한국식품위생안전성학회지 (Journal of Food Hygiene and Safety)

  • 제38권6호
  • /
  • Pages.430-441
  • /
  • 2023
  • /
  • 1229-1153(pISSN)
  • /
  • 2465-9223(eISSN)
한국식품위생안전성학회 (The Korean Society of Food Hygiene and Safety)
권호 표지
DOI QR코드

DOI QR Code

ICR 마우스 모델을 이용한 녹용 추출물의 생화학적 평가 및 급성 경구 독성을 포함한 세포 독성 효과

Biochemical Assessment of Deer Velvet Antler Extract and its Cytotoxic Effect including Acute Oral Toxicity using an ICR Mice Model

  • 투고 : 2023.12.07
  • 심사 : 2023.12.15
  • 발행 : 2023.12.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

녹용은 수많은 연구에서 면역력 강화를 포함한 영양 및 의학적 가치를 입증하였으며 전통적인 약으로 널리 사용되고 있다. 본 연구는 녹용 추출물 (sample 1: 생녹용 추출물, sample 2: 건녹용 추출물, sample 3: 동결 건조 추추물)의 일반성분과 우론산, 황산화 글리코사미노글리칸, 시알릭산, 콜라겐을 포함한 유효성분을 조사하고, 액체 크로마토그래피quadrupole-time-of-light mass spectrometry (UPLC/QTOFMS)를 사용하여 녹용 추출물의 화학 성분을 분석하는데 목적이 있다. 또한, HT22 해마 세포, BV2 미세아교세포, RAW264.7 대식세포 및 HaCaT 케라틴 세포를 사용하여 MTT 분석을 통해 녹용 추출물의 세포 독성 효과 평가와 암컷과 수컷 ICR 마우스에 녹용 추출물을 각각 (0, 500, 1000, 2000 mg/kg) 경구투여 하여 급성 독성평가를 실시하였다. 투여후에는 OECD 가이드라인에 따라 마우스의 일반독성, 생존율, 체중 변화, 사망률, 임상 징후 및 부검 결과를 관찰하였다. 결과적으로 녹용 추출물은 HaCaT 케라틴 세포에서 세포 독성 효과가 없었으며, 건녹용 추출물에서는 HT22 해마 세포에서 500 ㎍/mL, RAW264.7 대식세포의 경우 1000 ㎍/mL 에서, 동결건조추출물에서는 RAW264.7 세포와 BV2 미세아교세포의 경우 500 ㎍/mL 및 1000 ㎍/mL 농도에서 세포 독성을 가지고 있음을 보였다. 그러나 마우스를 이용한 급성 독성 평가에서는 녹용 추출물 시료를 처리한 모든 마우스에서 사망률, 임상 징후 및 부검 결과 특이사항이 없었으며 이는 LD50이 2000 mg/kg 이상으로 사료된다. 그러나 인간에 대한 안전성에 대한 충분한 증거를 확보하기 위해서는 동물과 사람에 대한 추가적인 연구가 필요하다.

Velvet antler is widely used as a traditional medicine, and numerous studies have demonstrated its tremendous nutritional and medicinal values including immunity-enhancing effects. This study aimed to investigate different deer velvet extracts (Sample 1: raw extract, Sample 2: dried extract, and Sample 3: freeze-dried extract) for proximate composition, uronic acid, sulfated glycosaminoglycan, sialic acid, collagen levels, and chemical components using ultra-performance liquid chromatography-quadrupole-time-of-light mass spectrometry. In addition, we evaluated the cytotoxic effect of the deer velvet extracts on BV2 microglia, HT22 hippocampal cells, HaCaT keratinocytes, and RAW264.7 macrophages using the cell viability MTT assay. Furthermore, we evaluated acute toxicity of the deer velvet extracts at different doses (0, 500, 1000, and 2000 mg/kg) administered orally to both male and female ICR mice for 14 d (five mice per group). After treatment, we evaluated general toxicity, survival rate, body weight changes, mortality, clinical signs, and necropsy findings in the experimental mice based on OECD guidelines. The results suggested that in vitro treatment with the evaluated extracts had no cytotoxic effect in HaCaT keratinocytes cells, whereas Sample-2 had a cytotoxic effect at 500 and 1000 ㎍/mL on HT22 hippocampal cells and RAW264.7 macrophages. Sample 3 was also cytotoxic at concentrations of 500 and 1000 ㎍/mL to RAW264.7 and BV2 microglial cells. However, the mice treated in vivo with the velvet extracts at doses of 500-2000 mg/kg BW showed no clinical signs, mortality, or necropsy findings, indicating that the LD50 is higher than this dosage. These findings indicate that there were no toxicological abnormalities connected with the deer velvet extract treatment in mice. However, further human and animal studies are needed before sufficient safety information is available to justify its use in humans.

