Protective effect of apple polyphenols against stress-provoked influenza viral infection in restraint mice

This study was conducted to investigate the effects of apple polyphenol extract (APE) against influenza virus in mice loaded with restraint stress. The high-performance liquid chromatography (HPLC) fingerprint of APE was recorded, and the percentage composition of polyphenols was determined as 81.7%. Our results showed that restraint stress significantly promoted the mortality and duration of complications of mice infected with the H1N1 virus. However, oral administration of APE (100 and 200 mg/kg) improved the survival rates and prolonged living time of stressed mice infected with influenza virus in a dose-dependent manner. APE was further found to significantly improve the number of immunocytes, ratio of CD4 helper cells, secretion of IL-2, and capabilities of natural killer (NK) cytotoxicity (LU10/spleen) in spleens of restraint-stressed mice. In addition, APE also significantly decreased the level of lipid peroxidation and increased oxygen radical absorbance capacity (ORAC) in splenocytes. These results indicated that the protective effects of APE on mice infected with influenza virus were related to the alleviation of stress-induced impairment of immune functions and its antioxidant property might contribute to the immune recovery.     

Components and activity of polysaccharides from coarse tea

Coarse tea contained a high content of polysaccharide complex. Composed of polysaccharide and protein, the polysaccharide complex from tea (TPS) belonged to glycoprotein with the molecular weight () of (10.7-11.0) x 10(4). When mice (7 weeks old, C57BL/8) were injected with TPS, the levels of blood glucose (BG) in normal mice and model mice with high BG were decreased significantly by averages of 13.54 and 22.18%, respectively. The antibody concentration (OD(413 nm)) in the mice injected with 2.4 mg/mL TPS was increased evidently by 44.93% (p < 0.01). TPS treatment was beneficial not only for the subsequent production of interleukin (IL) 2 in spleen cells of adjuvant arthritis (AA) rats but also because it prohibited the body from producing too much IL-1 in AA rats. Treatment of diabetes with coarse tea in both China and Japan may be related to TPS and the content of TPS in coarse tea.     

H5N1亚型高致病性禽流感病毒A/Duck/FuJian/01/02株感染性克隆构建

摘 要：1999-2002年本试验室从我国南方健康鸭体内分离到21株H5N1亚型禽流感病毒,对其进行系统地生物学特性和遗传演化分析发现,其中A/Duck/FuJian/01/02(简DKFJ)和A/Duck/Guangxi/53/02(简DKGX) 属于同一基因型, 对鸡都呈高致病性,但对哺乳动物模型Balb/c小鼠的致病性明显不同: DKGX对小鼠呈低致病性（MLD50>106.5）,DKGX株反向基因操作系统本试验室已经建立,而DKFJ对小鼠呈高致病性（MLD50<100.5）。为了研究这两株病毒对哺乳动物模型Balb/c小鼠致病性差异的分子机制,本研究构建了对DKFJ的8质粒反向基因操作系统,并通过细胞转染技术成功拯救了该病毒（R-DKFJ）。R-DKFJ在对哺乳动物模型Balb/c小鼠的致病性保持了与亲本野生毒（W-DKFJ, MLD50<100.5）一致的生物学特性,并且对小鼠呈全身感染,即106EID50鼻腔感染小鼠后第三天在脑、脾、肾、肺脏等脏器检测到病毒。DKFJ反向基因操作系统的成功建立为阐明H5N1亚型禽流感病毒对哺乳动物模型Balb/c小鼠的致病机制等方面奠定了基础。     

