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研究生:江姿儀
研究生(外文):Tzu-Yi Chiang
論文名稱:生脈散對體內外藥物代謝酶的影響
論文名稱(外文):Effects of Sheng-Mai-San on drug-metabolizing enzymes in vitro and in vivo
指導教授:蕭崇瑋翁芸芳
指導教授(外文):Chung-Wai ShiauYune-Fang Ueng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:73
中文關鍵詞:生脈散小腸肝臟CYP3A
外文關鍵詞:Sheng-Mai-SanIntestineliverCYP3A
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國家圖書館學位論文延後公開申請書........................i
論文電子檔著作授權書...................................ii
論文審定同意書........................................iii
誌謝.................................................iv
中文摘要..............................................v
英文摘要..............................................vi
目錄..................................................vii
圖目錄................................................viii
表目錄................................................xiii
縮寫..................................................xiv
第一章-緒論...........................................1
1.1生脈散 (Sheng-Mai-San, SMS).......................1
1.2硝苯地平 ( nifedipine )...........................3
1.3藥物代謝酶........................................4
1.4 細胞色素P450 (cytochrome P450)....................5
1.5第二相反應酵素 (phase II reaction enzyme)..........6
1.6中西藥物交互作用 ( herb-drug interaction )..........7
第二章-材料方法.......................................9
2.1化學藥物及劑............................................................................................9
2.2中藥處理.......................10
2.3酒精萃取.......................10
2.4實驗動物處理.......................11
2.5採血及血液處理.......................11
2.6微粒體 (microsome) 及細胞質液 (cytosol) 製備........11
2.7蛋白質含量測定 ( Lowry test )......................12
2.8 Cytochrome P450 含量測定......................13
2.9 NADPH-P450 reductase活性測定.....................13
2.10 7-Ethoxyresorufin O-deethylation (EROD) 活性測定及
7-Methoxyresorufin O-demethylation (MROD) 活性測定....13
2.11 7-Pentoxyresorufin O-deallkylation (PROD) 活性測定..14
2.12 Warfarin 7-hydroxylation活性測定..........14
2.13 Dextromethorphan O-demethylation (DMOD) 活性測定...14
2.14 Chlorzoxazone 6-hydroxylation活性測定...........15
2.15 Nifedipine Oxidation (NFO) 活性測定......15
2.16 Uridine 5’-diphosphate-glucuronosyltransferase (UGT) 活性測定..15
2.17 Glutathione S-transferase (GST) 活性測定........16
2.18 NADH-quinone oxidoreductase (NQO) 活性測定…………………...16
2.19 Immunoblot analyses免疫墨點法分析…………………………..…...16
2.20肝腎功能血液檢測……………………….……………………..…….17
2.21科學中藥化學成分分析…………………………………….……..….17
2.22透析實驗………………………………………………………..……..18
2.23 NFO半數抑制濃度 (IC50) 分析……………………………………18
2.24 Pharmacokinetic analysis藥物動力學分析……………………..……18
2.25實驗儀器…………………………………………………………..…..19
2.26實驗分析………………………………………………………..…..…19
第三章-結果........................................20
3.1在體外試驗中生脈散酒精萃取物對肝臟和小腸微粒體NFO活性影響…20
3.2生脈散對大鼠體內代謝酶的劑量相性………………………20
3.3生脈散對肝臟和小腸P450, NQO, UGT, GST的活性影響......21
3.4 Immunoblot及酵素動力學分析生脈散處理之肝臟和小腸NFO活性..21
3.5五味子對大鼠體內代謝酶的影響……………………………...……...22
3.6五味子對肝臟和小腸微粒體NFO活性影響…………………...…….22
3.7五味子、人參、麥門冬對肝臟和小腸微粒體NFO活性影響…....…22
3.8澱粉對肝臟和小腸藥物代謝酶的影響…………………………...…...23
3.9 UPLC分析生脈散光圖譜…………………………………………...…23
3.10在體外試驗中人參、麥門冬、五味子酒精萃取物對肝臟、小腸微
粒體以及基因重組人類CYP3A4對NFO活性影響………….....…..24
3.11在體外試驗中純化合物對肝臟、小腸微粒體以及基因重組人類 CYP3A4的NFO活性影響……………………………………………24
3.12透析試驗前後對methylophiopogonanone A抑制基因重組人類 CYP3A4的NFO活性及NADPH-P450 還原酶的影響…………...…...25
3.13時間變化與Methylophiopogonanone A對基因重組人類CYP3A4
抑制NFO活性的影響…………………………………...26
3.14時間變化與Methylophiopogonanone B對基因重組人類CYP3A4
抑制NFO活性的影響……………………………………………...…...26
第四章-討論.........27
第五章-結論....................................34
第六章-參考文獻..........35

圖目錄
Scheme 1. The effects of herbal components in SMS on CYP3A………………….…2
Scheme 2. Structures of pure compounds……………………….43
Figure 1. Linear relationship between protein concentrations and intestinal NFO activities and the inhibitory effects of the ethanolic extract of Sheng-Mai-San (SMS) on rat hepatic and intestinal NFO activities in vitro………………………………….44
Figure 2. Effects of Sheng-Mai-San (SMS) on liver/body weight ratio in rats……...45
Figure 3. Effects of SMS oral administration on hepatic and intestinal NFO activities……………………………………………………………………………...46
