臺灣博碩士論文加值系統

前胡(Peucedanum praeruptorum Dunn)為繖形科(Umbelliferae)植物，具有降氣化痰、散風清熱的功效，主產於中國安徽、浙江、湖南、四川等地區。本研究之初篩試驗中，於前胡乾燥根部的甲醇抽出物顯示具有顯著誘發脂質分解的能力，因此本實驗的目的是自前胡乾燥根中，分離出具有相關活性的天然抽出物。
將前胡乾燥根的甲醇抽出物分別以正己烷、乙酸乙酯及水進行部份劃分，分別得到正己烷層抽出物、乙酸乙酯層抽出物及水層抽出物。再將最具脂質分解活性的正己烷層抽出物以silica gel層析管柱進行分離，劃分為Fr1~Fr24。其中將活性效果為正己烷層1.77倍的分劃層Fr22以C18逆向層析管柱，由10% 至 100%甲醇濃度梯度沖提進行分離，獲得Fr22-1~Fr22-9。取其中生物活性與Fr22相近的Fr22-4進一步以C18逆向高效能層析儀進行分離，分為七個次分劃層Fr22-4-3-1~ Fr22-4-3-7；而生物活性為Fr22之1.22倍的Fr22-7則經由Sephadex LH-20層析管柱分離為Fr22-7-1~ Fr22-7-5。將最具活性的Fr22-7-1進而以C18逆向高效能層析儀分離，可得Fr22-7-1-1~Fr22-7-1-4。
將各層抽出物進行誘發脂質分解測試，並以偵測大鼠脂肪細胞的甘油釋放量評估脂質分解程度。結果顯示以 Fr22-4-3-2及Fr22-7-1-3抽出物活性分別為空白對照組的3.58倍及3.81倍，為最具活性效果的分劃層。而以PKA、PKC及PKG抑制劑分別處理脂肪細胞，結果發現PKA抑制劑(0.1 mM H89)具有顯著抑制前胡抽出物所誘導的脂質分解能力，故推測前胡抽出物是經由PKA的訊息傳遞路徑達到脂質分解的作用。

Peucedanum praeruptorurn Dunn (Umbelliferae) has been used for treatment of respiratory disease. Our preliminary lipolysis screening showed that the methanol extract of root tissues of P. praeruptorum Dunn exhibits high activity. Therefore, this study aims to identify the active fractions from the bioactive extract.
Three fractions, n-hexane extract, ethyl acetate extract, and aqueous extract, were obtained from sequentially partitioning fractionation of the methanol crude extract. Among these extracts, n-hexane extract contained ingredients with the most effective lipolysis potential, was followed by fractionation by silica gel column chromatography using gradient elution solvent: n-hexane to ethyl acetate, acetone and methanol respectively, collecting twenty four subfractions (Fr1~Fr24). The Fr22 subfraction with a 1.77-fold activity larger than n-hexane extract, was further purified by RP-C18 column with 10% to 100% methanol gradient elution, and nine subfractions Fr22-1~Fr22-9 were obtained. The Fr22-4 subfraction, with nearly same activity as Fr22 subfraction, being applied in C18 RP-HPLC column followed by acetonitrile gradient separation. The Fr22-7 subfraction with a 1.22-fold activity larger than Fr22 subfraction, was further isolated by chromatography on Sephadex LH-20 by ethanol elution, and five subfractions Fr22-7-1~ Fr22-7-5 were collected. Among these subfractions, Fr22-7-1 showed the highest lipolysis activity comparison with others subfractions. Then Fr22-7-1 subtraction was applied onto a C18 RP-HPLC column, and four subfractions Fr22-7-1-1~Fr22-7-1-4 were obtained.
Finally, the lipolysis activities and pathway of these fractions were evaluated. The lipolysis assay in the study was performed using primary adipocyte isolated from rat adipocyte tissue. The release of glycerol from rat adipocytes incubated with plant extracts were quantified as used as the criterion of lipolysis efficiency. Our results showed that markedly activity existed in the fractions of Fr22-4-3-2 (with a 3.58-fold activity larger than control) and Fr22-7-1-3 (with a 3.81-fold activity larger than control). And the extracts lipolysis effect was inhibited by 0.1 mM PKA inhibitor H89, but not by PKC and PKG inhibitors, respectively. That mean the P. praeruptorum Dunn extract directly stimulates lipolysis in rat adipocytes through activation of PKA pathway.

