한약재품질표준화연구사업단 맥문동 ( 麥門冬 ) Liriopis Tuber 생약연구과

한약재품질표준화연구사업단 맥문동 ( 麥門冬 ) Liriopis Tuber 생약연구과

Fig.1 ( ) ( )

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

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 < >..

(12D1001-A01BKX1105A) 100 g 70% Et (1L), (1L) reflux 95 3 2.. [ ]

[ ] (mg) 3 4 5 6 7 8 9 10 11 12D1001-01 spicatoside A 465.6 201.4 111.4 281.9 51.1 66.9 1178. 3 (mg) (mg) (mg) 5.1 319.5 1.0 *20 12D1001-02 ophiopogonin D 54.4 54.4 8.4 2.0 1.0 *20 12D1001-03 12D1001-04 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 36.3 36.3 3.3 1.0 1.0 *20 55.3 55.3 7.4 12D1001-05 spicatoside C 19.2 19.2-12D1001-06 25(S)-ruscogenin 1- -[4 -acetyl-α-l-rham nopyranosyl(1 2)] [β-d-xylopyranosyl(1 3)] -β-d-fucopyranosyl-3 -- α-l-rhamnopyran oside 7.1 7.1 5.4 12D1001-07 lirioprolisoside A 66.0 66.0 7.3 12D1001-08 1-n-butyl-β-D-fructo pyranoside 92.9 92.9 5.3 12D1001-09 C-32-3 ( ) 102.8 102.8-12D1001-10 ruscogenin 1--[α-L-rhamnopyra nosyl(1 4)] -β-d-fucopyranosyl-3 --α-l-rhamnopyrano side 35.6 35.6 5.4

12D1001-11 ruscogenin 1-- -β-d-fucopyranosyl(1 4)-α-L-rhamnopyran oside 40.2 40.2 - - -

(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

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

(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 (4.6 250 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 (%) 0.0 59 41 program 60.0 56 44

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)

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, %)

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) 12D1001 0.0024 N/D 0.0042 12D1003 0.0016 N/D 0.0028 12D2001 0.0018 0.0025 0.0022 12D2002 0.0022 N/D 0.0019 12D2003 0.0029 0.0018 0.0031 12D2004 0.0022 0.0016 0.0027 12D2005 0.0031 0.0027 N/D 12D2006 0.0019 N/D 0.0026 12D2007 0.0051 0.0030 N/D 12D2008 0.0070 0.0048 0.0015 12D2009 0.0031 0.0019 N/D 12D2010 0.0024 0.0012 0.0019 12D2011 0.0058 0.0033 N/D 12D2012 0.0045 0.0014 0.0055 12D2013 0.0032 0.0000 0.0053 12D2014 0.0039 0.0027 N/D 12D2015 0.0046 0.0028 0.0032 12D2016 0.0047 0.0021 0.0039 12D2018 0.0071 0.0042 0.0011 12D2019 0.0032 0.0024 0.0000 12D2021 0.0032 0.0020 0.0035 12D2022 0.0025 N/D 0.0040 12D2023 0.0037 0.0030 N/D 12D2038 0.0027 0.0015 N/D 12D2039 0.0027 N/D 0.0024 12D2040 0.0018 N/D 0.0025 12D2041 0.0020 N/D 0.0033 12D2042 0.0026 N/D 0.0043 12D2044 0.0015 0.0016 0.0018 12D2045 0.0038 0.0015 0.0045 12D2046 0.0017 N/D 0.0024 12D2047 0.0019 0.0015 0.0015 12D2048 0.0036 0.0017 0.0047 12D2049 0.0016 N/D 0.0021 12D2050 0.0015 0.0015 0.0009 12D2051 0.0023 0.0019 0.0027 12D2052 0.0013 0.0018 0.0011 12D2053 0.0009 0.0011 0.0010 12D2054 0.0024 0.0011 0.0041 12D2055 0.0043 0.0023 0.0029 12D2056 0.0025 0.0013 0.0021 12D2057 0.0029 0.0011 0.0038 12D2058 0.0031 0.0023 0.0030 12D2059 0.0027 0.0014 N/D 12D2060 0.0028 N/D 0.0046 (3)

