簡易檢索 / 詳目顯示

研究生: 吳炫梅
Wu, Hsuan-Mei
論文名稱: 龜鹿二仙膠抗氧化能力與對關節細胞活性的影響
Effects of Guilu Erxian Glue on Antioxidant Capacity and Joint Cell Activity
指導教授: 吳忠信
Wu, Chung-Hsin
口試委員: 沈賜川
Shen, Szu-Chuan
連家瑩
Lien, Chia-Ying
吳忠信
Wu, Chung-Hsin
口試日期: 2023/06/10
學位類別: 碩士
Master
系所名稱: 生技醫藥產業碩士學位學程
Graduate Program of Biotechnology and Pharmaceutical Industries
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 40
中文關鍵詞: 龜鹿二仙膠胜肽溶離試驗生物確效骨質疏鬆關節退化
英文關鍵詞: Guilu Erxian Gum, Peptides, Dissociation assay, Bio-Validation, Osteoporosis, Joint Degeneration
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202300613
論文種類: 學術論文
相關次數: 點閱:112下載:15
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 龜鹿二仙膠是由龜板和鹿角等重要藥材,再加上枸杞、人參等多種天然成分熬製而成的中藥製劑。它的功效在於強化骨骼和減緩關節退化的症狀,常被民間視為補筋骨的良方。然而,龜鹿二仙膠在市場上的價格差異極大,品質也參差不齊,時常發生以假亂真的情況。為此,我們希望針對龜鹿二仙膠成分中的龜板和鹿角建立中藥指紋圖譜,並且檢視龜板與鹿角溶離胜肽的抗氧化能力以及對於關節滑膜細胞(synoviocyte, HIG-82)的活性,希望藉此確立龜鹿二仙膠的成分品質,並且從龜鹿二仙膠篩選出有效緩解關節退化的成分,提供科學中藥廠家研製成有效緩解關節退化的健康食品或是藥物。我們首先與科學中藥廠家合作,建立傳統中藥龜鹿二仙膠的中藥成分指紋圖譜;然後選取科學中藥廠家提供的龜板與鹿角溶離胜肽成分,利用DPPH抗自由基實驗檢視龜板與鹿角以及溶離後胜肽成分的抗氧化能力;以及採用關節滑膜細胞,利用MTT細胞存活試驗檢視龜板與鹿角溶離胜肽對關節滑膜細胞的生長存活率,藉此篩選出有效緩解關節退化的溶離胜肽成分。本實驗結果顯示:從龜板與鹿角的13種溶離胜肽成分中,發現從鹿角萃取的7種溶離胜肽成分的抗氧化能力較佳,對於關節滑膜細胞的細胞生長存活情形也最佳,至於其他從龜板萃取的6種溶離胜肽成分的抗氧化能力則較差,對於關節滑膜細胞的細胞生長存活情形也較鹿角萃取的7種溶離胜肽來得差。從胺基酸序列的分析發現抗氧化能力佳以及對於關節滑膜細胞的細胞生長有利的溶離胜肽成分,均含有丙胺酸(Alanine,Ala)-絲胺酸(Serine,Ser)-半胱胺酸(Cysteine,Cys)的序列片段。換句話說,從鹿角溶離胜肽對於抗氧化能力以及關節滑膜細胞的細胞生長,都比龜板溶離胜肽來得效果佳。因此,本論文推論從鹿角溶離胜肽應該比較有機會研製成有效緩解關節退化的健康食品或是藥物。

