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1 한약재품질표준화연구사업단 인동 ( 忍冬 ) Lonicerae Folium et Caulis 생약연구과

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3 KP 11 Lonicera japonica Thunberg 0.1% CP 2015 JP 16 Lonicera japonica Thunberg chlorogenic acid 0.10% Lonicera japonica Thunberg - β α - 3 -

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

5 Lonicera japonica Thunberg (Leaf and Stem) - 5 -

6 Lonicera japonica Lonicera japonica ( ),, ( ), ( ), ( ), ( ), ( ),, ( ),, ( ),, 70% EtOH ( %) 16 g (16%) 12.5 g (12.5%) 38.8 g (38.8%) 17.6 g (17.6%) 39.3 g (39.3%) 14.0 g (14.0%) 17.7 g (17.7%) 13.2 g (13.2%), 70% EtOH

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8 - 8 -

9 - 9 -

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12 (mg) hydrocarbon alcohol (docosanol + tetracosanol) (1) 3, *hydrocarbon alcohol (docosanol + tetracosanol + eicosanol) (2) 4, *24(S )-cycloart-25-en-3β,24-diol (3) 5, *trilinolein (4) 20, *n-hexacosanol (5) 212, *obtusifoliol (6) 2, *gramisterol (7) 2, *citrostadienol (8) 3, *β-sitosterol (9) 1,126, *DOP[bis(2-ethylhexyl)phthalate] (10) 30, fatty acids (11) 46, *coniferaldehyde (12) 10, *caffeic acid docosanoyl ester (13) 13, *pomolic acid (14) 8, *ursolic acid (15) 20, *9α-hydroxypinoresinol (16) 5, *euscaphic acid (17) 10, *hederagenin (18) 10, secologanin dimethyl acetal (19) 256, *daucosterol (20) 98, *23-hydroxytormentic acid (21) 16, caffeic acid (22) 7, *9,12,13-trihydroxyoctadeca-10(E),15(Z)-dienoic acid (23) 17, *pinellic acid (24) 10, *7,8-threo-4,7,9,3',9'-pentahydroxy-3-methoxy-8-O-4'-neolignan (25) 2, **5-demethxyl-3-methoxyisolariciresinol (26) 2, *pinoresinol 4-O-glucoside (27) 105, sweroside (28) 1,232, *coniferylaldehyde 4-O-glucoside (29) 4, *linarin (30) 2, *coniferin (31) 120,

13 loganin (32) 6,526, *loganic acid (33) 304, *demethylsecologanol (34) 2,

14 (compound number) hydrocarbon alcohol (docosanol + tetracosanol) (1) *hydrocarbon alcohol (docosanol + tetracosanol + eicosanol) (2) *24(S)-cycloart-25-en-3β,24-diol (3) *trilinolein (4) *n-hexacosanol (5) *obtusifoliol (6) *gramisterol (7) *citrostadienol (8)

15 *β-sitosterol (9) *DOP[bis(2-ethylhexyl)phthalate] (10) fatty acids (11) *coniferaldehyde (12) *caffeic acid docosanoyl ester (13) *pomolic acid (14) *ursolic acid (15) *9α-hydroxypinoresinol (16)

16 *euscaphic acid (17) *hederagenin (18) secologanin dimethyl acetal (19) *daucosterol (20) *23-hydroxytormentic acid (21) caffeic acid (22)

17 *9,12,13-trihydroxyoctadeca-10(E),15(Z)-dienoic acid (23) *pinellic acid (24) *7,8-threo-4,7,9,3',9'-pentahydroxy-3-methoxy -8-O-4'-neolignan (25) **5-demethxyl-3-methoxy isolariciresinol (26) *pinoresinol 4-O-glucoside (27) sweroside (28) *coniferylaldehyde 4-O-glucoside (29) *linarin (30)

18 *coniferin (31) loganin (32) *loganic acid (33) *demethylsecologanol (34) β

19 α β α β β α

20 Lonicerae Folium

21 Table 2. TLC condition Compounds Mobile phase Detection TLC plate coniferin, loganic acid, demethylsecologanoside, sweroside, loganin EtOAc : MeOH : H 2 O (5.2 : 2.8 : 0.8) H 2 SO 4, MeOH : H 2 O (55 : 45) FeCl 3, I 2 Normal (Silica) Reverse (ODS) <FeCl3> <H 2 SO 4 > <I 2 > Fig. 4. Thin layer chromatography in silica gel plate of 24 Lonicerae Folium samples Marker compounds; 1. coniferin 2. loganic acid 3. demethylsecologanoside 4. sweroside 5. loganin (6. Uijeongbu, gyeonggi 7. Hong-cheon, gang-won 8. Gimpo, gyeonggi 9. Cheong-song, gyung book 10. Pocheon, gyeonggi 11. Wansan, yeongcheon 12. Hwasan, yeongcheon 13. Yong-mun, yangpyung 14. Sang-ju, gyung book 15. An-dong, gyung book 16. Yuseong, daejeon 17. Ganghwa 18. Dong-gu, daejeon 19. Gimcheon, gyung book 20. China 21. Gang-won 22. Bong-hwa, gyung book (stem) 23. Gyung-ju, gyung book (stem) 24. Gyung-ju, gyung book (leaf) 25. Seoul (stem) 26. Seoul (leaf) 27. Sandong, china (stem) 28. Sandong, china (stem) 29. Yeongcheon (stem))

