摘要
车前科(Plantaginaceae)车前属(genera Plantago)植物共有约265种，广布于全世界，是很多地区居民熟知的历史悠久的疗伤草药。
车前子为车前科车前属植物大粒车前Plantago asiatica L．的干燥成熟种子，具有药蔬兼用、医食同源之性，被国家卫生部列为可用于保健食品的物品。本文以江西省吉安产大粒车前子为主要研究对象，在考察车前子有关生物活性的基础上，应用现代分析分离技术和研究方法，重点对其活性成分多糖类、黄酮类和苯乙醇苷类进行了提取分离、含量测定、结构表征和体外活性研究，试图探寻车前子活性成分与其功效之间的关系，为拓展车前子新的食用和医用空间、合理利用车前子奠定坚实的理论基础。现将本文主要研究结果归纳如下：
1．首先对溶剂法和超临界流体萃取法提取车前子总黄酮的工艺分别进行了研究。
确立了溶剂法最佳工艺条件为：乙醇浓度60％、固液比1：30、提取温度90℃、提取3次、每次2h。车前子总黄酮的提取率可达2.61％。各提取因素对总黄酮提取率的影响程度由大到小依次为：乙醇浓度＞温度＞提取次数＞固液比。乙醇浓度对车前子总黄酮提取率的影响显著(P＜0.05)，而温度有一定的影响(0.05＜P＜0.10)，固液比和提取次数在选取的水平范围内对总黄酮提取率影响不明显。
确立了超临界提取工艺对车前子总黄酮的最佳提取条件为：每克车前子原料中加入2.5mL无水乙醇作为夹带剂，在温度45℃、压力30MPa的条件下提取2次。超临界提取各因素对总黄酮提取率的影响大小顺序依次为：提取次数＞提取压力＞夹带剂用量＞提取温度，提取次数和提取压力对总黄酮提取率具有显著性影响(P＜0.05)，夹带剂用量有一定影响(0.05＜P＜0.10)，温度在选取的水平中影响不明显。车前子总黄酮的提取率为1.58％，低于溶剂法提取，但杂质含量低。
2．其次，建立了车前子中多糖和苯乙醇苷类的含量测定方法。
建立了以木糖作标准曲线，采用苯酚-硫酸法进行车前子多糖含量测定的方法。通过对葡萄糖、木糖和车前子多糖分别采用苯酚-硫酸法、蒽酮-硫酸法显色的扫描吸收光谱数据进行分析，发现苯酚-硫酸法更适合测定车前子中多糖的含量，用木糖作标准曲线得出的结果更能准确反映真实的多糖含量值。对苯酚-硫酸法测定车前子中多糖含量的测定条件优化为：2mL样液先加3％苯酚溶液0.5mL，混匀后加浓硫酸4mL，迅速摇匀后，室温放置35min，在480nm处测定其吸光度。平均回收率为98.9％，RSD=1.79％(n=6)。此方法是一种测定车前子多糖含量的理想方法，亦可为其他类多糖测定提供依据。
建立了反相高效液相色谱法同时测定车前子中毛蕊花苷和异毛蕊花苷含量的方法。苯乙醇苷类是车前子主要有效成分，但药典中没有记载其含量的测定方法。本文采用反相高效液相色谱法在Waters Symmitry C_(18)(5μm，250mm×4.6mm)色谱柱上以甲醇：体积分数0.1％甲酸溶液(v40：v60)为流动相，流动相流速为0.6mL／min，检测波长为330nm，同时测定车前子中毛蕊花苷和异毛蕊花苷的含量。毛蕊花苷线性范围为0.2～2.0μg，线性回归方程为y=2E+06x+101494，相关系数r=0.9982。异毛蕊花苷线性范围为0.2～2.0μg，线性回归方程为y=2E+06x+8565.1，r=0.9957。样品的平均回收率分别为毛蕊花苷99.7％、异毛蕊花苷101％。测得大粒车前子中毛蕊花苷含量为8.65mg／g，异毛蕊花苷含量为1.36mg／g。该方法快速简捷，无需对样品进行太多柱前处理，精密度高，重现性好，可为该药材及制剂的质量控制提供依据。
3．再次，对车前子多糖、黄酮和苯乙醇苷组分的分离纯化和结构鉴定进行了研究。
对车前子多糖提取液的纯化和组分分离进行了研究。先确立了车前子多糖提取液中有效脱除游离蛋白的方法。将三氟乙酸法、盐酸法、Sevage法、酶解和Sevage法联用分别应用于脱除车前子多糖提取液中的蛋白，结果表明四种方法对车前子多糖提取液中蛋白的脱除效果相差不大，而酶解和Sevage法联用在保留较高蛋白脱除率的同时，能获得最高的多糖得率，故此法较其他方法更适合于车前子多糖提取液中蛋白质的脱除。然后采用Sephacryl-400葡聚糖凝胶层析柱(25XK×60cm)与大分子纯化系统AKTA Purifier 100联用对车前子精制多糖进行纯化，得到一个主要的多糖组分，经高效凝胶渗透色谱检测发现其色谱峰为单一对称色谱峰，该组分在示差检测器上监视到的多糖吸收峰和紫外检测到的蛋白质特征吸收峰(280nm)的最大值位置基本重叠，表明该组分可能是糖和蛋白的复合物。收集该组分，命名为SPG(Semen Plantaginis Glycoprotein)。以已知分子量的T-10、T-40、T-70标准葡聚糖和蓝色葡聚糖为标准作标准曲线，经HPGPC法测定，其分子量约为1461100道尔顿。
建立了液-液萃取富集有效成分和大孔树脂吸附技术纯化车前子乙醇提取液的方法。车前子乙醇提取液经有机溶剂乙醚、乙酸乙酯和水饱和正丁醇依次萃取后，对所得各萃取部分进行体外抗氧化活性检测，发现正丁醇萃取部分的抗氧化活性最高，表明在用乙醚萃除初提液中的脂溶性物质后，可用水饱和正丁醇萃取富集抗氧化有效成分。本文还对非极性树脂D101、弱极性树脂AB-8、极性树脂NKA-2和NKA-9四种树脂纯化总黄酮的效果进行了比较，结果发现AB-8型大孔树脂对车前子总黄酮的吸附率和解吸率均较高，适合用来纯化车前子黄酮，可使总黄酮含量从纯化前的4％提高到30％以上，达到较好的纯化效果。
