莲藕中白桦脂酸的微生物转化及其产物的抗肿瘤活性研究

李会伟; 段金廒; 刘培; 张森; 江曙; 陶伟伟

莲藕中白桦脂酸的微生物转化及其产物的抗肿瘤活性研究

南京中医药大学，江苏省中药资源产业化过程协同创新中心，中药资源产业化与方剂创新药物国家地方联合工程研究中心，中药品质与效能国家重点实验室培育，江苏南京 210023

计量
- 文章访问数: 1406
- HTML全文浏览量: 3
- PDF下载量: 1086
- 被引次数: 0
出版历程
- 收稿日期: 2017-01-05
- 修回日期: 2017-02-23
- 刊出日期: 2017-03-10

Biotransformation of Betulinic Acid From Nelumbinis Rhizoma and Cytotoxicity Determination of the Fermentation Products Against Cancer Cell Lines

摘要

摘要: 目的对莲藕中的白桦脂酸进行微生物转化，并对转化产物分离纯化，解析其结构特征，评价其抗肿瘤活性，以提高莲藕资源的利用率。方法采用HPLC-PDA法测定莲藕根茎不同部位白桦脂酸的含量；筛选具有转化作用的菌种，采用UPLC-Q-TOF/MS方法对发酵产物进行分析；采用硅胶柱层析法分离转化产物，NMR和MS法解析发酵产物结构特征；利用细胞分子生物学技术测定衍生物抗肿瘤活性。结果莲藕不同部位（藕尖、藕肉、藕皮、藕节、藕尾）中白桦脂酸含量以藕节中最高，为0.621%，筛选得到一株具有转化白桦脂酸的菌种Bacillus amyloliquefaciens FJ18，该菌转化藕节中的白桦脂酸（1），获得2个转化产物，分别为3β-hydroxy-lup-20(29)-en-28-oic acid 28-O-(4-oxy-4-oxobutanoic acid-4-O-β-D-glucopyranosyl) ester（2）和28-O-β-D-glucopyranosyl-3β-hydroxy-lup-20(29)-en-28-oate（3），其中化合物2为新化合物。体外细胞毒性实验表明转化产物对A375、Hela、U251、SH-SY5Y和MCF-7等肿瘤细胞均有一定的细胞毒活性。结论通过微生物发酵从藕节中获得具有抗肿瘤活性的白桦脂酸转化产物，其中一个为新化合物。
- 白桦脂酸 /
- 生物转化 /
- 莲藕 /
- 解淀粉芽孢杆菌
Abstract: OBJECTIVE Betulinic acid (BA) from the waste of Nelumbinis Rhizoma (NR) was introduce sugar groups at specific positions by biotransformation. The fermentation products were isolated and purified. Their structural characterization and antitumor activities were also investigated， which may improve utilization of NR. METHODS The content levels of BA in different parts of NR were analyzed by the modified HPLC-PDA method. With the transformation ability of strains screened， the transformation products were analyzed using a UPLC-QTOF-MS method， isolated and purified by silica gel column chromatography repeatedly. The structure characteristics were analyzed by NMR and MS. Cell viability was evaluated by the methylthiazolyl tetrazolium (MTT) assay. RESULTS The content of BA(1) in the nodus was measured to be 0.621%， the highest in the parts of NR. The strain， identified as Bacillus amyloliquefaciens FJ18， was used in the biotransformation. A novel glycosylation derivative of betulinic acid (BA) derivative， 3β-hydroxy-lup-20(29)-en-28-oic acid 28-O-(4-oxy-4-oxobutanoic acid-4-O-β-D-glucopyranosyl) ester， together with a known derivative， 28-O-β-D-glucopyranosyl-3β-hydroxy-lup-20(29)-en-28-oate were obtained by bioconversion of BA. Their structures were determined on the basis of spectroscopic data. The biotransformation products showed in vitro cytotoxic activity against A375 (melanoma)， HeLa (cervical carcinoma)， U251 (glioma)， SH-SY5Y (neuroblastoma) and MCF-7 (breast cancer) cell lines. CONCLUSION The biotransformation of betulinic acid to the derivatives featuring D- glucopyranosyl moieties was accomplished. The in vitro cytotoxicity study revealed that the derivatives exhibit potent cytotoxic activity against cancer cell lines.
- betulinic acid /
- biotransformation /
- Nelumbinis Rhizoma /
- Bacillus amyloliquefaciens

