Adjuvant Effects Of Different Extra Fractions Of Atractylodes Macrocephala Koidz

Atractylodis macrocephalae Koidz. is a natural plant in family of Compositae, The rhizome of the plant (RAM) has been utilized as a digestive stimulator in traditional Chinese medicine. Our previous study has demonstrated that oral administration of a decoction made from RAM has significantly increased immune responses to vaccines against FMD in mice. In the present thesis, adjuvant activity of different extracts from RAM were studied in order to screening for the adjuvant fractions, and then its effect on the systemic and intestinal mucosal immune response to FMD vaccine in mice was investigated.1. Extraction and purification of polysaccharide and volatile oil from the rhizome of Atractylodis macrocephalae Koidz.(RAM)Objective To extract and purify the polysaccharide and volatile oil from RAM. Methods Three experiments were designed and described as follows. In experiment A, the rhizome of RAM (100g) was ground into powder and then extracted with boiling water two times under reflux for2h each time. The aqueous portion was filtered through filter paper. The filtrate was concentrated under reduced pressure. Four volumes of95%ethanol were added to the supernatant, and kept overnight at4℃. The resulting precipitate was dissolved in distilled water, subjected to Macroporous Adsorption Resin column D101, and then washed with water.The collected elute was concentrated, dialyzed against distilled water (cut-off Mw7000Da) and lyophilized to afford a total RAM polysaccharide (RAMPS). Total sugar content was estimated by the phenol-sulfuric acid analysis using glucose as a standard. In experiment B, RAMPS (20mg) were hydrolyzed with15ml of2M TFA at110℃for6h to release component monosaccharides. The hydrolyzed monosaccharides (inositol as the internal standard) were derivatized to acetylated aldononitriles and isothermally separated by gas chromatography (GC) in an Agilent6890N system equipped with a flame-ionization detector (FID) and a DB-5capillary column (30.0m×0.32mm× 0.25μm). In experiment C, the rhizome of RAM (200g) was ground into powder and then extracted with petroleum ether at70℃two times under reflux for70min each time. The filtrate was concentrated under reduced pressure until no solvent left to obtain volatile oil of RAM. Results and Conclusions The yield of RAMPS in this experiment was4.2%, The polysaccharide contained in RAMPS was86.2%. GC quantitative analysis with derivatization revealed that RAMPS was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose with the molar ratio of1.00:2.49:2.07:4.94:11.33:1.35.The extraction rates of volatile oil was1.185%.2. Adjuvant effects of oral administration of RAMPS、volatile oil and atractylenolide I against FMD vaccine in miceObjective To screen for the ingredient(s) contributing the adjuvant activity of RAM. Methods In experiment A, Forty-two female ICR mice were randomly divided into seven groups with six mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or RAMPS (6.25,12.5,25,50or100mg) or RAM decoction (250mg). Blood samples were collected3weeks after the booster immunization for detection of of FMDV-specific antibody response. In experiment B, Forty-eight female ICR mice were randomly divided into eight groups with six mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or tween-80(2%) or volatile oil of RAM (1,2,4,8or16mg) or RAM decoction (250mg). Blood samples were collected3weeks after the booster immunization for detection of of FMDV-specific antibody response. In experiment C, Forty-eight female ICR mice were randomly divided into eight groups with six mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or DMSO (0.5%) or atractylenolide I (0.05,0.1,0.2,0.4or0.8mg) or RAM decoction (250mg). Blood samples were collected3weeks after the booster immunization for detection of of FMDV-specific antibody response. Results and Conclusions Oral administration of RAMPS tended to enhance serum sepcific IgG response against FMDV immunization, neither volatile oil nor atractylenolide I have been found adjuvant properties in our experiments. Therefore, the adjuvant activities of RAM may be attributed to RAMPS.3. Adjuvant effects of oral administration of RAMPS to FMD vaccine