22 <FeCl3> <H 2 SO 4 > <I 2 > Fig. 5. Thin layer chromatography in ODS plate of 24 Lonicerae Folium samples Marker compounds; 1. coniferin 2. loganic acid 3. demethylsecologanoside 4. sweroside 5. loganin (6. Uijeongbu, gyeonggi 7. Hong-cheon, gang-won 8. Gimpo, gyeonggi 9. Cheong-song, gyung book 10. Pocheon, gyeonggi 11. Wansan, yeongcheon 12. Hwasan, yeongcheon 13. Yong-mun, yangpyung 14. Sang-ju, gyung book 15. An-dong, gyung book 16. Yuseong, daejeon 17. Ganghwa 18. Dong-gu, daejeon 19. Gimcheon, gyung book 20. China 21. Gang-won 22. Bong-hwa, gyung book (stem) 23. Gyung-ju, gyung book (stem) 24. Gyung-ju, gyung book (leaf) 25. Seoul (stem) 26. Seoul (leaf) 27. Sandong, china (stem) 28. Sandong, china (stem) 29. Yeongcheon (stem)) Table 3. The optimal condition of sample preparation Extracting method Solvent Time (Ultrasonication) 70% EtOH 30 μ μ μ μ μ μ

23 Table 4. Analytical conditions of HPLC/DAD Column Shiseido Capcell Pak ODS UG120 ( mm, 5 μm) 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. μl Time (min) Solvent (%B) 0 15 Gradient elution DAD detector 250 nm μ

24 Table 5. Optimum analytical conditions of HPLC Column Optimapak C18 ( mm) Mobile phase A: 10% aqueous methanol with 0.1% formic acid B: 90% aqueous methanol with 0.1% formic acid Flow rate 1.0 ml/min Column temp. C Inj. vol. 10 μl Time (min) Solvent (%B) 0 0 Gradient elution (A) (B) Figure 6. HPLC chromatogram of mixed standard (A) and samples (B) of Lonicerae Folium et Caulis; 1. coniferin, 2. loganic acid, 3. sweroside, 4. loganin, 5. methylparaben

25 μ μ μ μ μ μ μ μ μ μ μ

26 Fig. 7. Distribution of (a) coniferin, (b) loganic acid, (c) sweroside and (d) loganin for Lonicerae Folium et Caulis samples

27 α α α α α α μ α α μ μ μ μ α

28 α α

29 γ

30 β β β α β β β β α β β β β α β β α β α β β α β α β β α

31 β

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33 Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Column Optimapak C18 ( mm) A: 10% aqueous methanol with 0.1% formic acid Mobile phase B: 90% aqueous methanol with 0.1% formic acid Flow rate 1.0 ml/min Column temp. C Inj. vol. 10 μl Time (min) Solvent (%B) 0 0 Gradient elution

34 Ÿ Ÿ Ÿ Ÿ α

35 γ

36 ( ) ( ) coniferin coniferin [ ]. loganic acid loganic acid (0.36%) [ ]. sweroside sweroside (0.41%) [ ]. loganin loganin (0.14%) [ ]. KP. coniferin, loganic acid, sweroside, loganin, coniferin

37 (Lonicerae Folium) KP Lonicerae Folium et Caulis Ÿ Lonicera japonica Thunberg JP Lonicerae Folium Cum Caulis Ÿ Lonicera japonica Thunberg CP Lonicerae Japonicae Caulis Ÿ Lonicera japonica Thunberg coniferin, loganic acid, sweroside, loganin 1 g 0.2 ml (methylparaben, 1 mg/ml) 9.8 ml 70% 30 ultrasonication 0.2 μm membrane filter 10 μl Detector ( ) Column ( ) Flow rate ( ) UV (254 nm) Optimapak C18 ( mm) 1.0 ml/min Column temperature ( ) - Injection volume ( ) 10 μl Mobile phase ( ) Gradient (, B%) A : 10% aqueous methanol with 0.1% formic acid B : 90% aqueous methanol with 0.1% formic acid 0 15 : 0-30%, : 30 70%, : 70%, : % coniferin loganic acid sweroside loganin % % % % KP ChP JP KP loganin 0.1% chlorogenic acid 0.1% loganic acid 0.36%, sweroside 0.41%, loganin 0.14%

