Identification of involatile chemical components from Moutai-flavored distiller’s grains / 中国药科大学学报

@#In order to study the involatile chemical components in Moutai-flavored distiller’s grains, the Moutai-flavored distiller’s grains were extracted with 75% ethanol, followed by extraction with petroleum ether, ethyl acetate, and n-butanol. Silica gel, ODS, sephadex LH-20, and preparative HPLC were used to separate and identify the petroleum ether and ethyl acetate layers.ESI-MS and NMR were used to identify the compounds, which were respectively identified as pentadecanoic acid (1), palmitic acid (2), trans-2-decenoic acid (3), n-nonyl octadecanoate (4), ethyl octadecanoate (5), ethyl linoleate (6), luric acid (7), 1, 3-dicaprylyl-2-linoleylglycerin (8), cyclic (phenylalanine-proline) (9), cyclo-(proline-leucine) (10), 3, 6-bis-(2-methylpropyl)-2,5-dione piperazine (11), 4-hydroxyphenethyl alcohol (12), 2,4-dihydroxybenzoic acid (13), stigmasterol (14), 2-furancarboxylic acid (15), valine (16), L-alanine acyl-L-proline (17), dihydroquercetin (18), 5, 7, 3'', 4''-tetrahydroxyflavonoids (19), quercetin (20), and naringenin (21). Compounds 1-21 were isolated from distiller’s grains for the first time.

Exploration of ²⁴¹Am measurement in fecal samples / 中国辐射卫生

@#<b>Objective</b> To preliminarily study and establish a method for measurement of the transuranic nuclide ²⁴¹Am in fecal samples, and to provide technical support for internal radiation monitoring of staff. <b>Methods</b> Fecal samples were collected with a self-made stool sampler and treated with a self-made carbonization and ashing furnace. DGA resin was used to separate and purify ²⁴¹Am from fecal samples. With ²⁴³Am as the tracer, the orthogonal method was used for condition optimization. <b>Results</b> The optimum conditions for separation and purification were: the acidity of HNO₃ added into the column, 6 mol/L; column flow rate, 0.6 mL/min; and the volume of analytical solution,12 mL. The method based on inductively coupled plasma mass spectrometry showed a detection limit of 9.79×10⁻⁴ Bq for ²⁴¹Am in fecal samples, which was satisfactory and feasible. <b>Conclusion</b> This method fills the vacancy of ²⁴¹Am measurement in fecal samples to some extent, which is of practical significance for internal radiation monitoring and protection for analysts.

Structures Characteristics and Bioactivity of Polysaccharide CALB-2 from Aurantii Fructus / 中国实验方剂学杂志

Objective:To isolate and purify a polysaccharide CALB-2 fraction from Aurantii Fructus，and analyze its basic chemical structure, morphological characteristics and bioactivity. Method:A refined CALB-2 was obtained from Aurantii Fructus by hot water extraction，then separated and purified by ion exchange resin，ion exchange agarose gel and propylene dextran gel to obtain homogeneous polysaccharide CALB-2. The molecular mass of CALB-2 was determined by high performance liquid chromatography （HPLC）. Monosaccharide composition analysis of CALB-2 was conducted by methylation analysis and Smith degradation. Structural analysis and morphological characterization were conducted by infrared scanning （IR） and scanning electron microscopy （SEM） analysis. Antioxidant activity of CALB-2 was studied by using H2O2-induced cardiomyocyte oxidative damage model. Result:CALB-2 was a homogeneous polysaccharide and the molecular weight of CALB-2 was estimated to be 3.57×107 Da，which was proved to be a kind of highly branched acidic polysaccharides in IR analysis, methylation analysis and Smith degradation, mainly present in form of 1→3，4 bonds. Through SEM observations，we indicated that the molecular morphology of CALB-2 was amorphous solid. The in vitro activity test showed that CALB-2 had obvious protective effects on injury of H9c2 myocardial cells induced by H2O2. Conclusion:CALB-2 is a kind of homogeneous polysaccharide extracted from Aurantii Fructus， with an anti-cardiomyocyte oxidative damage effect, laying a theoretical foundation for further study of Aurantii Fructus polysaccharides．