in miceObjective To investigate the effect of RAMPS on the immune responses to a commercial FMD vaccine in mice. Methods Thirty-five female ICR mice were randomly divided into five groups with7mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or RAMPS (0.025,0.05or0.1g) or RAM decoction (0.25g). Blood samples were collected3weeks after the booster immunization for detection of IgG titers, the IgG subclasses and western blot analysis. Splenocytes were harvested for determination of lymphocyte proliferation and cytokines mRNA expression. Results After oral administration for4days of RAMPS, immunization of a commercial FMD vaccine induced significantly higher serum specific IgG and the IgG isotype responses in association with up-regulated serum IFN-y and IL-5. In addition, RAMPS significantly increased splenocyte proliferative responses to ConA, LPS and FMDV, as well as mRNA expression of Thl/Th2cytokines (IFN-y/IL-4) and transcription factors (T-bet/GATA-3) by splenocytes. Conclusions Oral administration of RAMPS can enhance the activities of T and B cells and promote a balanced Th1/Th2immune responses against FMD in mice. Considering its natural origin and low side effects, RAMPS could be used as an effective adjuvant to FMD vaccine.4. Effects of oral administration of RAMPS on the intestinal mucosal immunity of miceObjective To investigate the effect of RAMPS on the intestinal mucosal immune syetem of mice. Methods Fifty-six female ICR mice were randomly divided into four groups with14mice in each. Two groups of the animals were subcutaneous!y injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or RAMPS0.05g.The other two groups were not immunized with vaccine, but had been orally administered at the same time.One week after first immunization and two weeks after the booster immunization, half mice of every group were sacrificed. Blood samples were collected for detection of antigen-specific antibody response. Intestine duodenum of mice were seperated. Fecal samples were collected from individual mice and extracted by making a1:10suspension (wt/vol) with stool diluent. The suspension was vortexed and centrifuged for20min at12000g. The supernatant was collected for detection of Total IgA levels. Part of the duodenum was grinded with liquid nitrogen for determination of cytokine mRNA expression. Tissue paraffin sections were prepared and stained with haematoxylin and eosin (HE) for analysis of intraepithelial lymphocytes (IEL) and immunohistochemical analysis were also performed for analysis of IgA+plasma cells. Results Oral administration of RAMPS at the dose of0.05g/d for4days could improve serum antigen-specific antibody responses as well as total IgA levels, mRNA expression of TGF-β, IL-6, TNF-α, the area of IgA+plasma cells and the number of IELs in the duodenum of mice. Conclusions Oral administration of RAMPS significantly increased systemic as well as gut mucosal immunity in mice immunized with FMD vaccine. 5. Effect on PRRS of a macrophage cell line of mice by RAMPSObjective To observe the effect of RAMPS on the activation of PRRs in a macrophage cell line of mice. Methods Raw-Blue is a macrophage cell line (Raw264.7), which was stably transfected with plasmid (pNifty-2), which can be induced by activated NF-κB to secrete SEAP protein. Raw-Blue cells were stimulated with RAMPS and APS at different final concentration (0、1、48、16、25、50、100、200μg/ml) for18h, then the SEAP in supernate were detected by substrate. Results and Conlusions There was no significantly ascensus of SEAP in the supernate, thus RAMPS and APS could not activate the signal pathways of NF-κB, showed no effects on PRRs(TLRs or NLRs or RLRs).All together, neither volatile oil nor atractylenolide I have been found adjuvant properties in our experiments.But oral administration of RAMPS tended to enhance serum sepcific IgG response against FMDV immunization, the activity of T and B cells of the mice and a balanced Th1/Th2immune responses against FMD, as well as the intestinal mucosal immune responses. Therefore, the oral adjuvant activities of RAM may be attributed to the fraction of polysaccharide (RAMPS). But the adjuvant activity of RAMPS was not mediated by TLRs or NLRs or RLRs, the mechanisms need to be further investigated.