생약학회지 Kor. J. Pharmacogn. 45(4) : 300 314 (2014) LC-MS/MS 를이용한천왕보심단 ( 天王補心丹 ) 의함량분석및항산화효능연구 서창섭 김온순 신현규 * 한국한의학연구원한약방제연구그룹 Quantitative Determination and Antioxidant Effects of Cheonwangbosimdan Chang-Seob Seo, Ohn Soon Kim, and Hyeun-Kyoo Shin* Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea Abstract An ultra-performance liquid chromatography-electrospray ionization-mass spectrometer (UPLC-ESI-MS) method was established for the simultaneous quantification of eighteen marker compounds in traditional Korean formula, Cheonwangbosimdan (CWBSD). In addition, we evaluated the antioxidant effects of CWBSD. Eighteen marker components were separated on a UPLC BEH C 18 analytical column (2.1 100 mm, 1.7 µm) and kept at 45 o C by gradient elution with 0.1% (v/ v) formic acid in water and acetonitrile as mobile phase. The flow rate was 0.3 ml/min and the injection volume was 2.0 µl. The antioxidant activities of CWBSD were assessed by measuring free radical scavenging activities on 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1-1-diphenyl-2-picrylhydrazyl (DPPH). The calibration curves of all analytes showed good linearity (correlation coefficient 0.9937) within the test ranges. The limits of detection and quantification for the 18 marker compounds were 0.01-4.71 ng/ml and 0.03-14.13 ng/ml, respectively. The contents of the 18 compounds in CWBSD extract ranged from none to 1701.00 µg/g. The CWBSD showed the radical scavenging activity in a dose-dependent manner. The concentration required for 50% reduction (RC 50 ) against ABTS and DPPH radicals were 149.42 µg/ml and 339.24 µg/ml. Key words Cheonwangbosimdan, Quantitative determination, Antioxidant effects, LC-MS/MS 천왕보심단 ( 天王補心丹 ) 은 世醫得效方 1) 에처음으로수록된처방으로생건지황 ( 生乾地黃, 酒洗, Rehmanniae Radix), 황련 ( 黃蓮, 酒炒, Coptidis Rhizoma), 석창포 ( 石菖蒲, Acori Graminei Rhizoma), 인삼 ( 人蔘, Ginseng Radix Alba), 당귀 ( 當歸, 酒洗, Angelicae Gigantis Radix), 오미자 ( 五味子, Schizandrae Fructus), 천문동 ( 天門冬, Asparagi Tuber), 맥문동 ( 麥門冬, Liriope Tuber), 백자인 ( 柏子仁, Thujae Semen), 산조인 ( 酸棗仁, Zizyphi Semen), 현삼 ( 玄蔘, Scrophulariae Radix), 백복신 ( 白茯神, Hoelen), 단삼 ( 丹蔘, Salviae Miltiorrhizae Radix), 길경 ( 桔梗, Platycodi Radix) 및원지 ( 遠志, Polygalae Radix) 등 15 종한약재로구성되어있으며, 심장이비정상적으로뛰며불안, 초조및건망증의증상을보이는神志不安과虛煩失眠, 手足心熱, 口舌生瘡및大便乾結등의증상을보이는陰虛血少를치료하는방제로사용되었다. 2) 본처방은혈관확장, 3) 혈압강하, 3) 항치매, 4-6) 항산화 6) * 교신저자 (E-mail) : hkshin@kiom.re.kr (Tel): +82-42-868-9464 및항우울 7) 효과등다양한실험적생리활성연구가진행및보고되었으며, 뇌경색후발생하는심인성심장질환환자의증상개선에대한증례보고가보고되었다. 8) 따라서저자들은본연구에서는임상에서不眠에널리사용되는처방의하나인天王補心丹을선정하여이에대한효율적인품질관리에대한기초자료를제공하고자주요성분에대한함량분석을실시하였다. 본처방의구성생약의성분에관한연구는생건지황 ( 生乾地黃 ) 의 aucubin 과 catalpol 등과같은 iridoid glycosides, 9) 황련 ( 黃蓮 ) 의 berberine 등과같은 alkaloids, 10) 석창포 ( 石菖蒲 ) 의 β-asarone 등과같은 phenylpropanoids, 11) 인삼 ( 人蔘 ) 의 ginsenosid Rb 1 과 Rg 1 등과같은 triterpenoid saponins, 12) 당귀 ( 當歸 ) 의 nodakenin 등과같은 coumarins, 13) 오미자 ( 五味子 ) 의 schizandrin 과 gomisin A 등과같은 lignans, 14) 천문동 ( 天門冬 ) 의 5-hydroxymethyl-2- furaldehyde(5-hmf) 등과같은 furan derivatives, 15) 맥문동 ( 麥門冬 ) 의 spicatoside A 와 B 등과같은 saponins, 16) 백자인 ( 柏子仁 ) 의 myricetin 등과같은 flavonoids, 17) 산조인 ( 酸棗仁 ) 300
