한약재품질표준화연구사업단 맥문동 ( 麥門冬 ) Liriopis Tuber 생약연구과

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1 한약재품질표준화연구사업단 맥문동 ( 麥門冬 ) Liriopis Tuber 생약연구과

2 Fig.1 ( ) ( )

3 Table 1. The origin of the Liriopis Tuber in KP, ChP and JP β

4 α β α βα αβ α aster saponin Hb methyl ester H H H H H H H liriope glycoside Lm-3 aster saponin Hd methyl ester H H H H liriope glycoside Ls-2 H H H H H H H H liriope glycoside Ls-3 liriope glycoside Ls-4 < >..

5 (12D1001-A01BKX1105A) 100 g 70% Et (1L), (1L) reflux [ ]

6 [ ] (mg) D spicatoside A (mg) (mg) (mg) *20 12D ophiopogonin D *20 12D D prosapogenin Ⅲ of spicatoside A pennogenin 3--β -D-xylopyranosyl (1 3)-α -L-arabinopyranosyl (1 3)-[4 -acetyl-α -L-rhamnopyranosyl (1 2)]-6 -acetyl-β -D-glucopyranoside * D spicatoside C D (S)-ruscogenin 1- -[4 -acetyl-α-l-rham nopyranosyl(1 2)] [β-d-xylopyranosyl(1 3)] -β-d-fucopyranosyl-3 -- α-l-rhamnopyran oside D lirioprolisoside A D n-butyl-β-D-fructo pyranoside D C-32-3 ( ) D ruscogenin 1--[α-L-rhamnopyra nosyl(1 4)] -β-d-fucopyranosyl-3 --α-l-rhamnopyrano side

7 12D ruscogenin 1-- -β-d-fucopyranosyl(1 4)-α-L-rhamnopyran oside

8 (compound number) CH3 H H CH 2 H CH 3 H H H H CH 3 CH3 H H HCH2 H CH 2 H H β α α β H CH3 H CH3 H CH 3 H H CH3 α β β α H CH 3 CH 3 H H H CH 3 H CH3 CH 2 CH 2 CH 2 CH 3 H H CH 2 β CH3 H CH3 H H α β α CH3 CH3 H CH3 H βα

9 βα α β CH3 H H H CH3 H H CH2 spicatoside A(1) H CH3 spicatoside C(2) H H CH 2 pennogenin 3--β-D-xylopyranosyl (1 3)-α-L-arabinopyranosyl (1 3)-[4 -acetyl-α-l-rhamnopyranosyl (1 2)]-6 -acetyl-β-d-glucopyranoside(3) I.S. : Butyl paraben Fig. 1. Structure of marker compounds

10 (2) ) TLC (normal phase) TLC ) LC-MS - ) HPLC/ELSD (1) Table 2. ptimum analytical conditions of HPLC/ELSD for 70% Me EtAc extract of Liriopis Tuber Column ZRBAX Eclipse Plus C18 ( mm, 5 μm) Mobile phase A : Water B : Acetonitrile Flow rate 1.0 ml/min Column temp. 35 Detector ELSD Gas pressure 1.5 bar ELSD temp. 30 Inj. vol. 40 μl Gradient Time (min) A (%) B (%) program

A; (2) Spicatoside C; (3)Pennogenin -β-d-xylopyranosyl(1

2)]-6'-acetyl-β-D-glucopyra noside; (4) Lirioprolioside A; (5) Prosapogenin Ⅲ

11 Fig. 2. Thin layer chromatography of 91 Liriope platyphylla and phiopogon japonicus samples. (normal phase, color developing method using 10% sulfuric acid) (1) Spicatoside A; (2) Spicatoside C; (3)Pennogenin -β-d-xylopyranosyl(1 3)-α-Lrabinopyranosyl(1 3)-[4"-acetyl-α-L-rhamnopyranosyl(1 2)]-6'-acetyl-β-D-glucopyra noside; (4) Lirioprolioside A; (5) Prosapogenin Ⅲ of spicatoside A; (6) Liriopeside B; (7) 25(S)-Ruscogenin1--[4"-acetyl-α-L-rhamnopyranosyl(1 2)]-[β-D-xylopyranosyl(1 3 )]-β-d-fucopyranosyl 3--α-L-rhamnopyranoside.(12D1001~12D2042, 12D2044~12D2071; Liriope platyphylla and 12D2043, 12D1002~12D2091; phiopogon japonicus)

