Verbascoside And Isoverbascoside Isolated From Semen Plantaginis Induced Phenotypic And Functional Maturation Of Dendritic Cells

Semen plantaginis is the dried ripe seeds of Plantago asiatica L. or Plantago depressa Willd (Fam. Flantaginaceae). Plantaginaceae consists of approximately 270 species within three genera. In China, only Plantago asiatica L—one of the three genara was found with about 17 species. However, only two species are listed in the Chinese Pharmacopeia which are Plantago asiatica L. and Plantago depressa willd. It is well known that Plantago asiatica L are mainly cultivated in Ji'an and Tai'he areas in Jiangxi Province. Plantago asiatica L. have been used since ancient times as a diuretic, an anti-inflammatory and an anti-asthmatic drug in China.Current research had revealed that Semen plantaginis contains a variety of chemical ingredients, including iridoids, flavonoids, phenylpropanoid glucoside and polysaccharides et.al. In order to illustrate the possible pharmacological mechanism of those medical functions of Plantago asiatica L. from the aspect of immunology, two kind of phenylpropanoid glucoside - verbascoside and isoverbascoside, were isolated from semen plantaginis., which were found to be pharmacologically active. Furthermore, a screening model of immunological activity of phytochemicals by using dendritic cells as the target cells was established to investigate the effects of those two phenylpropanoid glucoside on the phenotypic and functional maturation of DCs. The purpose of this study was to make a preliminary investigation of the immunomodulation mechanism of the two components and to provide theoretical foundation for the exploration of products made from semen plantaginis. Moreover, the screening model established in our study will provide a new approach for the bioactivity-screening studies. The main research results obtained in this dissertation are concluted as follows:1. The determination methods of the contents of the phenylethanoid glycosides in Semen Plantaginis were established.Two phenylethanoid glycoside compounds were isolated and purified from Semen Plantaginis using the AB-8 macroreticular resin absorption and the prepared high performance liquid chromatography (HPLC) technology. The contents of verbascoside and isoverbascoside in Semen Plantaginis were determined as 8.65 mg/g and 1.36 mg/g respectively. Both the purity of verbascoside and isoverbascoside were above 95%. The content of endotxin in verbascoside and isoverbascoside were 0.92 ng/mg and 1.27 ng/mg respectively.2. The screening model of immunological activity was established.Based on the classical literature and the current study, we induced the murine bone marrow to immature dendritic cells by rmGM-CSF and rmIL-4. The percentage of the DCs in the culture cells was above 80%. The result showe that the methord was a simple and stabiliy, and which could used for bioactivity-screening studies.3. Investigate the immunological activity of verbascoside and isoverbascoside by the bioactivity-screening model.We observed the DCs after stimulated by verbascoside and isoverbascoside with inverted microscopic, finded that these colonies were covered with many sheet-like processes typical of DCs. The effects of both compounds on proliferation of dendritic cells in vitro were studied by using MTT assay and the results showed that both them could significantly stimulate the proliferation of dendritic cells within or without rmGM-CSF and rmIL-4. Flow cytometer showed that both verbascoside and isoverbascoside could profoundly increase the percentage of CD11c~+MHCII~+DCs, enhanc the expression levels of MHCII on verb-DCs or isov-DCs surfaces which gated on CDllc~+DCs, and decrease the uptake dextran by verb-DCs, isov-DCs or LPS-stimulated DCs compare to unstimulated DCs. The lymphocyte proliferation induced by dendritic cells which stimulated by verbascoside and isoverbascoside were determined by MTT assay, the results showed that they could enhance the ability of DCs to induce proliferation of primed T cells in response to OVA as compared to the control group, which suggested they could enhance antigen-presenting ability of DCs. Cumulatively, the results shown for the first time that verbascoside and isoverbascoside might impair phenotypic and function maturation of BMDCs in vitro, suggesting the additional mechanism of its immunomodulatory and anti-inflammatory activity.