한약재품질표준화연구사업단 고삼 ( 苦參 ) Sophorae Radix 생약연구과

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KP 11 Sophorae Radix Sophora flavescens Solander ex Aiton oxymatrine, matrine 1.0% CP 2015 Sophorae Flavescentis Radix Sophora flavescens Aiton - JP 16 Sophorae Radix Sophora flavescens Aiton - - 2 -

α oxymatrine matrine Sophora flavescens Aiton - 3 -

( %) 70%EtOH 143.2 g (28.64%) MeOH 2.3 kg (23%) - 4 -

(mg) trans-hexadecyl ferulic acid cis-octadecyl ferulic acid β-sitosterol trans-hexadecyl sinapic acid ( )-4-Hydroxy-3-methoxy-(6aR, 11aR)-8,9-methylenedioxyptercarpan ( )-Maackiain Xanthohumol Formononetin Desmethylanhydroicaritin, (2S)-2'-Methoxykurarinone (2S)-3β,7,4'-Trihydroxy-5-methoxy-8 -(γ,γ-dimethylallyl)-flavanone (2S)-7,4'-Trihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone Umbelliferone Kuraridin Trifolirhizin, β-sitosterol glucoside Lupenone Genistein Kurarinone Matrine Oxymatrine 8-Lavandulylkaempferol Kurarinol - 5 -

(compound number) O O 14 R = H trans-hexadecyl ferulic acid (1) R = OCH 3 trans-hexadecyl sinapic acid (4) R OH OCH 3 O OH O 16 OCH 3 cis-octadecyl ferulic acid (2) R1 = H β-sitosterol (3) R1 = Glc β-sitosterol glucoside (16) R1 = CH 3 ( )-4-Hydroxy-3-methoxy-(6aR, R2 = OH 11aR)-8,9-methylenedioxyptercarpan (5) R1 = H R2 = H ( )-Maackiain (6) R1 = Glc R2 = H Trifolirhizin (15) R1 = A R2 = H Xanthohumol (7) R1 = B R2 = OH Kuraridin (14) R1 = C R2 = OH Kuraridinol (24) R1 = H R2 = OCH 3 Formononetin (8) R1 = OH R2 = OH Genistein (18) R1 = A Desmethylanhydroicaritin (9) R1 = B 8-Lavandulylkaempferol (22) - 6 -

R1 = B R2 = OCH 3 R3 = H R1 = A R2 = H R3 = OH R1 = A R2 = H R3 = H R1 = B R2 = OH R3 = H R1 = C R2 = OH R3 = H (2S)-2'-Methoxykurarinone (10) (2S)-3β,7,4'-Trihydroxy-5-methoxy-8-( γ,γ-dimethylallyl)-flavanone (11) (2S)-7,4'-Trihydroxy-5-methoxy-8-(γ, γ-dimethylallyl)-flavanone (12) Kurarinone (19) Kurarinol (23) Umbelliferone (13) Lupenone (17) Matrine (20) Oxymatrine (21) - 7 -

β βγ γ γ γ β - 8 -

R 1 O H O O H O O R1=Glucose trifolirhizin kurarinone β - 9 -

O O 16 OH OCH 3 lupenone cis-octadecyl ferulic acid R 1 O O H O H O O R1=Glucose umbelliferone trifolirhizin genistein desmethylanhydroicartin formononetin (-)-maackianin (+)-matrine kuraridin HO β-sitosterol (+)-kurarinione O OH N H propranolol (IS) - 10 -

Table 4. The optimal condition of sample preparation Extracting method Solvent Time (Reflux) MeOH 120 2 g pulverized sample added 100 ml MeOH reflux for 120 min filter filter with membrane filter (0.45 μm) HPLC analysis (20 μl) Fig. 3. Overall analytical procedures of extracts from Sophorae Radix - 11 -

Table 3-1. Optimum analytical conditions of HPLC/UV for trifolirhizin, kurarinone HPLC condition Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Mobile phase A : H 2 O B : Acetonitrile Flow rate 1.0 ml/min Inj. vol. 20 μl Total run time 40 min Time (min) Solvent (%A) Gradient elution 0-40 10-70 UV contidion wavelength 254 nm Fig. 4-1. Typical chromatogram of compounds from Sophorae Radix - 12 -

μ μ μ μ - 13 -

Table 3-2. Optimum analytical conditions of HPLC-MS/MS for lupenone, cis-octadecyl ferulic acid, umbelliferone, trifolirhizin, genistein, desmethylanhydroicartin, formononetin, (-)-maackianin, (+)-matrine, kuraridin, β-sitosterol, (+)-kurarinione HPLC condition HPLC-MS contidion Column Agilent Zorbax Rx C18 5μ (2.1 x 150mm) Mobile phase A : 5 mm ACN B : 5 mm HCOOH DIW Flow rate 0.2 ml/min Column temp. 40 C Inj. vol. 20 μl Time (min) Solvent (%A) 0-4 48 60 Gradient elution 4-4.5 60 90 4.5-7 90 90 7-7.5 90 50 7.5-10 50-50 Turbo gas temperature 350 C, 5.5kV Fig. 4-2. HPLC-MS/MS chromatograms : (+)-Matrine, kuraridin, (+)-kurarinione, Desmethylanhydroicaritin, propranolol (IS) - 15 -

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Fig. 5. Analytical results(μg/g) of the active compounds in Sophorae Radix 1. SF-K-1:, ; 2. SF-K-2:, ; 3. PL-K-3:, ; 4. PL-K-4:, ; 5. SF-C-1:, ; 6. SF-C-3:, ; 7. SF-C-2:,, ; 8. SF-C-7:, ; 9. SF-C-9:, ; 10. SF-C-10:,. - 17 -

