Study On Extraction,Purification,Structural Characterization,and Biological Activity Of Polysaccharides From Mori Fructus

Mori Fructus,also known as mulberry,is the mature fruit of the mulberry tree(Morus alba Linn.),a plant of the Moraceae family,and is one of the fruits commonly eaten by people.It is included in the first part of the Chinese Pharmacopoeia 2020 edition and in the catalogue of ingredients of the same origin as medicine and food.Studies have shown that mulberries are beneficial to human health,which may be related to the phenolic,amino acid,sugar and other compounds they contain.Mori Fructus polysaccharide(MFP)is one of the effective medicinal active ingredients in mulberry,with anti-inflammatory,anti-tumour,antioxidant,hypoglycaemic and hypolipidaemic activities.The current research on mulberry polysaccharides is mainly focused on crude mulberry polysaccharides,and there is a lack of research on the structural characterization and biological activity of purified mulberry polysaccharides.Meanwhile,extraction of polysaccharides is a key link in the research and utilization of natural polysaccharide resources,but there are fewer reports on the effects of extraction methods on structure and biological activity.To address this status quo,this study investigated the effects of extraction methods on the physicochemical properties,structural characteristics and biological activities of mulberry polysaccharides using mulberry as raw material,and then screened out the best active mulberry polysaccharides and further separated and purified them to obtain mulberry polysaccharides with homogeneous relative molecular weights.The structure of the mulberry polysaccharides was then analyzed using ion chromatography,methylation,scanning electron microscopy and thermogravimetry.Finally,the antioxidant activity and hypoglycemic activity of the purified mulberry polysaccharides were evaluated by in vitro experiments,and the main factors for their biological activity were discussed.The main studies and results are as follows:1.In this study,six fractions of crude polysaccharides(named: MFP-I,MFP-II,MFP-III,MFP-IV,MFP-V,MFP-VI)were obtained from mulberry using cold water extraction,hot water extraction,boiling water extraction and continuous assisted ultrasonic extraction with different concentrations of ethanol solvent(50%,25%,0%).The results showed that MFP-III had the highest polysaccharide yield;MFP-V had the lowest.The total sugar content of MFP-III was significantly higher than the other polysaccharide fractions(P<0.05),while MFP-II had the lowest total sugar content.All six fractions of mulberry polysaccharides were acidic polysaccharides,with MFP-V having the highest glyoxylate content and MFP-III the lowest.anthocyanin content of MFP-III was significantly lower than the other fractions(P<0.05),while MFP-IV had the highest anthocyanin content.2.The analysis of the monosaccharide composition showed that the six fractions of mulberry polysaccharides were mainly composed of glucose,followed by mannose,rhamnose,galactose and arabinose.MFP-III had the highest glucose content,MFP-II increased the proportion of arabinose and MFP-VI increased the content of rhamnose.MFP-V had higher content of rhamnose,galactose,galacturonic acid and arabinose than the other fractions.UV spectroscopic analysis showed that the six fractions of mulberry polysaccharides contained no nucleic acid material,but a small amount of protein.Infrared spectroscopy analysis showed that the obtained polysaccharides all had the typical infrared spectral characteristics of polysaccharides,all contained α-configuration glycosidic bonds and β-configuration glycosidic bonds,and all were in the form of pyranose.The results of NMR analysis verified that the mulberry polysaccharides obtained from the six extraction methods contained bothα-glucopyranose and β-glucopyranose.Thermodynamic analysis showed that the MFP-I structure was unstable by heat in the temperature range of 20～800 ℃;the MFP-V structure was more stable by heat.The results of scanning electron microscopy showed that the mulberry polysaccharides extracted by different methods had different apparent size and shape structures.MFP-I and MFP-III polysaccharides had a high degree of molecular cross-linking within the polysaccharide,with a dense structure and both had a thin film-like surface.MFP-II and MFP-V consisted