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pissn 19-1153 / eissn 465-93 J. Food Hyg. Saf. Vol. 34, No. 1, pp. 5~57 (019) https://doi.org

1 pissn / eissn J. Food Hyg. Saf. Vol. 34, No. 1, pp. 5~57 (019) Journal of Food Hygiene and Safety Available online at 참당귀지상부추출물의지표성분 decursin 의분석법개발및검증 김희연 * 이기연 김태희 박아름 노희선 김시창 안문섭강원도농업기술원농식품연구소 Development and Validation of Analytical Method for Decursin in Aerial Parts of Angelica gigas Nakai Extract Hee-Yeon Kim*, Ki-Yeon Lee, Tae-Hee Kim, A-Reum Park, Hee-Sun Noh, Si-Chang Kim, and Mun-Seob Ahn Agriproduct Processing Experiment Station, Gangwon-do Agricultural Research and Experiment Services, Chuncheon, Korea (Received November 9, 018/Revised January 4, 019/Accepted February 1, 019) ABSTRACT - Method development and validation of decursin for the standardization of Angelica gigas Nakai as a functional ingredient and health food were accomplished. The quantitative determination method of decursin as a marker compound of aerial parts of Angelica gigas Nakai extract (AAGE) was optimized by HPLC analysis using a C18 column (3 150 mm, 3 µm) with 0.1% TFA in water and acetonitrile as the mobile phase at a flow rate of 0.5 ml/ min and detection wavelength of 330 nm. The HPLC/PDA method was applied successfully to quantification of the marker compound in AAGE after validation of the method with linearity, accuracy, and precision. The method showed high linearity in the calibration curve at a coefficient of correlation (R ) of and the limit of detection and limit of quantitation were µg/ml and µg/ml, respectively. Relative standard deviation (RSD) values of data from intra- and inter-day precision were less than 1.10% and 1.13%, respectively. Recovery of decursin at 0.5, 1, 5 and 10 µg/ml were 9.38 ~ %. These results suggest that the developed HPLC method is very useful for the determination of marker compound in AAGE to develop a health functional material. Key words : Angelica gigas Nakai, Decursin, Validation, HPLC 참당귀는산형과 (Umbellifera) Angelica 속에속하는다년생초본으로우리나라와중국동북부지역에서자생하며건조된뿌리가생약재로사용된다. 국내에서는강원도평창, 경북봉화등지에서재배되며개화기전의뿌리가식용및약용으로사용된다 1). 참당귀는약성이따뜻하고무독한생약재로부인과질환및고혈압, 빈혈, 어혈등에효과가있으며면역강화, 항암및항산화효과, 간질환 ) 3) 4,5) 및혈류개선효과 6,7) 가있는것으로알려져있다. 참당귀에관한연구로당뇨유발모델에서의참당귀추출물의당뇨개선효과 8), 참당귀잎, 줄기, 뿌리추출물의생리활성효과, 발효당귀의생리활성물질및효과등이보고되었다. 