本文選題：中國(guó)沙棘葉 + 西藏沙棘葉　； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：研究目的中國(guó)沙棘(Hippophae rhamnoides L.subsp.sinensisRousi),胡頹子科沙棘屬植物沙棘(Hioppophae rhamnoides L.)的一個(gè)變種,自1977年被首次載入《中國(guó)藥典》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.)與沙棘(H.rhamnoides L.)相近,起初被合并在沙棘種內(nèi)作異名,后因其形態(tài)特征、地理分布和生長(zhǎng)環(huán)境都特殊,學(xué)者把它歸為獨(dú)立的種,是藏醫(yī)、蒙醫(yī)的習(xí)用藥材,具有補(bǔ)肺,活血的功效,用于治月經(jīng)不調(diào)、子宮病、胃病、肺結(jié)核、胃潰瘍等,西藏北部的群眾還用其治肝炎。沙棘作為一種藥食同源植物,具有豐富的生物活性成分和營(yíng)養(yǎng)物質(zhì)。因《藥典》中沙棘以果實(shí)入藥,故一般開(kāi)發(fā)產(chǎn)品利用的原料是其果實(shí),而沙棘葉被當(dāng)做廢料遺棄;黃酮類成分是沙棘相關(guān)產(chǎn)品質(zhì)量及其理化鑒別的重要衡量指標(biāo),而研究證實(shí)其在葉中含量最高,100 g干葉中為310-1238 mg(均值為792 mg,鮮果中均值為533 mg),每公頃的沙棘葉經(jīng)計(jì)算,其年產(chǎn)量可多達(dá)5噸,廢棄不用造成了極大的資源浪費(fèi);除此之外,相較于果實(shí),沙棘葉還具有較高的產(chǎn)量、較長(zhǎng)的收集周期、簡(jiǎn)單的生產(chǎn)加工工藝以及易于儲(chǔ)藏等優(yōu)勢(shì),通過(guò)對(duì)其葉中成分進(jìn)行深入研究、合理利用,其發(fā)展前景是誘人的。因此,立足資源優(yōu)勢(shì),本課題進(jìn)行了以下研究:(1)建立中國(guó)沙棘和西藏沙棘葉中化學(xué)成分快速鑒定的分析方法,總結(jié)其質(zhì)譜裂解規(guī)律,分析比較兩沙棘葉中成分組成特征;(2)將LC-MS分析技術(shù)與代謝組學(xué)數(shù)據(jù)處理技術(shù)相結(jié)合,構(gòu)建中國(guó)沙棘葉和西藏沙棘葉的區(qū)分模型并尋找造成兩沙棘葉差異的特征性成分。(3)收集不同產(chǎn)地中國(guó)沙棘和西藏沙棘葉,建立兩沙棘葉中黃酮苷的含量測(cè)定方法,分析、比較兩沙棘葉中成分含量上的特征;(4)采用H202誘導(dǎo)PC12細(xì)胞建立氧化損傷模型,通過(guò)倒置顯微鏡觀察細(xì)胞形態(tài),MTT法測(cè)定了解PC12細(xì)胞損傷情況和存活率,初步分析中國(guó)沙棘和西藏沙棘葉不同極性部位以及沙棘總黃酮(TFH)對(duì)氧化損傷后的PC12細(xì)胞的保護(hù)作用,為其在神經(jīng)退行性疾病臨床治療方面奠定基礎(chǔ)。研究方法1.運(yùn)用超高效液相串聯(lián)四級(jí)桿飛行時(shí)間質(zhì)譜的聯(lián)用儀器(UPLC/Q-TOF-MS)建立中國(guó)沙棘和西藏沙棘葉中化學(xué)成分快速鑒定的分析方法,優(yōu)化質(zhì)譜條件,使得各成分裂解的碎片穩(wěn)定,裂解信息全面;總結(jié)兩沙棘葉中成分的質(zhì)譜裂解規(guī)律,分析比較兩沙棘葉中成分組成上的特征。2.實(shí)驗(yàn)首先基于超高效液相線性離子阱-串聯(lián)靜電場(chǎng)軌道阱質(zhì)譜聯(lián)用技術(shù)(UPLC-LTQ-Orbitrap-MS)對(duì)兩沙棘葉中成分進(jìn)行充分解析獲得盡可能多的化合物,繼而采用代謝組學(xué)數(shù)據(jù)處理手段對(duì)實(shí)驗(yàn)數(shù)據(jù)做降維處理以及包括PCA和PLS-DA等在內(nèi)的統(tǒng)計(jì)學(xué)分析,用于兩沙棘葉中差異性成分的研究以及兩沙棘葉的鑒定。3.采用甲醇回流提取方法對(duì)多產(chǎn)地中國(guó)沙棘和西藏沙棘葉進(jìn)行提取,運(yùn)用高效液相(HPLC)建立兩沙棘葉中黃酮苷的定量方法,并對(duì)樣品進(jìn)行測(cè)定,分析不同產(chǎn)區(qū)兩沙棘葉中成分含量上的差異。4.通過(guò)分析不同濃度以及不同作用時(shí)間下,H202對(duì)PC12細(xì)胞存活的影響,建立H202誘導(dǎo)的PC12細(xì)胞氧化損傷模型,初步分析中國(guó)沙棘和西藏沙棘不同極性部位以及沙棘總黃酮(TFH)對(duì)氧化損傷后的PC12細(xì)胞的保護(hù)作用。結(jié)果與結(jié)論1.