키워드

과제정보

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (No. 321033-3).

참고문헌

  1. Zhang, H., Wanwimolruk, S., Coville, P.F., Schofield, J.C., Williams, W., Haines, S.R., Suttie, J.M., Toxicological evaluation of New Zealand deer velvet powder. Part I: acute and subchronic oral toxicity studies in rats. Food Chem. Toxicol.,38, 985-990 (2000). https://doi.org/10.1016/S0278-6915(00)00099-5
  2. Xia, P., Liu, D., Jiao, Y., Wang, Z., Chen, X., Zheng, S., Fang, J., Hao, L., Health Effects of Peptides Extracted from Deer Antler. Nutrients, 14, 4183 (2022).
  3. Chen, J.C., Hsiang, C.Y., Lin, Y.C., Ho, T.Y., Deer antler extract improves fatigue effect through altering the expression of genes related to muscle strength in skeletal muscle of mice. Evid.- based Complement. Altern. Med., 2014, 1-10 (2014).
  4. Kim, S.H., Ameer, K., Oh, J.H., Park, M.K., Characterization of hot water extract from Korean deer velvet antler (Cervus canadensis Erxleben). Korean J. Food Preserv., 27, 725-733 (2020). https://doi.org/10.11002/kjfp.2020.27.6.725
  5. Huo, Y.S., Huo, H., Zhang, J., The contribution of deer velvet antler research to the modern biological medicine. Chin. J. Integr. Med., 20, 723-728 (2014). https://doi.org/10.1007/s11655-014-1827-1
  6. Jeon, B.T., Cheong, S.H., Kim, D.H., Park, J.H., Park, P.J., Sung, S.H., Thomas, D.G., Kim, K.H., Moon, S.H., Effect of antler development stage on the chemical composition of velvet antler in elk (Cervus elaphus canadensis). Asian Australas. J. Anim. Sci., 24, 1303-1313 (2011). https://doi.org/10.5713/ajas.2011.10412
  7. Limmatvapirat, C., Rodhetbhai, P., Somsakraksanti, K., Danpongprasert, P., Poonsub, S., Krongrawa, W., Limmatvapirat, S., Meepan, M., Chemical constituents, antioxidant activities, and element concentrations of rusa deer velvet antler extracts. J. Chem., 2020, 1-8 (2020).
  8. Kim, Y.A., Kim, S.W., Lee, M.H., Lee, H.K., Hwang, I.H., Comparisons of chemical composition, flavor and bioactive substances between Korean and imported velvet antler extracts. Food Sci. Anim. Resour., 41, 386-401 (2021). https://doi.org/10.5851/kosfa.2021.e4
  9. Lee, S.R., Jeon, B.T., Kim, S.J., Kim, M.H., Lee, S.M., Moon, S.H., Effects of antler development stage on fatty acid, vitamin and GAGs contents of velvet antler in spotted deer (Cervus nippon). Asian Australas. J. Anim. Sci., 20, 1546-1550 (2007). https://doi.org/10.5713/ajas.2007.1546
  10. Jeon, B.T., Kim, K.H., Cheong, S.H., Kang, S.K., Park, P.J., Kim, D.H., Jung, H.S., Park, J.H., Thomas, D.G., Moon, S.H., Effects of growth stage and position within the beam in the structure and chemical composition of sika deer (Cervus nippon) antlers. Anim. Prod. Sci., 52, 51-57 (2012). https://doi.org/10.1071/AN11183
  11. Shi, B., Li, G., Wang, P., Yin, W., Sun, G., Wu, Q., Yu, G., Effect of antler extract on corticosteroid-induced avascular necrosis of the femoral head in rats. J. Ethnopharmacol., 127, 124-129 (2010). https://doi.org/10.1016/j.jep.2009.09.036
