Extraction And Preparation Of Polysaccharide From Ziziphi Spinosae Semen And Its Immunomodulatory Activity

Zizyphi Spinosae Semen?ZSS?is the dried seeds of Ziziphus jujuba Mill var.spinosa?Bunge?Hu ex.H.F.Chou?Rhamnaceae?,which as the medicine and food are homologous.And it was often used for treatment of fright palpitations,insomnia and dreaminess.Clinically,it is often used to treat physical deficiency,sweating,fatigue,sleeplessness,thirst due to body injury,palpitation,dreaminess and other symptoms.ZSS is commonly distributed in northwest,northeast and north China.And it contains saponins,triterpenes,flavonoids,alkaloids,fatty acids,polysaccharides and other components.However,the current research on ZSS mainly focuses on flavonoids,saponins and other compounds,while the research report on polysaccharides is rare.However,there are more and more researches on plant polysaccharides,and some studies have shown that plant polysaccharides exhibit significant biological activity in anti-tumor,anti-hepatitis,regulating glucose metabolism,and low toxicity,which has become one of the hot spots in the research field of medicine and health food in recent years.Therefore,the ZSS polysaccharide has the broad development application foreground.In this study,response surface methodology?RSM?was used to optimize the ultrasonic extraction and decolorization process of ZSS polysaccharide.Then,the physicochemical properties and chemical structure of ZSS polysaccharides were characterized by various analytical techniques.The immunomodulatory activity of ZSS polysaccharides was investigated by using RAW264.7 mouse monocyte macrophage as cell model.The mechanism of immunoregulation in vivo was preliminarily explored.The main findings of this paper were as follows:?1?In this study,the polysaccharide was extracted from ZSS with ultrasound assisted extraction method.Box-Behnken design?BBD?and response surface methodology?RSM?were applied to obtain the optimal conditions for perfect yield of ZSS polysaccharide:extraction temperature 52.5°C,extraction time 21.2 min,extraction power 134.9 W and ratio of liquid to solid 26.3 mL/g.The extraction yield was 1.05%.?2?The ZSS polysaccharide decoloration process was optimized by the method of Response Surface optimization.The results showed that the optimized decolorization parameters with D101 macroporous adsorption resin were 2mg/mL for the sample concentration,3.5 mL for the volume,and 4 BV/h for the elution flow rate.Under the condition that the value of polysaccharide decolorization rate and retention rate were61.32%and 87.05%,respectively,and the OD value was 0.56?the predicted value of0.55?.Thus,the optimized decoloration method is feasible.?3?HPLC-ELSD method determined that the polysaccharide mainly contains two kinds of polysaccharides with molecular weights of 10000 kDa and 2.34 kDa,respectively.Monosaccharide composition analysis was determined by HPLC using precolumn derivatization,and the results revealed that the purified polysaccharide contains glucuronic acid?0.89%?,mannose?15.42±0.08%?,rhamnose?18.36%?,glucose?0.20%?,galactose?35.49%?and xylose?29.33%?.Infrared spectra showed that the polysaccharides had the characteristic absorption peaks of polysaccharides in that wavelength range of 3300³500 cm^-1?2800³000 cm^-1?1200¹700 cm^-1 and 600¹100 cm^-1.The analysis by NMR spectroscopy?¹H-NMR?showed that the chemical shifts of anomeric H^-11 were larger than 4.9 ppm,indicating that the polysaccharide was mainly composed of?-configuration.These results are in accordance with FTIR and optical rotation analyses.The signals in the range of 0.8-1.5 ppm,demonstrating that rhamnosyl residues were linked with various types?1-linked,2-linked,4-linked or 2,4-linked?in the ZSS polysaccharide.In addition,the signals at 1.8-2.2 ppm suggested that some O-acetyl groups might be contained in the polysaccharide.?4?The effect of ZSS polysaccharides on the proliferation of RAW264.7 cells was detected by MTT.The results showed that the low concentration?5?g/mL?of ZSS polysaccharides can significantly promote the proliferation of RAW264.7 cells.The results of Griess and Western blot test showed that ZSS polysaccharide could significantly promote the release of NO and the expression of stress response protein?COX-2 and iNOS?in RAW264.7 cells.In addition,ZSS polysaccharides can significantly promote the phosphorylation of I?B-?and ERK protein,which indicated that the polysaccharide has a certain immunomodulatory effect.