Vol. 45, No. 4, 2014 301 의 spinosin과 6-feruloylspinosin 등과같은 flavonoids, 18) 현삼 ( 玄蔘 ) 의 cinnamic acid 등과같은 phenylpropanoids 19) 및 harpagide와 (E)-harpagoside 등과같은 iridoid glycosides, 20) 백복신 ( 白茯神 ) 의 pachymic acid와 dehydropachymic acid 등과같은 triterpenoid, 21,22) 단삼 ( 丹蔘 ) 의 salvianolic acid 등과같은 phenylpropanoids 23) 및 tanshinone IIA 등과같은 diterpenoids, 23) 길경 ( 桔梗 ) 의 platycodin D 등과같은 triterpenoid saponins 24) 및원지 ( 遠志 ) 의 3,4,5-trimethoxycinnamic acid 등과같은 phenylpropanoids 계열 25) 이분리보고되었다. 이들성분중본연구에서는생건지황 ( 生乾地黃 ) 의 aucubin 과 catalpol, 석창포 ( 石菖蒲 ) 의 β-asarone, 인삼 ( 人蔘 ) 의 ginsenoside Rb 1 과 ginsenoside Rg 1, 당귀 ( 當歸 ) 의 nodakenin, 오미자 ( 五味子 ) 의 schizandrin과 gomisin A, 천문동 ( 天門冬 ) 의 5-HMF, 백자인 ( 柏子仁 ) 의 myricetin, 산조인 ( 酸棗仁 ) 의 spinosin, 현삼 ( 玄蔘 ) 의 cinnamic acid, harpagide 및 (E)- harpagoside, 단삼 ( 丹蔘 ) 의 salvianolic acid B와 tanshinone IIA, 길경 ( 桔梗 ) 의 platycodin D 및원지 ( 遠志 ) 의 3,4,5- trimethoxycinnamic acid 등 18종에대하여질량분석기가결합된초고성능액체크로마토그래피 (ultra-performance liquid chromatography-electrospray ionization-mass spectrometer; UPLC-ESI-MS) 를이용하여함량분석과항산화효능을검색하였다. 재료및방법 실험재료 Table I 과같이본실험에사용된天王補心丹을구성하는 15 종의한약재는모두광명당제약 (Ulsan, Korea) 에서규격품을구입하여동국대학교한의과대학이제현교수 (Gyeongju, Korea) 로부터감정후사용하였다. 본처방을이루는구성한약재들의표본 (2012-KE34-1~2012- KE34-15) 은한국한의학연구원한약방제연구그룹에보관하였다시약및기기 Aucubin( 98.0%), cinnamic acid( 98.0%), ginsenoside Rb 1 ( 98.0%), schizandrin( 98.0%) 및 gomisin A( 98.0%) 는 Wako Pure Chemical Industries, Ltd. (Osaka, Japan) 로부터구입하였으며, catalpol( 98.0%), spinosin ( 98.0%), ginsenoside Rg 1 ( 98.0%) 및 (E)-harpagoside ( 98.0%) 는 Chengdu Biopurify Phytochemicals(Chengdu, China) 에서구입하였다. Harpagide( 98.0%), salvianolic acid B ( 98.0%), platycodin D( 98.0%) 및 tanshinone IIA( 98.0%) 는 ChemFaces(Wuhan, China) 에서구입하였으며, 5-HMF ( 98.0%) 와 3,4,5-trimethoxycinnamic acid( 98.0%) 는 Sigma- Aldrich(St. Louis, MO, USA) 에서구입하였다. Nodakenin ( 98.0%), myricetin( 97.8%) 및 β-asarone( 98.0%) 은 NPC Bio Technology(Yeongi, Korea), TCI(Tokyo, Japan) 및 ROTH(Karlsruhe, Germany) 로부터각각구입하였다. 이들의화학적구조는 Fig. 1과같다. 항산화효능평가를위한 dimethyl sulfoxide(dmso), 2,2'-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid(abts) 및 2,2-diphenyl-2-picrylhydrazyl (DPPH) 는 Sigma-Aldrich(St. Louis, MO, USA) 에서구입하였다. LC-MS/MS 분석을위한메탄올, 아세토나이트릴및물은 J.T. Baker(Phillipsburg, NJ, USA) 에서구입하였으며, 개미산은 Sigma-Aldrich(St. Louis, MO, USA) 에서구입하여사용하였다. Table I. Composition of CWBSD Scientific name Herbal medicine Origin Amount(g) Rehmannia glutinosa