Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

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

DOI QR Code

Development of Method using LC-ESI-MS/MS and KASP for Identification of Gymnema sylvestre in Food

식품에서 당살초 판별을 위한 LC-ESI-MS/MS 분석법과 KASP 마커 개발

  • Park, Boreum (Advanced Analysis center, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Lee, Sun Hee (Advanced Analysis center, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Eom, Kwonyong (Food Additives and Packaging Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Noh, Eunyoung (Drug Research Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Moon Han, Kyoung (Advanced Analysis center, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Hwang, Jinwoo (Advanced Analysis center, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Kim, Hyungil (Advanced Analysis center, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Baek, Sun Young (Advanced Analysis center, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety)
  • 박보름 (식품의약품안전처 식품의약품안전평가원 첨단분석센터) ;
  • 이선희 (식품의약품안전처 식품의약품안전평가원 첨단분석센터) ;
  • 엄권용 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 첨가물포장과) ;
  • 노은영 (식품의약품안전처 식품의약품안전평가원 의료제품연구부 의약품연구과) ;
  • 한경문 (식품의약품안전처 식품의약품안전평가원 첨단분석센터) ;
  • 황진우 (식품의약품안전처 식품의약품안전평가원 첨단분석센터) ;
  • 김형일 (식품의약품안전처 식품의약품안전평가원 첨단분석센터) ;
  • 백선영 (식품의약품안전처 식품의약품안전평가원 첨단분석센터)
  • Received : 2022.04.04
  • Accepted : 2022.04.19
  • Published : 2022.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

Known for its effectiveness in weight loss and diabetes prevention, Gymnema sylvestre products can be found in the US, Japanese, and Indian markets. However, the recommended dosage or safety of these products has not yet been proven. Therefore, development of an analytical method for detecting the content of Gymnema sylvestre in food products is required. Accordingly, this study proposes an analysis method that can examine Gymnema sylvestre in food using LC-ESI-MS/MS and KASP (Kompetitive Allele-Specific PCR) markers. In LC-ESI-MS/MS, a simultaneous analysis method for gymnemic acid and deacylgymnemic acid was optimized using negative ionization mode, and its validation test was completed for solid and liquid samples. In addition, KASP markers were prepared by finding the specific SNP of G. sylvestre in ITS2 and matK through DNA barcodes. The two KASP markers returned positive FAM fluorescence result when combined with G. sylvestre, and this aspect was confirmed in raw G. sylvestre as well. The applicability of the method was tested on 21 different food and healthy functional products containing G. sylvestre purchased on the internet. As a result, although there was a difference in the ratios of gymnemic acid and deacylgymnemic acid in LC-ESI-MS/MS, the index component was detected in all 21 products samples. In the KASP analysis, 9 products returned positive FAM result, and the rest of the products were found to be containing G. sylvestre extract. This study is the first study to use the dual system of LC-ESI-MS/MS and KASP for the analysis of G. sylvestre. The study has confirmed that these two methods are applicable to the examine G. sylvestre content in food products.

당살초(Gymnema sylvestre)는 다이어트와 당뇨에 효과가 있다고 알려지면서 미국, 일본, 인도에서 다양한 제품들이 판매, 유통되고 있다. 하지만 권장용량이 불분명하고 안전성이 입증되지 않았다. 따라서 식품에서 당살초를 확인할 수 있는 분석법 개발이 필요하다. 이 연구에서는 LC-ESI-MS/MS와 KASP 마커를 이용해 식품 속에서 당살초를 확인할 수 있는 분석법을 마련하였다. LC-ESI-MS/MS에서는 negative 이온화 모드에서 gymnemic acid 와 deacylgymnemic acid를 동시 분석법을 최적화 하였으며 고체시료와 액체시료에 대한 유효성 검증 마쳤다. 또한 염기서열 분석을 통해서 ITS2와 matK에서 당살초 특이적인 SNP를 찾아 KASP 마커를 제작하였다. 제작한 2개의 KASP 마커는 당살초와 결합해 FAM 형광 양성을 나타내며 당살초 원물에서 이러한 양상을 확인하였다. 인터넷을 통해 구매한 21개의 당살초 함유 식품 및 건강기능식품에 대해 적용성 검토를 진행하였다. LC-ESI-MS/MS에서 gymnemic acid 와 deacylgymnemic acid의 비율 차이는 있지만 21개의 제품 모두에서 지표성분이 검출되었다. KASP 분석에서는 9개의 제품에서 FAM 양성이 나타났으며 분석이 되지 않은 제품들은 추출물인 것으로 밝혀졌다. 이 연구는 LC-ESI-MS/MS와 KASP를 이중 시스템으로 당살초를 분석한 첫 번째 연구이며, 2개의 분석법이 식품에서 당살초 판별에 적용되는 것을 확인하였다.

