Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp / 生物工程学报

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-β-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

A new method of microglia sorting and functional characteristics of spinal microglia in aged rats / 中国应用生理学杂志

Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×106, aged rats(0.243±0.023)×106). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1β of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.

Three new ursane-type triterpenoids from Rosmarinus officinalis and their biological activities / 中国天然药物

Three new ursane-type triterpenoids, 3-oxours-12-en-20, 28-olide (1), 3β-hydroxyurs-12-en-20, 28-olide (2) and 3β-hydroxyurs-11, 13(18)-dien-20, 28-olide (3), were isolated from a potent anti-inflammatory and antibacterial fraction of the ethanolic extract of Rosmarinus officinalis. Their structures were elucidated by a combination of extensive 1D- and 2D-NMR experiments, MS data and comparisons with literature reports. Compounds 1-3 exhibited significantly inhibitory effects on nitric oxide production in lipopolysaccharide-activated mouse RAW264.7 macrophages, but no antibacterial activity was found at a concentration of 128 μg·mL-1.

Isolation and purification of neuraminidase from influenza vaccine and reference substance preparation / 中华微生物学和免疫学杂志

Objective:To establish a method for isolation and purification of neuraminidase from influenza vaccine and to prepare reference substance for quantitative detection of neuraminidase.Methods:Functional magnetic particles with specific affinity for neuraminidase were prepared. The method for separation and purification of neuraminidase was established based on the magnetic particles. The separation and purification conditions were optimized. The purity of neuraminidase was analyzed and the specificity was verified. The enzyme activity was determined and the protein was quantified.Results:The functional magnetic particles modified with 4-aminophenanthroline were successfully prepared and the method for isolation and purification of neuraminidase based on the magnetic particles was established. The purity of neuraminidase was 98.7%. The concentrations of neuraminidase isolated and purified from the monovalent stock solution of H1N1, H3N2, B/Victoria and B/Yamagate vaccines were 71.50, 100.58, 64.11 and 37.68 μg/ml, respectively, and the enzyme activity remained.Conclusions:The method for isolation and purification of influenza virus neuraminidase was established and the corresponding reference substance was prepared.

Isolation, structural identification of secondary metabolites from Pseudomonas aeruginosa HBD-12 with antibacterial and antitumor activities / 生物工程学报

To screen strains with antibacterial and antitumor activity, pregnenolone was used as the sole carbon source for screening bacteria from soil. Based on bacteriostatic activity assay, Pseudomonas aeruginosa HBD-12 was found to be effectively inhibiting the growth of Escherichia coli, Bacillus thuringiensis, Penicillium digitatum and Penicillium italicum, and its fermentation broth was separated and purified using column chromatography. Then, structure of the obtained monomeric compounds was analyzed by spectrum analysis, and their antitumor activity was measured using HTRF kinase detection kit. The isolated monomeric compounds 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline had significant antitumor activity. At 20 μg/mL, 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline inhibited 78.39±2.29% and 60.34±8.35% Aurora kinase A, respectively. Therefore, the secondary metabolites of Pseudomonas aeruginosa HBD-12 have the potential to develop antibacterial and antitumor drugs.

Isolation, purification and structural characterization of Bletilla striata polysaccharides and its antitumor activity / 中草药

Objective: Bletilla striata polysaccharide (BSP-1) extracted from the stem tubers of B. striata was purified to study the structural properties and antitumor activity. Methods: The crude polysaccharide was fractionated by DEAE-cellulose column chromatography, and three fractions were obtained including BP-1, BP-2, and BP-3, respectively, BP-1 was further purified by SephadexG-200 column chromatography, and a sub-fraction named BSP-1 was obtained. Subsequently, HPGPC, IR spectroscopy, gas chromatography (GC), GC coupled to mass spectrometry (GC-MS) and methylation analysis, and 1H- and 13C-NMR were employed to characterize the structural properties of the polysaccharide fractions. Moreover, the anticancer activity was investigated in vivo. Results: The results showed that the molecular weight of BSP-1 were 4.72 × 105. BSP-1 was consisted of mannose (Man) and glucose (Glc) residues. The backbone of BSP-1 was composed of β-1,4-linked Man and Glc residues at the end with a molar ratio of 8:1. BSP-1 could inhibit the tumor proliferation of HepG2-bearing mice. The tumor weight of mice was significantly inhibited by BSP-1 with inhibitory rate of 66.42%. Conclusion: Structural characterization of BSP-1 provides the significant reference for the further development and elucidation of polysaccharides from B. striata as new drugs.

