Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

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

DOI QR Code

In vivo Genotoxicity Assessment of Matrine and the Water Extract of Sophorae Radix Using a Comet Assay

  • Heo, Seongmoo (Department of Biotechnology and The Research Institute for Basic Sciences, Hoseo University) ;
  • Lee, Jiyeon (Department of Biotechnology and The Research Institute for Basic Sciences, Hoseo University) ;
  • Jeon, Hyoungwoon (Department of Biotechnology and The Research Institute for Basic Sciences, Hoseo University) ;
  • Kim, Myoungjun (Department of Biotechnology and The Research Institute for Basic Sciences, Hoseo University) ;
  • Chung, Young-Shin (Department of Biotechnology and The Research Institute for Basic Sciences, Hoseo University)
  • Received : 2021.01.25
  • Accepted : 2021.04.01
  • Published : 2021.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

The genotoxic potentials of the water extract of Sophorae Radix (WSR) and matrine as a major compound were investigated in murine hepatocytes and blood cells using single-cell gel electrophoresis. According to the test guidelines of OECD TG 489, an in vivo alkaline Comet assay was performed using male mice. The treatment doses of the WSR were 1,000 and 2,000 mg/kg body weight to include the limiting dose. The three test doses for matrine were 50, 100, and 200 mg/kg, and the maximum dose was estimated from a dose range-finding test. The negative control and test articles were administered three times by oral gavage, and the positive control (ethyl methanesulfonate, EMS) was administered twice. Within 3-4 h after the last treatment, the liver tissue and blood were collected, and single-cell electrophoresis was performed under alkaline conditions. The in vivo Comet assay showed no changes in body weights during the treatments with the test articles. The WSR and matrine did not induce significant DNA damage in the liver and blood cells of mice compared to the vehicle treatment. In conclusion, matrine and the WSR may not have genotoxic potential.

본 연구는 고삼 물추출물과 주 성분인 마트린의 유전독성 유발능을 마우스 간세포 및 혈구세포에서 체내 단세포전기영동 시험법을 사용하여 평가하였다. OECD TG 489에서 정하고 있는 체내 알카리코멧 시험법(in vivo alkanine Comet assay)을 수컷 마우스를 사용하여 수행하였다. 고삼물추출물의 투여 용량은 한계 용량을 포함하도록 체중 대비 1,000와 2,000 mg/kg으로 하였다. 마트린의 세 투여용량은 50, 100, 200 mg/kg으로 정하였고, 최고 투여용량은 용량 설정시험을 통하여 설정하였다. 음성대조물질과 시험물질은 경구로 3회 투여하였고, 양성대조물질(ethyl methanesulfonate, EMS)은 2회 투여하였다. 마지막 투여 후 3-4시간 내에 간조직과 혈액을 채취하였고 단세포전기영동을 알카리 조건에서 수행하였다. 코멧 시험 결과, 고삼 물추출물과 마트린에 의한 체중의 변화가 관찰되지 않았으며, 간세포와 혈구세포에서 통계적으로 유의성 있는 유전자 손상을 유발하지 않았다. 따라서, 본 연구조건에서 고삼 물추출물과 마트린은 유전독성 유발능이 없는 것으로 사료된다.

Keywords

Acknowledgement

This research was supported by the Academic Research Fund of Hoseo University in 2017 (2017-0350).

