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

- 2 -

- 3 -

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 -

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 -

(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 12-7 -

(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 -

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

α α-pinene m-cresol osthole sabinene Fig. 1-1. Marker compounds of Osterici seu Notopterygii Radix et Rhizoma - 10 -

bisabolangelone oxypeucedanin O O O O imperatorin isoimperatorin Fig. 1-2. Marker compounds of Osterici seu Notopterygii Radix et Rhizoma - 11 -

O'-glucopyranocyl-cimifugin marmesinin 3'-hydroxymarmesinin oxypeucedanin O O O O imperatorin isoimperatorin bisabolangelone Fig. 1-3. Marker compounds of Osterici seu Notopterygii Radix et Rhizoma - 12 -

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 - 280 (3min) Interface temp. 280 Scanning range 40-160 amu Ionization voltage 70 ev - 13 -

- 14 -

Fig. 4-1. TIC and mass spectrum - 15 -

Table 4-2. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) 70% EtOH 3 250 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. 3-1. Overall analytical procedures of extracts from Osterici seu Notopterygii Radix et Rhizoma - 16 -

Table 3-2. Optimum analytical conditions of HPLC for bisabolangelone, oxypeucedanin, imperatorin, isoimperatorin Column Develosil RPAQUEOUS C30 (4.6 250mm, 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. 4-2. HPLC chromatograms of biological active compounds (A) Standard mixture and (B) 70% EtOH extract from Ostericum koreanum by UV detection at 254 nm - 17 -

μ μ - 18 -

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. 3-2. Overall analytical procedures of extracts from Osterici seu Notopterygii Radix et Rhizoma - 19 -

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 0-20 50-80 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 - 20 -

- 21 -

- 22 -

Fig. 4-3. ESI-MS/MS spectrum - 23 -

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

- 25 -

- 26 -

κ κ μ μ β κ μ ω - 27 -

β α - 28 -

- 29 -

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 - 30 -

Mass spectrometer condition Split ratio 30:1 Injection temp. 270 Oven temp. 60 (3min) - 15 /min - 280 (3min) Interface temp. 280 Scanning range 40-160 amu Ionization voltage 70 ev Column Develosil RPAQUEOUS C30 (4.6 250mm, 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 0-20 50-80 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 - 31 -

Ÿ Ÿ Ÿ - 32 -

- 33 -

( ) () Oxypeucedanin Oxypeucedanin Oxypeucedanin Imperatorin Imperatorin Isoimperatorin Isoimperatorin Bisabolangelone Oxypeucedanin. - 34 -

(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 (4.6 250mm, 5μm) 1.0 ml/min Injection volume ( ) 10 μl Mobile phase ( ) Acetonitrile : H2O = 60 : 40 9 6 Oxypeucedanin - KP CP JP KP - - - 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 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%) 0-20 50-20% 9 6 Oxypeucedanin - KP CP JP KP - - - 36 -

- 37 -

성상 White crystal 이명 CAS number 737-52-0 분자식 C 16 H 14 O 5 (MW 286) 녹는점 ( ) 141 143 구조 - 38 -

δ δ - 39 -

Column Develosil RPAQUEOUS C30 (4.6 250mm, 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 - 40 -

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% - 41 -

Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Oxypeucedanin 50-1000 0.9983 0.0129-0.2384 Intra-day (n=5) Inter-day (n=5) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy 50 67.14 0.64 134.29 68.56 1.09 137.11 Oxypeucedanin 250 249.37 2.66 99.75 248.15 1.01 99.26 1000 1018.33 0.72 101.83 1027.45 0.83 102.75-42 -

Compounds Analytical condition Mean Accuracy(%) CV(%) Oxypeucedanin Column mobile phase combination Develosil (C30) 479.76 95.95 1.55 Hypersil (C18) 468.41 93.68 1.15 55% ACN 486.09 97.22 0.72 60% ACN 485.57 97.11 0.87 65% ACN 481.53 96.31 2.17-43 -

- 44 -

성상 White crystal 이명 CAS number 482-44-0 분자식 C 16 H 14 O 4 (MW 270) 녹는점 ( ) 101 102 구조 - 45 -

δ δ - 46 -

Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Imperatorin 50-1000 0.9987 0.0210-0.1233 Intra-day (n=5) Inter-day (n=5) Compounds nominal conc. (μg/ml) Observed conc. Precision Accuracy Observed Conc. Precision Accuracy 50 55.39 1.16 110.77 55.98 0.83 111.97 Imperatorin 250 253.30 2.52 101.32 257.67 1.16 103.07 1000 1019.03 0.64 101.90 1024.72 0.99 102.47-49 -

Compounds Analytical condition Mean Accuracy(%) CV(%) Imperatorin Column mobile phase combination Develosil (C30) 482.89 96.58 1.68 Hypersil (C18) 453.87 90.71 0.57 55% ACN 482.35 96.47 0.66 60% ACN 484.42 96.88 1.75 65% ACN 477.26 95.45 1.20-50 -