The Purification, Structure Elucidation And Activities Of Polysaccharides, Flavonoids And Phenylethanoid Glycosides In Semen Plantaginis

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 CO₂ 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 60ï¼…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 2.61ï¼…under the optimal condition.The optimal experimental parameters of the CO₂ 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 1.58ï¼…, 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-H₂SO₄ 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-H₂SO₄ colorimetry and the anthrone-H₂SO₄ colorimetry respectively, wereinvestigated and compared with each other. The results showed that the coloredcompounds of xylose and Semen Plantaginis polysaccharides reacted tophenol-H₂SO₄ 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-H₂SO₄ colorimetry method with xylose as a standard wasmore near to the true content. The optimum conditions and operating steps are asfollows. Firstly, 3ï¼…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 9.02ï¼…against the linear regression equation of the xylose standard. Theaverage recovery rate of the xylose standard was 98.9ï¼…with 1.79ï¼…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 C₁₈ (5μm, 250mm×4.6mm) column at 25℃. 0.1ï¼…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 99.7ï¼…. The linearity ofisoverbascoside was found in the range of 0.2～2.0μg, r=0.9957, and the averagerecovery was 101ï¼…. 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 4ï¼…to 30ï¼…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 (0.2ï¼…of therefined polysaccharides from Semen Plantaginis) is better than high concentrationgroups (0.4ï¼…and 0.8ï¼…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.