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생약학회지 Kor. J. Pharmacogn. 45(3) : 240 248 (2014) LC-MS/MS 를이용한桂枝茯 丸의동시분석및항산화효능연구 서창섭 김온순 신현규 * 한국한의학연구원한약방제연구그룹 Quantitative Analysis and Antioxidant Effects of Gyejibokryeong-hwan Chang-Seob Seo, Ohn Soon Kim, and Hyeun-Kyoo Shin* Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 305-811, Korea Abstract Gyejibokryeong-hwan (GJBRH) has been used for treatment of patients with climacteric syndrome. In this study, an ultra-performance liquid chromatography-electrospray ionization-mass spectrometer (UPLC-ESI-MS) method was established for the simultaneous quantification of seven marker compounds in GJBRH extract. In addition, we assessed the antioxidant effects of GJBRH. All analytes were separated by gradient elution using two mobile phases on a UPLC BEH C 18 column and maintained at 45 o C. The antioxidant activities of GJBRH were evaluated by measuring free radical scavenging activities on 2,2'-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and 1-1-diphenyl-2-picrylhydrazyl (DPPH). The inhibitory effects on low-density lipoprotein (LDL) oxidation were evaluated by the formation of thiobarbituric acid relative substances (TBARS) and relative electrophoretic mobility (REM). Regression equations of the seven compounds were acquired with r 2 values 0.9988. The amounts of the seven compounds, amygdalin, albiflorin, paeoniflorin, coumarin, cinnamic acid, cinnamaldehyde, and paeonol in GJBRH water extract were 21.71, 2.16, 17.17, 1.97, 0.40, 0.78, and 3.42 mg/g, respectively. The GJBRH showed the radical scavenging activity in a dose-dependent manner. The concentration required for 50% reduction (RC 50 ) against ABTS and DPPH radicals were 54.18 µg/ml and 79.53 µg/ml. Furthermore, GJBRH reduced the oxidation properties of LDL induced by CuSO 4. Key words Gyejibokryeong-hwan, Quantitative analysis, Antioxidant effects, LC-MS/MS 桂枝茯丸은張仲景의 金要略 에최초로수록된처방으로桂枝 (Cinnamomi Ramulus), 茯笭 (Poria Sclerotium), 牧丹皮 (Moutan Radicis Cortex), 芍藥 (Paeoniae Radix) 및桃仁 (Persicae Semen) 의 5 종한약재로구성되어있으며, 하복부의瘀血로인한痛症을치료하는기본방제로알려져있다. 1) 본처방은항암, 2-4) 진통, 항염, 항경련및근이완, 5,6) 항혈전, 7) 항동맥경화, 8,9) 항혈소판 10) 및신장보호 11) 효과등과같은다양한실험적생리활성연구가진행및보고되었으며, 교통사고등외상으로유발된瘀血변증에대한효과, 12) 원발성생리통에대한치료효과 1,13) 및여성의폐경후삶의질개선효과 14,15) 등의증례보고와임상연구도보고되었다. 본연구에서는골반혈관의염증, 월경및하혈등과같은鬱血로인한下服桶, 月經不順및月經痛등의부인과질 * 교신저자 (E-mail) : hkshin@kiom.re.kr (Tel): +82-42-868-9464 환에널리사용되는대표처방인桂枝茯丸을선정하여이에대한효율적인품질관리에대한기초자료를제공하고자주요성분에대한함량분석을실시하였다. 본처방의구성생약의성분에관한연구는桂枝의 coumarin, cinnamic acid 및 cinnamaldehyde 등과같은 coumarin 계열 16,17) 과茯笭의 pachymic acid와 dehydropachymic acid 등과같은 triterpenoid 계열 18,19) 이분리보고되었다. 또한牧丹皮는 phenol 성분인 paeonol, 芍藥은 monoterpenoid 성분인 albiflorin과 paeoniflorin 및桃仁은 cyanoglucoside 계열의 amygdalin 등이대한약전 10개정 20) 에지표물질로수록되어있다. 이와같이처방을구성하는각각의생약성분에대하여고성능액체크로마토그래피를이용한분석법 21-26) 은많이보고가되어있으나복합제제인처방의분석에대한연구는미비한실정이다. 따라서본연구에서는구성생약의주요성분들, 즉桂枝의 coumarin, cinnamic acid 및 cinnamaldehyde, 牧丹皮의 paeonol, 芍藥의 albiflorin과 240

