한약재품질표준화연구사업단 금은화 ( 金銀花 ) Lonicerae Flos 생약연구과
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KP 11 Lonicerae Flos Lonicera japonica Thunberg - CP 2015 JP 16 Lonicerae Japonicae Flos Lonicerae Folium Cum Caulis Lonicera japonica Thunberg - Lonicera japonica Thunberg - - 3 -
β β α α α β β α α α α α β β α α β β α β β α β - 4 -
O HO OH HO OH O O OH OH rutin chlorogenic acid Lonicera japonica Thunburg 70%EtOH ( %) 70%EtOH - 5 -
(mg) n-nonacosane 44 n-hexacosanol 80 monopalmitin 10 β-sitosterol 62 daucosterol 3,153-6 -
luteolin 175 esculetin 2 protocatechuic acid 10 azelaic acid 7 caffeic acid 5 flavoyadorinin-b 16 4,5-dicaffeoylquinic acid 3 cerebroside 3 4,5-dicaffeoylquinic acid methyl ester vanilic acid 4-O-β-D- (6-benzoylglucopyranoside) 4 7 7-ketologanin 3 secologanin dimethyl acetal 118 uracil 6 luteolin 7-O-glucoside 617 rutin 10 3,5-dicaffeoylquinic acid methyl ester 1,899 methyl chlorogenate 16 isoquercitrin 19 3,5-dicaffeoylquinic acid 513 rhoifolin 3 centauroside 70 sucrose 1,068 epivogeloside 3 chlorogenic acid 400, - 7 -
(compound number) n-nonacosane (1) monopalmitin (3) β-sitosterol (4) daucosterol (5) luteolin (6) esculetin (7) protocatechuic acid (8) azelaic acid (9) caffeic acid (10) flavoyadorinin-b (11) - 8 -
R = H 4,5-dicaffeoylquinic acid (12) R = CH 3 4,5-dicaffeoylquinic acid methyl ester (14) cerebroside (13) **vanilic acid 4-O-β-D- (6-benzoylglucopyranoside) (15) 7-ketologanin (16) secologanin dimethyl acetal (17) uracil (18) luteolin 7-O-glucoside (19) R = Rhamnose rutin (20) R = H isoquercitrin (23) - 9 -
R = CH 3 3,5-dicaffeoylquinic acid methyl ester (21) R = H 3,5-dicaffeoylquinic acid (24) O R = CH 3 methyl chlorogenate (22) HO OR O OH R = H chlorogenic acid (29) HO O OH OH rhoifolin (25) centauroside (26) sucrose (27) epivogeloside (28) - 10 -
β β β - 11 -
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chlorogenic acid sweroside luteolin 7-O-glucoside (E)-aldosecologanin 3,5-dicaffeoylquinic acid - 13 -
Table 2. TLC condition Compound Mobile phase Detection TLC plate 26 EtOAc : MeOH : H 2 O (8 : 2 : 1) H 2 SO 4 silica gel plate FeCl 3 50% MeOH ODS plate I 2 TLC pattern analysis in silica gel plate - 14 -
TLC pattern analysis in ODS plate Fig. 2. Thin layer chromatography of 26 Lonicerae Flos samples - 15 -
Table 4. The optimal condition of sample preparation Extracting method Solvent Time (ultrasonication) 70% EtOH 60 pulverized sample added 70% EtOH ultrasonication for 60 min filter with membrane filter (0.20 μm) Aliquot 1 ml added 10 g MeOH 15% acetonitrile HPLC analysis (20 μl) Fig. 3. Overall analytical procedures of extracts from Lonicerae Flos - 16 -
Table 3. Optimum analytical conditions of HPLC/DAD for chlorogenic acid, sweroside, luteolin 7-O-glucoside, (E)-aldosecologanin, 3,5-dicaffeoylquinic acid HPLC condition UV contidion Column Capcell Pak ODS UG120 (5 μm, 4.6 mm 150 mm) Mobile phase A : H 2 O with 0.1% formic acid B : acetonitrile with 0.1% formic acid Flow rate 0.3 ml/min Column temp. 25 C Inj. vol. 20 μl Time (min) Solvent (%A) Gradient elution 0-10 10-30 30-35 35-40 85-80 80-65 65 65 65-85 wavelength 250 nm - 17 -
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μ - 20 -
α β κ α κ - 21 -
β α κ β α β α β α κ κ β α κ - 22 -
α α κ - 23 -
κ α κ κ μ μ - 24 -
Ÿ Ÿ Ÿ Ÿ β β Ÿ Ÿ HPLC condition Column Capcell Pak ODS UG120 (5 μm, 4.6 mm 150 mm) Mobile phase A : H 2 O with 0.1% formic acid B : acetonitrile with 0.1% formic acid Flow rate 0.3 ml/min Column temp. 25 C Inj. vol. 20 μl Time (min) Solvent (%A) Gradient elution 0-10 85-80 - 25 -
