한약재품질표준화연구사업단 단삼 ( 丹參 ) Salviae Miltiorrhizae Radix 생약연구과

Size: px

Start display at page:

Download "한약재품질표준화연구사업단 단삼 ( 丹參 ) Salviae Miltiorrhizae Radix 생약연구과"

창숙 독고
5 years ago
Views:

1 한약재품질표준화연구사업단 단삼 ( 丹參 ) Salviae Miltiorrhizae Radix 생약연구과

2 - 1 -

3 KP 11 CP 2015 Salvia miltiorrhizae Radix Salviae Miltiorrhizae Radix et Rhizoma Salvia miltiorrhiza Bunge Salvia miltiorrhiza Bunge salvianolic acid 4.1% JP

4 salvianolic acid B tanshinone IIA - 3 -

5 Salvia miltiorrhiza Bunge ( %), 70%EtOH 2.37 kg (13.58%) - 4 -

6 (mg) dansenspiroketallactone 79 tanshinone ⅡA , - 5 -

7 tanshinoneⅠ , cryptotanshinone 5024, 15,16-dihydrotanshinoneⅠ , salvianolic acid B rosmarinic acid methyl lithospermate 13 monomethyl lithospermate B 38 dimethyl lithospermate B 450 methyl salvianolate C

8 (compound number) dansenspiroketallactone (1) O O O tanshinone ⅡA (2) tanshinoneⅠ (3) cryptotanshinone (4) 15,16-dihydrotanshinoneⅠ (5) R1=R2=H salvianolic acid B (6) R1=CH3, R2=H (or R1=H, R2=CH3) monomethyl lithospermate B (9) R1=CH3, R2=CH3 dimethyl lithospermate B (10) rosmarinic acid (7) - 7 -

9 9 -methyl lithospermate (8) methyl salvianolate C (11) - 8 -

10 rosmarinic acid salvianolic acid B 15,16-dihydrotanshinone I cryptotanshinone O O O tanshinone I tanshinone IIA 4-hydroxycinnamic acid (I.S.) Fig Marker compounds of salvia miltiorrhiza root and internal standard - 9 -

11 rosmarinic acid salvianolic acid B 15,16-dihydrotanshinone I cryptotanshinone O O O tanshinone I tanshinone IIA Imperatorin (I.S.) Fig Marker compounds of salvia miltiorrhiza root and internal compound

12 Table 4. The optimal condition of sample preparation Extracting method Solvent Time (sonication) 75% MeOH 30 Pulverized Salvia miltriorrhiza 100 mg Add 75% methanol 50 ml Sonication for 30 min Extract Centrifuge for 15 min (3,000rpm) Filter through 0.2-um membrane filter Aliquots, 1mL Add 2 ml of I.S (Imperatorin 320 ng/ml) Add mobile phase mL Flask (Test solution) Aliquots, 10.0uL LC/MS/MS Analysis Fig. 3. Analytical procedure of Pulverized Salvia miltriorrhiza

13 Table 3-1. Optimum analytical conditions of HPLC/UV for rosmarinic acid, salvianolic acid B, 15,16-dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA HPLC condition UV contidion Column Waters YMC ( mm) Mobile phase A : H 2 O : HCOOH = 100 : 1 B : MeOH : CH 3 CN : HCOOH = 100 : 75 : 1 Flow rate 1.0 ml/min Column temp. 20 C Inj. vol. 10 μl Time (min) Solvent (%A) Gradient elution wavelength 280nm Fig. 4-1.HPLC chromatogram of standard mixture (A) and sample (B) of Salvia miltiorrhizae Radix. 1 rosmarinic acid, 2 salvianolic acid B, 3 15,16-dihydrotanshinone I, 4 cryptotanshinone, 5 tanshinone I, 6 tanshinone IIA, 7 p-hydroxycinnamic acid