目錄 i
中文摘要 I
Abstract III
圖目次 V
前言 1
一、 前胡 1
二、 脂肪組織 9
三、 參與脂質分解的蛋白質 10
四、 脂質分解的機轉 12
五、 減少脂肪累積的天然物 14
六、 研究目的 17
材料與方法 18
一、 材料及儀器 18
二、 中藥樣本之篩選及萃取 19
三、 體外脂肪細胞分解測試 22
四、 MTT Assay測定細胞存活率 25
五、 前胡抽出物之薄層層析(TLC)定性分析 26
六、 前胡抽出物之高效能液相層析儀(HPLC)定性分析 26
七、 統計分析 27
結果 28
一、 前胡乾燥根經過partition後各層抽出物對脂質分解之影響 28
二、 前胡指標性成分對脂質分解之影響 28
三、 正己烷抽出物經silica gel層析管柱進行分離後各分劃層對脂質分解之影響 29
四、 不同濃度之Fr22誘導脂質分解效果及其細胞毒性 29
五、 PKA、PKC、PKG抑制劑對Fr22誘導脂質分解之影響 30
六、 Fr22經C18逆向層析管柱分離各分劃層對脂質分解之影響 31
七、 利用HPLC C18逆向層析管柱劃分Fr22-4各分劃層對脂質分解之影響 31
八、 前胡抽出物Fr22-4-3-2之TLC與HPLC定性分析 32
九、 Fr22-7以Sephadex LH-20層析管柱分離各分劃層對脂質分解之影響 32
十、 利用HPLC C18逆向層析管柱劃分Fr22-7-1各分劃層對脂質分解之影響 33
十一、 前胡抽出物Fr22-7-1-3之HPLC與TLC定性分析 33
十二、 前胡抽出物對大鼠細胞的毒性反應 34
十三、 前胡抽出物於不同時間點誘發脂質分解之效果 34
十四、 前胡抽出物誘發脂質分解後之細胞形態 35
討論 36
結論 42
參考文獻 43
圖 52
附錄一 68

Ⅰ. 張貴君，現代實用中藥鑑別技術，人民衛生出版社，北京， pp. 590-593，2000。
Ⅱ. 趙文達，中華人民共和國藥典中藥材外形組織粉末圖解，中國醫藥科
技出版社，北京， pp. 379，1998。
Ⅲ. 林宗旦、林宗平、林景彬，中藥藥理學，華香園，臺北， pp. 584-5，1996。
Ⅳ. 邱德文、吳家榮、夏同衍，本草綱目彩色藥圖，薪傳出版社，臺北， pp. 135，1991。
Ⅴ. 姜明燕、常天輝、徐業杰，中藥白花前胡對麻醉貓急性心肌梗死的保護作用，中國醫藥大學學報， 33(1)： 22-3，2004。
1.Hansen JB, Kristiansen K. Regulatory circuits controlling white versus brown adipocyte differentiation. Biochem J. 2006 Sep; 398(2): 153-68.
2.Hauner H. The new concept of adipose tissue function. Physiol Behav. 2004 Dec; 83(4): 653-8.
3.Almahbobi G, Williams LJ, Hall PF. Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells. J Cell Sci. 1992 Feb; 101(2): 383-93.
4.Wang S, Soni KG, Semache M, Casavant S, Fortier M, Pan L, Mitchell GA. Lipolysis and the integrated physiology of lipid energy metabolism. Mol Genet Metab. 2008 Nov; 95(3): 117-26.
5.Langin D. Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome. Pharmacol Res. 2006 Jun; 53(6): 482-91.
6.Holm C. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem Soc Trans. 2003 Dec; 31(6): 1120-4.
7.Anthonsen MW, R?圢nstrand L, Wernstedt C, Degerman E, Holm C. Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro. J Biol Chem. 1998 Jan; 273(1): 215-21.
8.Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr, Londos C. Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro. Proc Natl Acad Sci U S A. 1992 Sep; 89(18): 8537-41.
9.Clifford GM, Kraemer FB, Yeaman SJ, Vernon RG. Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation during the lactation cycle of the rat. Metabolism. 2001 Nov; 50(11): 1264-9.
