한약재품질표준화연구사업단 갈근 ( 葛根 ) Puerariae Radix 생약연구과

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1 한약재품질표준화연구사업단 갈근 ( 葛根 ) Puerariae Radix 생약연구과

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3 KP 11 Puerariae Radix Pueraria lobata Ohwi puerarin 2% daidzin 0.3% CP 2015 Puerariae Lobatae Radix Pueraria lobata (Willd.) Ohwi - JP 16 Pueraria Radix Pueraria lobata Ohwi puerarin 2% - 3 -

4 puerarin daidzin Pueraria lobata Ohwi - 4 -

5 ( %) 70%EtOH g (28.64%) MeOH 2.5 kg (20.8%) - 5 -

6 (mg) Lupenone Lupeol Glyceryl-1-tetracosanoate Puerarol Coumestrol Daidzein, Genistein, (+)-Puerarol B-2-O-glucopyranoside Ononin Genistin Daidzin, 3'-Methoxypuerarin Puerarin, 3'-Hydroxypuerarin Daidzein 8-C-apiosyl (1 6)-glucoside Allantoin - 6 -

7 (compound number) Lupenone (1) Lupeol (2) Glyceryl-1-tetracosanoate (5) R1 = A Puerarol (3) R1 = H Coumestrol (4) (+)-Puerarol B-2-O-glucopyranoside (8) R1 = H, R2 = H, R3 = H, R4 = H, R5 = OH R1 = OH, R2 = H, R3 = H R4 = H, R5 = OH R1 = H, R2 = Glc, R3 = H, R4 = H, R5 = OCH3 R1 = OH, R2 = Glc, R3 = H, R4 = H, R5 = OH R1 = H, R2 = Glc, R3 = H, R4 = H, R5 = OH R1 = H, R2 = H, R3 = Glc, R4 = OCH3, R5 = OH R1 = H, R2 = H, R3 = Glc, R4 = H, R5 = OH R1 = H, R2 = H, R3 = Glc, R4 = OH, R5 = OH R1 = H, R2 = H, R3 = Api(1 6) glc, R4 = H, R5 = OH Daidzein (6) Genistein (7) Ononin (14) Genistin (16) Daidzin (15) 3'-Methoxypuerarin (13) Puerarin (9) 3'-Hydroxypuerarin (11) Daidzein 8-C-apiosyl (1 6)-glucoside (12) Allantoin (10) - 7 -

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9 puerarin daidzein 8-C-Api-Glc daidzin daidzein genistein Fig. 1. Marker compounds of Puerariae Radix - 9 -

10 Table 4. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) MeOH g pulverized sample added 100 ml MeOH reflux for 120 min dilution (X10) filter filter with membrane filter (0.45 μm) HPLC analysis (5 μl) Fig. 3. Overall analytical procedures of extracts from Puerariae Radix

11 Table 3-1. Optimum analytical conditions of HPLC/UV for puerarin, daidzein 8-C-Api-Glc, daidzin, daidzein, genistein HPLC condition Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Mobile phase A : Acetonitrile B : H 2 O Flow rate 1.0 ml/min Inj. vol. 5 μl Total run time 40 min Time (min) Solvent (%A) Gradient elution UV contidion wavelength 254 nm Fig Typical chromatogram of compounds from Puerariae Radix. 1. puerarin; 2. daidzein 8-C-apiosyl(1-6)glucoside; 3. daidzin 4. daidzein; 5. genistein

12 μ μ μ

13 Table 4. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) MeOH g pulverized sample added 100 ml MeOH reflux for 120 min dilute (X100) filter filter with membrane filter (0.45 μm) HPLC analysis (5 μl) Fig. 3. Overall analytical procedures of extracts from Puerariae Radix

14 Table 3-2. Optimum analytical conditions of HPLC-MS/MS for puerarin, daidzein 8-C-Api-Glc, daidzin, daidzein, genistein Column Xterra C18 (150 x 2.1 mm, 3μ) HPLC condition Mobile phase 100% MeOH Flow rate 0.3 ml/min Column temp. 35 C Inj. vol. 5 μl collision gas flow 4 L/min HPLC-MS contidion curtain gas flow 10 L/min ion source gas flow 16 L/min Turbo gas temperature 350 C, kv ( 음이온모드 ) MS operation parameters

15 Fig HPLC-MS chromatogram of MeOH extract of Puerariae Radix

16 Fig. 5. Analytical results(μg/g) of the active compounds in Puerariae Radix. PL-K-1:, ; PL-K-2: ; PL-K-3: ; PL-K-5:, ; PL-K-7: ; PL-C-1: () ; PL-C-2: (; ) ; PL-C-3: ; PL-C-6: ( ) ; PL-C-8: ; PL-C-9:. PL-C-10:

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18 α γ γ β β

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21 μ κ

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23 Ÿ Ÿ Ÿ HPLC condition Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Mobile phase A : Acetonitrile B : H 2 O Flow rate 1.0 ml/min Inj. vol. 5 μl Total run time 40 min Time (min) Solvent (%A) Gradient elution UV contidion wavelength 254 nm

24 Column Xterra C18 (150 x 2.1 mm, 3μ) HPLC condition Mobile phase 100% MeOH Flow rate 0.3 ml/min Column temp. 35 C Inj. vol. 5 μl collision gas flow 4 L/min HPLC-MS contidion curtain gas flow 10 L/min ion source gas flow 16 L/min Turbo gas temperature 350 C, kv ( 음이온모드 ) MS operation parameters Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ

25 Ÿ

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27 ( ) () puerarin (,,,, puerarin (2%) ) daidzin (0.3%) daidzein (,,,, ) puerarin, daidzin,

28 (Puerariae Radix) KP 11 Puerariae Radix Pueraria lobata Ohwi ChP 2015 Puerariae Lobatae Radix Pueraria lobata (Willd.) Ohwi JP 16 Pueraria Radix Pueraria lobata Ohwi puerarin, daidzin (150 mesh ) 2.0 g 100 ml 120 methanol 10 μl MeOH 10 membrane filter (0.45 μm) 5 μl injection HPLC. Detector () UV (254 nm) Column ( ) Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Flow rate () 1.0 ml/min Injection volume ( ) 5 μl Mobile phase ( ) Gradient (, A%) A : Acetonitrile B : H 2 O Time (min) Solvent (%A) puerarin - KP ChP JP KP puerarin 2% daidzin 0.3% puerarin 2%

29 (Puerariae Radix) KP 11 Puerariae Radix Pueraria lobata Ohwi ChP 2015 Puerariae Lobatae Radix Pueraria lobata (Willd.) Ohwi JP 16 Pueraria Radix Pueraria lobata Ohwi puerarin, daidzin (150 mesh ) 2.0 g 100 ml 120 methanol 10 μl MeOH 10 membrane filter (0.45 μm) 5 μl injection HPLC. Detector () Column ( ) Xterra C18 (150 x 2.1 mm, 3μ) Flow rate () 0.3 ml/min Column temperature ( ) 35 C Injection volume ( ) 5 Mobile phase ( ) 100% MeOH 5 7 puerarin - KP ChP JP KP puerarin 2% daidzin 0.3% puerarin 2%

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31 성상 White amorphous powder CAS number 분자식 C 21 H 20 O 9 (MW ) 녹는점 ( ) 구조

32 δ δ λ

33 Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Detector UV (254 nm) Injection volume 5 μl Flow rate 1.0 ml/min Mobile phase Time(min) Acetonitrile H 2 O 0 10% 90% 10 10% 90% 40 52% 48% Typical chromatogram of compounds from Puerariae Radix. 1. puerarin; 2. daidzein 8-C-apiosyl(1-6)glucoside; 3. daidzin 4. daidzein; 5. genistein

34 Column Xterra C18 (150 x 2.1 mm, 3μ) Detector Temp. 30 Injection volume 5 Flow rate 0.3 ml/min Mobile phase 100% MeOH HPLC-MS chromatogram of MeOH extract of Puerariae Radix

35 Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Puerarin Intra-day (n=5) Inter-day (n=5) Compounds conc. (μg/ml) Precision Accuracy Precision Accuracy Puerarin

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38 성상 Colorless needles CAS number 분자식 C 21 H 20 O 9 (MW ) 녹는점 ( ) 구조

39 δ δ λ

40 Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Detector UV (254 nm) Injection volume 5 μl Flow rate 1.0 ml/min Mobile phase Time(min) Acetonitrile H 2 O 0 10% 90% 10 10% 90% 40 52% 48% Typical chromatogram of compounds from Puerariae Radix. 1. puerarin; 2. daidzein 8-C-apiosyl(1-6)glucoside; 3. daidzin 4. daidzein; 5. genistein

41 Column Xterra C18 (150 x 2.1 mm, 3μ) Detector Temp. 30 Injection volume 5 Flow rate 0.3 ml/min Mobile phase 100% MeOH HPLC-MS chromatogram of MeOH extract of Puerariae Radix

42 Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Daidzin Intra-day (n=5) Inter-day (n=5) Compounds conc. (μg/ml) Precision Accuracy Precision Accuracy Daidzin ,

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한약재품질표준화연구사업단 고삼 ( 苦參 ) Sophorae Radix 생약연구과

한약재품질표준화연구사업단 고삼 ( 苦參 ) Sophorae Radix 생약연구과 - 1 - KP 11 Sophorae Radix Sophora flavescens Solander ex Aiton oxymatrine, matrine 1.0% CP 2015 Sophorae Flavescentis Radix Sophora flavescens Aiton - JP 16