为深入了解纯化后车前子黄酮液的主要化学成分，便于后续单体分离实验的顺利进行，对纯化液进行了液相色谱．质谱联用技术分析，发现其化学成分主要是苯乙醇苷类(毛蕊花苷、异毛蕊花苷和大车前苷)、黄酮类(芹菜素的葡萄糖苷和双糖苷)和环烯醚萜类(京尼平苷酸)。
采用AB-8型大孔树脂吸附结合制备液相色谱技术从车前子中分离纯化得到2个黄酮苷类单体和2个苯乙醇苷类单体，经对其UV、IR、ESI-MS和NMR波谱数据进行分析，鉴定为野漆树苷、芹菜素-7-O-葡萄糖苷、毛蕊花苷和异毛蕊花苷。尚未见有关野漆树苷在车前属中被发现、芹菜素-7-O-葡萄糖苷在车前子中被发现的文献报道。研究表明，大孔树脂吸附结合制备液相色谱技术适合于大批量高效地分离植物有效成分。
4．最后，对车前子成分的体外活性进行了研究。
在本实验室付志红博士发现了车前子水提物有免疫促进作用的实验基础上，研究了车前子精制多糖对环磷酰胺致免疫抑制小鼠免疫功能的影响。研究发现，45℃低温提取和沸水浴提取的车前子多糖SPL和SPB均对免疫抑制小鼠的免疫功能有影响，且影响效果接近；不同浓度的大粒车前子精制多糖可显著提高免疫抑制小鼠腹腔巨噬细胞的吞噬百分率、促进淋巴细胞转化、促进溶血素抗体分泌、促进脾脏T淋巴细胞分泌IL-2(P＜0.05)，使之接近正常小鼠的免疫水平，且低剂量组(0.2％车前子多糖)效果好于高剂量组(0.4％和0.8％车前子多糖)。说明车前子多糖有较强的免疫增强作用，提取温度对车前子多糖活性的影响不大。
此外，本文采用对小鼠骨髓来源的树突状细胞的增殖影响实验研究了毛蕊花苷和异毛蕊花苷的免疫活性，结果发现它们具有良好的免疫活性。树突状细胞(Dendritic Cells，DCs)是机体功能最强的专职抗原提呈细胞，毛蕊花苷或异毛蕊花苷单独使用均可明显刺激BM-DCs增殖，有类细胞因子作用；与细胞因子联用时，均可显著增强细胞因子对BM-DCs的增殖作用。但在浓度过高时，会有一定的细胞毒性作用。毛蕊花苷和异毛蕊花苷能促进树突状细胞的增殖，增强树突状细胞的活力和生命力，可能是车前子具有免疫调节活性的另一作用机理。
通过体外抗氧化功能实验发现，车前子乙醇提取物具有较强的清除DPPH自由基活性和抑制邻苯三酚自氧化能力，活性与样品使用浓度呈明显的量效关系。从车前子乙醇提取物中分离得到的单体毛蕊花苷、异毛蕊花苷、野漆树苷具有很强的体外超氧阴离子自由基清除能力、羟自由基清除能力和卵黄脂蛋白脂质过氧化抑制能力。研究表明这三种物质具有优良的抗氧化特性，对车前子的抗衰老、抗炎症等功效起着重要作用。
Plantaginaceae, which consists of three genera and about 265 species widelyspread worldwide, is well-known as a kind of old-line therapeutic herbal medicine. InChina, only the Plantago genus, with about 17 species, has been found. According toChinese Pharmacopeia, only two species, Plantago asiatica L. and Plantago depressaWilld, are used medically. Semen plantaginis, dried mature seeds of Plantagoasiatica L. or Plantago depressa Willd, are one of the oldest and most popularclassical Chinese traditional medicines used in ancient China. Semen Plantaginis hasthe dual usages both as medicine and as food. It is an approved raw material forhealth care products by the Minister of Public Health, China. In Jiangxi Province, Ji'an and Tai'he are the major bases for Plantago asiatica L. plantation.
The researches on the pharmacological effects, components and bioactivities ofPlantago herbs in foreign countries and regions, especially in Europe and America, are in-depth and detailed. But the studies on Semen Plantaginis in China are notsystematized. Therefore, the seeds of Plantago asiatica L. from Ji'an were chosen tobe the subject for this study. The isolation, purification and in vitro bioactivities ofthe main effective component