γ γ γ γ - 18 -

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μ μ - 20 -

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Ÿ β βγ γ γ γ β Ÿ β Ÿ HPLC condition Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Mobile phase A : H 2 O B : Acetonitrile Flow rate 1.0 ml/min - 23 -

Inj. vol. 20 μl Total run time 40 min Time (min) Solvent (%A) Gradient elution 0-40 10-70 UV contidion wavelength 254 nm HPLC condition HPLC-MS contidion Column Agilent Zorbax Rx C18 5μ (2.1 x 150mm) Mobile phase A : 5 mm ACN B : 5 mm HCOOH DIW Flow rate 0.2 ml/min Column temp. 40 C Inj. vol. 20 μl Time (min) Solvent (%A) 0-4 48 60 4-4.5 Gradient elution 4.5-7 7-7.5 7.5-10 Turbo gas temperature 350 C, 5.5kV 60 90 90 90 90 50 50-50 Ÿ Ÿ Ÿ γ γ - 24 -

γ γ - 25 -

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( ) () trifolirhizin ( ) trifolirhizin kurarinone () kurarinone matrine (, ) matrine, oxymatrine (1.0% ) matrine, oxymatrine,. - 27 -

(Sophorae Radix) KP 11 Sophorae Radix Sophora flavescens Solander ex Aiton CP 2015 Sophorae Flavescentis Radix Sophora flavescens Aiton JP 16 Sophorae Radix Sophora flavescens Aiton matrine, oxymatrine (150 mesh ) 2.0 g 100 ml 120 methanol membrane filter (0.45 μm) 20 μl injection HPLC. Detector () Column ( ) Flow rate () Injection volume ( ) Mobile phase ( ) Gradient (, A%) 4 6 UV (254 nm) Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) 1.0 ml/min 20 μl A : H 2 O B : Acetonitrile Time (min) Solvent (%A) 0-40 10-70 trifolirhizin, kurarinone - KP CP JP KP oxymatrine, matrine 1.0% - - 28 -

(Sophorae Radix) KP 11 Sophorae Radix Sophora flavescens Solander ex Aiton CP 2015 Sophorae Flavescentis Radix Sophora flavescens Aiton JP 16 Sophorae Radix Sophora flavescens Aiton matrine, oxymatrine (150 mesh ) 2.0 g 100 ml 120 methanol membrane filter (0.45 μm) 20 μl injection HPLC. Detector () Column ( ) Agilent Zorbax Rx C18 5μ (2.1 x 150mm) Flow rate () 0.2 ml/min Column temperature ( ) Injection volume ( ) Mobile phase ( ) Gradient (, A%) 4 6 40 C 20 A : 5 mm ACN B : 5 mm HCOOH DIW Time (min) Solvent (%A) 0-4 48 60 4-4.5 4.5-7 7-7.5 7.5-10 trifolirhizin, kurarinone - KP CP JP KP oxymatrine, matrine 1.0% - 60 90 90 90 90 50 50-50 - 29 -

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성상 colorless powder 이명 Lupanidine CAS number 519-02-8 분자식 C 15 H 24 N 2 O (MW 284.36) 녹는점 ( ) 77 구조 - 31 -

δ α β β β β δ ν - 32 -

Column Agilent Zorbax Rx C18 5μ (2.1 x 150mm) Detector Temp. 40 Injection volume 20 Flow rate 0.2 ml/min Mobile phase Time(min) 5 mm ACN 5 mm HCOOH DIW 0 48% 52% 4 60% 40% 4.5 90% 10% 7 90% 10% 7.5 50% 50% 10 50% 50% HPLC-MS/MS chromatograms : (+)-Matrine, kuraridin, (+)-kurarinione, Desmethylanhydroicaritin, propranolol (IS) - 33 -

Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Matrine 0.9994 0.9998 1.3065 1.3143 0.4797 0.7679-34 -

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성상 Colorless powder 이명 CAS number 6807-83-6 분자식 C 22 H 22 O 10 (MW 446) 녹는점 ( ) 142-144 구조 - 36 -

δ δ - 37 -

Column Agilent Eclipse XD8-C18 (5 m, 4.6 X 150 mm) Detector UV (254 nm) Injection volume 20 μl Flow rate 1.0 ml/min Mobile phase Time(min) H 2 O Acetonitrile 0 10% 90% 40 70% 30% Typical chromatogram of compounds from Sophorae Radix - 38 -

Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) Trifolirhizin 5-100 0.9994 13889-15718 0.1 0.25-39 -

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성상 Colorless powder 이명 CAS number 34981-26-5 분자식 C 26 H 30 O 6 (MW 438) 녹는점 ( ) 115-117 구조 - 41 -

δ δ ν - 42 -

Column Agilent Zorbax Rx C18 5μ (2.1 x 150mm) Detector Column temp. 40 C Flow rate 0.2 ml/min Time(min) 5 mm ACN 5 mm HCOOH DIW 0 48% 52% 4 60% 40% Mobile phase 4.5 90% 10% 7 90% 10% 7.5 50% 50% 10 50% 50% HPLC-MS/MS chromatograms : (+)-Matrine, kuraridin, (+)-kurarinione, Desmethylanhydroicaritin, propranolol (IS) - 44 -

Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) kurarinione 0.5-10 0.9998 37335-1374 0.05 0.12 Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (ng/ml) LOQ (ng/ml) kurarinione 0.9993-0.9999 0.5405 0.5599 0.0282 0.0736-45 -