Expression, purification and NETs degradation activity of human DNase I / 中国药科大学学报

@#In order to study the effect of human deoxyribonuclease I(DNase I)on neutrophil extracellular traps(NETs)degradation, genetic engineering bacteria E. coli Rosetta(DE3)/pET32a-His-DNase I was constructed. The fusion protein His-DNase I was induced by lactose, and purified by Ni-affinity chromatography. Mouse neutrophils were extracted and stimulated with phorbol-12-myristate-13-acetate(PMA)to form NETs. The degradation activity of the fusion protein on NETs was detected by SytoxGreen and fluorescence microscopy. The results showed that the purified His-DNase I had high nuclease activity. This study provided the research foundation for further exploration of the clinical application of DNase I.

Study on Separation and Purification of Total Flavonoids from Polygonum hydropiper Linn. by Macroporous Resin / 中国中医药信息杂志

Objective To study the separation and purification of total flavonoids from Polygonum hydropiper Linn. by macroporous adsorption resin; To investigate the effects of various factors in the process of static and dynamic adsorption on effects of adsorption and desorption to determine the optimum capability. Methods The total flavonoids from Polygonum hydropiper Linn. was extracted; the contents of flavonoids of ethyl acetate part (FEA) and flavonoids of n-butanol part (FNB) were detected; the adsorption experiment of FEA and FNB was conducted by macroporous adsorption resin. D101, AB-8, DM130, and XDA-8 macroporous resin were used to optimize the separation and purification process. Results The XDA-8 macroporous resin was selected to enrich total flavonoids from Polygonum hydropiper Linn. The optimum process was: adjusting the pH value of FEA flavonoids to 6, the concentration of sample was 750 g/mL, eluting with 75% ethanol to elute 5 bed volumes at a flow rate of 1 bed per hour; adjusting the pH value of FNB flavonoids to 6, the concentration of sample was 1 mg/mL, eluting with 60% ethanol to elute 5 bed volumes at a flow rate of 1 bed per hour. Conclusion XDA-8 macroporous adsorption resin is helpful to separate and purify the total flavonoids of Polygonum hydropiper Linn.by the best process.

Studies on preparation and properties of Staphylococcus nuclease / 中国药科大学学报

In order to study the relationship between Staphylococcal nuclease(SNase) and diabetes mellitus,genetic engineering bacteria E.coli BL21/pET28a-His-SNase was constructed,the expression of soluble extracellular protein SNase was induced and the a preliminary research was made on it.An expression vector pET28a-His-SNase plasmid containing the His-SNase gene was constructed and transformed into E.coli BL21 (DE3) competent cells.The protein was induced by lactose and purified by ultrasound destruction and Ni-affinity chromatography,respectively.It was then analyzed by SDS-PAGE and Western blot.The enzymatic properties for SNase has been preliminary studied as well.Results indicated that the purity of the correctly expressed fusion protein HisSNase was over 85％.SNase showed good activity within a wide range of pH and good heat resistance.This experiment might be a foundation work for the further study on the relationship between SNase and with diabetes.

Separation and Purification of Haemocoagulase from Agkistrodon Blomhoffii in Changbai Mountain / 中国药师

Objective:To establish a method for the separation and purification of haemocoagulase from agkistrodon blomhoffii in Changbai Mountain. Methods: An enzyme component with clotting activity was isolated from the venin of agkistrodon blomhoffii in Changbai Mountain by gel filtration, anion-exchange chromatography and heparin-Sepharose affinity chromatography. SDS-Page and RP-HPLC were used to determine its purity, SDS-Page was applied to study its action mode with bovine fibrinogen, HPSEC was used to determine the molecular weight, an IEF method was employed to detect its isoelectric point, and Lowry method was used for the deter-mination of protein concentration. Results:One haemocoagulase was purified from agkistrodon in Changbai Mountain. SDS-Page dis-played one band, and HPLC showed one single chromatographic peak. The haemocoagulase acted only on α chain of fibrinogen. Its molecular weight was 32. 2kD with isoelectric point of 5. 21. The enzyme had clotting activity in vitro. Conclusion:The method can be used for the separation and purification of haemocoagulase from agkistrodon blomhoffii in Changbai Mountain.

