한약재품질표준화연구사업단 대계 ( 大薊 ) Cirsii Herba 생약연구과
KHP Cirsii Herba Cirsium japonicum DC. var. ussuriense (regel) Kitamura - CP2015 Cirsii Japonici Herba Cirsium setosum (Willd.) MB. linarin 0.70% JP16 - - - - 2 -
α α β β - 3 -
Sample information - 4 -
- 5 -
(mg) p-hydroxybenzoic acid (1) 60 - protocatechuic acid (2) 60 - vanillic acid (3) 60 - caffeic acid (4) 60 - chlorogenic acid (5) 60 - apigenin (6) 60, luteolin (7) 1070, luteolin-7--glucopyranoside (8) 130, luteolin-5--glucopyranoside (9) 7700, pectolinarin (10) 60, lupeol (11) 85 - apigenin 7--glucuronide (12) 50 - diosmin (13) 500 - diosmetin (14) 70 - hispidulin 7--neohesperidoside (15) 150, apigenin 7--glucoside (16) 20 - - 6 -
H H (compound number) H p-hydroxybenzoic acid (1) H protocatechuic acid (2) H H vanillic acid (3) H H H caffeic acid (4) H C 2 H H H H H H chlorogenic acid (5) H apigenin (6) H H H H luteolin (7) H H H H luteolin-7--glucopyranoside (8) H H H H - 7 -
H H H luteolin-5--glucopyranoside (9) H H H H H H CH3 H CH 3 H H pectolinarin (10) H H H 3 C CH 2 CH 3 CH 3 H CH 3 lupeol (11) H H H H 3 C CH3 H H H apigenin 7--glucuronide (12) H H H H H H H H H H H H H diosmin (13) H H diosmetin (14) H H H H H H H H hispidulin 7--neohesperidoside (15) H H H H H apigenin 7--glucoside (16) H H H H - 8 -
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μ μ μ μ μ - 10 -
Table6. TLC condition Compounds Mobile phase Detection TLC plate luteolin 5--glucoside, luteolin7--glucoside, hispidulin 7--neohesperidoside, luteolin, CHCl 3 : MeH 10% Normal (5 : 1) (Silica) pectolinarin, apigenin Table7. The optimal condition of sample preparation Extracting method Solvent Time reflux MeH 60 min μ μ μ μ μ 0.1 g pulverized sample added 1 ml MeH 60 min reflux membrane filter (0.2 μm) Aliquot 25 μl HPLC analysis (10 μl) Fig4. verall analytical procedures of extracts - 11 -
Table8. HPLC analysis condition 대계기존 HPLC 분석조건 Time(min) A (0.1 % trifluoro acetic acid) % B (ACN) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 55 60 90 10 Detector UV (254 nm) Column Agilent Edipse XD8 C18 (5 μm, 4.6 150 mm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ Device Gilson UV/VIS-151, Gilson 321 Pump, Gilson 231 XL Autosampler Fig. 5. HPLC chromatogram of MeH extract (A) and standard mixture (B). (1. luteolin 5--glucoside, 2. luteolin 7--glucoside,3. hispidulin 7--neohesperidoside, 4. luteolin, 5. pectolinarin, 6. apigenin) - 12 -
Table9. HPLC analysis condition 대계 HPLC 분석조건수정 Time(min) A (0.1 % trifluoro acetic acid) % B (ACN) % Mobile phase 0 10 90 40 35 65 Detector UV (270 nm) Column Agilent Edipse XD8 C18 (5 μm, 4.6 150 mm) Flow rate 1 ml/min Column temp. 40 Injection volume 10 μ Device Gilson UV/VIS-151, Gilson 321 Pump, Gilson 231 XL Autosampler Fig. 6. HPLC chromatogram of MeH extract (A and B) and standard mixture (C). (1. luteolin 5--glucoside, 2. luteolin 7--glucoside,3. hispidulin 7--neohesperidoside, 4. luteolin, 5. pectolinarin, 6. apigenin, MP methyparaben, PP, propylparaben) - 13 -
Fig7. Content Fig8. Pattern analysis - 14 -
α Fig9. hepatoprotective - 15 -
Fig9. Anticonvulsant effects Fig10. Memory impairment - 16 -
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μ - 18 -
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μ μ μ - 20 -
Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ - 21 -
Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ α - 22 -
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( ) ( ug / g) MTT assay, N assay, luteolin 5--glucoside RS assay, ELISA assay - luteolin 7--glucoside - - hispidulin 7--neohesperidoside - - - luteolin, -. pectolinarin,, - MTT assay, N assay, RS assay, ELISA assay. apigenin MTT assay, N assay, RS assay, ELISA assay - MTT assay, N assay, RS assay, ELISA assay. - 25 -
(Cirsii Herba) - - Detector ( ) Column ( ) Shimadzuu UV/VIS SPD-20A, UV (270 nm) Agilent Eclipse XD8- C18 (5 μm, 4.6 250 mm) Folw rate ( ) 1.0 ml/min Column temperature ( ) 40 Injection volume ( ) 1.0 μl Mobile phase ( ) ACN 10 % Gradient (, B%) ACN 10 % ~ 40 %, 대계의지표성분함량은각시료들간에일련의관련 성을보이지않아패턴분석을시행할수없다. - KHP CP JP KP - linarin 0.70% - 각시료들간에일련의관련성을보이지않아패턴분석을시행할수없다. - 26 -
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H H H H H H H - 28 -