12D2061 0.0048 0.0036 N/D 12D2062 0.0013 N/D 0.0027 12D2064 0.0028 0.0014 N/D 12D2065 0.0044 0.0034 N/D 12D2066 0.0026 0.0012 N/D 12D2067 0.0029 0.0016 N/D 12D2068 0.0025 0.0015 N/D 12D2069 0.0025 N/D 0.0036 12D2070 0.0027 0.0014 N/D 12D2071 0.0026 0.0011 N/D Average 0.0030 0.0015 0.0020 SD 0.0013 0.0012 0.0017 RSD(%) 44.2263 80.0315 83.8115 * 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

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.

βα α β β α α β βα α β

β αα β

Table 5. The regulation content of Liriope platyphylla. Compounds Content Mean RSD SD criteria (w/w%) (%) (w/w%) Spicatoside A (1) 0.0030 0.0013 44.2263 0.0009 Spicatoside C (2) 0.0015 0.0012 80.0315 0.0010 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) 0.0020 0.0017 83.8115 0.0011 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.

γ γ γ

κ

β β

κ κ β κ κ

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

Ÿ α β μ μ Ÿ Ÿ - Ÿ γ

γ γ Ÿ

japonicus

μ μ μ μ

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

성상 이명 White amorphous powder 25(S)-ruscogenin-l--β-D-glucopyranosyl(1 2)-[β-D- xylopyranosyl(1 3)]-β-D-fucopyranoside CAS number 128397-47-7 분자식 C 44 H 70 17 (870) 용해도 soluble in methanol 녹는점 ( ) 243-245 구조 CH3 H H CH 2 H

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%

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

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)

δ 83.4 (C-1), 37.8 (C-2), 68.7 (C-3), 44.2 (C-4), 140.2 (C-5), 124.7 (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), 113.2 (C-22), 31.4 (C-23), 28.6 (C-24), 34.9 (C-25), 75.2 (C-26), 17.1 (C-27), 100.9 (fuc-1), 79.3 (fuc-2), 83.4 (fuc-3), 72.7 (fuc-4), 71.5 (fuc-5), 17.6 (fuc-6), 106.7 (xyl-1), 75.6 (xyl-2), 78.7 (xyl-3), 71.1 (xyl-4), 67.7 (xyl-5), 105.4 (glc-1), 76.9 (glc-2), 79.1 (glc-3), 72.5 (glc-4), 78.8 (glc-5), 63.8 (glc-6)

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%

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 40 360 0.9981 12.00 28.00 Compounds Peak area ratio (peak area/is area) Retention time (min) Mean SD RSD (%) Mean SD RSD (%) Spicatoside A 1.01 0.00 0.42 27.13 0.09 0.33 Compounds Spiked Intra-day (n=3) Inter-day (n=3)

Spicatoside A conc. (μg/ml) bserved conc. (μg/ml) SD Precision RSD(%) Accuracy (%) bserved Conc. (μg/ml) SD Precision RSD(%) Accuracy 40.00 39.92 0.27 0.68 99.81 38.49 0.48 1.25 96.23 120.00 122.12 0.26 0.21 101.77 117.57 1.20 1.02 97.97 180.00 179.79 0.47 0.26 99.89 176.57 1.56 0.88 98.10 (%) 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 24800 10.22 1.59 14.74 0.93 Shiseido 33695 14.09 1.69 20.68 0.93 Waters 24261 13.94 1.65 17.14 0.89 30 20420 9.25 1.35 9.27 0.99 35 23756 10.30 1.59 14.72 0.95 40 26403 11.01 1.86 19.02 0.94 45 25013 11.55 2.10 22.21 0.92 μ