Optimization of Enzymatic Extraction Process of Polysaccharides from Pseudostellaria heterophylla Fibrous Roots by Response Surface Methodology and Its Pilot Application

[Objectives]To study and optimize the process conditions of enzymatic hydrolysis technology for extracting polysaccharides from Pseudostellaria heterophylla fibrous roots and its application in workshop pilot tests.[Methods]P.heterophylla fibrous roots were taken as the matrix material,and Box Behnken design was used to analyze the extraction time,composite enzyme addition amount,and liquid-solid ratio for response surface optimization experiments,and then applied to the pilot extraction of P.heterophylla fibrous roots.[Results]Response surface analysis showed that all factors had a significant impact on the experimental indicators.The optimal extraction process conditions for polysaccharides from P.heterophylla fibrous roots were extraction time of 2.7 h,compound enzyme addition of 2.5%,and liquid-solid ratio of 32.The yield of polysaccharides from P.heterophylla fibrous roots was 4.83%.The water extraction process of P.heterophylla fibrous roots extraction pilot was used as the control group for response surface optimization of the pilot experiment.The optimization results showed that the extraction time was 3 h,the amount of composite enzyme added was 2.5%,and the liquid-solid ratio was 28.The polysaccharide yield was 4.75%,an increase of 4.63%compared to the control group.[Conclusions]This paper could provide feasibility for the innovation of enzy-matic hydrolysis technology for P.heterophylla fibrous roots and its workshop pilot practice application,as well as a reference for the industrial application of its medicinal resources.

Extraction and purification of the polysaccharides in Hippohaere rhamnoides L.

The different extraction technology and purification technology ofHippohpae rhamoides polysaccharides were researched in the paper. The best method of papain extraction were obtained, the ratio of papain 2%, pH at 5.5, temperature at 45℃ and extraction time of 20 min were suitable for papain extraction. The highest content of Hippohpae rhamoides polysaccharides was 44.28 mg·g^-1. The optimum process of ultrasonic extraction were obtained, namely extracted for 55 min at 480 W with the material ratio of 1：20. The highest content of Hippohpae rhamoides polysaccharides was 48.63 mg·g^-1. The results showed that the ultrasonic and papain extraction together was the best method, the content was 54.30 mg·g^-1. After the removing protein, pigment and dialysis. Two fraction were separated from the purified Hippohpae rhamoides by DEAE-cellulose chromatography, the main fraction was collected finally. The fraction was identified by Sepharose CL-4B gel filtration. Ultraviolet spectrometry, freeze-thawing analysis showed that fraction was purified. Its molecular weight was probably 109.4 ku.

Optimal ultrasonic extraction condition and determination of polysaccharides in Tilia amurensis flowers

Seven extracting temperatures （25, 35, 45, 55, 65, 75, 85℃） and extracting time （30, 40, 50, 60, 70, 80, 90 min） were designed for selecting the optimal extracting time and extracting temperature for ultrasonic extraction of T. amurensis flowers. Polysaccharides from T. amurensis flowers were isolated and determined by spectrophotometry. Results show that the optimal ultrasonic temperature was 75℃ and ex tracting time 52 min. The content of polysaccharides in T. amurensis flowers measured by anthrone--H2SO4 colorimetry under 580 nm, was 9.74% with 0.47% of relative standard deviation （RSD, n=3）. This study demonstrated that ultrasonic extraction method was simple, and the color of the treated samples was stable in 4 h. The average recovery value for the polysaccharides measured was 99.48%±1.01%, with 0.112% of RSD （n=3）.

Extraction, purification and antioxidant activity of polysaccharides from bamboo leaves

Ultrasonic extraction （UE） was employed for the extraction of bamboo leaf polysaccharides （BLP）. The influential parameters of UE procedure including extraction time, ultrasonic power and solid/liquid ratio were optimized by orthogonal experiments. DEAE-cellulose col- umn chromatography was applied to purify BLP and then the radical scavenging activity of BLP was also evaluated. Optimal extraction con- ditions were： extraction time .of 15 min, ultrasonic power of 300 W, and solid/liquid ratio of 1：15. Four kinds of polysaccharides were obtained by DEAE-cellulose colunm chromatography; the maximum superoxide radical scavenging rate （20.4%） of BLP was inferior to that of vitamin C （Vc, the control） and the hydroxyl radical scavenging rate （50%） was equivalent to that of Vc.