10.Shen WJ, Sridhar K, Bernlohr DA, Kraemer FB. Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein. Proc Natl Acad Sci U S A. 1999 May; 96(10): 5528-32.
11.Jenkins-Kruchten AE, Bennaars-Eiden A, Ross JR, Shen WJ, Kraemer FB, Bernlohr DA. Fatty acid-binding protein-hormone-sensitive lipase interaction. Fatty acid dependence on binding. J Biol Chem. 2003 Nov; 278(48): 47636-43.
12.Shen WJ, Liang Y, Hong R, Patel S, Natu V, Sridhar K, Jenkins A, Bernlohr DA, Kraemer FB. Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase. J Biol Chem. 2001 Dec; 276(52): 49443-8.
13.Blanchette-Mackie EJ, Dwyer NK, Barber T, Coxey RA, Takeda T, Rondinone CM, Theodorakis JL, Greenberg AS, Londos C. Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes. J Lipid Res. 1995 Jun; 36(6): 1211-26.
14.Londos C, Brasaemle DL, Gruia-Gray J, Servetnick DA, Schultz CJ, Levin DM, Kimmel AR. Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells. Biochem Soc Trans. 1995 Aug; 23(3): 611-5.
15.Greenberg AS, Egan JJ, Wek SA, Moos MC Jr, Londos C, Kimmel AR. Isolation of cDNAs for perilipins A and B: sequence and expression of lipid droplet-associated proteins of adipocytes. Proc Natl Acad Sci U S A. 1993 Dec; 90(24): 12035-9.
16.Servetnick DA, Brasaemle DL, Gruia-Gray J, Kimmel AR, Wolff J, Londos C. Perilipins are associated with cholesteryl ester droplets in steroidogenic adrenal cortical and Leydig cells. J Biol Chem. 1995 Jul; 270(28): 16970-3
17.Clifford GM, Londos C, Kraemer FB, Vernon RG, Yeaman SJ. Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes. J Biol Chem. 2000 Feb; 275(7): 5011-5.
18.Richards JS, Rolfes AI. Hormonal regulation of cyclic AMP binding to specific receptor proteins in rat ovarian follicles. Characterization by photoaffinity labeling. J Biol Chem. 1980 Jun; 255(11): 5481-9.
19.Sengen?嫳 C, Berlan M, De Glisezinski I, Lafontan M, Galitzky J. Natriuretic peptides: a new lipolytic pathway in human adipocytes. FASEB J. 2000 Jul; 14(10): 1345-51.
20.Sengenes C, Bouloumie A, Hauner H, Berlan M, Busse R, Lafontan M, Galitzky J. Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. J Biol Chem. 2003 Dec; 278(49): 48617-26.
21.Galvin-Parton, P.A., Chen, X., Moxham, C.M., Malbon, C.C. Induction of Gα(q)-specific antisense RNA in vivo causes increased body mass and hyperadiposity. J Biol Chem. 1997 Feb; 272(7): 4335-41.
22.Flechtner-Mors M, Jenkinson CP, Alt A, Adler G, Ditschuneit HH. In vivo alpha(1)-adrenergic lipolytic activity in subcutaneous adipose tissue of obese subjects. J Pharmacol Exp Ther. 2002 Apr; 301(1): 229-33.
23.Manganiello VC, Murata T, Taira M, Belfrage P, Degerman E. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys. 1995 Sep; 322(1): 1-13.
24.Degerman E, Belfrage P, Manganiello VC. Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3). J Biol Chem. 1997 Mar; 272(11): 6823-6.
25.Rahn T, R?圢nstrand L, Leroy MJ, Wernstedt C, Tornqvist H, Manganiello VC, Belfrage P, Degerman E. Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. J Biol Chem. 1996 May; 271(19): 11575-80.
26.Xue B, Greenberg AG, Kraemer FB, Zemel MB. Mechanism of intracellular calcium ([Ca2+]i) inhibition of lipolysis in human adipocytes. FASEB J. 2001 Nov; 15(13): 2527-9.
27.Olas B, Wachowicz B, Saluk-Juszczak J, Zieliński T, Kaca W, Buczyński A. Antioxidant activity of resveratrol in endotoxin-stimulated blood platelets. Cell Biol Toxicol. 2001; 17(2): 117-25.