9) 10) 참당귀의약효성분으로 coumarin 계열의 decursin, decursinol, decursinol angelate, nodakenin 등이함유되어있으며그중주성분은 pyrancoumarin 계열의 decursin과 *Correspondence to: Hee Yeon Kim, Agriproduct Processing Experiment Station, Gangwon-do Agricultural Research and Experiment Services, Chuncheon, Gangwon-do 403, Korea Tel : , Fax : heeya80@korea.kr decursinol angelate이다. 참당귀의주요약효성분중 11,1) decursin의전립선암치료제개발에관한연구와 decursin 13) 유도체의아토피피부염및 ovalbumin에의한천식의폐염증유발억제효과에관한연구가보고되었다. 참당귀 14) 에서분리된 decursin의인지개선효과가확인되었으며 15) 참당귀에서분리된 decursin을비롯한 coumarin계화합물의우수한항염효과가확인되었다. 또한, decursin과 decursinol 16) angelate는 lipid droplet의축적을억제시켜동맥경화증과같은혈액순환장애의예방및치료효과가있으며 bovine ) 망막세포의 aldose reductase를억제시켜망막의 sorbitol 축적을방지하여당뇨로인한합병증을예방할수있다고보고되었다. 참당귀는생약재로쓰이는뿌리의생리활성 17) 및유효성분분석에관한연구가대부분이며참당귀지상부에관한연구로당뇨모델에서참당귀잎추출물의항당뇨효과 18) 와참당귀지상부추출물의생리활성 19) 에관한연구가보고되었으나유효성분및기능성소재활용방안에관한연구는미흡한실정이다. 참당귀의유효성분인 decursin이뿌리뿐만아니라지상부에도함유되어있는것으로확인되었으며이는기능성식품소재로서참당귀지 9) 5

2 Development and Validation of Analytical Method for Decursin in Aerial Parts of Angelica gigas Nakai Extract 53 상부의활용가치가높다고판단된다. 지표성분은원재료에존재하는해당성분의농도와효능간의상관관계가불분명할경우원재료를대표하는성분으로건강기능식품개발에있어서원료의표준화를위한지표성분분석법의설정과정및설정된분석법의과학적인검증과정이요구된다. 따라서, 본연구에서는건 0) 강기능성식품소재개발을목적으로참당귀지상부추출물의표준화를위하여 decursin을지표성분으로선정하고 HPLC분석을통하여 decursin의분석법확립및유효성검정을수행하였다. Materials and Methods 참당귀지상부추출물조제및 HPLC 분석본실험에사용된참당귀지상부는 017년강원도평창에서재배되었으며수확후이물질을제거하고건조한다음분쇄하여추출물조제시료로사용하였다. 참당귀지상부건조시료를 60% 에탄올로추출하여동결건조하였을시동결건조된시료의수분흡수로인하여균일한추출분말이생산되지않아분무건조를진행하였다. 참당귀지상부건체시료 5 kg에 60% 에탄올 50 L를첨가하여 70 C에서 4시간동안순환추출하고 10 μm 사이즈필터 o 에여과시켰다. 여과된추출액을 70 C에서최종추출액의 o 양이 5 L가될때까지농축하였으며농축액에 304 g의덱스트린을첨가한다음분무건조하여추출분말을얻었다. 참당귀지상부건조시료에대한분무건조추출분말의수율은평균약 14% 이었다. 분무건조로조제된 3 lot의추출분말을각각 0.1 g씩정확하게칭량하고메탄올 100 ml 을가하여최종농도가 1 mg/ml이되게재용해한다음 0.45 μm membrane filter에통과시켜 HPLC 분석용시료로사용하였으며모든분석은 3반복실시하였다. 표준용액의조제 Decursin 표준품 1 mg에 10 ml 메탄올을첨가하고용해시켜 100 μg/ml의농도로표준원액을조제하였다. 이표준원액을단계적으로희석하여 0.1, 0.5, 1, 5, 10 μg/ml 농도의표준용액을조제하여 HPLC 분석에사용하였으며분석된 peak 면적값에 0.97을곱하여표준품의순도를보정하였다. 분석시약본실험에사용된 decursin ( 97%, HPLC) 은 Sigma Co. (St Louis, MO, USA) 의제품을사용하였고추출및 HPLC 분석에사용된용매는 Merck Co. (Kenilworth, NJ, USA) 의 HPLC 등급을사용하였다. HPLC 분석참당귀지상부추출액및 decursin 표준용액의분석은 Table 1. HPLC analysis condition of decursin Classification Condition Instrument Nano Space SI- (Shiseido, Japan) Column Cadenza CD-C 18 (150 3 mm, 3 µm) Column temp o C 50 Mobile phase Eluent A: 0.1% TFA Eluent B: acetonitrile Injection volume 5 µl Detector UV 330 nm Run time 5 min Gradient table Time (min) Flow rate (µl) %A %B Initial Nano Space SI- HPLC (Shiseido, Japan) 를사용하였다. 