Purification of total flavonoids extracted from Astragali Complanati Semen using D-101 macroporous adsorption resin / 中草药

Objective: To optimize the purification technology of total flavonoids from Astragali Companati Semen (ACS) by macroporous adsorption resin. Methods: The process parameters of water decoction extraction of ACS and purification of total flavonoids extracted from ACS by D-101 macroporous adsorption resin were optimized by orthogonal experiment design method. Results: The optimal extraction process were as follows: water decoction extract for three times, each time 0.5 h, water addition of 10, 6, and 6 times amount of the medicine respectively. The optimized technology conditions of D-101 macroporous adsorption resin were as follows: The flavonoids concentration of sample liquid of ACS was about 1.0 mg/mL, the diameter ratio was 1∶5, the sample flow rate was 1 BV/h, the rate of sample weight was 0.6 g/mL (herbs/resin); The velocity of water elution was 1 BV/h and its elution volume was 4 BV; The elution concentration, elution velocity, and elution volume were 70%, 1 BV/h, and 5 BV, respectively. The content paste rate of made ACS of total flavonoids was 2.65%, the mass fraction of total flavonoids was 56.24%, and the process transfer rate was 68.37%. Conclusion: The method is simple and feasible for purification of total flavonoids extracted from ACS.

Bioinformatic analysis and characterization of myxobacteria laccase-like multicopper oxidases / 生物工程学报

Laccase is a widely-used environment-friendly copper-containing oxidase found in many plants, insects and fungi. Recently, more and more laccases are also found in bacteria. Myxobacteria are an important bacteria resource. However, myxobacteria are much more difficult to isolate and purify than other bacteria. We used bioinformatic approach to screen myxobacteria proteomes available in NCBI. Based on conserved sequences of four copper binding sites in multicopper oxidase, 30 potential laccase sequences were obtained. Among them, nine genes were synthesized and expressed in Escherichia coli BL21 (DE3). Seven proteins showed laccase activity when tested with traditional laccase substrates. One protein, named rSC-2, was chosen for further research because it exhibited the highest activity towards 2,6-dimethyl phenol (DMP). The molecular weight of rSC-2 was 57 kDa. Its specific activity to DMP was 0.27 U/mg. The optimal temperature and the optimal pH were 60 ℃ and 7.0, respectively. About 50% of the original activity was retained after incubation at 60 ℃ and pH 7.0-8.0 for 1 h. Metals showed different effects on rSC-2. rSC-2 activity was enhanced by several metalsat concentration of 1 mmol/L, such as Ca²⁺ and Mn²⁺. With a higher concentration of 5 mmol/L, the activity of rSC-2 was apparently inhibited. This is the first report of bioinformatics screening myxobacteria laccases in combination with expression in E. coli.

Polar Chemical Constituents from Prunella vulgaris L. and Their Anti-human Breast Tumor Activities / 中国药学杂志

OBJECTIVE: To study the polar chemical constituents from Prunella vulgaris L. METHODS: Silica gel, reverse-phase octadecylsilyl(ODS), Sephadex LH-20 chromatographic methods, MCI and HPLC were applied to isolate and purify compounds. MS and NMR methods were used to determine the structures of the compounds. Furthermore, the cytotoxicity of these chemical components for MCF-7, MDA-MB-231 and MCF-10A cell lines was measured by MTT method. RESULTS: A total of 12 compounds were isolated from the fruits of P. vulgaris and their structures were identified as methyl 3,4,α-trihydroxypropionate(1), danshensu(2), methyl rosmarinate(3),3,4-dihydroxybenzoic acid(4), quercetin-3-O-glycopyranoside(5), hyperoside(6), 2α,3α,19α,24-tetrahydroxylurs-12-en-28-oic acid(7), 2α, 3α, 24-trihydroxyolean-12-en-28-oic acid(8), cytidine(9), daucosterol(10), (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11,14-triol(11), and 2α,3α,24-trihydroxyolean-12,20(30)-dien-28-oic acid(12). The results of antitumor assay indicated that compound 2,3,5 and 6 significantly inhibited the activity of MCF-7, compound 3 could inhibit the activity of MDA-MB-231, but all of them also significantly inhibited the activity of normal cell lines MCF-10A. CONCLUSION: Compounds 9 and 11 are isolated from the genus of Prunella L. for the first time. Some chemical constituents form Prunella L. show certain anti-breast cancer activity.