Optimization of Polysaccharides Extraction from Physalis alkekengi L.Peel and Its Effect on the Expression of Inflammation-Related Proteins in SW620 Cells

Objective:To establish an optimized aqueous extraction process for polysaccharides from Physalis alkekengi L.peel and to preliminarily explore its in vitro anti-inflammatory activity against colorectal cancer SW620 cells.Methods:A single-factor test combined with orthogonal test analysis was used to evaluate the effects of the material-to-liquid ratio,extraction temperature,and extraction time on the yield of polysaccharides from Physalis alkekengi L.peel.The antioxidant activity of the polysaccharides was assessed by analyzing their free radical scavenging ability in vitro,and the anti-inflammatory effect was evaluated using SW620 cells.Results:The optimal extraction conditions were a material-to-liquid ratio of m(g):V(mL)=1:30,an extraction temperature of 100℃,and an extraction time of 40 minutes,with a predicted polysaccharide yield of 25.7%.The polysaccharides from Physalis peruviana peel effectively scavenged DPPH,superoxide anion,and hydroxyl radicals.After treatment with Physalis peruviana polysaccharides,the levels of IL-1β,IL-18,and TNF-αin the cell culture medium were significantly reduced,and the phosphorylation level of P65 protein in SW620 cells was decreased.Conclusion:This extraction method is stable and reliable,and the prepared Physalis alkekengi L.polysaccharides exhibit significant in vitro antioxidant and anti-inflammatory activities.This study provides a theoretical basis for developing drugs for the prevention and treatment of colorectal cancer.

Optimization of Ultrasonic-Assisted Extraction Process of Polysaccharides from American Ginseng and Evaluation of Its Immunostimulating Activity

Ultrasonic-assisted extraction （UAE） of American ginseng polysaccharides （AGP） was investigated using response surface methodology. Three-factor-three-level Box-Behnken design was employed to optimize the ultrasonic power, extraction time and ratio of water to raw material to obtain a high AGP yield. The analysis of variance and response surface plots indicated that ultrasonic power was the most important factor affecting the extraction yield. The optimal conditions were ultrasonic power 400 W, extraction time 71 min, and ratio of water to raw material 33 mL g-1. Under these conditions, the yield of AGP was 8.09%, which was agreed closely to the predicted value. Gas chromatography （GC） analysis showed that AGP was composed of arabinose, rhamnose, galactose, glucose, and galacturonic acid. Fourier transform infrared spectra revealed the general characteristic absorption peaks of AGP. In addition, AGP exhibited good immunostimulating activities by up-regulating the production of nitric oxide and cytokines. Compared with hot water extraction, UAE required shorter extraction time and gave a higher extraction yield, without changing the structure and immunostimulating activity of AGP. The results indicated that UAE could be an effective and advisable technique for the large scale production of plant polysaccharides.

Ultrasonic Extraction of Polysaccharides from Laminaria japonica and Their Antioxidative and Glycosidase Inhibitory Activities

In the present study, ultrasonic extraction technique （UET） is used to improve the yield of polysaccharides from Lami- naria japonica （LJPs）. And their antioxidative as well as glycosidase inhibitory activities are investigated. Box-Behnken design （BBD） combined with response surface methodology （RSM） is applied to optimize ultrasonic extraction for polysaccharides. The optimized conditions are obtained as extraction time at 54 min, ultrasonic power at 1050 W, extraction temperature at 80℃ and ratio of material to solvent at 1：50 （g mL-1）. Under these optimal ultrasonic extraction conditions, an actual experimental yield （5.75% ＋ 0.3%） is close to the predicted result （5.67%） with no significant difference （P〉0.05）. Vitro antioxidative and glycosidase inhibitory activities tests indicate that the crude polysaccharides （LJP） and two major ethanol precipitated fractions （LJP1 and LJP2） are in a concentration-dependent manner. LJP2 （30%-60% ethanol precipitated polysaccharides） possesses the strongest α-glucosidase in- hibitory activity and moderate scavenging activity against hydroxyl radicals （66.09% ±2.19%, 3.0 mg mL-l）. Also, the inhibitory activity against a-glucosidase （59.08% ± 3.79%, 5.0 mg mL-1） is close to that of acarbose （63.99% ± 3.27%, 5.0 mg mL-l）. LJP 1 （30% ethanol precipitated polysaccharides） exhibits the strongest scavenging activity against hydroxyl radicals （99.80%q-0.00%, 3.0mg mL-1） and moderate a-glucosidase inhibitory activity （47.76%± 1.92%, 5.0 mgmL-1）. LJP shows the most remarkable DPPH scav- enging activity （66.20%±0.11%, 5.0mgmL-1） but weakest a-glucosidase inhibitory activity （37.77%±1.30%, 5.0mgmL-1）. How- ever, all these LJPs exert weak inhibitory effects against a-amylase. These results show that UET is an effective method for extract- ing bioactive polysaccharides from seaweed materials. LJP 1 and LJP2 can be developed as a potential ingredient in hypoglycemic agents or functional food for the management of diabetes. This study provides scientific evidence and advances in the preparation technology and a hypoglycemic activities evaluation method for seaweed polysaccharides, especially glycosidase inhibition in com- bination with an antioxidative activity evaluation method.

