Purification and identification of polysaccharide derived from Chlorella pyrenoidosa

The conditions for extracting and purifying polysaccharides from Chlorella pyrenoidosa, including intensity and duration of ultrasound, the temperature and incubation time, and ethanol concentration, were investigated through an orthogonal design of L₁₆(4⁵) in this work. High performance liquid chromatography (HPLC) and gas chromatography (GC) were used to characterize the compounds in C. pyrenoidosa. The highest yield of 44.8 g kg⁻¹ was achieved at 400 W of ultrasound for 800 s and then followed by incubation in water bath at 100 °C for 4 h in 80% ethanol. Two polysaccharide fractions (S1 and S2) were separated from the extracts of C. pyrenoidosa using Sepharose 4B column chromatography. The average molecular weights (M_w) of S1 and S2 were 81,877 Da and 1749 Da, respectively. Gas chromatographic (GC) traces of the hydrolyzed polysaccharides showed that most of the majority of monosaccharide in both fractions was mannose (78.0% and 76.5% of relative mass from S1 and S2, respectively) with low levels of glucose (13.2% and 8.4% of relative mass from S1 and S2, respectively). The Fourier-transform infrared spectra (FT-IR) of S1 and S2 revealed typical characteristics of polysaccharides. Both samples had the characteristics of hydroxyl groups, weak C–H band and α-pyranoses; however, only S2 had a carboxyl group.     

Preparation,identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa

Ultrafiltration membranes of different pore size were applied to fractionate Chlorella pyrenoidosa polysaccharides (CPPS) and the main fraction could be separated by a membrane with nominal molecular weight cut-off (NMWCO) of 30 kDa. Ultrafiltration parameters of 40 °C 14.0 psi were optimized for obtaining the main fraction. The resulting sample was further purified by anion-exchange chromatography and size exclusion chromatography, and two distinctive polysaccharides, CPPS Ia and IIa were recovered. CPPS IIa had infrared spectral characteristic of polysaccharides similar to CPPS Ia, and the symmetrical stretching peak at 1408–1382 cm⁻¹ was an indication of the presence of carboxyl groups. The peak molecular weights were 69658 Da and 109406 Da, for CPPS Ia and CPPS IIa, respectively. Both CPPS Ia and IIa were composed of rhamnose, mannose, glucose, galactose and an unknown monosaccharide. Galactose (relative mass 46.5%) was the predominant monosaccharide of CPPS Ia and in CPPS IIa, rhamnose (37.8%) was predominant. CPPS Ia and IIa presented significantly higher antitumor activity against A549 in vitro than did a blank control, in a dose-dependent manner. Both fractions might be useful for developing natural safe antitumor drugs from C. pyrenoidosa resources.     

Structural characterisation of acid- and alkali-soluble polysaccharides in the fruiting body of Dictyophora indusiata and their immunomodulatory activities

The acid- and alkali-soluble polysaccharides (DIPs I and II) were extracted from the fruiting body of Dictyophora indusiata in this work. DIP I comprised of Glc (glucose), Fru (fructose) and Man (mannose), whereas DIP II was of Glc and Fru. Glc was the dominant monosaccharide in both DIPs with relatively molar percentage of >60%, which constructed the backbone of polysaccharide chain. The analysis of the glycosidic linkages indicated that Glc occurred as Glc 1 → 6 in DIP II. The immunological assays showed that both DIPs I and II had a noticeable effect on the hemolysis antibody level in the tested dosage range. However, DIP I could improve the weight of thymus organ and phagocytosis of monocyte. DIP II could restore delayed-type hypersensitivity reaction to dinitrofluorobenzene (DNFB), improving the activity of natural killer cells and the proliferation of splenocytes at high dose.     

