한약재품질표준화연구사업단 작약 ( 芍藥 ) Paeoniae Radix 생약연구과

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KP 11 CP 2015 JP 16 Paeoniae Radix Paeoniae Radix Alba Paeoniae Radix Rubra Paeoniae Radix Paeonia lactiflora Pallas paeoniflorin, albiflorin 2.3% Paeonia lactiflora Pallas - Paeonia lactiflora Pallas Paeonia veitchii Lynch Paeonia lactiflora Pallas - paeoniflorin 2% - 3 -

β paeoniflorin albiflorin Paeonia lactiflora Pallas - 4 -

( %), 70%EtOH 2.642 kg (14.67%) - 5 -

(mg) α-tocopherol 65 paeonol 59, β-amyrin 3 24-methylenecycloartanol 6 palmitic acid 23 β-sitosterol 132 daucosterol 6 -O-palmitate 353 paeonia cerebroside 34 betulinic acid 160 oleanolic acid 100 hederagenin 1,000 daucosterol 9 3,3'-di-O-methylellagic acid 13 3,4'-di-O-methylellagic acid 12 benzoic acid 70 aromadendrin 6 30-norhederagenin 19 lactiflorin 51-6 -

p-hydroxybenzoic acid 16 methyl gallate 15 benzoylpaeoniflorin 3,100 30-norarjunolic acid 7 arjunolic acid 4 mudanpioside C 45 12,20(29)-dien-28-oic acid 10 1-O-β-D-glucosylpaeonisuffrone 10 paeonidanin 20 1-O-β-D-glucosyl-8-O-benzoyl paeonisuffrone 45 paeoniflorin 17,000, albiflorin 1,200 oxypaeoniflorin 578 mudanpioside E 28 adenosine 30-7 -

(compound number) R = H benzoic acid (1) R = OH p-hydroxybenzoic acid (2) methyl gallate (3) R = CH 3, R1 = H *3,3'-di-O-methylellagic acid (4) R = H, R1 = CH 3 *3,4'-di-O-methylellagic acid (5) paeonol (6) *aromadendrin (7) *α-tocopherol (8) paeoniflorin (9) - 8 -

albiflorin (10) lactiflorin (11) benzoylpaeoniflorin (12) HO HO HO HO OH O O O O O O OH H oxypaeoniflorin (13) *mudanpioside C (14) - 9 -

*mudanpioside E (15) HO HO HO OH O O O O O R R= H **1-O-β-D-glucosyl-8-O-benzoyl paeonisuffrone (16) R= OCH 3 **paeonidanin (17) O *1-O-β-D-glucosylpaeonisuffrone (18) R= H R1= CH 3 R2= H R= H R1= COOH R2= H R= OH R1= COOH R2= H R= OH R1= COOH R2= OH *β-amyrin (19) oleanolic acid (20) hederagenin (21) 30-norhederagenin (22) R= CH 3 R1= H *arjunolic acid (23) R= CH 3 R1= OH *30-norarjunolic acid (24) R= OH R1= H *3β,4β,23-trihydroxy-24,30-dinorolean -12,20(29)-dien-28-oic acid (25) *24-methylenecycloartanol (26) - 10 -

betulinic acid (27) R= H β-sitosterol (28) R= glucose daucosterol (29) *daucosterol 6 -O-palmitate (30) *paeonia cerebroside (31) *adenosine (32) *palmitic acid (33) β β β β - 11 -

β α β β β β β β β α β - 12 -

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paeoniflorin (+)-catechin paeonol albiflorin benzoic acid hederagenin 2-ethoxybenzamide (IS) (HPCL-UV) simvastatin (IS) (HPLC-MS/MS) Fig. 1. Marker compounds of Paeoniae Radix and internal standard - 14 -

Table 4. The optimal condition of sample preparation Extracting method Solvent Time (sonication) 70% EtOH 1 100 mg pulverized sample added 20 ml 70% EtOH sonication for 60 min filter with membrane filter (17 mm, 0.45 μm) HPLC analysis (10 μl) Fig. 3. Overall analytical procedures of extracts from Paeoniae Radix - 15 -