12 Fig. 3. HPLC/ELSD chromatogram of Liriope platyphylla extracts and phiopogon japonicus extracts.(a) standard compounds; (B) 12D2001, (C) 12D2005 and (D) 12D2006 of Liriope platyphylla extracts;(e) 12D2084, (F) 12D2075 and (G) 12D2079 of phiopogon japonicus extracts. (I.S. butyl paraben; (1) spicatoside A; (2) spicatoside C; (3) pennogenin 3--β-D-xylopyranosyl(1 3)-α-L-arabinopyranosyl(1 3)-[4"-acetyl-α-L-rhamnopyranosyl(1 2)] -6'-acetyl-β-D-glucopyranoside.) Table 3. Assay results (w/w, %) of the marker compounds, (Liriope platyphylla) contents (w/w, %)

13 Sample number Spicatoside A (1) Spicatoside C (2) Pennogenin 3--β-D-xylopyranosyl (1 3)-α-L-arabinopyranosyl(1 3)-[4"-ace tyl-α-l-rhamnopyranosyl (1 2)]-6'-acetyl-β-D-glucopyranoside) 12D N/D D N/D D D N/D D D D N/D 12D N/D D N/D 12D D N/D 12D D N/D 12D D D N/D 12D D D D D D N/D D N/D 12D N/D 12D N/D D N/D D N/D D N/D D D D N/D D D D N/D D D D D D D D D D D N/D 12D N/D (3)

14 12D N/D 12D N/D D N/D 12D N/D 12D N/D 12D N/D 12D N/D 12D N/D D N/D 12D N/D Average SD RSD(%) * N/D : not detected Table 4. Assay results (w/w, %) of the marker compounds, (phiopogon japonicus.) contents (w/w, %) Pennogenin 3--β-D-xylopyranosyl (1 3)-α-L-arabinopyranosyl(1 3)-[4"-ace Sample Spicatoside A Spicatoside C tyl-α-l-rhamnopyranosyl number (1) (2) (1 2)]-6'-acetyl-β-D-glucopyranoside) (3) phiopogon japonicus Average N/D - N/D - N/D - SD RSD(%) * N/D : not detected

15 Fig. 4. Content of marker compounds: (1) spicatoside A; (2) spicatoside C; (3) pennogenin 3--β-D-xylopyranosyl(1 3)-α-L-arabinopyranosyl(1 3)- [4"-acetyl-α-L-rhamnopyranosyl(1 2)]-6'-acetyl-β-D-glucopyranoside). βα α β β αα β.

16 βα α β β α α β βα α β

17 β αα β

18 Table 5. The regulation content of Liriope platyphylla. Compounds Content Mean RSD SD criteria (w/w%) (%) (w/w%) Spicatoside A (1) Spicatoside C (2) Pennogenin 3--β-D-xylopyranosyl(1 3)-α-Larabinopyranosyl(1 3)-[4"-acetyl-α-L-rhamnopyranos yl(1 2)]-6'-acetyl-β-D-glucopyranoside (3) The effects of 70% Et extract of Morus alba (A), 70% Et extract of phinopogan japonicus (B) or spicatoside A (C) on the scopolamine-induced memory impairments in the passive avoidance task.

