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In the field of research and development of biomedicine and drugs, the technologies about the extraction, isolation and purification of proteins play an important role. In recent years, the research on proteins has achieved significant progress, and new understanding of various properties of proteins has been achieved. At the same time, the development of physical, chemical and engineering technologies has also promoted the continuous improvement and promotion of extraction and isolation methodology. This paper reviews the technologies of extraction, isolation and purification according to the sources and classification of protein products.
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OBJECTIVE: To study the chemical constituents and their proliferation activity on rat osteoblasts of Semen Sojae Praeparatum. METHODS: The compounds were isolated by chromatography on silica gel column, MCI and sephadex LH-20 column. Their structures were elucidated by spectral analysis. The proliferation test was performed by using rat osteoblasts with MTT assays in vitro. RESULTS: Twelve compounds were obtained and identified as daidzin(1), daidzein(2), genistein(3), genistin(4), glycitein(5), glycitin(6), apigenin(7), β-sitosterol(8), stigmasterol(9), campesterol(10), syringaldehyde(11), syringic acid(12), respectively. CONCLUSION: Compounds 5-11 are isolated from Semen Sojae Praeparatum for the first time. The five isoflavones show proliferation activity on rat osteoblasts, and aglycones proved to show better activity than glycosides.
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OBJECTIVE: To study the chemical constituents of the total flavonoids from Sophora flavescens and establish a method for simultaneous determination of seven compounds. METHODS: The compounds were isolated by chromatography on silica gel and ODS column and their structures were elucidated by spectroscopic analysis. The samples were analyzed on a Dikma C18 column (4.6 mm × 250 mm, 5 μm) ; gradient elution was performed using mobile phase composed of methanol (A) and water (B); the detection was carried out using a photodiode array detector at 280 nm. RESULTS: Seven compounds were isolated and their structures were identified as kuratidine (1), sophoraflavanone G (2), kurarinone (3), isoanhydroicaritin (4), isoxanthohumol (5), formononetin (6), and trifolirhizin (7). The calibration curve was linear within 8.70-87.00, 44.25-442.50, 128.10-1 281.00, 9.40-94.00, 48.40-484.00, 14.20-142.00, and 25.70-257.00 μg·mL-1 for kuraridine, sophoraflavanone G, kurarinone, isoanhydroicaritin, isoxanthohumol, formononetin, and trifolirhizin, respectively (r > 0.999 0), and the extraction recoveries varied from 95% to 105%. CONCLUSION: The main chemical components contributing to antibacterial activity of total flavonoids may be sophoraflavanone G, kurarinone, and isoxanthohumol. The method is simple, rapid, accurate, and can be used simultaneously to determine the contents of the seven active ingredients.
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Objective To study the effect of Qige San ethyl acetate extract on the proliferation of human esophageal carcinoma Eca109 cells and to explore its possible mechanism. Methods Inhibitory effects of Qige San ethyl acetate extract on the proliferation of esophageal carcinoma Eca109 cells were detected by MTT assay, cell apoptosis was detected by flow cytometer, and the expression of protein STAT3, Bcl-2 and Caspase 9 was detected by Western blotting method. Results In the range of 10~100 μg/mL, Qige San ethyl acetate extract inhibited the proliferation of Eca109 cells effectively (P<0.05) . Compared with the control group, Qige San ethyl acetate extract in the concentrations of 1.47, 33.26, 75.52 μg/mL significantly increased the apoptotic rate of Eca109 cells within 48h ( P<0.01). And Western blotting results showed that the ex pression levels of STAT3 and Bcl-2 were reduced, and Caspase 9 was increased with the increase of drug concentration. Conclusion Qige San ethyl acetate extract could significantly inhibit the proliferation of Eca109 cells and induce cell apoptosis, and its mechanism is probably associated with the inhibition of signal transducers and activators of transcription 3 (STAT3) signaling pathway.
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Objective To study the optimum decoloration technological conditions of crude polysaccharides from Cordyceps militaris Link. (PCM) by macroporous resin. Methods With the decolorization rate and retention rate of PCM as indexes, static adsorption factor experiment was used for the optimization of seven kinds of macroporous resin. And then the optimum conditions of decolorization of PCM were investigated by the static/dynamaic adsorption single-factor experiments. Results D941 macroporous resin is the best. The optimum decoloration technological conditions were as follows:initial concentration of the sample was 50 mg/mL, pH value was 5.5, operating temperature was 45℃, the flow rate of dynamaic adsorption was 2.5 BV/h, and the sample solution volume was 5 bed volume per hour. Under the optimal conditions, decolorization rate and retention rate of PCM were ( 93.9 ± 3.4) % and (91.7 ± 2.2) %, respectively. Conclusion The resin D941 is suitable for the decoloration of PCM. The optimized method is feasible and reliable, having the prospects for popularizing.
