PDF 무료 다운로드

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 -

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 -

Lonicera japonica Thunberg (Leaf and Stem) - 5 -

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 - - - 6 -

- 7 -

- 8 -

- 9 -

- 10 -

- 11 -

(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, - 12 -

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

(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) - 14 -

*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) - 15 -

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

*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) - 17 -

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

α β α β β α - 19 -

Lonicerae Folium - 20 -

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> 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 14 15 16 17 18 19 20 21 1 2 3 4 5 22 23 24 25 26 27 28 29 <H 2 SO 4 > 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 14 15 16 17 18 19 20 21 <I 2 > 1 2 3 4 5 22 23 24 25 26 27 28 29 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 22 23 24 25 26 27 28 29 1 2 3 4 5 14 15 16 17 18 19 20 21 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)) - 21 -

<FeCl3> 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 14 15 16 17 18 19 20 21 1 2 3 4 5 22 23 24 25 26 27 28 29 <H 2 SO 4 > 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 14 15 16 17 18 19 20 21 <I 2 > 1 2 3 4 5 22 23 24 25 26 27 28 29 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 22 23 24 25 26 27 28 29 1 2 3 4 5 14 15 16 17 18 19 20 21 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 - 22 -

Table 4. Analytical conditions of HPLC/DAD Column Shiseido Capcell Pak ODS UG120 (4.6 150 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 10 20 30 35 35 40 35 15 DAD detector 250 nm μ - 23 -

Table 5. Optimum analytical conditions of HPLC Column Optimapak C18 (4.6 250mm) 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 15 30 25 70 30 35 70 100 (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 - 24 -

μ μ μ μ μ μ μ μ μ μ μ - 25 -

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

α α α α α α μ α α μ μ μ μ α - 27 -

α α - 28 -

γ - 29 -

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 - 30 -

β - 31 -

- 32 -

Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Ÿ Column Optimapak C18 (4.6 250mm) 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 15 30 25 70-33 -

30 35 70 100 Ÿ Ÿ Ÿ Ÿ α - 34 -

γ - 35 -

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

(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 (4.6 250 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%, 15 25 : 30 70%, 25 30 : 70%, 30 35 : 70-100% coniferin loganic acid sweroside loganin 92 0.0191% 0.3640% 0.2360% 0.2710% KP ChP JP KP loganin 0.1% chlorogenic acid 0.1% loganic acid 0.36%, sweroside 0.41%, loganin 0.14% - 37 -

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

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 531-29-3 분자식 C 16 H 22 O 8 (MW 342) 용해도 녹는점 ( ) 구조 - 39 -

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

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% - 41 -

Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) coniferin 1-20 Y = 0.18180X + 0.02239 0.99 0.557 1.687 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) coniferin 0.85 0.00 0.05 14.33 0.25 1.74 Compounds coniferin Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%) 2 101.19 0.45 100.62 0.45 4 100.08 0.20 100.18 0.26 8 99.90 0.23 100.72 0.27-42 -

Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido 30260.00 ± 510.49 21.31 ± 0.13 1.18 ± 0.01 6.93 ± 0.13 Column phenomenex 39417.00 ± 514.85 19.34 ± 0.02 1.66 ± 0.00 2.69 ± 0.03 coniferin Column temp. Agilent 30822.00 ± 556.90 17.69 ± 0.05 1.51 ± 0.01 3.07 ± 0.00 25 31956.67 ± 458.50 17.62 ± 0.13 1.74 ± 0.01 2.70 ± 0.01 30 31946.00 ± 844.56 17.81 ± 0.15 1.61 ± 0.01 2.90 ± 0.02 35 29825.00 ± 596.92 17.41 ± 0.15 1.49 ± 0.01 3.15 ± 0.03 40 27732.00 ± 936.31 17.07 ± 0.27 1.37 ± 0.01 3.40 ± 0.03 45 26748.00 ± 102.68 16.83 ± 0.07 1.26 ± 0.01 3.66 ± 0.02 μ - 43 -

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

CAS number 22255-40-9 분자식 C 16 H 24 O 10 (MW 376) 용해도 녹는점 ( ) 구조 - 45 -

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

Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) loganic acid 1-20 Y = 0.02735X + 0.00282 1.00 0.327 0.990 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) loganic acid 0.52 0.00 0.09 15.90 0.29 1.81 Compounds loganic acid Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%) 2 105.89 3.15 102.50 1.99 4 100.87 1.96 100.88 0.96 8 100.00 0.56 100.10 0.58-48 -

Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido 39002.00 ± 560.89 5.87 ± 0.02 1.48 ± 0.01 1.25 ± 0.00 Column phenomenex 44325.33 ± 553.77 4.09 ± 0.02 1.88 ± 0.01 1.13 ± 0.00 loganic acid Column temp. Agilent 37342.33 ± 403.20 4.75 ± 0.05 1.79 ± 0.01 1.18 ± 0.00 25 38317.00 ± 281.51 5.66 ± 0.06 2.09 ± 0.01 1.20 ± 0.01 30 37773.00 ± 1178.10 5.21 ± 0.08 1.69 ± 0.42 1.19 ± 0.00 35 36147.67 ± 637.27 4.68 ± 0.05 1.76 ± 0.01 1.39 ± 0.36 40 34245.33 ± 348.33 4.15 ± 0.03 1.61 ± 0.01 1.17 ± 0.00 45 31441.67 ± 1056.21 3.58 ± 0.01 1.46 ± 0.01 1.16 ± 0.00 μ - 49 -

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

성상 white powder 이명 β CAS number 14215-86-2 분자식 C 16 H 22 O 9 (MW 358) 용해도 녹는점 ( ) 구조 - 51 -

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

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% - 53 -

Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) sweroside 1-20 Y = 0.09848X + 0.00084 0.99 0.657 1.990 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) sweroside 2.28 0.00 0.03 22.05 0.27 1.21 Compounds sweroside Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%) 2 100.55 0.57 101.14 0.86 4 99.81 0.16 100.27 0.48 8 99.39 0.16 99.61 0.14-54 -

Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido 57276.26 ± 624.36 10.94 ± 0.07 2.49 ± 0.01 1.38 ± 0.00 Column phenomenex 76078.33 ± 830.74 11.77 ± 0.03 2.82 ± 0.00 1.31 ± 0.01 sweroside Column temp. Agilent 57332.67 ± 132.40 12.21 ± 0.06 2.81 ± 0.01 1.32 ± 0.00 25 67969.33 ± 51.64 9.74 ± 0.12 3.10 ± 0.01 1.25 ± 0.00 30 59198.33 ± 1405.63 10.24 ± 0.12 2.94 ± 0.01 1.29 ± 0.01 35 56038.67 ± 762.36 10.82 ± 0.07 2.77 ± 0.01 1.32 ± 0.00 40 53077.67 ± 531.74 11.28 ± 0.03 2.61 ± 0.01 1.36 ± 0.01 45 49995.00 ± 1187.00 11.77 ± 0.13 2.46 ± 0.01 1.40 ± 0.00 μ - 55 -

- 56 -

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

CAS number 18524-94-2 분자식 C 17 H 26 O 10 (MW 390) 용해도 녹는점 ( ) 구조 - 58 -

δ - 59 -

δ ν α β α - 60 -

Analytes Linear range (μg/ml) Conversion equation r 2 LOD (ng/ml) LOQ (ng/ml) loganin 1-20 Y = 0.06499X + 0.01462 0.99 1.098 3.327 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) loganin 1.13 0.00 0.13 23.39 0.29 1.22 Compounds loganin Spiked amount (μg) Intra-day (n=5) Inter-day (n=5) Recovery (%) RSD (%) Recovery (%) RSD (%) 2 100.02 0.69 100.76 0.65 4 99.64 0.45 100.75 0.67 8 101.64 1.74 100.13 0.78-62 -

Compounds Analytical condition Theoretical plate (N) Resolution capacity factor separation factor Shiseido 92897.00 ± 1168.34 4.04 ± 0.03 2.70 ± 0.01 1.09 ± 0.00 Column phenomenex 123533.67 ± 1908.80 3.55 ± 0.01 3.00 ± 0.00 1.06 ± 0.00 loganin Column temp. Agilent 72175.33 ± 0.00 3.40 ± 0.01 3.02 ± 0.01 1.08 ± 0.01 25 103215.33 ± 4064.81 2.96 ± 0.02 3.28 ± 0.01 1.06 ± 0.01 30 84204.33 ± 1351.77 3.22 ± 0.01 3.14 ± 0.01 1.07 ± 0.00 35 71067.67 ± 170.42 3.40 ± 0.02 2.98 ± 0.01 1.08 ± 0.00 40 62570.67 ± 2104.56 3.49 ± 0.02 2.83 ± 0.01 1.08 ± 0.00 45 60890.67 ± 896.68 3.55 ± 0.02 2.67 ± 0.01 1.09 ± 0.00 μ - 63 -