Rehmanniae Radix Andong, Korea 15.000 Coptis japonica Coptidis Rhizoma China 7.500 Acorus gramineus Acori Graminei Rhizoma Jeju, Korea 3.750 Panax ginseng Ginseng Radix Alba Yeongju, Korea 1.875 Angelica gigas Angelicae Gigantis Radix Bonghwa, Korea 1.875 Schisandra chinensis Schizandrae Fructus Samcheok, Korea 1.875 Asparagus cochinchinensis Asparagi Tuber China 1.875 Liriope platyphylla Liriope Tuber Miryang, Korea 1.875 Thuja orientalis Thujae Semen China 1.875 Zizyphus jujuba Zizyphi Semen China 1.875 Scrophularia buergeriana Scrophulariae Radix Uiseong, Korea 1.875 Poria cocos Hoelen Pyeongchang, Korea 1.875 Salvia miltiorrhiza Salviae Miltiorrhizae Radix China 1.875 Platycodon grandiflorum Platycodi Radix Muju, Korea 1.875 Polygala tenuifolia Polygalae Radix China 1.875 Total amount 48.750
302 Kor. J. Pharmacogn. Fig. 1. Chemical structures of 18 marker compounds of CWBSD. 시료조제를위한추출기는경서기계산업의초고속진공저온농축추출기 (COSMOS660, Incheon, Korea) 를사용하여추출하였다. 본처방의함량분석에사용된 UPLC 는 pump, digasser, column oven 및 autosampler 로구성된 Waters 의 ACQUITY UPLC system(milford, MA, USA) 를사용하였으며, 질량분석기는 ESI source 가장착된탠덤사중극자질량분석기 (ACQUITY TQD LC-MS/MS, Waters, Milford, MA, USA) 를사용하여분석하였다. 데이터는 Waters MassLynx software(version 4.1, Milford, MA, USA) 를사용하여수집및처리하였다. 天王補心丹물추출물조제 天王補心丹을구성하는한약재를 Table I 과같이같은비율로배합한후총시료양을 5.0 kg(1 첩 48.75 g 102.5 배 ) 으로맞춘후초고속진공저온추출기 (Cosmos 660, Kyungseo Machine Co., Incheon, Korea) 에넣고, 물을시료의 10 배 (50 L) 로첨가하여 100 o C 에서 2 시간전탕한후표준체 (No. 270, 53 µm, Chung Gye Sang Gong Sa, Seoul, Korea) 를이용하여여과하였다. 여과액은동 결건조기 (PVTFD100R, IlShinBioBase, Dongduchun, Korea) 를사용하여동결건조하여 1065.1 g(21.3%) 의추출물을얻었다. 표준액및검액의조제 Aucubin, catalpol, harpagide, 5- HMF, spinosin, nodakenin, myricetin, salvianolic acid B, ginsenoside Rg 1, 3,4,5-trimethoxycinnamic acid, platycodin D, (E)-harpagoside, cinnamic acid, ginsenoside Rb 1, schizandrin, β-asarone, gomisin A 및 tanshinone IIA 18종의표준품에대한표준용액은메탄올을이용하여 100.0 µg/ml 의농도로조제한후 4 o C에보관하면서사용전에희석하여사용하였다. 또한 LC-MS/MS 정량분석을위하여동결건조된추출물 100 mg을정확히취한후물을넣어 5mL 로맞춘후 5분간초음파추출하였다. 추출액에 0.1 ml을정확히취한후물을넣어 10 ml로 100배희석후 0.22 µm 멤브레인필터하여검액으로하였다. UPLC 및 LC-MS/MS 분석조건 天王補心丹내주요성분의분리와검출은 Waters(Milford, MA, USA) 사의질
Vol. 45, No. 4, 2014 303 량분석기가결합된 ACQUITY TQD LC-MS/MS를사용하여분석하였다. 주요성분의분리는 UPLC BEH C 18 (2.1 100 mm, 1.7 µm, Waters, Milford, MA, USA) 칼럼을사용하여분리하였으며, 칼럼온도는 45 o C를유지하였다. 이동상, 유속및주입량은 Table II와같다. 또한각성분의 MS 및 MS/MS 분석조건은 Table II와같으며양이온모드와음이온모드에서검출하였다. 최적의 MS 검출을위해 capillary voltage(3.3 kv), extract voltage(3 V), source temperature(120 o C), RF lens(0.3 V), desolvation temperature(300 o C), desolvation gas(600 L/h), cone gas(50 L/h) 및 collision gas(0.14 ml/min) 등에대한조건 (Table II) 과각성분의 precursor ion, product ion, cone voltage 및 collision energy 등에대한최적의조건 (Table III) 을설정하여다중반응탐색법 (multiple reaction monitoring) 을이용하여정량을실시하였다. 