  12. Zhang, L.Q., Wang, J., Li, T., Li, P.Y., Wang, Y.H., Yang, M., Liu, J.P., Liu, J.H., Determination of the chemical components and phospholipids of velvet antler using UPLC/QTOF-MS coupled with UNIFI software. Exp. Ther. Med., 17, 3789-3799 (2019). https://doi.org/10.3892/etm.2019.7372
  13. Cheng, S.L., Jian, Y.L., Chen, C.M., Liu, B.T., Relationships between antioxidants and quality characteristics from velvet antlers of formosan sambar deer. Korean J. Food Sci. Anim. Resour., 37, 542-551 (2017). https://doi.org/10.5851/kosfa.2017.37.4.542
  14. Zhao, L., Mi, Y., Guan, H., Xu, Y., Mei, Y., Velvet antler peptide prevents pressure overload-induced cardiac fibrosis via transforming growth factor (TGF)-β1 pathway inhibition. Eur. J. Pharmacol., 783, 33-46 (2016). https://doi.org/10.1016/j.ejphar.2016.04.039
  15. Sui, Z., Zhang, L., Huo, Y., Zhang, Y., Bioactive components of velvet antlers and their pharmacological properties. J. Pharm. Biomed. Anal., 87, 229-240 (2014). https://doi.org/10.1016/j.jpba.2013.07.044
  16. Zheng, K., Li, Q., Lin, D., Zong, X., Luo, X., Yang, M., Yue, X., Ma, S., Peptidomic analysis of pilose antler and its inhibitory effect on triple-negative breast cancer at multiple sites. Food Funct., 9, 7481-7494 (2020).
  17. Tang, Y., Fan, M., Choi, Y.J., Yu, Y., Yao, G., Deng, Y., Moon, S.H., Kim, E.K., Sika deer (Cervus nippon) velvet antler extract attenuates prostate cancer in xenograft model. Biosci. Biotechnol. Biochem., 83, 348-356 (2019). https://doi.org/10.1080/09168451.2018.1537775
  18. Wang, P., Sun, T.F., Li, G., Zhang, H.M., Liu, F.J., Gao, Z.H., Cao, S.N., Sun, G.D., Du, H.T., Wang, C.A., Wang, D.D., Shi, B., Lin, L., The separation of antler polypeptide and its effects on the proliferation and Osteogenetic differentiation of bone marrow mesenchymal stem cells. Evid. based Complement. Altern. Med., 2020, 1-10 (2020).
  19. He, J., Li, X., Wang, Z., Bennett, S., Chen, K., Xiao, Z., Zhan, J., Chen, S., Hou, Y., Chen, J., Wang, S., Xu, J., Lin, D., Therapeutic anabolic and anticatabolic benefits of natural Chinese medicines for the treatment of osteoporosis. Front. Pharmacol., 10, 1344 (2019).
  20. Zang, Z.J., Tang, H.F., Tuo, Y., Xing, W.J., Ji, S.Y., Gao, Y., Deng, C.H., Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice. Asian J. Androl., 18, 613-619 (2016). https://doi.org/10.4103/1008-682X.166435
  21. Xin, J.L., Zhang, Y., Li, Y., Zhang, L.Z., Lin, Y., Zheng, L.W., Protective effects of Cervus nippon Temminck velvet antler polypeptides against MPP+-induced cytotoxicity in SH-SY5Y neuroblastoma cells. Mol. Med. Rep., 16, 5143-5150 (2017). https://doi.org/10.3892/mmr.2017.7303
  22. Xiao, X., Li, L., Xu, S., Mao, M., Pan, R., Li, Y., Wu, J., Huang, L., Zheng, X., Evaluation of velvet antler total protein effect on bone marrow-derived endothelial progenitor cells. Mol. Med. Rep., 16, 3161-3168 (2017). https://doi.org/10.3892/mmr.2017.7019
  23. Ding, Y., Ko, S.C., Moon, S.H., Lee, S.H., Protective effects of novel antioxidant peptide purified from alcalase hydrolysate of velvet antler against oxidative stress in chang liver cells in vitro and in a zebrafish model in vivo. Int. J. Mol. Sci., 20, 5187 (2019).