Separation, purification and immunomodulatory activity of polysaccharides from Rubus sachalinensis / 中草药

Objective: To extract, separate and purify homogeneous polysaccharides from Rubus sachalinensis and study on monosaccharide component and immunomodulatory activity. Methods: The crude polysaccharides of Rubus sachalinensis (RSP) were extracted by hot water. The Cellulose DE-52 and Sephadex G-100 columns were used to separate and purify homogeneous polysaccharides. The relative molecular mass was analyzed by high-performance gel permeation chromatography, and the monosaccharide composition and structure were preliminarily identified by GC, IR and NMR. The effects on proliferation function of mice spleen lymphocyte proliferation were determined by CCK-8, and the effects on the release capacity of IL-2, IFN-γ and TNF-α were determined by the ELISA kit. Results: Two homogeneous polysaccharides, RSP1-1 and RSP1-2, were separated and purified, with molecular weights of 13 227 and 9 343 determined by HPGPC. They mainly contained arabinose, mannose, glucose and galactose, with the mole ratios at 9.5:7.0:10.3:18.6 and 5.7:11.1:10.3:14.2, respectively. The structure of RSP1-1and RSP1-2 was analyzed by IR and NMR, and RSP1-1 might mainly contain α-1,3-Ara, β-1,4Gal, β-1,6-Glc, β-1,3-Man, and RSP1-2 might mainly contain β-1,4-Gal, β-1,6-Glc, β-1,3-Man. At 5-200 μg/mL, RSP1-1and RSP1-2 stimulated proliferation of spleen lymphocytes (P < 0.05) and promoted lymphocytes to secrete IFN-γ and TNF-α. At 5 μg/mL, RSP1-1and RSP1-2 promoted lymphocytes to secrete IL-2. Conclusion: RSP1-1and RSP1-2 are natural homogeneous polysaccharides that are obtained from this plants for the first time. Its purity and structure were further characterized by IR and NMR. These two homogeneous polysaccharides promoted the proliferation of splenic lymphocyte in different degrees and promoted lymphocytes to secrete IL-2, IFN-γ and TNF-α that all possessing immunomodulating activity.

Isolation and Identification of Chemical Constituents from Rhizomes of Dendropanax proteus / 中国实验方剂学杂志

The chemical constituents from 95% ethanol extract of Dendropanax proteus rhizomes and their anti-inflammatory activities were investigated. These compounds in 95% ethanol extract of D. proteus rhizomes were isolated and purified by silica gel column chromatography, medium-pressure liquid chromatography, preparative liquid chromatography, etc. Their structures were elucidated based on the spectral data and physicochemical properties. All the compounds were tested for their ability to inhibit lipopolysaccharide (LPS) induced nitric oxide production in the murine microglia BV2 cell line. Nine compounds were isolated from the ethyl acetate fraction of 95% ethanol extract of D. proteus rhizomes, and identified as (-)-syringaresinol (1), (+)-(7S,8S)-1',4-dihydroxy-3,3',5'-trimethoxy-7',8,9'-trinor-8,4'-oxyneoligna-7,9-diol (2), erythro-guaiacylglycerol-β-O-4'-coniferyl ether (3), threo-guaiacylglycerol-β-O-4'-coniferyl ether (4), coniferyl alcohol (5), 7-O-ethylguaiacylglycerol (6), vanillin (7), syringaldehyde (8), and excoecanol B (9). Compounds 2 and 4 showed neuritis inhibitory activity against microglial inflammation factor, their half inhibitory concentrations (IC50) were 5.85, 7.29 μmol ·L-1, respectively. Compounds 1-6,8-9 are isolated from this plant for the first time, compounds 2 and 4 exhibit the potent inhibitory activity.