컬럼은 Cadenza CD-C 18 (150 3mm, 3μm, Imtakt, Kyoto, Japan) 을사용하였고, 0.1% TFA와 100% acetonitrile을이동상으로사용하여시료주입량 5μL, 컬럼온도 50 C, 유속 500 μl/min으로 5분동안분석하였 o 다 (Table 1). 분석법의검증 (method validation) 참당귀지상부추출물의기능성식품원료개발을위한벨리데이션은 의약품등분석법의벨리데이션가이드 ( 식품의약품안전처 ) 를바탕으로 decursin의농도별표준용액을 HPLC로분석하여특이성 (specificity), 직선성 (linearity), 검출한계 (limit of detection, LOD), 정량한계 (limit of quantification, LOQ), 정밀성 (precision), 정확성 (accuracy) 을측정하였다. 특이성은시료내에존재할수있는불순물, 분해물등의영향을받지않고해당성분을특이적으로측정할수있는정도를나타내며특이성검정을위하여 decursin의농도별표준용액과참당귀지상부추출액을 HPLC로분석하고측정되는머무름시간과크로마토그램을비교하였다. 직선성은 decursin 표준용액의농도에비례하여직선적인측정값을얻어낼수있는능력으로농도별표준용액의 peak 면적과농도간회귀분석에의한표준검량선을작성하고산출된검량선의결정계수 (coefficient of determination, R ) 를통하여직선성을확인하였다. Decursin 표준용액의검량선에서기울기와 y절편의표준편차를구하고아래의식에근거하여정량한계 (LOQ) 와검출한계 (LOD) 를산출하

3 54 Hee-Yeon Kim, Ki-Yeon Lee, Tae-Hee Kim, A-Reum Park, Hee-Sun Noh, Si-Chang Kim, and Mun-Seob Ahn 였다. (LOQ = 10 σ/s, LOD = 3.3 σ/s, σ : y절편의표준편차, S : 검량선의기울기 ) 정확성은일내분석 (intra-day) 과일간분석 (inter-day) 을통하여측정하였다. 정확성은농도별표준용액의일내분석과일간분석을통하여측정된값 (measured value) 이참값 (true value) 에근접한정도를백분율로나타내었으며일내분석은농도별표준용액을 1일 3구간에서분석하였고일간분석은일내분석과정을 3일동안반복분석하여변이성을확인하였다. 일내분석및일간분석시각구간마다 3농도 3회반복측정하였다. 정밀성은일내분석과일간분석에서얻어진표준용액의평균값과표준편차를근거로상대표준편차를측정하여평가하였으며상대표준편차 (relative standard deviation, RSD) 는표준편차를평균으로나누어백분율로나타내었다. 회수율은농도별로조제된지표성분과함량이확인된시료를혼합후분석하여회수되는지표성분의양으로측정하였다. Decursin 표준정량곡선에서직선성이확인된 0.5, 1, 5, 10 μg/ml 농도의표준용액에참당귀추출물을혼합하여 HPLC로 3회반복분석하고측정된값으로부터회수율과상대표준편차를산출하였다. 토그램상에서표준용액및참당귀지상부추출물의 decursin peak 머무름시간은 15.7분대로동일하였으며 spectrum도일치하는것으로본분석법의특이성을검정하였다 (Fig. 1). 직선성, 정량한계, 검출한계 Decursin 표준용액을 0.1, 0.5, 1, 5, 10 μg/ml로희석한다음 HPLC로분석하여작성된표준검량선은 y = 31,340x 19,638로나타났으며상관계수 (R ) 는 으로매우 우수한직선성을나타내었다 (Fig. ). 정량한계 (LOQ) 와검출한계 (LOD) 를산출하기위하여 decursin 표준용액을 0.05, 0.05, 0.1, 0.5, 1 μg/ml로희석한다음 HPLC로분석하여작성된표준검량선은 y = 74,355x + 4,400.1로나타났으며상관계수 (R ) 는 이었다. 이표준검량선으로부터 Results and Discussion 특이성 HPLC 분석결과, 참당귀지상부추출물내에혼합되어있을가능성이있는다른성분들로부터 decursin만선택적으로측정되었으며다른물질의간섭없이성분이분리되는것을확인하였다. HPLC분석을통하여얻어진크로마 Fig.. PDA spectrums of blank (A), decursin (B) and AAGE (C). AAGE : aerial parts of Angelica gigas Nakai extract. Fig. 1. HPLC Chromatograms of blank (A), decursin (B) and AAGE (C). AAGE : aerial parts of Angelica gigas Nakai extract. Fig. 3. Calibration curve of decursin standard solution.