28.Wung BS, Hsu MC, Wu CC, Hsieh CW. Resveratrol suppresses IL-6-induced ICAM-1 gene expression in endothelial cells: effects on the inhibition of STAT3 phosphorylation. Life Sci. 2005 Dec; 78(4): 389-97.
29.Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC, Pezzuto JM. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997 Jan; 275(5297): 218-20.
30.Picard F, Kurtev M, Chung N, Topark-Ngarm A, Senawong T, Machado De Oliveira R, Leid M, McBurney MW, Guarente L. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature. 2004 Jun; 429(6993): 771-6.
31.Zhu J, Yong W, Wu X, Yu Y, Lv J, Liu C, Mao X, Zhu Y, Xu K, Han X, Liu C. Anti-inflammatory effect of resveratrol on TNF-alpha-induced MCP-1 expression in adipocytes. Biochem Biophys Res Commun. 2008 May; 369(2): 471-7.
32.Mojzisov?? G, Kuchta M. Dietary flavonoids and risk of coronary heart disease. Physiol Res. 2001 Feb; 50(6): 529-35.
33.Ohkoshi E, Miyazaki H, Shindo K, Watanabe H, Yoshida A, Yajima H. Constituents from the leaves of Nelumbo nucifera stimulate lipolysis in the white adipose tissue of mice. Planta Med. 2007 Oct; 73(12): 1255-9.
34.Li Z, Li J, Mo B, Hu C, Liu H, Qi H, Wang X, Xu J. Genistein induces cell apoptosis in MDA-MB-231 breast cancer cells via the mitogen-activated protein kinase pathway. Toxicol In Vitro. 2008 Oct; 22(7): 1749-53.
35.Szkudelska K, Nogowski L. Genistein--a dietary compound inducing hormonal and metabolic changes. J Steroid Biochem Mol Biol. 2007 Jun; 105(5): 37-45.
36.Moon HS, Lee HG, Seo JH, Guo DD, Kim IY, Chung CS, Kim TG, Choi YJ, Cho CS. Lipolysis is stimulated by PEGylated conjugated linoleic acid through the cyclic adenosine monophosphate-independent signaling pathway in 3T3-L1 cells: activation of MEK/ERK MAPK signaling pathway and hyper-secretion of adipo-cytokines. J Cell Physiol. 2008 Feb; 214(2): 283-94.
37.Hu J, Zhou D, Chen Y. Preparation and antioxidant activity of green tea extract enriched in epigallocatechin (EGC) and epigallocatechin gallate (EGCG). J Agric Food Chem. 2009 Feb; 57(4): 1349-53.
38.Moon HS, Chung CS, Lee HG, Kim TG, Choi YJ, Cho CS. Inhibitory effect of (-)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells. Obesity (Silver Spring). 2007 Nov; 15(11): 2571-82.
39.Chang H, Chu XY, Zou J, Chang TH. Effect of dl-praeruptorin A on desmin and vimentin content in rat ischemia/reperfusion myocardiocytes. Chin Med J (Engl). 2007 Dec; 120(24): 2256-9.
40.Zhang JX, Fong WF, Wu JY, Yang M, Cheung HY. Pyranocoumarins isolated from Peucedanum praeruptorum as differentiation inducers in human leukemic HL-60 cells. Planta Med. 2003 Mar; 69(3): 223-9.
41.Fong WF, Zhang JX, Wu JY, Tse KW, Wang C, Cheung HY, Yang MS. Pyranocoumarin (+/-) -4''-O-acetyl-3''-O-angeloyl-cis-khellactone induces mitochondrial-dependent apoptosis in HL-60 cells.Planta Med. 2004 Jun; 70(6): 489-95.
42.Lu M, Nicoletti M, Battinelli L, Mazzanti G. Isolation of praeruptorins A and B from Peucedanum praeruptorum Dunn. and their general pharmacological evaluation in comparison with extracts of the drug. Farmaco. 2001 Jul; 56(7): 417-20.
43.Ishii H, Okada Y, Baba M, Okuyama T. Studies of coumarins from the Chinese drug Qianhu, XXVII: structure of a new simple coumarin glycoside from Bai-Hua Qianhu, Peucedanum praeruptorum. Chem Pharm Bull (Tokyo). 2008 Sep; 56(9): 1349-51.