Isolation and Structural Elucidation of Chemical Constituents of Corydalis Rhizoma / 中国实验方剂学杂志

Objective: In order to clarify the characteristic chemical constituents and furnish applicable information to the basic research and quality control research related to the chemistry of traditional Chinese medicines for Corydalis Rhizoma,this paper investigated the chemical constituents of Corydalis Rhizoma extensively. Method: The dried-up and pulverized plant materials were extracted using 95% EtOH as solvent,the EtOH extract was fractionated using different solvents to afford the EtOAc-soluble and n-BuOH-soluble portion,respectively,among others. These two portions were subjected to procedures of isolation and purification on silica gel or ODS column chromatographies to afford monomers. 1D and 2D NMR and MS methods,along with comparison with the data of literatures,were used to identify the structures. Result: Twelve compounds,all belonging to alkaloids,were isolated and identified as d-corydaline(1),tetrahydrocoptisine(2),tetrahydropalmatine(3),tetrahydrocolumbamine(4),corybulbine(5),tetrahydrojatrorrhizine(6),dehydrocorydaline(7),dehydroglaucine(8),8-oxodihydrocoptisine(9),protopine(10),taxilamine(11),and pontevedrine(12). Of these compounds,the structure of 12 was a revised structure which was assigned by combined examinations of their 1D and 2D NMR spectra and MS data. Conclusion: Compounds 6 and 11 were reported from Corydalis Rhizoma for the first time. The structure of pontevedrine was verified as 1,2,9,10-tetramethoxy-6-methyl-4H-dibenzo[de,g]quinoline-4,5(6H)-dione.

Proteolytic Enzymes Screening for Enzymolysis Preparation Technology of Active Parts of Periplaneta americana against Liver Fibrosis / 中国药房

OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and  purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.

Isolation and Identification of Chemical Constituents from Goodyera schlechtendaliana / 中国实验方剂学杂志

Objective:To isolate and purify the chemical constituents from Goodyera schlechtendaliana, and lay a foundation for further research on the effective materials of the plant. Method:The dried grass of G. schlechtendaliana(10 kg)was heated by 95% EtOH for reflux extraction for 4 times at 85℃. The extract liquids were combined, decompressed and concentrated to obtain the crude extract. The crude extract was then suspended in distilled water and extracted with petroleum ether,EtOAc and n-BuOH in turn to obtain corresponding fractions. Silica gel,Sephadex LH-20,ODS and semi-preparation high performance liquid chromatography (HPLC) were used for the separation and purification of EtOAc and n-BuOH fractions. The structures of compounds obtained were identified by 1H-NMR,13C-NMR,MS,physicochemical properties and reference literature. Result:Ten compounds were isolated and identified as 2,5-dihydroxy-4-methoxy-9,10-dihydroxyphenanthrene (1),1,3,5-trimethoxybenzene (2),protocatechuic acid (3),p-hydroxybenzaldehyde (4),p-hydroxybenzoic acid (5),quercetin (6),vanillin(7),daucosterol (8),adenine (9) and adenosine (10). Conclusion:Compounds 1-10 were obtained from the genus of Goodyera for the first time.

Isolation of a novel disintegrin containing Arg-Gly-Asp tripeptide sequence from Gloydius brevicaudus venom and investigation of its biological activities / 药学学报

