中国沙棘和西藏沙棘叶中成分比较研究及其活性初步筛选

发布时间：2018-06-22 04:15

本文选题：中国沙棘叶 + 西藏沙棘叶　；参考：《北京中医药大学》2017年硕士论文

【摘要】：研究目的中国沙棘(Hippophae rhamnoides L.subsp.sinensisRousi),胡颓子科沙棘属植物沙棘(Hioppophae rhamnoides L.)的一个变种,自1977年被首次载入《中国药典》至今,具有健脾消食,止咳祛痰,活血散瘀等功效。西藏沙棘(H.tibetana Schlecht.)与沙棘(H.rhamnoides L.)相近,起初被合并在沙棘种内作异名,后因其形态特征、地理分布和生长环境都特殊,学者把它归为独立的种,是藏医、蒙医的习用药材,具有补肺,活血的功效,用于治月经不调、子宫病、胃病、肺结核、胃溃疡等,西藏北部的群众还用其治肝炎。沙棘作为一种药食同源植物,具有丰富的生物活性成分和营养物质。因《药典》中沙棘以果实入药,故一般开发产品利用的原料是其果实,而沙棘叶被当做废料遗弃;黄酮类成分是沙棘相关产品质量及其理化鉴别的重要衡量指标,而研究证实其在叶中含量最高,100 g干叶中为310-1238 mg(均值为792 mg,鲜果中均值为533 mg),每公顷的沙棘叶经计算,其年产量可多达5吨,废弃不用造成了极大的资源浪费;除此之外,相较于果实,沙棘叶还具有较高的产量、较长的收集周期、简单的生产加工工艺以及易于储藏等优势,通过对其叶中成分进行深入研究、合理利用,其发展前景是诱人的。因此,立足资源优势,本课题进行了以下研究:(1)建立中国沙棘和西藏沙棘叶中化学成分快速鉴定的分析方法,总结其质谱裂解规律,分析比较两沙棘叶中成分组成特征;(2)将LC-MS分析技术与代谢组学数据处理技术相结合,构建中国沙棘叶和西藏沙棘叶的区分模型并寻找造成两沙棘叶差异的特征性成分。(3)收集不同产地中国沙棘和西藏沙棘叶,建立两沙棘叶中黄酮苷的含量测定方法,分析、比较两沙棘叶中成分含量上的特征;(4)采用H202诱导PC12细胞建立氧化损伤模型,通过倒置显微镜观察细胞形态,MTT法测定了解PC12细胞损伤情况和存活率,初步分析中国沙棘和西藏沙棘叶不同极性部位以及沙棘总黄酮(TFH)对氧化损伤后的PC12细胞的保护作用,为其在神经退行性疾病临床治疗方面奠定基础。研究方法1.运用超高效液相串联四级杆飞行时间质谱的联用仪器(UPLC/Q-TOF-MS)建立中国沙棘和西藏沙棘叶中化学成分快速鉴定的分析方法,优化质谱条件,使得各成分裂解的碎片稳定,裂解信息全面;总结两沙棘叶中成分的质谱裂解规律,分析比较两沙棘叶中成分组成上的特征。2.实验首先基于超高效液相线性离子阱-串联静电场轨道阱质谱联用技术(UPLC-LTQ-Orbitrap-MS)对两沙棘叶中成分进行充分解析获得尽可能多的化合物,继而采用代谢组学数据处理手段对实验数据做降维处理以及包括PCA和PLS-DA等在内的统计学分析,用于两沙棘叶中差异性成分的研究以及两沙棘叶的鉴定。3.采用甲醇回流提取方法对多产地中国沙棘和西藏沙棘叶进行提取,运用高效液相(HPLC)建立两沙棘叶中黄酮苷的定量方法,并对样品进行测定,分析不同产区两沙棘叶中成分含量上的差异。4.通过分析不同浓度以及不同作用时间下,H202对PC12细胞存活的影响,建立H202诱导的PC12细胞氧化损伤模型,初步分析中国沙棘和西藏沙棘不同极性部位以及沙棘总黄酮(TFH)对氧化损伤后的PC12细胞的保护作用。结果与结论1.基于UPLC/Q-TOF-MS技术,结合质谱数据、文献数据以及与标准品比对,从两沙棘叶中分析、推断出35个化合物,其中,29个化合物为两沙棘叶共有成分,剩余的6个成分中,2个成分为中国沙棘叶特有,其余为西藏沙棘叶独有。结果表明,中国沙棘和西藏沙棘叶中成分组成上大体一致,主要是以槲皮素、山奈酚、异鼠李素为苷元和糖结合形成的黄酮醇苷类物质,糖基主要有葡萄糖、鼠李糖等,因此碎片离子以丢失[M-H-162]-和[M-H-146]-居多;对于黄酮母核,主要发生C环的RDA裂解和一些小分子碎片的丢失,如CO,CHO,C302,CO2等。总结沙棘叶中成分的质谱裂解特征,发现负离子模式,糖基化位置不同,苷元离子(Y0-)和自由基苷元离子[Y0-H]-丰度不同:黄酮醇3-O-单糖苷产生的[Yo-H]-·丰度强,黄酮醇7-O-单糖苷产生的Y0-丰度强;对于黄酮3,7-二-O-双糖苷类成分,可通过3-O苷键的均裂,失去自由基形式的糖,例如-G1c,形成[Y30-H]-离子;3,7位同时丢失糖基,形成[Y0-2H]-离子;除此之外,质量数不同的糖基,其连接位置可通过[M-H]-离子及特征离子[Y30-H]-·与[Y0-2H]-的质量数之差得到推断。利用Mass1ynx 4.1软件经去噪、基线校正、重叠峰解析及峰对齐获得了两种沙棘叶中35种化学成分的相对定量信息,结果表明,对于两沙棘叶的主要成分而言,中国沙棘约为西藏沙棘含量的1.5倍,6个特异性成分含量相对主成分而言含量很低。2.基于UPLC-LTQ-Orbitrap-MS分析技术,根据分子离子峰、保留时间及裂解碎片信息,并与文献报道的化学成分及对照品进行比对,共确定及推断出41个成分。运用代谢组学数据处理手段对数据进行主成分分析(PCA)及偏最小二乘判别分析(PLS-DA),其中,PCA 分析模型 R2X = 69.2%,Q2=53.6%;PLS-DA 分析模型 R2Y=99.9%,Q2 =98.5%,成功构建了中国沙棘叶和西藏沙棘叶的区分模型;此外,采用PLS-DA对数据做进一步分析,对VIP(变量性重要投影)1的成分做配对t检验且以p0.05的化合物作为两沙棘叶中差异性成分,经统计分析及综合前期鉴定的化合物,从中国沙棘和西藏沙棘叶中共分析得到10个特征性差异成分。3.基于HPLC法建立了不同产地的两沙棘叶中黄酮苷的含量测定方法,结果表明,不同产地的两沙棘叶中黄酮苷的含量存在较大差异。中国沙棘叶中黄酮苷含量均值为1.13%,而西藏沙棘叶中黄酮苷含量变化较大,于0.6%-1.4%之间波动,猜测该测定结果可能与西藏沙棘的原始生境相关,其常常分布于海拔5000米以上的高寒地区,而本实验中样品采集地的温度、光照、海拔以及紫外辐射等均与其原始生境相差较大,对其黄酮类成分的合成与积累产生较大影响。4.分析不同浓度以及不同作用时间下H2O2对PC12细胞存活率影响,实验结果表明,H2O2能够呈剂量和时间依赖性的损伤PC12细胞并降低其生存率,且采用300 μM的H2O2处理PC12细胞4 h时,和对照组相比,细胞活力下降至54.67%,表明造模成功。研究中国沙棘和西藏沙棘不同极性部位以及沙棘总黄酮(TFH)对氧化损伤后的PC12细胞的保护作用,结果表明,中国沙棘和西藏沙棘叶不同极性部位成分都能够不同程度的抑制H2O2氧化损伤导致的细胞活力下降,其活性顺序为:正丁醇部位乙醇部位乙酸乙酯部位,而且中国沙棘乙酸乙酯部位活性明显高于其他各极性部位。测定各极性部位中总黄酮含量,表明沙棘氧化应激活性与总黄酮含量呈正相关。将沙棘总黄酮(TFH)含量富集至29.63%,可显著提高氧化损伤细胞的生存率。其中,0.4,0.25 mg/mlTFH保护组同损伤组间有显著差异(p0.05),对细胞有明显的保护作用,最大保护率达35.87%。