Table 3-1. Optimum analytical conditions of HPLC/UV for paeoniflorin, (+)-catechin, paeonol, albiflorin, benzoic acid, hederagenin, gallic acid methyl ester HPLC condition UV contidion Column Hypersil Gold column (4.6 150 mm, 5 μm) Mobile phase (A) 1/15 M KH 2 PO 4 buffer/acetonitrile (80/20 v/v, ph 7.4) (B) 1/15 M KH 2 PO 4 buffer/acetonitrile (20/80 v/v, ph 7.4) Flow rate 1.0 ml/min Column temp. 30 C Inj. vol. 10 μl run time 15 min Time (min) Solvent (%A) 0-1 0 50 Gradient elution 1-6 50 60 6-9 60 100 9-14 14-14.1 100 100 100-0 wavelength 230 nm Fig. 4-1. Hypersil Gold column A) reference components B) 70% EtOH HPLC/UV chromatogram : 1. benzoic acid, 2. (+)-catechin, 3. albiflorin, 4. paeoniflorin, 5. paeonol and the internal standard, 2-ethoxybenzamide - 16 -

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Table 3-2. Optimum analytical conditions of HPLC/MS/MS for paeoniflorin, (+)-catechin, paeonol, albiflorin, benzoic acid, hederagenin, gallic acid methyl ester Column Capcell pak C18 UG (2.0 150 mm, 5 μm) Mobile phase 1 mm Ammonium acetate/acetonitrile = 20/80 (ph 4.5 with acetic acid) Flow rate 0.2 ml/min Column temp. 40 C Inj. vol. 20 μl run time 5 min HPLC/MS/MS condition MRM ion transition - 19 -

Fig. 4-2. LC-MS/MS chromatograms ; A) paeonol, B) (+)-catechin, C) benzoic acid, D) gallic acid methyl ester, E) albiflorin, F) hederagenin, G) paeoniflorin, H) simvastatin (I.S) - 20 -

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α α α α γ γ α α - 24 -

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α β β α β α - 26 -

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Ÿ β β Ÿ ββ β α Ÿ Ÿ β β Ÿ Ÿ HPLC/MS/MS condition Column Capcell pak C18 UG (2.0 150 mm, 5 μm) Mobile phase 1 mm Ammonium acetate/acetonitrile = 20/80-28 -

(ph 4.5 with acetic acid) Flow rate 0.2 ml/min Column temp. 40 C Inj. vol. 20 μl run time 5 min MRM ion transition Ÿ Ÿ Ÿ - 29 -

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α γ - 31 -

( ) () paeoniflorin paeoniflorin (,,) paeoniflorin, albiflorin albiflorin albiflorin (, ) (2.3% ) paeonol paeonol (, ) paeoniflorin, albiflorin,. - 32 -

(Paeoniae Radix) KP Paeoniae Radix Paeonia lactiflora Pallas ChP Paeoniae Radix Alba Paeoniae Radix Rubra Paeonia lactiflora Pallas Paeonia lactiflora Pallas Paeonia veitchii Lynch JP Paeoniae Radix Paeonia lactiflora Pallas paeoniflorin, albiflorin 100mg 70% EtOH 20 ml 60 0.45 m membrane filter 10 μl Detector () Column ( ) Flow rate () Column temperature ( ) Injection volume ( ) Mobile phase ( ) Gradient (, B%) UV (230nm) Hypersil Gold column (4.6 150 mm, 5 μm) 1.0 ml/min 30 C 10 μl (A) 1/15 M KH2PO4 buffer/acetonitrile (80/20 v/v, ph 7.4) (B) 1/15 M KH2PO4 buffer/acetonitrile (20/80 v/v, ph 7.4) Time (min) Solvent (%A) 0-1 0 50 1-6 50 60 6-9 60 100 9-14 100 100 14-14.1 100-0 7 10 paeoniflorin, albiflorin - KP CP JP KP paeoniflorin, albiflorin 2.3% paeoniflorin 2% - 33 -

(Paeoniae Radix) KP Paeoniae Radix Paeonia lactiflora Pallas ChP Paeoniae Radix Alba Paeoniae Radix Rubra Paeonia lactiflora Pallas Paeonia lactiflora Pallas Paeonia veitchii Lynch JP Paeoniae Radix Paeonia lactiflora Pallas paeoniflorin, albiflorin 100mg 70% EtOH 20 ml 60 0.45 m membrane filter 10 μl Detector () Column ( ) Capcell pak C18 UG (2.0 150 mm, 5 μm) Flow rate () Column temperature ( ) Injection volume ( ) 0.2 ml/min 40 C 20 μl Mobile phase ( ) 7 10 1 mm Ammonium acetate/acetonitrile = 20/80 (ph 4.5 with acetic acid) paeoniflorin, albiflorin - KP CP JP KP paeoniflorin, albiflorin 2.3% paeoniflorin 2% - 34 -