Optimal separation and purification of extract in Sanao Sustained-release Tablets with macroporous resins by multi index comprehensive score / 中草药

Objective: To explore the optimal separation and purification of extract in Sanao Sustained-release Tablets. Methods: Taking the adsorption ratio and desorption ratio of ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, and glycyrrhizinate as evaluation indexes to optimize the purification process of Sanao Sustained-release Tablets by multi index comprehensive score. Results: Macroporous resin HPD 300 had the best adsorption and desorption properties. In the course of adsorption, the optimum concentration of the sample liquid was 1.67 g crude drugs of per milliliter, the resin column size ratio was 1: 7, the concentration of sample solution was 0.6 g/mL crude drug, the sample flow rate was 4.0 BV/h. In the course of elution, 2 BV deionized water was used and the resin column chromatography was eluted with 5 BV of 70% EtOH by flow rate of 3 BV/h. Conclusion: Macroporous resin HPD 300 is suitable to separate and purify the extract from Sanao Sustained-release Tablets.

Separation and purification of emodin-8-O-β-D-glucopyranoside from Polygoni Cuspidati Rhizoma et Radix by column and high-speed counter-current chromatography / 中草药

Objective: To develop an effective and rapid method for the preparation of emodin-8-O-β-D-glucopyranoside (EG) reference substance from Cuspidati Rhizoma et Radix (PCRR). Methods: The target fraction was isolated by macroporous resin column chromatography and ODS column chromatography, and then the target compound was purified by high-speed counter-current chromatography (HSCCC). The structure was identified by ESI-MS and NMR spectral techniques, and its purity was determined by HPLC analysis using the main component self-comparison with no correction factor method. Results: A solvent system that consisted of dichloromethane-ethyl acetate-methanol-water (8:1:6:5) was applied to the separation based on assessment using HPLC partition coefficient method. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase. The reference substance of EG was prepared, and its purity was up to 98% in line with the standard of quantitative analysis. Conclusion: The method is suitable for the preparation of EG from PCRR, which provides a basis for the quality control of natural product and their preparations.

Seperation and purification of daidzein by agar gel microspheres bonded β-cyclodextrin study on and its chromatographic mechanism / 中草药

Objective: Daidzein was efficiently purified by agar gel microspheres bonded β-cyclodextrin (AG-β-CD). Methods: Using agar as raw material, after emulsification, crosslinking, and bonding β-CD as functional group, AG-β-CD was synthesized for the purification of daidzein, and the purification process was determined and proved with mobile phase, flow rate, and loading capacity of microspheres. The structure of daidzein was identified by MS and NMR, AG-β-CD was chromatographically evaluated with daidzein, EGCG, and puerarin as the following tripartition such as difference of retention behavior on C18 reversed phase column chromatography, molecular simulation by autoDOCK4.0, and retention time curves on AG-β-CD with different contents of acetonitrile. Results: The main component of soybean isoflavone was daidzein (57.14%). The loading quantity of AG-β-CD was 1.33 mg/mL, flow rate was 2 BV/h, eluted by 2 BV of 20% ethanol, 1.33 BV of 40% ethanol, and 6-7 BV of 70% ethanol, the content was ≥ 95%, purity of daidzein (96.98%) was obtained with 97.86% yield. Chromatographic mechanism research showed that AG-β-CD had hydrophilic interaction chromatography and reversed-phase chromatography. Conclusion: AG-β-CD is capable of highly efficient purification of daidzein.

Study on Chemical Constituents from Petroleum Ether Fraction ofLiparis nervosa / 世界科学技术-中医药现代化

This article was aimed to study the chemical constituents of the petroleum ether fraction of Liparis nervosa.Chemicalcompoundswereisolatedandpurifiedthroughvariouschromatographytechniques.The accurate structures of chemical compounds were confirmed with spectral data and literatures. The results showed that7chemicalconstituentswereisolatedfromthepetroleumetherfractionofLiparis nervosa,whichwere moscatin (1), batatasin Ⅲ (2), bergapten (3), isoimpinellin (4), xanthotoxin (5), imperatorin (6) and β-sitosterol (7). It was concluded that chemical constituents 3-7 were isolated from this genus for the first time. And chemical constituents 1-7 were isolated from this plant for the first time.