4 Development and Validation of Analytical Method for Decursin in Aerial Parts of Angelica gigas Nakai Extract 55 Table. Correlation coefficients of the calibration curves, and limit of detection (LOD) and limit of quantification (LOQ) of decursin Concentration range LOD LOQ Slope Intercept Correlation coefficient(r ) 0.1 ~ 10 31,340-19, Table 3. Intra- and inter-day precision and accuracy of decursin Concentration.5 Intra-day ) Inter-day 3) ) Relative standard deviation. ) Intra-day : tree times per day. 3) Inter-day : one time analysis of decursin per day for 3 days. 4) Each data was obtained by triple analysis (n=3). Mean± SD.54 ± ) 4.68 ± ± ± ± ± 0.05 Accuracy (%) RSD 1) (%) 산출된기울기와 y절편의표준편차로계산된정량한계 (LOQ) 는 μg/ml이었고검출한계 (LOD) 는 μg/ml인것으로나타났다 (Table ). 정량한계는검체중정확성과정밀성을가진정량값으로나타낼수있는분석대상물질의최저농도를의미하며검출한계는검체내존재하는분석대상물질의검출가능한최저농도를의미한다. 따라서 1) 참당귀지상부추출물에함유되어있는 decursin의함량은 μg/ml 농도수준까지정량이가능하며 μg/ml 농도수준까지검출이가능하다. Yang등의연구에서 ) decursin의정량한계는 0.08 μg/ml, 검출한계는 0.03 μg/ml 수준인것과비교하였을때본연구의 decursin 분석법이더낮은농도의미량성분까지정량및검출이가능하다고판단된다. 정확성, 정밀성평가정확성은농도별표준용액의일내분석과일간분석을통하여측정된값 (measured value) 이참값 (true value) 에근접한정도를백분율로나타내었으며측정된값들로부터상대표준편차 (RSD) 를구하여정밀성을검정하였다. 일간분석의정확성및상대표준편차는각각 ~ %, 0.53 ~ 1.10% 이었으며일내분석의정확성및상대표준편차는각각 97.3 ~ %, 0.6 ~ 1.15% 이었다 (Table 3). 각각의분석에서의상대표준편차는모두.0% 이하로나타나 decursin 분석법의정확성및정밀성이있음을알수있었다. Table 4. Recovery and relative standard deviation of measured amount for decursin Concentration Recovery (%) RSD ) (%) 1) ± ) ± ± ± ) Concerntration of decursin standard. ) Relative standard deviation. 3) Each data was obtained by triple analysis (n=3) Table 5. Amount of decursin in aerial parts of Angelica gigas Nakai extract Lot No. Concentrations (%, mean ± SD) ± ) ± ± ) Each data was obtained by triple analysis (n=3). 용액의검출농도차이를구하고이값을참당귀지상부추출물만분석하여검출된농도값으로나누어측정하였다. 표준품농도별지표성분의회수율은 9.38 ~ % 의범위였으며분석오차 10% 이내로측정되었다. 지표성분회수율에대한상대표준편차는모두 0.5% 이내인것으로나타나 decursin 분석법의정확성을확인할수있었다 (Table 4). 회수율측정회수율은참당귀지상부추출물이혼합된농도별표준용액의검출농도와추출물이혼합되지않은농도별표준 참당귀지상부추출물의 decursin 함량본연구의분석법검증을통하여 decursin에대한 HPLC 분석법이참당귀지상부추출물에함유되어있는지표성분

5 56 Hee-Yeon Kim, Ki-Yeon Lee, Tae-Hee Kim, A-Reum Park, Hee-Sun Noh, Si-Chang Kim, and Mun-Seob Ahn 의정량에적용할수있는특이성, 우수한직선성, 정확성및정밀성등이있음을확인하였다. 본분석법으로참당귀지상부분무건조분말시료의 decursin 평균함량은 0.359% 이었으며, 기준규격을 80 ~ 10% 로설정하였을때기준 3) 값은 0.87 ~ 0.431% 인것을확인할수있었다 (Table 5). Acknowledgement 본연구는중소벤처기업부와한국산업기술진흥원의 지역주력산업육성사업 (P0007) 로수행된연구결과입니다. 국문요약 본연구에서는건강기능성식품소재개발을목적으로참당귀지상부추출물의표준화를위하여 decursin을지표성분으로선정하고 HPLC분석을통하여 decursin의분석법을확립하고유효성검정을수행하였다. 유효성검정결과, decorsin 표준용액과참당귀지상부추출물의 retention time 및 spectrum이일치하는특이성을확인하였다. 표준검량선의상관계수 (R ) 는 로매우우수한직선성을 나타내었으며정량한계 (LOQ) 는 μg/ml이었고검출한계 (LOD) 는 μg/ml이었다. Decursin의 intra-day 분석에서정확성및상대표준편차는각각 ~ %, 0.53 ~ 1.10%, inter-day 분석에서정확성및상대표준편차는각각 97.3 ~ %, 0.6 ~ 1.15% 인것으로나타나본시험법의정확성및정밀성을확인할수있었다. 