한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과

Size: px

Start display at page:

Download "한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과"

지애 현
5 years ago
Views:

1 한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과

2 - 2 -

3 - 3 -

4 KP 11 Osterici seu Notopterygii Radix et Rhizoma Ostericum koreanum Maximowicz, Notopterygium incisum Ting, Notopterygium forbesii Boissier - CP 2015 Notopterygii Rhizoma et Radix Notopterygium incisum Ting ex H.T. Chang Notopterygium franchetii H. de Boiss. - JP 16 Notopterygii Rhizoma Notopterygium incisum Ting, Notopterygium forbesii Boissier - β - 4 -

5 α β α β β δ α α δ Oxypeucedanin Imperatorin Ostericum koreanum Maximowicz ( %) 70%EtOH 72.8g (29.1%) MeOH 1.68kg (21.57%) - 5 -

6 - 6 -

7 (mg) Oxypeucedanin 7,800 Isoimperatorin 3,420 Imperatorin 1,850, O-glucopyranosyl-cimifugin 1,140 Marmesinin 318 3'-hydroxy marmesinin 71 Scopolin 25 m-cresol 3,215 Osthole 181, Sabinene 98 Bisabolangelone 1,524 α-pinene

8 (compound number) Oxypeucedanin (1) O Isoimperatorin (2) O O O Imperatorin (3) O-glucopyranosyl-cimifugin (4) Marmesinin (5) 3'-hydroxy marmesinin (6) Scopolin (7) - 8 -

9 m-cresol (8) Osthole (9) Sabinene (10) Bisabolangelone (11) α-pinene (12) α - 9 -

10 α α-pinene m-cresol osthole sabinene Fig Marker compounds of Osterici seu Notopterygii Radix et Rhizoma

11 bisabolangelone oxypeucedanin O O O O imperatorin isoimperatorin Fig Marker compounds of Osterici seu Notopterygii Radix et Rhizoma

12 O'-glucopyranocyl-cimifugin marmesinin 3'-hydroxymarmesinin oxypeucedanin O O O O imperatorin isoimperatorin bisabolangelone Fig Marker compounds of Osterici seu Notopterygii Radix et Rhizoma

13 Table 3-1. Optimum analytical conditions of CG/MS for α-pinene, m-cresol, osthole, sabinene Column DB-1MS (30 m 0.32 m 0.2 μm Film) Flow rate 1.0 ml/min Gas chromatograph condition Mass spectrometer condition injection vol. 1.0 μl Average velocity 37 cm/sec Split ratio 30:1 Injection temp. 270 Oven temp. 60 (3min) - 15 /min (3min) Interface temp. 280 Scanning range amu Ionization voltage 70 ev

14 - 14 -

15 Fig TIC and mass spectrum

16 Table 4-2. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) 70% EtOH g sample added 1L 70% EtOH reflux for 3hr filter with filter paper (No.2) vacuum evaporation and freeze drying add acetonitrile : H 2 0 = 60 : 40 filter with membrane filter (0.45 μm) 2times HPLC analysis Fig Overall analytical procedures of extracts from Osterici seu Notopterygii Radix et Rhizoma

17 Table 3-2. Optimum analytical conditions of HPLC for bisabolangelone, oxypeucedanin, imperatorin, isoimperatorin Column Develosil RPAQUEOUS C30 ( mm, 5μm) HPLC conditon UV contidion Mobile phase Acetonitrile : H2O = 60 : 40 Flow rate 1.0 ml/min run time 25 min Inj. vol. 10 μl wavelength 254 nm Fig HPLC chromatograms of biological active compounds (A) Standard mixture and (B) 70% EtOH extract from Ostericum koreanum by UV detection at 254 nm

18 μ μ

19 Table 4-3. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) 70% EtOH 7hr Dried O. koreanum (30g) cutting 300mL 70% EtOH Extraction filter with paper Evaporation add 25mL H2O freezing dry Dried Extract Fig Overall analytical procedures of extracts from Osterici seu Notopterygii Radix et Rhizoma

20 Table 3-3. Optimum analytical conditions of ESI-MS/MS for O'-glucopyranocyl-cimifugin, marmesinin, 3'-hydroxymarmesinin, oxypeucedanin, imperatorin, isoimperatorin, bisabolangelone Column Luna 5u C18(2) 100, 150 1mm 5micron Mobile phase A : 5% ACN (0.1% formic acid) B : 95% ACN (0.1% formic acid) LC condition Flow rate 50 μl/min Inj. vol. 1 μl Time (min) Solvent (%A) Gradient elution UV condition wavelength 254nm Ionization source ESI (electrospray ionization) Polarity Positive, Negative Mass spectrometer conditon Analyze type Ion trap analyze Mass range 100~1000m/z For MS/MS Scan mode Isolation width 2.0m/z, collision E 35% Activation Q 0.25, Activation time 30msec

21 - 21 -

22 - 22 -

23 Fig ESI-MS/MS spectrum

24 Fig. 5. Simultaneous quantification of bioactive components from extract of O. koreanum

25 - 25 -

26 - 26 -

27 κ κ μ μ β κ μ ω

28 β α

29 - 29 -

30 Ÿ Ÿ α Ÿ α Ÿ Column DB-1MS (30 m 0.32 m 0.2 μm Film) Gas chromatograph condition Flow rate 1.0 ml/min injection vol. 1.0 μl Average velocity 37 cm/sec