灵芝生物转化蛋白核小球藻发酵粗多糖的抑瘤实验

在灵芝基础培养基中添加小球藻粉,灵芝能对小球藻细胞进行生物破壁,同时进行牛物转化：藻粉添加量以10g／L为最佳,产生的多糖最高达0．29mg／L。提取转化的多糖,并与灵芝多糖、小球藻提取多糖的混合物进行抗肿瘤实验：结果表明,产生的转化多糖抗肿瘤效果最好,仡最适剂量（0．2g／kg·d）时,抑瘤率为76．2％。     

小球藻多肽的制备、表征及抗氧化活性研究

目的 优化蛋白核小球藻多肽的提取工艺,探究其蛋白与多肽的结构差异及多肽的抗氧化性能,深入开发其蛋白资源。方法 分别用高压均质法、热碱法、酶解法提取小球藻蛋白;结合酶解和发酵的方法,用单因素实验优化提取小球藻中多肽;采用扫描电镜、红外光谱等表征方法测定其蛋白与多肽的物理性能;结合3种抗氧化实验,研究其多肽的抗氧化性能。结果 蛋白的最佳提取方法为热碱法;固定酶解条件,多肽的最佳发酵条件为发酵温度37℃,发酵液pH 5.0,发酵时间24 h,菌接种量为3%;蛋白和多肽的形貌都呈现不规则片状;蛋白的粒径为54.13±0.17 μm,多肽的粒径为15.60±0.74 μm;蛋白与多肽的二级结构具有显著性差异;综合考虑多肽的抗氧化性能和成本,多肽的最佳浓度为6 mg/mL。结论 采用酶解辅助发酵小球藻蛋白质的方式,可以获得无异味、且抗氧化性能优异的小球藻多肽。     

Purification, antitumour and immunomodulatory activity of water-extractable and alkali-extractable polysaccharides from Solanum nigrum L.

The water-extractable and the alkali-extractable polysaccharides from Solanum nigrum L. (named SNLWP and SNLAP, respectively) and their four polysaccharide sub-fractions (SNLWP-1, SNLWP-2, SNLAP-1 and SNLAP-2), were isolated and purified by ethanol precipitation, dialysis, anion-exchange and gel filtration chromatography. Antitumour and immunomodulatory activity of the polysaccharides was evaluated by determining the survival time, the ascites volume, the weight of immune organ and the level of cytokine (IL-2, IL-10 and IFN-γ) of H₂₂-bearing mice, respectively. The results showed that SNLWP-1, SNLAP-1 and SNLAP-2 had significant antitumour and immunomodulatory activity, whereas SNLWP-2 hardly demonstrated the activity. SNLWP-1 contained galactose and arabinose as main sugar components, and both SNLAP-1 and SNLAP-2 were rich in xylose, galactose and arabinose. SNLWP-2 was rich in glucose. In conclusion, SNLWP-1, SNLAP-1 and SNLAP-2 have potent antitumour activity which may be associated with their potent immunostimulating effect and monosaccharide composition.     

超声辅助热水浸提小球藻多糖及抗氧化活性测定

选取超声时间、提取温度和提取时间为变量,在单因素试验的基础上,利用响应面优化超声辅助热水浸提小球藻多糖的提取条件。利用傅里叶红外变换（FTIR）对提取的多糖进行结构表征,并测定了其体外抗氧化活性。结果表明,超声辅助热水浸提小球藻多糖的最佳条件为：超声时间52 min,提取温度97 ℃,提取时间3.0 h。此优化条件下,多糖得率为5.30%。FTIR结果显示该多糖含有糖醛酸,为吡喃糖。同时该多糖对1,1-二苯基-2-三硝基苯肼（DPPH）、OH自由基有显著清除作用,半抑制浓度（IC50值）分别为4.83 mg/mL、0.77 mg/mL。试验结果表明超声辅助热水浸提法可有效提取小球藻多糖并保留多糖活性。     

