J Korean Soc Food Sci Nutr 한국식품영양과학회지 46(9), 1091~1096(2017) https://doi.org/10.3746/jkfn.2017.46.9.1091 목단피추출물의 Oxypaeoniflorin 및 Paeoniflorin 의분석법개선및검증 최승현 1 * 유창길 2 * 황지현 3 이기쁨 3 이영진 2 이부용 3 이옥환 1 1 강원대학교식품생명공학과 2 차의과학대학교통합의학대학원 3 차의과학대학교식품생명공학과 Modification and Validation of Analytical Method for Oxypaeoniflorin and Paeoniflorin in Moutan Cortex Radicis Extract Seung-Hyun Choi 1*, Chang-Kil Yoo 2*, Ji-Hyun Hwang 3, Gi-Bbeum Lee 3, Young-Jin Lee 2, Boo-Yong Lee 3, and Ok-Hwan Lee 1 1 Department of Food Science and Biotechnology, Kangwon National University 2 Graduate School of Integrative Medicine and 3 Department of Food Science and Biotechnology, CHA University ABSTRACT The aim of this study was the validation of a modified analytical method for determination of oxypaeoniflorin and paeoniflorin in Moutan Cortex Radicis extract. For validation of the analytical method, we modified established analytical methods and validated improvement. For validation, the specificity, linearity, precision, accuracy, limit of detection (LOD), and limit of quantification of oxypaeoniflorin and paeoniflorin were measured by high performance liquid chromatography. The results show that the correlation coefficients of the calibration curve for oxypaeoniflorin and paeoniflorin were 1.0000 and 0.9998, respectively. The LOD for oxypaeoniflorin and paeoniflorin were 0.23 μg/ml and 0.2 μg/ml, respectively. The inter-day and intra-day precision values of oxypaeoniflorin and paeoniflorin were 0.70 3.19% and 1.74 2.43%, and 0.32 0.92% and 0.62 2.28%, respectively. The inter-day and intra-day accuracies of oxypaeoniflorin and paeoniflorin were 98.33 102.11% and 97.72 118.12%, and 98.44 101.6% and 97.10 112.00%, respectively. Therefore, the analytical method was validated for the detection of oxypaeoniflorin and paeoniflorin in Moutan Cortex Radicis. Key words: Moutan Cortex Radicis, oxypaeoniflorin, paeoniflorin, HPLC-PDA, method validation 서 목단피 (Moutan Cortex Radicis) 는작약과목단 ( 모란, Paeonia suffruticosa Andrews) 의뿌리껍질로서한방에서예로부터해열, 양혈, 구어, 진통및항균등의효능을가지고있어널리사용되고있다 (1-6). 목단피의생리활성으로는동맥경화억제, 관절염, 항염증, 혈소판응집억제, 패혈증억제, 항산화및 tyrosinase 억제효과, 히스타민유리억제, TNF-α 생성저해, 항당뇨효과등의생리활성이보고되고있다 (7-13). 