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OBJECTIVE: To study immunological activity and chemical constituents of fermentation product of the roots of Cynanchum auriculatum Royle ex Wight. METHODS: The effect of fermentation product of the roots of C. auriculatum on the spleen index and thymus index of the immunocompromised rat was measured by weighing and phagocytosis of chicken red blood cells, silicagel, Sephadex LH-20 and recrystallization were used for isolation and purification.The structures are identified by physicochemical properties and spectral data analysis, and the separated compounds were identified by HPLC. RESULTS: The fermentation product of the roots of Cynanchum auriculatum Royle ex Wight auricular leaf can improve the spleen and thymus index of mice and is superior to the alcohol extract of Cynanchum auriculatum Royle ex Wight. The results of phagocytosis of chicken red blood cells by peritoneal macrophages in mice are positive. Eleven compounds Compounds were isolated from the fermentation product in the roots of Cynanchum auriculatum Royle ex Wight, which were identified as 2,4-dihydroxyacetophenon(1), 2,5-dihydroxyacetophenone(2), baishouwubenxophenone(3), caudatin(4), kidjolanin(5), periplogenin(6),gallic acid(7),oleanolic acid(8), deacylmetaplexigenin(9),sorbitol(10) and linoleic acid(11). CONCLUSION: The fermentation product of the roots of Cynanchum auriculatum Royle ex Wight have the effect of improving immune function.Compounds 7,10,11 are the substances produced during the fermentation process.
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Objective:To establish a method for determining the immunological activity of ribonucleic acid. Methods: Leucocyte adherence inhibition test ( LAI) was applied, and the important parameters of LAI including the mouse strain, drug concentration, treatment time, content of buffer solution and cell density were researched. The immunological activity of RNAⅠ, Ⅱand Ⅲ was re-spectively determined by the method. Results:Stable and reliable parameters were obtained: the sample concentration was 10 mg· ml-1 , the treatment time was 2 hours, Ca2+ and Mg2+ were necessary for the buffer solution, and the cell density was about 4 × 107 cell·ml-1 . The strain of mouse showed no effect on the results. As a result, the determination method for immunological activity was established. Using the method, the immunological activity of RNA Ⅰ,Ⅱand Ⅲ was determined 3 times, and the results met the re-quirements with RSD below 20%. Conclusion:The method is suitable for determining the immunological activity of RNA.
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Objectives: To investigate the anti-inflammatory components from the leaves of Liriodendron chinensis. Methods: The 95% alcohol extract from the leaves of L. chinensis was subjected to column chromatography, and the structures of purified compounds were determined by spectral methods. The bioassay was performed through the inhibitory effects on the inflammatory cells activated by lipopolysaccharide (LPS). Results: Nine compounds were isolated, including octacosanoic acid (1), stearic acid (2), (2 R)-2-hydroxy-N-[(2 S,3 S,4 R,8 E)-1,3,4-tri-hydroxyicos-8-en-2-yl]tetracosanamide (3), (2 R)-2-hydroxy-N-[(2 S,3 S,4 R,8 E)-1-O-ß-D-glucopyranosyloxy-3,4-dihydroxyoctadec-8-en-2-yl]eicosanamide (4), (2 R)-2-hydroxy-N-[(2 S,3 S,4 R,8 E)-1-O-ß-Dglucopyranosyloxy-3,4-dihydroxyoctadec-8-en-2-yl]hexadecanamide (5), dicentrinone (6), liriodenine (7), daucosterol (8), and liriodendritol (9) and among which five compounds could significantly lower the content of nitric oxide (NO) from peritoneal macrophages of rats induced by LPS and reduce the splenic lymphocyte proliferation in mice. This is the first report on the occurrence of ceramides and dicentrinone in the plants of Liriodendron Linn. Conclusion: The five compounds are likely to be anti-inflammatory compounds concerning to their pronounced inhibitory action on the activated inflammatory cells. This assessment might provide a basis for searching the potent active compounds used for the treatment of inflammation.
