한약재품질표준화연구사업단 갈근 ( 葛根 ) Puerariae Radix 생약연구과
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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 -
puerarin daidzin Pueraria lobata Ohwi - 4 -
( %) 70%EtOH 143.2 g (28.64%) MeOH 2.5 kg (20.8%) - 5 -
(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 -
(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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puerarin daidzein 8-C-Api-Glc daidzin daidzein genistein Fig. 1. Marker compounds of Puerariae Radix - 9 -
Table 4. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) MeOH 120 2 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 - 10 -
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 0-10 10-40 10 10 10-52 UV contidion wavelength 254 nm Fig. 4-1. Typical chromatogram of compounds from Puerariae Radix. 1. puerarin; 2. daidzein 8-C-apiosyl(1-6)glucoside; 3. daidzin 4. daidzein; 5. genistein - 11 -
μ μ μ - 12 -
Table 4. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) MeOH 120 2 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 - 13 -
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, -4500 kv ( 음이온모드 ) MS operation parameters - 14 -
Fig. 4-2. HPLC-MS chromatogram of MeOH extract of Puerariae Radix. - 15 -
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:. - 16 -
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α γ γ β β - 18 -
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μ κ - 21 -
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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 0-10 10-40 10 10 10-52 UV contidion wavelength 254 nm - 23 -
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, -4500 kv ( 음이온모드 ) MS operation parameters Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ - 24 -
Ÿ - 25 -
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( ) () puerarin (,,,, puerarin (2%) ) daidzin (0.3%) daidzein (,,,, ) puerarin, daidzin,. - 27 -
(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) 0-10 10-40 Solvent (%A) 10 10 10-52 5 7 puerarin - KP ChP JP KP puerarin 2% daidzin 0.3% puerarin 2% - 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 () 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% - 29 -
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성상 White amorphous powder CAS number 3681-99-0 분자식 C 21 H 20 O 9 (MW 416.38) 녹는점 ( ) 187-189 구조 - 31 -
δ δ λ - 32 -
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 - 33 -
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. - 34 -
Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Puerarin 5-400 0.9999 13320 18146 1 2.5 Intra-day (n=5) Inter-day (n=5) Compounds conc. (μg/ml) Precision Accuracy Precision Accuracy 5 8.48 1.20 7.28 3.13 Puerarin 10 1.03 4.03 3.57 7.68 20 3.87 1.68 0.46 1.96-35 -
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성상 Colorless needles CAS number 552-66-9 분자식 C 21 H 20 O 9 (MW 416.38) 녹는점 ( ) 235-237 구조 - 38 -
δ δ λ - 39 -
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 - 40 -
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. - 41 -
Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Daidzin 5-400 0.9999 12105 46 0.5 1.25 Intra-day (n=5) Inter-day (n=5) Compounds conc. (μg/ml) Precision Accuracy Precision Accuracy 5 1.24 5.60 6.74 1.64 Daidzin 10 3.41 8.33 1,96 6.44 20 1.11 4.27 1.61 2.51-42 -
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