TLR4-mediated activation of macrophages by the polysaccharide fraction from Polyporus umbellatus(pers.) Fries

Aim of the Study

Zhu Ling (Polyporus umbellatus) is well-known to reduce the risk of a variety of diseases. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using polysaccharides prepared from Polyporus umbellatus (PPS).

Materials and methods

Splenocyte proliferation was analyzed with ³H-TdR incorporation method. Nitric oxide (NO) was measured by Griess method and cytokines of culture supernatants was detected by enzyme linked immunosorbent assay (ELISA). The fluoresceinamine-labeled PPS (Flu-PPS) and dextran (Flu-dextran) were prepared by the cyanogen bromide activation method. The cell-binding activity of Flu-PPS was analyzed with FACS and confocal microscopy. NF-κB activity was measured by ELISA assay.

Results

We found that PPS is able to strongly upregulate the functions of macrophages such as Nitric oxide (NO) production and cytokine expression. Compared with C3H/HeJ group, PPS significantly stimulated the proliferation of splenocytes and the production of TNF-α, IL-1β and NO of peritoneal macrophages from C3H/HeN mice. The function blocking antibodies to TLR-4, but not TLR-2 and CR3, markedly suppressed PPS-mediated TNF-α and IL-1β production. Flow cytometric and confocal laser-scanning microscopy analysis shown that fluorescence-labeled PPS (f-PPS) can bind specifically to the target cells, and the binding can blocked by unlabeled PPS and anti-TLR4, but not anti-TLR2 and CR3 monoclonal antibodies. Nuclear translocation and DNA binding activity of NF-κB was significantly induced by PPS.

Conclusions

Therefore, our data suggest that PPS may exert its immunostimulating potency via TLR-4 activation of signaling pathway.     

Macrophage activation induced by Orbignya phalerata Mart

Babassu is the popular name of Orbignya phalerata Mart. [Arecaceae (Palmae)], which fruits mesocarp has been used in Brazil as medicine for the treatment of pains, constipation, obesity, leukemia, rheumatism, ulcerations, tumors and inflammations. In this study, we investigated the effect of babassu mesocarp flour aqueous extract (BM) on C3H/HePas mice peritoneal cellular migration and macrophage activation by measuring the nitric oxide (NO), hydrogen peroxide (H(2)O(2)) and tumor necrosis factor (TNF) release, spreading activity and major histocompatibility complex (MHC) class II expression. Our results demonstrate that BM injected once ip in mice at 10 and 20 mg/kg increased the cellular influx to the peritoneal cavity, the MHC class II expression and the spreading ability, and also induced the production of NO, TNF and H(2)O(2). The increase in NO-production and MHC expression was also observed after the addition of BM to resident macrophage cultures (100 microg/ml). Thus, BM-treatment was able to activate peritoneal macrophages in vitro and in vivo inducing the production of inflammatory and cytotoxic metabolites, which could justify the popular use of babassu mesocarp in the treatment of tumor diseases, but not in inflammatory pathologies.     

肉苁蓉低分子糖对巨噬细胞激活作用的研究

探究肉苁蓉低分子糖对RAW264.7小鼠巨噬细胞的激活作用及潜在机制。该实验将RAW264.7细胞分为正常对照组、细菌脂多糖(LPS)阳性对照组、肉苁蓉低分子糖处理组,其中肉苁蓉低分子糖的给药浓度为3.91~62.5 g·L-1。采用中性红法检测巨噬细胞吞噬活性,一氧化氮(NO)试剂盒检测NO的释放量,Western blot法检测巨噬细胞激活相关蛋白(TNF-α,IL-6,IKKβ,p-IKKβ,IκBα,p-IκBα,NF-κB,p-NF-κB)的表达。结果显示,肉苁蓉低分子糖对巨噬细胞具有激活作用,具体表现在:其可显著提高RAW264.7细胞NO的释放量,并促进细胞因子TNF-α和IL-6的表达。同时,其还可显著提高NF-κB的磷酸化及其上游关键信号蛋白IKKβ和IκBα的磷酸化水平。另外,低分子糖中的甘露醇可以使巨噬细胞的吞噬活性显著提升。以上结果表明,肉苁蓉低分子糖对巨噬细胞具有激活作用,其潜在机制是通过NF-κB信号通路来实现的,甘露醇可能是其中的一种关键活性单体成分。     

Neo-clerodane diterpenes from Salvia herbacea

Chemical investigation of the aerial parts of Salvia herbacea led to the isolation of eight new neo-clerodane diterpenes (1-8), named tehuanins A-H, and three known compounds. The structures of these compounds were determined by analysis of their spectroscopic data. Three of the new diterpenes possess a 1,8-epoxy group (1-3). This unusual structural feature was confirmed by X-ray diffraction of 1. The structure of the previously isolated 1α,10α-epoxysalviarin was revised. The absolute configuration of 6 was established by X-ray diffraction analysis of its bromo derivative 6a. Cytotoxic and anti-inflammatory activities of these diterpenes were examined. None of the compounds were considered to be cytotoxic; however, compound 7 exhibited anti-inflammatory activity comparable to that of indomethacin.     

