Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

Improvement and Validation of an Analytical Method for Quercetin-3-𝑜-gentiobioside and Isoquercitrin in Abelmoschus esculentus L. Moench

오크라 분말의 Quercetin-3-𝑜-Gentiobioside 및 Isoquercitrin의 분석법 개선 및 검증

  • Han, Xionggao (Department of Food Biotechnology and Environmental Science, Kangwon National University) ;
  • Choi, Sun-Il (Department of Food Biotechnology and Environmental Science, Kangwon National University) ;
  • Men, Xiao (Department of Food Biotechnology and Environmental Science, Kangwon National University) ;
  • Lee, Se-jeong (Department of Food Biotechnology and Environmental Science, Kangwon National University) ;
  • Jin, Heegu (Department of Food Science and Biotechnology, College of Life Science, CHA University) ;
  • Oh, Hyun-Ji (Department of Food Science and Biotechnology, College of Life Science, CHA University) ;
  • Cho, Sehaeng (Syspang Co. Ltd) ;
  • Lee, Boo-Yong (Department of Food Science and Biotechnology, College of Life Science, CHA University) ;
  • Lee, Ok-Hwan (Department of Food Biotechnology and Environmental Science, Kangwon National University)
  • 한웅호 (강원대학교 식품환경융합학과) ;
  • 최선일 (강원대학교 식품환경융합학과) ;
  • 문효 (강원대학교 식품환경융합학과) ;
  • 이세정 (강원대학교 식품환경융합학과) ;
  • 진희구 (차의과학대학교 식품생명공학과) ;
  • 오현지 (차의과학대학교 식품생명공학과) ;
  • 조세행 ((주)씨스팡) ;
  • 이부용 (차의과학대학교 식품생명공학과) ;
  • 이옥환 (강원대학교 식품환경융합학과)
  • Received : 2022.02.25
  • Accepted : 2022.03.30
  • Published : 2022.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

This study aimed to investigate the validation and modify the analytical method to determine quercetin-3-𝑜-gentiobioside and isoquercitrin in Abelmoschus esculentus L. Moench for the standardization of ingredients in development of functional health products. The analytical method was validated based on the ICH (International Conference for Harmonization) guidelines to verify the reliability and validity there of on the specificity, linearity, accuracy, precision, detection limit and quantification limit. For the HPLC analysis method, the peak retention time of the index component of the standard solution and the peak retention time of the index component of A. esculentus L. Moench powder sample were consistent with the spectra thereof, confirming the specificity. The calibration curves of quercetin-3-𝑜-gentiobioside and isoquercitrin showed a linearity with a near-one correlation coefficient (0.9999 and 0.9999), indicating the high suitability thereof for the analysis. A. esculentus L. Moench powder sample of a known concentration were prepared with low, medium, and high concentrations of standard substances and were calculated for the precision and accuracy. The precision of quercetin-3-𝑜-gentiobioside and isoquercitrin was confirmed for intra-day and daily. As a result, the intra-day precision was found to be 0.50-1.48% and 0.77-2.87%, and the daily precision to be 0.07-3.37% and 0.58-1.37%, implying an excellent precision at level below 5%. As a result of accuracy measurement, the intra-day accuracy of quercetin-3-𝑜-gentiobioside and isoquercitrin was found to be 104.87-109.64% and the daily accuracy thereof was found to be 106.85-109.06%, reflecting high level of accuracy. The detection limits of quercetin-3-𝑜-gentiobioside and isoquercitrin were 0.24 ㎍/mL and 0.16 ㎍/mL, respectively, whereas the quantitation limits were 0.71 ㎍/mL and 0.49 ㎍/mL, confirming that detection was valid at the low concentrations as well. From the analysis, the established analytical method was proven to be excellent with high level of results from the verification on the specificity, linearity, precision, accuracy, detection limit and quantitation limit thereof. In addition, as a result of analyzing the content of A. esculentus L. Moench powder samples using a validated analytical method, quercetin-3-𝑜-gentiobioside was analyzed to contain 1.49±0.01 mg/dry weight g, while isoquercitrin contained 1.39±0.01 mg/dry weight g. The study was conducted to verify that the simultaneous analysis on quercetin-3-𝑜-gentiobioside and isoquercitrin, the indicators of A. esculentus L. Moench, is a scientifically reliable and suitable analytical method.

