Extraction,Isolation,Purification,and Biological Activities Of Polysaccharides From Ginkgo Biloba L.Leaves

Ginkgo biloba L.,which belongs to the genus Ginkgo in the family of Ginkgoaceae,is widely distributed in the world.G.biloba has been existed on earth since 200 million years and has a long life,thus it has the reputation of "living fossil".G.biloba leaf is one of the plant-derived medicinal materials.G.biloba extract has been recorded in the traditional herbal handbook in ancient China,which has been used as a traditional Chinese herbal medicine for treatment of asthma for thousands of years.Thereinto,G.biloba leaf is a potential resource for functional food.The active ingredients in G.biloba leaf mainly include polyphenols,flavonoid glycosides,polysaccharides,amino acids,ginkgolic acids,terpenoids and various trace elements.An increasing number of modern works has showed that G.biloba leaf has many health-promoting activities,such as antioxidant,anti-inflammatory,antidiabetic,hepatoprotective,antimicrobial,antihypertensive and anticancer activities.Polysaccharide,a natural macromolecular compound,is widely found in animals,plants and microorganisms.It has antioxidant,anti-tumor,immune regulation,blood sugar lowering,blood lipid lowering and other biological activities.It has become a updated attentive study area in the fields of food science and natural products.In recent years,polysaccharides,regarded as one of the important active ingredients in G.biloba leaves,have attracted more and more researchers' interests.However,the research on G.biloba leaf polysaccharide(GBPS)is still not in-depth.Therefore,this work focuses on the extraction,separation and purification,physicochemical properties,antioxidant activity,immunomodulatory activity,in vitro simulated digestion and fermentation of GBPS,which can provide a certain theory and scientific basis for the utilization of GBPS.The main research contents and results in this work are as follows:(1)Extraction optimization,purification,and the preliminary characterizationof GBPSIn this study,the effects of extraction temperature,extraction time,ratio of water to raw and extraction cycles on the yields of GBPS were carried out based on single-factor tests.Three factors including extraction temperature(80?),extraction time(3 h)and ratio of water to raw(26 mL/g),which significantly affects the yields of GBPS,and their proper ranges were obtained.Furthermore,a three-level,three-variable Box-Behnken design(BBD)method was carried out to investigate the optimal levels for the extraction of GBPS.The result showed that optimum extraction condition was as as follows:extraction temperature 86?,extraction time 3 h and ratio of water to raw material 22 mL/g analyzed by the software of Design Expert version 8.0.6.The yield of GBPS was 9.08±0.74%.The crude GBPS was further purified on a DEAE Sepharose Fast Flow column to yield GBPS-2 and GBPS-3,and the yields of GBPS-2 and GBPS-3 were 9.45%±1.67%and 32.87%± 3.11%,respectively.The carbohydrate,uronic acid,protein,and total polyphenol contents of GBPS-2 and GBPS-3 were determined using the phenol-sulfuric acid method,the m-hydroxydiphenyl-sulfuric acid method,the Bradford method and the Folin-Ciocalteu method,respectively.The results showed that the carbohydrate contents in GBPS-2 and GBPS-3 were 71.46±1.93%and 44.32 ± 1.18%,respectively,GBPS-2 and GBPS-3 were typical acidic polysaccharides with high uronic acid contents of 12.49 ± 0.98%and 38.37±1.62%respectively.The measured protein and total polyphenol contents were<0.3%.The molecular weights and monosaccharide compositions of GBPS-2 and GBPS-3 were evaluated by HPLC,the result showed the molecular weights of GBPS-2 and GBPS-3 were 672 and 723 kD,respectively.Furthermore,GBPS-2 was composed of Man,Rha,GlcA,GalA,Glc,Gal,and Ara(molar ratio:0.08:0.12:0.16:0.06:0.11:1.00:0.32),whereas GBPS-3 was composed of Man,Rha,GlcA,GalA,Gal and Ara(molar ratio:0.92:1.00:0.83:0.11:0.42:0.23).(2)The antioxidant activities of GBPS-2 and GBPS-3In this chapter,antioxidant activities of GBPS-2 and GBPS-3 were evaluated by chemical antioxidant methods.The results showed that both GBPS-2 and GBPS-3 had strong abilities to scavenge ABTS and superoxide anion free radicals,inhibit oxidation of?-carotene-linoleate and chelate iron ion,but had limited abilities to scavenge DPPH and hydroxyl radicals,furthermore,the total antioxidant capacity and reducing power were significantly lower than those of the positive control.At the same time,PC 12 cell model was used to evaluate the antioxidant activities of GBPS-2 and GBPS-3 at the cellular level.The results showed that GBPS-2 and GBPS-3 had significantly protective effects on H2O2-mediated cell oxidative damage.At the same time,GBPS-2 and GBPS-3 could decrease the levels of LDH and MDA secreted by PC12 cells and increase the activities of SOD,CAT and GSH-Px.The results of chemical antioxidant activities and cell antioxidant activities showed that the antioxidant activity of GBPS-3 was better than that of GBPS-2.