苦参-蛇床子药对提取物对白念珠菌VVC临床株的抑制作用研究

中国真菌学杂志　 2019, Vol. 14 　Issue (3): 147-153.

苦参-蛇床子药对提取物对白念珠菌VVC临床株的抑制作用研究

施高翔^1,2, 姜晶晶^1,2, 汪云霞^1,2, 赵婷^1,2, 邵菁^1,2, 汪天明^1,2, 汪长中^1,2,3

1. 安徽中医药大学中西医结合学院;
2. 安徽省中医药科学院中西医结合研究所;
3. 中药复方安徽省重点实验室, 合肥 230012

收稿日期:2018-12-15 出版日期:2019-06-28 发布日期:2019-06-28
通讯作者: 汪长中,E-mail:ahwcz63@sina.com E-mail:ahwcz63@sina.com
作者简介:施高翔(1990-),男(汉族),硕士,讲师.E-mail:ssjlsgx@163.com
基金资助:
安徽省自然科学基金项目（1708085QH214）

Inhibitory effect of extract of Sophorae Flavescentis Radix-Cnidii Fructus Couplet medicines against Candida albicans isolate from VVC

SHI Gao-xiang^1,2, JIANG Jing-jing^1,2, WANG Yun-xia^1,2, ZHAO Ting^1,2, SHAO Jing^1,2, WANG Tian-ming^1,2, Wang Chang-zhong^1,2,3

1. Institute of Integrated Traditional Chinese and Western Medicine, Anhui University of Traditional Chinese Medicine;
2. Institute of Integrated Traditional Chinese and Western Medicine, Anhui Academy of Traditional Chinese Medicine;
3. Key Laboratory of Chinese Medicinal Formula of Anhui province, Hefei 230012, China

Received:2018-12-15 Online:2019-06-28 Published:2019-06-28

摘要/Abstract

摘要：

目的本文着重研究苦参-蛇床子药对提取物（Extract of Sophorae Flavescentis Radix-Cnidii Fructus Couplet medicines，ESCC）对白念珠菌VVC临床株的抑制作用。方法实验通过微量液基稀释法检测ESCC对白念珠菌的MIC₈₀；XTT减低法检测ESCC对白念珠菌VVC临床株细胞增殖活性的影响；倒置显微镜分别在4 h、8 h、12 h观察ESCC对白念珠菌VVC临床株酵母-菌丝二相性转换的影响；扫描电子显微镜观察ESCC对白念珠菌VVC临床株生物膜形成的影响；微孔板观察ESCC对白念珠菌VVC临床株成熟生物膜的影响；qRT-PCR检测ESCC对白念珠菌VVC临床株菌丝和生物膜形成相关基因的影响。结果实验结果显示，ESCC具有一定的抗真菌作用，MIC₈₀在512~1024 μg/mL之间，可显著降低白念珠菌VVC临床株细胞增殖活性；ESCC可抑制白念珠菌VVC临床株酵母-菌丝二相性转换，并可影响成熟生物膜的完整性。PCR结果显示ESCC可显著降低ALS1、ASL3、HWP1等基因转录水平。结论本实验研究表明，苦参-蛇床子1：1药对水提物可通过下调白念珠菌菌丝和生物膜形成相关基因转录水平，从而抑制酵母-菌丝二相性转换，影响其代谢活性，抑制生物膜的形成，并可破坏完整生物膜的完整性，从而起到抗真菌作用。

关键词: 苦参, 蛇床子, 药对, 白念珠菌, 菌丝, 生物膜

Abstract:

Objective To investigate the antifungal activity of extract of Sophorae Flavescentis Radix-Cnidii Fructus Couplet medicines against Candida albicans. Methods Microbroth dilution method was performed to determine the minimal inhibitory concentration (MIC). XTT reduction assay was used to evaluate the metabolic activity of C. albicans. Inverted microscopy and scanning electron microscopy (SEM) were used to observe C. albicans yeast-hypha conversion and biofilms respectively. Quantitative real time PCR (qRT-PCR) was performed to measure the expression of hypha and biofilms-related genes. Results ESCC showed significant antifungal effect and the MIC₈₀ range was 512~1024 μg/mL. The metabolic activity of C. albicans was inhibited by ESCC, and induced incomplete three-dimensional structure of C. albicans biofilms. And expression of hypha and biofilms related genes was down-regulated with ESCC exposure. Conclusion This study demonstrated that ESCC showed prominent anti-fungal activity via affecting yeast-hypha conversion and biofilms of C. albicans.

Key words: Sophorae Flavescentis Radix, Cnidii Fructus, Couplet medicines, Candida albicans, hypha, bioflims

中图分类号:

R379.4

施高翔, 姜晶晶, 汪云霞, 赵婷, 邵菁, 汪天明, 汪长中. 苦参-蛇床子药对提取物对白念珠菌VVC临床株的抑制作用研究[J]. 中国真菌学杂志, 2019, 14(3): 147-153.

SHI Gao-xiang, JIANG Jing-jing, WANG Yun-xia, ZHAO Ting, SHAO Jing, WANG Tian-ming, Wang Chang-zhong. Inhibitory effect of extract of Sophorae Flavescentis Radix-Cnidii Fructus Couplet medicines against Candida albicans isolate from VVC[J]. Chinese Journal of Mycology, 2019, 14(3): 147-153.