groups, i.e., polysaccharides, flavonoids andphenylethanoid glycosides in Semen Plantaginis, were investigated by applyingmodem analytical and separate technology and research methods. Some new resultswere obtained. These results will serve as the theoretical foundation for correctcomments on the relationship between the components and the physiologicalfunctions of the seeds of Plantago asiatica L., for the expanded usages of this plantboth in medical field and in food industries, and for the reasonable utility of SemenPlantaginis. The main research results obtained in this dissertation are summarized asfollows:
1. Firstly, the solvent extraction technology and the CO_2 supercritical fluidextraction technology of total flavonoids in Semen PIantaginis were optimized byorthogonal experiments respectively.
The experiment results show that the optimal experimental parameters of the solvent extraction technology are 60％ethanol, 90℃of the extraction temperature, 1:30 of the ratio of solid/liquid, extraction for three times, 2 h each time. The effecton the extraction rate of the concentration of ethanol is remarkable (P＜0.05), and theeffect of the extraction temperature is definite (0.05＜P＜0.10). The effect of both theratio of solid/liquid and the extraction times are not visible in the range of theexperimental levels. The yield of the total flavonoids extracted from SemenPlantaginis exceeds 2.61％under the optimal condition.
The optimal experimental parameters of the CO_2 supercritical fluid extractiontechnology are 45℃of the extraction temperature, 30 MPa of the extraction pressure, 2.5 mL/(g materials) of the ethanol dosage, extraction for two times, 0.5 h each time.The effect on the extraction rate of both the extraction times and the extractionpressure are remarkable (P＜0.05), and the effect of the dosage of ethanol is definite(0.05＜P＜0.10). The effect of the extraction temperature is not visible in the range ofthe selected levels. The results show that the yield of the total flavonoids extractedfrom Semen Plantaginis is 1.58％, which is lower than the yield by the solventextraction technology but the content of the impurities is also lower.
2. Secondly, the determination methods of the contents of the polysaccharidesand the phenylethanoid glycosides in Semen PIantaginis were established.