Snake venom has special pharmacological activities and contains a array of small polypeptides that can antagonize integrins, therefore called disintegrins. Disintegrins can block integrin-dependent platelet aggregation, tumor growth, and tumor metastasis. A disintegrin fraction was isolated and purified from the venom of snake Gloydius brevicaudus (GBV). Its physical and chemical properties were characterized, and its biological activities were investigated. The crude venom of GBV were isolated by Superdex 75 gel filtration chromatography. The anti-platelet aggregation activity of the fractions was screened by the Born method. The fraction that shown anti-platelet activity was further purified with Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion exchange chromatography, and Lichrospher C18 reversed-phase chromatography respectively. The purity of the active component was analyzed with SDS-PAGE (Tris-Tricine system) and high-performance liquid-phase chromatography (HPLC), with protein concentration determined by the Bradford method. The molecular weight was evaluated by the gel imaging method and mass spectrometry, and the isoelectric point was measured by disc isoelectric focusing electrophoresis. The protease activity was measured with the Rick method. The phospholipase A activity was determined by the automatic potentiometric titration method. Amino acid sequencing results were subjected to homology comparison using the BLAST program. Seven fractions (Ⅰ-Ⅶ) were isolated from GBV by gel filtration chromatography on Superdex 75 column. The fraction Ⅳ inhibited the platelet aggregation induced by ADP with molecular weight lower than 10 000 Da, suggesting a disintegrin component. A disintegrin named GBV-Ⅳ4 was purified from the fraction by Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous shown as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) with molecular weight 8 746 Da as calculated by Image Master VDS system. The isoelectric point of GBV-Ⅳ4 was 6.3 by disc polyacrylamide gel electrophoresis. GBV-Ⅳ4 exhibited no detectable phospholipase A2 (PLA2) activity with the pH-stat technique or proteinase activity according to the method of Rick. GBV-Ⅳ4 is composed of 70 amino acids with RGD (Arg-Gly-Asp) active region and a molecular weight of 7 442 Dalton as assayed by Mass Spectrography. Characterization of GBV-Ⅳ4 is consistent with meta-chain disintegrin (70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank, GBV-Ⅳ4 has high homology with other disintegrins. We concluded that GBV-Ⅳ4 is a novel disintegrin contained RGD. GBV-Ⅳ4 showed dose-dependent inhibition of ADP- or thrombin-induced platelet aggregation with IC50 0.339 or 0.577 μg·mL-1 respectively. In conclusion, a new disintegrin derived from the GBV snake venom and named GBV-Ⅳ4 containing RGD tripeptide sequence could inhibit platelet aggregation.

Isolation and characterization of phospholipase A1 from Vespa tropica ducalis / 中草药

Objective: To find an effective method to isolated phospholipase from Vespa tropica ducalis and characterize its biological activities to support the pathogenic mechanism research and officinal value exploitation in the future. Methods: The component with phospholipase activity was isolated by successive gel filtration (Sephadex G-75, supferfine) and heparin affinity chromatography steps (Hitrap Heparin HP). The protein was identified by peptide mass fingerprinting, N-terminal amino acid determination and blast analysis, as well as phospholipase A1 (PLA1) activity monitor. Plasma recalification time test was employed to detect the effect of Vtp32 on coagulation. Results: A protein with phospholipase activity was orderly separated and purified from V. tropica ducalis venom using gel filtration and heparin affinity chromatography. The purified protein was homogenous on the SDS-PAGE gel with relative molecular mass of 32 000, so it was termed as Vtp32. Peptide mass fingerprinting assay and N-terminal amino acid sequence blast result revealed that Vtp32 showed high homologous with PLA1 from wasp of Vespa genus. In addition, Vtp32 hydrolyzed the sn-1 ester linkage of phospholipids. These results indicated that Vtp32 was PLA1 from V. tropica ducalis. Vtp32 hydrolyzed phosphatidylcholine, and the hydrolysis product can lyse human erythrocytes. Vtp32 delayed the recalification time of human plasma and hence had anti-coagulation activity. Conclusion: PLA1 is widely existed in the venom from V. tropica ducalis. Gel filtration followed by heparin affinity chromatography is an effective isolation strategy for the purification of PLA1. The results show that V. tropica ducalis PLA1 has hemolytic and anticoagulative activity.

Isolation and purification of polysaccharides from leaves of Eriobotrya japonica and its monosaccharide composition / 中草药

Objective: To determine the relative molecular weight (Mw), monosaccharide composition, and structures of polysaccharides from the leaves of Eriobotrya japonica. Methods: The polysaccharides (PEL60) were extracted from the leaves of E. japonica with hot water, followed by precipitating with ethanol, freezing, and drying. PEL60-A was purified by DEAE-Sepharose Fast Flow ion column and Sephacryl S 500 High-Resolution gel column chromatography from PEL60 of the leaves of E. japonica. The purity and molecular weight of PEL60-A was detected by high performance liquid chromatography (HPLC), and the monosaccharide composition and molecular ratio of the PEL60-A was analyzed by gas chromatography-mass spectrometry (GC-MS). Results: The PEL60-A, with Mw of 2.69 × 105. The composition of monosaccharide was mainly L-rhamnose and L-fucose and a small amount of D-xylose, D-mannose, D-glucose, and D-galactose with the molar ratio of 0.781.000.150.090.110.19. Conclusion: The study on isolation, purification, and preliminary identification of monosaccharide composition from leaves of E. japonica provided the important scientific theoretical basis for the fine processing of the leaves of E. japonica.

