Structure and identification of immune fragments of acid-degraded oligosaccharides from active Astragalus polysaccharides APS-Ⅱ / 药学学报

We previously reported that active Astragalus polysaccharides APS-Ⅱ generate strong immune activity. Here we establish the optimal method for APS-II acid degradation. After preliminary structural studies and separation and preparation of the degradation products, the oligosaccharide active center with the strongest immune activity was identified by in vitro immune cell culture experiments. The optimum acid degradation conditions for APS-II were determined by a single factor experiment and an orthogonal experiment. Astragalus oligosaccharides prepared under the optimal conditions were subjected to structural analysis by hydrophilic interaction chromatography - electrospray ionization source - high resolution time-of-flight mass spectrometry. The products were separated and oligosaccharide fragments with different degrees of polymerization were isolated by preparative purification chromatography. Finally, fragments of the immunologically active centers were identified by in vitro immune cell cultures from multiple perspectives. The results show that the optimal acid hydrolysis conditions for APS-Ⅱ are hydrolysis temperature 80 ℃, trifluoroacetic acid concentration 1.0 mol·L-1, hydrolysis time 1 h. The degradation conditions have good repeatability. The degradation product is a six-carbon aldehyde glycan structure with the main chain 1→4 connected. The immune activity screening experiment for six oligosaccharide fragments showed that larger molecular weight oligosaccharides have stronger immune-promoting effects. It is speculated that the immunologically active center of Astragalus oligosaccharide is located in the sugar chain of DP9-DP19. The animal welfare and the experimental process in this study follow the requirements of the Animal Ethics Committee of Shanxi University. This result suggests a foundation for the structural characterization and structure-activity relationship research of Astragalus oligosaccharides, and may promote the development of Astragalus oligosaccharide drugs.

Establishment of enzymatic hydrolysis method of active Astragalus polysaccharides APS-Ⅱ and study on the immune activity of oligosaccharides after its degradation / 药学学报

italic>Astragalus polysaccharides are the main immunomodulatory substances in Astragali Radix. The structure of polysaccharides is difficult to accurately determine, which limits the in-depth study of the molecular mechanism of Astragalus polysaccharides in vivo. "Polysaccharide receptor theory" believes that there are one or more oligosaccharide fragment "active centers" in immunologically active polysaccharide molecules. Therefore, the degradation of Astragalus polysaccharides into oligosaccharides and the study of the active centers of polysaccharides at the oligosaccharide level provide new ideas in the study of the structure and mechanism of Astragalus polysaccharides. This article adopts endo-α-1,4-glucanase enzymatic hydrolysis, and determines the best degradation conditions through single factor test and orthogonal test to degrade the immunologically active polysaccharide APS-Ⅱ (10 kDa component) into oligomers with different degrees of polymerization. Then through the preparation of polyacrylamide gel chromatography and specific immune and non-specific immune cell tests, the immune activity screening of different oligosaccharide components is carried out. The animal welfare and the experimental process in this study follow the requirements of the Animal Ethics Committee of Shanxi University. The results showed that compared with the immunologically active polysaccharide APS-Ⅱ, different oligosaccharide components have obvious differences in different immunological activities. This paper studies the different immunological activities of Astragalus polysaccharides at the level of oligosaccharides, laying a foundation for further elucidating the structure and function of Astragalus polysaccharides, enriching the theory of polysaccharide receptors, and providing new ideas for the development of Astragalus polysaccharides.

Study on in vitro Antibacterial，Anti-inflammatory and Immunopotentiating Activity of An ’erning Granules / 中国药房

OBJECTIVE： To evaluate the in vitro antibacterial， anti-inflammatory and immunopotentiating activity of An’erning granules. METHODS： Micro-dilution method was used to determine the minimum inhibitory concentration（MIC）of An’erning granules （1.562 5-100 mg/mL） on 10 kinds of common pathogenic bacteria. Using RAW 264.7 cells （mono-nuclear macrophage） as objects， LPS was used to replicate inflammation model. MTT assay was used to investigate the effects of An’erning granules （0.312 5-20 mg/mL） on proliferation and the release of NO of macrophage. Using spleen lymphocyte of BALB/c mice as objects， MTT assay was used to investigate the effects of An’erning granules （0.312 5-20 mg/mL） on lymphocyte proliferation. RESULTS： MIC of An’erning granules to Epidermis staphylococcus and Streptococcus pneumoniae was 6.25 mg/mL； MIC of An’erning granules to Proteus vulgaris， Klebsiella pneumoniae， Bacillus subtilis and Escherichia coli （standard strain） were 12.5 mg/mL； MIC of An’erning granules to Pseudomonas aeruginosa， Enterococcus faecalis and Bacillus cereus were 25 mg/mL； MIC of An’erning granules to E. coli （clinical isolates） and Salmonella typhia were 50 mg/mL. Compared with LPS model group， An’erning granules could significantly reduce the LPS-induced release of NO in RAW 264.7 cells at the dose of 0.312 5-1.25 mg/mL （P＜0.05）. Compared with blank control group， An’erning granule had no interference on the proliferation of RAW 264.7 cells and spleen lymphocytes of mice or had a tendency to promote proliferation. It could significantly promote lymphocyte proliferation at doses of 0.625 and 0.312 5 mg/mL （P＜0.05）. CONCLUSIONS： An’erning granules not only have certain in vitro inhibitory effects on clinical common Gram-positive and Gram-negative pathogenic bacteria， but also have anti-inflammatory and immunization enhancement effects.