References

  1. Sun, M.G., Cao, H.Y., Sun, L., Dong, S., Bian, Y.Q., Han, J., Zhang, L.J., Ren, S., Hu, Y.Y., Liu, C.H., Xu, L.M., Liu, P., Antitumor activities of kushen: Literature review. Evid. Based Complement Alternat. Med., 2012, 373219 (2012).
  2. Yu, Q.Q., Cheng, N.N., Ni, X.J., Identifying 2 prenylflavanones as potential hepatotoxic compounds in the ethanol extract of Sophora flavescens. J. Food Sci., 78, T1830-T1834 (2013). https://doi.org/10.1111/1750-3841.12275
  3. Ding, Y., Tian, R.H., Kinjo, J., Nohara, T., Kitagawa, I., Three new oleanene glycosides from Sophora flavescens. Chem. Pharm. Bull., 40, 2990-2994 (1992). https://doi.org/10.1248/cpb.40.2990
  4. Cheng, H., Xia, B., Zhang, L., Zhou, F., Zhang, Y.X., Ye, M., Hu, ZG.., Li, J., Li, J., Wang, Z.L., Li, C., Guo, Q.S., Matrine improves 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. Pharmacol. Res., 53, 202-208 (2006). https://doi.org/10.1016/j.phrs.2005.11.001
  5. Zhang, Y.F., Wang, S.Z., Li, Y.Y., Xiao, Z.Y., Hu, Z.L., Zhang, J.P., Sophocarpine and matrine inhibit the production of TNF-alpha and IL-6 in murine macrophages and prevent cachexia-related symptoms induced by colon26 adenocarcinoma in mice. Int. Immunopharmacol., 8, 1767-1772 (2008). https://doi.org/10.1016/j.intimp.2008.08.008
  6. Kuroyanagi, M., Arakawa, T., Hirayama, Y., Hayashi, T., Antibacterial and antiandrogen flavonoids from Sophora flavescens. J. Nat. Prod., 62, 1595-1599 (1999). https://doi.org/10.1021/np990051d
  7. Son, J.K., Park, J.S., Kim, J.A., Kim, Y.S., Chung, S.R., Lee, S.H., Prenylated flavonoids from the roots of Sophora flavescens with tyrosinase inhibitory activity. Planta Medica, 69, 559-561 (2003). https://doi.org/10.1055/s-2003-40643
  8. Han, J., Sun, M.G., Cui, Y.M., Wang, T., Zhang, W.H., Guo, M.C., Zhou, Y., Liu, W., Zhang, M.F., Duan, J.F., Xiong, S.D., Yao, M.H., Yan, X.Q., Kushen flavonoids induce apoptosis in tumor cells by inhibition of NF-κB activation and multiple receptor tyrosine kinase activities. Phytother. Res., 21, 262-268 (2007). https://doi.org/10.1002/ptr.2065
  9. Zhang, H., Chen, L.L., Sun, X.P., Yang, Q.J., Wan, L.L., Guo, C., Matrine: A promising natural product with various pharmacological activities. Front. Pharmacol., 11, 588 (2020). https://doi.org/10.3389/fphar.2020.00588
  10. Zhang, Y., Zhang, H., Yu, P.F., Liu, Q., Liu, K., Duan, H.Y., Luan, G.L., Yagasaki, K., Zhang, G.Y., Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration. Cytotechnology, 59, 191-200 (2009). https://doi.org/10.1007/s10616-009-9211-2
  11. Xiao, X., Ao, M., Xu, F., Li, X., Hu, J.L., Wang, Y., Li, D.X., Zhu, X.Q., Xin, C.L., Shi, W., Effect of matrine against breast cancer by downregulating the vascular endothelial growth factor via the Wnt/beta-catenin pathway. Oncol. Lett., 15, 1691-1697 (2018).
  12. Cao, J., Wei, R.J., Yao, S.K., Matrine has pro-apoptotic effects on liver cancer by triggering mitochondrial fission and activating Mst1-JNK signaling pathways. J. Physiol. Sci., 69, 185-198 (2019). https://doi.org/10.1007/s12576-018-0634-4
  13. Huang, M., Xin, W., Matrine inhibiting pancreatic cells epithelialmesenchymal transition and invasion through ROS/NF-kappaB/MMPs pathway. Life Sci., 192, 55-61 (2018). https://doi.org/10.1016/j.lfs.2017.11.024
  14. Zhang, J.W., Su, K., Shi, W.T., Wang, Y., Hu, P.C., Wang, Y., Wei, L., Xiang, J., Matrine inhibits the adhesion and migration of BCG823 gastric cancer cells by affecting the structure and function of the vasodilator-stimulated phosphoprotein (VASP). Acta Pharmacol. Sin., 34, 1084-1092 (2013). https://doi.org/10.1038/aps.2013.15
  15. Chang, C., Liu, S.P., Fang, C.H., He, R.S., Wang, Z., Zhu, Y.Q., Jiang, S.W., Effects of matrine on the proliferation of HT29 human colon cancer cells and its antitumor mechanism. Oncol. Lett., 6, 699-704 (2013). https://doi.org/10.3892/ol.2013.1449