44.Ishii H, Okada Y, Baba M, Okuyama T. Studies of coumarins from the Chinese drug Qianhu, XXVII: structure of a new simple coumarin glycoside from Bai-Hua Qianhu, Peucedanum praeruptorum. Chem Pharm Bull (Tokyo). 2008 Sep; 56(9): 1349-51.
45.Chang HT, Okada Y, Ma TJ, Okuyama T, Tu PF. Two new coumarin glycosides from Peucedanum praeruptorum. J Asian Nat Prod Res. 2008 Jun; 10(6): 577-81.
46.Chang H, Okada Y, Okuyama T, Tu P. 1H and 13C NMR assignments for two new angular furanocoumarin glycosides from Peucedanum praeruptorum. Magn Reson Chem. 2007 Jul; 45(7): 611-4.
47.Liu R, Feng L, Sun A, Kong L. Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed counter-current chromatography. J Chromatogr A. 2004 Nov; 1057(2): 89-94.
48.Lafontan M. Inhibition of epinephrine-induced lipolysis in isolated white adipocytes of aging rabbits by increased alpha-adrenergic responsiveness. Lipid Res. 1979 Feb; 20(2): 208-16.
49.Gettys TW, Rohlfs EM, Prpic V, Daniel KW, Taylor IL, Collins S. Age-dependent changes in beta-adrenergic receptor subtypes and adenylyl cyclase activation in adipocytes from Fischer 344 rats. Endocrinology. 1995 May;136(5): 2022-32.
50.Lemaure B, Touch?? A, Zbinden I, Moulin J, Courtois D, Mac?? K, Darimont C. Administration of Cyperus rotundus tubers extract prevents weight gain in obese Zucker rats. Phytother Res. 2007 Aug; 21(8): 724-30.
51.Gauthier MS, Miyoshi H, Souza SC, Cacicedo JM, Saha AK, Greenberg AS, Ruderman NB. AMP-activated protein kinase is activated as a consequence of lipolysis in the adipocyte: potential mechanism and physiological relevance. J Biol Chem. 2008 Jun; 283(24): 16514-24.
52.Okazaki H, Osuga J, Tamura Y, Yahagi N, Tomita S, Shionoiri F, Iizuka Y, Ohashi K, Harada K, Kimura S, Gotoda T, Shimano H, Yamada N, Ishibashi S. Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases. Diabetes. 2002 Dec; 51(12): 3368-75.
53.Chang MS, Lee WS, Chen BC, Sheu JR, Lin CH. YC-1-induced cyclooxygenase-2 expression is mediated by cGMP-dependent activations of Ras, phosphoinositide-3-OH-kinase, Akt, and nuclear factor-kappaB in human pulmonary epithelial cells. Mol Pharmacol. 2004 Sep; 66(3): 561-71.
54.Huang SH, Shen WJ, Yeo HL, Wang SM. Signaling pathway of magnolol-stimulated lipolysis in sterol ester-loaded 3T3-L1 preadipocyes. J Cell Biochem. 2004 Apr; 91(5): 1021-9.
55.Suganuma M, Fujiki H, Suguri H, Yoshizawa S, Hirota M, Nakayasu M, Ojika M, Wakamatsu K, Yamada K, Sugimura T. Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proc Natl Acad Sci U S A. 1988 Mar; 85(6): 1768-71.
56.Haystead TA, Sim AT, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan; 337(6202): 78-81.
57.Clifford GM, McCormick DK, Londos C, Vernon RG, Yeaman SJ. Dephosphorylation of perilipin by protein phosphatases present in rat adipocytes. FEBS Lett. 1998 Sep; 435(1): 125-9.
58.He J, Jiang H, Tansey JT, Tang C, Pu S, Xu G. Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes. Biochim Biophys Acta. 2006 Feb; 1761(2): 247-55.
59.Ahn IS, Do MS, Kim SO, Jung HS, Kim YI, Kim HJ, Park KY. Antiobesity effect of Kochujang (Korean fermented red pepper paste) extract in 3T3-L1 adipocytes. J Med Food. 2006 Spring; 9(1): 15-21.
60.Yin J, Gao Z, He Q, Zhou D, Guo Z, Ye J. Role of hypoxia in obesity-induced disorders of glucose and lipid metabolism in adipose tissue. Am J Physiol Endocrinol Metab. 2009 Feb; 296(2): E333-42.