一株对小鼠具有高致病性的H5N1亚型禽流感病毒的拯救

禽流感病毒(Avianinfluenzavirus)正在逐渐获得突破种间屏障感染哺乳动物的能力,揭示其获得此种能力的分子机制已经成为禽流感病毒的研究热点。A/Chicken/Guangdong/04(H5N1)是一株高致病性禽流感病毒,同时对BALB/c小鼠也具有高致病力。实验通过反向遗传操作技术对该病毒进行拯救,获得了拯救毒R-A/Chicken/Guangdong/04(R-CG)。R-CG与其亲本毒A/Chicken/Guangdong/04(W-CG)在胚半数感染量(EID50)、细胞培养半数感染量(TCID50)、对SPF鸡和BALB/c小鼠致病力等生物学特性保持一致。即R-CG与W-CG对鸡都为高致病力,静脉接种指数分别为2.88和2.91;R-CG与W-CG一样,以106EID50鼻腔感染BALB/c小鼠后,均能引起小鼠死亡,在小鼠脑、肺、肾和脾脏中都能分离到病毒,说明拯救的病毒与亲本毒一样,都可在小鼠体内有效复制,对小鼠具有高致病性。本实验成功地拯救了A/Chicken/Guangdong/04,拯救病毒R-CG可作为背景毒株,为研究禽流感病毒突破种间屏障感染哺乳动物的分子机制奠定了实验基础。     

Nonenzymatic C-glycosylation of flavan-3-ols by oligo- and polysaccharides

Model reactions between the polysaccharide amylose and the polyphenol (-)-epicatechin followed by partial enzymatic hydrolysis of the reaction products formed led to the detection of mono- and oligo-C-glucosylated flavan-3-ols by means of LC-MS/MS experiments. To confirm the structure of these putative flavan-3-ol/oligosaccharide conjugates, (-)-epicatechin was reacted with maltose and maltotriose, respectively, giving rise to a series of previously unreported flavan-3-ol/maltose and flavan-3-ol/maltotriose conjugates, namely, (-)-epicatechin-8-C-beta-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranoside, (-)-catechin-8-C-beta-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranoside, (-)-catechin-6- C-beta-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranoside, (-)-catechin-8-C-beta-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranoside, (-)-catechin-6-C-beta-D-glucopyranosyl-(4-->1)- O-alpha-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranoside, and (-)-epicatechin-6/8-C-beta-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranosyl-(4-->1)-O-alpha-D-glucopyranoside. Furthermore, quantitative analysis of flavan-3-ol-C-glucosides in an enzymatic total hydrolysate using a newly developed stable isotope dilution assay (SIDA) enabled a first insight into the yield of the formation of polyphenol/polysaccharide cross-links, for example, an amount of 14.0, 9.0, and 0.15 micromol of flavan-3-ol-6-C-beta-D-glucopyranoside, flavan-3-ol-8-C-beta-D-glucopyranoside, and flavan-3-ol-6- C,8-C-beta-D-glucopyranoside were per mmol (-)-epicatechin when reacted with amylose.     

Effect of Application of Acidified Porous Hydrate Calcium Silicate and Porous Hydrated Calcium Silicate on the Growth of Rice Plants (Oryza sativa L.)

The effect of the application of acidified porous hydrate calcium silicate (APS) in nursery bed soil and porous hydrate calcium silicate (PS) in paddy fields on the growth of rice plants (Oryza sativa L. cv. Hitomebore) was examined in 2002 and 2003. The results revealed the following: 1) Shoot dry weight of rice seedlings increased by APS treatment in nursery bed soil. The tiller number of rice plants after transplanting in both years also increased by APS treatment in nursery bed soil, and in 2003, the tiller number in the treatment with a combination of APS in nursery bed soil and PS in paddy fields was significantly higher than that in the other treatments until the maximum tiller number stage. Furthermore, the root length of rice plants 14 d after transplanting increased by APS treatment in nursery bed soil. 2) Silicon concentration in the soil solution significantly increased by PS treatment in paddy fields, and the concentration of dissolved carbon oxide increased by APS treatment in nursery bed soil. 3) Only in the APS treatment the rice yield was 341 g m^?2, while 400 and 450 g m^?2 in the PS and both APS and PS treatments, respectively, in 2003. Percentages of ripened grains in the plots without PS treatment ranged from 57 to 63%, respectively, while, those in the PS treated plots were 82%. The numbers of panicles and ripened grains in both APS and PS treatments were the highest among the treatments. Based on the above results, we concluded that both APS in nursery bed soil and PS in paddy field treatments were effective in improving the silicon nutrition and growth of rice plants, and that this effect was enhanced by a combination of treatments with the two.     

