臺灣博碩士論文加值系統

C型肝炎病毒(Hepatitis C virus)屬於黃熱病毒科(flaviviridae) 的成員，是引起人類肝炎之主要病毒，全球約有一億七千萬人遭受感染，是全球公共衛生與健康的重要議題。近年針對病毒非結構蛋白(viral non-structural proteins)作用設計出的直接作用抗病毒藥物(direct-acting antivirals; DAAs)，雖可大幅改善過去干擾素-α(PEGylated interferon-alpha)及雷巴威林(ribavirin)的治療效果，但是仍存在一些限制，例如：高成本、藥物交互作用(drug-drug interactions ; DDIs)、預防HCV感染的措施以及抗藥突變株出現等問題。因此，開發出新的先導化合物來補充或擴大目前的治療方案，特別是作用於早期病毒進入(early entry)機制的抗病毒藥物，有助於改善及管理C型肝炎病人所產生的問題。本篇研究分析黃連(Rhizoma Coptidis; RC)甲醇萃取物，透過HCVcc及HCVpp進行一系列的實驗，例如：抗病毒試驗、病毒侵入試驗、給藥時間點試驗等，藉以了解生物活性物質抗HCV的作用機制。此外，也透過黃連主要指標活性物質小蘗鹼(berberine; BBR)，對HCV早期病毒進入階段的影響進行探討，以及利用分子嵌合模擬分析，預測藥物與HCV醣蛋白之間的交互作用。實驗結果發現，黃連甲醇萃取物及小蘗鹼(berberine; BBR)的CC50分別為168.9±1.06 g/ ml、82.75±0.27 M; EC50分別為20.07±1.08 g /ml、7.87±1.10 M; SI分別為8.42、10.51。黃連甲醇萃取物及小蘗鹼(berberine; BBR)的濃度分別為50 g /mL及20 g /mL時，不會引起明顯的細胞毒性。此外也發現，黃連甲醇萃取物及小蘗鹼可阻斷病毒附著、進入及融合作用，證明了黃連甲醇萃取物及小蘗鹼是透過干擾HCV早期病毒進入步驟(HCV early viral entry steps)方式抑制病毒。最後也發現黃連甲醇萃取物可以抑制其他基因型的C型肝炎病毒，包含：基因型2b（J8 / JFH1），3a（S52 / JFH1）和7a（QC69 / JFH1），表示黃連甲醇萃取物具有抑制HCV泛基因型的能力。在分子嵌合(molecular docking)實驗結果方面，發現小蘗鹼(berberine; BBR)具有與HCV醣蛋白交互作用的潛力，進而干擾HCV早期進入的步驟(early entry stage)。本研究為首度發現黃連甲醇提取物和其主要指標活性成分小蘗鹼(berberine; BBR)，具有強效抑制HCV侵入細胞的能力，具有發展成抗C型肝炎病毒新藥的候選藥物。

HCV is an important threat to public health with approximately 170 million carriers worldwide. Although the introduction of direct-acting antivirals (DAAs) has remarkably improved the therapeutic effects, high costs, drug-drug interactions (DDIs), and development of drug-resistant mutants may limit their use. Further development of new lead compounds to supplement the current treatment options is urgently needed. We investigated the anti-HCV activity of the methanolic extract from Rhizoma Coptidis (RC) and their related active compound berberine (BBR). In this study, dose-response analysis, time-of-drug-addition assay, and synchronized infection analysis on early viral entry were used to identify the effective dosage and target window of RC against HCV infection. In addition, the effect of berberine (BBR) on the early stage of HCV entry was also examined. Molecular docking was performed using Autodock Vina (Scripps, LLC; La Jolla, CA, USA) to predict the free energy associations of bound conformations between ligand and protein targets. The 50 % cytotoxic concentration (CC50), the 50 % effective concentration (EC50), and the selective index (SI) of the Rhizoma Coptidis and BBR were determined to be 168.9±1.06 g/ mL、20.07±1.08 g /mL; 8.42 and 82.75±0.27 M、7.87±1.10 M; 10.51, respectively. Our results showed that RC and BBR could inhibit HCV infection dose-dependently and up to 50 μg/ml and 20 μg/ml without inducing significant cytotoxicity. Our data showed that the methanolic extract of RC and BBR robustly inhibited HCV infection. Specifically, RC mainly targeted the HCV early viral entry steps such as attachment and entry/fusion to the host cells. Furthermore, RC inhibits multiple HCV genotypes, include: genotypes 2b (J8/JFH1), 3a (S52/JFH1), and 7a (QC69/JFH1). The data showed that molecular docking analysis pointed at potential interaction with HCV glycoprotein E2. This study is the first to discover the methanolic extract of Rhizoma Coptidis and its bioactive substance berberine as potent HCV entry inhibitors, which could be useful for developing novel antiviral agents against HCV.

