Optimisation of enzyme-assisted extraction of Erythronium sibiricum bulb polysaccharide and its effects on immunomodulation

Glycoconjugate Journal - In this study, polysaccharides of Erythronium sibiricum bulb were extracted using enzyme-assisted extraction technology and then optimised by response surface methodology....     

Immunomodulatory effects of polysaccharides isolated from Hericium erinaceus on dendritic cells

Hericium erinaceus (H. erinaceus; HE) polysaccharides (HE-PS) have been shown to have immunomodulatory activity. We found that the bioactive components of β-glucan derivatives consisted of 20% in HE-PS. We used an analytic platform for investigating the effects of HE-PS on the maturation of rat dendritic cells (DCs), which are derived from rat bone marrow hematopoietic cells (BMHCs). The results showed that treatment with 50 μg/mL HE-PS changed the morphology of the DCs to an active form in parallel with a significant two fold increase in MHC class II and CD80/86 surface antigens compared to the control. Furthermore, endocytosis by the DCs was significantly reduced at the same dosage. IL-12, IFN-γ and IL-10 cytokine secretion was significantly increased by 2.7, 1.5 and 1.6-fold, respectively, compared to the control after treatment with 50 μg/mL of HE-PS. This study used a powered analysis platform to show that HE-PS induces DCs activation and modulates the T_H1 immune response. Thus, HE-PS has potential as an immunopotentiating agent that could be further developed in the health food industry.     

类短命植物新疆猪牙花解剖结构及其生态适应性的研究

新疆猪牙花是分布于新疆阿勒泰地区西伯利亚落叶松林下的一种多年生高山类短命植物。解剖学研究结果表明:其地下鳞叶中薄壁组织发达,是重要的储藏器官,为该植物每年长达10个月的休眠提供了充足的水分和营养物质的储存空间。茎由表皮、基本组织以及3～4轮散生的维管束组成;茎表皮细胞具厚角质层,能有效降低体内水分的散失;发达的维管束为其在冰冷的土壤中有效地吸收水分和养分,实现碳水化合物由地下器官向地上器官的转移和物质重新分配提供了有力保障。发达韧皮纤维为支撑顶部的花、果器官提供了必要的机械支持。叶片大而薄,有助于增大光合器官的面积;叶表皮角质层不明显,可以减少对日光的反射;叶肉有海绵组织与"拟栅栏组织"的分化,后者的细胞呈长柱形,其长轴方向与叶表皮方向平行,多层排列,能有效提高叶绿体对光能的捕获效率,是该植物对林下弱光生境长期适应的结果。花被片中维管束密集,发达的维管组织对维持花被片薄壁细胞的膨压,维持花被片较长时间的强烈反折状态以增加被昆虫访花的机会,提高昆虫传粉效率,促进繁殖成功具有关键作用;中轴胎座,胚珠多数;柱头与花柱中空,内表面具分泌细胞,有利于花粉管的快速萌发和迅速伸长。     

Anatomy and its ecological adaptation of an ephemeroid Erythronium sibiricum in Xinjiang

新疆猪牙花是分布于新疆阿勒泰地区西伯利亚落叶松林下的一种多年生高山类短命植物.解剖学研究结果表明:其地下鳞叶中薄壁组织发达,是重要的储藏器官,为该植物每年长达10个月的休眠提供了充足的水分和营养物质的储存空间.茎由表皮、基本组织以及3～4轮散生的维管束组成;茎表皮细胞具厚角质层,能有效降低体内水分的散失;发达的维管束为其在冰冷的土壤中有效地吸收水分和养分,实现碳水化合物由地下器官向地上器官的转移和物质重新分配提供了有力保障.发达韧皮纤维为支撑顶部的花、果器官提供了必要的机械支持.叶片大而薄,有助于增大光合器官的面积;叶表皮角质层不明显,可以减少对日光的反射;叶肉有海绵组织与“拟栅栏组织”的分化,后者的细胞呈长柱形,其长轴方向与叶表皮方向平行,多层排列,能有效提高叶绿体对光能的捕获效率,是该植物对林下弱光生境长期适应的结果.花被片中维管束密集,发达的维管组织对维持花被片薄壁细胞的膨压,维持花被片较长时间的强烈反折状态以增加被昆虫访花的机会,提高昆虫传粉效率,促进繁殖成功具有关键作用;中轴胎座,胚珠多数;柱头与花柱中空,内表面具分泌细胞,有利于花粉管的快速萌发和迅速伸长.     