Purple carrot (Daucus carota L.) polyacetylenes decrease lipopolysaccharide-induced expression of inflammatory proteins in macrophage and endothelial cells

Carrots ( Daucus carota L.) contain phytochemicals including carotenoids, phenolics, polyacetylenes, isocoumarins, and sesquiterpenes. Purple carrots also contain anthocyanins. The anti-inflammatory activity of extracts and phytochemicals from purple carrots was investigated by determining attenuation of the response to lipopolysaccharide (LPS). A bioactive chromatographic fraction (Sephadex LH-20) reduced LPS inflammatory response. There was a dose-dependent reduction in nitric oxide production and mRNA of pro-inflammatory cytokines (IL-6, IL-1beta, TNF-alpha) and iNOS in macrophage cells. Protein secretions of IL-6 and TNF-alpha were reduced 77 and 66% in porcine aortic endothelial cells treated with 6.6 and 13.3 microg/mL of the LH-20 fraction, respectively. Preparative liquid chromatography resulted in a bioactive subfraction enriched in the polyacetylene compounds falcarindiol, falcarindiol 3-acetate, and falcarinol. The polyacetylenes were isolated and reduced nitric oxide production in macrophage cells by as much as 65% without cytotoxicity. These results suggest that polyacetylenes, not anthocyanins, in purple carrots are responsible for anti-inflammatory bioactivity.     

Characterization of Aloeride, a new high-molecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity

We have characterized a new immunostimulatory polysaccharide called Aloeride from commercial aloe vera (Aloe barbadensis) juice. Aloeride is between 4 and 7 million Da, and its glycosyl components include glucose (37.2%), galactose (23.9%), mannose (19.5%), and arabinose (10.3%). At 0.5 microg/mL Aloeride increased NF-kappa B directed luciferase expression in THP-1 human monocytic cells to levels 50% of those achieved by maximal concentrations (10 microg/mL) of LPS. Aloeride induced the expression of the mRNAs encoding IL-1beta and TNF-alpha to levels equal to those observed in cells maximally activated by LPS. Acemannan, the major carbohydrate component from aloe, used at 200 microg/mL in the macrophage assay resulted in negligible NF-kappa B activation. Analysis of acemannan and Aloeride using size-exclusion chromatography suggests that the low activity of acemannan is due to trace amounts of Aloeride. Although Aloeride comprises only 0.015% of the aloe juice dry weight, its potency for macrophage activation accounts fully for the activity of the crude juice.     

Specific immunomodulatory and secretory activities of stevioside and steviol in intestinal cells

Stevioside, isolated from Stevia rebaudiana, is a commercial sweetener. It was previously demonstrated that stevioside attenuates NF-kappaB-dependent TNF-alpha and IL-1beta synthesis in LPS-stimulated monocytes. The present study examined the effects of stevioside and its metabolite, steviol, on human colon carcinoma cell lines. High concentrations of stevioside (2-5 mM) and steviol (0.2-0.8 mM) decreased cell viability in T84, Caco-2, and HT29 cells. Stevioside (2 mM) potentiated TNF-alpha-mediated IL-8 release in T84 cells. However, steviol (0.01-0.2 mM) significantly suppressed TNF-alpha-induced IL-8 release in all three cell lines. In T84 cells, steviol attenuated TNF-alpha-stimulated IkappaB --> NF-kappaB signaling. Chloride transport was stimulated by steviol (0.1 mM) > stevioside (1 mM) at 30 min. Two biological effects of steviol in the colon are demonstrated for the first time: stimulation of Cl(-) secretion and attenuation of TNF-alpha-stimulated IL-8 production. The immunomodulatory effects of steviol appear to involve NF-kappaB signaling. In contrast, at nontoxic concentrations stevioside affects only Cl(-) secretion.     