参考文献

[1] Sardi JC, Scorzoni L, Bernardi T, et al. Candida species:current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options[J]. J Med Microbiol, 2013, 62(Pt 1):10-24.
[2] DenningDW,Kneale M,Sobel JD, et al. Global burden of recurrent vulvovaginal candidiasis:a systematic review[J]. Lancet Infect Dis, 2018, 18(11):e339-e347.
[3] 晏亮, 邓淑文, 潘炜华, 等. 我国外阴阴道念珠菌病致病菌菌种分布及体外药敏研究现状[J]. 中国真菌学杂志, 2018, 13(1):46-52.
[4] Cronin S,Chandrasekar PH. Safety of triazole antifungal drugs in patients with cancer[J]. J Antimicrob Chemother,2010, 65(3):410-416.
[5] Kolve H, Ahlke E, Fegeler W, et al. Safety, tolerance and outcome of treatment with liposomal amphotericin B in paediatric patients with cancer or undergoing haematopoietic stem cell transplantation[J]. J Antimicrob Chemother,2009, 64(2):383-387.
[6] 吴岚, 周曾同, 周永梅, 等. 苦参碱对白色念珠菌生物膜的体外抑制作用[J]. 上海口腔医学, 2008, 18(4):415-418.
[7] 杨利林, 郜洁, 丘维钰, 等. 基于中医传承辅助平台的念珠菌性阴道炎外用治疗方剂组方规律分析[J]. 时珍国医国药, 2018, 29(5):1251-1253.
[8] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards:Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved Standard,edn 3; Document M27-A3. Wayne, PA:Clinical and Laboratory Standards Institute; 2009
[9] Martins CHG,Pires RH, Cunha AO, et al. Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm[J]. Fungal Biol. 2016, 120(4):530-537.
[10] Staniszewska M, Bondaryk M, Swoboda-Kopec E, et al. Candida albicans morphologies revealed by scanning electron microscopy analysis[J]. Braz J Microbiol,2013, 44(3):813-821.
[11] Xie Z, Thompson A, Kashleva H, et al. A quantitative real-time RT-PCR assay for mature C. albicans biofilms[J]. BMC Microbiol, 2011, 11:93.
[12] Khan M, Ahmed J,Gul A, et al. Antifungal susceptibility testing of vulvovaginal Candida species among women attending antenatal clinic in tertiary care hospitals of Peshawar[J]. Infect Drug Resist. 2018, 11:447-456.
[13] Takalkar D, Desai N. Nanolipid gel of an antimycotic drug for treating vulvovaginal candidiasis-development and evaluation[J]. AAPS PharmSciTech,2018, 19(3):1297-1307.
[14] 吕春艳, 吕邵娃, 李国玉, 等. 中药复方性味与组分配伍药理效应的研究进展[J]. 中国中药杂志, 2018, 43(6):1099-1103.
[15] 宋佳, 高晓霞, 田俊生, 等. 中药药对配伍机制的现代研究[J]. 中草药, 2017, 48(21):4367-4374.
[16] 范瑞强, 袁娟娜. 复发性外阴阴道念珠菌病中西医结合治疗专家共识[J]. 中国真菌学杂志, 2017, 12(6):325-327.
[17] 宋倩倩, 毕焕洲. 中医药治疗外阴阴道假丝酵母菌病简况[J]. 实用中医内科杂志, 2013, 30(3):122-123.
[18] 王慧, 陆兔林, 李金慈, 等. 舒乐洗剂治疗外阴阴道念珠菌病的体内外实验研究[J]. 临床合理用药杂志, 2015, 8(2):33-35.
[19] 刘一婷,杜敏. 苦参栓联合中药内服治疗念珠菌性阴道炎临床研究[J]. 亚太传统医药, 2017, 13(6):154-155.
[20] 艾芸, 刘建利,刘竹兰, 等. 苦参与蛇床子配伍过程中化学成分变化研究[J]. 中草药, 2007, 38(2):205-207.
[21] 郭晓蕾, 朱思潮, 张晨, 等. 苦参碱氧化苦参碱与蛇床子提取物联合抗菌作用研究[J]. 中华中医药学刊, 2011, 29(6):1398-1400.
[22] 钟嘉丽, 梁丹灵, 李丹. 基于网络药理学观察苦参与蛇床子抗皮肤瘙痒的协同作用机制[J]. 广州中医药大学学报, 2017, 34(5):742-752.
[23] Caldara M,Marmiroli N. Tricyclic antidepressants inhibit Candida albicans growth and biofilm formation[J]. Int J Antimicrob Agents,2018, 52(4):500-505.
[24] Zarnowski R, Sanchez H, Covelli AS, et al. Candida albicans biofilm-induced vesicles confer drug resistance through matrix biogenesis[J]. PLoS Biol,2018, 16(10):e2006872.
[25] Hoyer LL, Green CB, Oh SH, et al. Discovering thesecerts of the Candida albicans agglutinin-like sequence(ALS) gene family-a sticky pursuit[J]. Med Mycol, 2008, 46(1):1-15.
[26] Cleary IA,Reinhard SM, Miller CL, et al. Candida albicans adhersion Als3p is dispensable for virulence in the mouse model of disseminated candidiasis[J]. Microbiology, 2011, 157(6):1806-1815.
[27] Freire F, de Barros PP, Pereira CA, et al. Photodynamic inactivation in the expression of the Candida albicans genes ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 in biofilms[J]. Lasers Med Sci,2018, 33(7):1447-1454.