검량선작성 Aucubin, catalpol, harpagide, 5-HMF, spinosin, nodakenin, myricetin, salvianolic acid B, ginsenoside Rg 1, 3,4,5-trimethoxycinnamic acid, platycodin D, (E)- harpagoside, cinnamic acid, ginsenoside Rb 1, schizandrin, β-asarone, gomisin A 및 tanshinone IIA 등 18종의표준품에대한검량선은 10, 50, 100 및 500 ng/ml 의농도로희석하여피크면적과농도에대해서작성하였다. 작성된검량선은상관계수 (r 2 ) 를구하여직선성을판단하였다. 검출한계 (limit of detection, LOD) 와정량한계 (limit of quantification, LOQ) 는신호대잡음비인 3과 10으로계산하였다. Table II. Conditions for LC-MS/MS analysis of CWBSD HPLC condition Column ACQUITY UPLC BEH C 18 (2.1 100 mm, 1.7 µm) Flow rate 0.3 ml/min Injection volume 2.0 µl Column temperature 45 o C Sample temperature 5 o C Time(min) A(%) 1 B(%) 2 0 80 20 0.1 80 20 Mobile phase 14.0 5 95 15.0 0 100 15.1 80 20 18.1 80 20 MS condition Capillary voltage(kv) 3.3 Extract voltage(v) 3.0 Source temperature( o C) 120 RF lens(v) 0.3 Desolvation temperature( o C) 300 Desolvation gas(l/h) 600 Cone gas(l/h) 50 Collision gas(ml/min) 0.14 1 0.1%(v/v) formic acid in water Acetonitrile Table III. Chromatographic retention time, MRM parameters, cone voltage, and collision energy for 18 compounds Compound Mode Retention time(min) Molecular weight(da) Precursor ion(m/z) Product ion(m/z) Cone voltage(v) Collision energy(ev) Aucubin(1) [M-H] - 0.80 346.3 345.1 183.4 30 10 Catalpol(2) [M-H] - 0.80 362.3 361.3 199.1 30 10 Harpagide(3) [M-H] - 0.80 364.3 363.2 183.1 33 15 5-HMF(4) [M+H] + 1.06 126.1 126.9 108.8 20 10 Spinosin(5) [M+H] + 1.37 608.5 609.6 327.1 40 25 Nodakenin(6) [M+H] + 1.85 408.4 409.4 247.2 30 15 Myricetin(7) [M+H] + 2.23 318.2 319.2 153.1 40 30 Salvianolic acid B(8) [M-H] - 2.35 718.2 717.5 519.2 30 15 Ginsenoside Rg 1 (9) [M-H] - 2.69 801.01 800.4 637.0 50 20 3,4,5-Trimethoxycinnamic acid(10) [M+H] + 3.08 238.2 239.2 221.1 20 10 Platycodin D(11) [M-H] - 3.38 1225.3 1224.1 469.4 45 48 (E)-Harpagoside(12) [M-H] - 3.39 494.4 493.4 345.6 33 18 Cinnamic acid(13) [M+H] + 3.74 148.1 149.1 130.9 20 10 Ginsenoside Rb 1 (14) [M-H] - 4.34 1108.6 1107.4 178.8 50 45 Schizandrin(15) [M+H] + 6.48 432.2 433.4 415.1 25 13 β-asarone(16) [M+H] + 6.57 208.1 209.1 194.0 20 15 Gomisin A(17) [M+H] + 7.10 416.4 417.4 399.4 20 10 Tanshinone IIA(18) [M+H] + 10.44 294.3 295.3 249.2 35 20
304 Kor. J. Pharmacogn. 항산화능측정 ABTS 라디칼소거능측정 ABTS 라디칼을이용한항산화능측정은 ABTS + cation decol orization assay 방법을 96 well plate 에맞게수정하여실시하였다. 7 mm ABTS 와 2.45 mm potassium perulfate 를최종농도로혼합하여실온인암소에서 24 시간동안방치하여 ABTS + 를형성시킨후 743 nm 에서 0.7 의흡광도값을갖도록 phosphate buffer saline(pbs, ph 7.4) 로희석하였다. 96 well plate 에 ABTS + 용액과시료를혼합하여실온에서 5 분간반응시킨후, microplate reader(benchmark Plus, Bio-Rad. USA) 를사용하여 734 nm 에서흡광도를측정하였다. 시료의항산화능은시료를녹인용매인 PBS 를대조군으로하여대조군에대한라디칼소거능을백분율로나타내었다. 