CTAB-silica Method for DNA Extraction and Purification from Castanea mollissima and Ginkgo biloba

A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.

A Study on the Ultrasonic Extraction and Antioxidant Activity of Lychee Pericarp Polysaccharides

The aim was to optimize ultrasonic extraction conditions and study antioxidant activities of lychee pericarp polysaccharides. The effects of extraction temperature,time,ultrasonic power,solid-liquid ratio on polysaccharides yield were investigated by single-factor experiment and orthogonal experiment to obtain optimum extraction conditions; the antioxidant activities of lychee pericarp polysaccharides were evaluated by the total antioxidant activity. Optimum extraction process was under extraction temperature of 80 ℃,extraction time of 2. 5 h,ultrasonic power of 180 W,solid-liquid ratio of 1∶ 25 g / m L; the total antioxidant activity was increased with the increasing concentration of polysaccharides. The extraction process is simple and efficient. The extraction rate of lychee pericarp polysaccharides with antioxidant activities is high.

Optimization of Extraction Process,Analysis of Polysaccharides from Morchella septimelata

Morchella septimelata polysaccharide(MSP)possesses multiple biological activities.In this study,double enzyme method was used to extract polysaccharides from Morchella septimelata.The basis of single factor experiment and response surface design was used to optimize the extraction process of polysaccharides.The optimal extraction conditions of polysaccharide were as follows:extraction time 2 h,extraction temperature 40.5℃,solid-liquid ratio 1:15(w/v),and the amount of double enzyme(cellulase:β-glucanase,mass ratio 1:2)2.0%.Under these conditions,the extraction yield of polysaccharide reached(14.86±0.12)%.

Extraction,Physiochemical Properties and Antibacterial Activities of Polysaccharides from Auricularia auricular

[Objective] To explore the optimal conditions for extracting polysaccharide from Auricularia auricular, and to study its physiochemical properties and antibacterial activities. [ Method] The extraction conditions for Auricularia auricular polysaccharides were optimized by orthogonal test, and its physiochemical characteristics and antagonistic activities on bacteria and molds were tested. I-Result] The optimized condition was with sample to liquid ratio of 1 ： 50, temperature of 90 ℃ and extraction time of 2.5 h. Under the optimized condition, the yield of pelysaccharide was 7.3%, with polysaccharide content of 77.81%. Containing aldose and amylase, the polysaccharides of Auncu/aria auricular was soluble in water. The inhibitory effect on bacteria was significantly stronger than on molds that the minimum diameter of inhibition zone for bacteria is 12.7 mm, while 7.9 mm for the molds. [ Conclusion] This study provides scientific basis for the development and utilization of Auricularia auricular polysaccharides.

PURIFICATION AND PARTIAL CHARACTERIZATION OF POLYSACCHARIDES FROM THE POLLEN OF TYPHA ANGUSTIFILIA L.

Three polysaccharides TAA,TAB and TAC were purified from the pollen of Typha An- gustifilia L..TAA is mainly composed of α-L-arabinofuranose,β—D—galactose and α—D—galac- turonic acid.TAB and TAC contain the backbone mainly composed of (1→5)—linked—L—arabi- nosyl residue.

Study on the Optimization of Extraction Technology of Rose laevigate Michx. Polysaccharides and Its in vitro Antioxidant Activity

[Objectives]This study was conducted to optimize the optimal process conditions for extracting polysaccharides from Rose laevigate Michx.with hot water.[Methods]The extraction rate of R.laevigate was used as an evaluation index,and extraction temperature,material-to-liquid ratio,extraction time and extraction times were selected as influencing factors.On the basis of single factor tests,L 9(34)orthogonal tests were carried out for optimization,and the content of polysaccharides was determined by the phenol-sulfuric acid method.The in vitro antioxidant activity of the extracted R.laevigate polysaccharides was studied.[Results]The effects of various influencing factors on the extraction rate of R.laevigate polysaccharides ranked as material-liquid ratio>extraction temperature>extraction times>extraction time.The optimal extraction process was extraction temperature 80℃,extraction time 2 h,and material-to-liquid ratio 1∶25,extraction times 3 times,under which the extraction rate was(9.55±0.12)%.R.laevigate polysaccharides had certain scavenging capacities of DPPH free radicals and hydroxyl free radicals,and its scavenging capacity of DPPH free radicals was better than that of hydroxyl free radicals.[Conclusions]This study provides a theoretical basis for the optimization of the extraction process of R.laevigate polysaccharides and the in-depth development and application of polysaccharides.