  24. Zha, E., Li, X., Li, D., Guo, X., Gao, S., Yue, X., Immunomodulatory effects of a 3.2 kDa polypeptide from velvet antler of Cervus nippon Temminck. Int. Immunopharmacol., 16, 210-213 (2013). https://doi.org/10.1016/j.intimp.2013.02.027
  25. Broeder, C.E., Percival, R., Quindry, J., Panton, L., Wills, T., Browder, K.D., Earnest, C., Almada, A., Haines, S.R., Suttie, J.M., The effects of New Zealand deer antler velvet supplementation on body composition, strength, and maximal aerobic and anaerobic performance. Advances in antler science and product technology., 28, 161-165 (2004).
  26. Yoo, J., Lee, J., Zhang, M., Mun, D., Kang, M., Yun, B., Kim, Y.A., Kim, S., Oh, S., Enhanced γ-aminobutyric acid and sialic acid in fermented deer antler velvet and immune promoting effects. J. Anim. Sci. Technol., 64, 166-182 (2022). https://doi.org/10.5187/jast.2021.e132
  27. Tu, G., 1988. Pharmacopoeia of the people's republic of China, People's Medical Publishing House, Beijing, China, pp. 536.
  28. Association of Official Analytical Chemists (AOAC), 1990. AOAC official methods of analysis, 15th ed, AOAC, Arlington, VA, USA, pp. 171.
  29. Cesaretti, M., Luppi, E., Maccari, F., Volpi, N., A 96-well assay for uronic acid carbazole reaction. Carbohydr. Polym., 54, 59-61 (2003). https://doi.org/10.1016/S0144-8617(03)00144-9
  30. Farndale, R.W., Buttle, D.J., Barrett, A.J., Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. ActaGen. Subj., 883, 173-177 (1986). https://doi.org/10.1016/0304-4165(86)90306-5
  31. Warren, L., The thiobarbituric acid assay of sialic acids. J. Biol. Chem., 234, 1971-1975 (1959). https://doi.org/10.1016/S0021-9258(18)69851-5
  32. Jeon, B.T., Kim, M.H., Lee, S.M., Moon, S.H., Effects of dietary protein level on dry matter intake, and production and chemical composition of velvet antler in spotted deer fed forest by-product silage. Asian Australas. J. Anim. Sci., 19, 1737-1741 (2006). https://doi.org/10.5713/ajas.2006.1737
  33. Kolar, K., Colorimetric determination of hydroxyproline as measure of collagen content in meat and meat products: NMKL collaborative study. J. Assoc. Off. Anal. Chem., 73, 54-57 (1990).
  34. Zhang, Z.Y., Liu, X.F., Hou, Y.T., Wang, R.R., Li, P.F., Zhao, C.W., Quality control of chinese velvet antler by profiling of pesticide residues and heavy metals. Appl. Ecol. Environ. Res., 17, 10731-10742 (2019).
  35. Hu, Y., Wan, L., Zhang, J., Yang, F., Cao, J., Rapid determination of pesticide residues in Chinese materia medica using QuEChERS sample preparation followed by gas chromatography-mass spectrometry. Acta Pharm. Sin. B., 2, 286-293 (2012). https://doi.org/10.1016/j.apsb.2012.03.005
  36. Xue, P., Yao, Y., Yang, X.S., Feng, J., Ren, G.X., Improved antimicrobial effect of ginseng extract by heat transformation. J. Ginseng Res., 41, 180-187 (2017). https://doi.org/10.1016/j.jgr.2016.03.002