[Abstract]:Hippophae rhamnoides L.subsp.sinensisRousi (Seabuckthorn), a variety of Seabuckthorn (Hioppophae rhamnoides L.) of the genus seabuckthorn, was first loaded into the Chinese Pharmacopoeia (China Pharmacopoeia) for the first time since 1977. It has the efficacy of invigorating spleen and dissipating food, relieving cough and eliminating phlegm, promoting blood circulation and dispersing blood stasis. Tibet Seabuckthorn (H.tibetana Schlecht.) and Seabuckthorn (H.rhamnoide) S L.) is similar, initially merged in the Seabuckthorn species as a special name, after its morphological characteristics, geographical distribution and growth environment are special, the scholar classified it as an independent species, is a Tibetan medicine, Mongolian medicine used medicinal herbs, with the effect of lung filling, blood activating, used to treat menstruation, uterine disease, stomach disease, tuberculosis, gastric ulcer, and so on. The people in northern Tibet are also treated with their treatment. Seabuckthorn, as a kind of medicine and food homologous plant, has abundant bioactive components and nutrients. As the Pharmacopoeia of Hippophae rhamnoides in the Pharmacopoeia, the raw material used for the development of the product is its fruit, and the leaves of the Seabuckthorn are abandoned as waste, and flavonoids are the important measure of the quality and physicochemical identification of Hippophae rhamnoides. The study confirmed that the content of the leaves was the highest in the leaves, and the 100 g dry leaves were 310-1238 Mg (mean 792 mg and 533 mg in fresh fruit). The annual output of seabuckthorn leaves per hectare could be as many as 5 tons, and the waste did not cause great waste of resources; in addition to this, the leaves of seabuckthorn also had a higher yield and a longer collection cycle than the fruit. The development prospects of the components in the leaves are attractive. Therefore, based on the advantages of resources, the following research has been carried out in this subject: (1) to establish an analytical method for rapid identification of chemical components in the leaves of seabuckthorn and Tibet Hippophae rhamnoides in China, and to sum up the law of mass spectrometric fragmentation. The composition characteristics of the leaves of two Hippophae rhamnoides were analyzed and compared. (2) combining the LC-MS analysis technique with the metabolic data processing technology, the distinguishing model of the leaves of seabuckthorn and Tibet Hippophae rhamnoides was constructed and the characteristic components of the leaves of two Seabuckthorn were found. (3) collecting the leaves of seabuckthorn and Tibet Seabuckthorn from different habitats, and establishing the flavonoids in the leaves of two Seabuckthorn The content determination method of glucoside, analysis and comparison of the characteristics of components in the leaves of two Hippophae rhamnoides; (4) H202 induced PC12 cells were induced to establish an oxidative damage model, and the cell morphology was observed by inverted microscope. The damage and survival rate of PC12 cells were measured by MTT method, and the different polar parts of seabuckthorn and Tibet seabuckthorn leaves and seabuckthorn were preliminarily analyzed. The protective effect of total flavonoids (TFH) on PC12 cells after oxidative damage lay the foundation for the clinical treatment of neurodegenerative diseases. 1. the method of rapid identification of chemical components in Chinese Hippophae rhamnoides and Tibet Hippophae rhamnoides leaves was established by using super high performance liquid phase tandem four time mass spectrometry (UPLC/Q-TOF-MS), and the method of rapid identification of Chinese Hippophae rhamnoides and Tibet Hippophae rhamnoides leaves was established. By optimizing the mass spectrum conditions, the fragmentation of the components is stable and the fragmentation information is comprehensive; the mass spectrometric fragmentation of the components of the two sea buckthorn leaves is summarized, and the characteristics of the composition of the components of the two