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Objective To compare the advantages and disadvantages of the four DNA extraction methods according to the endophytic diversity in the roots, stems, and leaves of mulberry analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) , and by taking the yield, purity and PCR amplification as indexes. Methods Four common methods, i.e., cetyltrimethyl ammonium bromide ( CTAB) , sterile phosphate buffered saline (SPBS) vibration, liquid nitrogen grinding (LNG) , and KIT methods, were used to extract the total DNA from different tissues of mulberry, and then were compared based on the diversity analysis results for endophyte by PCR-DGGE. Results From the roots and stems of mulberry, we got the highest concentration of DNA by LNG extraction method, and got the lowest concentration by SPBS extraction method. But for the leaves of mulberry, the results of the four extraction methods were completely opposite to those for the roots and stems. For different tissues of mulberry, the purity of DNA extracted by KIT method was the best. According to the endophytic bacteria diversity analyzed by 16S rDNA PCR-DGGE, the appropriate method for extraction of DNA was LNG or CTAB, but was not KIT. And according to the results of endophytic fungi diversity analyzed by ITS PCR-DGGE, the best extraction method was KIT, and the unsuited methods varied from the tissues of mulberry. Conclusion The optimum DNA extraction method for mulberry varies from the tissues of mulberry and endophtic bacteria.
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Objective To screen the active components of Bushen Huoxue Decoction ( BSHXD) involved in promoting the proliferation of bone marrow mesenchymal stem cells ( MSCs). Methods BSHXD and its subdivisions were extracted with petroleum ether, ethyl acetate, water-free ethanol and water respectively. MSCs were isolated and cultured by the bone marrow adherent method. At the third passage, MSCs were identified by the specific surface markers with immunofluorescence, and their osteogenic and adipogenic differentiation were tested by alizarin red staining and oil red “O” staining. After treated with the extracts of BSHXD and its subdivisions at gradient concentrations for 24 hours, cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay for the screening of active components and optimal concentration. MTT assay was used to describe the growth curve of MSCs treated with the most effective components, and cell cycle was analyzed by flow cytometry. Results Compared with the blank control group, the extracts of BSHXD and its subdivisions could protect MSCs from death to various degrees. Of all the extracts, the ethyl acetate extract of Bushen Division ( BSD) , ethyl acetate extract of BSHXD, ethyl acetate extract of Huoxue Division ( HXD) had the strongest effect, and the effect was dose-dependent, 100 μg/mL being the optimal active concentration while having no any cytotoxic reaction. The results of MTT assay revealed that BSD extracts promoted the proliferation of MSCs significantly and was the most effective component, and then came BSHXD. The results of flow cytometry indicated that BSD extract had the most strongest effect on increasing the amount of MSCs at proliferative phase, and then came BSHXD. Conclusion BSD ethyl acetate extract is the active component of BSHXD for promoting the proliferation of MSCs, showing an effect on increasing the proportion of MSCs at proliferative phase.
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Objective To evaluate the inhibitory activity of Herba Taraxaci extract on Escherichia coli DH5α (E. coli DH5α) and to investigate proteomic response of E. coli. Methods Medicinal powder of Herba Taraxaci was extracted with the solvents of different polarity ( n-hexane, ethyl acetate, distilled water) , and then the obtained 8 different extracts were subjected to thin layer chromatography ( TLC) analysis. Microdilution method was performed to detect the minimum inhibitory concentration ( MIC) of different extracts and the growth curves were described. The protein expression profiles of E . coli treated with the extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis ( SDS-PAGE) and two dimensional electrophoresis (2-DE) . Results Water decoction of Herba Taraxaci could obviously suppress the growth of E. coli with a MIC of 1.95 mg/mL. The different extractions exhibited no antibacterial activity except ethyl acetate phase 3 with a MIC of 0.13 mg/mL, which was equal to 19.23 mg/mL of crude drugs. The results of TLC analysis showed that chlorogenic acid was undetectable in n-hexane extract and ethyl acetate phase 1 extract, and ethyl acetate phase 2 and 3 extracts showed obviously increased spots. The results of SDS-PAGE and 2-DE showed that water decoction of Herba Taraxaci had inhibitory effect on the expression of functional protein. The results of 2-DE showed that after treatment with ethyl acetate phase 3 at the concentration of 2 × MIC for 21 hours, the amount of protein spots were 92 less than those of the blank control group, the spots of E. coli DH5α soluble protein with expression amount down-regulated doubly were 24, and those with expression amount up-regulated doubly were 19. Ethyl acetate phase 3 extract had an effect on down-regulating the protein expression of E. coli DH5α soluble protein pH3-10, and water decoction of Herba Taraxaci had inhibitory effect on E. coli DH5αprotein expression. Conclusion Herba Taraxaci has significant antibacterial activity on E. coli DH5α, and the water-soluble fraction of chlorogenic acid and caffeic acid might be the active components. The possible antibacterial mechanism may be related with the regulation of bacterial protein expression.