蛋白核小球藻的培养及应用研究

选用密闭气升式反应器进行自养培养蛋白核小球藻,通过单因素优化获得碳源二氧化碳的通气量2%,氮源硝酸钠为10 g/L;通过对蛋白核小球藻培养基的均匀设计,获得了适宜的培养基组成：磷酸氢二钾0.6 g/L,硫酸镁0.2 g/L,硝酸钠11 g/L,硫酸钾0.8 g/L,柠檬酸铁胺0.006 g/L,氯化钙0.05 g/L,维生素B10.11 g/L,EDTANa20.01 g/L,钨酸钠0.08×10–3 g/L,氯化镍0.02×10–3 g/L;实验采用中间白光四周彩光的合适光源,用除氧剂使氧气维持在20%左右,获得了8.648 g/L的干菌体。经分析,蛋白核小球藻蛋白质含量高达58 g/100 g,同时含有丰富的氨基酸和矿物质元素。用蛋白核小球藻替代艾草粉,制作出了蛋白核小球藻青团。     

灵芝转化小球藻发酵液粗多糖对荷瘤小鼠免疫功能的影响

在灵芝基础培养基中添加蛋白核小球藻粉,灵芝能对小球藻细胞进行生物破壁,同时进行生物转化。提取转化多糖,与基础培养基所产多糖进行比较,研究对荷瘤动物免疫功能的影响。结果表明,产生的转化多糖能显著提高荷瘤小鼠白细胞的数量、脾细胞增殖能力和单核巨噬细胞吞噬能力等免疫学功能功能,对其他免疫调节功能也有一定的提高作用,从而显著地提高了抑瘤效果。     

米糠多糖及硫酸酯化米糠多糖的体外免疫应答和抗肿瘤活性研究

以小鼠黑色素瘤细胞(B16)为抗肿瘤模型,通过MTT比色法评价米糠多糖及硫酸酯化米糠多糖对B16增殖抑制能力。以小鼠巨噬细胞Raw264.7细胞系为免疫模型,通过MTT比色法评价Raw264.7增殖能力,中性红吞噬试验评价巨噬细胞活性,Griess方法检测一氧化氮(NO)释放量,以及酶联免疫(ELISA)检测肿瘤坏死因子(TNF-α)分泌量,考察米糠多糖及硫酸酯化米糠多糖的免疫活性。研究发现:米糠粗多糖(RBP)和米糠多糖纯化组分(RBP2a)主要通过增强机体的免疫功能而间接抑制肿瘤细胞。质量浓度为250μg/m L时,RBP和RBP2a样品组的NO释放量分别为对照组的4.67、6.36倍,TNF-α分泌量为对照组的441.1、465.5倍;RBP直接组和间接组的B16抑制率分别为8.16%、45.55%,间接组的B16抑制率比直接组增长458%。硫酸酯化米糠多糖(SRBP-B,SRBP-D,SRBP2a-B)一方面可以直接抑制B16增殖,质量浓度为1 000μg/m L时,对B16抑制率达73.65%、65.53%、78.43%,另一方面也可通过免疫途径提高NO和TNF-α等细胞因子释放,进一步提高抗肿瘤活性。但高浓度SRBP-B,SRBP-D,SRBP2a-B能抑制Raw264.7增殖,在500μg/m L时,Raw264.7存活率仅为83.26%、81.8%、79.78%。     

海水小球藻抗菌多糖的分离纯化

海水小球藻抗菌粗多糖以DEAE-纤维素52离子交换层析分离,得到一个A1洗脱峰。经纯度鉴定,A1为均一组分,总糖含量为89.2%,硫酸根含量为12.0%,含有葡萄糖、半乳糖、阿拉伯糖、甘露糖、鼠李糖、核糖等单糖组分。红外光谱显示海水小球藻多糖具有多糖类物质的一般特征,是一个硫酸化程度较高的多糖。     

Isolation and characterization of antitumor polysaccharides from the marine mollusk Ruditapes philippinarum