HTML全文

参考文献(20)

[1]	ALI-SEYED M， JANTAN I， VIJAYARAGHAVAN K， et al. Betulinic acid: recent advances in chemical modifications， effective delivery， and molecular mechanisms of a promising anticancer therapy[J] . Chem Biol Drug Des， 2016， 87: 517-536.
[2]	ZHANG DM， XU HG， WANG L， et al. Betulinic acid and its derivatives as potential antitumor agents[J] . Med Res Rev， 2015， 35: 1127-1155.
[3]	CSUK R. Betulinic acid and its derivatives: a patent review (2008-2013)[J] . Expert Opin Ther Pat， 2014， 24: 913-923.
[4]	GAUTHIER C， LEGAULT J， LAVOIE S， et al. Synthesis and cytotoxicity of bidesmosidic betulin and betulinic acid saponins[J] . J Nat Prod， 2009， 72: 72-81.
[5]	SCHWARZ S， SIEWERT B， XAVIER NM， et al. A "natural" approach: synthesis and cytoxicity of monodesmosidic glycyrrhetinic acid glycosides[J] . Eur J Med Chem， 2014， 72: 78-83.
[6]	THIBEAULT D， GAUTHIER C， LEGAULT J， et al. Synthesis and cytotoxicity of lupane-type triterpenoid glyceryl esters[J] . Bioorg Med Chem Lett， 2012， 22: 4735-4739.
[7]	BARATTO LC， PORSANI MV， PIMENTEL IC， et al. Preparation of betulinic acid derivatives by chemical and biotransformation methods and determination of cytotoxicity against selected cancer cell lines[J] . Eur J Med Chem， 2013， 68: 121-131.
[8]	CHATTERJEE P，PEZZUTO JM， KOUZI SA. Glucosidation of betulinic acid by Cunninghamella species[J] . J Nat Prod， 1999， 62: 761-763.
[9]	YANG X， YANG G. HPLC determination of betulinic acid in semen Ziziphi Spinosae[J] . Drug Anal， 2006， 5: 683-685.
[10]	Doukyu N， Aono R. Screening and application of organic solvent resistant-microorganisms[J] . Biosci Indust， 2003， 61: 17-22.
[11]	CHEPHA T， CADAU M， GIRIN P， et al. Monitoring of ascorbate at a constant rate in cell culture: Effect on cell growth[J]. In Vitro Cell Dev-An， 2001， 37(1):26-30.
[12]	UTAGAWA T， MORISAWA H， MIYOSHI T， et al. A novel and simple method for the preparation of adenine arabinoside by bacterial transglycosylation reaction[J] . Febs Lett， 1980， 109(2): 261-3.
[13]	MARAGOS CM， WANG JM， HRABIE JA， et al. Nitric oxide/Nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via Nitric oxide release[J] . Cancer Res， 1993， 53(3): 564-568.
[14]	HAUPT Y， ROWAN S， SHAULIAN E， et al. Induction of apoptosis in HeLa cells by trans-activation-deficient p53[J] . Gene Dev， 1995， 9(17): 2170-2183.
[15]	ZHANG H， KONG X， KANG J， et al. Oxidative stress induces parallel autophagy and mitochondria dysfunction in human glioma U251 cells[J] . Toxicol Sci， 2009， 110(2): 376-388.
[16]	ZUCO V， SUPINO R， RIGHETTI SC， et al. Selective cytotoxicity of betulinic acid on tumor cell lines， but not on normal cells[J] . Cancer Lett， 2002， 175(1): 17-25.
[17]	CAMPLING BG， PYM J， BAKER HM， et al. Chemosensitivity testing of small cell lung cancer using the MTT assay[J] . Br J Cancer， 1991， 63(1): 75-83.
[18]	WOO PC， LAU SK， TENG JL， et al. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories[J] . Clin Microbiol Infect， 2008， 14(10): 908-934.
[19]	WANG Y， ZHANG CL， LIU YF， et al. Hepatoprotective Triterpenoids and Saponins of Schefflera kwangsiensis[J] . Planta Med， 2014， 80: 215-222.
[20]	GAUTHIER C， LEGAULT J， LEBRUN M， et al. Glycosidation of lupane-type triterpenoids as potent in vitro cytotoxic agents[J] . Bioorgan Med Chem， 2006， 14: 6713-6725.