of irregular rough surfaces and small particles of different shapes.MFP-IV and MFP-VI had a sheet-like dense form,with a relatively smooth surface structure is relatively smooth and the lamellar structure is stable.The results of the Congo red test showed that none of the six mulberry polysaccharides contained a three-stranded helical structure.3.The results of in vitro antioxidant activity tests showed that,in the concentration range of 0.1～0.5 mg/m L,the scavenging activities of the six fractions of mulberry crude polysaccharides against DPPH radicals were MFP-IV>MFP-I>MFP-VI>MFP-V>MFP-II>MFP-III in order;the scavenging abilities against ABTS radicals were MFP-IV>MFP-I>MFP-VI>MFP-II>MFP-III in order.When the polysaccharide was concentrated at 0.5 mg/m L,MFP-IV showed the highest DPPH radical scavenging rate and ABTS radical scavenging rate,close to that of ascorbic acid(VC),and the best antioxidant activity.The results of the in vitro hypoglycaemic activity test showed that in the concentration range of 2～10 mg/m L,the inhibition ability of the six fractions of mulberry polysaccharides on α-glucosidase activity was in: MFP-III>MFP-I>MFP-IV>MFP-V>MFP-II>MFP-VI;the inhibition ability on α-amylase activity was in the following order:MFP-III>MFP-V>MFP-IV>MFP-VI>MFP-I>MFP-II.MFP-III showed the strongest inhibitory effect on α-glucosidase and α-amylase activities and the best hypoglycemic activity.4.The best extraction fraction was selected as 50% ethanol ultrasound-assisted extraction(MFP-III)by combining the physicochemical properties,structural characteristics and biological activity evaluation of polysaccharides.The three eluted fractions were separated by gradient elution of MFP-III with different concentrations of Na Cl solution(0 mol/L,0.05 mol/L and 0.1 mol/L)through a DEAE cellulose-52 anion exchange column.Among them,the distilled water elution fraction had the highest sugar content.The distilled water elution fraction was collected and separated on a Sephadex G-100 dextran gel to obtain a relatively homogeneous elution peak with a yield of 12%.5.The results of HPGPC analysis showed that only a single peak appeared in the HPGPC chromatogram of MFP-III with good peak symmetry,indicating that the purity of the purified polysaccharide from MFP-III was relatively high.Based on the dextran standard curve,the Mp of MFP-III purified polysaccharide was calculated to be 9225 Da,Mw was 10353 Da and Mn was 7847 Da.Chemical analysis showed that the total sugar content of MFP-III purified polysaccharide was higher than that of MFP-III crude polysaccharide,and the glyoxylate content of MFP-III was significantly lower than that of MFP-III crude polysaccharide.The monosaccharide composition of the MFP-III purified polysaccharides was rockulose(1.40%),rhamnose(1.10%),arabinose(7.30%),galactose(7.40%),glucose(45%),N-acetyl-D-glucosamine(1.40%),xylose(1.60%),mannose(1.80%),fructose(33%).UV spectral analysis showed that MFP-III was protein and nucleic acid free.Infrared spectroscopy showed that MFP-III had the typical absorption peaks characteristic of polysaccharides,and that β-glycosidic bonds and pyranose rings were present in MFP-III.The methylation results indicated that MFP-III was a fructan with glycosidic bond linkages mainly →1)-Fruf-(2→(27.10%)and Glcp-(1→(25.10%).6.At a polysaccharide concentration of 5 mg/m L,the scavenging rate of DPPH radical and ABTS radical by purified MFP-III was significantly lower than that of MFP-III crude polysaccharide(P<0.001).When the polysaccharide concentration was10 mg/m L,the purified MFP-III showed significantly higher inhibition ofα-glucosidase and α-amylase than the crude MFP-III polysaccharide(P<0.001)and increased hypoglycemic activity.This study demonstrated that different extraction methods had different effects on the physicochemical properties,structural characteristics and biological activities of mulberry polysaccharides.Mulberry polysaccharides were further purified by gel with higher purity,increased total sugar content,decreased protein,anthocyanin and glyoxylate content,lower molecular weight,mainly composed of fructose and glucose,and the glycosidic bond linkages were mainly →1)-Fruf-(2→ and Glcp-(1→.The purified mulberry polysaccharide had significantly lower antioxidant activity but increased hypoglycaemic activity,indicating that it may be a potential anti-diabetic natural drug.