中文摘要………………………………………………………..…....….7
英文摘要(abstract)……………………………………………..….……9
第一章 緒論……………………………………………………...….…11
第一節 C型肝炎流病學及治療現況介紹…………………..…..…11
第二節 C型肝炎病毒介紹…………………………………..….….13
第三節 慢性C型肝炎之治療進展與限制瓶頸……….……..……16
第四節 生藥黃連(Rhizoma Coptidis)之簡介……………….......….20
第二章 研究目的、方向與設計……………………………..……..…23
第一節 研究目的……………………………………….….……..…23
第二節 研究方向及原理…………………………………..…….….24
2.2.1. 第一期：生藥黃連(Rhizoma Coptidis; RC)的基原鑑定、病毒及細胞製備、細胞毒性及抗病毒試驗…..…..24
2.2.2. 第二期/第一階段：探討黃連甲醇萃取物與小蘗鹼(berberine, BBR)在C型肝炎病毒感染生命週期之影響…………….……..………….……………………....25
2.2.3. 第二期/第二階段：探討小蘗鹼(berberine, BBR)對宿主細胞之影響………………..……………………26
2.2.4. 實驗設計流程圖(flow chart of study design)…...………..27
第三章 材料與方法……………………………………………………28
第一節 黃連甲醇萃取物及小蘗鹼(berberine, BBR)製備……..…..28
第二節 黃連HPLC指紋圖譜分析及小蘗鹼(berberine; BBR)
含量測定…………….……….……....………………..…...29
第三節 細胞培養與測試病毒株製備……………………………....30
3.3.1. 細胞株/cell line……………………………………….…..30
3.3.2. 病毒株/virus strain………………………………….…….30
第四節 細胞毒性及抗病毒試驗…………………………………....31
3.4.1. 細胞毒性試驗(cytotocixity assay)………………….…….31
3.4.2. 抗病毒試驗(antiviral assay)………………………….…...31
第五節 給藥時間點試驗………………………………………...….32
3.5.1. 先投予藥物試驗(pre-treatment assay)………………..….32
3.5.2. 藥物和病毒同時給予試驗(co-addition treatment assay)..32
3.5.3. 感染後再投藥試驗(post-infection treatment assay)……..33
第六節 病毒侵入試驗…………..………………..……………...….34
3.6.1. 無細胞依附(cell-free attachment)病毒顆粒試驗………...34
3.6.2. 抑制病毒吸附(attachment)試驗………..………………...34
3.6.3. 抑制病毒融合(entry/fusion)試驗………..……………….35
第七節 藥物影響宿主細胞進入因子(entry factors)試驗..………...36
第八節 病毒結合細胞膜試驗…………………………………...….37
第九節 C型肝炎病毒假顆粒(pseudoparticles)感染試驗……....….38
3.9.1. C型肝炎病毒假顆粒製備(preparation of HCV pseudoparticles; HCVpp)…..………………………..…...38
3.9.2. HCVpp感染性試驗(HCVpp infectivity assay)…………..38
第十節 病毒複製試驗……………………….……………………...39
第十一節 抑制多基因型C型肝炎病毒試驗………………......….40
第十二節 干擾素拮抗作用試驗………………………….……..….41
第十三節 初代人類肝細胞感染HCVcc之抗病毒能力分析….….42
第十四節 藥物分子嵌合(molecular docking)模擬分析……………43
第十五節 統計分析……………………………………………..…..44
第四章 結果……………………………………………………………45
第一節 生藥黃連藥材基源鑑定及黃連甲醇萃取物抑制C型肝炎
病毒早期進入宿主細胞之機制……..……………..……....45
4.1.1. 生藥黃連藥材HPLC指紋圖譜及小蘗鹼(berberine, BBR)
含量分析結果………….…..............……………………...45
4.1.2. 黃連甲醇萃取物抑制C型肝炎病毒感染宿主細胞能力..45
4.1.2.1. 黃連甲醇萃取物對細胞毒性及抗HCV能力分析結果.45
4.1.2.2. 黃連甲醇萃取物不同給藥時間點試驗分析結果……..46
4.1.2.3. 黃連甲醇萃取物抗C型肝炎病毒侵入試驗分析