활성비교를위하여 vitamin C 를사용하였다. ABTS radical scavenging activity =(1 A sample /A control ) 100 DPPH 라디칼소거능측정 DPPH 라디칼을이용한항산화능측정은 96 well plate 을이용하여실시하였다. 96 well plate 에 0.15 mm 의 DPPH 용액과시료를혼합하여실온에서 30 분간반응시킨후, 517 nm 에서흡광도를측정하였다. 시료의항산화능은시료를녹인용매인 DMSO 를대조군으로하여대조군에대한라디칼소거능을백분율로나 타내었다. 활성비교를위하여 vitamin C 를사용하였다. DPPH radical scavenging activity =(1 A sample /A control ) 100 결과및고찰 LC-MS/MS 분석조건확립 天王補心丹을구성하는구성생약의주요성분인 aucubin, catalpol( 이상생건지황 ), β- asarone( 석창포 ), ginsenoside Rb 1, ginsenoside Rg 1 ( 이상인삼 ), nodakenin( 당귀 ), schizandrin, gomisin A( 이상오미자 ), 5-HMF( 천문동 ), myricetin( 백자인 ), spinosin( 산조인 ), cinnamic acid, harpagide, (E)-harpagoside( 이상현삼 ), salvianolic acid, tanshinone IIA( 이상단삼 ), platycodin D ( 길경 ) 및 3,4,5-trimethoxycinnamic acid( 원지 ) 등 18 종성분에대하여 LC-MS/MS 를이용하여동시정량을실시하였다. 역상 UPLC 조건에서 UPLC BEH C 18 (2.1 100 mm, 1.7 µm) 칼럼을이용하여성분의분리를위해이동상으로물 - 메탄올, 물 - 아세토나이트릴, 산이함유된물 - 메탄올및산이함유된물 - 아세토나이트릴등을이용하여성분들의검출을시도하였다. 그결과최적의이동상조건을산이함유된물 - 아세토나이트릴로결정하였으며, 산으로피크꼬리끌림현상을줄이고각성분의이온에대한검출감도를향상시키기위하여 0.1% 개미산을첨가하였다. 이와같이최적의이 Table IV. Linearities, regression equation, correlation coefficients, LOD, and LOQ for 18 marker compounds Compound Linear range (ng/ml) Regression equation Correlation coefficient LOD (ng/ml) LOQ (ng/ml) Aucubin(1) 10-500 y = 0.71x 5.56 0.9993 0.88 2.64 Catalpol(2) 10-500 y = 0.62x 4.73 0.9992 4.71 14.13 Harpagide(3) 10-500 y = 0.85x + 4.52 0.9937 0.82 2.47 5-HMF(4) 10-500 y = 27.24x + 153.92 0.9993 0.99 2.96 Spinosin(5) 10-500 y = 15.11x 47.22 0.9999 0.17 0.51 Nodakenin(6) 10-500 y = 51.46x 195.46 0.9995 0.46 1.39 Myricetin(7) 10-500 y = 2.66x 39.00 0.9961 0.98 2.93 Salvianolic acid B(8) 10-500 y = 4.55x 65.06 0.9967 0.52 1.57 Ginsenoside Rg 1 (9) 10-500 y = 0.52x 3.37 0.9971 1.11 3.33 3,4,5-Trimethoxycinnamic acid(10) 10-500 y = 58.80x + 6.39 0.9999 0.11 0.32 Platycodin D(11) 10-500 y = 2.06x 8.05 0.9995 0.96 2.89 (E)-Harpagoside(12) 10-500 y = 7.91x 9.18 1.0000 0.43 1.30 Cinnamic acid(13) 10-500 y = 16.81x 63.47 0.9996 0.87 2.62 Ginsenoside Rb 1 (14) 10-500 y = 0.99x + 4.04 0.9997 0.94 2.81 Schizandrin(15) 10-500 y = 205.12x + 603.88 0.9996 0.06 0.17 β-asarone(16) 10-500 y = 125.96x + 747.07 0.9993 0.01 0.03 Gomisin A(17) 10-500 y = 92.58x + 461.60 0.9993 0.13 0.40 Tanshinone IIA(18) 10-500 y = 141.03x + 1792.30 0.9966 3.85 11.55
Vol. 45, No. 4, 2014 305 Fig. 2. Q1 (A) and Q3 (B) mass spectra of 18 standard compounds. Aucubin(1), catalpol(2), harpagide(3), 5-HMF(4), spinosin(5), nodakenin(6), myricetin(7), salvianolic acid B(8), ginsenoside Rg 1 (9), 3,4,5-trimethoxycinnamic acid(10), platycodin D(11), (E)- harpagoside(12), cinnamic acid(13), ginsenoside Rb 1 (14), schizandrin(15), β-asarone(16), gomisin A(17), and tanshinone IIA(18).
306 Kor. J. Pharmacogn. Fig. 2. Continued.
Vol. 45, No. 4, 2014 307 Fig. 2. Continued.