이런목단피의주요성분으로는 paeonol, paeonoside 등의페놀류, oxypaeoniflorin, paeoniflorin 등의 monoterpene 배당체, tetragalloyglucose 등의탄닌류등을함유한것으로알려져 Received 22 June 2017; Accepted 26 July 2017 Corresponding author: Ok-Hwan Lee, Department of Food Science and Biotechnology, Kangwon National University, Chuncheon, Gangwon 24341, Korea E-mail: loh99@kangwon.ac.kr, Phone: +82-33-20-644 * These authors contributed equally to this work. 론 있다 (14-16). 이중 oxypaeoniflorin 과 paeoniflorin 은 hyaluronidase 억제효과 (17) 등의생리활성을가지고있는것으로보고되었다. 최근건강식품에대한소비자의관심및니즈가증가하고있고다양한소재로제품이개발되고있으며, 목단피의경우다양한생리활성효능이알려져있기때문에목단피추출물을이용하여개별인정형건강기능식품개발시원료의표준화를위한유용성분에대한검출방법및분석법검증에대한연구가필요하다. 건강기능식품을개발하기위해서는기능성및안전성을과학적으로입증해야하며기능성원료에대한표준화가필요하다 (18). 표준화란원재료의생산부터제조과정전반에사용된기술및정보를관리함으로써천연물질에함유된고유성분의변동을최소화시켜품질을일정하게유지하는것을말하며, 표준화의일반적인지표로는지표성분을이용한다. 지표성분의확인을통하여기능성원료의기능성이일정하게유지되는것을확인할수있으므로이를확인하기위하여공인된분석방법또는정밀한분석방법을사용하여야하며, 기준규격을설정하기위해분석방법의타당성및신뢰성이검증되어야한다 (19). 현재 high-
1092 최승현 유창길 황지현 이기쁨 이영진 이부용 이옥환 performance liquid chromatography-photodiode array (HPLC-PDA), high-performance liquid chromatography-mass spectrometry(hplc-ms) 등을이용한목단피중유용성분들의분석법이일부보고되어있으나 (20-2), 유효성검증 (method validation) 이시행되지않아분석법의타당성및신뢰성을확보하기힘든실정이다. 따라서본연구에서는목단피추출물의건강기능식품원료로개발시원료의표준화를위하여 oxypaeoniflorin 및 paeoniflorin의분석법개발및검증에대한연구를실시하였다. 재료및방법실험재료본실험에서사용한목단피 (Moutan Cortex Radicis) 는중국안휘성에서 6년이상재배된목단의뿌리를채취하였으며, 잔뿌리와목심부를제거하고껍질부분만무황처리한뒤이를건조시켜사용하였다. 표준물질 oxypaeoniflorin과 paeoniflorin은 MedChem Express Co.(Monmouth Junction, NJ, USA) 에서구입하였다. 용매로사용한 dimethyl sulfoxide(dmso) 와 formic acid는 Junsei Chemical(Tokyo, Japan) 에서구입하였으며 acetonitrile은 J.T. Baker (Phillipsburg, NJ, USA) 에서구입하여사용하였다. Oxypaeoniflorin 및 paeoniflorin 분석목단피에함유된 oxypaeoniflorin 및 paeoniflorin의분석을위한시료는다음과같이제조하였다. 채취된목단피를 1차선별을통하여줄기및흙을제거한후세척하고 1차건조를진행하였다. 이를거심작업을거치고일정크기로절단을한후 2차건조를거치고입고검사를진행한다음 2차선별을통하여이물질및가루를제거하였다. 이렇게선별한목단피시료 00 g을깨끗하게세척한후 단계정수필터로정수된물 8,000 ml에 3시간탕전하여추출물을제조하였다. 제조된목단피추출물의추출수율은 12.27±0.1% 로확인되었다. 조추출물을 filter paper(whatman No. 3, Whatman, Maidstone, UK) 를이용하여여과한후에회전식진공농축기 (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) 를이용하여농축한다음, 동결건조기 (Ilshin BioBase Co., Ltd., Gyeonggi, Korea) 를이용하여동결건조물로제조하여실험에사용하였다. Oxypaeoniflorin과 paeoniflorin의 HPLC 분석은 Xu 등 (22) 의분석방법을변형하여최적분석방법을확립한후실시하였으며, 두개의표준물질을동시분석하였다. 