Keywords

Acknowledgement

본 연구는 2019년도 식품의약품안전처의 연구개발비(18181부분연422)로 수행되었으며, 이에 감사드립니다.

References

  1. Kanetkar, P., Singhal, R., Kamat, M., Gymnema sylvestre: a memoir. J. Clin. Biochem. Nutr. 41, 77-81 (2007). https://doi.org/10.3164/jcbn.2007010
  2. Gupta, P., Sujata, G.S., Pratibha, S.P., A miracle fruit plant-Gymnema sylvestre R. Br. (Retz) Pharmacieglobale. Int. J. Comprehens. Pharm., 3, 1-12 (2012).
  3. Marakis, G., Ziegenhagen, R., Lampen, A., Hirsch-Ernst, K.I., Risk assessment of substances used in food supplements:the example of the botanical Gymnema sylvestre. EFSA J., 16, e16083 (2018).
  4. Ministry of Food and Drug Safety, (2022, February 21). Food Code; Available from: http://www.foodsafetykorea.go.kr/foodcode/01_03.jsp?idx=9.
  5. Yoshikawa, K., Amimoto, K., Arihara, S., Matsuura, K., Structure studies of new antisweet constituents from Gymnema sylvestre. Tetrahedron Lett., 30, 1103-1106 (1989). https://doi.org/10.1016/S0040-4039(01)80371-3
  6. Yoshikawa, K., Amimoto, K., Arihara, S., Matsuura, K., Gymnemic acid V, VI and VII from gur-ma, the leaves of Gymnema sylvestre R.Br.. Chem. Pharm. Bull., 37, 852-854 (1989). https://doi.org/10.1248/cpb.37.852
  7. Yoshikawa, K., Nakagawa, M., Yamamoto, R., Arihara, S., Matsuura, K., Antisweet natural products. V. Structures of gymnemic acids VIII-XII from Gymnema sylvestre R.Br.. Chem. Pharm. Bull., 40, 1779-1782, (1992). https://doi.org/10.1248/cpb.40.1779
  8. Lee, Y.C., Park, J.S., Kim, S.D., Yang, H.R., Kim, E.H., Yi, Y.J., Cho, S.J., Jo, H.B., Kim, H.J., Chae, Y.Z., Simultaneous determination of non-steroidal anti-inflammatory drugs and corticosteroids added to foods as adulterants using LC-ESI-tandem mass spectrometry. J. Fd. Hyg. Safety, 28, 247-251 (2013). https://doi.org/10.13103/JFHS.2013.28.3.247
  9. Kress, W. J., Plant DNA barcodes: Applications today and in the future. J. Syst. Evol., 55, 291-307 (2017). https://doi.org/10.1111/jse.12254
  10. Nair, S., Keshavachandran, R., Molecular diversity in chakkarakolli (Gymnema sylvestre R. Br.) assessed through isozyme and RAPD analysis. Trop. Agric. 44, 31-36 (2006).
  11. Osman, M.A., Dhawan, S.S., Bahl, J.R., Darokar, M.P., Khanuja, S.P.S, AFLP marking and polymorphism among progenies of Gymnema sylvestre: an important medicinal Plant of India. Nat. Prod. Commun., 6, 1679-1682 (2011).
  12. Shahnawaz, M., Zanan, R.L., Wakte, K.V., Mathure, S.V., Kad, T.D., Deokule, S.S., Nadaf, A.B., Genetic diversity assessment of Gymnema sylvestre (Retz.)R. Br. ex Sm. populations from Western Ghats of Maharashtra, India. Genet. Resour. Crop. Evol. , 59, 125-134 (2012). https://doi.org/10.1007/s10722-011-9757-7
  13. Krishna, R.B., Reddy, reddy S.R., Vijayalaxmi, M., Anugna, K., Divyavani, N., Reddy, K.J., Molecular characterization of 17 accessions of Gymnema sylvestre R. Br. using rapd markers. Int. J. of Pharma Bio Sci., 3, B126-135 (2012).
  14. Saeed T, Shahzad A, Ahmad N, Parveen S, High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in Gymnema sylvestre. Plant Cell Tiss. Org., 134, 163-168 (2018). https://doi.org/10.1007/s11240-018-1409-6
  15. Kalariya K.A., Meena R.P., Poojara L, Shahi D, Patel S, Characterization of squalene synthase gene from Gymnema sylvestre R. Br.. Beni Suef Univ. J. basic Appl. Sci, 10, 6 (2021). https://doi.org/10.1186/s43088-020-00094-4