苍耳4种溶剂提取物除草活性的研究

以油菜(Brassica campestris L.)、黄瓜(Cucumis sativus L.)、小麦(Triticum aestivum L.)和高粱(Sorghum vulgare Pers.)4种作物种子为供试材料,用种子萌发法测定了苍耳(Xanthium sibiricum Patrin)全株的石油醚、乙酸乙酯、丙酮和苯4种溶剂提取物的除草活性。结果表明,苍耳丙酮提取物对4种作物生长的抑制活性最高,其次为乙酸乙酯提取物和苯提取物,石油醚提取物对4种作物生长的抑制活性最低。不同作物幼根和幼茎对4种溶剂提取物的敏感性不同,油菜和高粱的幼根对苍耳丙酮提取物极为敏感,5 g.L-1苍耳丙酮提取物对油菜和高粱幼根生长的抑制率分别为90.7%和92.1%。     

Immunomodulatory effects of Pseudomonas aeruginosa quorum sensing small molecule probes on mammalian macrophages

Pseudomonas aeruginosa produces the quorum sensing signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). This natural product not only coordinates production of virulence factors by the bacterium, but also has immunomodulatory effects on the host organism. Immunomodulatory small molecules are valuable for immunology research and are potential therapeutics for autoimmune diseases such as rheumatoid arthritis, and immunosuppressive drugs following organ transplants. We describe the total synthesis of OdDHL using solid-supported reagents and scavengers, which has the potential to be used for automated analogue synthesis. OdDHL and four analogues were tested for their ability to activate or inhibit release of the pro-inflammatory mediators tumour necrosis factor alpha (TNFalpha) and nitric oxide (NO) from equine or murine macrophages (immune cells). Two of the analogues showed substantial immunomodulatory activity with these macrophages. One analogue showed differing species selectivity, being a potent antagonist in mouse cells, but a partial agonist in horse-derived macrophages. These compounds have the therapeutic potential to be used for protecting animals from bacterial septic shock.     

Antioxidant effects and cytotoxicity of three purified polysaccharides from Ligusticum chuanxiong Hort.

Water-soluble crude polysaccharide LC was obtained from Ligusticum chuanxiong Hort. by boiling-water extraction and ethanol precipitation. The major polysaccharides LCA, LCB, and LCC were isolated and purified from the crude polysaccharide LC, and the monosaccharide components, average molecular weight and the main skeleton were analyzed and determined. The antioxidant properties of LCA, LCB, and LCC were extensively investigated with several biochemical methods, and the antiproliferative activities were also involved. Experimental results showed that all purified polysaccharides exhibited antioxidation and cytotoxicity, and LCB has the highest antioxidant and cytotoxic activity among them. It is possible that LCB is explored as a novel potential antioxidant and cytotoxic natural bioactive macromolecule.     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

Stimulatory effects of purified macrophage colony-stimulating factor on murine resident peritoneal macrophages

A purified preparation of macrophage colony-stimulating factor (M-CSF) free of interferon and endotoxin activity was studied for its effects on resident murine peritoneal macrophages. M-CSF was found to induce profound morphologic alterations in resident macrophages. These changes included a marked increase in cell size, membrane ruffling, and cytoplasmic vacuolization. Further, after 72 hr of incubation with 1000 U/ml of M-CSF, there were significant increases in macrophage DNA synthesis as measured by autoradiography (P less than 0.001), and in macrophage monolayer protein content (P less than 0.01). None of these changes was seen in control macrophages or those exposed to recombinant interferon-gamma (IFN). Low activity levels of the ectoenzymes 5'-nucleotidase (5'NTD) and alkaline phosphodiesterase I (APD) have been associated with certain macrophage functions, particularly the expression of tumor cytotoxicity. Macrophage monolayers exposed to M-CSF demonstrated an unaltered level of 5'NTD activity from controls and a significantly increased level of APD activity (P less than 0.01) and did not demonstrate an increased ability to kill tumor cells, as measured by the 51Cr-release assay. On the other hand, IFN caused significant decreases in both 5'NTD (P less than 0.05) and APD (P less than 0.01) and also induced marked tumoricidal activity in macrophage monolayers. These results indicate that purified M-CSF induces highly specific alterations in the functional activity and morphologic appearance of resident macrophages and these changes are distinct from those induced by IFN.     