    The main medicinal materials of Guilu Erxian Gum (GEG) include tortoise plastron and antlers with adding wolfberry and ginseng to strengthen bone density and slow down the degeneration of joints. In view of the fact that Taiwanese folks often use GEG to replenish their muscles and bones, but the market price varies greatly, the quality is different, and the fake is often reported. To this end, we hope to establish a fingerprint of traditional Chinese medicine for the tortoise plastron and antlers in GEG, and to examine the antioxidant capacity and the activity of synovial cells (synoviocyte, HIG-82) in dissolved peptides from tortoise plastron and antler. We hoped to establish the quality of GEG, and screen out dissolved peptides from GEG that can effectively relieve joint degeneration, and provide traditional Chinese medicine (TCM) manufacturers to develop healthy food or medicine that can effectively relieve joint degeneration. We first cooperated with TCM manufacturers to establish the fingerprints of GEG. Then we selected the dissolved peptides from tortoise plastron and antler that provided by TCM manufacturer, and used the DPPH anti-free radical assay to examine the antioxidant capacity in these dissolved peptides. We used the MTT cell survival assay to examine survival rate of joint synoviocytes in these dissolved peptides. We hoped to screen out the dissolved peptides that can effectively relieve joint degeneration. The results of our experiment showed that among 13 dissolved peptides from tortoise plastron and antlers, Seven dissolved peptides components extracted from antlers have been found that have better antioxidant capacity, and better cell growth and survival rate of joint synoviocytes. As for the other six dissolved peptides extracted from tortoise plastron, the antioxidant capacity is poor, and the cell growth and survival of joint synoviocytes are also worse than dissolved peptides from antlers. From the analysis of the amino acid sequence, it was found that the dissolved peptide components with good antioxidant capacity and beneficial to the cell growth of joint synoviocytes all contain sequence of Alanine (Ala)-Serine (Ser)-Cysteine (Cys). In other words, dissolved peptides from deer antlers have a better effect on antioxidant capacity and cell growth of synoviocytes than dissolved peptides from tortoise plastron. We suggested that dissolved peptides from antlers may have a better chance to develop into healthy food or medicine that can effectively relieve joint degeneration.

    第一章緒論 1 第一節 研究背景1 第二節 文獻探討 3 第三節 研究目的 7 第二章 研究材料與方法 9 第一節 龜鹿二仙膠指紋圖譜分析與溶離胜肽配製 9 第二節 龜板與鹿角溶離胜肽清除氧化自由基能力的DPPH的測定 10 第三節 龜板與鹿角溶離胜肽對關節滑膜細胞生長情形的MTT測定 11 第四節 統計與資料分析 12 第三章 研究結果 13 第一節 龜鹿二仙膠的指紋圖譜 13 第二節 龜板膠、鹿角膠與龜鹿二仙膠的抗氧化能力比較 14 第三節 龜板與鹿角溶離胜肽的胺基酸序列 14 第四節 龜鹿溶離胜肽的抗氧化能力比較 14 第五節 龜鹿溶離胜肽對關節滑膜細胞的活性與生長 15 第四章 討論 16 第五章 結論 22 參考文獻 23 附錄 實驗圖表 32

    Buendia, I., Michalska, P., Navarro, E., Gameiro, I., Egea, J., & León, R. (2016). Nrf2-ARE pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacology & therapeutics, 157, 84–104. https://doi.org/10.1016/j.pharmthera.2015.11.003

    Cheung, H.Y., Cheung, C.S. (1998) Nutritive value of plastron extracts and its effects on the differentiation of cancer cell. Hong Kong Pharm. J., 7, pp. 104-107.

    Chonco, L., Landete-Castillejos, T., Serrano-Heras, G., Serrano, M. P., Pérez-Barbería, F. J., González-Armesto, C., García, A., de Cabo, C., Lorenzo, J. M., Li, C., & Segura, T. (2021). Anti-tumour activity of deer growing antlers and its potential applications in the treatment of malignant gliomas. Scientific reports, 11(1), 42. https://doi.org/10.1038/s41598-020-79779-w

    Chou, Y. J., Chuu, J. J., Peng, Y. J., Cheng, Y. H., Chang, C. H., Chang, C. M., & Liu, H. W. (2018). The potent anti-inflammatory effect of Guilu Erxian Glue extracts remedy joint pain and ameliorate the progression of osteoarthritis in mice. Journal of orthopaedic surgery and research, 13(1), 259. https://doi.org/10.1186/s13018-018-0967-y