38 CH 2 Cl 2, CH 2 Cl 2 -MeOH

39 성상 white powder 이명 [3-[(4-β-D-glucopyranosyloxy)-3-methoxyphenyl]-2-propenol,4-hydroxy-3 -methoxy-1-(γ-hydroxypropenyl)benzene-4-d-glucoside CAS number 분자식 C 16 H 22 O 8 (MW 342) 용해도 녹는점 ( ) 구조

40 δ δ ν α λmax (log ε) (MeOH) 212 (sh, 3.45), 258 (3.26), 265 (sh, 3.23) nm

41 Column Shiseido Capell Pak ODS UG 120 C18 (5 μm, 150 mm 4.6 mm) Detector DAD (254 nm) Temp. 35 Injection volume - Flow rate 0.3 ml/min Mobile phase Time(min) H 2 O with 0.1% formic acid MeOH with 0.1% formic acid 0 80% 20% 15 65% 135% 25 35% 65% 30 35% 65% 35 80% 20%

42 Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) coniferin 1-20 Y = X Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) coniferin Compounds coniferin Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%)

43 Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido ± ± ± ± 0.13 Column phenomenex ± ± ± ± 0.03 coniferin Column temp. Agilent ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 μ

44 CH 2 Cl 2, CH 2 Cl 2 -MeOH loganic acid 성상 white powder 이명 β

45 CAS number 분자식 C 16 H 24 O 10 (MW 376) 용해도 녹는점 ( ) 구조

46 δ β α δ ν α β α λmax (log ε) (MeOH)

47 Column Shiseido Capell Pak ODS UG 120 C18 (5 μm, 150 mm 4.6 mm) Detector DAD (254 nm) Temp. 35 Injection volume - Flow rate 0.3 ml/min Mobile phase Time(min) H 2 O with 0.1% formic acid MeOH with 0.1% formic acid 0 80% 20% 15 65% 135% 25 35% 65% 30 35% 65% 35 80% 20%

48 Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) loganic acid 1-20 Y = X Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) loganic acid Compounds loganic acid Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%)

49 Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido ± ± ± ± 0.00 Column phenomenex ± ± ± ± 0.00 loganic acid Column temp. Agilent ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.00 μ

50 CH 2 Cl 2, CH 2 Cl 2 -MeOH sweroside

51 성상 white powder 이명 β CAS number 분자식 C 16 H 22 O 9 (MW 358) 용해도 녹는점 ( ) 구조

52 δ δ ν α β α λmax (log ε) (MeOH) 240 (3.95) nm

53 Column Shiseido Capell Pak ODS UG 120 C18 (5 μm, 150 mm 4.6 mm) Detector DAD (254 nm) Temp. 35 Injection volume μl Flow rate 0.3 ml/min Mobile phase Time(min) H 2 O with 0.1% formic acid MeOH with 0.1% formic acid 0 80% 20% 15 65% 135% 25 35% 65% 30 35% 65% 35 80% 20%

54 Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) sweroside 1-20 Y = X Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) sweroside Compounds sweroside Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%)

55 Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido ± ± ± ± 0.00 Column phenomenex ± ± ± ± 0.01 sweroside Column temp. Agilent ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.00 μ

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57 CH 2 Cl 2, CH 2 Cl 2 -MeOH CH 2 Cl 2 -MeOH 성상 white powder 이명 β

58 CAS number 분자식 C 17 H 26 O 10 (MW 390) 용해도 녹는점 ( ) 구조

59 δ

60 δ ν α β α

61 Column Shiseido Capell Pak ODS UG 120 C18 (5 μm, 150 mm 4.6 mm) Detector DAD (254 nm) Temp. 35 Injection volume μl Flow rate 0.3 ml/min Mobile phase Time(min) H 2 O with 0.1% formic acid MeOH with 0.1% formic acid 0 80% 20% 15 65% 135% 25 35% 65% 30 35% 65% 35 80% 20%

62 Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) loganin 1-20 Y = X Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) loganin Compounds loganin Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%)

63 Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido ± ± ± ± 0.00 Column phenomenex ± ± ± ± 0.00 loganin Column temp. Agilent ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.00 μ

한약재품질표준화연구사업단 금은화 ( 金銀花 ) Lonicerae Flos 생약연구과

한약재품질표준화연구사업단 금은화 ( 金銀花 ) Lonicerae Flos 생약연구과 - 2 - KP 11 Lonicerae Flos Lonicera japonica Thunberg - CP 2015 JP 16 Lonicerae Japonicae Flos Lonicerae Folium Cum Caulis Lonicera japonica Thunberg - Lonicera