  16. Bai, S.M., Chen, T., Yu, X., Luo, M., Chen, X.J., Lin, C.H., Lai, Y.M., Huang, H., The specific killing effect of matrine on castration-resistant prostate cancer cells by targeting the Akt/FoxO3a signaling pathway. Oncol. Rep., 37, 2819-2828 (2017). https://doi.org/10.3892/or.2017.5510
  17. Ma, K., Huang, M.Y., Guo, Y.X., Hu, G.Q., Matrine-induced autophagy counteracts cell apoptosis via the ERK signaling pathway in osteosarcoma cells. Oncol. Lett., 12, 1854-1860 (2016). https://doi.org/10.3892/ol.2016.4848
  18. Vardarli, A.T., Duzgun, Z., Erdem, C., Kaymaz, B.T., Eroglu, Z., Cetintas, V.B., Matrine induced G0/G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia (T-ALL) cells. Bosn. J. Basic Med. Sci., 18, 141-149 (2018).
  19. Zhang, Y.F., Liu, M.F., Sun, H.R., Yin, K.M., Matrine improves cognitive impairment and modulates the balance of Th17/Treg cytokines in a rat model of Aβ1-42 -induced Alzheimer's disease. Cent. Eur. J. Immunol., 40, 411-419 (2015). https://doi.org/10.5114/ceji.2015.56961
  20. Zhao, P., Zhou, R., Zhu, X.Y., Hao, Y.J., Li, N., Wang, J., Niu, Y., Sun, T., Li, Y.X., Yu, J.Q., Matrine attenuates focal cerebral ischemic injury by improving antioxidant activity and inhibiting apoptosis in mice. Int. J. Mol. Med., 36, 633-644 (2015). https://doi.org/10.3892/ijmm.2015.2260
  21. Huang, W.C., Chan, C.C., Wu, S.J., Chen, L.C., Shen, J.J., Kuo, M.L., Chen, M.C., Liou, C.J., Matrine attenuates allergic airway inflammation and eosinophil infiltration by suppressing eotaxin and Th2 cytokine production in asthmatic mice. J. Ethnopharmacol., 151, 470-477 (2014). https://doi.org/10.1016/j.jep.2013.10.065
  22. Liu, Z.W., Zhang, Y., Tang, Z.G., Xu, J., Ma, M.J., Pan, S., Qiu, C., Guan, G.C., Wang, J.K., Matrine attenuates cardiac fibrosis by affecting ATF6 signaling pathway in diabetic cardiomyopathy. Eur. J. Pharmacol., 804, 21-30 (2017). https://doi.org/10.1016/j.ejphar.2017.03.061
  23. Guo, S., Gao, C.Y., Xiao, W.T., Zhang, J., Qu, Y.S., Li, J., Ye, F., Matrine protects cardiomyocytes from ischemia/reperfusion injury by regulating HSP70 expression via activation of the JAK2/STAT3 pathway. Shock, 50, 664-670 (2018). https://doi.org/10.1097/SHK.0000000000001108
  24. Shi, D., Zhang, J.J., Qiu, L., Li, J.Z., Hu, Z.L., Zhang, J., Matrine inhibits infiltration of the inflammatory Gr1(hi) monocyte subset in injured mouse liver through inhibition of monocyte chemoattractant Protein-1. Evid. Based Complement Alternat. Med., 2013, 580673 (2013).
  25. Yang, Y,S., Dong, Q.M., Li, R.H., Matrine induces the apoptosis of fibroblast like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway. Int. J. Mol. Med., 39, 307-316 (2017). https://doi.org/10.3892/ijmm.2016.2843
  26. Khan, A., Shal, B., Naveed, M., Shah, F.A., Atiq, A., Khan, N.U., Kim, Y.S., Khan, S., Matrine ameliorates anxiety and depression-like behaviour by targeting hyperammonemia-induced neuroinflammation and oxidative stress in CCl4 model of liver injury. Neurotoxicology, 72, 38-50 (2019). https://doi.org/10.1016/j.neuro.2019.02.002
  27. Che, J.H., Yun, J.W., Kim, Y.S., Kim, S.H., You, J.R., Jang, J.J., Kim, H.C., Kim, H.H., Kang, B.C., Genotoxicity and subchronic toxicity of Sophorae Radix in rats: Hepatotoxic and genotoxic potential. Regul. Toxicol. Pharmacol., 71, 379-387 (2015). https://doi.org/10.1016/j.yrtph.2015.01.012
  28. Lu, Z.G., Li, M.H., Wang, J.S., Wei, D.D., Liu, Q.W., Kong, L.Y., Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine and sophocarpine, in zebrafish (Danio rerio) embryos/larvae. Reprod. Toxicol., 47, 33-41 (2014). https://doi.org/10.1016/j.reprotox.2014.05.015