Modulation of cytokine secretion by garlic oil derivatives is associated with suppressed nitric oxide production in stimulated macrophages

We previously described that garlic oil derivatives differentially suppress the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in activated macrophages. In the present study, we investigated the effects of the garlic derivatives, diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS), on cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and the association between modulation of cytokines and inhibition of NO production was also assessed. The results indicated that these garlic compounds had different effects on the secretion of activated cytokines, including proinflammatory tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, as well as the antiinflammatory, IL-10. DAS inhibited the production of all stimulated cytokines in a concentration-dependent manner, and the inhibition was closely associated with the suppression of NO and PGE(2) production. DADS repressed the production of stimulated TNF-alpha and IL-10 and increased the production of activated IL-1beta and, to a lesser extent, IL-6; but only the decreased IL-10 production was associated with DADS-induced NO inhibition. Yet, the DAS- and DADS-suppressed NO production was independent of TNF-alpha. AMS, on the other hand, slightly suppressed the stimulated TNF-alpha but enhanced IL-10 production, and such modulation was closely associated with the decrease in NO production.     

香菇多糖对鲤鱼（cyprinus carpio）离体培养免疫细胞的活性调节作用*

采用Percoll密度离心等技术,对鲤鱼的头肾巨噬细胞和外周血白细胞进行分离纯化,并离体培养。体外暴露不同浓度的黄芪多糖（APS）和香菇多糖（LNT）后,分别采用 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)法测定它们对鲤鱼外周血白细胞增殖的影响;NBT(nitroblue tetrazolium)还原法和Griess试剂显色法测定对头肾巨噬细胞的呼吸爆发的影响;实时定量PCR法测定头肾巨噬细胞细胞因子IL-1β诱导表达的影响。结果显示,香菇多糖能显著诱导巨噬细胞的氧爆发活性;其低浓度时对细胞的氮呼吸爆发活性无显著影响,随着作用浓度的增加表现为高浓度抑制作用;香菇多糖可以显著促进鲤鱼外周血白细胞的增殖,且能显著增强IL-1β的体外诱导表达。结果表明香菇多糖对离体培养鲤鱼免疫细胞有明显活性作用,对鲤鱼非特异性免疫和特异性免疫具有促进作用,是一种有潜力的鱼用免疫增强剂。     

Isolation and characterization of two flavonoids, engeletin and astilbin, from the leaves of Engelhardia roxburghiana and their potential anti-inflammatory properties

Engeletin, a flavonoid compound, was isolated from the leaves of Engelhardia roxburghiana for the first time, along with astilbin, another flavonoid. The chemical structures of engeletin and astilbin were confirmed by (1)H and (13)C nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectra, and their anti-inflammatory activities were studied in lipopolysaccharide (LPS)-stimulated mouse J774A.1 macrophage cells. LPS induced the inflammatory state in macrophage cells and increased mRNA expressions of pro-inflammatory cytokines. Engeletin and astilbin exhibited remarkable inhibitory effects on interleukin (IL)-1β and IL-6 mRNA expression. Significant inhibition of LPS-mediated mRNA expressions were also seen in LPS binding toll-like receptor (TLR)-4, pro-inflammatory cytokine tumor necrosis factor (TNF)-α, IL-10, chemoattractant monocyte chemotactic protein (MCP)-1, and cyclooxygenase (COX)-2 genes. The reduced expression of these cytokines may alleviate immune response and reduce inflammatory activation, indicating that engeletin and astilbin may serve as potential anti-inflammatory agents.     