308 Kor. J. Pharmacogn. Fig. 2. Continued.
Vol. 45, No. 4, 2014 309 Fig. 2. Continued. 동상을이용하여 ESI 방법의양이온모드와음이온모드에서 Table II와 III의조건과같이분석을실시하였으며, 칼럼온도는 45 o C, 유속은분당 0.3 ml로하여모든성분을 11 분이내로분리하였다. 검량선, 검출한계및정량한계작성 LC-MS/MS를이용하여농도에대한피크면적값에대하여 18종성분의검량선작성결과상관계수 (r 2 ) 값이 0.99이상으로양호한직선성을나타내었으며, 검출한계와정량한계는 0.01-4.71 ng/ ml와 0.03-14.13 ng/ml로나타났다 (Table IV). 天王補心丹추출물중주요성분의함량분석 확립된 LC- MS/MS 분석방법에의해天王補心丹의주요성분에대한피크확인을실시하였다. 그결과 5-HMF, spinosin, nodakenin, myricetin, 3,4,5-trimethoxycinnamic acid, cinnamic acid, schizandrin, β-asarone, gomisin A 및 tanshinone IIA 등 10종은 m/z 126.9, 609.5, 409.4, 319.2, 239.2, 148.9, 433.3, 209.2, 417.4 및 295.3에서 [M+H] + 형태의분자이온피크를확인하였으며, aucubin, catalpol, harpagide, salvianolic acid B, ginsenoside Rg 1, platycodin D, (E)-harpagoside 및 ginsenoside Rb 1 등 8종의성분은 m/z 345.3, 361.3, 363.3, 717.5, 799.2, 1224.3, 493.4 및 1107.5 에서 [M H] 형태의분자이온피크를각각확인하였다 (Table III). LC- MS/MS를이용한이들성분들의정량분석을위하여 precursor ion(q1) 과 product ion(q3) 설정은다음과같다 (Fig. 2). Aucubin, catalpol 및 ginsenoside Rg 1 의 MRM 조건은 m/z 345.1, 361.3 및 800.4(precursor ion) 에서 glucose 1분자가각각이탈된 [M H Glc] 형태의 m/z 183.4, 199.1 및 637.0(product ion) 에서각각의피크를확인하였다. 26,27) Harpagide는 m/z 363.2에서 1분자의 glucose와물분자가떨어진 [M H Glc H 2 O] 형태의 m/z 183.1에서피크를확인하였으며, 28) 5-HMF는 m/z 126.9에서물분자 1개가이탈된 [M+H H 2 O] + 형태의 m/z 108.8에서분자이온피크를확인하였다. 29) Spinosin은 m/z 609.6에서 1분자의 glucose와 C 4 H 6 O 4 가떨어진 [M+H C 4 H 6 O 4 ] + 형태의 m/z 327.1에서피크를확인하였으며, 30) nodakenin은 m/z 409.4에서 glucose가떨어진 [M+H Glc] + 형태의 m/z 247.2에서분자이온피크를확인하였다. 31) Myricetin과 salvianolic acid B는 m/z 319.2와 717.5에서 C 8 H 6 O 4 와 C 9 H 9 O 5 가각각떨어진 [M+H C 8 H 6 O 4 ] + 와 [M H C 9 H 9 O 5 ] 형태의 m/z 153.1과 519.2에서피크를각각확인하였으며, 32-34) platycodin D는 28번탄소에결합된 Ara-Rha-Xyl-Api에서 90 Da이이탈된 m/z 469.4에서분자이온피크를확인하였다. 35) 3,4,5- Trimethoxycinnamic acid, cinnamic acid, schizandrin 및
310 Kor. J. Pharmacogn. gomisin A 는 m/z 239.2, 148.9, 433.4 및 417.4 에서 1 개의물분자가이탈된 [M+H H 2 O] + 형태의 m/z 221.1, 130.9, 415.1 및 399.4 에서분자이온피크를확인하였다. 36-38) (E)- Harpagoside 는 m/z 493.4 에서 cinnamic acid 가이탈된 [M H cinnamic acid] 형태로 m/z 345.6 에서확인하였으며, 39) ginsenoside Rb 1 은 [Glc H] 형태의 m/z 178.8 에서분자이 Fig. 3. Total ion chromatogram of 18 marker compounds (A) and CWBSD sample (B) by LC-MS/MS MRM mode. Aucubin(1), catalpol(2), harpagide(3), 5-HMF(4), spinosin(5), nodakenin(6), myricetin(7), salvianolic acid B(8), ginsenoside Rg 1 (9), 3,4,5-trimethoxycinnamic acid(10), platycodin D(11), (E)-harpagoside(12), cinnamic acid(13), ginsenoside Rb 1 (14), schizandrin(15), β-asarone(16), gomisin A(17), and tanshinone IIA(18).