    Chunhua, M., & Hongyan, L. (2017). Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice. International journal of biological macromolecules, 99, 648–654. https://doi.org/10.1016/j.ijbiomac.2017.03.024

    Chunhui, Y., Wenjun, C., Hui, W., Liquan, S., Changwei, Z., Tianzhu, Z., & Wenhai, Z. (2017). Pilose antler peptide protects osteoblasts from inflammatory and oxidative injury through EGF/EGFR signaling. International journal of biological macromolecules, 99, 15–20. https://doi.org/10.1016/j.ijbiomac.2017.02.056

    Dong, Y., Liu, L., Shan, X., Tang, J., Xia, B., Cheng, X., Chen, Y., & Tao, W. (2018). Pilose antler peptide attenuates LPS-induced inflammatory reaction. International journal of biological macromolecules, 108, 272–276. https://doi.org/10.1016/j.ijbiomac.2017.11.176

    Du, F., Zhao, H., Yao, M., Yang, Y., Jiao, J., & Li, C. (2022). Deer antler extracts reduce amyloid-beta toxicity in a Caenorhabditis elegans model of Alzheimer's disease. Journal of ethnopharmacology, 285, 114850. https://doi.org/10.1016/j.jep.2021.114850

    Gu, Y., Lu, X., Jiang, G., Deng, J., Tang, S., Li, S. (2007) Comparison of the nourishing-yin functions of different types of tortoise shell. Lishizhen Med. Mater. Med. Res. 18: 1417–1418.

    Guo, Y., Zhou, Q., Liu, P., Wang, Y., Fang, J., Wang, B. (1998) The research of pilose antler polypeptides promoting osteoblast precursor cells and chondrocytes proliferation. J Chinese J Biochem Pharmaceutics 19: 74–75.

    Gyamfi, M. A., Yonamine, M., & Aniya, Y. (1999). Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries. General pharmacology, 32(6), 661–667. https://doi.org/10.1016/s0306-3623(98)00238-9

    He, G., Zhao, Q., Zhao, Y., Zong, Y., Gu, S., Li, M., Li, R., & Sun, J. (2022). 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. Pharmaceutical biology, 60(1), 1077–1087. https://doi.org/10.1080/13880209.2022.2068617

    Ho, T. J., Lin, J. H., Lin, S. Z., Tsai, W. T., Wu, J. R., & Chen, H. P. (2023). Isolation, Identification, and Characterization of Bioactive Peptides in Human Bone Cells from Tortoiseshell and Deer Antler Gelatin. International journal of molecular sciences, 24(2), 1759. https://doi.org/10.3390/ijms24021759

    Hu, W., Qi, L., Tian, Y. H., Hu, R., Wu, L., & Meng, X. Y. (2015). Studies on the purification of polypeptide from sika antler plate and activities of antitumor. BMC complementary and alternative medicine, 15, 328. https://doi.org/10.1186/s12906-015-0845-7

    Jiang, N., Zhang, S., Zhu, J., Shang, J., & Gao, X. (2015). Hypoglycemic, hypolipidemic and antioxidant effects of peptides from red deer antlers in streptozotocin-induced diabetic mice. The Tohoku journal of experimental medicine, 236(1), 71–79. https://doi.org/10.1620/tjem.236.71

    Kim, K. H., Kim, K. S., Choi, B. J., Chung, K. H., Chang, Y. C., Lee, S. D., Park, K. K., Kim, H. M., & Kim, C. H. (2005). Anti-bone resorption activity of deer antler aquaacupunture, the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong) in adjuvant-induced arthritic rats. Journal of ethnopharmacology, 96(3), 497–506. https://doi.org/10.1016/j.jep.2004.09.039