  37. Berridge, M.V., Tan, A.S., Characterization of the cellular reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch. Biochem. Biophys., 303, 474-482 (1993). https://doi.org/10.1006/abbi.1993.1311
  38. Organisation for Economic Co-operation and Development (OECD), 2008. Guidelines for testing of chemical. Acute oral toxicity: up-and-down-procedure, No. 425. OECD, Paris, TX, USA.
  39. Bird, P., Flannery, J., Crowley, E., Agin, J., Goins, D., Jechorek, R., Evaluation of the 3MTM PetrifilmTM Rapid Aerobic Count Plate for the Enumeration of Aerobic Bacteria: Collaborative Study, First Action 2015.13. J. AOAC Int., 99, 664-675 (2016). https://doi.org/10.5740/jaoacint.15-0260
  40. Brown, R.D., 1983. Nutrition and antler development in white-tailed deer. Antler development in Cervidae: A proceedings of the first international symposium of the Caesar Kleberg Wildlife Research Institute, College of Agriculture, Texas A&I University, Kingsville, TX, USA.
  41. Sunwoo, H.H., Sim, L.Y.M., Nakano, T., Hudson, R.J., Sim, J.S., Glycosaminoglycans from growing antlers of wapiti (Cervus elaphus). Can. J. Anim. Sci., 77, 715-721 (1997). https://doi.org/10.4141/A97-033
  42. Choi, H.K., Kim, K.H., Kim, K.H., Kim, Y.S., Lee, M.W., Whang, W.K., Metabolomic differentiation of deer antlers of various origins by 1H NMR spectrometry and principal components analysis. J. Pharm. Biomed. Anal., 41, 1047-1050 (2006). https://doi.org/10.1016/j.jpba.2006.01.036
  43. Chapman, D.I., Antlers-bones of contention. Mammal Rev. 5, 121-172 (1975). https://doi.org/10.1111/j.1365-2907.1975.tb00194.x
  44. Jeon, B., Kim, S., Lee, S., Park, P., Sung, S., Kim, J., Moon, S., Effect of antler growth period on the chemical composition of velvet antler in sika deer (Cervus nippon). Mamm. Biol., 74, 374-380 (2009). https://doi.org/10.1016/j.mambio.2008.07.005
  45. Hunter, G.K., Role of proteoglycan in the provisional calcification of cartilage: A review and reinterpretation. Clin. Orthop. Rel. Res., 262, 256-280 (1991). https://doi.org/10.1097/00003086-199101000-00035
  46. Sunwoo, H.H., Nakano, T., Hudson, R.J., Sim, J.S., Chemical composition of antlers from wapiti (Cervus elaphus). J. Agric. Food Chem., 43, 2846-2849 (1995). https://doi.org/10.1021/jf00059a014
  47. Ha, Y.W., Jeon, B.T., Moon, S.H., Toyoda, H., Toida, T., Linhardt, R.J., Kim, Y.S., Characterization of heparan sulfate from the unossified antler of Cervus elaphus. Carbohydr. Res., 340, 411-416 (2005). https://doi.org/10.1016/j.carres.2004.11.011
  48. Goss, R., 1983. Developmental anatomy of antlers. Deer antlers: Regeneration, function and evolution, Academics Press, New York, NY, USA, pp. 33-171.
  49. Salek, R.M., Steinbeck, C., Viant, M.R., Goodacre, R., Dunn, W.B., The role of reporting standards for metabolite annotation and identification in metabolomic studies. GigaScience., 2, 13 (2013).
  50. Li, Z.H., Zhao, W.H., Zhou, Q.L., Experimental study of velvet antler polypeptides against oxidative damage of osteoarthritis cartilage cells. Chin. J. Orthop. Trauma., 24, 245-248 (2011).