結果……………………………………………………..47
4.1.2.4. 黃連甲醇萃取物影響C型肝炎病毒與宿主細胞
結合試驗分析結果………………………………....…48
4.1.2.5. 黃連甲醇萃取物抑制C型肝炎病毒假顆粒感染
宿主細胞能力分析結果…………..…………………..48
4.1.2.6. 黃連甲醇萃取物影響宿主細胞進入因子(entry
factors)分析結果…………….……......…...……...…...49
4.1.2.7. 黃連甲醇萃取物抑制多基因型C型肝炎病毒試
驗分析結果……………………..………………………50
第二節 小蘗鹼(berberine, BBR)透過標的病毒E2醣蛋白
方式抑制C型肝炎病毒的進入階段分析結果….....….…51
4.2.1. 小蘗鹼(berberine, BBR)的細胞毒性及抗病毒試驗
分析結果…………….……………………………………51
4.2.2. 小蘗鹼(berberine, BBR)抑制C型肝炎病毒侵入
分析結果……………………………………………...….51
4.2.3. 小蘗鹼(berberine, BBR) 抑制C型肝炎病毒生命
週期分析結果………..…………………………………..52
4.2.4. 小蘗鹼(berberine, BBR)影響宿主細胞進入因子(entry
factors)分析結果…………………......……………..……53
4.2.5. 小蘗鹼(berberine, BBR) 抑制C型肝炎病毒假顆粒
感染宿主細胞能力分析結果………..………………..….54
4.2.6. 小蘗鹼(berberine, BBR)與C型肝炎病毒醣蛋白E2
活性位置嵌合(docking)的結構解析結果………………...54
4.2.7. 小蘗鹼(berberine, BBR)抑制HCVCC病毒感染
初代人類肝臟細胞(PHHs) 效果分析結果…….………55
第五章 討論………………………………………………………...….57
第一節 黃連藥材基源鑑定及黃連甲醇萃取物抑制C型
肝炎病毒早期進入宿主細胞之機制…………………..…57
第二節 小蘗鹼(berberine, BBR)透過標的病毒E2醣蛋
白方式抑制C型肝炎病毒的進入階段…………………...62
第六章 結論…………………………………………………..……….65
第七章 未來展望………………………………………………………67
第八章 參考文獻………………………………………………………69
圖、表
圖1、小蘗鹼(berberine; BBR)檢量線………..………….....……….78
圖2、黃連藥材高效液相色層分析(HPLC)化學成分指紋
圖譜…………………………………….…………………….79
圖3、黃連藥材與科學文獻之高效液相色層分析(HPLC)
化學成分指紋圖譜…………..………………………...…….80
圖4. 黃連甲醇萃取物對Huh-7.5肝癌細胞中HCVcc的
抗病毒活性試驗…………..………..…………..………........81
圖5. 給藥時間點試驗……………..……………………………......82
圖6. 黃連甲醇萃取物抑制病毒entry階段試驗……………......83
圖 7. 黃連甲醇萃取物抑制多基因型C型肝炎病毒試驗……......85
圖 8. 小蘗鹼(berberine, BBR)細胞毒殺和抗病毒活性試驗..……..86
圖 9. 小蘗鹼(berberine, BBR)對HCV早期進入階段(early
stage)的影響…..…....………..…………………………..…87
圖10. 小蘗鹼(berberine, BBR)對HCV病毒複製和誘導
抗病毒反應的影響………..……….……………………….88
圖11. 小蘗鹼(berberine, BBR)對細胞受體表現之影響…….......…90
圖12. 小蘗鹼(berberine, BBR)對攜帶HCV醣蛋白E1/E2
(HCV glycoproteins E1/E2)的逆轉錄病毒假顆粒
(HCVpp)感染試驗….……………….……...………………91
圖13. 分子嵌合(molecular docking)技術試驗……………...……92
圖 14. 小蘗鹼(berberine, BBR)對遭病毒感染的初代人類
肝細胞(PHHs)之影響………………..…..…………………93
圖15. 黃連甲醇萃取物抗病毒效應試驗…………………...……...94
表1. 黃連甲醇萃取物選擇性指數(selective index)分析……….…95
研究業績與成果…………………………………………………….….96

1. De oliveria andrade, L.J., D'Oliveira, A., Melo, R.C., De Souza, E.C., Silva, C.A.C., Parana, R. Association between hepatitis C and hepatocellular carcinoma. International Journal of Infectious Diseases. 2009, 1:33-37.
