한약재품질표준화연구사업단 작약 ( 芍藥 ) Paeoniae Radix 생약연구과

Size: px

Start display at page:

Download "한약재품질표준화연구사업단 작약 ( 芍藥 ) Paeoniae Radix 생약연구과"

혜성 요
5 years ago
Views:

1 한약재품질표준화연구사업단 작약 ( 芍藥 ) Paeoniae Radix 생약연구과

2 - 2 -

3 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 -

4 β paeoniflorin albiflorin Paeonia lactiflora Pallas - 4 -

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

6 (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

7 p-hydroxybenzoic acid 16 methyl gallate 15 benzoylpaeoniflorin 3, 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

8 (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 -

9 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 -

10 *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)

11 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

12 β α β β β β β β β α β

13 - 13 -

14 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

15 Table 4. The optimal condition of sample preparation Extracting method Solvent Time (sonication) 70% EtOH 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

16 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 ( 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) Gradient elution wavelength 230 nm Fig 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

17 - 17 -

18 Table 4. The optimal condition of sample preparation Extracting method Solvent Time (sonication) 70% EtOH 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

19 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 ( 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

20 Fig 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)

21 - 21 -

22 5-22 -

23 - 23 -

24 α α α α γ γ α α

25 - 25 -

26 α β β α β α

27 - 27 -

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

29 (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

30 - 30 -

31 α γ

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

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 m membrane filter 10 μl Detector () Column ( ) Flow rate () Column temperature ( ) Injection volume ( ) Mobile phase ( ) Gradient (, B%) UV (230nm) Hypersil Gold column ( 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) paeoniflorin, albiflorin - KP CP JP KP paeoniflorin, albiflorin 2.3% paeoniflorin 2%

34 (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 m membrane filter 10 μl Detector () Column ( ) Capcell pak C18 UG ( mm, 5 μm) Flow rate () Column temperature ( ) Injection volume ( ) 0.2 ml/min 40 C 20 μl Mobile phase ( ) mm Ammonium acetate/acetonitrile = 20/80 (ph 4.5 with acetic acid) paeoniflorin, albiflorin - KP CP JP KP paeoniflorin, albiflorin 2.3% paeoniflorin 2%

35 - 35 -

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

37 δ δ

38 Column Hypersil Gold column ( 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% % 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

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

40 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)

41 Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Paeoniflorin 1,000-50, Intra-day (n=5) Inter-day (n=5) Compounds conc. (ng/ml) Precision Accuracy Precision Accuracy Paeoniflorin

42 Compounds Analytical condition Theoretical plate (N) Resolution Selectivity Temperature Paeoniflorin Flow rate Gradient profile ml/min ml/min ml/min (1) (2) (3)

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

44 - 44 -

45 - 45 -

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

47 δ δ δ

48 Column Hypersil Gold column ( 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% % 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

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

50 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)

51 Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) albiflorin 1,000-50, Intra-day (n=5) Inter-day (n=5) Compounds conc. (ng/ml) Precision Accuracy Precision Accuracy Albiflorin

52 Compounds Analytical condition Theoretical plate (N) Resolution Selectivity Temperature Albiflorin Flow rate Gradient profile ml/min ml/min ml/min (1) (2) (3)

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

54 - 54 -

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