The Extraction and Purification of Stevioside from Stevia

tevioside is a natural sweetener extracted from stevia,which is widely used in the food industry and the pharmacy.Centre on the extraction and purification of stevioside,using ADS-7 resin to extract stevioside through one single step,greatly shorten the process and solve the problems of lower yield and higher raw materials cost existed in the general process.

Polysaccharides in Sipunculus nudus: Extraction Condition Optimization and Antioxidant Activities

Marine organisms constitute unlimited resource of bioactive substances due to their high biodiversity and represent a valuable source of new compounds. This study optimized the alkali-extraction conditions and antioxidant activities of soluble polysaccharides from the body wall of Sipunculus nudus. The effects of solid-liquid ratio, extraction duration, extraction temperature, and alkali concentration on the yield of S. nudus polysaccharides(SNP) were examined, according to which the optimal combination of extraction parameters was obtained by an orthogonal test. The relative influencing importance of different extraction parameters on the yield of SNP followed the order as solid-liquid ratio > extraction temperature > alkali concentration > extraction duration. The highest extraction yield, 1.98%, was achieved under an optimal extraction condition: temperature 60℃, solid-liquid ratio 1:6 g mL^(-1), duration 5 h, and alkali(Na OH) mass fraction 6%. The in vitro antioxidant activities examination showed that extracted SNP under this optimized condition had strong power in reducing certain hydroxyl and superoxide radical scavenging abilities. The promising results showed that extracted SNP could be a potent natural antioxidant.

Isolation,Purification and Bioactivities of Polysaccharides from Irpex lacteus

Irpex lacteus has been widely used for treating chronic glomerulonephritis as a traditional Chinese medicine.Seven water-soluble polysaccharide fractions（ILN I,ILN II,ILN III,ILA I,ILA II,ILB I and ILB II） were isolated and purified from Irpex lacteus by hot-water extraction,deproteinization,decolorization,dicthy laminoethyl（DEAE）-cellulose ion exchange and sephadex G100 chromatographies,respectively.The average molecular weights and monosaccharide composition of these polysaccharide fractions greatly differed from each other.The antitumor and antinephritis activities of the seven polysaccharide fractions were evaluated.It was found that ILN III displayed significant inhibition effects on both humar hepatocellular liver carcinoma（HepG2） and hentietta lacks（HeLa） tumor cells with IC 50 values of 60.95 and 99.95 μg/mL,respectively.ILA I exhibited significant inhibition effects on murine mesangial cells（HBZT-1） with an IC 50 value of 185.06 μg/mL.The inhibition effects of other polysaccharide fractions on these three cells were significantly different.These results suggest that the polysaccharide fractions isolated from Irpex lacteus have potential antitumor and antinephritis activities.

Influence of Different Extraction and Purification Methods on Astragalus Polysaccharides and Pharmacological Evaluation

Objective To clarify the influence on component and pharmacological action of Astragalus polysaccharides(APS) as complementary therapeutic agents prepared by different extraction and purification techniques.Methods Components of APS prepared by different extraction and purification techniques were analyzed,and these APS were used for synergy and attenuation of chemotherapy,radiotherapy treatment with H22 liver cancer and Lewis lung cancer of tumor-bearing mice,and also used for the regulation of immune function to immunosuppression mice.Results Experimental data were analyzed by means of statistical method to get pharmaco-result:A3(extracted by microwave assistance and purified by membrane separation)>A4(extracted by refluxing and purified by membrane separation)>A1(extracted by refluxing and no purification)≈A2(extracted by microwave assistance and no purification).There were no significant differences on pharmacodynamic action between A1 and A2.However,compared with A1 and A2,it was worth noting that A3 and A4 exhibited good pharmacodynamic action.Then A3-in and A4-in,the samples in dialyzer after dialysis,were separated and purified to get homogeneous APS,which were the principal constituents of APS in dialyzer,with the molecular weight(Mw) of 7669 and 14142 determined by HPGPC,respectively.The average Mw of APS outside of the dialyzer,A3-out was 3102 and A4-out 3256,which were the main compositions of A3 and A4,accounted for 79.63% and 53.92%,respectively.Conclusion APS with Mw about 5000 Da exhibit better antitumor effect and immunological activity.Refluxing,microwave assistance extractions,and membrane enrichment techniques bring different cases on Mw distribution,components and pharmacodynamic action,and obviously exhibit relationship among component,Mw distribution,and pharmacological action.

Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil

Microorganism DNA of rhizosphere soil from Pinus koraiensis and Pinus sylvestriformis were extracted by proteinase K based on SDS method, CTAB method, PVP （polyvinylpolypyrrolidone） method, and freezing and thawing method and the crude DNA from rhizosphere soil were purified by dialysis method, silver beads absorption method, and squeezing DNA gel method. The results of different extracting and purifying methods were compared and evaluated. Results indicated that the best method of extraction for microorganism DNA in rhizosphere soil was proteinse K based on SDS method with high salt concentration of 1.0% （w/v） NaCl, which could effectively eliminate humic acids and other impurities. The dialysis method was suitable to purify DNA from rhizosphere soil because of effectively removing brown matters and humic acids and the purified products were suited to PCR amplification. Squeezing DNA gel method was also a good purification method with the advantage of inexpensive in cost and efficient in use.

Effects of Different Extraction and Purification Methods on the Acquisition of Phycoerythrin from Porphyridium purpureum

Phycoerythrin, as the main light-harvesting antenna in Porphyridium purpureum, exists at the outermost end of the phycobilisome. It has advantages of good fluorescence intensity, anti-oxidation, scavenging free radicals, and high chroma, so it has been widely used in food, cosmetics, pharmaceuticals, and other industries. In this study, the effects of different extraction(ultrasonic breaking method, bead grinding method, liquid nitrogen grinding method, and freezing-thawing method) and purification methods(salting out method, ultrafiltration method, and combination of salting out and ultrafiltration method) on the acquisition of phycoerythrin from P. purpureum were studied, and the characteristics of phycoerythrin in the P. purpureum were identified. The results showed that the freezing-thawing method could extract phycoerythrin from the powder of P. purpureum to the utmost extent, and the concentration of the extracted phycoerythrin was up to 0.036 g/L. The salting out method could most effectively purify phycoerythrin, and the purity index was 2.216. The identification of phycoerythrin by ultraviolet absorption spectroscopy and fluorescence spectroscopy indicated that the phycoerythrin had the maximum absorption peak at 545 nm, and the maximum Stokes shift was up to 79 nm. Due to its high fluorescence characteristics, it can be used as a fluorescent marker in the fields of molecular biology and clinical medicine, and can also be used as a good photosensitizer in tumor therapy.

Structural characterization of three acidic polysaccharides from Opuntia dillenii Haw.fruits and their protective effect against hydrogen peroxide-induced oxidative stress in Huh-7 cells

Three novel acidic polysaccharide fractions(OFPP-1,OFPP-2,OFPP-3)with different m olecular weights(803.7,555.1 and 414.5 k Da)were isolated from the peeled Opuntia dillenii Haw.fruits by alkali-extraction,graded alcohol precipitation and column chromatography.Structural analysis indicated that OFPPs were pectic polysaccharides consisting of rhamnose,arabinose and galactose residues.The backbone of OFPP-1 consisted of a repeating unit→6-α-D-Galp A-(1→2)-α-L-Rhap-(1→with T-α-D-Galp A-(1→6)-α-D-Galp A-(1→4)-α-D-Glcp-(1→,T-β-D-Xylp-(1→6)-α-D-Galp A-(1→4)-α-D-Glcp-(1→or T-α-D-Galp A-(1→3)-α-L-Araf-(1→as the side chains.The backbone of OFPP-2 consisted of a disaccharide repeating unit→2)-α-L-Rhap-(1→4)-β-D-Galp A-(1→with T-β-L-Araf-(1→as the branches substituted at the O-4 position of→2,4)-α-LRhap-(1→.Whereas the backbone of OFPP-3 was→2,4)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-β-L-Araf-(1→or→2,4)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-β-D-Galp A-(1→,which was branched at the O-4 position of→2,4)-α-L-Rhap-(1→.Moreover,these three polysaccharide fractions could protect Huh-7 cells against H2O2-induced oxidative stress to different extents by decreasing the MDA content and increasing the SOD,CAT,GSH-Px activities and the GSH level in the Huh-7 cells.These results suggest that OFPPs have the potential to be used as natural antioxidants.