独山瓜馥木抗结核杆菌成分及机制研究

【作者】 邓伟；

【导师】 杨再昌；

【作者基本信息】 贵州大学 ， 药物化学， 2017， 硕士

【摘要】 结核病是由结核分枝杆菌引起的慢性传染性疾病,其致死率仅次于艾滋病,对患者身体健康造成了严重的影响。近年来由于耐药性结核杆菌和结核病与艾滋病双重感染的出现,无疑给结核病的治疗带来了更加严峻的挑战,研发新型抗结核药物刻不容缓。结核杆菌持留状态或称休眠状态是导致结核病疗程长、疗效不佳、复发及难以根治的根本原因之一。结核杆菌休眠后,对一线抗结核药物形成表型耐药,长期潜伏在患者体内,当机体免疫功能降低时,休眠型结核杆菌复苏,使患者再次发病。所以我们萌生了一种思路,即先复苏休眠型结核杆菌然后再联用抗痨药物对其进行杀灭,从而达到根治结核的目的。本实验在前期的研究基础上,采用活性跟踪分离方法,从独山瓜馥木石油醚萃取物分离得到能促进休眠型结核杆菌复苏的化合物GF-01,被鉴定为β-谷甾醇-9,12-十六碳二烯酸酯。化合物GF-01在浓度为16μg/m L时开始显示促复苏活性,呈明显的量效关系。异烟肼、乙胺丁醇、利福平和吡嗪酰胺单独用药时,不能杀死休眠型结核杆菌,化合物GF-01(32μg/mL)与异烟肼联合用药,培养14、21d后检测,休眠型结核杆菌被杀死,化合物GF-01可增强利福平对休眠菌的杀灭作用,但分别与乙胺丁醇和吡嗪酰胺联合时,均表现为拮抗作用。化合物GF-01能激活休眠型结核杆菌的KatG酶,使细胞内O2含量升高,导致休眠型结核杆菌复苏。另外,发现独山瓜馥木的乙酸乙酯萃取物对复制型结核杆菌具有明显的抑制作用,其MIC值小于1 mg/mL,通过活性跟踪分离,从中分离得到的GF-02,被鉴定为5-羟甲基糠醛,对复制型结核分枝杆菌具有抑制作用,MIC为80μg/mL,且该化合物与异烟肼、利福平均表现出协同作用。5-羟甲基糠醛虽然抗结核杆菌的活性并不特别强,但其抗结核杆菌活性属首次发现,是抗结核杆菌的新骨架。对4种5-羟甲基糠醛结构类似物进行体外抗结核杆菌活性试验,发现2,5-呋喃二甲醇、5-羟甲基-2-呋喃甲酸具有抗结核杆菌活性,初步构效关系表明,抗结核杆菌结构母核为α-呋喃甲醇,为进一步研发新型抗结核药物提供了契机。更多还原

【Abstract】 Tuberculosis(TB)is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis complex(MTBC),its mortality is ranking below AIDs,which seriously endangers human health and increases the family burden.Because of the emergence of drug resistance and the co-infection of Human immunodeficiency virus-Tuberculosis(HIV-TB),making the effective therapy of tuberculosis to face more serious challenges.So it is urgent to research the novel anti-tuberculosis drugs at present.Mycobacterium tuberculosis has the unique property of becoming persistent or dormant for very long periods,it is the major reason that leads a long course of treatment regimens,low efficiency,easy recurrence and difficult to cure.When the Mycobacterium tuberculosis stays dormancy,making Mycobacterium tuberculosis to first-line anti-TB drugs phenotypic resistance and latency for long time,but when the human body immunity drops,the dormant Mycobacterium tuberculosis will recover and the body become infection again.In this paper,the study is based on the previous research.Compound GF-01,with resuscitating activity on dormant Mycobacterium tuberculosis,was isolated from Fissistigma cavaleriei root by bioassay-guided method.It was identified asβ-sitosterol-9,12-hexadecadienoate by physical and spectroscopic methods.Compound GF-01 showed activity to resuscitate dormant Mycobacterium tuberculosis into active state in a dose-dependent manner with minimum effective concentration of 16 μg/mL.The dormant Mycobacterium tuberculosis was insensitive to isoniazid,ethambutol,rifampin,and pyrazinamide in single drug.At the concentration of 32 μg/m L,compound GF-01 could help isoniazid to kill the dormant Mycobacterium tuberculosis after 14 and 21 days of exposure,and it can increase the killing activity of rifampin against dormant Mycobacterium tuberculosis.However,among two-drug combinations,compound GF-01-ethambutol and compound GF-01-pyrazinamide,exhibited antagonistic action on dormant Mycobacteriumtuberculosis.Compound GF-01 could activate the KatG activity of dormant Mycobacterium tuberculosis,which caused the content of O2 in dormant Mycobacterium tuberculosis cells at a high level to trigger the resuscitation of dormant Mycobacterium tuberculosis.Modified-Lowenstein-Jensen-Medium was used to evaluate the anti-Mycobacterium tuberculosis activity of the Fissistigma cavaleriei fraction.The active compound GF-02 were isolated from the active fraction of Fissistigma cavaleriei root by silica gel column chromatography.We found that the ethyl acetate fraction of Fissistigma cavaleriei root ethanol extracts showed activity to inhibit Mycobacterium tuberculosis,the minimum effective concentration less than 1mg/ml.And the compound GF-02,it was identified as 5-hydroxymethylfurfural(5-HMF).Through the activity test,the GF-02 showed activity to inhibit replicating Mycobacterium tuberculosis with minimum effective concentration of 80 μg/ml,and also,the GF-02 showed asynergistic effect with isoniazid and rifampici,indicated that the GF-02 could reduce the degree of isoniazid and rifampicin resistance,so as to reduce the clinical dosage.GF-02 was isolated from the plant for the first time.Althoug the activity of GF-02 anti-Mycobacterium tuberculosis is not particularly strong,but its anti-Mycobacterium tuberculosis activity is the first discovery,and it is the new structure of anti-Mycobacterium tuberculosis molecular.In order to evaluate preliminary the structure-activity relationship of5-hydroxymethylfurfural,we carry out the anti-Mycobacterium tuberculosis activity test of four 5-hydroxymethylfurfural analogues,The results showed that2,5-dihydroxymethylfura and 5-hydroxymethyl-2-furancarboxylic acid had anti-Mycobacterium tuberculosis activity,so we can get conclusion that the active nucleus structure is furfuryl alcohol.It provides an opportunity to find new anti-TB drugs for the further.更多还原