14 - 13 -

15 α

16 Table 4. The optimal condition of sample preparation Extracting method Solvent Time (sonication) 75% MeOH 30 Pulverized Salvia miltriorrhiza 100 mg Add 75% methanol 50 ml Sonication for 30 min Extract Centrifuge for 15 min (3,000rpm) Filter through 0.2-um membrane filter Aliquots, 1mL Add 2 ml of I.S (Imperatorin 320 ng/ml) Add mobile phase mL Flask (Test solution) Aliquots, 10.0uL LC/MS/MS Analysis Fig. 3. Analytical procedure of Pulverized Salvia miltriorrhiza

17 Table 3-2. Optimum analytical conditions of HPLC/MS/MS for wogonin-7-o-glucuronide methyl ester, wogonin, baicalein, 5,7,2',5'-tetrahydroxy-8,6'-dimethoxyflavone, 5,7,2',6'-tetrahydroxyflavone, baicalin, chrysin Value S/W setting Ion source voltage (kv) 5.5 Temp ( C) 350 HPLC -MS/MS operational parameter Nebulizer gas flow (Ion spray flow) (L/min) Auxilliary gas flow (L/min) Curtain gas flow (L/min) Collision gas pressure (Tor) Fig Typical chromatogram of standard mixture (A) and sample (B)

18 - 17 -

19 Figure 5. Analytical results(w/w %) of the marker compounds in Salvia miltiorrhizae Radix

20 - 19 -

21 - 20 -

22 - 21 -

23 - 22 -

24 α α κ

25 μ μ μ μ

26 μ α α α α α α κ

27 α α β

28 Ÿ Ÿ Ÿ Ÿ HPLC condition UV contidion Column Waters YMC ( mm) Mobile phase A : H 2 O : HCOOH = 100 : 1 B : MeOH : CH 3 CN : HCOOH = 100 : 75 : 1 Flow rate 1.0 ml/min Column temp. 20 C Inj. vol. 10 μl Time (min) Solvent (%A) Gradient elution wavelength 280nm

29 Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ

30 α

31 ( ) () salvianolic acid B (KP 4.1% ) (ChP 3% ) tanshinone ⅡA (,,, ) tanshinone ⅡA (ChP 0.2% ) tanshinone Ⅰ (, CNS,,,, ) cytotanshinone (, CNS,,, 15,16-dihydrotanshinone I (,,, ) salvianolic acid B,

32 (Salviae Miltiorrhizae Radix) KP Salvia miltiorrhizae Radix Ÿ Salvia miltiorrhiza Bunge JP Ÿ ChP Salvia miltiorrhizae Radix Ÿ Salvia miltiorrhiza Bunge salvianolic acid B 100 mg 75% MeOH 50 ml 30 sonication 0.2 um membrane filter 1 ml internal standard 2 ml (96.5 ppm) mobile phase 100 ml 10 ul injection Detector () Column ( ) Flow rate () Column temperature ( ) UV (280nm) Waters YMC ( mm) 1.0 ml/min 20 C Injection volume ( ) 10 μl Mobile phase ( ) Gradient (, A%) 2 14 A : H 2 O : HCOOH = 100 : 1 B : MeOH : CH 3 CN : HCOOH = 100 : 75 : 1 Time (min) Solvent (%A) salvianolic acid B 0.2% KP CP JP KP salvianolic acid B 4.1%

33 (Salviae Miltiorrhizae Radix) KP Salvia miltiorrhizae Radix Ÿ Salvia miltiorrhiza Bunge JP Ÿ ChP Salvia miltiorrhizae Radix Ÿ Salvia miltiorrhiza Bunge salvianolic acid B 100 mg 75% MeOH 50 ml 30 sonication 0.2 um membrane filter 1 ml internal standard 2 ml (96.5 ppm) mobile phase 100 ml 10 ul injection Detector () Column ( ) Flow rate () Column temperature ( ) Luna C18 column ( mm, 3u, 100A ) 0.15 ml/min 40 C Injection volume ( ) 10 μl A : 90% MeOH (with 1% acetic acid, 0.1% Mobile phase ( ) 2 14 formic acid) B : 10% MeOH (with 1% acetic acid, 0.1% formic acid) salvianolic acid B 0.2% KP CP JP KP salvianolic acid B 4.1%