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Objective: To optimize the extraction process for polysaccharides from the stems of Acanthopanax gracilistylus by smashing tissue extraction (STE) and to further investigate their cytotoxicity and immunological activities. Methods: Extraction process for the polysaccharides from the stems of A. gracilistylus was optimized by using the spherical symmetrical design test, and three factors were considered (material-liquid ratio, extraction temperature, and extraction time). The test compared STE with two traditional extraction methods, reflux extraction and ultrasonic extraction. The bioassay tests of cytotoxicity and immunological activities on polysaccharides extracted were studied in vitro. Results: The optimal extraction conditions for polysaccharides from the stems of A. gracilistylus were as follows: material-liquid ratio was 1:13.2, the extraction temperature was 80°C, and the extraction time was 420 s. With the best extraction conditions, the yield of polysaccharides from the stems of A. gracilistylus was 0.78%. The yield by STE was higher than those by reflux extraction and ultrasonic extraction in this research. In addition, the bioassay results showed the polysaccharides had no significant toxicity on RAW 264.7 cells at the dose of 10-20 μg/mL and facilitated the secretion of nitric oxide (NO) in the cells supernatant at the dose of 10-40 μg/mL in vitro. Conclusion: These results establish a steady and convenient extraction technology for polysaccharides from the stems of A. gracilistylus using STE method, indicating the polysaccharides have immunological activity to some extent, which provides a theoretical basis for the reasonable development of A. gracilistylus resources.
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Aim: To explore the relationship between structure and immunological activity of marine polysaccha-ride YCP,the physicochemical property and immunological activity of the YCP-derived fragment were studied.Methods: YCP was hydrolyzed by α-amylase from human saliva.The hydrolysate was purified to obtain an polysaccharide fragment by gel filtration chromatography.The physicochemical properties of this YCP-derived fragment was characterized by HPLC,FT-IR and TLC.In addition,changes of phagocytic activity,production of reactive nitrogen and macrophage binding were investigated.Results: The relative molecular weight of YCP-de-rived fragment was approximately 6.6 × 10~3.The monosaccharide composition and FT-IR of the YCP-derived frag-ment were identical to YCP.No significant effect of the YCP-derived fragment on NO production and murine mac-rophage phagocyte were observed.And this fragment was not able to compete the binding between YCP and mac-rophages.Conclusion: The remarkable decrease of immunological activity of YCP-derived fragments degraded byα-amylase of human saliva suggests that the complete structure and high molecule weight of YCP are essential for its immuno-modulatory activity.
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Objective To get recombinant F1 antigen (rF1) and to construct the detection dipstick of plague antibody. Methods The cafl gene removing the signal peptide coding sequence was cloned into plasmid pET32a ( +) by double-digested sites of BamHI and Not I. Recombinant plasmid caf1-pET32a(+) was transformed into BL21 (DE3) and the rFl was expressed. Expression products were purified by affinity chromatography. Dual detection dipstick of plague antibody was constructed with purified rF1 and natural F1, and evaluated with 528 human serum samples of Zhejiang province. Results The fusion protein rF1 of 35.5 KD was expressed by BL21 strains containing caf1-pET32a( + ). The sensitivity of rF1 showed equivalent to or higher than the natural Fl antigen in detecting plague antibody. It seemed that there was a better consistency of 97.9% (k= 0.466) when 528 human sera was detected by rF1 and natural F1. Conclusion We successfully extracted the rF1 with good immunological activity that might be used to detecting Yersinia pestis.
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Flavonoids are known to be effective scavengers of free radicals. In particular, proanthocyanidins are flavonoids that possess cardiovascular protection, antioxidative activities, and immunomodulatory activities. Here, we evaluated proanthocyanidin contents in the total polyphenolic compounds of pine needle extracts prepared by hot water, ethanol, hexane, hot water-hexane (HWH), and hot water-ethanol (HWE). Analysis of each extract indicated that the ethanol extract contained the highest proanthocyanidin concentration. The HWH and hexane extracts also contained relatively high concentrations of proanthocyanidin. On the other hand, proanthocyanidin content analyses out of the total polyphenolic compounds indicated that the HWH extract contained the highest content. These results suggest that HWH extraction is a suitable method to obtain an extract with a high level of pure proanthocyanidins and a relatively high yield. The HWH extract possessed superior activity in diverse antioxidative analyses such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferrous ion chelating (FIC), and ferric-ion reducing power (FRAP) assays. In addition, upon assessing the effects of the pine needle extracts on macrophages (Raw 264.7 cell), the HWH extract exhibited the highest activity. In this study, we discerned an efficient extraction method to achieve relatively pure proanthocyanidins from pine needles and evaluated the biological functions of the resulting extract, which could potentially be used for its efficacious components in functional food products.