当归多糖对小鼠免疫功能的调节作用

目的:研究当归多糖对小鼠免疫功能的影响.方法:ig氢化可的松复制免疫抑制模型;测定胸腺、脾脏重量并计算脏器系数;碳粒廓清法测定单核巨噬细胞吞噬功能;测定血清溶血素IgG、IgM含量;MTT法测定细胞增殖.结果:在10～100mg·kg-1剂量范围内,当归多糖能对抗氢化可的松引起的小鼠脾萎缩,而对胸腺影响不大;显著增加氢化可的松免疫抑制小鼠的碳粒廓清率;而对小鼠血清溶血素IgG、IgM的生成有较强的抑制作用;当归多糖还能显著增强小鼠脾细胞和腹腔巨噬细胞的增殖.结论:当归多糖能增强非特异性免疫功能,而对体液免疫功能有抑制作用.     

猴头菇子实体中的主要多糖成分

目的对猴头菇子实体中的多糖进行分离、纯化和组成分析。方法猴头菇依次经过水提取和碱提取,所得粗多糖以DEAE-celluose色谱、Sephadex凝胶滤过色谱和Fehling试剂沉淀等方法进一步分离纯化,高效凝胶滤过法(HPGPC)鉴定纯度及相对分子质量,气相色谱和IR光谱分析多糖的比旋光度和化学组成。结果从猴头菇子实体中提取得3部分粗多糖CPW、CPB1和CPB2,经分离纯化得到5种多糖HEP-1～HEP-5,其中HEP-1和HEP-4为杂多糖,HEP-3和HEP-5为葡聚糖,HEP-2为酸性多糖。结论猴头菇子实体分到的5种多糖主要含以D-葡萄糖为主的中性糖,以葡聚糖含量最多,其中HEP-1、HEP-2为新类型的真菌多糖。     

Polybrominated diphenyl ethers from the Indonesian sponge Lamellodysidea herbacea

Four new (1-4) and 10 known polybrominated diphenyl ethers (5-14) have been isolated from the title sponge. The structures of the new entities were elucidated by interpretation of spectroscopic data and chemical transformations. These metabolites showed potent antimicrobial activity against Bacillus subtilis and moderate/weak cytotoxicity against NBT-T2 rat bladder epithelial cells. The major constituent 14 was treated under debromination conditions to give eight derivatives, which were subjected to a structure-activity relationship study. The results indicated that the presence of two phenolic hydroxyl groups and bromines at C-2 and/or C-5, as in 2, is important for the exhibition of antibacterial activity.     

Anticonvulsant activities of ethanolic extract and aqueous fraction isolated from Delphinium denudatum

Dried roots of Delphinium denudatum Wall. are a popular folk remedy for the treatment of epilepsy in the traditional Unani system of medicine in the sub-continent. We carried out anticonvulsant screening of the ethanolic extract (EE) and aqueous fraction (AF) of this plant utilising the maximal electroshock (MEST) and subcutaneous pentylenetetrazole (scPTZ), bicuculline (scBIC), picrotoxin (scPTX) and strychnine (scSTN) tests for anticonvulsant activity. EE had weak dose-dependent anticonvulsant effects on seizures induced by PTZ and BIC. AF exhibited dose-dependent activity against hind limb tonic extension phase (HLTE) of MEST and comparatively stronger anticonvulsant activity against seizures induced by PTZ and BIC. The results suggest the presence of potent anticonvulsant compounds in AF of D. denudatum and deserve further investigation for isolation of active compounds and elucidation of the mechanism of anticonvulsant action.     

Inhibition of sustained repetitive firing in cultured hippocampal neurons by an aqueous fraction isolated from Delphinium denudatum

In this report we investigated the effects of the aqueous fraction (AF) isolated from Delphinium denudatum on sustained repetitive firing in cultured neonatal rat hippocampal pyramidal neurons. Blockade of SRF is one of the basic mechanisms of antiepileptic drugs (AED) at the cellular level.