基于UPLC/Q-TOF-MS技術(shù),結(jié)合質(zhì)譜數(shù)據(jù)、文獻(xiàn)數(shù)據(jù)以及與標(biāo)準(zhǔn)品比對(duì),從兩沙棘葉中分析、推斷出35個(gè)化合物,其中,29個(gè)化合物為兩沙棘葉共有成分,剩余的6個(gè)成分中,2個(gè)成分為中國(guó)沙棘葉特有,其余為西藏沙棘葉獨(dú)有。結(jié)果表明,中國(guó)沙棘和西藏沙棘葉中成分組成上大體一致,主要是以槲皮素、山奈酚、異鼠李素為苷元和糖結(jié)合形成的黃酮醇苷類物質(zhì),糖基主要有葡萄糖、鼠李糖等,因此碎片離子以丟失[M-H-162]-和[M-H-146]-居多;對(duì)于黃酮母核,主要發(fā)生C環(huán)的RDA裂解和一些小分子碎片的丟失,如CO,CHO,C302,CO2等�？偨Y(jié)沙棘葉中成分的質(zhì)譜裂解特征,發(fā)現(xiàn)負(fù)離子模式,糖基化位置不同,苷元離子(Y0-)和自由基苷元離子[Y0-H]-豐度不同:黃酮醇3-O-單糖苷產(chǎn)生的[Yo-H]-·豐度強(qiáng),黃酮醇7-O-單糖苷產(chǎn)生的Y0-豐度強(qiáng);對(duì)于黃酮3,7-二-O-雙糖苷類成分,可通過(guò)3-O苷鍵的均裂,失去自由基形式的糖,例如-G1c,形成[Y30-H]-離子;3,7位同時(shí)丟失糖基,形成[Y0-2H]-離子;除此之外,質(zhì)量數(shù)不同的糖基,其連接位置可通過(guò)[M-H]-離子及特征離子[Y30-H]-·與[Y0-2H]-的質(zhì)量數(shù)之差得到推斷。利用Mass1ynx 4.1軟件經(jīng)去噪、基線校正、重疊峰解析及峰對(duì)齊獲得了兩種沙棘葉中35種化學(xué)成分的相對(duì)定量信息,結(jié)果表明,對(duì)于兩沙棘葉的主要成分而言,中國(guó)沙棘約為西藏沙棘含量的1.5倍,6個(gè)特異性成分含量相對(duì)主成分而言含量很低。2.基于UPLC-LTQ-Orbitrap-MS分析技術(shù),根據(jù)分子離子峰、保留時(shí)間及裂解碎片信息,并與文獻(xiàn)報(bào)道的化學(xué)成分及對(duì)照品進(jìn)行比對(duì),共確定及推斷出41個(gè)成分。運(yùn)用代謝組學(xué)數(shù)據(jù)處理手段對(duì)數(shù)據(jù)進(jìn)行主成分分析(PCA)及偏最小二乘判別分析(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%,成功構(gòu)建了中國(guó)沙棘葉和西藏沙棘葉的區(qū)分模型;此外,采用PLS-DA對(duì)數(shù)據(jù)做進(jìn)一步分析,對(duì)VIP(變量性重要投影)1的成分做配對(duì)t檢驗(yàn)且以p0.05的化合物作為兩沙棘葉中差異性成分,經(jīng)統(tǒng)計(jì)分析及綜合前期鑒定的化合物,從中國(guó)沙棘和西藏沙棘葉中共分析得到10個(gè)特征性差異成分。3.基于HPLC法建立了不同產(chǎn)地的兩沙棘葉中黃酮苷的含量測(cè)定方法,結(jié)果表明,不同產(chǎn)地的兩沙棘葉中黃酮苷的含量存在較大差異。中國(guó)沙棘葉中黃酮苷含量均值為1.13%,而西藏沙棘葉中黃酮苷含量變化較大,于0.6%-1.4%之間波動(dòng),猜測(cè)該測(cè)定結(jié)果可能與西藏沙棘的原始生境相關(guān),其常常分布于海拔5000米以上的高寒地區(qū),而本實(shí)驗(yàn)中樣品采集地的溫度、光照、海拔以及紫外輻射等均與其原始生境相差較大,對(duì)其黃酮類成分的合成與積累產(chǎn)生較大影響。4.分析不同濃度以及不同作用時(shí)間下H2O2對(duì)PC12細(xì)胞存活率影響,實(shí)驗(yàn)結(jié)果表明,H2O2能夠呈劑量和時(shí)間依賴性的損傷PC12細(xì)胞并降低其生存率,且采用300 μM的H2O2處理PC12細(xì)胞4 h時(shí),和對(duì)照組相比,細(xì)胞活力下降至54.67%,表明造模成功。研究中國(guó)沙棘和西藏沙棘不同極性部位以及沙棘總黃酮(TFH)對(duì)氧化損傷后的PC12細(xì)胞的保護(hù)作用,結(jié)果表明,中國(guó)沙棘和西藏沙棘葉不同極性部位成分都能夠不同程度的抑制H2O2氧化損傷導(dǎo)致的細(xì)胞活力下降,其活性順序?yàn)?正丁醇部位乙醇部位乙酸乙酯部位,而且中國(guó)沙棘乙酸乙酯部位活性明顯高于其他各極性部位。測(cè)定各極性部位中總黃酮含量,表明沙棘氧化應(yīng)激活性與總黃酮含量呈正相關(guān)。將沙棘總黃酮(TFH)含量富集至29.63%,可顯著提高氧化損傷細(xì)胞的生存率。其中,0.4,0.25 mg/mlTFH保護(hù)組同損傷組間有顯著差異(p0.05),對(duì)細(xì)胞有明顯的保護(hù)作用,最大保護(hù)率達(dá)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%.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284;R285