Vol. 45, No. 3, 2014 241 paeoniflorin 및桃仁의 amygdalin 등 7종의성분을대상으로질량분석기가결합된초고성능액체크로마토그래피 (ultraperformance liquid chromatography-electrospray ionizationmass spectrometer; UPLC-ESI-MS) 를이용하여함량분석을실시하고본처방에대한항산화효과와 LDL 산화억제효과를통한동맥경화질환의예방및치료의적용가능성을검토하였다. 재료및방법 실험재료 Table I과같이본실험에사용된桂枝茯丸을구성하는 5종의한약재는모두광명당제약 (Ulsan, Korea) 에서규격품을구입하여동국대학교한의과대학이제현교수 (Gyeongju, Korea) 와대전대학교한의과대학서영배교수 (Daejeon, Korea) 등 2인의전문가로부터감정후사용하였다. 본처방을이루는구성한약재들의표본 (2012-KE31-1~2012-KE31-5) 은한국한의학연구원한약방제연구그룹에보관하였다. 시약및기기 Amygdalin, coumarin, 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) 에서구입하였으며, albiflorin, paeoniflorin, cinnamic acid, cinnamaldehyde 및 paeonol은 Wako Pure Chemical Industries, Ltd.(Osaka, Japan) 로부터구입하였다. 이들표준물질의순도는모두 98.0% 이상이었다. LC-MS/MS 분석을위한메탄올, 아세토나이트릴및물은 J.T. Baker(Phillipsburg, NJ, USA) 에서구입하였으며, 개미산은 Sigma-Aldrich(St. Louis, MO, USA) 에서구입하여사용하였다. 7종의성분분리를위해 pump, digasser, column oven 및 autosampler로구성된 Waters의 ACQUITY UPLC(Milford, MA, USA) 를사용하였으며, 질량분석기는 ESI source가장착된탠덤사중극자질량분석기 (ACQUITY TQD LC-MS/ MS, Waters, Milford, MA, USA) 를사용하여분석하였다. 데이터는 Waters MassLynx software(version 4.1, Milford, MA, USA) 를사용하여수집및처리하였다. 桂枝茯丸물추출물조제 桂枝茯丸을구성하는한약재를 Table I과같이같은비율로배합한후총시료양을 5.0 kg으로맞춘후초고속진공저온추출기 (Cosmos 660, Kyungseo Machine Co., Incheon, Korea) 에넣고, 물을시료의 10배 (50 L) 로첨가하여 100 o C에서 2시간전탕한후표준체 (No. 270, 53 µm, Chung Gye Sang Gong Sa, Seoul, Korea) 를이용하여여과하였다. 여과액은동결건조기 (PVTFD100R, IlShinBioBase, Dongduchun, Korea) 를사용하여동결건조하여 11.8% 의수율인약 591 g의추출물을얻었다. 표준액의조제 Amygdalin, albiflorin, paeoniflorin, coumarin, cinnamic acid, cinnamaldehyde 및 paeonol 7종의표준품에대한표준용액은무게를정확하게측정한후메탄올로녹여모두 0.1 mg/ml의농도로조제한후 4 o C에보관하면서사용전에희석하여사용하였다. 검액의조제 LC-MS/MS 정량분석을위하여동결건조된추출물 100 mg을정확히취한후물을넣어 5 ml로맞춘후 5분간초음파추출하였다. 추출액에대하여 1000배희석후 0.22 µm 멤브레인여과하여검액으로하였다. UPLC 및 LC-MS/MS 분석조건 桂枝茯丸내주요성분인 amygdalin, albiflorin, paeoniflorin, coumarin, cinnamic acid, cinnamaldehyde 및 paeonol의함량분석을위해 Waters(Milford, MA, USA) 사의질량분석기가결합된 UPLC를사용하여분석하였다. 각성분의분리를위한칼럼은 UPLC BEH C 18 (2.1 100 mm, 1.7 µm, Waters, Milford, MA, USA) 칼럼을이용하였으며, 칼럼온도는 45 o C를유지하였다. 이동상은 0.1% 개미산이함유된물 (A) 과아세토나이트릴 (B) 를이용하여기울기용매조건으로흘려주었으며, 유속은 0.3 ml/ 분, 주입량은 2.0 µl였다 (Table II). 또한각성분의 MS분석을위해 ESI가결합된탠덤사중극자질량분석기 (ACQUITY TQD, Waters, Milford, MA, USA) 를사용하여양이온모드와음이온모드에서검출하였다. 