분석에사용한기기는 Waters 26 Separation Module HPLC system과 Waters 996 Photodiode Array Detector(Waters Co., Milford, MA, USA) 로조건은 Table 1과같으며분석용 column은 Capcell pak C 18 MG(4.6 mm 20 mm,.0 μm, Shiseido, Tokyo, Japan) 를사용하였다. Table 1. HPLC conditions of oxypaeoniflorin and paeoniflorin analysis for Moutan Cortex Radicis extract Instrument Conditions Column Capcell pak C 18 MG (.0 μm, 4.6 mm 20 mm) Column temp. 2 C Time (min) A 1) (%) B 2) (%) Mobile phase (gradient) Detector Flow rate Injection volume Run time 0 2 30 31 3 1) 0.1% formic acid in water. 2) 0.1% formic acid in acetonitrile. 84 Waters 996 Photodiode Array Detector (24 nm) 1.0 ml/min 10 μl 3 min 16 표준용액및시험용액의조제 Oxypaeoniflorin 및 paeoniflorin 표준물질을각각 10 mg을취한후 10 ml 정용플라스크를이용하여 1,000 μg/ ml의농도가되도록 DMSO 로표선까지정용하여이를 stock solution으로하였다. Working solution은제조된 stock solution을이용하여 3.12, 6.2, 12., 2, 0, 100, 200 μg/ml가되도록 DMSO로희석하여사용하였다. 시험용액은동결건조된시료 0 mg을칭량한후 0 ml 정용플라스크를이용하여 1,000 μg/ml의농도가되도록 DMSO로표선까지채운다음, 이를 0.4 μm syringe filter(whatman) 로여과하여시험용액으로하였다. 분석법의유효성검증분석법의유효성검증은 International Conference for Harmonization(ICH) 가이드라인 (26) 을근거로하여개발된분석법의특이성 (specificity), 직선성 (linearity), 정밀성 (precision), 정확성 (accuracy), 검출한계 (limit of detection, LOD) 및정량한계 (limit of quantitation, LOQ) 를이용하여분석법의유효성을검증하였다. 특이성표준물질 oxypaeoniflorin, paeoniflorin 및목단피추출물을 HPLC로분석하여얻은 chromatogram을비교하여 oxypaeoniflorin 및 paeoniflorin이선택적으로분리가되는지확인하였으며 PDA spectrum을확인하여동일한 spectrum을나타내는지확인하였다. 직선성 Oxypaeoniflorin 및 paeoniflorin 표준물질을 3.12, 6.2, 12., 2, 0, 100, 200 µg/ml의농도로제조하여 HPLC를이용하여 3회반복측정하였으며, 각표준물질의
목단피지표성분의분석법개선및검증 1093 peak에대한면적과농도비의관계를표시하는검량선을작성하고작성한검량선으로부터얻어진상관계수 (correlation coefficient, R 2 ) 값을통하여직선성을확인하였다. 정밀성및정확성농도를알고있는목단피추출물에표준용액 oxypaeoniflorin과 paeoniflorin을 6.2, 2, 100 μg/ml의농도를각각첨가하여일내 (intra-day) 정밀성및정확성을확인하기위하여하루에 3회반복하여 HPLC로분석하였으며, 일간 (inter-day) 정밀성및정확성을확인하기위하여 3일간반복하여 HPLC로분석하였다. 분석하여얻어진 peak의머무름시간 (retention time, RT) 과 PDA spectrum을비교하여정성을하고작성한검량선에시험용액의 peak 면적을대입하여 oxypaeoniflorin과 paeoniflorin의농도를계산하였다. 각결과값의표준편차를결과값의평균으로나눈비인상대표준편차 (relative standard deviation, RSD) 로일내및일간정밀성을확인하였으며, 정확성은다음식을이용하여첨가한농도에대비하여회수된농도를계산함으로써회수율을구해정확성을확인하였다. C f: Concentration of spiked sample C u: Concentration of sample C a: Concentration of standard 다 (Fig. 1). 각물질의최대흡수파장인 29, 233 nm 및기존보고된논문의분석파장인 24 nm에따른두물질의 peak 면적을비교하여최적면적값을나타내는파장값을확인한결과, 24 nm에서두물질의최적 peak 면적값을나타내어최적분석파장은 24 nm로설정하였다. He 등 (24) 에의하면 oxypaeoniflorin 및 paeoniflorin을 24 nm에서분석하였다고보고하였으며, 이와유사한경향을나타내었다. 특이성확인특이성은추출물, 불순물등이혼합되어있는시료에서분석대상물질을선택적으로측정할수있는능력을말한다. 표준용액과목단피추출물의 chromatogram을비교하여 oxypaeoniflorin과 paeoniflorin peak를확인한결과 Fig. 2와같이다른성분의간섭없이선택적으로분리되었음을확인하였다. 