31 Mass spectrometer condition Split ratio 30:1 Injection temp. 270 Oven temp. 60 (3min) - 15 /min (3min) Interface temp. 280 Scanning range amu Ionization voltage 70 ev Column Develosil RPAQUEOUS C30 ( mm, 5μm) HPLC conditon UV contidion Mobile phase Acetonitrile : H2O = 60 : 40 Flow rate 1.0 ml/min run time 25 min Inj. vol. 10 μl wavelength 254 nm Column Luna 5u C18(2) 100, 150 1mm 5micron Mobile phase A : 5% ACN (0.1% formic acid) B : 95% ACN (0.1% formic acid) LC condition Flow rate 50 μl/min Inj. vol. 1 μl Time (min) Solvent (%A) Gradient elution UV condition wavelength 254nm Ionization source ESI (electrospray ionization) Polarity Positive, Negative Mass spectrometer conditon Analyze type Ion trap analyze Mass range 100~1000m/z For MS/MS Scan mode Isolation width 2.0m/z, collision E 35% Activation Q 0.25, Activation time 30msec Ÿ

32 Ÿ Ÿ Ÿ

33 - 33 -

34 ( ) () Oxypeucedanin Oxypeucedanin Oxypeucedanin Imperatorin Imperatorin Isoimperatorin Isoimperatorin Bisabolangelone Oxypeucedanin

35 (Ostericum Root) Osterici seu KP Notopterygii Radix et Rhizoma Ÿ Ostericum koreanum Maximowicz, Ÿ Notopterygium incisum Ting, Ÿ Notopterygium forbesii Boissier ChP Notopterygii Rhizoma et Radix Ÿ Ÿ Notopterygium incisum Ting ex H.T. Chang Notopterygium franchetii H. de Boiss. JP Notopterygii Rhizoma Ÿ Ÿ Notopterygium incisum Ting, Notopterygium forbesii Boissier Oxypeucedanin 250g 70% EtOH 1L 3 No.2 filter paper acetonitrile : H 2 0 = 60 : 40 membrane filter (0.45 μm) Detector () Column ( ) Flow rate () UV (254 nm) Develosil RPAQUEOUS C30 ( mm, 5μm) 1.0 ml/min Injection volume ( ) 10 μl Mobile phase ( ) Acetonitrile : H2O = 60 : Oxypeucedanin - KP CP JP KP

36 (Ostericum Root) Osterici seu KP Notopterygii Radix et Rhizoma Ÿ Ostericum koreanum Maximowicz, Ÿ Notopterygium incisum Ting, Ÿ Notopterygium forbesii Boissier ChP Notopterygii Rhizoma et Radix Ÿ Ÿ Notopterygium incisum Ting ex H.T. Chang Notopterygium franchetii H. de Boiss. JP Notopterygii Rhizoma Ÿ Ÿ Notopterygium incisum Ting, Notopterygium forbesii Boissier Oxypeucedanin Detector () UV (254 nm) Column ( ) Luna 5u C18(2) 100, 150 1mm 5micron Flow rate () 50 μl/min Injection volume ( ) Mobile phase ( ) 1 μl A : 5% ACN (0.1% formic acid) B : 95% ACN (0.1% formic acid) Gradient (, B%) % 9 6 Oxypeucedanin - KP CP JP KP

37 - 37 -

38 성상 White crystal 이명 CAS number 분자식 C 16 H 14 O 5 (MW 286) 녹는점 ( ) 구조

39 δ δ

40 Column Develosil RPAQUEOUS C30 ( mm, 5μm) Detector UV (254 nm) Injection volume 10 μl Flow rate 1.0 ml/min Mobile phase Acetonitrile : H 2 O = 60 : 40 HPLC chromatograms of biological active compounds (A) Standard mixture and (B) 70% EtOH extract from Ostericum koreanum by UV detection at 254 nm

41 Column Luna 5u C18(2) 100, 150 1mm 5micron Detector UV (254 nm) Injection volume 1 μl Flow rate 50 L/min Mobile phase Time(min) 5% ACN (0.1% formic acid) 95% ACN (0.1% formic acid) 0 50% 50% 20 80% 20%

42 Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Oxypeucedanin Intra-day (n=5) Inter-day (n=5) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy Oxypeucedanin

43 Compounds Analytical condition Mean Accuracy(%) CV(%) Oxypeucedanin Column mobile phase combination Develosil (C30) Hypersil (C18) % ACN % ACN % ACN

44 - 44 -

45 성상 White crystal 이명 CAS number 분자식 C 16 H 14 O 4 (MW 270) 녹는점 ( ) 구조

46 δ δ

47 Column Develosil RPAQUEOUS C30 ( mm, 5μm) Detector UV (254 nm) Injection volume 10 μl Flow rate 1.0 ml/min Mobile phase Acetonitrile : H 2 O = 60 : 40 HPLC chromatograms of biological active compounds (A) Standard mixture and (B) 70% EtOH extract from Ostericum koreanum by UV detection at 254 nm

48 Column Luna 5u C18(2) 100, 150 1mm 5micron Detector UV (254 nm) Injection volume 1 μl Flow rate 50 L/min Mobile phase Time(min) 5% ACN (0.1% formic acid) 95% ACN (0.1% formic acid) 0 50% 50% 20 80% 20%

49 Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Imperatorin Intra-day (n=5) Inter-day (n=5) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy Imperatorin

50 Compounds Analytical condition Mean Accuracy(%) CV(%) Imperatorin Column mobile phase combination Develosil (C30) Hypersil (C18) % ACN % ACN % ACN

한약재품질표준화연구사업단 단삼 ( 丹參 ) Salviae Miltiorrhizae Radix 생약연구과

한약재품질표준화연구사업단 단삼 ( 丹參 ) Salviae Miltiorrhizae Radix 생약연구과 - 1 - KP 11 CP 2015 Salvia miltiorrhizae Radix Salviae Miltiorrhizae Radix et Rhizoma Salvia miltiorrhiza Bunge Salvia miltiorrhiza Bunge salvianolic