Effect of steaming process on the structural characteristics and antioxidant activities of polysaccharides from Polygonatum sibiricum rhizomes

Polygonatum sibiricum (P. sibiricum) rhizomes are widely used as a tonic and functional food, and are often processed to enhance their tonic function by repeated steaming and drying. As the most important constituent, the polysaccharide from P. sibiricum rhizomes (PSP) has demonstrated various activities, but the alteration of structural characteristics and activities of the purified PSPs during steaming process was rarely investigated. To well understand the effect of steaming process on the polysaccharides of P. sibiricum, neutral polysaccharides from P. sibiricum rhizomes (PSP0?~?PSP9) after steaming were first isolated and purified, and then the chemical properties and antioxidant activities were determined. The results showed that the molar ratios of monosaccharides in PSPs were different. The molecular weights of PSPs were increased significantly after the fourth steaming. Morphological studies showed that the surface of PSPs became much tighter during the steaming process. Fourier transform infrared spectroscopy spectra displayed the polysaccharides had similar backbones and chemical groups. Furthermore, the antioxidant activity of PSPs was measured through radical scavenging tests. It was found that the radical scavenging activity of PSPs was elevated strikingly after steaming, and increased gradually with numbers of steaming process. The biological and chemical variance of PSPs revealed considerable segregation of PSP0, PSP1?~?PSP4 and PSP5?~?PSP9. In conclusion, our results proposed the fourth time as the optimal number of steaming to extract functional polysaccharide from P. sibiricum rhizomes.

     

Immunopontentiating and antitumor activities of the purified polysaccharides from Phellodendron chinese SCHNEID

The polysaccharide fractions were isolated and purified from Phellodendron chinese SCHNEID, and antitumor activities were examined at dosages of 2, 5 and 10 mg/100 g. F-7 and F-8 showed the highest tumor inhibitory activities (inhibition ratio 96.4 and 98.2% in 2 mg/100 g), and in dose of 5 mg/100 g, the inhibitory ratios were 95.3 and 97.5%, respectively. Furthermore, 10 mg/100 g of intraperitoneal (i.p.) injection gave 97.3 and 98.7% of inhibition. In oral administration, the inhibitory activities were not markedly observed, indicating that the polysaccharides are directly acting to immune system. Also the polysaccharides increased the number of circulating blood leukocytes and total peritoneal exudate cells. Although implantation of tumor cells greatly decreased the productivity of antibody (antibody-mediated) and T lymphocyte reactivity (delayed-type) as 6.3 from 9.3 and 5.9 from 7.7, represented by the increase of footpad thickness, respectively. The polysaccharides elevated the reactivity of T lymphocyte in tumor-bearing mice, which were rapidly recovered by discontinuance of sample treatments. Especially, F-2, F-5, F-7 and F-8 remarkably recovered the decreased sensitivity. When the effects on thymidylate synthase (TS) and thymidine kinase (TK) activities were determined, TS activities in the F-2 and F-7-treated mice were markedly suppressed to 73.7% and 79.5% of that in the control (p < 0.01), while there was little difference in TK activity with a slight decrease in F-2 only. However, in i.p. injection, TS activities in the F-2, F-5, F-7 and F-8-treated mice were markedly suppressed to 83% to 85% of that in the control (p < 0.01). Furthermore, there were also significant differences in TK activities in F-2, F-5, F-7 and F-8-treated mice (p < 0.05). These results clearly indicated that the i.p. injection is much effective to suppress tumor growth than oral administration.     

四种野生食用菌粗多糖的抗氧化活性

利用DPPH自由基清除法、羟基自由基(OH.)清除法、铁离子鳌合能力及测定还原能力等方法对鸡油菌Cantharellus cibarius、变绿红菇Russula virescens、蜜环菌Armillaria mellea和棕灰口蘑Tricholoma myomyces等4种食用菌的粗多糖进行了抗氧化活性评价。结果显示,4种真菌粗多糖均不同程度地具有抗氧化活性。在DPPH自由基清除试验中,棕灰口蘑和蜜环菌表现出很强的活性,其EC50值分别为1.35 g/L、1.53 g/L;棕灰口蘑和蜜环菌对羟基自由基的清除能力也要优于其他2种食用菌,其EC50值分别为0.65 g/L、0.78 g/L;棕灰口蘑的铁离子螯合能力明显优于其他3种测试菌,其EC50值为1.69 g/L;在还原力方面同样是棕灰口蘑活性最强,蜜环菌次之,其EC50值为1.05 g/L、1.37 g/L。     