2.Feeney, E.R., Chung, R.T. Antiviral treatment of hepatitis C. Journal of
British Medicine. 2014, 348:3308-3320.
3. 衛生福利部，國家消除C肝政策綱領。2018，4-47。
https:// www.mohw.gov.tw/
4. Shepard, C.W., Finelli, L., Alter, M.J. Global epidemiology of hepatitis C virus infection. Lancet infectious diseases. 2005, 5:558-567.
5. World Health Organization. Hepatitis C: fact sheet. http://www.who.int/mediacentre/factsheets/fs164/en/.
6. World Health Assembly. Draft global health sector strategies on viral
hepatitis.
http://apps.who.int/gb/ebwha/pdf_files/WHA69/A69_32-en.pdf?ua=1
7. World Health Organization. Global health sector strategy on viral
hepatitis 2016-2021:towards ending viral hepatitis. http://apps.who.int/iris/bitstream/10665/246177/1/WHO-HIV-2016
.06eng.pdf.
8. 李欣融。慢性C型肝炎治療之發展-淺談DAA藥品及三期試驗設
計重點。當代醫藥法規月刊。2019, 99:1-20。
9. Poordad, F., McCone, J.J., Bacon, B.R. Boceprevir for untreated chronic HCV genotype1 infection. New England Journal of Medicine. 2011,364:1195-1206.
10. Soriano, V., Labarga, P., Barreiro, P., Fernandez-Montero, J.V., De
Mendoza, C., Esposito, I., Benitez-Gutierrez, L., Pena, J.M. Drug
interactions with new hepatitis C oral drugs. Expert Opinion of Drug
Metabolism and Toxicology. 2015, 11:333-341.
11. Zoulim, F., Liang, T.J., Gerbes, A.L., Aghemo, A., Deuffic-Burban, S.,
Dusheiko, G., Fried, M.W., Pol, S., Rockstroh, J.K., Terrault, N.A.,
Wiktor, S. Hepatitis C virus treatment in the real world: optimizing
treatment and access to therapies. Gut. 2015, 64:1824-1833.
12. Verna, E.C., Brown, R.S. Hepatitis C and liver transplantation:
enhancing outcomes and should patients be retransplanted. Clinics in
Liver Disease. 2008, 12:637-659.
13. Xiao, F., Fofana, I., Thumann, C., Mailly, L., Alles, R., Robinet, E.,
Meyer, N., Schaeffer, M., Habersetzer, F., Doffoel, M., Leyssen, P.,
Neyts, J., Zeisel, M.B., Baumert, T.F. Synergy of entry inhibitors with
direct-acting antivirals uncovers novel combinations for prevention
and treatment of hepatitis C. Gut. 2015, 64:483-494.
14. Douam, F., Lavillette, D., Cosset, F.L. The mechanism of HCV entry
into host cells. Journal of Translational Medicine. 2015, 129:63-107.