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) 등에대한조건을설정 Table I. Composition of Gyejibokryeong-hwan Herb Scientific name Herbal medicine Original region Amount(g) 桂枝 Cinnamomum cassia Cinnamomi Ramulus Vietnam 3.75 茯笭 Poria cocos Poria Sclerotium Pyeongchang, Korea 3.75 牧丹皮 Paeonia suffruticosa Moutan Radicis Cortex Jecheon, Korea 3.75 芍藥 Paeonia lactiflora Paeoniae Radix Uiseong, Korea 3.75 桃仁 Prunus persica Persicae Semen South Africa 3.75 Total amount(g) 18.75

242 Kor. J. Pharmacogn. Table II. Conditions for LC-MS/MS analysis of Gyejibokryeonghwan HPLC condition Column ACQUITY UPLC BEH C 18 (100 2.1 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 Mobile phase Time(min) A(%) 1 B(%) 2 0 80 20 0.1 80 20 3.0 5 95 4.0 5 95 4.1 80 20 6.0 80 20 MS condition Capillary voltage(kv) 3.3 Extract voltage(v) 3.0 Source temperature( C) 120 RF lens(v) 0.3 Desolvation 300 temperature( C) 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 2 Acetonitrile 하여 multiple reaction monitoring(mrm) 모드를적용하여정량을실시하였다 (Table II). 검량선작성 Amygdalin, albiflorin, paeoniflorin, coumarin, cinnamic acid, cinnamaldehyde 및 paeonol 등 7 종의표준품에대한검량선은 5, 10, 50, 100 및 500 ng/ml 의농도로희석하여피크면적과농도에대해서작성하였다. 작성된검량선은상관계수 (r 2 ) 를구하여직선성을판단하였다. 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 를대조군으로하여대조군에대한라디칼소거능을백분율로나타내었다. 활성비교를위하여 vitamin C 를사용하였다. DPPH radical scavenging activity=(1 A sample /A control ) 100 LDL 산화억제활성측정 Cu 2+ 에의한 LDL의산화 반응은 LDL(Biomedical Technologies, Stoughton, MA, USA) 100 µg과시료를혼합한후 25 µm CuSO 4 를첨가함으로써시작하였고, 37 o C에서 6시간동안반응시켰다. 반응후 thiobarbituric acid reacting substance(tbars) 의형성과 relative electrophoretic mobility (REM) 을측정하였다. TBARS 측정 산화반응이끝난 LDL 50 µg을취하여 TBARS Assay Kit(BioAssay Systems, Hayward, CA, USA) 를이용하여제조사의 protocol에따라측정하였다. 측정된흡광도값을 malondialdehyde(mda) 표준곡선에대입시켜 MDA 양으로환산하였다. REM 측정 산화반응이끝난 LDL 10 µg을 0.8% agarose gel에 loading하여 TAE buffer(40 mm Tris, 40 mm acetic acid 및 1mM EDTA) 에서 100 V로 30분동안전기영동한후, Coomassie brilliant blue R-250로염색하여이동거리를측정하였다. 통계처리실험값은 mean±s.e.m. 으로표시하였다. 실험결과에대한통계학적유의성검정은 ANOVA 검정을적용하였으며, Dunnet s multiple comparison test 를이용하여 p-value 가 0.05 미만일경우유의한것으로판정하였다. 결과및고찰 LC-MS/MS 분석조건확립桂枝茯丸의구성약재중桂枝의 coumarin, cinnamic acid 및 cinnamaldehyde, 牧丹皮의 paeonol, 芍藥의

Vol. 45, No. 3, 2014 243 Fig. 1. Chemical structures of 7 marker compounds of Gyejibokryeong-hwan. Table III. Linearities, regression equation, and correlation coefficients for 7 marker compounds Compound Linear range(ng/ml) Regression equation Correlation coefficient Amygdalin 5 500 y = 2.88x 23.52 0.9988 Albiflorin 5 500 y = 18.94x 63.08 0.9995 Paeoniflorin 5 500 y = 2.64x 26.62 0.9990 Coumarin 5 500 y = 93.41x + 535.23 0.9994 Cinnamic acid 5 500 y = 41.14x + 105.39 0.9997 Cinnamaldehyde 5 500 y = 3.57x + 4.79 0.9991 Paeonol 5 500 y = 9.30x + 10.49 0.9998 albiflorin 과 paeoniflorin 및桃仁의 amygdalin 등 7 종의성분을 LC-MS/MS 를이용하여동시정량을실시하였다. 