The effects of aqueous fraction (0.2–0.6 mg/ml) were compared with the prototype antiepileptic drug, phenytoin (PHT). Using the whole cell current-clamp technique, sustained repetitive firing was elicited in neurons by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1–0.6 nA current strength. Similar to phenytoin, aqueous fraction reduced the number of action potentials (AP) per pulse in a concentration-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and V_max (velocity of upstroke) of action potentials. The V_max and amplitude of the first action potential was not affected by phenytoin, while aqueous fraction exhibited concentration-dependent reduction. At 0.6 mg/ml aqueous fraction reduced V_max to 58–63% and amplitude to 16–20% of the control values. The blockade of sustained repetitive firing by aqueous fraction was reversed with hyperpolarization of membrane potential (−65 to −75 mV) while depolarization of membrane potential (−53 to −48 mV) potentiated the block.

The results suggest that aqueous fraction blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to phenytoin. However, unlike phenytoin, which interacts preferably with the inactive state of the Na⁺ channel, the compounds present in aqueous fraction apparently also interact with the resting state of the Na⁺ channels as suggested by dose-dependent reduction of V_max and amplitude of first AP. We conclude that aqueous fraction contains potent anticonvulsant compounds.     

In vivo anti-tussive activity and structural features of a polysaccharide fraction from water extracted Withania somnifera

Aim of the study

Anti-tussive drugs are amongst the most widely used medications worldwide; however no new class of drugs has been introduced into the market for many years. The present study aims at evaluating the structural features and in vivo anti-tussive activity of a polysaccharide fraction from water extracted Withania somnifera.

Materials and methods

Herein, we have analyzed water extracted material of Withania somnifera using chemical, chromatographic, spectroscopic and biological methods.

Results

A polysaccharide fraction (F3) containing arabinosyl, galactosyl and galacturonosyl residues were obtained by anion exchange chromatography of the water extracted material. This polymer is branched and contained (1,5)-/(1,3,5)-linked arabinofuranosyl, (1,3)-/(1,6)-/(1,3,6)-linked galactopyranosyl residues together with small amount of terminal rhamnopyranosyl and terminal arabinofuranosyl residues. Peroral administration of this pectic arabinogalactan in a dose of 50 mg kg⁻¹ body weight (b.w.) decreased the number of cough efforts induced by citric acid in guinea pigs like that of codeine.

Conclusions

This study provides a scientific basis for the past and present ethnomedical uses of this plant.     

Free radical scavenging and antioxidant enzymes activation of polysaccharide extract from Nostoc sphaeroides

Nostoc sphaeroides Kuetzing has been used as a traditional medicine in China to treat a variety of ailments. This research identified the antioxidant activities of polysaccharide extract from Nostoc sphaeroides. The extract, which contains 46.2% carbohydrates, exhibited an effective scavenging capability on superoxide radical, hydroxyl radicals in non site-specific as well as site-specific assays, and also performed lipid peroxidation inhibition in a dose-dependent manner. Polysaccharide extract had no 1,1-diphenyl-2-picrylhydrazyl radical scavenging potential at all test concentrations. Activities of superoxide dismutase, catalase, and glutathione peroxidase in human embryo kidney 293 cells were increased effectively when Nostoc sphaeroides extract was applied. These results suggested that the use of N. sphaeroides in treating ailments may be based on the antioxidant capacities of polysaccharide composition.     

An Asparagus polysaccharide fraction inhibits MDSCs by inducing apoptosis through toll‐like receptor 4

Despite decades of research, malignant tumors are extremely difficult to eliminate with conventional methods. Although surgical resection potentially eradicates the problem, only a few cases are suitable for operation, and other approaches often involve harmful consequences. Revolutionary methods are desperately needed to improve patient outcomes and diminish harmful side effects. Myeloid‐derived suppressor cells (MDSCs), downregulators of the innate and adaptive immune systems, have been widely studied over the past 2 decades. MDSCs inhibit the antitumor immune response by suppressing T cell proliferation, cytokine production, and tumor cell killing. With MDSCs becoming novel targets in cancer therapy, our research has focused on the anti‐MDSC function of Asparagus polysaccharide (AP), extracted from asparagus, a traditional Chinese herb. In this study, we have used MDSCs isolated from the spleen of mice with colon cancer as an in vitro model to assess the efficacy of AP. Treatment of MDSCs with AP significantly decreased cell proliferation and induced apoptotic cell death through a toll‐like receptor 4 dependent way. Subsequent studies showed that the AP treatment enhanced the expression of Bax and Caspase‐9 and inhibited the expression of Bcl‐2, suggesting that AP induced apoptosis in the MDSCs via the intrinsic pathway. Altogether, the results showed that AP exhibited a significant anti‐MDSC activity and attenuated suppression of the antitumor immune response, thereby indicating its potential use in cancer therapy.     