또한, 목단피추출물을분석하였을때표준용액의머무름시간과추출물의두물질의머무름시간이일치한것을확인하였으며, 표준용액과목단피추출물의 PDA spectrum 결과에서도동일한 spectrum을나타내었으며이를통하여본시험법의특이성을확인하였다 (Fig. 1). A 검출한계및정량한계 Oxypaeoniflorin 및 paeoniflorin의검출한계및정량한계는검량선의기울기와반응의표준편차에근거하는방법을사용하였으며다음식을이용하여확인하였다. σ: The standard deviation of the response S: The slope of the calibration curve B 결과및고찰 Oxypaeoniflorin 및 paeoniflorin 의크로마토그램목단피의두성분을동시에분석할수있는동시분석조건이요구된다. 기존의보고된분석법 (22) 을재현한결과분석시간이길고반복측정시압력의안정화시간이짧아기기의안정화가제대로이루어지지않아재현성이낮은문제점을보였다. 따라서기기조건을일부변경하여최적분석조건을확립하였다 (Table 1). HPLC를이용하여 oxypaeoniflorin 및 paeoniflorin 표준물질을분석하였을때 oxypaeoniflorin은 29 nm에서최대흡수파장을나타내었으며, paeoniflorin은 233 nm에서최대흡수파장을나타내었 Fig. 1. PDA spectrums of oxypaeoniflorin (A) and paeoniflorin (B) in STD, spiked sample, and Moutan Cortex Radicis extract.
1094 최승현 유창길 황지현 이기쁨 이영진 이부용 이옥환 A B Fig. 2. HPLC chromatograms of oxypaeoniflorin and paeoniflorin. Standard (A), Moutan Cortex Radicis extract (B). 검량선을이용한직선성확인 Oxypaeoniflorin 및 paeoniflorin 표준용액을 3.12, 6.2, 12., 2, 0, 100, 200 µg/ml 농도로단계적으로희석하여 HPLC로분석한결과, 표준검량선이 oxypaeoniflorin 및 paeoniflorin 각각 y=303x-7337, y=246x- 1113으로나타났으며상관계수 (R 2 ) 값은 oxypaeoniflorin 1.0000, paeoniflorin 0.9998로나타나우수한직선성을보였다 (Fig. 3). 정밀성및정확성농도를알고있는목단피추출물에표준용액을각각저농도 (6.2 μg/ml), 중간농도 (2 μg/ml), 고농도 (100 μg/ml) 로첨가한뒤 HPLC로분석하여분석하였을때, 각측정결과값사이의근접성을확인하여정밀성을평가하였고, 회수율을측정하여정확성을평가하였다. 정밀성의결과는상대표준편차 (RSD) 로확인하였다. Oxypaeoniflorin 및 paeoniflorin의정밀성은 Table 2와같이일간정밀성에서각각 Fig. 3. Calibration curve of oxypaeoniflorin and paeoniflorin standard solution. Table 2. Precision and accuracy of oxypaeoniflorin and paeoniflorin analysis for Moutan Cortex Radicis extract Analytes Concentration (ppm) Mean±SD (ppm) RSD (%) Recovery (%) Oxypaeoniflorin Paeoniflorin Intra-day Inter-day Intra-day Inter-day 1) Value are mean±sd in triplicate (n=3). 6.2 2.0 6.2 2.0 6.2 2.0 6.2 2.0 6.31±0.06 1) 24.61±0.23 101.6±0.32 6.24±0.20 24.8±0.17 102.11±0.81 7.00±0.16 24.1±0.1 97.10±1. 7.38±0.18 7.7±0.4 113.4±0. 0.92 0.92 0.32 3.19 0.70 0.80 2.28 0.62 1.60 2.43 2.10 1.74 100.92 98.44 101.6 99.91 98.33 102.11 112.00 98.03 97.10 118.12 97.98 97.72