Figure 4. Effects of SMS treatment on hepatic and intestinal NFO activities……….47
Figure 5. Effects of Sheng-Mai-San (SMS) and Kampo SMS treatments on hepatic and intestinal NFO activity………………………………………………………..…48
Figure 6. Effects of SMS treatment on hepatic and intestinal contents or activities of P450, cytochrome b5, CPR, NQO, UGT and GST…………………………………...49
Figure 7. Effects of SMS treatment on CYP3A protein levels………………………50
Figure 8. The catalytic kinetics of intestinal NFO in rats……………………………51
Figure 9. Effects of Schisandrae Fructus on liver/body weight ratio in rat………….52
Figure 10. Effects of Schisandrae Fructus treatment on hepatic and intestinal contents or activities of P450, CPR, cytochrome b5…………………………………………53
Figure 11. Effects of Schisandrae Fructus treatment on hepatic and intestinal NFO activities……………………………………………………………………………54
Figure 12. Effects of Ginseng Radix (GR), Ophiopogonis Radix (OR), Schisandrae Fructus (SF) and Mix group treatments on hepatic and intestinal NFO activities…...55
Figure 13. Effects of starch treatment on hepatic and intestinal contents or activities of P450, CPR, cytochrome b5……………………………………………………...…56
Figure 14. Effects of starch treatment on hepatic and intestinal NFO activities…….57
Figure 15. The chromatograms of Sheng-Mai-San in UPLC analysis with detection of absorbance at 203nm, 254nm………58
Figure 16. The inhibition of nifedipine oxidation activities of Radix Ginseng, Fructus Schisandrae, Radix Ophiopogonis extract on rat liver, intestinal microsomes and recombinant human CYP3A4 with preincubation in the presence of a NADPH generating system in vitro………………………60
Figure 17. The inhibition of NFO activities of ginsenoside Rb1, ginsenoside Rd, panaxatriol, protopanaxatriol, ruscogenin, schisantherin A, arctigenin, methylophiopogonanone A, and methylophiopogonanone B on rat liver microsomes with 10 min pre-incubation………………..61
Figure 18. The Inhibition of NFO activity of ginsenoside Rb1, ginsenoside Rd, panaxatriol, protopanaxatriol, ruscogenin, schisantherin A, schisandrin B, arctigenin, methylophiopogonanone A, and methylophiopogonanone B on rat intestinal microsomes with 10 min pre-incubation……………..62
Figure 19. The Inhibition of NFO activity by ginsenoside Rb1, ginsenoside Rd, panaxatriol, protopanaxatriol, ruscogenin, schisantherin A, schisandrin A, schisandrin B, schisandrol A, arctigenin, methylophiopogonanone A, and methylophiopogonanone B on recombinant human CYP3A4 with 10 min pre-incubation……………………………………………………………...……………..63
Figure 20. The influence of dialysis on nifedipine oxidation and CPR activities in vitro…………………………….………64
Figure 21. The inhibition of nifedipine oxidation activities with 0, 3, 10, 25, 50, 100 uM methylophiopogonanone A and with pre-incubation in 0, 10,15,30 minutes on recombinant human CYP3A4……………………..65
Figure 22. The inhibition of nifedipine oxidation activity with 0, 5, 10, 25, 50, 100 uM methylophiopogonanone B and with pre-incubation in 0, 10, 30 minutes on recombinant human CYP3A4………………………………………..66

表目錄
Table1. The contents of dried component herbs in SMS and Kampo SMS…………67
Table 2. Effects of SMS 3-week treatment on the body weights in rats…………......68
Table 3. Effects of Schisandrae Fructus (SF) 3-week treatment on the body weights in rats……………………………………69
Table 4. Serum biochemical markers for monitoring hepatic and renal functions after 3-week oral treatments with SMS and its component herbs…………………………70
Table 5. Effects of 3-week SMS, SF, starch treatments on hepatic P450 activities in rats. EROD, MROD, PROD, DMOD, warfarin 7-hydroxylation and chlorzoxazone 6-hydroxylation activities were detected……………………………………………….71
Table 6. Effects of 3-week herbal component treatments on hepatic phase II drug-metabolizing enzyme activities in rats……………………………………………….72
Table 7. Effects of 3-week herbal component treatments on intestinal phase II drug-metabolizing enzyme activities in rats……………………………………………….73
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