본 연구에서는 오크라를 이용하여 건강기능식품 개발 시원료의 표준화를 위하여 HPLC-PDA를 이용하여 지표성분 quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 기존 보고된 분석법을 개선하고 분석법에 대한 유효성 검증을 실시하였다. 분석법의 유효성 검증은 ICH 가이드라인에 근거하여 특이성, 직선성, 정확성, 정밀성, 검출한계 및 정량한계를 통해 신뢰성 및 타당성을 검증하였다. HPLC를 이용한 분석방법에서 표준용액의 지표성분 피크 머무름 시간과 오크라 분말 시료의 지표성분 피크 머무름 시간 및 spectrum의 확인결과 모두 일치하므로 특이성을 확인하였다. Quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 검량선은 1에 가까운 높은 상관계수 값(0.9999, 0.9999)으로 우수한 직선성을 확인할 수 있었으며 분석에 적합함을 알 수 있었다. 농도를 알고 있는 오크라 분말 시료에 표준물질을 저, 중, 고농도로 제조한 후 첨가하여 정밀성 및 정확성을 계산하였다. Quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 정밀성은 일내, 일간 정밀성으로 확인하였으며, quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 일내 정밀성은 각각 0.50-1.48%, 0.77-2.82% 수준으로 확인되었으며, 일간 정밀성은 0.07-3.37%, 0.58-1.37% 수준으로 5% 이하의 우수한 정밀성을 보였다. 정확성 측정결과 quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 일내 정확성은 104.87-109.64%, 108.50-109.70%를 나타내었으며, 일간 정확성은 106.69-111.08%, 106.85-109.06% 수준으로 우수한 정확성을 나타내었다. Quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 검출한계는 각각 0.24 ㎍/mL, 0.16 ㎍/mL이었고 정량한계는 0.71 ㎍/mL, 0.49 ㎍/mL로 나타내어, 낮은 농도에서도 검출이 가능함을 확인하였다. 확립된 분석법은 특이성, 직선성, 정밀성, 정확성, 검출한계 및 정량한계에 대한 분석법 검증결과가 모두 우수한 분석법임을 증명하였다. 또한 검증된 분석법을 이용하여 오크라 분말 시료 중 quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 함량분석 결과, quercetin-3-𝑜-gentiobioside은 1.49±0.01 mg/dry weight g, isoquercitrin은 1.39±0.01 mg/dry weight g의 함량을 함유하고 있는 것으로 분석되었다. 본 연구는 HPLC-PDA를 이용한 오크라의 지표성분인 quercetin-3-𝑜-gentiobioside 및 isoquercitrin의 동시 분석방법이 과학적으로 신뢰성이 있는 적합한 분석방법임이 검증되었다.

Keywords

Acknowledgement

이 논문은 한국연구재단의 재원(NRF-2017R1D1A3B06028469) 및 2021년도 교육부의 재원(NRF-2021R1A6A1A03044242)과 한국연구재단의 4단계 두뇌한국21 사업(4단계 BK21 사업)(4299990913942)의 지원으로 수행되었으며 이에 감사드립니다.