목단피지표성분의분석법개선및검증 10 Table 3. Correlation coefficients of the calibration curves, and limit of detection (LOD) and limit of quantification (LOQ) of oxypaeoniflorin and paeoniflorin analysis for Moutan Cortex Radicis extract Analytes Oxypaeoniflorin Paeoniflorin Range (μg/ml) 3.13 200 3.13 200 Slope 302.8 24.0 Intercept -7336.88-1112.61 Correlation coefficient (R 2 ) 1.0000 0.9998 LOD (μg/ml) 0.23 0.2 LOQ (μg/ml) 0.71 0.77 Table 4. Content of oxypaeoniflorin and paeoniflorin in Moutan Cortex Radicis extract Compounds (mg/dry weight g) Sample Oxypaeoniflorin Paeoniflorin Moutan Cortex 6.43±0.20 1) 20.2±0.37 Radicis extract 1) Value are mean±sd in triplicate (n=3). 0.70~3.19%, 1.74~2.43% 를나타내었으며일내정밀성에서는 0.32~0.92%, 0.62~2.28% 로 % 이하의우수한정밀성을나타내었다. 정확성은회수율을측정하여나타내었다. Table 2와같이 oxypaeoniflorin 및 paeoniflorin의일간정확성은 98.33 ~102.11%, 97.72~118.12% 를나타내었으며, 일내정확성은 98.44~101.6%, 97.10~112.00% 로우수한정확성을나타내었다. 검출한계및정량한계검출한계와정량한계는 ICH 가이드라인에근거하여분석한결과 oxypaeoniflorin 및 paeoniflorin의검출한계는각각 0.23 μg/ml, 0.2 μg/ml로측정되었고, 정량한계는각각 0.71 μg/ml, 0.77 μg/ml로나타났다 (Table 3). 이상의결과를볼때목단피의 oxypaeoniflorin 및 paeoniflorin 은 HPLC를이용하여동시분석이가능하며정량분석이가능한것으로나타났다. 목단피추출물의 oxypaeoniflorin 및 paeoniflorin 함량분석확립된분석법을이용하여목단피추출물내 oxypaeoniflorin 및 paeoniflorin의함량분석결과는 Table 4와같이 oxypaeoniflorin 6.43±0.20 mg/dry weight g, paeoniflorin 20.2±0.37 mg/dry weight g의함량을가지고있는것으로분석되었다. 요 목단피의 oxypaeoniflorin 및 paeoniflorin의함량분석및원료의표준화를위하여분석법의개발및검증을실시하였다. 기존의보고된분석법을개선하여분석법을개발하고확립된분석법에대한분석법검증을실시하였다. 분석법검증은특이성, 직선성, 정확성, 정밀성, 검출한계및정량한계를통하여분석법의신뢰성을검증하였다. HPLC를이용한분석방법에서표준용액의머무름시간과목단피추출물 약 의머무름시간이일치하였으며, 동일한 spectrum을나타내는것을확인하여분석법의특이성을검증하였다. Oxypaeoniflorin 및 paeoniflorin의검량선은상관계수값이각각 1.0000, 0.9998로나타나우수한직선성을보여주어분석에적합함을확인하였다. 농도를아는시료에인위적으로저농도, 중간농도, 고농도의표준물질을첨가하여정밀성및정확성을계산하였다. Oxypaeoniflorin 및 paeoniflorin의정밀성은일간정밀성, 일내정밀성으로확인하였으며, oxypaeoniflorin 및 paeoniflorin의일간정밀성은각각 0.70~ 3.19%, 1.74~2.43% 수준으로확인되었으며, 일내정밀성은 0.32~0.92%, 0.62~2.28% 수준으로 % 이하의우수한정밀성을보였다. 정확성측정결과 oxypaeoniflorin 및 paeoniflorin의일간정확성은 98.33~102.11%, 97.72~ 118.12% 를나타내었으며, 일내정확성은 98.44~101.6%, 97.10~112.00% 수준으로우수한정확성을나타내었다. Oxypaeoniflorin 및 paeoniflorin의검출한계는각각 0.23 μg/ml, 0.2 μg/ml였고정량한계는 0.71 μg/ml, 0.77 μg/ ml로나타내어, 저농도에서도검출이가능함을확인하였다. 분석법검증결과, 확립된분석법은특이성, 직선성, 정밀성, 정확성, 검출한계및정량한계가모두우수한분석법임을검증하였다. 또한, 검증된분석법을이용하여목단피추출물시료중 oxypaeoniflorin 및 paeoniflorin의함량을분석한결과 oxypaeoniflorin 6.43±0.20 mg/dry weight g, paeoniflorin 20.2±0.37 mg/dry weight g의함량을가지고있는것으로확인되었다. 본연구결과목단피의지표성분인 oxypaeoniflorin 및 paeoniflorin의 HPLC를이용한동시분석방법이적합한분석방법임이검증되었다. REFERENCES 1. Bae KH. 2000. The medicinal plants of Korea. Kyo-Hak Publishing Co., Ltd., Seoul, Korea. p 364. 