A suitable method for the determination of the contents of polysaccharides inSemen Plantaginis by the phenol-H_2SO_4 colorimetry was established, and xylose wasused as a standard. The UV/Vis scan spectra of colored compounds, which were thereaction products of glucose, xylose and Semen Plantaginis polysaccharides via thephenol-H_2SO_4 colorimetry and the anthrone-H_2SO_4 colorimetry respectively, wereinvestigated and compared with each other. The results showed that the coloredcompounds of xylose and Semen Plantaginis polysaccharides reacted tophenol-H_2SO_4 reagent were more stable and had the similar maximal wavelength. Itmeans that the determined value of the content of polysaccharides in SemenPlantaginis by the phenol-H_2SO_4 colorimetry method with xylose as a standard wasmore near to the true content. The optimum conditions and operating steps are asfollows. Firstly, 3％phenol solution (0.5 mL) is added into the sample solution (2mL). Then sulfuric acid (4 mL) is added in. After shaking up rapidly and sufficiently, the mixture is stayed for 35 min at room temperature. Finally the absorbency at 480nm is determined. The content ofpolysaccharides in Semen Plantaginis wasevaluated as 9.02％against the linear regression equation of the xylose standard. Theaverage recovery rate of the xylose standard was 98.9％with 1.79％of RSD (n=6).This method is suitable for the determination of polysaccharides in Semen Plantaginis.It can also be regarded as the reference for the determination of polysaccharides withhigh pentoses content in other plants.
Phenylethanoid glycosides are the main effective components in SemenPlantaginis. But the method of determining their contents in Semen Plantaginishasn't been recorded in Chinese Pharmacopoeia. A method for determiningverbascoside and isoverbascoside in Semen Plantaginis simultaneously by RP-HPLCwas established in this thesis. The determination was carried out on a WatersSymmitry C_(18) (5μm, 250mm×4.6mm) column at 25℃. 0.1％HCOOH: MeOH(v40: v60) was used as mobile phase at a flow rate of 0.6 mL/min and the detectionwavelength was set at 330 nm. The linearity of verbascoside was found in the rangeof 0.2～2.0μg, r=0.9982, and the average recovery was 99.7％. The linearity ofisoverbascoside was found in the range of 0.2～2.0μg, r=0.9957, and the averagerecovery was 101％. The contents of verbascoside and isoverbascoside in SemenPlantaginis were determined as 8.65 mg/g and 1.36 mg/g respectively. This method issimple, reliable and accurate. It is suitable for the determination of verbascoside andisoverbascoside in Semen Plantaginis and even suitable for the quality control of themedicine material Semen Plantaginis and its preparations.
3. Thirdly, the purification and structure elucidation of the polysaccharides, theflavonoids and the phenylethanoid glycosides in Semen Plantaginis were studied.
The method of effectively deproteinizing from the crude polysaccharides ofSemen Plantaginis was established. Four reagents, i.e., trifluoroacetic acid, hydrochloric acid, the Sevage reagent and papain associated with the Sevage reagent, were applied to remove the protein from the crude polysaccharides of SemenPlantaginis. It was found that the deproteinizing abilities of them were similar to eachother. But the highest recovery rate of polysaccharides was obtained afterdeproteinized by hydrolysis with papain and then disposal with the Sevage reagent, while the removal rate of protein was reserved at high degree. This method is moreproper to remove the proteins in the crude polysaccharides of Semen Plantaginis thanother methods. The Sephacryl-400 gel column (25XK×60 cm) chromatography inseries with the AKTA purifier 100 biological macromolecule purified system wasused to purify Semen Ptantaginis polysaccharides. A main component was obtainedand named SPG (Semen Plantaginis Glycoprotein). It was found that SPG'smaximum polysaccharide and protein absorptions were overlapped, implying that thiscomponent is a glycoprotein. The result of high performance gel permeationchromatography (HPGPC) indicates that it is a homogeneous glycoprotein component.The molecular weight (Mw) of SPG was determined by HPGPC. The Mw was foundto be 1461100 Da against the calibration curve of Dextran standards (T-2000, T-70, T-40 and T-10) and glucose.
The method to purify the ethanol extract of Semen Plantaginis by theliquid-liquid extraction and macroreticular resins absorption technology wasestablished. The in vitro antioxidative activities of different solvent extracts of theethanol extract of Semen PIantaginis were compared by examining the decolorationof the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and the elimination of superoxide anionradicals generated from autoxidation of pyrogallol. It was found that the antioxidativeactivity of the n-butanol extract fraction was most closed to the ethanol extract thanthe ether fraction, the ethyl acetate fraction and the residual water solution. It meantthat n-butanol is the best solvent to extract the main functional components from theethanol extract after removing fat-soluble substances by ether. The performance ofabsorption and desorption for flavonoids from Semen Plantaginis with four types ofmacroreticular resins AB-8, D101, NKA-2, NKA-9 were also determined in order topurify Semen Plantaginis flavonoids. The results showed that the AB-8 resin had thehighest ratio of absorption and desorption and it was the most suitable absorbent. Thecontent.of flavonoids was raised from 4％to 30％after absorption and separation bythis resin and was preferable. The main components of the purified solution offlavonoids of Semen Plantaginis were determined by LC-MS. It was found that themain components in the purified solution were three phenylethanoid glycosides(verbascoside, isoverbascoside and plantamajoside), two flavonoids (the glycosides of apigenin with one or two moiety of saccharides) and one iridiods (geniposidic acid), etc.