표준품농도별지표성분의회수율은 9.38 ~ % 의범위였으며분석오차 10% 이내로측정되었다. 이는본연구에서수행된 HPLC 분석법이참당귀지상부추출물의지표성분인 decursin을분석하고표준화하는데적합한시험법임을의미한다. Reference 1. Cho, M.G., Bang, J.K., Chae, Y.A.: Comparison of volatile compounds in plant parts of Angelica gigas Nakai and A. acutiloba Kitagawa. Korean J. Med. Crop Sci., 11, (003).. Son, C.Y., Beak, I.H., Song, G.Y., Kang, J.S., Kwon, K.I.: Pharmacological effect of decursin and decursinol angelate from Angelica gigas Nakai. Yakhak Hoeji, 53, (009). 3. Kim, S.B., Kim, Y.H., Lee, C.W., Park, S.M., Ahn, K.S., Kim, I.H., Kim, H.M.: Characteristic immunostimulation by angelan isolated from Angelica gigas Nakai. Immunopharmacol, 40, (1998). 4. Kim, E.Y., Baik, I.H., Kim, J.H., Kim, S.R., Rhyu, M.R.: Screening of the antioxidant activity of some medicinal plants. Korean J. Food Sci. Technol., 36, (004). 5. Park, K.W., Choi, S.R., Shon, M.E., Jeong, I.Y., Kang, K.S., Lee, S.T., Shim, K.H., Seo, K.I.: Cytotoxic effects of decursin from Angelica gigas Nakai in human cancer cells. J. Korean Soc. Food Sci. Nutr., 36, (007). 6. Oh, S.H., Cha, Y.S., Choi, D.S.: Effects of Angelica gigas Nakai diet on lipid metabolism, alcohol metabolism and liver function of rats administered with chronic ethanol. J. Korean Soc. Agric. Chem. Biotechnol., 4, 9-33 (1999). 7. Kim, C.H., Kwon, M.C., Han, H.G., Na, C.S., Kwak, H.G., Choi, G.P., Park, U.Y., Lee, H.Y.: Skin-whitening and UVprotective effects of Angelica gigas Nakai extracts on ultra high pressure extraction process. Korean J. Medicinal Crop Sci., 16, (008). 8. Park, M.J., Kang, S.J., Kim, A.J.: Hypoglycemic effect of Angelica gigas Nakai extract in streptozotocin-induced diabetic rats. Korean J. Food Nutr.,, (009). 9. Heo, J.S., Cha, J.Y., Kim, H.W., Ahn, H.Y., Eom, K.E., Heo, S.J., Cho, Y.S.: Bioactive materials and biological activity in the extracts of leaf, stem mixture and root from Angelica gigas Nakai. J. Life Sci., 0, (010). 10. Cha, J.Y., Kim, H.W., Heo, J.S., Ahn, H.Y., Eom, K.E., Heo, S.J., Cho, Y.S.: Ingredients analysis and biological activity of fermented Angelica gigas Nakai by Mold. J. Life Sci., 0, (010). 11. Kim, K.M., Jung, J.Y., Hwang, S.W., Kim, M.J., Kang, J.S.: Isolation and purification of decursin and decusinol angelate in Angelica gigas Nakai. J. Korean Soc. Food Sci. Nutr., 38, (009). 1. Lee, S.H., Kang, S.S., Shin, K.H.: Coumarins and a pyrimidine from Angelica gigas roots. Nat. Prod. Sci., 8, (00). 13. Yim, D., Singh, R.P., Agarwal, C., Lee, S., Chi, H., Agarwal, R.: A novel anticancer agent, decursin, induces G 1 arrest and apoptosis in human prostate carcinoma cells. Cancer Res., 65(3), (005). 