  51. Zhang, Z., Liu, X., Duan, L., Li, X., Zhang, Y., Zhou, Q., The effects of velvet antler polypeptides on the phenotype and related biological indicators of osteoarthritic rabbit chondrocytes. Acta Biochim. Pol., 58, 297-302 (2011).
  52. Wu, F., Li, H., Jin, L., Li, X., Ma, Y., You, J., Li, S., Xu, Y., Deer antler base as a traditional Chinese medicine: a review of its traditional uses, chemistry and pharmacology. J. Ethnopharmacol., 145, 403-415 (2013). https://doi.org/10.1016/j.jep.2012.12.008
  53. Suh, S.J., Kim, K.S., Lee, A.R., Ha, K.T., Kim, J.K., Kim, D.S., Lee, Y.C., Kim, M.S., Kwon, D.Y., Kim, C.H., Prevention of collagen-induced arthritis in mice by Cervus korean TEMMINCK var. mantchuricus Swinhoe. Environ. Toxicol. Pharmacol., 23, 147-153 (2007). https://doi.org/10.1016/j.etap.2006.08.001
  54. Fraser, A., Haines, S., Stuart, E., Scandlyn, M., Alexander, A., Somers-Edgar, T., Rosengren, R., Deer velvet supplementation decreases the grade and metastasis of azoxymethane-induced colon cancer in the male rat. Food Chem. Toxicol., 48, 1288-1292 (2010). https://doi.org/10.1016/j.fct.2010.02.024
  55. Kang, C.Z., Guo, L.P., Zhou, T., Zhao, D., Kang, L.P., He, Y.L., Wang, S., Zhou, L.Y., Discussion on present situation of study on pesticide residues in Chinese herbal medicines. Zhongguo Zhong Yao Za Zhi., 41, 155-159 (2016).
  56. Yang, Y.H., Dou, X.W., Kong, W.J., Yang, M.H., Chen, S.L., Xiao, Q., Status of pesticide registration and residue analysis for traditional Chinese medicine in China. Zhongguo Zhong Yao Za Zhi., 38, 4238-4245 (2013).
  57. Lee, S.D., Jung, J.Y., Choi, K.H., Lee, J.T., Park, H.M., Shin, H.T., Risk analysis and estimating consumption of heavy metal from intake of oriental medicines. J. Environ. Health Sci., 36, 14-19 (2010).
  58. He, G., Zhao, Q., Zhao, Y., Zong, Y., Gu, S., Li, M., Li, R., Sun, J., Deer antler based active ingredients have protective effects on LPS/d-GalN-induced acute liver injury in mice through MAPK and NF-κB signalling pathways. Pharm. Biol., 60, 1077-1087 (2022). https://doi.org/10.1080/13880209.2022.2068617
  59. Reigner, B.G., Blesch, K., Estimating the starting dose for entry into humans: principles and practice. Eur. J. Clin. Pharmacol., 57, 835-845 (2002). https://doi.org/10.1007/s00228-001-0405-6
  60. Nair, A.B., Jacob, S., A simple practice guide for dose conversion between animals and human. J. Basic and clin. Phar (JBCP)., 7, 27-31 (2016). https://doi.org/10.4103/0976-0105.177703
  61. Lee, J., Jeong, J.S., Cho, K.J., Moon, K.N., Kim, S.Y., Han, B., Kim, Y.S., Jeong, E.J., Chung, M.K., Yu, W.J., Developmental and reproductive toxicity assessment in rats with KGC-HJ3, Korean Red Ginseng with Angelica gigas and Deer antlers. J. Ginseng Res., 43, 242-251 (2019). https://doi.org/10.1016/j.jgr.2017.12.004
  62. Barbour, E.K., Nabbut, N.H., Hamadeh, S.K., Al-Nakhli, H.M., Bacterial identity and characteristics in healthy and unhealthy respiratory tracts of sheep and calves. Vet. Res. Commun., 21, 421-430 (1997). https://doi.org/10.1023/A:1005855318665