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성상 White fine powder 이명 CAS number 23180-57-6 분자식 C 23 H 28 O 11 (MW 480) 녹는점 ( ) 구조 - 36 -

δ δ - 37 -

Column Hypersil Gold column (4.6 150 mm, 5 μm) Detector UV Temp. 30 Injection volume 10 μl Flow rate 1.0 ml/min Mobile phase Time(min) 1/15 M KH 2 PO 4 buffer/acetonitrile 1/15 M KH 2 PO 4 buffer/acetonitrile (80/20 v/v, ph 7.4) (20/80 v/v, ph 7.4) 0 0% 100% 1 50% 50% 6 60% 40% 9 100% 0% 14 100% 0% 14.1 0% 100% Hypersil Gold column A) reference components B) 70% EtOH HPLC/UV chromatogram : 1. benzoic acid, 2. (+)-catechin, 3. albiflorin, 4. paeoniflorin, 5. paeonol and the internal standard, 2-ethoxybenzamide - 38 -

Column Capcell pak C18 UG (2.0 150 mm, 5 μm) Detector Temp. 40 Injection volume 20 μl Flow rate 0.2 ml/min MS/MS conditon MRM ion transition - 39 -

LC-MS/MS chromatograms A) paeonol, B) (+)-catechin, C) benzoic acid, D) gallic acid methyl ester, E) albiflorin, F) hederagenin, G) paeoniflorin, H) simvastatin (I.S) - 40 -

Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Paeoniflorin 1,000-50,000 0.9996 0.00002 0.0056 1000 Intra-day (n=5) Inter-day (n=5) Compounds conc. (ng/ml) Precision Accuracy Precision Accuracy 1000 0.71 119.01 8.48 99.10 2000 1.17 113.21 9.19 102.75 Paeoniflorin 5000 6.07 103.64 3.38 102.92 10000 1.41 97.80 2.04 96.76 50000 4.03 98.26 2.54 97.01-41 -

Compounds Analytical condition Theoretical plate (N) Resolution Selectivity 28 51924.3 Temperature 30 48143.9 Paeoniflorin Flow rate Gradient profile 32 51953.9 0.9 ml/min 45545.3 1.0 ml/min 48143.9 1.1 ml/min 49082.6 (1) 45367.4 (2) 48143.9 (3) 52109.7-42 -

Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Paeoniflorin 50-600 50 Intra-day (n=5) Inter-day (n=5) Compounds conc. (ng/ml) Precision Accuracy Precision Accuracy 50 5.89 86.57 Paeoniflorin 100 2.30 101.79 600 4.84 94.13-43 -

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성상 White powder 이명 CAS number 39011-90-0 분자식 C 23 H 28 O 11 (MW 480) 녹는점 ( ) 구조 - 46 -

δ δ δ - 47 -

Column Capcell pak C18 UG (2.0 150 mm, 5 μm) Detector Temp. 40 Injection volume 20 μl Flow rate 0.2 ml/min MS/MS conditon MRM ion transition - 49 -

LC-MS/MS chromatograms A) paeonol, B) (+)-catechin, C) benzoic acid, D) gallic acid methyl ester, E) albiflorin, F) hederagenin, G) paeoniflorin, H) simvastatin (I.S) - 50 -

Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) albiflorin 1,000-50,000 0.9996 0.00002 0.0026 1000 Intra-day (n=5) Inter-day (n=5) Compounds conc. (ng/ml) Precision Accuracy Precision Accuracy 1000 2.31 115.60 6.71 96.08 2000 3.81 105.16 2.05 92.70 Albiflorin 5000 6.61 95.75 4.92 92.34 10000 1.05 89.95 3.42 88.90 50000 3.05 88.07 4.31 88.64-51 -

Compounds Analytical condition Theoretical plate (N) Resolution Selectivity 28 23055.5 Temperature 30 23850.3 Albiflorin Flow rate Gradient profile 32 25438.9 0.9 ml/min 22087.3 1.0 ml/min 23850.3 1.1 ml/min 23373.5 (1) 23662.2 (2) 23850.3 (3) 23588.3-52 -

Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Albiflorin 50-600 50 Intra-day (n=5) Inter-day (n=5) Compounds conc. (ng/ml) Precision Accuracy Precision Accuracy 50 5.91 93.60 albiflorin 100 6.51 104.45 600 3.49 99.76-53 -

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