet X Y 1 y = x 28827, R 2 = (1) Reproducibility 1~7 HPLC-ELSD 5-HMF 8 HPLC-UV 280nm catalpol 9 catalpol HPLC- UV 206nm intraday interday / RSD, % 3 HPLC-UV UV ELSD UV UV ELSD Table 3 Reproducibility of the HPLC method Reproducibility (RSD %) Compounds Intraday(n=6) Interday(n=6) Peak ratio Peak area R.T. Peak ratio Peak area R.T and 4 were doublets. Peak area was calculated by the sum of doublet. Retention time was calculated by the mean of the doublet.

11 157 Glucose glucose RSD % RSD 4.01% 5.05% RSD 2.44% 2.75% glucose glucose 2~7 RSD RSD 1.01~3.38 RSD 1.24~3.33 4% 1~7 RSD 0.51~1.09% 0.69~1.25% HPLC-UV 8 9 RSD % % RSD % % (2) (Recovery) 20 μ L % (3) Detection limit 20 μ L signal/noise=3/1 4 1~7 ELSD 0.2~2 μg/ml 5-HMF UV 0.05 μg/ml ELSD Gain 1~12 Gain=7 Gain (4) isomaltotriose manninotriose manninotriose isomaltotriose Glc α1 6 Glc α1 6 Glc manninotriose Gal α1 6 Gal α1 6 Glc ELSD ELSD UV Table 4 Recoveries and detection limits of the HPLC method Compounds Recovery (%) μg/ml detection limit ng

12 158 Isomaltotriose manninotriose α1 6 Hypercarb ELSD doublet ELSD isomaltotriose manninotriose isomaltotriose manninotriose 0.781mg/mL isomaltotriose manninotriose iosmaltotriose mg/mL HPLC manninotriose iosmaltotriose 99.1:100:98.4 RSD manninotriose RSD 2.1% 0.2x 0.4x 0.8x isomaltotriose % isomaltotriose manninotriose A1~A8 B1~B3 C1~C D1~D8 F1 F2 E1 E2, E3 HPLC UV ELSD catalpol stachyose manninotriose catalpol 4

13 159 (a) (b) 9 (Area x ) (x0.5) (x 10) 3 8 (x 10) (x 0.5) 6 ( x 0.5) Fig. 3 Patterns of the compositions ( 1-9 ) in (a) Rehmanniae Radix and (b) Rehmanniae Preparata Radix (*9 was shown as peak areas) Table 5 Content of Maker substances 1-9 in Rehmanniae Radix Collected Samples ** (peak area) mg/g A A A A A A A A B B B C C C Mean* SD * Mean was calculated except C2; - : not detected ** 9 was shown as peak areas

14 160 Table 6 Content of Maker substances 1-9 in Rehmanniae Preparta Radix mg/g Collected Sample D D D D D D D D E E E F F Mean* SD * Mean was calculated except E1, F1; - : not detected catalpol A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 C1 C2 C3 Fig. 4 Catalpol content in Rehmanniae Radix calculated by peak area Material source A- Medical and Pharmaceutical Industry Technology and Development Center B-Taipei drug market C- Mainland China drug market

15 HMF mg/g D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 F1 F2 Fig. 5 5-HMF content in Rehmanniae Preparta Radix mg/g Material source D-Taipei drug market E-Mainland China drug market F-provided by a doctor 5-HMF 5 catalpol 5-HMF defructosylation raffinose series oligosaccharide defructose oligosaccharide 5 Tomoda stachyose 64.9% % 29 Kubo 5 S K 5 Kubo galactose glucose fructose Kubo 5 S K Kubo Kubo S stachyose Kubo stachyose 35.1% 33.3% C2 stachyose 9.24% 6 Kubo 5 Kubo fructose raffinose series oligosaccharide Kubo 3 4 defructose

16 162 oligosaccharide 5 6 raffinose series F1 E1 catalpol 6-10 catalpol catalpol HPLC 4 C2 catalpol A7 catalpol 5 HPLC catalpol 5-HMF 0.05 ± 0.02 mg/g 5 5-HMF 2.50 ± 1.84 mg/g 50 5-HMF 5-HMF 5-HMF E1 5 2mg/g 5-HMF 5-HMF A B 7 6 (1) A A 9*[4/(4+6)] C2 E1 0.05~ ~8.99 C2 A 3.9 E 1 A 1.55 F1 A A 9*[4/(4+6)] (2) B B 4 6 log(4/6) 4<6 B<0 4>6 B>0-2.24~ ~3.07 C2 B -0.12

17 163 Table 7 The values of Indicator A and B of Rehmannia samples Sample Indicator A Indicator B Sample Indicator A Indicator B A D A D A D A D A D A D A D A D B E1* B E B E C F1* C2* F C AVG AVG SD SD * Not calculated when getting mean value Indicator B Indicator A A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 C1 C2 C3 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 F1 F2 Fig. 6 The values of indicator A and B of Rehmannia samples. A~C, Rehmanniae Radix D~F, Rehmanniae Preparta Radix

18 164 0 E1 B F1 B (3) A B A B B 4/ ~ ~ A 4/4+6 0~1 9 0~9 9 A 0 9 F1 8 (4) A B A B % A 0~1.57 B -2.42~-0.34 A 8.09~9 B 1.04~3.28 catalpol 5-HMF catalpol 5-HMF HPLC Hypersil Hypercarb porous graphitic carbon 100x2.1mm 5µm ELSD ELSD catalpol 5-HMF A B A 0~1.57 B -2.42~-0.34 A 8.09~9 B 1.04~ pp