Aqueous purified extract (PE) was isolated from the crude extract (CE) of the marine mollusk, Ruditapes philippinarum. Two water-soluble polysaccharides, PEF1 and PEF2, were purified from PE. The chemical structures were determined using FTIR, GC, HPLC and ¹³C NMR. The results indicated that PEF1 and PEF2 were homoglucan–protein complexes with a protein content of 26.0 and 8.2%, and their average molecular weights were about 2.0 × 10⁶ and 5.0 × 10³, respectively. The glucan moiety of PEF1 was mainly a (1 → 6)-branched (1 → 4)-α-d-glucan, while that of PEF2 contained (1 → 4)-α-d-glucan and (1 → 6)-β-d-glucan. PE fractions contained large quantities of glutamic acid and aspartic acid, but no cysteine. The antitumor activities were tested both in vitro and in vivo. PE showed significantly the highest tumoricidal activity against human hepatoma SMMC-7721 cells, and PEF1 had better cytotoxicity than PEF2. There was no observed cytotoxicity in human hepatocyte HL-7702 cells within the experimental concentration range for all PE fractions. PE showed antitumor activity against solid tumor Sarcoma 180 in a dose-dependent manner. It also demonstrated stimulating effect on murine lymphocyte proliferation induced by concanavalin A. The results demonstrated that the presence of bound protein, compositional glucan and moderate molecular mass would be helpful to the enhancement of antitumor activities. This study suggested that the polysaccharides isolated from the marine mollusk had a potential application as natural antitumor and immunomodulator agents.     

Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis

Exopolysaccharide (EPS) was isolated and purified from Lactococcus lactis subsp. Lactis culture broth. Selenium chloride oxide (SeCl₂O) was added to the EPS to synthesize selenium-exopolysaccharide (Se-EPS). The in vitro and in vivo antioxidant and in vivo immunomodulatory activity of EPS and Se-EPS were compared. EPS and Se-EPS scavenged superoxide anions and hydroxyl radicals. They also increased catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, while decreasing malondialdehyde (MDA) levels in serum and in the livers of mice. Se-EPS showed stronger in vitro and in vivo antioxidant activity than were shown by EPS. The in vivo immunoenhancement activity of EPS and Se-EPS induced by cyclophosphamide (CY) treatment in immunosuppressed mice was researched. EPS and Se-EPS treatments increased macrophage phagocytosis, spleen and thymus indices and haemolytic complement activity (HC₅₀). Se-EPS showed stronger immunomodulatory activity than did EPS.     

芜菁酸性多糖体内抗肿瘤活性研究

目的：探究芜菁酸性多糖一组分（BRAP-1）对非小细胞肺癌H226细胞荷瘤小鼠的影响。方法：将60只小鼠分为模型组（0.1 mL/10 g）、阳性药物顺铂组[3 mg/（kg·2 d）]、BRAP-1低剂量组[50 mg/（kg·d）]、BRAP-1中剂量组[100 mg/（kg·d）]和BRAP-1高剂量组[200 mg/（kg·d）]。试验期间观察小鼠肿瘤生长情况,计算抑瘤率、脏器指数;酶联免疫法检测各组小鼠血清中的白细胞介素18（IL-18）、IL-1β和肿瘤坏死因子α （TNF-α）含量;实时荧光定量核酸扩增法（q-PCR）、免疫印迹试验检测IL-18、IL-1β、受体相互作用蛋白1（RIP-1）、RIP3、混合谱系激酶结构域样蛋白（MLKL）基因表达情况。结果：与模型组相比较,BRAP-1低、中剂量组抑瘤效果明显（P＜0.05）,阳性组和BRAP-1高剂量组抑瘤率效果显著（P＜0.01）;模型组与其他各组相比较,阳性组和BRAP-1高剂量组小鼠血清中IL-1β含量显著增加（P＜0.01）,IL-18、TNF-ɑ在阳性组中明显降低（P＜0.05）,BRAP-1各剂量组中显著增加（P＜0.01）;q-PCR、免疫印迹试验结果显示,与模型组相比IL-1β随给药剂量增加mRNA相对表达量减少（P＜0.01）,而IL-18、MLKL、RIP1、RIP3的mRNA及蛋白相对表达量增加（P＜0.01）。结论：BRAP-1可以通过调节免疫细胞因子水平,上调坏死性凋亡相关蛋白MLKL、RIP1、RIP3抑制肺鳞癌H226细胞体内生长。     