Isolation, purification, and structural elucidation of polysaccharide CALB-1 from Aurantii Fructus and study on its immunoregulatory activity / 中草药

To separate and purify the crude polysaccharide from Aurantii Fructus to obtain CALB-1 and to study the structure of CALB-1 and immunoregulatory activity. A refined CALB-1 was obtained from F. aurantii by hot water extraction, then separated and purified by ion exchange resin and ion exchange agarose gel. The molecular weight of CALB-1 was measured by HPLC. The chemical structure and molecular morphology of CALB-1 were determined by IR, periodate oxidation, methylation analysis, scanning electron microscopy (SEM), and atomic force microscope (AFM). The immunoregulatory activity of CALB-1 was evaluated by splenocyte proliferation and mononuclear-macrophage phagocytic function in hypo-immunologic mice. CALB-1 was a homogeneous polysaccharide measured by HPLC and the molecular weight of CALB-1 was estimated to be 3.28 × 107. Chemical and spectroscopic analyses illustrated that CALB-1 was highly branched acidic polysaccharides, contained Ara, Man, and Gal as monosaccharide constitutes, and it consisted of backbone chain of 1→ and 1→4 linkages. Through SEM and AFM observations, we indicated that the molecular morphology of CALB-1 was amorphous solid. Besides, CALB-1 significantly stimulated the splenocyte proliferation in vitro and improved the K value of expurgation index and α value of phagocytic index in hypo-immunologic mice. CALB-1 is highly branched acidic polysaccharides and uniform relative molecular mass. Moreover, CALB-1 could present the certain immune regulation in vivo and in vitro. Our study provides a theoretical basis for the development and utilization of CALB-1.

The research of separation and purification technology for effective parts f rom Zhizichi decoction by macroporous absorption resina / 药学实践杂志

Objective To optimize separation and purification technology of Zhizichi decoction by macroporous resina . Methods The content of total iridoid glycosides and total isoflavones were determined by ultraviolet spectrophotometry .Tak-ing the content of total iridoid glycosides and total isoflavones as indexes ,the effect of the concentration of sample solution , medicinal herbs on the amount of sample ,concentration and volume of eluent was investigated by single factor test .Results The optimum separation and purification technology was as following :the concentration of sample solution was 0 .1 g/ml ,the volume ratio of resin to material drug was 2∶1 ,the adsorption time was 2 h ,and the sample was firstly eluted with 1 BV wa-ter ,then 6 BV 20% ethanol and 60% ethanol ,and the eluent was collected .Conclusion This optimized separation and purifi-cation technology was reasonable and stable ,and it could be extended to large-scale production applications .

Optimization of Macroporous Resin Purification Process for Total Flavonoids in Papaya / 中国药师

Objective:To optimize the macroporous resin separation process for total flavonoids in papaya. Methods:The content of total flavonoids in papaya was selected as the index, and the resin model, sample solution concentration, ratio of diameter and height, the flow rate of adsorption, type and volume of eluent, type and volume of impurity removing solvent, elution velocity and the other parameters were investigated. Results:The optimal purification process was as follows: the macroporous resin type was D-140, the sample solution concentration was 0. 1 g·ml-1 , the sample volume was 2BV, the ratio of diameter and height was 1∶9, washing the impurities with 3BV water, eluting with 3BV 10% ethanol first followed by 3BV 50% ethanol with 2BV·h-1 , and collecting 50%ethanol elution. The total flavonoids content was 52%. Conclusion:The optimized process can separate and purify the total flavonoids in papaya effectively.