HPLC conditions for the analysis of Cirsii Herba. Time(min) A (ACN) % B (0.1% H 3 P 4 in H 2 ) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 5510 60 90 Detector UV (254 nm) Column RP C18 (4.6 150 mm 5 μm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ - 29 -
δ δ - 30 -
Parameters LC/MS/MS System: API 2000 coupled with Shimadzu 10Avp Column: gemini C18 150 2 mm, 3 micron Solvent system: ACN : 0.5 % Formic acid = 70 : 30 Running time: 5 min Volume injection : 20 μl Flow rate: 0.25 ml/min Ion mode: ESI positive MRM - 31 -
No. Compound MRM transition Q1 DP FP EP CE CXP Source Tem. 1 luteolin 5--glucoside 449.2 287.0 946 6.0 370.0 5.0 35.0 10.0 400-32 -
2-33 -
H H H H H H H - 34 -
HPLC conditions for the analysis of Cirsii Herba. Time(min) A (ACN) % B (0.1% H 3 P 4 in H 2 ) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 5510 60 90 Detector UV (254 nm) Column RP C18 (4.6 150 mm 5 μm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ - 35 -
δ δ - 36 -
Parameters LC/MS/MS System: API 2000 coupled with Shimadzu 10Avp Column: gemini C18 150 2 mm, 3 micron Solvent system: ACN : 0.5 % Formic acid = 70 : 30 Running time: 5 min Volume injection : 20 μl Flow rate: 0.25 ml/min Ion mode: ESI positive MRM - 37 -
No. Compound MRM transition Q1 DP FP EP CE CXP Source Tem. 2 luteolin 7--glucoside 449.2 287.0 1563 96.0 370.0 6.5 29.0 10.0 400-38 -
3 hispidulin 7--neohesperidose hispidulin 7--neohesperidose - 39 -
α β H H H H H H H H H H - 40 -
HPLC conditions for the analysis of Cirsii Herba. Time(min) A (ACN) % B (0.1% H 3 P 4 in H 2 ) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 5510 60 90 Detector UV (254 nm) Column RP C18 (4.6 150 mm 5 μm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ - 41 -
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Parameters LC/MS/MS System: API 2000 coupled with Shimadzu 10Avp Column: gemini C18 150 2 mm, 3 micron Solvent system: ACN : 0.5 % Formic acid = 70 : 30 Running time: 5 min Volume injection : 20 μl Flow rate: 0.25 ml/min Ion mode: ESI positive MRM - 43 -
4 luteolin luteolin - 44 -
H H H H - 45 -
HPLC conditions for the analysis of Cirsii Herba. Time(min) A (ACN) % B (0.1% H 3 P 4 in H 2 ) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 5510 60 90 Detector UV (254 nm) Column RP C18 (4.6 150 mm 5 μm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ - 46 -
δ δ - 47 -
Parameters LC/MS/MS System: API 2000 coupled with Shimadzu 10Avp Column: gemini C18 150 2 mm, 3 micron Solvent system: ACN : 0.5 % Formic acid = 70 : 30 Running time: 5 min Volume injection : 20 μl Flow rate: 0.25 ml/min Ion mode: ESI positive MRM - 48 -
No. Compound MRM transition Q1 DP FP EP CE CXP Source Tem. 4 luteolin 287.1 153.1 359 76.0 370.0 12.0 45.0 4.0 400-49 -
5 pectolinarin pectolinarin - 50 -
H H CH3 H CH 3 H H H H - 51 -
HPLC conditions for the analysis of Cirsii Herba. Time(min) A (ACN) % B (0.1% H 3 P 4 in H 2 ) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 5510 60 90 Detector UV (254 nm) Column RP C18 (4.6 150 mm 5 μm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ - 52 -
δ δ - 53 -
Parameters LC/MS/MS System: API 2000 coupled with Shimadzu 10Avp Column: gemini C18 150 2 mm, 3 micron Solvent system: ACN : 0.5 % Formic acid = 70 : 30 Running time: 5 min Volume injection : 20 μl Flow rate: 0.25 ml/min Ion mode: ESI positive MRM - 54 -
No. Compound MRM transition Q1 DP FP EP CE CXP Source Tem. 5 pectolinarin 623.3 315.1 408 51.0 370.0 8.0 37.0 10.0 400-55 -
6 apigenin apigenin - 56 -
H H H - 57 -
HPLC conditions for the analysis of Cirsii Herba. Time(min) A (ACN) % B (0.1% H 3 P 4 in H 2 ) % 0 10 90 20 35 65 Mobile phase 25 35 65 30 45 55 50 45 5510 60 90 Detector UV (254 nm) Column RP C18 (4.6 150 mm 5 μm) Flow rate 1 ml/min Column temp. 30 Injection volume 10 μ - 58 -
δ δ - 59 -
Parameters LC/MS/MS System: API 2000 coupled with Shimadzu 10Avp Column: gemini C18 150 2 mm, 3 micron Solvent system: ACN : 0.5 % Formic acid = 70 : 30 Running time: 5 min Volume injection : 20 μl Flow rate: 0.25 ml/min Ion mode: ESI positive MRM - 60 -
No. Compound MRM transition Q1 DP FP EP CE CXP Source Tem. 6 apigenin 271.6 153.1 838 76.0 370.0 12.0 43.0 4.0 400-61 -