Vol. 45, No. 4, 2014 311 온피크를확인하였다. 40) β-asarone은 m/z 209.1에서메틸기가이탈된물분자가떨어진 [M+H CH 3 ] + 형태의 m/z 194.0에서확인하였으며, 41) tanshinone IIA는 m/z 295.3에서 1개의물분자와 CO 분자가이탈된 [M+H H 2 O CO] + 형태의 m/z 249.2에서분자이온피크를확인하였다. 42) 이상과같이각성분의 precursor ion과 product ion을설정한후정량을실시하였으며, 설정된天王補心丹의 LC-MS/MS MRM 분석법에의해분석한결과 aucubin, catalpol, harpagide, 5- HMF, spinosin, nodakenin, myricetin, salvianolic acid B, ginsenoside Rg 1, 3,4,5-trimethoxycinnamic acid, platycodin D, (E)-harpagoside, cinnamic acid, ginsenoside Rb 1, schizandrin, β-asarone, gomisin A 및 tanshinone IIA 등 18 종의성분은 0.80, 0.80, 0.80, 1.06, 1.37, 1.85, 2.23, 2.35. 2.69, 3.08, 3.38, 3.39, 3.74, 4.34, 6.48, 6.57, 7.10 및 10.44 분에서각각검출되었다 (Table III 및 Fig. 3). 함량분석결과天王補心丹추출물중에서천문동 ( 天門冬 ) 의 5-HMF, 백자인 ( 柏子仁 ) 의 myricetin, 오미자 ( 五味子 ) 의 schizandrin 및단삼 ( 丹蔘 ) 의 tanshinone IIA 등 4종은검출이되지않았으며, 이들 4종을제외한 14종의성분함량은 6.23-1701.00 µg/g으로나타났다 (Table V). 天王補心丹추출물의항산화효능 天王補心丹의항산화효능을평가하고자추출물을농도별로조제한후 ABTS 와 DPPH 라디칼소거활성을측정하였다. ABTS 라디칼의 소거활성을비교한결과추출물의농도가증가함에따라 ABTS 라디칼소거활성이증가하는경향을보였다. 天王補心丹추출물 25, 50, 100, 200 및 400 µg/ml 농도에서각각 9.78, 20.31, 35.49, 65.21 및 94.87% 의라디칼소거활성을나타냈으며, 시료를첨가하지않은대조구의흡광도를 1/2 로감소시키는데필요한시료의양 (RC 50 ) 은 149.92 µg/ml 로관찰되었다. 양성대조군인 vitamin C 의 RC 50 값은 3.40 µg/ml 로관찰되었다 (Fig. 4A). DPPH 라디칼의소거활성또한 ABTS 라디칼의소거활성과유사하게농도의존적인증가를나타냈다. 추출물 25, 50, 100, 200 및 400 µg/ml 농도에서각각 4.02, 9.92, 17.61, 37.18 및 57.02% 의라디칼소거활성을나타냈으며, RC 50 는 339.24 µg/ml 로관찰되었다. 양성대조군인 vitamin C 의 DPPH 라디칼에대한 RC 50 값은 12.62 µg/ml 로관찰되었다 (Fig. 4B). 결 天王補心丹을구성하는 15가지구성생약의주요성분중 aucubin, catalpol, harpagide, 5-HMF, spinosin, nodakenin, myricetin, salvianolic acid B, ginsenoside Rg 1, 3,4,5-trimethoxycinnamic acid, platycodin D, (E)-harpagoside, cinnamic acid, ginsenoside Rb 1, schizandrin, β-asarone, gomisin A 및 tanshinone IIA 등 18종의성분에대하여 LC-MS/MS 론 Table V. Amount of 18 marker compounds in CWBSD Compound Amount(µg/g) Mean SD RSD(%) Source Aucubin(1) 39.00 1.50 3.85 Rehmanniae Radix Catalpol(2) 167.83 7.52 4.48 Rehmanniae Radix Harpagide(3) 14.50 0.50 3.45 Scrophulariae Radix 5-HMF(4) ND 1 - - Asparagi Tuber Spinosin(5) 34.17 3.25 9.52 Zizyphi Semen Nodakenin(6) 463.50 5.89 1.27 Angelicae Gigantis Radix Myricetin(7) ND - - Thujae Semen Salvianolic acid B(8) 1701.00 63.29 3.72 Salviae Miltiorrhizae Radix Ginsenoside Rg 1 (9) 19.83 0.76 3.85 Ginseng Radix Alba 3,4,5-Trimethoxycinnamic acid(10) 23.83 1.04 4.37 Polygalae Radix Platycodin D(11) 225.00 16.18 7.19 Platycodi Radix (E)-Harpagoside(12) 202.50 10.76 5.31 Scrophulariae Radix Cinnamic acid(13) 77.17 3.88 5.03 Scrophulariae Radix Ginsenoside Rb 1 (14) 25.33 1.26 4.97 Ginseng Radix Alba Schizandrin(15) ND - - Schizandrae Fructus β-asarone(16) 204.17 7.77 3.80 Acori Graminei Rhizoma Gomisin A(17) 6.23 0.25 4.04 Schizandrae Fructus Tanshinone IIA(18) ND - - Salviae Miltiorrhizae Radix 1 ND: not detected