Two flavonoid compounds and two phenylethanoid glycoside compounds wereisolated and purified from Semen Plantaginis using the AB-8 macroreticular resinabsorption and the prepared high performance liquid chromatography technology.They were identified as rhoifolin, apigenin-7-O-glucoside, verbascoside andisoverbascoside by UV, IR, ESI-MS (both negative and positive ionizations) andNMR spectra. There is no correlative report about rhoifolin andapigenin-7-O-glucoside from the seeds of PIantago asiatica L. Erenow, especiallyrhoifolin, a flavonoid of apigenin with a disaccharide moiety, has not been found inPlantago genus. The results showed that the combination of the macroreticular resinabsorption and the prepared HPLC technology is highly effective to isolate thebioactive components of plants.
4. Finally, the in vitro activities of the three components of Semen Plantaginiswere investigated.
The immunological effects of the refined polysaccharides from SemenPlantaginis on the low-immunity mouse model mice induced by Cyclohosphamide(Cy) were examined after Doctor Zhihong Fu of our laboratory found that the waterextract of Semen Plantaginis has the immunity regulating ability. The results showedthat both the two polysaccharides SPL and SPB, which were extracted at 45℃and atthe boiled temperature respectively, had the similar effects on the immunologicalfunction of the Cy induced low-immunity mouse. And the degrees of the effects werevery similar. It meant that different extraction temperatures won't visibly affect theimmunological effects of the refined polysaccharides. The different concentrations ofthe refined polysaccharides from Semen Plantaginis significantly strengthenedphagocytosis function of the abdominal phagocyte, improved the proliferation of thelymphocyte, heightened the level of hemolysin antibody and improved the productionof IL-2 by the splenic T lymphocyte in the Cy induced low-immunity mouse tonormal index (P＜0.05). And the effect of low concentration group (0.2％of therefined polysaccharides from Semen Plantaginis) is better than high concentrationgroups (0.4％and 0.8％of the refined polysaccharides from Semen Plantaginis). It indicated that the refined polysaccharides from Semen Plantaginis have strong effecton immunological function.
Moreover, the effects of verbascoside and isoverbascoside isolated from SemenPlantaginis on proliferation of murine bone marrow-derived dendritic cells (BM-DCs)were investigated using the tetrazoliun salt reduction (MTT) assay. The resultshowed that both verbascoside and isoverbascoside have significant stimulation onthe proliferation of murine bone marrow-derived dendritic cells (BM-DCs) in therange of experimental doses (P＜0.05). The stimulations of verbascoside evenexceeded the cytokine (rmGM-CSF+ rmIL-4) control group (P＜0.05). Furthermore, verbascoside and isoverbascoside can promote the accelerated effect of cytokine onthe proliferation of BM-DCs when they were used together with the cytokine. Butthey inhibited the accelerated effect of cytokine at high concentrations, i.e., 1000μg/mL. Verbascoside and isoverbascoside have the strong abilities to stimulating theproliferation of BM-DCs and enhancing the energy and life-force of BM-DCs. Itstands a good chance to be another functional mechanism of Semen Plantaginis toexhibit the immunity regulating ability.
The in vitro antioxidative activities of the ethanol extract and three purifiedcompounds isolated from Semen Plantaginis were evaluated. It was found that theethanol extract of Semen PIantaginis has strong abilities of scavenging DPPH radicalsand of restraining the autoxidation of pyrogallol. The purified compounds, i.e., verbascoside, isoverbascoside and rhoifolin all have very remarkable effects ofscavenging superoxide anion radical, scavenging hydroxyl radical and restraining thelipid peroxidation of yolk liporeotein. The correlativity of their effects and the dosesare positive. The results showed that verbascoside, isoverbascoside and rhoifolin haveexecutively antioxidative activities and play a very important role to the anti-agingand anti-inflammation functions of Semen Plantaginis.
引文
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