2. Kwon OG, Kim SH, Chun BY, Park CK, Son KH. 1999. Isolation of antimicrobial components from Moutan cortex. Korean J Pharmacogn 30: 340-344. 3. Kubo M, Matsuda H, Tani T, Arichi S, Kitagawa I. 1984. Studies on Moutan Cortex (Ⅶ): Inhibitory effects on histamine release from rat peritoneal mast cells in vitro. Shoyakugaku Zasshi 38: 276-278. 4. Yook CS, Lee SJ, Yoo SJ, Kim TH, Han YG, Lee SY, Moon YH, Han MW, Lee GS. 1981. Korean herbal medicine. Gyechuk Publishing Company, Seoul, Korea. p 184.. Herbal Medicine School Compilation Committee. 2012. Pharmacognosy. Dongmyeong Publishing Company, Seoul, Korea. p 23-2.
1096 최승현 유창길 황지현 이기쁨 이영진 이부용 이옥환 6. Choi YJ. 1991. Growing and using wild edible greens. Osung Publishing Co., Seoul, Korea. p 22-29. 7. Wang B, Pang Z, Zhang Q. 1994. Chemiluminescence in the study of paeonol. Chung Kuo Yao Hsueh Tsa Chih 29: 3-38. 8. Dai M, Zhi X, Peng D, Liu Q. 1999. Inhibitory effect of paeonol on experimental atherosclerosis in quails. Zhongguo Zhong Yao Za Zhi 24: 488-490. 9. Lin HC, Ding HY, Ko FN, Teng CM, Wu YC. 1999. Aggregation inhibitory activity of minor acetophenones from Paeonia species. Planta Med 6: -99. 10. Li G, Seo CS, Lee KS, Kim HJ, Chang HW, Jung JS, Song DK, Son JK. 2004. Protective constituents against sepsis in mice from the root cortex of Paeonia suffruticosa. Arch Pharmacal Res 27: 1123-1126. 11. Kim SH, Kim SA, Park MK, Kim SH, Park YD, Na HJ, Kim HM, Shin MK, Ahn KS. 2004. Paeonol inhibits anaphylactic reaction by regulating histamine and TNF-α. Int Immunopharmacol 4: 279-287. 12. You JK, Chung MJ, Kim DJ, Seo DJ, Park JH, Kim TW, Choe M. 2009. Antioxidant and tyrosinase inhibitory effects of Paeonia suffruticosa water extract. J Korean Soc Food Sci Nutr 38: 292-296. 13. Park S, Jun DW, Park CH, Jang JS, Park SK, Ko BS, Kim BJ, Choi SB. 2004. Hypoglycemic effects of crude extracts of Moutan Radicis Cortex. Korean J Food Sci Technol 36: 472-477. 14. Li Q. 1988. Advances in the pharmacology of the root bark of Paeonia suffruticosa. Zhong Cao Yao 19: 276-278. 1. Rho S, Chung HS, Kang M, Lee E, Cho C, Kim H, Park S, Kim HY, Hong M, Shin M, Bae H. 200. Inhibition of production of reactive oxygen species and gene expression profile by treatment of ethanol extract of Moutan Cortex Radicis in oxidative stressed PC12 cells. Biol Pharm Bull 28: 661-666. 