sea buckthorn leaves are compared with the ultra high performance liquid phase linear ion trap series electrostatic field track well mass spectrometry (UPLC-LTQ-Orbitrap-MS) technique (two) to two. The components of the Hippophae rhamnoides leaves are fully analyzed to obtain as many compounds as possible, and then using the metabonomics data processing methods to reduce the dimension of the experimental data and the statistical analysis including PCA and PLS-DA, and for the study of the difference components in the leaves of two Hippophae rhamnoides and the identification of the two seabuckthorn leaves by the method of methanol reflux extraction. The leaves of Hippophae rhamnoides and Tibet sea buckthorn were extracted from China. The quantitative method of flavonoid glycosides from two seabuckthorn leaves was established by high performance liquid phase (HPLC). The samples were determined and the difference of the content of the components in the leaves of two seabuckthorn in different producing areas was analyzed by the analysis of the effects of H202 on the survival of PC12 cells by analyzing the different concentration and the different action time, and establishing the H. 202 induced oxidative damage model of PC12 cells, preliminary analysis of the protective effect of different polar parts of seabuckthorn and Tibet sea buckthorn and total flavonoids of Hippophae rhamnoides (TFH) on the PC12 cells after oxidative damage. Results and conclusion 1. based on UPLC/Q-TOF-MS technology, combined with mass spectrometry data, literature data and comparison with standard products, the analysis from two Hippophae rhamnoides leaves is carried out. 35 compounds were broken out, of which 29 compounds were two seabuckthorn leaves. Among the remaining 6 components, 2 were Chinese Hippophae leaves and the rest were unique to the leaves of Tibet Hippophae rhamnoides. The results showed that the components of the leaves of seabuckthorn and Tibet sea buckthorn were generally consistent with the combination of quercetin, kaempferol and ISO rhamnolipin as glycosides and sugar. The formation of flavonol glycosides, sugar based mainly glucose, rhamnose, and so on, so the debris ions to lose [M-H-162]- and [M-H-146]- mostly; for the flavonoid nucleus, the main occurrence of C ring RDA cracking and some small molecular fragments loss, such as CO, CHO, C302, CO2 and so on. Summarize the characteristics of mass spectrometric fragmentation in the leaves of seabuckthorn, and find the negative ion mode, The abundance of glucoside ions (Y0-) and free radical glycosides are different: the flavonol 3-O- monoglycosides have strong [Yo-H]- abundance and strong Y0- abundance produced by the flavonol 7-O- monoglycosides; for the flavonoid 3,7- two -O- double glycosides, the free radical form of sugar, such as -G1c, can be lost through the split of the 3-O glycosides. Ion; 3,7 bit lost sugar base and formed [Y0-2H]- ion at the same time; in addition to this, the mass number of different glycosyl groups, its connection position can be deduced from the difference of the mass number of [M-H]- ions and characteristic ions [Y30-H]- and [Y0-2H]-. Using Mass1ynx 4.1 software to denoise, baseline correction, overlapping peak analysis and peak alignment to obtain 35 of the two kinds of seabuckthorn leaves The relative quantitative information of chemical composition shows that, for the main components of the two Hippophae rhamnoides leaves, Chinese Hippophae rhamnoides is about 1.5 times the content of Tibet sea buckthorn, and the content of 6 specific components is very