14. Yang, E.J., Song, G.Y., Lee, J.S., Yun, C.Y., Kim, I.S.: A novel (S)-(+)-Decursin derivative,(s)-(+)-3-(3,4-dihydroxyphenyl dihydroxyphen dihydroxyphenyl)-acrylic acid,-dimethyl- 8-oxo-3,4-dihydro-H, 8Hpyrano[3,-g]-chromen-3-ylester, In hibits ovalbumin-induced lung inflammation in a mouse model of asthma. Biol. Pharm. Bull., 3, (009). 15. Kang, S.Y., Lee, K.Y., Sung, S.H., Park, M.J., Kim, Y.C.: Coumarins isolated from Angelica gigas inhibit acetylcholinesterase: Structure-activity relationships. J. Nat. Prod., 64, (001). 16. Jung, Y.J., Choi, J.H., Jeong, W.S., Song, Y.S., Kang, J.S., Bi, K., Kim, M.J.: Anti-inflammatory activities of coumarins isolated from Angelica gigas Nakai on LPS-stimulated RAW 64.7 Cells. J. Food Sci. Nutr., 14, (009). 17. Jung, M.H., Lee, S.H., Ahn, E.M., Lee, Y.M.: Decursin and decursinol angelate inhibit VEGF-induced angiogenesis via suppression of the VEGFR--signaling pathway. Carcinogenesis., 30, (009). 18. Jeong, H.J., Hong, S.Y,, Lee, K.Y., Lee, J.H., Lim, S.H., Heo,

6 Development and Validation of Analytical Method for Decursin in Aerial Parts of Angelica gigas Nakai Extract 57 N.K., Kim, H.Y.: Diabetic Effect Of Angelica gigas Nakai Leaf Extracts in Diabetes Mellitus Type? Model Mice by High Fat-fed and Streptozotocin-treated Rats. J. Agricultural, Life and Environ. Sci., 7, 6-14 (015). 19. Lee, K.Y., Hong, S.Y., Jeong, H.J., Lee, J.H., Lim, S.H., Heo, N.K., Kim H.Y.: Biological activities of extract from aerial parts of Angelica gigas Nakai. J. Agricultural, Life and Environ. Sci., 7, 15- (015). 0. Kim, Y.J., Han, S.H., Jeon, J.Y., Hwang, M.H., Im, Y.J., Chae, S.W., Kim, M.G.: Method development of ellagic acid as marker compound for standardization of gochang bokbunja (Rubus coreanus Miquel) as functional ingredient. J. Korean Soc. Food Sci. Nutr., 41, (01). 1. Kwon, J.G., Sed, C., Choi, Y.H,, Choi, C.W., Kim, J.K., Jeong, W., Lee, J.E., O, K.H., Hong, S.S.: Validation of method determining coixol in Coix lachrymal-jobi var. mayuen roots extract. J. Korean Soc. Food Sci. Nutr., 46, (017).. Yang, H.J., Weon, J.B., Ma, J.Y., Ma, C.J.: Simultaneous determination of six bioactive components in Guibi-tang by HPLC-DAD. Kor. J. Pharmacogn., 41, (010). 3. KFDA.: Guideline of standard of functional ingredient for health functional food development. Korea Food and Drug Administration, Korea. pp (008).

한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과

한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과 - 2 - - 3 - KP 11 Osterici seu Notopterygii Radix et Rhizoma Ostericum koreanum Maximowicz, Notopterygium incisum Ting, Notopterygium