역상 UPLC 조건에서 UPLC BEH C 18 칼럼을이용하여모든성분의분리를위해물과아세토나이트릴의조건을이용하여 ESI 방법의양이온모드와음이온모드에서 Table II 와 III 의조건을이용하여검출하였다. 또한피크꼬리끌림현상을줄이고각성분의이온에대한검출감도를향상시키기위하여 0.1% 개미산을첨가하였다. 최적의이동상을이용하여기울기용매로분리를실시하였으며, 칼럼온도는 45 o C, 유속은분당 0.3 ml 로하여모든성분을 6 분이내로분리하였다. 검량선작성 LC-MS/MS 를이용하여농도에따른 ESI 방법의 MRM 모드에서 amygdalin, albiflorin, paeoniflorin, coumarin, cinnamic acid, cinnamaldehyde 및 paeonol 7 종의성분에대한피크면적과농도에따른검량선작성결과상관계수 (r 2 ) 값이 0.99 이상으로양호한직선성을각각나타내었다 (Table III). 桂枝茯丸추출물중주요성분의함량분석桂枝茯丸을설정된 LC-MS/MS 분석법으로 7종성분에대한함량을분석하였다. MS를이용한각성분의확인은 albiflorin, coumarin, cinnamic acid, cinnamaldehyde 및 paeonol 5종은양이온모드에서 m/z 480.9, 146.8, 148.9, 132.8 및 166.9의 [M+H] + 형태의분자이온피크를각각확인하였으며, amygdalin과 paeoniflorin은음이온모드에서 m/ z 456.1과 m/z 479.3의 [M-H] - 형태의분자이온피크를확인하였다 (Fig. 2). LC-MS/MS에서정량분석을위한 amygdalin의 MRM 조건은 m/z 456.1(precursor ion) 323.3(product ion), albiflorin 은 m/z 480.9 196.9, paeoniflorin은 m/z 479.3 121.0, coumarin은 m/z 146.8 90.9, cinnamic acid는 m/z 148.9 130.9, cinnamaldehyde는 m/z 132.8 115.0 및 paeonol은 m/z 166.9 120.8로설정한후정량을실시하였다. 이상과같이설정된桂枝茯丸의 LC-MS/MS MRM 분석법에의해분석한결과 amygdalin, albiflorin, paeoniflorin, coumarin, cinnamic acid, cinnamaldehyde 및 paeonol은 1.16분, 1.34 분, 1.39분, 2.15분, 2.26분, 2.53분및 2.63분에서각각검출

244 Kor. J. Pharmacogn. Fig. 2. Mass spectra of seven standard compounds. Amygdalin(A), albiflorin(b), paeoniflorin(c), coumarin(d), cinnamic acid(e), cinnamaldehyde(f), and paeonol(g). 되었다 (Table IV 및 Fig. 3). 함량분석결과桂枝茯丸추출물중桂枝의 coumarin, cinnamic acid 및 cinnamaldehyde 의함량이 1.97±0.09, 0.40±0.03 및 0.78±0.05 mg/g 으로각각검출되었으며, 牧丹皮의 paeonol 과桃仁의 amygdalin 이

Vol. 45, No. 3, 2014 245 Table IV. Chromatographic retention time, MRM parameters, cone voltage, and collision energy for the seven compounds Analyte Molecular weight(da) Ionization Mode Retention time(min) Precursor ion(m/z) Product ion(m/z) Cone voltage(v) Collision energy(ev) Amygdalin 457.4 [MH] 1.16 456.1 323.3 32 11 Albiflorin 480.4 [M+H] + 1.34 480.9 196.9 20 15 Paeoniflorin 480.4 [MH] 1.39 479.3 121.0 32 25 Coumarin 146.1 [M+H] + 2.15 146.8 90.9 30 20 Cinnamic acid 148.1 [M+H] + 2.26 148.9 130.9 20 10 Cinnamaldehyde 132.1 [M+H] + 2.53 132.8 115.0 25 15 Paeonol 166.1 [M+H] + 2.63 166.9 120.8 30 20 Fig. 3. Total ion chromatogram of seven marker compounds(a) and Gyejibokryeong-hwan sample(b) in positive ion(i and III) and negative ion(ii and IV) modes by LC-MS/MS MRM mode. Amygdalin(1), albiflorin(2), paeoniflorin(3), coumarin(4), cinnamic acid(5), cinnamaldehyde(6), and paeonol(7).