low relative to the principal component, which is based on the UPLC-LTQ-Orbitrap-MS analysis technology, according to the ion peak, the retention time and the fragmentation information, and the.2. A total of 41 components were determined and deduced from the comparison of the chemical components and the control products reported in the literature. The data were analyzed by the metabonomics data processing methods by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), in which the PCA analysis model R2X = 69.2%, Q2=53.6%; PLS-DA analysis model R2Y=99.9%, and Q2 =98.5% were successfully constructed. In addition, PLS-DA was used to analyze the data of the sea buckthorn leaves and Tibet Hippophae rhamnoides leaves. In addition, the component of VIP (variable important projection) 1 was paired t test and the compound of P0.05 was used as the difference component in the leaves of the two Hippophae rhamnoides. By statistical analysis and comprehensive preliminary identification, the Chinese Hippophae rhamnoides and the Tibet seabuckthorn leaf were divided. The content determination method of flavonoid glycosides from two seabuckthorn leaves from different habitats was established by the analysis of 10 characteristic difference components.3. based on HPLC. The results showed that the content of flavonoids in the leaves of two seabuckthorn leaves from different habitats was different. The content of flavonoids in the leaves of Chinese Hippophae rhamnoides was 1.13%, and the content of flavonoid glycosides in the leaves of Tibet Hippophae rhamnoides was greatly changed. It is assumed that the results may be related to the original habitat of Tibet seabuckthorn, which is often distributed in alpine areas above 5000 meters above sea level, and the temperature, illumination, altitude and ultraviolet radiation of the samples in this experiment are different from those of the original habitat, and the synthesis and accumulation of Flavonoids from the samples are more than that of the original habitats. The effect of H2O2 on the survival rate of PC12 cells under different concentrations and different action time was greatly affected by.4.. The experimental results showed that H2O2 could damage PC12 cells in dose and time dependent and reduce the survival rate, and when PC12 cells were treated with 300 u M H2O2 to treat PC12 cells for 4 h, the cell viability decreased to 54.67%, indicating that the model was successful. The protective effects of different polar parts of Hippophae rhamnoides and Tibet sea buckthorn (TFH) on PC12 cells after oxidative damage were studied. The results showed that the different polar parts of Chinese Hippophae rhamnoides and Tibet seabuckthorn leaves can inhibit the decrease of cell vitality caused by H2O2 oxidative damage to some extent. The activity sequence is: n-butanol site The ethyl acetate part of ethanol was significantly higher than that of the other polar parts. The determination of the total Huang Tong content in the polar parts showed that the oxidative stress activity of seabuckthorn was positively correlated with the total Huang Tong content. The concentration of total Huang Tong (TFH) in Seabuckthorn to 29.63% could significantly increase the survival rate of oxidative damaged cells. Among them, there was a significant difference between the 0.4,0.25 mg/mlTFH protection group and the injury group (P0.05), which had obvious protective effect on the cells, and the maximum protection rate was 35.87%.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285

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