15. Burnouf, T., Liu, C.H., Lin, L.T. Strategies to preclude hepatitis C virus entry. In: Allam, N. (Ed.), Advances in Treatment of Hepatitis C and B. 2017, IntechOpen, Rijeka, pp.251-284.
16. Moradpour, D., Penin, F., Rice, C.M. Replication of hepatitis C virus.
Nature Reviews Microbiology. 2007, 5:453-463.
17. Bartenschlager, R., Cosset, F.L., Lohmann, V. Hepatitis C virus
replication cycle. Journal of Hepatology. 2010, 53:583-585.
18. Yang, Z., Wang, X., Chi, X., Zhao, F., Guo, J., Ma, P., Zhong, J., Niu,
J., Pan, X., Long, G. Important role of apolipoprotein E exchange in
hepatitis C virus infection. Journal of Virology. 2016, 90:9632-9643.
19. Chuang, W.L., Yu, M.L., Dai, C.Y., Chang, W.Y. Treatment of chronic
hepatitis C in Southern Taiwan. Intervirology. 2006, 49: 99-106.
20. Lee, S.D., Yu, M.L., Cheng, P.N., Lai, M.Y., Chao, Y.C., Hwang, S.J.,
Chang, W.Y., Chang, T.T., Hsieh, T.Y., Liu, C.J., Chen, D.S.
Comparison of a 6-month course peginterferon alpha-2b plus ribavirin
and interferon alpha-2b plus ribavirin in treating Chinese patients with
chronic hepatitis C in Taiwan. Journal of Viral hepatitis. 2005, 12: 283-291.
21. Liu, C.H., Huang, C.F., Liu, C.J., Dai, C.Y., Liang, C.C., Huang, J.F.,
Hung, P.H., Tsai, H.B., Tsai, M.K., Chen, S.I., Lin, J.W., Yang, S.S.,
Su, T.H., Yang, H.C., Chen, P.J., Chen, D.S., Chuang, W.L., Yu, M.L.,
Kao, J.H. Pegylated interferon-alpha-2a plus ribavirin for treatment-
naïve Asian patients with hepatitis C virus genotype 1 infection: a
multicenter, randomized controlled trial. Annals of Internal Medicine.
2008, 47:1260-1269.
22. Yu, M.L., Dai, C.Y., Huang, J.F., Hou, N.J., Lee, L.P., Hsieh, M.Y., Chiu, C.F., Lin, Z.Y., Chen, S.C., Hsieh, M.Y., Wang, L.Y., Chang, W.Y., Chuang, W.L. A randomised study of peginterferon and ribavirin for 16 versus 24 weeks in patients with genotype 2 chronic hepatitis C. Gut. 2007, 56:553-559.
23. Centers for Disease Control and Prevention.2019, Viral hepatitis
preventive services.
https://www.cdc.gov/nchhstp/preventionthroughhealthcare/preventiv
eervices/ hepatitis.htm.
24. Bacon, B.R., Gordon, S.C., Lawitz, E. Boceprevir for previously
treated chronic HCV genotype1 infection. New England Journal of
Medicine. 2011, 364:1207-1217.
25. Das, D., Pandya, M. Recent advancement of direct-acting antiviral
agents (DAAs) in hepatitis C therapy. Mini Review Medicinal
Chemistry. 2018, 18:584-596.
26. Mohamed, A.A., El-Toukhy, N.E.R., Said, E.M., Gabal, H.M.R.,
AbdelAziz, H., Doss, W., El-Hanafi, H., El Deeb, H.H., Mahmoud,
S., Elkadeem, M., Shalby, H.S., Abd-Elsalam, S. Hepatitis C virus:
efficacy of new DAAs regimens. Infection Disord Drug Targets. 2019, 32, 1117-1127.
27. Zoulim, F., Liang, T., Gerbes, A.L., Aghemo, A., Deuffic-Burban,
S., Dusheiko, G., Fried, M.W., Pol, S., Rockstroh, J.K., Terrault, N.