Isolation and characterization in vitro and in vivo of pancreatic islets from NOD mice / 中国实验动物学报

Objective This study was conducted to establish a stable and highly efficient method for isolation and purification of pancreatic islets from NOD mice and to evaluate their characteristics in vitro and in vivo. Methods The is-lets were isolated from mouse pancreas using modified collagenase digestion and Ficoll density gradient centrifugation. The endocrine secretory function was assessed by insulin secretion in either low or high dose glucose stimulation. To evaluate the function of the graft,body weight and blood glucose were monitored,and IVGTT was performed. In addition,to assess sur-vival of the implanted islets,Pathology using HE staining and insulin immunostaining of the graft were performed. Results The average islet yield was 116 ± 12 islets/pancreas and purity was higher than 90%. Compared with islets from Kunming mice,the islets isolated from NOD mice were poorly responsive to glucose challenge. Blood glucose levels and body weight changes of the islet-transplanted diabetic mice were significantly improved compared with the sham-operated mice. In addi-tion,blood glucose levels in vivo after an IVGTT also significantly improved. However,these improvements were only main-tained for 2 weeks. Furthermore,HE staining and immunostaining assays demonstrated that there were insulin-positive cell clusters and lymphocyte infiltration in the graft-bearing kidney. Conclusions A large number of quality islets can be isola-ted and purified from NOD mice by using the modified mouse islet isolation method, which can be used to develop thera-peutic strategies to protect transplanted islets from rejection and autoimmune attack.

Research Methods for Structural Analysis of Lactic Acid Bacteria Induced Exopolysaccharides / 分析化学

Exopolysaccharides (EPSs), produced by lactic acid bacteria (LAB), have been used primarily to improve the quality and taste of food, also possess a variety of unique biological functions, such as immunoregulation and anti-tumor activities. The diversity of molecular structural characteristics of LAB-generated EPSs represents one of the main factors responsible for this plethora of functions. Accordingly, the structural analysis of the EPSs produced by LAB is both a prerequisite and basis for examining its functional and structure-activity relationships. In this article, we summarized the current progress of key methodologies involved in the structural analysis of LAB-generated EPSs, including their isolation, purification, primary structure and advances in structural research. A comprehensive discussion regarding the application of chemical analysis, instrument analysis and computer aided technology in the structure analysis of LAB-generated EPSs was provided. Further, the future development of the research on the structure of LAB-generated EPSs was presented.

Effects of total lignans of Schisandra chinensis on memory impairment in rats with Alzheimer's disease / 国际中医中药杂志

Objective To study the effects of total lignans of Schisandra chinensis on memory impairment in rats with Alzheimer's disease, and to discuss its mechanism of action. Methods The rats were randomly into the blank control group, the sham injury group, the pathological model group, the low dose group of Schisandra chinensis and the high dose group of Schisandra chinensis with 15 rats in each group. The animals were given medicine at 3 days after the model establishment. The ainimals of the pathological model group, the low dose group of Schisandra chinensis and the high dose group of Schisandra chinensis established the AD model by D-gal intraperitoneal injection of compound Aβ hippocampal localization injection. The low dose group of Schisandra chinensis and the high dose group of Schisandra chinensis were administrated by means of intragastric administration 1mL physiological saline containing Schisandra chinensis total lignans 50 mg×kg-1×d-1 and 100 mg×kg-1×d-1, and the blank control group, the sham injury group, the pathological model group were administrated 0.9% Sodium Chloride Injection 1 ml×d-1by means of intragastric administration. After two weeks of continuous administration, the behavioral tests were carried out on the experimental animals, and the expressions of caspase-3, Bax, Bcl-2 and mRNA in the hippocampus of rats were measured by RT-PCR. Results Compared with the pathological model group,the escape latency shortened of the low dose group and the high dose group of Schisandra chinensis were significantly shortened (P<0.05). The number of traversing platforms (3.82 ± 1.23, 5.82 ± 1.18 vs. 2.13 ± 1.48), the percentage of swimming time (39.43% ± 7.12%, 48.63% ± 7.15% vs. 59.98% ± 6.13%) of the low dose group and the high dose group of Schisandra chinensis significantly decreased (P<0.05). Compared with the pathological model group, the expression of caspase-3(6.11 ± 1.25,4.42 ± 1.13 vs.7.88 ± 1.28)and Bax mRNA(5.84 ± 1.62,4.63 ± 1.51 vs.7.54 ± 2.14) significantly decreased (P<0.05), and the expression of the Bcl-2 mRNA(1.73 ± 0.82, 3.04 ± 1.29 vs. 0.93 ± 0.62) wese increased (P<0.05). Conclusions The diatomite Soxhlet extraction method is suitable for the isolation and purification of total lignans from Schisandra chinensis, and the total lignans of Schisandra chinensis can up-regulate the expression of Bcl-2, down-regulate the expression of caspase-3 and Bax, inhibit the apoptosis of neurons induced by A beta, improve the memory impairment of AD animal models, and the intervention effect is dose dependent.