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Objective To improve the detecting sensitivity of serum specific IgE (sIgE) by improving the quality of coated antigen in shrimp. Methods The extracts from shrimp protein was prepared. Western blot assay was used to identify the major allergenic protein components. The protein components>55 ku were separated by Sephadex gel chromatography. SDS-PAGE technology was used to analyze proteins. Samples of shrimp protein and proteins>55 ku were used as the coat-ing antigen to coat 96 microplate respectively. Western blot assay and ELISA were used to evaluate preliminary sensitivity of the purified antigen for detecting sIgE. Results Immunoblot experiments showed that the protein>55 ku was the main aller-genic protein component of shrimp. Those >55 ku proteins were separated successfully by Sephadex gel chromatography, showing 10 identifiable bands in SDS-PAGE. Dot-pot immunoassay showed that proteins>55 ku used as coated antigens could improve the spots density of the weak serum. Meanwhile, the result of ELISA showed that sIgE detection value in-creased 92.9%in patients with shrimp allergy after coating effective antigens. Conclusion The detecting sensitivity of sIgE can be improved by using effective protein components of shrimp as coated antigens.
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Clinical islet transplantation is currently being explored as a treatment for its superiorities of lesser in vasive,lower risk,avoiding or preventing chronic complications.Because of the difficulty in isolation,purification and transplantation,the yield of islet is lower than needed;shortage of donor,difficulty in obtaining adequate islet cells for sustaining B cell mass and function over time,and immune rejection reactions are the hurdels to widespread application of islet transplantation.
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Objective To explore a simple, effective and stable method for the isolation and purification of Kupffer cells from rat liver, enabling further study on the structure and function of these cells in vitro. Methods After laparotomy, a catheter was inserted into the portal vein and secured with artery clamp. Then, the rat liver was perfused and digested with solution Ⅰ and solution Ⅱ containing 0.05% collagenase Ⅳ respectively. The cell suspension was centrifuged with isopycnic sedimentation in a two-step Percoll gradient to harvest Kupffer cells. The isolated Kupffer cells were purified by selective adherence after 30 min of cultivation, and identified by evaluation of phagocytosis of India ink and peroxidase staining with DAB through light and electron microscopy. Results It was verified that the viability of isolated Kupfffer cells was more than 90% through Trypan blue staining. Those Kupffer cells could attach to plastic quickly and phagocytose ink, and had the appearance of "fried eggs" in positive peroxidase staining with a purity of 95%. Under the light microscopy, the appearance of newly isolated Kupffer cells was round with uniform shape and size. After two days of culture, Kupffer cells appeared to distend with irregular or stellate shape. The typical features were observed in the transmission electron micrographs. There were numerous pseudopods and occasional cup-like indentations in the cell membrane of Kupffer cells. The cytoplasm contained numerous types of lysosomes and other phagocytotic vesicles. Conclusion The method for isolating and culturing Kupffer cells in this study is effective and stable, and the biological characters are preserved in the cultured cells.
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A new method based on adherence of cellulolytic bacteria to insoluble cellulose for isolation and purification of thermophilic cellulolytic anaerobes was reported, in which Hungate anaerobic operating techniques were used to roll tubes with insoluble cellulose powder as substrate.
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PFW was isolated from fruit body of Pleurotus ferulae by using hot water Proteins were removed from PFW with Sevag method, after DEAE cellulose chromatography, PF 1 and PF 2 were isolated from PFW The weight was determined by using multiangle laser photometer combined with GPC; the results showed: PF 1 contains two fractions, the weights (Mw) are 7 875?10 5 and 3 245?10 4, the Mw of PF 2 is 3 187?10 6 The components and structure of polysaccharides were analyzed by IR and GC The polysaccharides PF 1 consists of rhamnose, glucose, galactose, mannose; PF 2 consists of rhamnose, xylose, glucose, galactose, mannose; PF 1 contains ? glucoside and mannoside, PF 2 contains ? glucoside It was found polysaccharides have antitumor activity on the implanted Sarcoma 180 in mice