246 Kor. J. Pharmacogn. Table V. Amount of the 7 marker compounds in Gyejibokryeong-hwan Compound Amount(mg/g) Mean SD RSD(%) Source Amygdalin 21.71 0.67 3.07 Persicae Semen Albiflorin 2.16 0.05 2.20 Paeoniae Radix Paeoniflorin 17.17 0.40 2.31 Paeoniae Radix Coumarin 1.97 0.09 4.67 Cinnamomi Ramulus Cinnamic acid 0.40 0.03 8.25 Cinnamomi Ramulus Cinnamaldehyde 0.78 0.05 6.70 Cinnamomi Ramulus Paeonol 3.42 0.29 8.38 Moutan Radicis Cortex 3.42±0.29 및 21.76±0.67 mg/g 으로각각나타났다. 또한芍藥의 albiflorin 과 paeoniflorin 은 2.16±0.05 및 17.17±0.40 mg/g 으로검출되었다 (Table V). 桂枝茯桂枝茯 丸추출물의항산화활성丸의항산화활성을평가하고자추출물을농도 별로조제한후 ABTS와 DPPH 라디칼소거활성을측정하였다. ABTS 라디칼의소거활성을비교한결과추출물의농도가증가함에따라 ABTS 라디칼소거활성이증가하는경향을보였다. 桂枝茯丸추출물 25, 50, 100 및 200 µg/ml 농도에서각각 28.13, 52.29, 87.17 및 99.72% 의라디칼소거활성을나타냈으며, 시료를첨가하지않은대조구의흡광도를 1/2로감소시키는데필요한시료의양 (RC 50 ) 은 54.18 µg/ ml로관찰되었다. 양성대조군인 vitamin C의 RC 50 값은 3.66 µg/ml로관찰되었다 (Fig. 4A). DPPH 라디칼의소거활성또한 ABTS 라디칼의소거활성과유사하게농도의존적인증가를나타냈다. 추출물 25, 50, 100 및 200 µg/ml 농도에서각각 18.94, 37.63, 59.34 및 82.24% 의라디칼소거활성을나타냈으며, RC 50 는 79.53 µg/ml로관찰되었다. 양성대조군인 vitamin C의 DPPH 라디칼에대한 RC 50 값은 12.93 µg/ml로관찰되었다 (Fig. 4B). 桂枝茯丸추출물의 LDL 산화억제활성 LDL 산화억제활성을검색하고자 Cu 2+ 에의해 LDL 산 Fig. 4. Effects of Gyejibokryeong-hwan 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 GJBRH 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. Fig. 5. Effects of Gyejibokryeong-hwan on Cu 2+ -induced lipid peroxidation in LDLs. Indicated concentrations of GJBRH or vitamin C(50 µg/ml) and LDLs were incubated with CuSO 4 for 6 h at 37 C. The quantitative data were presented as mean±s.e.m. of triplicate experiments. **p<0.01 vs. OxLDL group.