Z0206细菌富硒多糖对小鼠的免疫调节作用研究

本试验通过建立环磷酰胺（CP）诱导的免疫抑制小鼠模型,研究了Z0206细菌富硒多糖的免疫调节功能。试验分为四个处理：Ⅰ组为对照组;Ⅱ组为CP组;Ⅲ组为细菌富硒多糖组;Ⅳ组为细菌富硒多糖预防组。结果表明,与对照组相比,细菌富硒多糖组小鼠免疫器官指数、NK细胞活性、血液溶血素水平（HC50）和脾细胞抗体形成数（PFC）均显著提高（p<0.05）,T细胞亚群增殖有所提高,但差异不显著（p>0.05）。与CP组相比,细菌富硒多糖预防组能使CP诱导的免疫抑制得到修复,改善试验小鼠免疫器官指数、T淋巴细胞和自然杀伤细胞（NK）活性、血液溶血素水平和脾细胞抗体形成数,差异显著（p<0.05）。提示：细菌富硒多糖能增强小鼠的免疫功能,对CP诱导的免疫抑制具有修复作用。     

Antiinflammatory properties of the muscadine grape (Vitis rotundifolia)

The muscadine grape possesses one of the highest antioxidant levels among fruits; yet, the effect of this fruit on mammalian metabolic systems has not received significant attention. To examine the antiinflammatory properties of the muscadine, grape skins were dried, pulverized, and extracted (10% w/v) with 50% ethanol. The extract was then tested in two different assays: the release of superoxide in phorbol myristate acetate-activated neutrophils and the release of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-beta), and interleukin-6 (IL-6)] by lipopolysaccharide-activated peripheral blood mononuclear cells. The release of superoxide and cytokines was inhibited by increasing concentrations of the extract. A 1:100 dilution of the extract inhibited superoxide release by approximately 60% while the release of TNF-alpha and IL-1beta was reduced at a dilution of 1:200 by approximately 15 and 90%, respectively (all P < 0.05). The inhibition pattern on the release of IL-6 was similar to that seen with TNF-alpha. In a related in vivo study, rats were fed a diet containing 5% (wt/wt) dried muscadine grape skins for 14 days and then were injected with carrageenan in the foot pad. After 3 h, paw edema was measured and the rats on the grape skin diet had approximately 50% less paw edema than controls (P < 0.05). These results demonstrate that the muscadine grape skin powder possesses significant in vitro and in vivo antiinflammatory properties.     

雅安藏茶茶多糖对~(60)Co-γ射线辐照损伤小鼠抗氧化和造血功能的防护作用

为探究雅安藏茶茶多糖对~(60)Co-γ射线辐射损伤小鼠抗辐射作用的影响,从雅安藏茶中提取茶多糖,用不同浓度(50、100、200 mg·kg-1·d-1)的茶多糖连续15 d灌胃小鼠,在第6天采用5Gy的~(60)Co-γ射线一次性全身辐照各组小鼠,灌胃第16天测定各组小鼠外周血细胞、肝脏组织总抗氧化能力(T-AOC)、总超强氧化物歧化酶(T-SOD)活性和总蛋白(TP)含量等指标。结果表明,与单纯辐照组相比,雅安藏茶茶多糖能显著提高辐照损伤小鼠的外周血细胞数量,极显著提高肝脏组织T-AOC、T-SOD活性和股骨骨髓DNA含量,极显著降低肝脏MDA含量,缓解了免疫器官胸腺的萎缩;且随着茶多糖浓度的增加,其抗辐射作用效果增强。综上,雅安藏茶茶多糖对~(60)Co-γ射线辐照损伤小鼠抗氧化功能和造血功能具有较强的防护作用,且具有一定的剂量效应。本研究结果为进一步深入探究茶多糖防辐射作用机理提供了新参考,也为雅安藏茶辐照保健功效研究提供了一定的理论依据。     

A matured fruit extract of date palm tree (Phoenix dactylifera L.) stimulates the cellular immune system in mice