  16. Winfield, M., Burridge, A., Ordidge, M., Harper, H., Wilkinson, P., Thorogood, D., Copas, L., Edwards, K., Barker, G., Development of a minimal KASP marker panel for distinguishing genotypes in apple collections, PLoS ONE, 15, e0242940 (2020). https://doi.org/10.1371/journal.pone.0242940
  17. Steele, K., Tulloch, M.Q., Burns, M., Nader, W., Developing KASP markers for identification of basmati rice varieties. Food Anal. Method, 14, 663-673 (2021). https://doi.org/10.1007/s12161-020-01892-3
  18. Zhao, S., Li, A., Li, C., Xia, H., Zhao, C., Zhang, Y., Hou, L., Wang, X.J., Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut. Electron J. Biotechn., 25, 9-12 (2017). https://doi.org/10.1016/j.ejbt.2016.10.010
  19. Grimm, K.D.S., Porter, L.D., Development and Validation of KASP markers for the identification of pea seedborne mosaic virus pathotype P1 resistance in Pisum sativum. Plant Dis., 1004, 1824-1830 (2020). https://doi.org/10.1094/PDIS-09-19-1920-RE
  20. Food and Drug Administration. "Guidelines for the validation of chemical methods in food, feed, cosmetics, and veterinary products." Food and Drug Administration: Silver Spring, MD, USA, 1-39 (2019).
  21. Guideline, ICH Harmonised Tripartite. "Validation of analytical procedures: text and methodology." Q2 (R1) (2005).
  22. Validation of Compendial Procedures <1225>, The United States Pharmacopeia, 32th Rev., and The National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc., (2009).
  23. Center, Cold Spring Harbor DNA Learning. "Using DNA barcodes to identify and classify living things." DNA Learning Center (2014).
  24. Dateo, G.P. and Long, L., Gymnemic acid, the antisaccharine principle of Gymnema sylvestre. Studies on isolation and heterogenesity of gymnemic acid A1. J. Agric. Food Chem., 21, 899-903 (1973). https://doi.org/10.1021/jf60189a030
  25. Khramov, V.A., Spasov, A.A., Samokhina, M.P., Chemical composition of dry extracts of Gymnema sylvestre leaves. Pharm. Chem. J., 42, 30-32 (2008).
  26. Tiwari, P., Mishra, B.N., Neelam, S., Sangwan, N.S., Phytochemical and pharmacological properties of Gymnema sylvestre: An important medicinal plant, Hindawi Publishing Corporation. BioMed. Res. Int., 2014, 18 (2014).
  27. Pandey, A.K., Yadav, S., Variation in gymnemic acid content and non-destructive harvesting of Gymnema sylvestre (Gudmar). Pharmacogn. Res., 2, 309-312 (2010). https://doi.org/10.4103/0974-8490.72330
  28. Baker, D.A., Dennis, Wm. Stevenson, Little DP, DNA barcode identification of black cohosh herbal dietary supplements. J. AOAC Int., 95, 1023-1034 (2012). https://doi.org/10.5740/jaoacint.11-261
  29. Masada-Atsumi, S., Onuma, M., Suenaga, E., Maruyama, T., Hishida, A., Kiuchi, F., Kobayashi, S., Goda, Y., Jakamatsuka, T., Genome-based authentication of black cohosh (Cimicifuga racemosa; Ranunculaceae) supplements available in the Japanese markets. Jpn. J. Food Chem. Safety, 20, 178-189 (2013).
  30. K. Shimizu, M. Ozeki, A. Iino, S. Nakajyo, N. Urakawa, and M.Atsuchi, "Structure-activity relationships of triterpenoid derivativesextracted from Gymnema inodorum leaves on glucoseabsorption." Jpn. J. Pharmacol., 86, 223-229 (2001). https://doi.org/10.1254/jjp.86.223
  31. Xie, J.T., Wang, A., Mehendale S., Wu J., Aung, H.H., Dey, L., Qiu, S., Yuan, C.S., Anti-diabetic effects of Gymnema yunnanense extract." Pharmacol. Res., 47, 323-329 (2003). https://doi.org/10.1016/S1043-6618(02)00322-5