Vol. 45, No. 3, 2014 247 LC-MS/MS를이용한함량분석을실시하였다. 분석결과桃仁과芍藥의주요성분인 amygdalin과 paeoniflorin이 21.71 mg/g과 17.17 mg/g으로다른성분에비해많이함유되어있음을확인하였다. 또한桂枝茯丸의 ABTS와 DPPH 라디칼제거를통한항산화활성은용량이높을수록높은활성을나타내었으며, LDL 산화억제활성역시고농도로처리할경우 LDL의산화정도를나타내는지표인 TBARS 와 REM이더감소됨을알수있었다. 이러한결과는桂枝茯丸이나이와관련된한약제제의품질관리를위한기초자료로활용될수있을것으로사료되며, 桂枝茯丸의우수한항산화활성과 LDL의산화억제활성을통해산화적스트레스에의한동맥경화질환의초기단계를예방하는데활용할수있을것으로기대된다. 사 사 본연구는한국한의학연구원에서지원하는 한약처방의과학적근거기반구축사업 (K14030) 에의해수행되었으며이에감사드린다. Fig. 6. Effects of Gyejibokryeong-hwan on electrophoretic mobility of Cu 2+ -induced oxidized LDLs. Indicated concentrations of GJBRH or vitamin C(50 µg/ml) and LDLs were incubated with CuSO 4 for 6 h at 37 o C. The electrophoretic mobility of LDLs was detected by agarose gel electrophoresis. (A) The stained gel is representative of three independent experiments. (B) Relative electrophoretic mobility (REM) indicated the distances moved from the origin were calculated and presented. **p<0.01 vs. OxLDL group. 화를유도하고, 추출물을처리하여 LDL 산화정도를비교분석하였다. TBARS assay 결과桂枝茯丸추출물의처리에의해농도의존적으로저해효과를나타냈으며, IC 50 값이 75.81 µg/ml으로계산되었다 (Fig. 5). Fig. 6은 Cu 2+ 에의해유도된산화 LDL의전기적이동차 (REM) 에대한桂枝茯丸의효과를나타내고있다. 桂枝茯丸을 37.5, 75 및 150 µg/ml 농도로처리하였을때산화 LDL의 REM이각각 19.73, 63.34 및 73.73% 감소되었다. 양성대조군인 vitamin C를처리한경우산화된 LDL의이동을 77.21% 억제하였다. 결 桂枝茯丸을구성하는桂枝, 茯笭, 牧丹皮, 芍藥및桃仁의 5가지구성생약중桂枝의 coumarin, cinnamic acid 및 cinnamaldehyde, 牧丹皮의 paeonol, 芍藥의 albiflorin과 paeoniflorin 및桃仁의 amygdalin 등 7종의성분에대하여 론 인용문헌 1. Nam, E. J., Lee, Y. K., Lee, D. N. and Kim, H. J. (2006) Clinical study on the effect of Gyebongnyeong capsule in the treatment of primary dysmenorrhea. Korean J. Physiol. Pathol. 20: 1364-1368. 2. Lee, S. K., Kim, H. G., Ahan, J. C., Chung, T. W., Moon, J. Y., Park, S. D., Kim, J. K., Choi, D. Y., Kim, C. H. and Park, W. H. (2003) Effect of the Geijibokryunghwan on human hepatocarcinoma cells. Korena J. Oriental Physiol. Pathol. 17: 568-573. 3. Hwang, D. S., Cho, J. H., Jang, J. B. and Lee, K. S. (2006) Inhibitory effects of Gaejibokryunghwan on cell proliferation in HeLa cells. J. Korean Oriental Med. 27: 23-35. 4. Yao, Z, and Shulan, Z. (2008) Inhibition effect of Guizhi-Fuling-decoction on the invasion of human cervical cancer. J. Ethnopharmacol. 120: 25-35. 5. Ha, D. J., Jeong, K. J. and Lee, K. N. (1995) Effect of Kaejibokryungwhan on analgesic, anti-inflammation, anti-convulsion, muscle relaxation and changes of body temperature through experimental animal. J. Korean Oriental Med. 16: 339-350. 6. Park, W. H., Joo, S. T., Park, K. K., Chang, Y. C. and Kim, C. H. (2004) Effects of the Geiji-Bokryung-Hwan on carrageenan-induced inflammation in mice and cyclooxygenase- 2 in hepatoma cells of HeG2 and Hep3B. Immunopharmacol. Immunotoxicol. 26: 103-112. 7. Yi, I. h., Lee, K. S. and Song, B. K. (2000) Effect of Guizibokryungwhan aqua-acupuncture on the intravascular coag-

248 Kor. J. Pharmacogn. ulation induced by endotoxin in rats. J. Oriental Gynecol. 13: 1-17. 8. Choi, M. H. and Park, W. H. (2006) Effect of the Geijibokryunghwan water extracts on stimulus-induced superoxide generation and tyrosyl phosphorylation in human neutrophils. Korean J. Oriental Physiol. Pathol. 20: 477-481. 9. Kim, B. J., Kim, Y. K., Park, W. H., Ko, J. H., Lee, Y. C. and Kim, C. H. (2003) A water-extract of the Korean traditional formulation Geiji-Bokryung-Hwan reduces atherosclerosis and hypercholesteremia in cholesterol-fed rabbits. Int. Immunopharmacol. 3: 723-734. 