珠子参化学成分及其活性成分的筛选研究

【作者】 张延妮；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2010， 博士

【摘要】 珠子参被称为太白七药的“八大金刚”之一,是太白中草药中的要药。《中华人民共和国药典》(2010年,一部)收录的珠子参原植物为五加科(Araliaceae)人参属(Panax)植物珠子参(Panaxjaponicus C.A. Mey. var. major (Burk.)C.Y.Wu et K.M.Feng)和羽叶三七(Panaxjaponicus C.A.Mey. var. bipinnatifidus (Seem.)C.Y.Wu et K.M.Feng)。长期以来,由于缺乏对珠子参的化学物质基础及活性成分的筛选研究,它的开发与利用也受到了一定的限制。本文对珠子参的皂苷、醇提物中脂肪酸、多糖及其中单糖组成、不同部位的脂溶性成分进行了定量分析,并对几种提取物的抗氧化和抗菌活性进行了考察,同时建立了基于兔心肌细胞膜的药物筛选方法并将其应用于珠子参醇提物脂溶性成分的筛选,以期为珠子参的合理用药和开发利用提供科学依据。本论文的主要研究内容和结果如下：1.不同提取方法对珠子参总皂苷得率的影响。超声法提取珠子参总皂苷的最佳工艺条件为：料液比1：20,乙醇浓度50%,提取温度50℃,提取时间30 min。超声提取与传统的提取方法(温浸法和回流法)相比,超声提取法提取珠子参总皂苷得率(8.30%)明显高于温浸法(5.06%)和回流法(6.30%)。2.珠子参中人参皂苷Re、Rf和Rb1含量的同时测定。建立了反相高效液相色谱同时测定珠子参中人参皂苷Re、Rf和Rb1含量的方法,实验条件为：流动相乙腈(A)-水(B),梯度洗脱条件：0-35 min,19%A；35-55 min,19%A-29%A；55-65 min,29%A；流动相流速为0.8 mL/min；进样量10μL；检测波长203 nm；柱温为室温。该法灵敏、准确、可靠。应用所建立的方法对珠子参中人参皂苷Re、Rf和Rb1的含量进行了分析,结果表明珠子参中含有丰富的人参皂苷Re、Rf和HRb1,其质量百分比含量分别为原料药的0.072%,1.341%和0.064%。3.珠子参总酚酸的提取及超声提取工艺的研究。正交实验确定的珠子参总酚酸超声提取的最佳工艺条件为：料液比1：20,乙醇浓度50%,提取温度60℃,提取时间30 min。在此条件下珠子参总酚酸得率为0.273%。4.珠子参醇提物中高级游离脂肪酸的含量测定。对脂肪酸的甲酯化方法进行了优化,采用气相色谱质谱联用技术建立了高级游离脂肪酸的含量测定方法,并应用于珠子参和羽叶三七醇提物的分析。两种醇提物中均鉴定出了6种主要的高级游离脂肪酸,分别是棕榈油酸、棕榈酸、亚油酸、油酸、α-亚麻酸和硬脂酸,它们的含量对于珠子参醇提物分别为0.53 mg/g,4.74 mg/g、2.31 mg/g、1.02 mg/g、0.12 mg/g和1.54 mg/g；对于羽叶三七醇提物分别为0.12mg/g、0.98mg/g、0.54mg/g、0.25mg/g、0.30mg/g和10.20mg/g。5.珠子参多糖中的单糖组成分析。优化了多糖的水解方法和单糖的衍生化方法；应用气相色谱-质谱联用技术对珠子参多糖中的单糖组成和相对含量(归一化含量)进行了分析,结果显示,珠子参多糖由鼠李糖(1.88%)、核糖(5.22%)、阿拉伯糖(2.21%)、木糖(3.59%)、甘露糖(4.08%)、葡萄糖(78.13%)和半乳糖(4.21%)7种单糖组成。6.珠子参不同部位的脂溶性成分。结果显示,珠子参根茎、茎与叶中的脂溶性成分有显著差别,实验结果为传统的用药经验即将根茎与叶分开入药提供了理论依据；在珠子参的不同部位中鉴定出了很多含量很高且具有重要药理活性的化合物,如十六烷酸、十八碳二烯酸、十八烷酸、镰叶芹醇、β-谷甾醇以及角鲨烯等,从物质基础上阐明了珠子参的脂溶性成分确实具有重要的药用价值。7.珠子参不同提取物的体外抗氧化活性研究。采用DPPH自由基清除实验和β-胡萝卜素亚油酸漂白实验对珠子参不同提取物体外抗氧化能力进行了综合评价。结果显示,珠子参的不同提取物都有一定的抗氧化能力,丙酮提取物具有优于其他提取物的较强的抗氧化活性,各提取物的DPPH自由基清除能力次序和总抗氧化能力的次序一致,依次为：丙酮提取物>甲醇提取物>总皂苷。8.珠子参不同提取物的抑菌活性研究。珠子参中各种提取物对大肠杆菌、金黄色葡萄球菌、铜绿假单孢杆菌、白色葡萄球菌、枯草芽胞杆菌、凝结芽胞杆菌以及蜡状芽胞杆菌等7种所试菌种均有不同程度的抑制和灭活作用,其中总皂苷的抑制效果最强。9.基于心肌细胞膜受体的生物学特性并借助GC-MS技术建立了旨在筛选治疗心血管疾病的药物筛选模型。建立了细胞膜ATP酶活性的测定方法并将其用于兔心肌细胞膜的分析借以间接考察细胞膜受体的活性变化规律,研究结果显示：保存在PBS缓冲液中的心肌细胞膜上的ATP酶的生物活性随着离体时间的流逝逐渐降低,但能持续一段时间(至少9h)保持活性,这在很大程度上反应了细胞膜受体生物活性的变化规律,提示心肌细胞膜在离体的一段时间内可用于药物筛选。将所建立的模型应用于珠子参醇提物脂溶性成分中潜在活性成分的筛选。将珠子参醇提物脂溶性成分分别与不同活性的心肌细胞膜一起温浴反应,然后分离细胞膜并借助GC-MS技术对温浴前后的提取液中的各组分进行分离和鉴定,通过对比组分种类和含量的变化来分析、筛选潜在的活性成分。结果显示,在目前的筛选条件下,珠子参醇提物脂溶性成分中有两种能够与活性心肌细胞膜特异性作用的组分,包括β-谷甾醇和一个未鉴定物质。实验结果为进一步改善分子水平的药物筛选模型奠定了一定的基础。更多还原

【Abstract】 Panax japonicus C. A. Mey. var. major (Burk.) C.Y.Wu et K.M.Feng, being listed among Eight KingKong of Taibai qiyao, is one of the main Chinese medicinal plants. There are two varieties of Panax(P. japonicus C. A. Mey. var. major (Burk.) C.Y.Wu et K.M.Feng and P. japonicus C. A. Mey. var. bipinnatifidus (Seem.) C.Y.Wu et K.M.Feng) recorded as the original plants in the Chinese Pharmacopoeia. Popular as it is in folk medicine, yet the application and exploitation of P. japonicus C. A. Mey. var. major are far from its due. The reason mainly lies in it that we lack in the studies on chemical substances and screening of the active components. In this study, total saponins, free fatty acids(FFAs) in ethanol extract, polysaccharide and its monosaccharide composition, liposoluble constituents in different parts from P. japonicus C. A. Mey. var. major were quantitative analyzed, and antioxidant and antibacterial activities of several