四种多孔菌子实体粗多糖抗肿瘤活性的比较研究

对粗毛纤孔菌、椭圆嗜蓝孢孔菌、火木层孔菌、木蹄层孔菌4种多孔菌子实体粗多糖成分的含量及其体内抗肿瘤活性进行了比较研究。结果表明粗毛纤孔菌子实体中的粗多糖含量为4.1%,高于其他3种多孔菌;同时,4种多孔菌子实体粗多糖对H22荷瘤小鼠均显示出一定的抗肿瘤活性,除木蹄层孔菌外,其他3种多孔菌给药剂量为500mg/mL和1 000mg/mL时抑瘤率均大于40%,其中粗毛纤孔菌子实体粗多糖抑瘤率最高,低剂量组（500mg/kg）为58.12%,高剂量组（1 000mg/kg）为47.75%。     

Immunomodulatory effects of honey cannot be distinguished from endotoxin

In recent years, the use of honey has re-emerged as a remedy for wound treatment. Effects of honey have been related to the presence of an unidentified component that induces release of inflammatory cytokines from monocytic cells. The present study was intended to further characterize the reported in vitro effects of honey. Our results show that natural honeys induce interleukin-6 release from Mono Mac 6 cells as well as release of reactive oxygen species from all-trans retinoic acid (ATRA) differentiated HL-60 cells. The natural honeys contained substantial amounts of endotoxin, and the responses observed in the cell based assays were similar to the responses induced by endotoxin alone. In addition, we determined that the immunomodulatory component present in the natural honeys was retained in the ultra filtrated fraction with a molecular weight greater than 20 kDa. The component was resistant to boiling and its immunomodulatory activity could be abrogated by the addition of polymyxin B. We speculate that the observed in vitro immunomodulatory effects of honey might solely be explained by the endotoxin content in the natural honeys.     

Degradation of pectic polysaccharides extracted from suspension cultures of carrot by purified exo-polygalacturonase

Highly purified exo-polygalacturonase was obtained from suspension cultures of carrot ( Daucus carota L. cv. Kintoki) by dialysis at pH 5.2, chromatography on DEAE-Sephadex A-50 and on Sephadex G-150, and preparative polyacrylamide disc gel electrophoresis. The enzyme did not attack the isolated carrot cell walls directly, but it had some effect on pectic polysaccharides extracted from the walls. The extracted polysaccharides were fractionated by DEAE-Sephadex A-50 column chromatography yielding four carbohydrate fractions. The major fraction (P-3) was then reacted with the exo-polygalacturonase. The enzyme treatment resulted in hydrolysis of approximately 18% of the glycosyl linkages of fraction P-3 with the release of galacturonic acids. The molecular size estimated by Bio-Gel A-5m gel filtration was not markedly affected by the enzyme action, but the percentage of galacturonosyl residues was clearly reduced. The specific activity of exo-polygalacturonase changed during the growth cycle, in relation to the cell growth.     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

Molecular characterization of four forms of phosphofructokinase purified from potato tuber

Four forms of phosphofructokinase (PFK) have been purified to apparent homogeneity from tubers of potato (Solanum tuberosum cv. Record). Each had a final specific activity of about 200 mumol.min-1.mg-1 protein. Similar forms of PFK were found in partially purified extracts from tubers and leaves of other potato cultivars and related wild species. The relative molecular masses of three forms of PFK were about 200,000 whereas that of the fourth PFK was greater than 800,000. The four forms of PFK contained different proportions of four polypeptides which had apparent relative molecular masses of 46,300, 49,500, 50,000, and 53,000. These polypeptides gave different patterns of peptide fragments after chemical and proteolytic cleavage. Western blots and immunoprecipitation studies using antibodies raised against the individual polypeptides showed that all four are associated with PFK. Thus, potato tubers contain four distinct forms of PFK that differ in their subunit composition.     

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A₂ (PLA₂), and BaltTX-II, an Asp49 catalytically active PLA₂ isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA₂ BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA₂, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA₂s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.