UV contidion 10-30 30-35 35-40 wavelength 250 nm 80-65 65 65 65-85 Ÿ Ÿ Ÿ Ÿ μ - 26 -
Ÿ - 27 -
α β - 28 -
( ) ( ) chlorogenic acid ( ) chlorogenic acid rutin ( ) methyl caffeate ( ) sweroside luteolin 7-O-glucoside 3,5-dicaffeoylquinic acid (E)-aldosecologanin chlorogenic acid. - 29 -
(Lonicerae Flos) KP 11 Lonicerae Flos Lonicera japonica Thunberg CP 2015 Lonicerae Japonicae Flos Lonicera japonica Thunberg JP 16 Lonicerae Folium Cum Caulis Lonicera japonica Thunberg chlorogenic acid 70% EtOH 60 0.2 μm membrane filter 1 ml 10 μg 15% 20 μl Detector ( ) Column ( ) Flow rate ( ) Column temperature ( ) UV (250 nm) Capcell Pak ODS UG120 (5 μm, 4.6 mm 150 mm) 0.3 ml/min 25 C Injection volume ( ) 20 μl Mobile phase ( ) Gradient (, A%) A : H2O with 0.1% formic acid B : acetonitrile with 0.1% formic acid Time (min) Solvent (%A) 0-10 85-80 10-30 30-35 35-40 chlorogenic acid - KP CP JP KP - - - 80-65 65 65 65-85 - 30 -
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성상 white to slightly yellowish-beige powder 이명 Chlorogenate CAS number 327-97-9 분자식 C 16 H 18 O 9 (MW 354) 녹는점 ( ) 205-209 구조 - 32 -
Column: Shiseido CAPCELL PAK C18 UG120 (5μm, 4.6 x 150 mm ) Mobile phase: A: CH 3 CN with 0.1% HCOOH B: H 2 O with 0.1% HCOOH Flow rate: 0.3 ml/min Gradient elution: 0-10 min 15-20% A, 10-30 min 20-35% A, 30-35min 35% A, 35-40min 35-15% A UV condition: 250 nm - 33 -
δ δ υ α β - 34 -
Column Capcell Pak ODS UG120 (5 μm, 4.6 mm 150 mm) Detector UV (250 nm) Temp. 25 Injection volume 20 μl Flow rate 0.3 ml/min Mobile phase Time(min) H 2 O with 0.1% formic acid acetonitrile with 0.1% formic acid 0 85% 15% 10 80% 20% 30 65% 35% 35 65% 35% 40 85% 15% - 35 -
Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (μg/ml) LOQ (μg/ml) Chlorogenic acid 4-30 0.997 0.01226-0.01638 0.06 0.17 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) Chlorogenic acid 0.34 0.55 11.59 1.04 Compounds Analytical condition Theoretical plate (N) Resolution Selectivity Chlorogenic acid Column Column oven temp. Shiseido CAPCELL PAK C18 UG120 15219.33 - Phenomenex Gemini 5μ C18 25633.00 - Agilent ZORBAX Eclipse XDB-C18 14669.67-20 17916.67-25 17221.67-30 16547.67-35 15266.67-40 14119.00 - μ - 36 -
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성상 light yellow powder 이명 cynaroside, luteoloside CAS number 5373-11-5 분자식 C 21 H 20 O 11 (MW 448) 녹는점 ( ) 구조 - 38 -
Column: Shiseido CAPCELL PAK C18 UG120 (5μm, 4.6 x 150 mm ) Mobile phase: A: CH 3 CN with 0.1% HCOOH B: H 2 O with 0.1% HCOOH Flow rate: 0.3 ml/min Gradient elution: 0-10 min 15-20% A, 10-30 min 20-35% A, 30-35min 35% A, 35-40min 35-15% A UV condition: 300 nm - 39 -
δ δ δ - 40 -
Column Capcell Pak ODS UG120 (5 μm, 4.6 mm 150 mm) Detector UV (250 nm) Temp. 25 Injection volume 20 μl Flow rate 0.3 ml/min Mobile phase Time(min) H 2 O with 0.1% formic acid acetonitrile with 0.1% formic acid 0 85% 15% 10 80% 20% 30 65% 35% 35 65% 35% 40 85% 15% - 41 -
Analytes luteolin 7-O-glucoside Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (μg/ml) LOQ (μg/ml) 1-20 0.997 0.05220-0.00121 0.03 0.1 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) luteolin 7-O-glucoside 0.67 0.43 23.40 0.66 Compounds Analytical condition Theoretical plate (N) Resolution Selectivity luteolin 7-O-glucoside Column Column oven temp. Shiseido CAPCELL PAK C18 UG120 90855.33 27.15 Phenomenex Gemini 5μ C18 121260.33 29.37 Agilent ZORBAX Eclipse XDB-C18 89549.67 27.20 20 98785.67 28.25 25 92642.33 27.22 30 86111.00 26.37 35 79101.33 25.27 40 72307.67 23.96 μ - 42 -