  29. Wang, X.Y., Liang, L., Chang, J.L., Yang, M.H., Li, Z.G., Toxicity of matrine in kunming mice. Nan Fang Yi Ke Da Xue Xue Bao, 30, 2154-2155 (2010) (in Chinese).
  30. Gu, Y.M., Lu, J.Y., Sun, W., Jin, R.M., Ohira, T.K., Zhang, Z., Tian, X.S., Oxymatrine and its metabolite matrine contribute to the hepatotoxicity induced by radix Sophorae tonkinesis in mice. Exp. Ther. Med., 17, 2519-2528 (2019).
  31. You, L.T., Yang, C.J., Du, Y.Y., Liu, Y., Chen. G.S., Sai, N., Dong, X.X., Yin, X.B., Ni, J., Matrine exerts hepatotoxic effects via the ROS-dependent mitochondrial apoptosis pathway and inhibition of Nrf2-mediated antioxidant response. Oxid. Med. Cell. Longev., 2019, 1045345 (2019).
  32. Yoon, W.H., Genotoxicological safety evaluation of the solvent extracts for medicinal herbs that are of highly domestic spendings. Korean J. Food Nutr., 26, 814-823 (2013). https://doi.org/10.9799/ksfan.2013.26.4.814
  33. Engen, A., Maeda, J.K., Wozniak, D.E., Brents, C.A., Bell, J.J., Uesaka, M., Aizawa, Y., Kato, T.A., Induction of cytotoxic and genotoxic responses by natural and novel quercetin glycosides. Mutat. Res. Genet. Toxicol. Environ. Mutagen., 784-785, 15-22 (2015). https://doi.org/10.1016/j.mrgentox.2015.04.007
  34. Chen, T., Mei, N., Fu, P.P., Genotoxicity of pyrrolizidine alkaloids. J. Appl. Toxicol., 30, 183-196 (2010). https://doi.org/10.1002/jat.1504
  35. Fan, P.H., Lou, H.X., Effects of polyphenols from grape seeds on oxidative damage to cellular DNA. Mol. Cell. Biochem., 267, 67-74 (2004). https://doi.org/10.1023/B:MCBI.0000049366.75461.00
  36. Chung, Y.S., Park, C.B., Evaluation of genotoxicity and 14-day repeat dose toxicity of water extract of the mixture of natural plants. Anemarrhena and Phellodendron. J. Food Hyg. Saf., 29, 383-390 (2014). https://doi.org/10.13103/JFHS.2014.29.4.383
  37. Lv, H., Ma, S., Wang, X., Shang, X., Simultaneous determination of co-formulated matrine and secnidazole in suppositories by reverse phase high performance liquid chromatography. Trop. J. Pharm. Res., 12, 413-418 (2013).
  38. Zhang, R., Hu, S., Chen, X., Bai, X., Dispersive liquid-liquid microextraction combined with high-performance liquid chromatography for the simultaneous analysis of matrine alkaloids in traditional chinese medicine. J. Chromatogr. Sci., 54, 1687-1693 (2016). https://doi.org/10.1093/chromsci/bmw114
  39. OECD (Organisation for Economic Cooperation and Development), (2016, July 29). OECD Guideline for the testing of chemicals: In vivo mammalian alkaline comet assay. Test guideline 489. Retrieved from https://www.oecd.org/env/test-no-489-in-vivo-mammalian-alkaline-comet-assay9789264264885-en.htm
  40. Yin, X.J., Liu, D.X., Wang, H., Zhou, Y., A study on the mutagenicity of 102 raw pharmaceuticals used in Chinese traditional medicine. Mutat. Res., 260, 73-82 (1991). https://doi.org/10.1016/0165-1218(91)90082-W
  41. Cho, H.J., Yoon, H.J., Park, K.H., Lee, J.B., Shim, C.K., Kim, J.H., Jeong, M.H., Oh, J.A., Kim, D.H., Paik, M.K., In vitro antimutagenic and genotoxic effects of Sophora Radix extracts. Korean J. Pestic. Sci., 17, 335-342 (2013). https://doi.org/10.7585/kjps.2013.17.4.335
  42. Cho, H.J., Park, K.H., Jeong, M.H., Park, S.J., Oh, J.A., Kim, W.I., Cho, N.J., Ryu, J.G., Paik, M.K., Genotoxicity of environment-friendly organic materials of plant origin in the micronucleus test using Chinese hamster lung cells. Korean J. Environ. Agric., 33, 138-143 (2014). https://doi.org/10.5338/KJEA.2014.33.2.138
  43. ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use), (2013, November 2). ICH guideline S2 (R1) on genotoxicity testing and data interpretation for pharmaceuticals intended for human use - Step 5. Retrieved from https://www.ema.europa.eu/en/documents/scientific-guideline/ichguideline-s2-r1-genotoxicity-testing-data-interpretation-pharmaceuticals-intended-human-use-step_en.pdf