Effect of concurrent chemoradiotherapy and radiotherapy alone on peripheral myeloid-derived suppressor and T regulatory cells in patients with nasopharyngeal cancer / 中华肿瘤杂志

Objective@#To investigate the percentage of myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) in peripheral blood of nasopharyngeal cancer (NPC) patients undergoing concurrent chemoradiotherapy or radiotherapy alone.@*Methods@#Sixty NPC patients who received radiotherapy or concurrent chemoradiotherapy from September 2012 to November 2015 and 20 healthy individuals were included in this study. For the patients, the blood samples were collected at four time points: pre-radiation (Pre-RT), reaching a dose of 40 Gy (RT-40 Gy), finishing radiation (RT-finish) and three months after finishing radiation (3m-post-RT). Flow cytometry was used to evaluate the percentage of Treg (CD4+ CD25+ CD127low/-) and MDSC (HLA-DR-CD11b+ CD33+ ) cells in peripheral blood.@*Results@#Treg and MDSC cells were present in peripheral blood lymphocytes of healthy individuals as a percentage of (7.50±1.62)% and (1.08±0.48)%, respectively. The proportions of peripheral Treg cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (8.42± 1.52)%, (9.10±1.57)%, (8.87±1.56)% and (7.31±1.43)%, respectively, showing a statistically significant difference between Pre-RT and the other groups (P<0.05). At Pre-RT point, the percentage of Treg cells in Stage Ⅲ-Ⅳ patients [(8.63±1.39)%] was higher than that in Stage Ⅰ-Ⅱ [(7.65±1.94)%, P=0.042]. Moreover, the proportions of peripheral MDSC cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (2.14±1.21)%, (4.08±1.90)%, (3.76±1.31)% and (1.52±0.88)%, respectively. The percentages of MDSC cells at RT-40 Gy and RT-finish points were significantly higher than those at Pre-RT, while the percentage of MDSC cells at 3m-post-RT was significantly lower than those at Pre-RT (P<0.05). At Pre-RT point, the percentage of MDSC cells in Stage Ⅲ-Ⅳ patients [(2.25±1.26)%] was higher than that in Stage Ⅰ-Ⅱ [(1.35±0.66)%, P=0.007]. At RT-finish point, the proportions of MDSC and Treg cells in patients with Ⅲ-Ⅳ grade of radiation induced oral mucositis [(4.41±1.27)% and (9.91±1.23)%] were significantly higher than those in Ⅰ-Ⅱ grade patients [(3.15±1.04)% and (8.41±1.52)%, both of P<0.05].@*Conclusions@#The proportions of MDSC and Treg cells in initial treated NPC patients are higher than healthy individuals, and they are also associated with the tumor stages. During the concurrent chemoradiotherapy and radiation, the percentage of MDSC and Treg cells is elevated, suggesting a decreased immune activity. The increase of MDSC and Treg cells is related to radiation induced oral mucositis.

Study on the Extraction Technology of Polysaccharides from Antlion and Its Immune Activity in vivo / 中国药房

OBJECTIVE:To optimize the extraction technology of polysaccharides from antlion and explore its effect on im-mune functions of mice. METHODS:Using content of polysaccharides as investigation index,the effects of extracting polysaccha-rides from antlion by water extraction method protease hydrolysis extraction(optimized by orthogonal test using extraction tempera-ture,enzyme dosage,extraction time as indexes),and diluted alkali extraction(optimized by orthogonal test using alkali concentra-tion,extraction temperature,extraction time as indexes)were compared. 128 KM mice were randomly divided into 4 groups,then randomly divided into control group(normal saline),polysaccharides low-dose,medium-dose,high-dose groups(20,40,80 mg/kg),8 in each group,iv in tail vein,0.2 mL/10 g,once a day,for 1 week,which were respectively used to determine the phago-cytosis percentage and phagocytic index of peritoneal macrophages,spleen and thymus index,lymphocyte transformation rate and serum hemolysin levels. RESULTS:The contents of polysaccharides by 3 methods were 14.48%,38.66%,30.62%,respectively. The content of polysaccharides by protease hydrolysis extraction was the highest,the optimal extraction technology were as follows as using 100 μg/g papain extracting 3 h under 40 ℃. Compared with control group,phagocytosis percentage,phagocytic index, spleen index in polysaccharides low-dose,medium-dose,high-dose groups were significantly increased (P0.05);serum hemoly-sin in polysaccharides medium-dose group was significantly increased (P<0.05). CONCLUSIONS:Protease hydrolysis extraction is suitable for the extraction of polysaccharides from antlion,the optimal technology is reliable. Polysaccharides from antlion show activity in enhancing mice non-specific immunity and humoral immunity.