    Kim, K. W., Song, K. H., Lee, J. M., Kim, K. S., Kim, S. I., Moon, S. K., Kang, B. S., Kim, D. S., Chung, K. H., Chang, Y. C., & Kim, C. H. (2008). Effects of TGFbeta1 and extracts from Cervus korean TEMMINCK var. mantchuricus Swinhoe on acute and chronic arthritis in rats. Journal of ethnopharmacology, 118(2), 280–283. https://doi.org/10.1016/j.jep.2008.04.010

    Kraan, P.M., Blaney Davidson, E.N., Blom, A., Berg, W.B. (2009) TGF-beta signaling in chondrocyte terminal differentiation and osteoarthritis: modulation and integration of signaling pathways through receptorSmads. J Osteoarthritis Cartilage 17: 1539–1545.

    Lee, T. H., Jung, H., Park, K. H., Bang, M. H., Baek, N. I., & Kim, J. (2014). Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells. Experimental biology and medicine (Maywood, N.J.), 239(10), 1325–1334. https://doi.org/10.1177/1535370214533883

    Li, L. Q., Baibado, J. T., Shen, Q., & Cheung, H. Y. (2017). Determination of the authenticity of plastron-derived functional foods based on amino acid profiles analysed by MEKC. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 1070, 23–30. https://doi.org/10.1016/j.jchromb.2017.10.036

    Li, X., Xie, X., Huang, C., Zhong, Y., Li, Y., Zhou, J., Chen, D. (2007) Repairing of oxidative damage to mesenchymal stem cell in rats and anti-lipid peroxidation by Plastrum testudinis ethanolic extract. Chin. Tradit. Herb. Drugs 38:1043–1046.

    Li, Y., Cui, X., Chen, D., Du, S., Li, H., Zhou, J. (2003) Effect of tortoises shell on neural stem cell following injuried spinal cord. Chin. J. Neuroanat. 19: 321–324.

    Liao, J. A., Yeh, Y. C., & Chang, Z. Y. (2022). The efficacy and safety of traditional Chinese medicine Guilu Erxian Jiao in the treatment of knee osteoarthritis: A systematic review and meta-analysis. Complementary therapies in clinical practice, 46, 101515.

    Lien, C. Y., Lu, C. W., Lin, Y. H., Wu, W. J., Hsu, C. H., Chuang, T. Y., Lin, K. F., Chuang, W. C., Lee, M. C., & Wu, C. H. (2021). Chinese Herbal Medicine, Guilu Erxian Glue, as Alternative Medicine for Adverse Side Effects of Chemotherapy in Doxorubicin-Treated Cell and Mouse Models. Evidence-based complementary and alternative medicine : eCAM, 2021, 5548968. https://doi.org/10.1155/2021/5548968

    Liu, C., Xia, Y., Hua, M., Li, Z., Zhang, L., Li, S., Gong, R., Liu, S., Wang, Z., & Sun, Y. (2020). Functional properties and antioxidant activity of gelatine and hydrolysate from deer antler base. Food science & nutrition, 8(7), 3402–3412. https://doi.org/10.1002/fsn3.1621

    Liu, Y., Li, H., Li, Y., Yang, M., Wang, X., & Peng, Y. (2021). Velvet Antler Methanol Extracts Ameliorate Parkinson's Disease by Inhibiting Oxidative Stress and Neuroinflammation: From C. elegans to Mice. Oxidative medicine and cellular longevity, 2021, 8864395. https://doi.org/10.1155/2021/8864395

    Luo, D., Li, H., Li, X., Zhou, J., Du, F., Wang, L. (2008) Determination of collagen in tortoise shell using HPLC with phenyl isothiocyanate derivatization. Chin. Tradit. Herb. Drugs 39: 851–852.

    Mao, C., Zhang, Y., Yan, W., & Zheng, X. (2008). Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi, 25(4), 897–902.