Purification technology of total flavonoids in Tricyrtis maculata produced in Shaanxi province / 中草药

Objective: To optimize the purification technology of total flavonoids in Tricyrtis maculata (TFTM) produced in Shaanxi province using macroporous adsorption resin. Methods: The content of TFTM was determined with ultraviolet spectrophotometry. Taking the content of total flavonoids purified and transferred by per gram of resin as the indicator, the processes of dynamic adsorption-desorption and static adsorption-desorption were observed, and five macroporous resins, AB-8, D-101, HPD-450, HPD-600, and HPD-700 were optimized. The parallel invastigations were carried out to study the effects of the concentration of extracted liquor, eluent dosage, ratio of diameter to height, and pH value of liquor on the purification technology of macroporous adsorption resin, and to determine the best technological condition. The verification was performed on the optimized extraction conditions. Results: The ratio of diameter to height was 1:5, the liquid concentration was 1.2 g/mL, the pH value of liquor was 6.47, the raw material to resin was 1:3, the concentration of eluent was 40%, the sample loading velocity was 1 BV/h, the eluant velocity was 1 BV/h, and the elution volume was 2 BV. Conclusion: The technology has the scientific rationality and could be used to enrich TFTM efffectly.

Production and properties of matallothionein in Cordyceps kyusyuensis under Zn2+ stress / 中草药

Objective: To explore the production and properties of matallothionein (MT) in Cordyceps kyusyuensis under the zinc ion (Zn2+) stress. Methods: By adding ZnSO4 to the culture, the production regularity of MT in C. kyusyuensis under Zn2+ stress was explored. Ultrasonic cell disruption, crude extraction, and gel column chromatography were employed to isolate and purify the MT. The methods of Coomassie brilliant blue, electron spray mass spectrometry (ESMS), Ellman's reagent colorimetry, atomic flame absorption spectrometry, silver saturation method with atomic absorption spectrometry analysis, and amino acid analyzer were used to determine the purity, molecular weight, sulfhydryl content, zinc content, MT content, and amino acid composition, respectively. In addition, the anti-oxidative activity was identified by scavenging capacity of hydroxyl radicals (HO·), DPPH radicals (DPPH·), and superoxide anion radicals (O2·̄). Results: The contents of Zn and MT in mycelia increased strikingly with the increase of Zn2+ concentration (0-15 g/L) in the culture medium. In 10 L fermenter, MT achieved the maximum yield up to 14.335 mg/g mycelium (wet weight) with Zn concentration of 15 g/L in 56 h, and the biomass reached 16.921 g/L. The C. kyusyuensis Zn-MT was obtained with Sephadex G-50 and Sephadex G-25 gel filtration, and dried by vacuum freeze-drying. The molecular weight of the protein was 6 750, one molecule Zn-MT binded six zinc atoms and contained 56 molecules of amino acid, which contained 20 molecules of cysteine and one molecule of histidine. The eliminating action of C. kyusyuensis Zn-MT for HO·, DPPH·, and O2·̄ was better than that of glutathione, and the eliminating action was different in O2·̄ > DPPH· > HO·. The concentration of half clearance rate was 71.49, 80.58, and 100.65 mg/L. Conclusion: According to ultraviolet scan pattern analysis, protein column chromatogram analysis, physicochemical properties, and anti-oxidative activity of C. kyusyuensis Zn-MT, it is similar with the characteristics of standard animal MT, and a certain concentration of zinc sulfate liquid fermentation of C. kyusyuensis could induce the synthesis of Zn-MT, which could provide the reliable theoretical basis for the industrial production.

Study on Separation and Purification of Total Flavones from Striga asiatica (L.) O. Ktze. With Polyamide / 中药新药与临床药理

Objective To study the technique condition for separating and purifying total flavones from Striga asiatica (L.) O. Ktze. by Polyamide. Methods Total flavones content, in the sample solution of Striga asiatica (L.)O. Ktze. , detected by UV spectrophotometry, is used as the index. Some technological parameters are observed by single factor observation. Results Polyamide has good absorbing effect on total flavones of Striga asiatica (L.) O. Ktze. The liquid concentration of its absorbing and separating technological condition is 1.12～2. 24 mg/mL and at the absorbing speed of 2BV/h. The elution effect of 95 % alcohol of 250 mL is the best. Conclusion This method is simple and feasi-ble, fit for separating and purifying total falvones from Striga asiatica (L.) O. Ktze.