19 γ γ γ

20 κ

21 β β

22 κ κ β κ κ

23 α β 8. β α α β α β β α β α β α βα Ÿ βα

24 Ÿ α β μ μ Ÿ Ÿ - Ÿ γ

25 γ γ Ÿ

26 japonicus

30 μ μ μ μ

31 Liriope platyphylla Extracted with Me Me extract Partitioned with Hexane, CH2Cl2 EtAc, Bu, Water Hexane fra. Aqueous layer CH2Cl2 fra. Silica gel C.C (hexane:etac, EtAc saturated with H 2 :Me) CH 2 Cl 2-32 fra. Silica gel C.C (CH 2 Cl 2 :Me) spicatoside A Aqueous layer EtAc fra. Aqueous layer Bu fra. Aqueous layer

32 성상 이명 White amorphous powder 25(S)-ruscogenin-l--β-D-glucopyranosyl(1 2)-[β-D- xylopyranosyl(1 3)]-β-D-fucopyranoside CAS number 분자식 C 44 H (870) 용해도 soluble in methanol 녹는점 ( ) 구조 CH3 H H CH 2 H

33 ZRBAX Eclipse Plus C18 (5 μm, 250 mm 4.6 mm) Mobile phase: A: Water, B:Acetonitrile gradient 41-44% Flow rate: 1.0 ml/min Detector: ELSD Spicatoside A 피크 모든피크면적의합 100 = 100.0%

35 흡착제실리카겔 GF 254 전개용매클로로포름 메탄올 물 (52 : 28 : 8, lower layer) 발색시약 10 % 황산시액 Rf값 0.3

36 δ 0.86 (3H, s, 18-CH 3 ), 1.07 (3H, d, J=7.2 Hz, 27-CH 3 ), 1.10 (3H, d, J=6.8 Hz, 21-CH 3 ), 1.35 (3H, s, 19-CH 3 ), 1.52 (3H, d, J=6.2 Hz, fucose-ch 3 ), 4.85 (1H, d, J=7.6 Hz, anomeric H), 5.28 (1H, d, J=7.6 Hz, anomeric H), 5.45 (1H, d, J=7.6 Hz, anomeric H)

37 δ 83.4 (C-1), 37.8 (C-2), 68.7 (C-3), 44.2 (C-4), (C-5), (C-6), 32.7 (C-7), 33.4 (C-8), 50.9 (C-9), 43.3 (C-10), 24.1 (C-11), 40.4 (C-12), 41.0 (C-13), 57.4 (C-14), 32.3 (C-15), 81.8 (C-16), 64.8 (C-17), 17.3 (C-18), 15.3 (C-19), 41.0 (C-20), 16.8 (C-21), (C-22), 31.4 (C-23), 28.6 (C-24), 34.9 (C-25), 75.2 (C-26), 17.1 (C-27), (fuc-1), 79.3 (fuc-2), 83.4 (fuc-3), 72.7 (fuc-4), 71.5 (fuc-5), 17.6 (fuc-6), (xyl-1), 75.6 (xyl-2), 78.7 (xyl-3), 71.1 (xyl-4), 67.7 (xyl-5), (glc-1), 76.9 (glc-2), 79.1 (glc-3), 72.5 (glc-4), 78.8 (glc-5), 63.8 (glc-6)

39 Column ZRBAX Eclipse Plus C18 (5 μm, 250 mm 4.6 mm) Detector ELSD Column temp. 35 ELSD temp. 30 Gas pressure. 1.5 bar Injection volume 40 μl Flow rate 1.0 ml/min Mobile phase Time(min) Water Acetonitrile 0 59% 41% 60 56% 44%

40 HPLC/ELSD chromatograms of 70% methanolic extracts and spicatoside A. (A), (B) and (C) of Liriope platyphylla extracts; I.S. Butylparaben; (1) Spicatoside A. Analytes Linear range (μg/ml) Correlation coefficient LD (ng/ml) LQ (ng/ml) Spicatoside A Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) Spicatoside A Compounds Spiked Intra-day (n=3) Inter-day (n=3)

41 Spicatoside A conc. (μg/ml) bserved conc. (μg/ml) SD Precision RSD(%) Accuracy (%) bserved Conc. (μg/ml) SD Precision RSD(%) Accuracy (%) System suitability and robustness Compounds Analytical condition Theoretical plate (N) Capacity factor (K') Separation factor (a) Resolution (Rs) Tailing Factor Spicatoside A Column Column Temp. Aglient Shiseido Waters μ

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