Studies On Extraction,Purification,Structural Characterization And Bioactivities Of Polysaccharides From Cynomorium Songaricum

Cynomorium songaricum Rupr.commonly called “elixir” and “desert ginseng”,is a kind of root parasitic fleshy spermatophyte in arid desert area.Fresh C.songaricum is not only edible,but also fed to livestocks.Its dried fleshy stem is used as a traditional Chinese and Mongolian medicinal herb.Modern pharmacological studies have shown that the extracts of C.songaricum present a variety of biological activities.There are many studies on the main components,flavonoids and terpenoids,in ethanol extracts,but few studies on polysaccharides in water extracts accordingly.Polysaccharides are important components of plant secondary metabolites and are closely related to the physiological functions of the organisms.Therefore,polysacchardie is one of the important material bases for the unique therapeutic effects of traditional Chinese medicine.In this paper,the physical,chemical and spectroscopic techniques were adopted to analysis and compare the five extraction methods.Next,scanning electron microscope(SEM),atom force microscope(AFM)and monosaccharide composition analytical techniques were utilized to comparatively study morphology and monosaccharide composition between the new enzyme assisted extraction and the traditional hot water extraction of Cynomorium songaricum polysaccharides(CSPs).Meanwhile,scavenging free radicals and reducing power tests in vitro were employed to evaluate the antioxidant activities of the two CSPs.Secondly,a series of studies on CSPs extracted by enzyme assisted method(CSP-PAE)was carried out.The extraction process parameters were optimized by response surface methodology.DEAE-52 cellulose anion-exchange and Sephadex G-200 gel permeation columns were utilized for isolation and purification.High performance gel permeation chromatography(HPGPC),monosaccharide composition analysis,methylation analysis,ultraviolet spectroscopy(UV),infrared spectroscopy(IR),gas chromatography-mass spectrometry(GC-MS)and nuclear magnetic resonance(NMR)were used to identify the chemical structures.Then,the splenic lymphocyte test model in vitro was employed to evaluate the two purified CSPs.The main results are summarized as follows:(1)Compared to the other four methods,the enzyme assisted extraction took on the advantages of high extraction efficiency,high purity and good water solubility.The yield of the enzyme assisted extraction method was the highest,nearly 2 times higher than the hot water extraction method(CSP-HWE).The carbohydrate content was 89.13%,the water solubility was good,and was free-of protein.CSP-PAE can be fully dissolved in aqueous solution.The molecule height was 6.5 nm,the molecule length was 500 nm.The monosaccharide composition analysis indicated that CSP-PAE was mainly composed of glucose,and a small amount of arabinose,mannose and galactose.(2)Based on a single factor experiment design,screened the main factors by Placket-Burman design,and optimized the extraction technology parameters by Box-Behnken design of response surface methodology,the optimal enzyme assisted extraction conditions for the yield of CSP-PAE were p H of 1.5,liquid-solid ratio of 108:1,and proteolysis temperature of 40 ℃,the predicted yield of CSP-PAE was 23.86%.Additional verification experiments were performed,an actural value of 23.63 ± 0.21% was obtained under the optimal extraction conditions,which was in good agreement with the predict value.(3)The antioxidant activities in vitro of CSP-PAE and CSP-HWE was compared.The results showed that CSP-PAE should not be used as an antioxidant,and the antioxidant activities might mainly come from polyphenols in CSP-HWE.CSP-HWE can significantly scavenge DPPH· and ABTS+· radicals,have strong reducing power,and have certain scavenging capacity for HO· and O2-· radicals.CSP-PAE have certain scavenging capacity for DPPH·,ABTS+·,HO· and O2-·radicals,and weak reducing power.(4)Two novel polysaccharides CSP1 and CSP2 were purified by a series of column chromatogranphy methods.The yields were 0.3% and 0.9% respectively,the carbohydrate contents were 98.68% and 96.14% respectively,and the average molecular weights were 18.33 k Da and 26.71 k Da respectively.The structural characterization of the two purified fractions were identified by spectroscopic analysis,monosaccharide composition,methylation analysis and NMR.CSP1 was found to be a highly branched heteroglucan with branching degree(DB)of 52.84%.The backbone of CSP2 was made up of α-1,4-D-Glcp,α-1,2-D-Glcp and α-1,3-D-Glcp branched at C-2 with terminal residue T-α-D-Glcp,C-3 and C-6 with terminal residue β-4-D-Glcp-OMe.The possible primary structure is as follows:CSP2 was a homoglucan with DB of 17.93%.The backbone of CSP1 was mainly composed of α-1,4-D-Glcp branched at C-6 with terminal residue β-4-D-Glcp-OMe and α-4-D-Glcp-OMe respectively.The possible primary structure is as follows:(5)Two polysaccharides CSP1 and CSP2 took on different immunomodulatory activities.Proved by splennocyte cytotoxicity and proliferation evaluation tests,CSP1 was cytotoxic to splenocytes in high concentration(greater than 10 μg/m L),showed the inhibitory effect of IL-2 with the reverse concentration gradient which was due to the reduced number of splenocyte secreted IL-2.In the same evaluation tests,CSP2 had the analogous effect of lymphocyte proliferation inducer,also showed the inhibitory effect of IL-2 with the reverse concentration gradient.Whereas,the effect of CSP2 on splenocyte proliferation was similar or same as that of IL-2 on cell activation and proliferation,which led to the negative feedback effect of IL-2 and the decrease in IL-2 levels.In conclusion,the enzyme assisted extraction method was first proposed in this study,which had the advantages of high CSPs yield,high purity of carbohydrate and good water solubility and so on.The optimization extraction parameters can be used to guide the production practice,and to improve the production efficiency.There were no significant antioxidant activities in CSP-PAE.Two novel glucans were obtained by a series of column chromatography,presented different immunological activities.CSP1 was highly branched α-heteroglucan,and CSP2 was small branched α-homoglucan.The above results can provide an important basis for the development of Cynomorium songaricum polysaccharide drugs and health products.