Characterization of Lactobacillus reuteri BCLR-42 and Lactobacillus plantarum BCLP-51 as novel dog probiotics with innate immune enhancing properties

Probiotics that are able to provide beneficial effects on animal health have become important ingredients of dog foods. This study was conducted to characterize the probiotic potentials of two strains, Lactobacillus reuteri BCLR-42 and Lactobacillus plantarum BCLP-51, that were derived from feces of healthy dogs and evaluated based on tolerance to low pH and bile acid, antimicrobial activities, enzyme profiles, sensitivity to antibiotics, and innate immune enhancing potentials. Both strains showed survival of more than 90% at pH 3 and 0.2% bile acid and exhibited broad antimicrobial activities against indicator bacteria. Moreover, both strains showed high sensitivity to antibiotics, except vancomycin, metronidazole, and gentamicin. The alkaline phosphatase was negligible (score 0), whereas they showed strong beta galactosidase activity (score range 5 or 3, respectively). The phagocytosis and oxidative burst activities of canine granulocytes were significantly enhanced in response to both strains. These results show that both strains have the capability to act as probiotics and the potential for application as ingredients in dog foods.

Study on Anti-HIV-1 Activity of Wuweilingqi Capsule in Vitro / 中国中医药信息杂志

Objective To study the activity of Wuweilingqi capsule alone and the synergy with other anti-HIV-1 inhibitors against various HIV-1 isolates,and investigate its immunological impacts for CD4+ and CD8+ T cells. Method Wuweilingqi capsule was extracted from a traditional Chinese medicine recipe which contains 5 herbs,it showed anti-retroviral activity in vitro in the cultures of human peripheral blood mononuclear cells (PBMC) and T cell lines (H9 and MT2). Results Wuweilingqi capsule inhibited HIV-1018a (an AZT-sensitive clinical isolate) in PBMC and HTLV-ⅢB (a HIV-1 lab isolate) in H9 and MT-2 T cell lines markedly,and the therapeutic indexes (TI) were 96,84 and 181 separately,but weakly inhibitory activity against HIV-1018c (an AZT-resistant clinical isolate) and chronically infective HTLV-ⅢB were demonstrated. Significant synergy against HIV-1018a and HIV-1018c replication between Wuweilingqi capsule and AZT,indinavir or T-20 were observed. In addition,Wuweilingqi capsule significantly increased PBMC proliferation,interferon-gamma secreting cell number with or without a stimulation of PHA or candida. Conclusion Although Wuweilingqi capsule did not show so strong anti-HIV-1 activity as known west drugs in clinical,its good synergy and significant increase of immune activity suggests that it will be an available and characteristic agent against HIV-1 replication.

Effects of Job's Tear(Yul-Moo) Extracts on Mouse Immune Cell Activation

Natural products are increasingly appreciated as a lead for drug discovery and development. A number of investigators have studied various activities of natural products and have found that they have not only nutritional effects but also beneficial properties to cure various diseases and to maintain good health. Job's Tear(Yul-Moo) is a grass crop that have long been used in traditional medicine and a nourishing food. Job's Tear has been reported to exhibit anti-inflammatory, stomachic, antiallergic activity, and antispastic effects and has been used in China for the treatment of warts, rheumatism, and neuralgia although its mechanism remains unclear. Previous results in our laboratory demonstrated that the ethanol extract and water extract of Job's Tear exerted an immune regulatory function on mice cells in vitro. The present study was performed to investigate the ex vivo effect of Job's Tear on immune function. Seven to eight weeks old mices(Balb/c) were fed ad libitum on chow diet and water extract of Job's Tear were orally administrated every other day for two or four weeks at two different concentrations (50 and 500mg/kg B.W.). Proliferation of mice spenocytes and antibody production to sheep red blood cells(SRBC) using hemolytic plague forming cell assay were used to indicate the immune activity. Splenocytes proliferation of Job's Tear with mitogen stimulation such as Con A and LPS was enhanced at 50 mg/kg B.W. concentrations compared to those of control group. In case of antibody production to sheep red blood cells, the number of antibody- secreting cells was increased by administration of 50mg/kg B.W. concentration in mice immunized as a T-dependent antigen. From the present study, Job's Tear water extracts may be suggested to stimulate the mice immune response by enhancing the splenocytes proliferation and the number of plague forming cells.

The lymphangial immunochemotherapy of the advanced gastric carcinoma before and after the operation / 临床外科杂志

Objective To study the action of the lymphangial immunochemotherapy on the advanced stomach cancer. Methods Group of therapy: To inject the chemotherapeutic medicine and biological response modifiers (BRM) absorbed by active carbon through the stomach mucosa by means of gastroscope and through the stomach serosa by means of surgical operation. Group of contrast: To inject the aqueous solution of the chemotherapeutic medicine and BRM through the arteria femoralis by means of the intervention. Then we examined the immune activity of the blood cell.Results There is a marked difference between the lymphangial immunochemotherapy and the blood immunochemotherapy in the immune activity of the blood cell. Conclusion The lymphangial immunochemotherapy can promote the apoptosis of the transferred cancer in the lymph system and enhance the immune activity of the blood cell.