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相關(guān)期刊論文 前10條

1 曹厚華;俞仲毅;;代謝組學(xué)與中醫(yī)證候的相關(guān)性研究進(jìn)展及液質(zhì)聯(lián)用方法的客觀評(píng)價(jià)[J];上海中醫(yī)藥大學(xué)學(xué)報(bào);2016年06期

2 牟丹;唐楠;黃原林;張同作;謝久祥;;沙棘、青海云杉、祁連圓柏總多酚含量的比較研究[J];湖北農(nóng)業(yè)科學(xué);2016年11期

3 錢學(xué)射;金敬紅;;沙棘的藥用研究與開(kāi)發(fā)[J];中國(guó)野生植物資源;2015年06期

4 馮欣欣;于文會(huì);柏慧敏;魏慶微;馬雋;姜曉文;;沙棘黃酮抗衰老作用及對(duì)大鼠非特異性免疫功能的影響研究[J];中獸醫(yī)醫(yī)藥雜志;2015年05期

5 王瑩;杜靜;;液質(zhì)聯(lián)用技術(shù)在中藥復(fù)方研究中的應(yīng)用[J];浙江中西醫(yī)結(jié)合雜志;2015年08期

6 張麗杰;孟鑫;陳忠新;楊連榮;;基于液質(zhì)聯(lián)用技術(shù)的中藥藥代動(dòng)力學(xué)研究進(jìn)展[J];黑龍江畜牧獸醫(yī);2015年13期

7 臧茜茜;鄧乾春;從仁懷;葛亞中;黃鳳洪;魏曉珊;陳鵬;張逸;;沙棘油功效成分及藥理功能研究進(jìn)展[J];中國(guó)油脂;2015年05期

8 孫冬梅;董玉娟;胥愛(ài)麗;羅文匯;江潔怡;;葛根枳i軟膠囊中黃酮類化合物的UPLC/Q-TOF-MS快速分析[J];中草藥;2015年07期

9 李e，

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