16. Hsu FL, Lai CW, Cheng JT. 1997. Antihyperglycemic effects of paeoniflorin and 8-debenzoylpaeoniflorin, glucosides from the root of Paeonia lactiflora. Planta Med 63: 323-32. 17. Jeong SJ, Ahn NH, Kim YC. 1998. Hyaluronidase inhibitors from Moutan Cortex Radicis. Korean J Pharmacogn 29: 44-47. 18. Kim YH, Bae DB, Park SO, Lee SJ, Cho OH, Lee OH. 2013. Method validation for the determination of eleutherosides and β-glucan in Acanthopanax koreanum. J Korean Soc Food Sci Nutr 42: 1419-142. 19. KFDA. 2008. Guideline for standard of health functional food. Korea Food & Drug Administration, Seoul, Korea. p 1-146. 20. Fujiwara H, Tabuchi M, Yamaguchi T, Iwasaki K, Furukawa K, Sekiguchi K, Ikarashi Y, Kudo Y, Higuchi M, Saido TC, Maeda S, Takashima A, Hara M, Yaegashi N, Kase Y, Arai H. 2009. A traditional medicinal herb Paeonia suffruticosa and its active constituent 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose have potent anti-aggregation effects on Alzheimer s amyloid β proteins in vitro and in vivo. J Neurochem 109: 1648-167. 21. Kim HG, Park G, Piao Y, Kang MS, Pak YK, Hong SP, Oh MS. 2014. Effects of the root bark of Paeonia suffruticosa on mitochondria-mediated neuroprotection in an MPTP-induced model of Parkinson s disease. Food Chem Toxicol 6: 293-300. 22. Xu SJ, Yang L, Zeng X, Zhang M, Wang ZT. 2006. Characterization of compounds in the Chinese herbal drug Mu-Dan- Pi by liquid chromatography coupled to electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 20: 327-3288. 23. Deng XM, Yu JY, Ding MJ, Zhao M, Xue XY, Che CT, Wang SM, Zhao B, Meng J. 2016. Liquid chromatographydiode array detector-electrospray mass spectrometry and principal components analyses of raw and processed Moutan Cortex. Pharmacogn Mag 12: 0-6. 24. He C, Peng B, Dan Y, Peng Y, Xiao P. 2014. Chemical taxonomy of tree peony species from China based on root cortex metabolic fingerprinting. Phytochemistry 107: 69-79. 2. Xiao C, Wu M, Chen Y, Zhang Y, Zhao X, Zheng X. 201. Revealing metabolomic variations in Cortex Moutan from different root parts using HPLC-MS method. Phytochem Anal 26: 86-93. 26. ICH Harmonised Tripartite Guideline. 200. Validation of analytical procedures: text and methodology Q2 (R1). International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Geneva, Switzerland. p 1-13.