34 - 33 -

35 성상 powder 이명 lithospermic aicd B CAS number 분자식 C 36 H 30 O 16 (MW 718) 녹는점 ( ) 102 구조

36 - 35 -

37 Column Waters YMC ( mm) Detector UV Temp. 20 Injection volume 10 μl Flow rate 1.0 ml/min Mobile phase Time(min) H 2 O : HCOOH = 100 : 1 MeOH : CH 3 CN : HCOOH = 100 : 75 : % 25% 1 60% 40% 14 60% 40% 22 40% 60% 23 11% 89% 40 11% 89% (A) standard mixture and (B) sample of Salvia miltiorrhizae Radix. 1 rosmarinic acid, 2 salvianolic acid B, 3 15,16-dihydrotanshinone I, 4 cryptotanshinone, 5 tanshinone I, 6 tanshinone IIA, 7 p-hydroxycinnamic acid

38 Column Detector Temp. Injection volume Flow rate Time(min) Mobile phase HPLC-MS/MS operational parameter Value S/W setting Ion source voltage (kv) 5.5 Temp ( C) 350 Nebulizer gas flow (Ion spray flow) (L/min) Auxilliary gas flow (L/min) 6.1 Curtain gas flow (L/min) Collision gas pressure (Tor) (A) Typical chromatogram of standard mixture and (B) sample

39 Analytes Linear range (mg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) salvianolic acid B Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) salvianolic acid B

40 Intra-day (n=3) Inter-day (n=3) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy salvianolic acid B Compounds Analytical condition Theoretical plate (N) Resolution Selectivity ymc Column shisheido sodex salvianolic acid B Column temp μ

41 Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) salvianolic acid B

42 - 41 -

43 성상 red needles 이명 CAS number 분자식 C 19 H 18 O 3 (MW 294) 녹는점 ( ) 구조

44 δ

45 Column Waters YMC ( mm) Detector UV Temp. 20 Injection volume 10 μl Flow rate 1.0 ml/min Mobile phase Time(min) H 2 O : HCOOH = 100 : 1 MeOH : CH 3 CN : HCOOH = 100 : 75 : % 25% 1 60% 40% 14 60% 40% 22 40% 60% 23 11% 89% 40 11% 89% (A) standard mixture and (B) sample of Salvia miltiorrhizae Radix. 1 rosmarinic acid, 2 salvianolic acid B, 3 15,16-dihydrotanshinone I, 4 cryptotanshinone, 5 tanshinone I, 6 tanshinone IIA, 7 p-hydroxycinnamic acid

46 Column Detector Temp. Injection volume Flow rate Time(min) Mobile phase HPLC-MS/MS operational parameter Value S/W setting Ion source voltage (kv) 5.5 Temp ( C) 350 Nebulizer gas flow (Ion spray flow) (L/min) Auxilliary gas flow (L/min) 6.1 Curtain gas flow (L/min) Collision gas pressure (Tor) (A) Typical chromatogram of standard mixture and (B) sample

47 Analytes Linear range (mg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) tanshinone IIA Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) tanshinone IIA

48 Intra-day (n=3) Inter-day (n=3) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy tanshinone IIA Compounds Analytical condition Theoretical plate (N) Resolution Selectivity ymc Column shisheido sodex tanshinone IIA Column temp μ

49 Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) tanshinone IIA Intra-day (n=3) Inter-day (n=3) Compounds Fortified conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy tanshinone IIA

한약재품질표준화연구사업단 작약 ( 芍藥 ) Paeoniae Radix 생약연구과

한약재품질표준화연구사업단 작약 ( 芍藥 ) Paeoniae Radix 생약연구과 - 2 - KP 11 CP 2015 JP 16 Paeoniae Radix Paeoniae Radix Alba Paeoniae Radix Rubra Paeoniae Radix Paeonia lactiflora Pallas paeoniflorin, albiflorin 2.3% Paeonia