(3)The immunostimulating activities of GBPS-2 and GBPS-3 in vitroIn this chapter,RAW264.7 cell was used to evaluate the immunoregulatory activities of GBPS-2 and GBPS-3.The results showed that GBPS-2 and GBPS-3 had no significant toxicity to RAW264.7 cells at the concentration of 50-200 ?g/mL,and GBPS-2 could significantly promote the proliferation of RAW264.7 cells at the concentration of 200 ?g/mL.GBPS-2 and GBPS-3 could significantly increase the phagocytosis of neutral red and acid phosphatase activity of RAW264.7 cells,and promote the secretions of NO,PGE2 and cytokines including TNF-?,IL-6 and IL-1?.The effects of GBPS-2 and GBPS-3 on mRNA expression of COX-2,iNOS,TNF-?,IL-6,and IL-1? were also examined using RT-qPCR.The results showed that GBPS-2 and GBPS-3 could significantly stimulate the expressions of COX-2,iNOS,TNF-?,IL-6,and IL-1? at mRNA level.Furthermore,Western Blot was used to evaluate the expression of COX-2 and iNOS at protein level.The results showed that GBPS-2 could significantly promote the expression of COX-2 and iNOS at protein level.Therefore,GBPS-2 and GBPS-3 had significantly immune promoting effects on RAW264.7 cells.At the same time,Western Blot was used to investigate the effect of GBPS-2 on the protein expressions of P65,I?B? and p-I?B? in RAW264.7 cells.The results showed that GBPS-2 significantly increased the protein expressions of p65 and p-I?B?and decreased the protein expression of I?B?,suggesting that GBPS-2 may activate the transcription and expression of cytokines through NF-?B pathway,thereby to enhance the immune activity of macrophages.(4)Digestive characteristics and fermentation of GBPS by gut microbiota in vitroThe simulated saliva,gastric and small intestinal digestive models were used to study the digestive characteristics of GBPS in vitro.The result showed that there was no significant change in molecular weights of GBPS before and after simulated saliva,gastric and small intestinal digestion,indicating that saliva,gastric juice and small intestinal juice had no digestive effect on GBPS.Furthermore,there was no significant increase in contents of reducing sugar after digestion,indicating that there was no new reducing end released after digestion.Therefore,GBPS could pass through the saliva,gastric and small intestinal digestive conditions and safely reach the large intestine.The interaction between GBPS and gut microbiota was further investigated using anaerobic fermentation model in vitro.The result showed that the response value of GBPS for molecular weight in HPLC decreased gradually after fermentation,and the contents of polysaccharide and reducing sugar in fermentation solution also decreased gradually with the increase of fermentation time,suggested that indigestible GBPS could be gradually broken down and utilized by gut microbiota.At the same time,GBPS could significantly reduce the pH of fermentation solution and promote the secretions of lactic acid,acetic acid,propionic acid and butyric acid by gut microbiota.Furthermore,GBPS could promote the proliferation of total bacteria,Bacteroides,Bifidobacterium and Lactobacillus.Therefore,GBPS has potentially probiotic activity.(5)The effects of GBPS-2 and GBPS-3 on the gut microbiotaIn this chapter,the anaerobic fermentation model of gut microbiota in vitro and 16S mRNA sequencing technology were used to further evaluate the prebiotic activities of GBPS-2 and GBPS-3 on gut microbiota.The Chao,ACE and Shannon indices of GBPS-2 samples were significantly higher than those of GBPS-3.The results showed that GBPS-2 was more helpful to improve the species and diversity of gut microbiota than GBPS-3.Meanwhile,principal component analysis(PCA),principal coordinate analysis(PCoA),non-metric multidimensional scale analysis(NMDS)and clustering analysis were used to evaluate the effects of GBPS-2 and GBPS-3 on structure and composition of gut microbiota.The results of PCoA,NMDS and clustering analysis showed that the structure of GBPS-3 and BLK groups was similar,whereas GBPS-2 and INL groups were similar,suggested that GBPS-3 showed better regulation activity of gut microbiota than GBPS-2.GBPS-3 and GBPS-2 could significantly promote the secretions of acetic acid,propionic acid and butyric acid.The contents of acetic acid and butyric acid in GBPS-2 group was significantly higher than those in GBPS-3 group,while the content of propionic acid in GBPS-3 group was significantly higher than that in GBPS-2 group.The results showed that both GBPS-2 and GBPS-3 had potential probiotic activities.