Isolation and purification of Codonopsis pilosula polysaccharide and its anti-oxidant activity / 中草药

Objective: To isolate and purify the Codonopsis pilosula polysaccharide (CPP) and investigate its structure characterization and anti-oxidant activity. Methods: CPP was extracted by heating reflux, crud polysaccharide was refined and then isolated by hollow fiber ultrafiltration experimental device, the anti-oxidant activity of polysaccharides was studied by in vivo and in vitro methods. Results: CPP was purified and obtained three ingredients named CPP1, CPP2, and CPP3. The anti-oxidant activity in vitro showed that CPP3 had the strongest scavenging ability on DPPH, hydroxyl radical, and superoxide anion. In vivo study showed that high dose group of CPP3 had obvious protective effect on the mice induced by D-galactose. Conclusion: CPP has obvious anti-oxidant function. This study provides the theoretical basis for the development of CPP functional food.

System isolation and purification of Isatidis Radix polysaccharides and determination of their compositions / 中草药

Objective: To isolate and purify the polysaccharides (glycoproteins) from IsatidisRadix (Banlangen) systematically and to study the composition of them. Methods: Crude polysaccharides precipitated by 80% ethanol from thewater extract of IsatidisRadix, which has theanti-viral activity, were fractionated by DEAE-Sepharose Fast Flow, Sephacryl-200, and high gel chromatography system sequentially. The composition of monosaccharides and amino acids of polysaccharides (glycoproteins) was then determined by HPLC with pre-column derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP) and o-phthalaldehyde (OPA), respectively. Results: Two of homogeneous polysaccharides named IRPS1A and IRPS1B and two of homogeneous glycoproteins named IRPS2A and IRPS3A were obtained from IsatidisRadix by systematical separation and purification. The monosaccharide composition of IRPS1A and IRPS1B was of arabinose, mannose, galactose, and glucose. IRPS2A contained galacturonic acid, glucose, galactose, and arabinose. While IRPS3A contained mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose. The amino acid compositions of IRPS2A and IRPS3A were 14 kinds of amino acid residues including Asp, Glu, Ser, His, Gly, Thr, Arg, Ala, Cys, Val, Phe, Iso, Leu, and Lys. Besides all, IRPS2A also contained Tyr. Conclusion: This strategy can be used for the isolation and purification of homogeneouspolysaccharides/glycoproteins from IsatidisRadixwhich provides a possible support for the elucidation of the structure and the pharmacologic action of IsatidisRadixpolysaccharides (glycoproteins).

Application of macroporous adsorptive resins in separation and purification of natural products / 中草药

Macroporous adsorptive resin is a class of organic polymer adsorbent which has good adsorption properties, developed in the late 1960s. The adsorption technology of macroporous adsorptive resin has more and more important application in the field of medicine. Recently, it has been widely used in the extraction, separation, and purification processes of the active ingredients of herbal. This paper reviewed the applications of macroporous adsorptive resin in the isolation and purification of the active monomer components from Chinese materia medica and the mixed and combined application of resin, so as to provide the references for the use of macroporous adsorptive resin in the separation of natural products.