10. Park, W. H., Kim, K. S., Kim, K. H., Kim, D. S. and Kim, C. H. (2003) The antiplatelet activity of Geiji-Bokryung-Hwan, Korean traditional formulation, is mediated through inhibition of phospholipase C and inhibition of TxB 2 synthetase activity. Int. Immunopharmacol. 3: 971-978. 11. Nakagawa, T., Goto, H., Hikiami, H., Yokozawa, T., Shibahara, N. and Shimada, Y. (2007) Protective effects of keishibukuryogan on the kidney of spontaneously diabetic WBN/ Kob rats. J. Ethnopharmacol. 110: 311-7. 12. Jo, H. G., Park, A. R., Kee, Y. B., Kang, D. H., Choi, J. B. and Sil, J. U. (2011) A clinical trial on the blood stasis and efficacy of Kyejibokryong-whan (Guizhifuling-wan) in the patients with motor vehicle accident. J. Oriental Rehab Med. 21: 45-55. 13. Cho, J. H. (2007) A pilot study of the difference between Gyejibongnyeong-hwan and Gyejibongnyeong-hwan combined acupuncture therapy on the primary dysmenorrhea. J. Oriental Obstetrics & Gynecol. 20: 161-168. 14. Park, J. M., Yang, J. M. and Kim, D. I. (2007) A clinical trial to verity the quality of life imporvement efficacy of Dangguijakyak-san and Gyejibongnyeong-hwan granulation in postmenopausal women. J. Oriental Obstetrics & Gynecol. 20: 213-228. 15. Cho, K. O., Kim, Y. S., Jung, W. S. and Kim, T. H. (2011) Effect of Gui-zhi-fu-ling-wan on hot flashes in young patients: A retrospective case series. J. Acupunct Meridian Stud. 4: 129-133. 16. Liang, K., Cui, S., Zhang, Q., Bi, K., Qian, Z. and Jia, Y. (2011) UPLC simultaneous determination of five active components in Cinnamomi Ramulus. Zhongguo Zhong Yao Za Zhi 36: 3298-3320. 17. Wen, K. C., Huang, C. Y. and Liu, F. S. (1992) Determination of cinnamic acid and paeoniflorin in traditional Chinese medicinal preparations by high-performance liquid chromatography. J. Chromatogr. 593: 191-199. 18. Li, G., Xu, M. L., Lee, C. S., Woo, M. H., Chang, H. W. and Son, J. K. (2004) Cytotoxicity and DNA topoisomerases inhibitory of constituents from the sclerotium of Poria cocos. Arch. Pharm. Res. 27: 829-833. 19. Hoang, L., Kwon, S. H., Kim, K. A., Hur, J. M., Kang, Y. H. and Song, K. S. (2005) Chemical standardization of Poria cocos. Kor. J. Pjarmacogn. 36: 177-185. 20. Korean Pharmacopoeia Committee (2013) The Korean Pharmacopoeia, 10th ed, 1129-1192. Shinilbooks, Seoul. 21. Xu, X. Y., Yi, Z. H., Zheng, Y. M. and Fu, S. Q. (2008) Study on quality standard of Poria cocos. Zhong Yao Cai 31: 597-599. 22. Che, S., Li, Q., Huo, Y. S., Chen, X. H. and Bi, K. S. (2010) RP-HPLC simultaneous determination of five triterpenoid acids in different parts of Poria cocos by UV wavelengths switch. Yao Xue Xue Bao 45: 494-497. 23. Jeong, S. Y., Zhao, B. T., Moon, D. C., Kang, J. S., Lee, J. H., Min, B. S., Son, J. K. and Woo, M. H. (2013) Quantitative analysis of bioactive marker compounds form Cinnamomi Ramulus and Cinnamomi Cortex by HPLC-UV. Nat. Prod. Sci. 19: 28-35. 24. Ding, Y., Wu, E., Chen, J., Nguyen, H. T., Do, T. H., Park, K. L., Bae, K. H., Kim, Y. H. and Kang, J. S. (2009) Quality evaluation of Moutan Cortex Radicis using multiple component analysis by high performance liquid chromatography. Bull. Korean. Chem. Soc. 30: 2240-2244. 25. Yang, L., Xu, S. J., Tian, R. T., Xie, P. S. and Wang, Z. T. (2007) HPLC fingerprinting of Radix Paeoniae Alba. Yao Xue Xue Bao 42: 71-74. 26. Koo, J. Y., Hwang, E. Y., Cho, S., Lee, J. H., Lee, Y. M. and Hong, S. P. (2005) Quantitative determination of amygdalin epimers from Armeniacae Semen by liquid chromatography. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 814: 69-73. (2014. 7. 11 접수 ; 2014. 8. 20 심사 ; 2014. 8. 27 게재확정 )