한약재품질표준화연구사업단 금은화 ( 金銀花 ) Lonicerae Flos 생약연구과

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1 한약재품질표준화연구사업단 금은화 ( 金銀花 ) Lonicerae Flos 생약연구과

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3 KP 11 Lonicerae Flos Lonicera japonica Thunberg - CP 2015 JP 16 Lonicerae Japonicae Flos Lonicerae Folium Cum Caulis Lonicera japonica Thunberg - Lonicera japonica Thunberg

4 β β α α α β β α α α α α β β α α β β α β β α β - 4 -

5 O HO OH HO OH O O OH OH rutin chlorogenic acid Lonicera japonica Thunburg 70%EtOH ( %) 70%EtOH - 5 -

6 (mg) n-nonacosane 44 n-hexacosanol 80 monopalmitin 10 β-sitosterol 62 daucosterol 3,

7 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) 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 -

8 (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 -

9 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 -

10 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)

11 β β β

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13 chlorogenic acid sweroside luteolin 7-O-glucoside (E)-aldosecologanin 3,5-dicaffeoylquinic acid

14 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

15 TLC pattern analysis in ODS plate Fig. 2. Thin layer chromatography of 26 Lonicerae Flos samples

16 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

17 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 wavelength 250 nm

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19 - 19 -

20 μ

21 α β κ α κ

22 β α κ β α β α β α κ κ β α κ

23 α α κ

24 κ α κ κ μ μ

25 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

26 UV contidion wavelength 250 nm Ÿ Ÿ Ÿ Ÿ μ

27 Ÿ

28 α β

29 ( ) ( ) chlorogenic acid ( ) chlorogenic acid rutin ( ) methyl caffeate ( ) sweroside luteolin 7-O-glucoside 3,5-dicaffeoylquinic acid (E)-aldosecologanin chlorogenic acid

30 (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 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) chlorogenic acid - KP CP JP KP

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32 성상 white to slightly yellowish-beige powder 이명 Chlorogenate CAS number 분자식 C 16 H 18 O 9 (MW 354) 녹는점 ( ) 구조

33 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, min 20-35% A, 30-35min 35% A, 35-40min 35-15% A UV condition: 250 nm

34 δ δ υ α β

35 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%

36 Analytes Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (μg/ml) LOQ (μg/ml) Chlorogenic acid Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) Chlorogenic acid Compounds Analytical condition Theoretical plate (N) Resolution Selectivity Chlorogenic acid Column Column oven temp. Shiseido CAPCELL PAK C18 UG Phenomenex Gemini 5μ C Agilent ZORBAX Eclipse XDB-C μ

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38 성상 light yellow powder 이명 cynaroside, luteoloside CAS number 분자식 C 21 H 20 O 11 (MW 448) 녹는점 ( ) 구조

39 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, min 20-35% A, 30-35min 35% A, 35-40min 35-15% A UV condition: 300 nm

40 δ δ δ

41 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%

42 Analytes luteolin 7-O-glucoside Linear range (μg/ml) Correlation coefficient Slope Intercept LOD (μg/ml) LOQ (μg/ml) Compounds Peak area ratio (peak area/is area) Retention time (min) Mean RSD (%) Mean RSD (%) luteolin 7-O-glucoside Compounds Analytical condition Theoretical plate (N) Resolution Selectivity luteolin 7-O-glucoside Column Column oven temp. Shiseido CAPCELL PAK C18 UG Phenomenex Gemini 5μ C Agilent ZORBAX Eclipse XDB-C μ

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- 2 - 한약재품질표준화연구사업단 인동 ( 忍冬 ) Lonicerae Folium et Caulis 생약연구과 - 2 - KP 11 Lonicera japonica Thunberg 0.1% CP 2015 JP 16 Lonicera japonica Thunberg chlorogenic acid 0.10% Lonicera japonica Thunberg - β α - 3