    Nardone, V., D'Asta, F., & Brandi, M. L. (2014). Pharmacological management of osteogenesis. Clinics (Sao Paulo, Brazil), 69(6), 438–446. https://doi.org/10.6061/clinics/2014(06)12

    Qu, M., Kang, Y. (2000) Study on biological characteristics of hypertrophic chondrocytes of damaged articular cartilage. J Chinese J Sports Med 19: 76–81

    Shin, I. S., Ahn, K. S., Shin, N. R., Lee, H. J., Ryu, H. W., Kim, J. W., Sohn, K. Y., Kim, H. J., Han, Y. H., & Oh, S. R. (2016). Protective effect of EC-18, a synthetic monoacetyldiglyceride on lung inflammation in a murine model induced by cigarette smoke and lipopolysaccharide. International immunopharmacology, 30, 62–68. https://doi.org/10.1016/j.intimp.2015.11.025

    Si, Y., Yao, Y., Ma, Y., Guo, Y., & Yin, H. (2020). Effectiveness and safety of Guilu Erxian Glue (a traditional Chinese medicinal product) for the treatment of postmenopausal osteoporosis: A protocol for systematic review and meta-analysis. Medicine, 99(29), e20773. https://doi.org/10.1097/MD.0000000000020773

    Tang, Y., Jeon, B. T., Wang, Y., Choi, E. J., Kim, Y. S., Hwang, J. W., Park, P. J., Moon, S. H., & Kim, E. K. (2015). First Evidence that Sika Deer (Cervus nippon) Velvet Antler Extract Suppresses Migration of Human Prostate Cancer Cells. Korean journal for food science of animal resources, 35(4), 507–514. https://doi.org/10.5851/kosfa.2015.35.4.507

    Tsai, C. C., Chou, Y. Y., Chen, Y. M., Tang, Y. J., Ho, H. C., & Chen, D. Y. (2014). Effect of the herbal drug guilu erxian jiao on muscle strength, articular pain, and disability in elderly men with knee osteoarthritis. Evidence-based complementary and alternative medicine : eCAM, 2014, 297458. https://doi.org/10.1155/2014/297458

    Wang, W., Zhang, J., Yang, X., & Huang, F. (2019). Hypoglycemic activity of CPU2206: A novel peptide from sika (Cervus nippon Temminck) antler. Journal of food biochemistry, 43(12), e13063. https://doi.org/10.1111/jfbc.13063

    Wu, F., Li, H., Jin, L., Li, X., Ma, Y., You, J., Li, S., & Xu, Y. (2013). Deer antler base as a traditional Chinese medicine: a review of its traditional uses, chemistry and pharmacology. Journal of ethnopharmacology, 145(2), 403–415. https://doi.org/10.1016/j.jep.2012.12.008

    Wu, M. H., Lee, T. H., Lee, H. P., Li, T. M., Lee, I. T., Shieh, P. C., & Tang, C. H. (2017). Kuei-Lu-Er-Xian-Jiao extract enhances BMP-2 production in osteoblasts. BioMedicine, 7(1), 2. https://doi.org/10.1051/bmdcn/2017070102

    Xia, P., Liu, D., Jiao, Y., Wang, Z., Chen, X., Zheng, S., Fang, J., & Hao, L. (2022). Health Effects of Peptides Extracted from Deer Antler. Nutrients, 14(19), 4183. https://doi.org/10.3390/nu14194183

    Xie, X., Li, X., Zhong, Y., Huang, C., Du, S., Li, Y., Chen, D. (2006) Study of antioxidant activities of Plastrum testudinis in Vitro. China Pharm. 17: 1368–1370.

