한약재품질표준화연구사업단 단삼 ( 丹參 ) Salviae Miltiorrhizae Radix 생약연구과
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KP 11 CP 2015 Salvia miltiorrhizae Radix Salviae Miltiorrhizae Radix et Rhizoma Salvia miltiorrhiza Bunge Salvia miltiorrhiza Bunge salvianolic acid 4.1% JP 16 - - - 2 -
salvianolic acid B tanshinone IIA - 3 -
Salvia miltiorrhiza Bunge ( %), 70%EtOH 2.37 kg (13.58%) - 4 -
(mg) dansenspiroketallactone 79 tanshinone ⅡA 3530.9, - 5 -
tanshinoneⅠ 3880.2, cryptotanshinone 5024, 15,16-dihydrotanshinoneⅠ 1252.8, salvianolic acid B 2567.9 rosmarinic acid 1500 9 -methyl lithospermate 13 monomethyl lithospermate B 38 dimethyl lithospermate B 450 methyl salvianolate C 280-6 -
(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 -methyl lithospermate (8) methyl salvianolate C (11) - 8 -
rosmarinic acid salvianolic acid B 15,16-dihydrotanshinone I cryptotanshinone O O O tanshinone I tanshinone IIA 4-hydroxycinnamic acid (I.S.) Fig. 1-1. Marker compounds of salvia miltiorrhiza root and internal standard - 9 -
rosmarinic acid salvianolic acid B 15,16-dihydrotanshinone I cryptotanshinone O O O tanshinone I tanshinone IIA Imperatorin (I.S.) Fig. 1-2. Marker compounds of salvia miltiorrhiza root and internal compound - 10 -
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 100.0-mL Flask (Test solution) Aliquots, 10.0uL LC/MS/MS Analysis Fig. 3. Analytical procedure of Pulverized Salvia miltriorrhiza - 11 -
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 (4.6 250 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) 0-1 75 60 Gradient elution 1-14 60 60 14-22 60 40 22 23 23 40 40 11 11 11 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. - 12 -
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α - 14 -
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 100.0-mL Flask (Test solution) Aliquots, 10.0uL LC/MS/MS Analysis Fig. 3. Analytical procedure of Pulverized Salvia miltriorrhiza - 15 -
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) 1.31 11 6.1 1.25 10 Collision gas pressure (Tor) 3.58 7 Fig. 4-2. Typical chromatogram of standard mixture (A) and sample (B). - 16 -
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Figure 5. Analytical results(w/w %) of the marker compounds in Salvia miltiorrhizae Radix. - 18 -
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α α κ - 23 -
μ μ μ μ - 24 -
μ α α α α α α κ - 25 -
α α β - 26 -
Ÿ Ÿ Ÿ Ÿ HPLC condition UV contidion Column Waters YMC (4.6 250 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) 0-1 75 60 Gradient elution 1-14 60 60 14-22 60 40 22 23 23 40 40 11 11 11 wavelength 280nm - 27 -
Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ - 28 -
α - 29 -
( ) () 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,. - 30 -
(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 (4.6 250 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) 0-1 75 60 1-14 14-22 22 23 23 40 salvianolic acid B 0.2% KP CP JP KP salvianolic acid B 4.1% - 60 60 60 40 40 11 11 11-31 -
(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 (2.0 150 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% - - 32 -
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성상 powder 이명 lithospermic aicd B CAS number 115939-25-8 분자식 C 36 H 30 O 16 (MW 718) 녹는점 ( ) 102 구조 - 34 -
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Column Waters YMC (4.6 250 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 : 1 0 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 - 36 -
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) 1.31 11 (L/min) Auxilliary gas flow (L/min) 6.1 Curtain gas flow (L/min) 1.25 10 Collision gas pressure (Tor) 3.58 7 (A) Typical chromatogram of standard mixture and (B) sample. - 37 -
Analytes Linear range (mg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) salvianolic acid B 0.2-800 0.9999 0.0393 0.0060 51.5 155 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) salvianolic acid B 0.3939 3.03 15.09 0.6008-38 -
Intra-day (n=3) Inter-day (n=3) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy 60.0 64.31 4.0.3 107.2 56.71 94.51 5.89 salvianolic acid B 150.0 144.38 4.01 96.3 144.28 96.19 4.21 300.0 294.19 3.01 98.1 303.52 101.17 4.46 Compounds Analytical condition Theoretical plate (N) Resolution Selectivity ymc 8274 5.78 Column shisheido 5418 2.47 sodex 3053 4.43 salvianolic acid B 25 7853 5.34 Column temp. 30 8813 6.05 35 8758 5.47 40 8288 6.62 μ - 39 -
Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) salvianolic acid B 7.5-1500 0.9996 0.0028-0.00444 2.30 7.5-40 -
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성상 red needles 이명 CAS number 568-72-9 분자식 C 19 H 18 O 3 (MW 294) 녹는점 ( ) 215-216 구조 - 42 -
δ - 43 -
Column Waters YMC (4.6 250 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 : 1 0 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 - 44 -
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) 1.31 11 (L/min) Auxilliary gas flow (L/min) 6.1 Curtain gas flow (L/min) 1.25 10 Collision gas pressure (Tor) 3.58 7 (A) Typical chromatogram of standard mixture and (B) sample. - 45 -
Analytes Linear range (mg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) tanshinone IIA 0.2-40 0.9999 0.1510-0.0154 32.4 108.1 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) tanshinone IIA 1.3929 0.3952 37.66 0.1027-46 -
Intra-day (n=3) Inter-day (n=3) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy 2.0 2.01 2.74 98.9 1.98 0.82 100.5 tanshinone IIA 4.0 3.95 1.36 100.2 4.01 2.94 98.7 6.0 6.00 2.76 99.5 5.97 1.41 100.1 Compounds Analytical condition Theoretical plate (N) Resolution Selectivity ymc 298134 21.26 Column shisheido 306002 17.15 sodex 296153 13.55 tanshinone IIA 25 308980 22.50 Column temp. 30 300496 21.49 35 295459 21.82 40 286093 20.38 μ - 47 -
Analytes Linear range (ng/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) tanshinone IIA 1.08-108 0.9998 0.0290 0.00218 0.31 1.08 Intra-day (n=3) Inter-day (n=3) Compounds Fortified conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy 2.70 2.52 4.20 93.3 2.55 6.09 94.4 tanshinone IIA 5.40 5.39 4.42 99.9 5.31 3.96 98.4 13.61 14.25 3.31 104.7 14.07 1.55 103.4 27.00 26.69 1.11 98.9 26.80 0.40 99.3-48 -