19 pp , pp , pp , Kubo M, Asano T, Matsuda H, Yutani S, Honda S. Studies on Radix Rehmanniae. The relation between changes of constituents and improvable effects on roots of Rehmannia glutinosa. Yakugaku Zasshi 116: , (2):71-72, Chang HM, Paul PH. Pharmacology and Applications of Chinese Materia medica. World Scientific, Singapore, 2nd Ed, pp , Tyler VE. Herbs of Choice: The therapeutic use of phytomedicinals. Pharmaceutical Products Press, New York, pp.37-41, Hamia B, Marak AB, Jos MM. Two herbivoredeterrent iridoid glycosides reduce the in-vitro growth of a specialist but not of a generalist pathogenic fugus of Plantago lanceolata L. Chemoecology 12: , pp : HPLC 29: : : :408-9, 447, Kitagawa I, Fukuda Y, Taniyama T, Yoshikawa M. Chemical studies on crude drug processing. X. On the constituents of rehmannia radix (4): comparison of the constituents of various rehmanniae radixes originating in China, Korea, and Japan. Yakugaku Zasshi 115: , Lee KS. A comparative study on the Rehmannia glutinosa Preparata's Index Component, 5-HMF. text-2.htm : , Lipniunas PH, Neville DCA, Trimble RB, Townsend RR. Separation of biosynthetic oligosaccharide branch isomers using highperformance liquid chromatography on a porous two-dimensional graphite stationary phase. Anal. Biochem. 243: , Davies MJ, Hounsell EF. Comparison of separation modes of high-performance liquid chromatography for the analysis of glycoproteinand proteoglycan-derived oligosaccharides. J. Chromatogr. A 720: , Thomsson KA, Karlsson NG, Hansson GC. Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins. J. Chromatogr. A 854: , Zhang J, Xie Y, Hedrick JL, Lebrilla CB. Profiling the morphological distribution of O-linked oligosaccharides. Anal. Biochem. 334:20-35,

20 Christie WW. Evaporative light-scattering detectors in lipid analysis. Lipid Technol 5:68-70, Shimadzu corporation. The Shimadzu ELSD-LT Evaporative Light Scattering detector Linearity and Sensitivity Performance for Selected Analytes chromato/elsd.html 25. Oppenheimer LE, Mourey TH. Examination of the concentration response of. evaporative light-scattering mass detectors. J. Chromatogr. 323: , To m o d a M, K a t o S, O n u m a M. Wa t e r- soluble constituents of Rehmanniae Radix. I. Carbohydrates and acids of Rehmannia glutinosa f. hueichingensis. Chem. Pharm. Bull. 19:1455, Tomoda M, Tanaka M, Kondo N. Water-soluble constituents of Rehmanniae Radix. I. On the Constituents of Roots of Rehmannia glutinosa var. purpurea Chem. Pharm. Bull. 19:2411, 1971.

21 167 J Chin Med 18(3,4): , 2007 A METHOD OF ANALYZING THE COMPOSITION OF REHMANNIA AND USING CARBOHYDRATE INDICATORS FOR DIFFERENTIATING REHMANNIAE RADIX AND REHMANNIAE PREPARATA RADIX Ching-Che Lin 1,2, Shuenn-Jyi Sheu *1,2 1 Brion Research Institute of Taiwan 2 Department of Chemistry, National Taiwan Normal University ( Received 4 th December 2007, accepted 3 th January 2008 ) Rehmannia is a commonly used Chinese medicine. According to different processing procedures, Rehamnnia is classified as Rehmanniae Radix and Rehmanniae Preparata Radix that have totally different pharmaceutical properties and clinical applications. The present study used Hypersil Hypercarb (porous graphitic carbon) as the column connected to the UV and ELSD detectors. Using 0.1% formic acid aqueous solution and methanol as the mobile phase, the composition of a total of 9 compounds, catalpol (9) in Rehmanniae Radix, 5-hydroxymethylfurfural (5-HMF) (8) in Rehmanniae Preparata Radix, and the various carbohydrates including glucose (1), sucrose (2), melibiose (3), manninotriose (4), raffinose (5), stachyose (6), and verbascose (7) in Radix were analyzed simultaneously within 60 minutes. This analytical method showed good reproducibility. The RSD values of the peak areas of the analysis of compounds 1~7 by ELSD ranged between 1.01~3.38% (intraday) and 1.24~3.33% (interday). The RSD values of the analysis of 8 and 9 were 1.01% and 1.23% (intraday) and 1.24% and 1.52% (interday). The major carbohydrates in Rehmanniae Radix and Rehmanniae Preparata Radix were determined to be stachyose (6) and manninotroiose (4), respectively. The data of the samples from 14 batches of Rehmanniae Radix and 13 batches of Rehmanniae Preparata Radix were used to determine the processing of Rehamnnia by indicators A (9*[4/(4+6)]) and B (log(4/6)). Based on the statistical data, the indicators A of 0~1.57 and B of -2.42~-0.34 indicated that the sample was collected from Rehmanniae Radix. On the other hand, the indicators A of 8.09~9 and B of 1.04~3.28 indicated that the sample was collected from the well processed Rehmanniae Preparata Radix. Key word: Rehmanniae Radix, Rehmanniae Preparata Radix, HPLC, ELSD, processing *Corresponding authors: Bor-Jinn Shien, Phone: , Fax: , borjinn@cycu.edu.tw; Yu- Ling Huang, Phone: ext. 6241, Fax: , ylhuang@nricm.edu.tw

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