    Xin, J. L., Zhang, Y., Li, Y., Zhang, L. Z., Lin, Y., & Zheng, L. W. (2017). Protective effects of Cervus nippon Temminck velvet antler polypeptides against MPP+‑induced cytotoxicity in SH‑SY5Y neuroblastoma cells. Molecular medicine reports, 16(4), 5143–5150. https://doi.org/10.3892/mmr.2017.7303

    Yang, Q., Lin, J. N., Sui, X., Li, H., Kan, M., Wang, J. F., Li, J., Zhang, Z., Liu, X. R., Ming, S. T., Qu, X. B., & Li, N. (2020). Antiapoptotic effects of velvet antler polypeptides on damaged neurons through the hypothalamic-pituitary-adrenal axis. Journal of integrative neuroscience, 19(3), 469–477. https://doi.org/10.31083/j.jin.2020.03.167

    Yao, B., Zhang, M., Leng, X., Liu, M., Liu, Y., Hu, Y., Zhao, D., & Zhao, Y. (2018). Antler extracts stimulate chondrocyte proliferation and possess potent anti-oxidative, anti-inflammatory, and immune-modulatory properties. In vitro cellular & developmental biology. Animal, 54(6), 439–448. https://doi.org/10.1007/s11626-018-0266-2

    Yun, C., Qian, W., Wu, J., Yuan, C., Jiang, S., & Lv, J. (2020). Pilose antler peptide promotes osteoblast proliferation, differentiation and mineralization via the insulin signaling pathway. Experimental and therapeutic medicine, 19(2), 923–930. https://doi.org/10.3892/etm.2019.8286

    Zang, Z. J., Tang, H. F., Tuo, Y., Xing, W. J., Ji, S. Y., Gao, Y., & Deng, C. H. (2016). Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice. Asian journal of andrology, 18(4), 613–619. https://doi.org/10.4103/1008-682X.166435

    Zhang, J. M., & An, J. (2007). Cytokines, inflammation, and pain. International anesthesiology clinics, 45(2), 27–37. https://doi.org/10.1097/AIA.0b013e318034194e

    Zhang, L. Z., Xin, J. L., Zhang, X. P., Fu, Q., Zhang, Y., & Zhou, Q. L. (2013). The anti-osteoporotic effect of velvet antler polypeptides from Cervus elaphus Linnaeus in ovariectomized rats. Journal of ethnopharmacology, 150(1), 181–186. https://doi.org/10.1016/j.jep.2013.08.029

    Zhang, W., Ke, C. H., Guo, H. H., & Xiao, L. (2021). Antler stem cells and their potential in wound healing and bone regeneration. World journal of stem cells, 13(8), 1049–1057. https://doi.org/10.4252/wjsc.v13.i8.1049

    Zhang, Z., Liu, X., Duan, L., Li, X., Zhang, Y., & Zhou, Q. (2011). The effects of velvet antler polypeptides on the phenotype and related biological indicators of osteoarthritic rabbit chondrocytes. Acta biochimica Polonica, 58(3), 297–302.

    Zhao, L., Li, J. H., Zhu, D. Z., & Ji, B. P. (2010). Principal component analysis of nutrients in five varieties of velvet antler (Cornu Cervi Pantotrichum). Guang Pu Xue Yu Guang Pu Fen Xi 30(9): 2571–2575.

    Zhao, S., Zuo, W., Chen, H., Bao, T., Liu, X., Sun, T., & Wang, S. (2019). Effects of pilose antler peptide on bleomycin-induced pulmonary fibrosis in mice. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 109, 2078–2083. https://doi.org/10.1016/j.biopha.2018.08.114

    Zheng, K., Li, Q., Lin, D., Zong, X., Luo, X., Yang, M., Yue, X., & Ma, S. (2020). Peptidomic analysis of pilose antler and its inhibitory effect on triple-negative breast cancer at multiple sites. Food & function, 11(9), 7481–7494. https://doi.org/10.1039/d0fo01531h

    Zhu, W., Wang, H., Zhang, W., Xu, N., Xu, J., Li, Y., Liu, W., & Lv, S. (2017). Protective effects and plausible mechanisms of antler-velvet polypeptide against hydrogen peroxide induced injury in human umbilical vein endothelial cells. Canadian journal of physiology and pharmacology, 95(5), 610–619. https://doi.org/10.1139/cjpp-2016-0196

    下載圖示
    QR CODE