Hypoglycemic activity of root water decoction, sesquiterpenoids, and one polysaccharide fraction from Psacalium decompositum in mice

The hypoglycemic activity of Psacalium decompositum (Asteraceae) was investigated in fasting healthy mice and alloxan-diabetic mice. The freeze-dried water decoction significantly reduced the blood glucose in normal mice (from 50.9 +/- 4.7 to 32.5 +/- 3.1 mg/dl) and in mild diabetic mice (from 208.5 +/- 13.0 to 52.3 +/- 7.0 mg/dl), 240 min after intraperitoneal administration (P < 0.005). This preparation also diminished fasting glycemia in severe diabetic mice, but the effects were minor (from 394.4 +/- 9.4 to 289.3 +/- 39.5 mg/dl). The main sesquiterpenoid constituents from P. decompositum roots, cacalol, cacalone and maturin, as well as the transformation product cacalol acetate, did not show a hypoglycemic effect on healthy mice. Nevertheless, two polysaccharide fractions (F1 and F3) obtained from the freeze-dried water extract significantly reduced the fasting glycemia in healthy mice. The best results were obtained with the F1 fraction.     

酶法提取紫背天葵多糖工艺的研究

目的研究纤维素酶法提取紫背天葵多糖的工艺。方法采用苯酚-硫酸法检测多糖含量,以多糖收率为指标选择适当的酶解温度、酶解时间和加酶量,通过正交试验确定酶法提取紫背天葵多糖的最佳条件。结果最佳提取工艺为:酶解温度为55℃,酶解时间3 h,加酶量3%。结论该工艺合理、简单,多糖提取率高。     

银杏叶多糖的抗肿瘤和免疫调节作用

研究了银杏叶多糖(Ginkgo biloba leaf polysaccharidel GBLP)小鼠S180实体瘤、腹水瘤的生长抑制作用和对荷瘤小鼠免疫调节作用。小鼠接种S180实体瘤或腹水瘤，分组饲喂不同剂量(0mg／kg、50mg／kg、100mg／kg、200mg／kg、400mg／kg体重）的GBLP，检测GBLP对小鼠S180实体瘤和腹水瘤生长的抑制作用以及对荷瘤小鼠脾脏指数和胸腺指数的影响。结果显示：GBLP荷瘤小鼠的脾脏指数和胸腺指数显增加；四个剂量的GBLP对S180实体瘤的抑瘤率分别为6．03％、12．99％、55．69％、62．89％；抑制S180腹水瘤的生长，并延长荷瘤小鼠的存活时间，表明GBLP具有较强的免疫调节活性和抗肿瘤作用。     

A preliminary study on extracellular polysaccharide isolated from fermented Tremella aurantia Schw. ex Fr.]

TaA, the extracellular polysaccharide purified from the filtrate of fermented Tremella aurantia, is a water-soluble neutral polysaccharide. Gas chromatography of its alditol acetates has shown that TaA is composed of mannose and xylose by the ratio of 2.85:1. The physical and chemical properties of TaA have been characterized.     

碱提灵芝多糖的分离纯化、结构表征及免疫活性评价

目的从赤芝Ganoderma lucidum中提取、分离和纯化获得碱提多糖组分,在表征其基本理化性质和精细结构的基础上,对其体外免疫调节活性进行研究。方法赤芝子实体干燥粉碎后,利用碱提醇沉法提取粗多糖,经Q-Sepharose Fast Flow离子交换色谱柱纯化得到灵芝多糖组分(LZJ-0.15)。采用PMP柱前衍生-高效液相色谱法(HPLC)及高效凝胶渗透色谱-十八角度激光光散射联用法(HPGPC-MALLS)测定LZJ-0.15的单糖组成及相对分子质量特征,并通过核磁共振氢谱(1H-NMR)、碳谱(13C-NMR)及二维谱(1H-1H COSY和1H-13C HSQC)对LZJ-0.15的精细结构进行表征。采用小鼠巨噬细胞RAW264.7吞噬中性红实验对灵芝多糖LZJ-0.15进行免疫活性评价。结果 LZJ-0.15的相对分子质量、分子半径及Mw/Mn分别为24 700、46.6 nm和1.019,其单糖组成分析显示以葡萄糖为主(92.3%),核磁谱图显示LZJ-0.15为→3)Glc(β1→及→6)Glc(β1→连接为主的葡聚糖。RAW264.7细胞吞噬中性红实验显示LZJ-0.15具有较好的免疫调节活性。结论赤芝碱提灵芝多糖相较于水提多糖组分具有较优的免疫活性,有望开发成重要的免疫调节剂。