312 Kor. J. Pharmacogn. 과학적근거기반구축사업 (K14030) 에의해수행되었으며이에감사드린다. 인용문헌 Fig. 4. Effects of CWBSD on ABTS and DPPH free radical scavenging activity. ABTS(A) or DPPH(B) radical solution was added to a 96-well plate containing of indicated concentrations of CWBSD or vitamin C. After 30 min of incubation, the absorbance(abts; 734 nm, DPPH; 517 nm) was measured using a microplate reader. The quantitative data were presented as mean±s.e.m. of triplicate experiments. 를이용한함량분석을실시하였다. 분석결과본한약처방에서는단삼 ( 丹蔘 ) 의주요성분인 salvianolic acid B 가가장많은 1701.00 µg/g 으로검출되었으며, 당귀 ( 當歸 ) 의 nodakenin(463.50 µg/g), 길경 ( 桔梗 ) 의 platycodin D(225.00 µg/g), 현삼 ( 玄蔘 ) 의 (E)-harpagoside(202.50 µg/g), 석창포 ( 石菖蒲 ) 의 β-asarone(204.17 µg/g) 및생건지황 ( 生乾地黃 ) 의 catalpol(167.83 µg/g) 등이다른성분에비해많이함유되어있음을확인하였다. 또한天王補心丹의 ABTS 와 DPPH 라디칼제거를통한항산화활성은용량이높을수록높은활성을나타내었다. 이러한분석결과는天王補心丹의품질관리와새로운생리활성연구를위한기초자료로활용될수있을것으로사료되며, 天王補心丹의항산화효능을통해산화적스트레스에의한다양한질환의초기단계를예방하는데활용할수있을것으로기대된다. 사 사 본연구는한국한의학연구원에서지원하는 한약처방의 1. Wei, Y. L. (1990) Seuideukhyobang, 217. Esdang, Seoul. 2. Herbal formulation Professors of Korean Medicine (2003) Herbal formulation, 329-331. Younglimsa, Seoul. 3. Kim, N. J., Kong, Y. Y. and Chang, S. W. (1988) Studies on the efficacy of combined preparation of crude drugs (XXX- VII). The effects of Chunwangboshimdan on the central nervous system and cardio-vascular system. Kor. J. Pharmacogn. 19: 208-215. 4. Lee, J. Y., Jung, I. C. and Lee, S. R. (2002) Effects of Chenwhangbosimdan (CWBD) on inhibition of impairment of learning and memory, and acetylcholinesterase in amnesia mice. J. Orient. Neuropsychiatry 13: 149-171. 5. Choi, K. W. and Jung, I. C. (2008) The effects of Chenwhangbosindan (CBD) hot water extract & ultra-fine powder on the Alzheimer's disease model. J. Orient. Neuropsychiatry 19: 77-93. 6. Jung, I. C. (2008) Effects of Chenwhangbosim-dan and Sungimjihwang-tang on protecting microglia and inhibiting acetylcholinesterase and oxidants. Korean J. Orient. Physiol. Pathol. 22: 120-125. 7. Park, J. H., Bae, C. W., Jun, H. S., Hong, S.Y. and Park, S. D. (2004) Antidepressant effect of chunwangboshimdan and its influence on monoamines. Korena J. Orient. Med. Prescrip. 12: 77-93. 8. Han, S. H., Jeong, S. H., Jeog, S. H., Shin, G. C. and Lee, W. C. (2002) Treated one case of neurogenic heart disease patient with Chunwangbosim-dan ( 天王補心丹 ). Korea J. Joongpoong 3: 75-80. 9. Kim, N. J., Jung, E. A., Kim, H. J., Sim, S. B. and Kim, J. W. (2000) Quality evaluation of various dried roots of Rehmannia glutinosa. Kor. J. Pharmacogn. 31: 130-141. 10. Ma, B. L., Ma, Y. M., Shi, R., Wang, T. M., Zhang, N., Wang, C. H. and Yang, Y. (2010) Identification of the toxic constituents in Rhizoma Coptidis. J. Ethnopharmacol. 128: 357-364. 11. Liao, J. F., Huang, S. Y., Jan, Y. M., Yu, L. L. and Chen, C. F. (1998) Central inhibitory effects of water extract of Acori Graminei Rhizoma in mice. J. Ethnopharmacol. 61: 185-193. 12. Shan, S. M., Luo, J. G., Huang, F. and Kong, L. Y. (2014) Chemical characteristics combined with bioactivity for comprehensive evaluation of Panax ginseng C.A.Meyer in different ages and seasons based on HPLC-DAD and chemometric methods. J. Pharm. Biomed. Anal. 89: 76-82. 13. Ahn, M. J., Lee, M. K., Kim, Y. C. and Sung, S. H. (2008) The simultaneous determination of coumarins in Angelica gigas root by high performance liquid chromatography-diode
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