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Biphenyl Compounds , Ethanol , Flavonoids , Free Radicals , Functional Food , Hand , Macrophages , Needles , Picrates , Proanthocyanidins , WaterABSTRACT
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.
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Mycobacterium tuberculosis is responsible for over 8 million cases of tuberculosis (TB) annually. Natural products may play important roles in the chemotherapy of TB. The immunological activity of Davilla elliptica chloroform extract (DECE) was evaluated in vitro by the determination of hydrogen peroxide (H2O2), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-alpha) release in peritoneal macrophages cultures. DECE was also tested for its antimycobacterial activity against M. tuberculosis using the microplate alamar blue assay. DECE (50, 150, 250 mug/ml) stimulated the production of H2O2 (from 1,79 ± 0,23 to 7,27 ± 2,54; 15,02 ± 2,86; 20,5 ± 2,1 nmols) (means ± SD), NO (from 2,64 ± 1,02 to 25,59 ± 2,29; 26,68 ± 2,41; 29,45 ± 5,87 mumols) (means ± SD) and TNF-alpha (from 2,44 ± 1,46 to 30,37 ± 8,13; 38,68 ± 1,59; 41,6 ± 0,90 units/ml) (means ± SD) in a dose-dependent manner and also showed a promising antimycobacterial activity with a minimum inhibitory concentration of 62,5 mug/ml. This plant may have therapeutic potential in the immunological and microbiological control of TB.
Subject(s)
Animals , Mice , Antibiotics, Antitubercular/pharmacology , Dilleniaceae/chemistry , Macrophages/microbiology , Mycobacterium tuberculosis/drug effects , Antibiotics, Antitubercular/isolation & purification , Hydrogen Peroxide/metabolism , Microbial Sensitivity Tests , Macrophages/metabolism , Nitric Oxide/biosynthesis , Plant Extracts/pharmacology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Objective To clone lexA gene,express and purify the repressor LexA from Pseudomonas aeruginosa(PA),prepare the polyclonal antibody against PC-1 protein in rabbits,detect immunological activity of LexA protein.Methods The genomic DNA was extracted from the PAO1,the gene fragment encoding the mature LexA was amplied by PCR.It was linked the vector pET32a(+)and expressed in the E.coli BL21(DE3).The expressed protein was purified by two steps of Ni2+ chelate affinity chromatography and gel filtration chromatography respectively.The purified LexA protein immune the rabbits by injection and prepare the polyclonal antibody against PC-1 protein.The immunological activity of expressed and purified LexA protein was detected by ELISA,and Western blot.Results The expressed fused protein was found in insoluble form,accounted for 45% of the total bacteria protein.The final purity was 98.97%,which was determined by the HPLC.The expressed and purified LexA protein had satisfactory immunological activity.
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Objective To extract and purify a polysaccharide SEP from eggs of sea urchin Strongylocentrotus nudus. and to determine its purity, molecular weight and immunological activity in vitro. Methods The orthogonal design was employed to obtain the best possible combination of the critical parameters for polysaccharide extraction. By ultrafiltration, DE-AE Sepharose Fast Flow anion-exchange chromatography and Sephacryl S-400 gel filtration chromatography, the deproteinated crude polysaccharide was purified. The homogeneity of SEP was proved by HPLC, polyacrylamide gel electrophoresis and paper chromatography. Its molecular weight was determined by HPGPC in reference to standVd T-series Dextran. Lymphocyte proliferation assay was made to investigate the immuno-modulating activity of SEP. Results and Conclusion The results indicated SEP was a homogeneous polysaccharide. Its molecular weight was about 1950KD. SEP increased remarkably spleen lymphocyte proliferation. The homogeneous polysaccharide SEP showed significantly immunological activity in vitro.