A., Wiktor, S. Hepatitis C virus treatment in the real world:optimizing
treatment and access to therapies. Gut. 2015, 64:1824-1833.
28. Gritsenko, D., Hughes, G. Ledipasvir/Sofosbuvir (harvoni):improving
options for hepatitis C virus infection. Pharmacy and Therapeutics.
2015, 40:256-276.
29. Stephen, J.P., Nicholas, H.O., Eve-Isabelle, P., Harel, D., Peter, F.,
Jean-Michel, P. Silymarin for hepatitis C virus infection. Antiviral
Therapy. 2013, 18:141–147.
30. Beinhardt, S., Rasoul-Rockenschaub, S., Scherzer, T.M., Ferenci, P.
Silibinin monotherapy prevents graft infection after orthotopic liver
transplantation in a patient with chronic hepatitis C. Journal of
Hepatology. 2011, 54:591-592.
31. Song, J.M. Anti-infective potential of catechins and their derivatives
against viral hepatitis. Clinical and Experimental Medicine Vaccine
Reserch. 2018, 7:37-42.
32. Yang, C.S., Wang, H. Cancer preventive activities of tea catechins.
Molecules. 2016, 21:16791698.
33. Lin, L.T., Chung, C.Y., Hsu, W.C., Chang, S.P., Hung, T.C., Shields, J., Russell, R.S., Lin, C.C., Li, C.F., Yen, M.H., Tyrrell, D.L., Lin, C.C., Richardson, C.D. Saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis C virus entry. Journal of epatology. 2015, 62:541-548.
34. Chung, C.Y., Liu, C.H., Burnouf, T., Wang, G.H., Chang, S.P.,
Jassey, A., Tai, C.J., Huang, C.J., Richardson, C.D., Yen,
M.H., Lin, C.C., Lin, L.T. Activity-based and fraction-guided
analysis of Phyllanthus urinaria identifies loliolide as a potent
inhibitor of hepatitis C virus entry. Antiviral Research. 2016, 130:58-
68.
35. Chung, C.Y., Liu, C.H., Wang, G.H., Jassey, A., Li, C.L., Chen, L.,
Yen, M.H., Lin, C.C., Lin, L.T. (4R,6S)-2-Dihydromenisdaurilide is
a butenolide that efficiently inhibits hepatitis C virus entry. Scientific
Reports. 2016, 6:29969-29980.
36. World Health Organization WHO Monographs on Selected Medicinal
Plants - Volume 1.
http://apps.who.int/medicinedocs/en/d/Js2200e/13.html#Js2200e.13
37. Meng, F.C., Wu, Z.F., Yin, Z.Q., Lin, L.G., Wang, R., Zhang, Q.W.
Rhizoma Coptidis and its main bioactive components: recent advances
in chemical investigation, quality evaluation and pharmacological
activity. Journal of Chinese Medicine. 2018, 13:13-30.
38. MOHW, Taiwan Herbal Pharmacopeia 2nd Edition, English Version
(Ministry of Health and Welfare, Taiwan). In 2016.
39. Muluye, R.A., Bian, Y., Alemu, P.N. Anti-inflammatory and
antimicrobial effects of heat-clearing Chinese herbs:A current
review. Journal of Traditional and Complementary Medicine. 2014,
4:93-98.
40. Grycová, L., Dostál, J., Marek, R. Quaternary protoberberine alkaloids. Phytochemistry. 2007, 68:150-175.
41. Chin, L.W., Cheng, Y.W., Lin, S.S., Lai, Y.Y., Lin, L.Y., Chou, M.Y.,
Chou, M.C., Yang, C.C. Anti-herpes simplex virus effects of berberine from Rhizoma Coptidis, a major component of a Chinese herbal medicine, Ching-Wei-San. Archives of Virology. 2010, 155:1933-1941.
42. Kim, H.Y., Shin, H.S., Park, H., Kim, Y.C., Yun, Y.G., Park, S., Shin,
H.J., Kim, K. In vitro inhibition of coronavirus replications by the
traditionally used medicinal herbal extracts, Cimicifuga rhizoma,
Meliae cortex, Rhizoma Coptidis, and Phellodendron cortex. Journal
of Clinival Virology. 2008, 41:122-128.