References

  1. Panneerselvam, K., Ramachandran, S., Sabitha, V., Naveen, K.R., Antidiabetic and antihyperlipidemic potential of Abelmoschus esculentus (L.) Moench. in streptozotocin-induced diabetic rats. J. Pharm. Bioallied Sci., 3, 397-402 (2011). https://doi.org/10.4103/0975-7406.84447
  2. Xia, F., Zhong, Y., Li, M., Chang, Q., Liao, Y., Liu, X., Pan, R., Antioxidant and Anti-Fatigue Constituents of Okra. Nutrients, 7, 8846-8858 (2015). https://doi.org/10.3390/nu7105435
  3. Yuan, K., Liao, H., Dong, W., Shi, X., Liu, H., Analysis and comparison of the active components and antioxidant activities of extracts from Abelmoschus esculentus L. Pharmacogn. Mag., 8, 156-161 (2012). https://doi.org/10.4103/0973-1296.96570
  4. Graham, J.O., Agbenorhevi, J.K., Kpodo, F.M., Total phenol content and antioxidant activity of okra seeds from different genotypes. Am. J. Food Nutr., 5, 90-94 (2017). https://doi.org/10.12691/ajfn-5-3-2
  5. Karim, M., Islam, M., Sarkar, S., Murugan, A., Makky, E., Rashid, S., Yusoff, M., Anti-amylolytic activity of fresh and cooked okra (Hibiscus esculentus L.) pod extract. Biocatal. Agric. Biotechnol., 3, 373-377 (2014). https://doi.org/10.1016/j.bcab.2014.07.006
  6. Tongjaroenbuangam, W., Ruksee, N., Chantiratikul, P., Pakdeenarong, N., Kongbuntad, W., Govitrapong, P., Neuroprotective effects of quercetin, rutin and okra (Abelmoschus esculentus Linn.) in dexamethasone-treated mice. Neurochem. Int., 59, 677-685 (2011). https://doi.org/10.1016/j.neuint.2011.06.014
  7. Daliu, P., Annunziata, G., Tenore, G.C., Santini, A., (2020). Abscisic acid identification in Okra, Abelmoschus esculentus L.(Moench): Perspective nutraceutical use for the treatment of diabetes. Nat. Prod. Res., 34(1), 3-9 (2020). https://doi.org/10.1080/14786419.2019.1637874
  8. Arapitsas, P., (2008). Identification and quantification of polyphenolic compounds from okra seeds and skins. Food Chem., 110(4), 1041-1045 (2008). https://doi.org/10.1016/j.foodchem.2008.03.014
  9. Xia, F., Zhong, Y., Li, M., Chang, Q., Liao, Y., Liu, X., Pan, R., Antioxidant and anti-fatigue constituents of okra. Nutrients, 7(10), 8846-8858 (2015). https://doi.org/10.3390/nu7105435
  10. Wu, D. T., Nie, X. R., Shen, D. D., Li, H. Y., Zhao, L., Zhang, Q., Qin, W., Phenolic compounds, antioxidant activities, and inhibitory effects on digestive enzymes of different cultivars of okra (Abelmoschus esculentus). Molecules, 25(6), 1276 (2020). https://doi.org/10.3390/molecules25061276
  11. Xia, F., Li, C., Zhao, N., Li, H., Chang, Q., Liu, X., Pan, R., Rapid determination of active compounds and antioxidant activity of okra seeds using Fourier transform near infrared (FT-NIR) spectroscopy. Molecules, 23(3), 550 (2018). https://doi.org/10.3390/molecules23030550
  12. Kim Y.H., Bae D.B., Park S.O., Lee S.H., Cho O.H., Lee O.H.. Method validation for the determination of eleutherosides and β-glucan in Acanthopanax koreanum. J. Korean Soc. Food Sci. Nutr., 42, 1419-1425 (2013). https://doi.org/10.3746/JKFN.2013.42.9.1419
  13. KFDA. 2008. Guideline for standard of health functional food. Korea Food & Drug Administration, Korea. p 1-146.
  14. Lin, Y., Lu, M.F., Liao, H.B., Li, Y.X., Han, W., Yuan, K., Content determination of the flavonoids in the different parts and different species of Abelmoschus esculentus L. by reversed phase-high performance liquid chromatograph and colorimetric method. Pharmacogn. Mag., 10(39), 278 (2014). https://doi.org/10.4103/0973-1296.137368
  15. Lu, Y., Demleitner, M.F., Song, L., Rychlik, M., Huang, D., Oligomeric proanthocyanidins are the active compounds in Abelmoschus esculentus Moench for its α-amylase and α-glucosidase inhibition activity. J. Funct. Foods, 20, 463-471 (2016). https://doi.org/10.1016/j.jff.2015.10.037
  16. Romdhane, M.H., Chahdoura, H., Barros, L., Dias, M.I., Correa, R.G., Morales, P., Ferreira, I.C., Chemical composition, nutritional value, and biological evaluation of Tunisian okra pods (Abelmoschus esculentus L. Moench). Molecules, 25(20), 4739 (2020). https://doi.org/10.3390/molecules25204739
  17. Sami, R., Alshehry, G., Ma, Y., Abdelazez, A., Benajiba, N.J.J.O.F., Evaluation of some specific components existences in okra (Abelmoschus esculentus L.(Moench)) cultivated from different areas. J. Food Nutr. Res., 7, 155-161 (2019).
  18. ICH Harmonised Tripartite Guideline. 2005. Validation of analytical procedures: text and methodology Q2(R1). International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use, p 1-13.
  19. Lee, J., Park, J., Lee, J., Suh, H. J., Lee, C., Optimization of analytical method for annatto pigment in foods. J. Food Hyg. Saf., 36(4), 298-309. (2021). https://doi.org/10.13103/JFHS.2021.36.4.298
  20. Hwang, K. H., Choi, W. H., Hu, S. J., Hwang, K. M., Improvement of analysis methods for fatty acids in infant formula by gas chromatography flame-ionization detector. J. Food Hyg. Saf., 36(1), 34-41. (2021). https://doi.org/10.13103/JFHS.2021.36.1.34