extracts were studied. The screening model for active compounds was established and applied to liposoluble constituents of alcoholic extract from P. japonicus C.A. Mey. var. major. The study would provide scientific information for the rational drug administration and further utilization.The main contents of this dissertation are as follows:1. Comparison between the extraction yields of total saponins with different methods from the rhizome of P. japonicus C. A. Mey. var. major. The best technological conditions for the ultrasonic-assisted extraction were obtained:ultrasonic time, ultrasonic temperature, ethanol concentration and ratio of gardenia to liquor were 30 min,50℃,50% and 1:20, respectively. The extraction yields obtained with ultrasonic-assisted extraction (8.30%) was considerably higher than those obtained with immersion (5.06%) and reflux method (6.30%)2. Simultaneous determination of ginsenoside R、Rf and Rb1 in the rhizome of P. japonicus C.A. Mey. var. major. RP-HPLC Simultaneous determination of ginsenoside Re、Rf and Rb1 was established, optimized experimental conditions were as follows: mobile phase was CH3CN (A)-H2O (B), the gradient elution was programmed 0-35 min,19%A; 35-55 min,19%A-29%A; 55-65 min,29%A; mobile phase was set at a flow rate of 0.8 mL/min; injection volume was set 10μL; the detection wavelength was set 203 nm. The method is sensitive, accurate and reliable. The method has been applied to the determination of ginsenoside Re、Rf and Rb1 from the rhizome of P. japonicus C. A. Mey. var. major. Experiment results revealed that the medicinal plant was abundant in ginsenoside, the percentage mass content of Re、Rf and Rb1 were 0.072%,1.341% and 0.064%, respectively.3. Study on optimization of the ultrasonic extraction conditions of total phenolic acids in rhizome of P. japonicus C. A. Mey. var. major, The best technological conditions were obtained by orthogonal experiment design. They were 60℃,30 min, ethanol concentration 50% and a ratio of gardenia to liquor of 1：20, respectively. The extraction yield of total phenolic acids under the optimized conditions was 0.273%.4. FFAs profile analysis in alcohol extract of rhizome of P. japonicus C. A. Mey. var. major. The method for the methyl esterification of FFAs was optimized, the quantitative method for the determination of FFAs by GC-MS was established and applied to the analysis of FFAs in alcohol extract of P. japonicus C. A. Mey. var. major and P. japonicus C.A.Mey. var. bipinnatifidus. Six main FFAs were identified in the alcohol extract of both medicinal plants; they are palmitoleic acid, palmitic acid, linoleic acid, linolenic acid, oleic acid and stearic acid. The content of FFAs in the alcohol extract from P. japonicus C. A. Mey. var. major is 0.53mg/g,4.74 mg/g、2.31 mg/g、1.02 mg、0.12mg/g、1.54 mg/g, respectively, The content of FFAs in the alcohol extract from P. japonicus C.A.Mey. var. bipinnatifidus 0.12mg/g、0.98mg/g、0.54mg/g、0.25mg/g、0.30mg/g、0.20mg/g, respectively.5. Study on the composition of monoses hydrolyzied from polysaccharide of the rhizome of P. japonicus C. A. Mey. var. major. The hydrolysis method for polysaccharide and the derivative method for monosaccharide were optimized. A GC-MS method was developed for identifying and quantifying(normalization method) the hydrolyzied monoses. The results revealed that 7 monoses were found