43. Lee, B.H., Chathuranga, K., Uddin, M.B., Weeratunga, P., Kim, M.S.,
Cho, W.K., Kim, H.I., Ma, J.Y., Lee, J.S. Rhizoma Coptidis extract
inhibits replication of respiratory syncytial virus in vitro and in vivo
by inducing antiviral state. Journal of Microbiology. 2017, 55:488-498.
44. Varghese, F.S., Thaa, B., Amrun, S.N., Simarmata, D., Rausalu, K.,
Nyman, T.A., Merits, A., McInerney, G.M., Ng, L.F.P., Ahola, T. The antiviral alkaloid berberine reduces chikungunya virus-induced mitogen-activated protein kinase signaling. Journal of Virology. 2016, 90:9743-9757.
45. Yan, Y.Q., Fu, Y.J., Wu, S., Qin, H.Q., Zhen, X., Song, B.M., Weng,
Y.S., Wang, P.C., Chen, X.Y., Jiang, Z.Y. Anti-influenza activity of
berberine improves prognosis by reducing viral replication in mice.
Phytotherapy Research. 2018, 32:2560-2567.
46. Enkhtaivan, G., Muthuraman, P., Kim, D.H., Mistry, B. Discovery
of berberine based derivatives as anti-influenza agent through blocking of neuraminidase. Bioorganic & Medicinal Chemistry. 2017, 25:5185- 5193.
47. Wang, H., Li, K., Ma, L., Wu, S., Hu, J., Yan, H., Jiang, J., Li, Y.
Berberine inhibits enterovirus 71 replication by downregulating the
MEK/ERK signaling pathway. Autophagy Virology Journal. 2017,
14:1-8.
48. Hayashi, K., Minoda, K., Nagaoka, Y., Hayashi, T., Uesato, S.
Antiviral activity of berberine and related compounds against human
Cytomegalovirus. Bioorganic & Medicinal Chemistry Letters. 2007,
17:1562-1564.
49. Luganini, A., Mercorelli, B., Messa, L., Palù, G., Gribaudo, G.,
Loregian, A. The isoquinoline alkaloid berberine inhibits human
cytomegalovirus replication by interfering with the viral immediate
early-2 (IE2) protein transactivating activity. Antiviral Research. 2019, 164:52-60.
50. Lin, L.T., Hsu, W.C., Lin, C.C. Antiviral natural products and
herbal medicines. Journal of Traditional and Complementary Medicine. 2014, 4:24-35.
51. Freedman, H., Logan, M.R., Law, J.L.M., Houghton, M. Structure and
function of the hepatitis C virus envelope glycoproteins E1 and E2:
antiviral and vaccine targets. ACS Infectious Diseases. 2016, 2:749-
762.
52. Troesch, M., Meunier, I., Lapierre, P., Lapointe, N., Alvarez, F.,
Boucher, M., Soudeyns, H. Study of a novel hypervariable region in
hepatitis C virus (HCV) E2 envelope glycoprotein. Virology. 2016,
352:357-367.
53. Abubakr, M., Mandal, S.C., Banerjee, S. Natural compounds against
flaviviral infections. Natural Product Communications. 2013, 8:1487-
1492.
54. Wu, T.J., Lu, J., Ni, H., Li, P., Jiang, Y., Li, H.J. Construction of an
optimized method for quality evaluation and species discrimination
of Coptidis Rhizoma by ion-pair high performance liquid
chromatography combined with response surface methodology.
Journal of Pharmaceutical and Biomedical Analysis. 2018, 153:152-
157.
55. 余祁暐、李盼。動物保健產品之管理法規與發展趨勢。生物經濟
2015, 44:58-66.
56. Haid, S., Novodomska, A., Gentzsch, J., Grethe, C., Geuenich, S.,
Bankwitz, D., Chhatwal, P., Jannack, B., Hennebelle, T., Bailleul, F.
A plant-derived flavonoid inhibits entry of all HCV genotypes into
human hepatocytes. Gastroenterology. 2012, 143:213–222.