The immunomodulatory effects of a hot water extract from matured fruit of the date palm tree (Phoenix dactylifera L.) were investigated in comparison to those of prune and fig fruit in mice. The number of spleen IFN-γ(+)CD4(+), IFN-γ(+)CD49b(+) and IL-12(+)CD11b(+) cells was highest in mice given the date extract-added diet. Polyphenols identified in the date extract, such as chlorogenic acid, caffeic acid, pelargonin and ferulic acid, stimulated IFN-γ mRNA expression significantly in mouse Peyer's patch cell cultures. Chlorogenic acid and caffeic acid also increased the number of IFN-γ(+)CD4(+) cells significantly, while some polyphenols increased the number of IFN-γ(+)CD49b(+) and IL-12(+)CD11b(+) cells significantly. On the other hand, a 70% ethanol-insoluble date extract treated with trypsin increased the number of IFN-γ(+)CD49b(+) and IL-12(+)CD11b(+) cells significantly. These results indicate that some polyphenols and polysaccharides present in date fruit stimulate the cellular immune system in mice.     

Antiallergic effect of milk fermented with lactic acid bacteria in a murine animal model

The objective of this study was to assess the antiallergic effect of fermented milk prepared, respectively, with Streptococcus thermophilus MC, Lactobacillus acidophilus B, Lactobacillus bulgaricus Lb, L. bulgaricus 448, and Bifidobacterium longum B6. Female BALB/c mice fed fermented milk were immunized intraperitoneally with ovalbumin (OVA)/complete Freund's adjuvant (CFA) to evaluate the immune response by observing the secretion of cytokines IL-2, IL-4, and IFN-gamma and serum antibody IgE. The results showed that supplementation with lactic acid bacteria fermented milk did not significantly change the IL-2 spontaneous and OVA-stimulated secretions of splenocytes. However, both spontaneous and OVA-stimulated secretions of splenocytes from mice fed lactic acid bacteria fermented milk showed significantly (P < 0.05) lower levels of IL-4 (Th2 cytokine) than those from OVA/CFA-immunized mice fed non-fermented milk (OVA/CFA-milk group). The spontaneous secretion of IFN-gamma (Th1 cytokine) by splenocytes from mice fed L. bulgaricus 448 or L. bulgaricus Lb fermented milk significantly increased as compared to that from the OVA/CFA-milk group. The results showed that the ratios of IFN-gamma to IL-4 of both spontaneous and OVA-stimulated secretions in splenocytes from mice fed lactic acid bacteria fermented milk increased significantly as compared to that of PBS- or OVA/CFA-milk groups. The serum levels of OVA-specific IgE in fermented milk fed groups, especially the group fed S. thermophilus MC fermented milk, were significantly lower than those in the OVA/CFA-milk group through a 6 week feeding experiment. The results showed that milk fermented with lactic acid bacteria demonstrated in vivo antiallergic effects on OVA/CFA-immunized mice via increasing the secretion ratio of IFN-gamma/IL-4 (Th1/Th2) by splenocytes and decreasing the serum level of OVA-specific IgE.     

Inhibition of Lipopolysaccharide (LPS)-induced inflammatory responses by Sargassum hemiphyllum sulfated polysaccharide extract in RAW 264.7 macrophage cells

Sargassum hemiphyllum , a kind of brown seaweed generally found along coastlines in East Asia, has long served as a traditional Chinese medicine. S. hemiphyllum has shown an anti-inflammatory effect; however, its mechanism has not been elucidated clearly. This study explored S. hemiphyllum for its biomedical effects. S. hemiphyllum sulfated polysaccharide extract (SHSP) was first prepared; the mouse macrophage cell line (RAW 264.7) activated by lipopolysaccharide (LPS) was used as a model system. The secretion profiles of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and NO, were found significantly to be reduced in 1-5 mg/mL dose ranges of SHSP treatments. RT-PCR analysis suggested SHSP inhibits the LPS-induced mRNA expressions of IL-β, iNOS, and COX-2 in a dose-dependent manner. At protein levels, Western blot analysis demonstrated a similar result for NF-κB (p65) in cytosol/nuclear. Taken together, the anti-inflammatory properties of SHSP may be attributed to the down-regulation of NF-κB in nucleus.