hydrolyzied from polysaccharide of P. japonicus C. A. Mey. var. major, they were rhamnose (1.88%),ribose (5.22%), arabinose (2.21%), xylopyranose (3.59%), mannose (4.08%), glucose (78.13%), galactose (4.21%)6. The liposoluble constituents from different parts of P. japonicus C. A. Mey. var. major. The results revealed that components from rhizome were quite different from those from leaves. Compounds composition analysis provided favorable evidence for the regularity of prescription about this medicinal plant, in which many pharmacologically valuable components with high content were found in different parts, such as hexadecanoic acid, linoleic acid, stearic acid, falcarinol,β-sitosterol, squalene, and so on. The results revealed that the liposoluble components from P. japonicus C. A. Mey. var. major did have high pharmaceutical value. 7. Study on the antioxidant activities of different extract from the rhizome of P. japonicus C. A. Mey. var. major. The antioxidant activities were evaluated with DPPH free radical scavenging assay andβ-carotene-linoleic acid assay. The results showed all the extracts tested all possessed antioxidant capacity, and the acetone extracts showed higher antioxidant activity than other extracts. The order of the capacity to scavenge DPPH free radical was in accord with that of total antioxidant capacity, ie. acetone extracts>methanol extracts>total saponins.8. Study on the antibacterial activities of different extracts from the rhizome of P. japonicus C. A. Mey. var. major. All extracts from P. japonicus C.A.Mey. var. major had showed different degrees of antibacterial activity on pathogenic bacteria tested, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus white, Bacillus subtilis, Bacillus coagulans and Bacillus cereus. The total saponins showed better bacterial activities.9. At the advantage of GC-MS techniques and special affinity for active receptors on CMCM, we established a drug discovery model for screening active compounds for cardiovascular diseases. An online method for determining ATPase bioactivity was so developed as to indirectly investigate the activities of acceptors on CMCM. experimental results revealed that the bioactivity of ATPase decreased with increasing lapse of time in vitro in buffer solution, while the ATPase on CMCM remained bioactive over a certain time (at least 9 h). It reflected the rule of the variation of the acceptor activities, which indicated that CMCM was eligible for pertinent medicine discovery. The screening model was applied to the liposoluble components of alcoholic extract of P. japonicus C.A.Mey. var. major. CMCM at different activity levels was incubated with the liposoluble components of the extract from rhizome of P. japonicus C.A. Mey. var. major, and then the individual liposoluble compounds underwent separation and identification on GC-MS. With probing the variation of the content of the identified compounds before and after the incubation, we preliminarily screened the potentially active compounds.Experimental results revealed that under the developed discovery model two compounds in the liposoluble components of the extract were found to have shown specific affinity, they wereβ-sitosterol and another unidentified compound.The experimental results laid the foundation for the further perfection of drug discovery model in molecular level.更多还原