农杆菌介导小球藻快速转化方法的建立与条件优化

基于根瘤农杆菌（Agrobacterium tumefaciens）介导的方法实现异养蛋白核小球藻快速遗传转化,并优化转化条件。结果表明,小球藻异养生长条件的碳氮组合配比为葡萄糖20 g/L和酵母粉2 g/L时,生长速率为自养的3～5倍;根瘤农杆菌GV3101和LBA4404均可成功实现对异养小球藻的遗传转化,且转化效率无显著性差异（P＞0.05）。最优转化条件为根瘤农杆菌GV3101初始OD600 nm值= 0.8,小球藻（Chlorella pyrenoidosa）浓度为107个/mL,各取100 μL混合涂布在pH=5.4、乙酰丁香酮浓度为200 μmol/L的CM平板上,24 ℃避光培养40 h后转到筛选平板,仅2 d转化子便清晰可见,数目达88个/106微藻。转化过程整体耗时仅5 d,普遍比自养微藻转化时间缩短3倍以上,为小球藻代谢工程改造提供技术手段,同时为其他可异养培养微藻的短时高效遗传转化提供科学依据。     

培养条件对蛋白核小球藻生长及油脂合成的影响

以富油蛋白核小球藻为出发藻株,研究自养、异养和混养培养模式对小球藻生物量和油脂含量的影响,以及异养发酵培养基葡萄糖质量浓度、氮源种类及质量浓度对小球藻生长的影响。结果表明,与自养和混养培养模式相比,采用异养发酵方式培养蛋白核小球藻可获得最大的生物量和油脂含量。通过气相色谱法测得异养蛋白核小球藻油主要脂肪酸为棕榈酸(36.07%)、油酸(34.26%)、亚油酸(20.17%)和亚麻酸(6.12%)。经单因素试验优化得到最适蛋白核小球藻生长异养发酵培养基的葡萄糖质量浓度为60 g/L,最适的氮源为酵母粉,质量浓度为4 g/L,在此条件下经192 h发酵,蛋白核小球藻生物量可达12.43 g/L,油脂产量为5.45 g/L。研究结果表明,异养发酵培养获得的蛋白核小球藻油是一种潜在且可再生的新油源。     

Anti-diabetic effects of polysaccharides from Opuntia monacantha cladode in normal and streptozotocin-induced diabetic rats

Opuntia was widely known for its use in herbal medicines to treat many diseases. In this work, the polysaccharides of Opuntia monacantha cladode (POMC) were extracted by distilled water and classified by ethanol solution with different concentrations. POMC could decrease the daily water consumption in streptozotocin-induced diabetic rats comparable to dimethylbiguanide, a commercial anti-diabetic drug. An increase to food intake was also shown for streptozotocin-induced diabetic rats administered by POMC. By determination of blood glucose (BG), total cholesterol (TC), total triglyceride (TG) and high density lipoprotein cholesterol (HDL) levels, it revealed that POMC had beneficial effects on the improvement in the control of blood glucose and serum lipid level. Daily treatment with 100–300 mg/kg body weight of POMC for four weeks not only brought a significant decrease on blood glucose level in streptozotocin-induced diabetic rats, but also enhanced the cardioprotective lipid HDL level (P < 0.05). The insulin level in streptozotocin-induced diabetic rats was not significantly affected by POMC and dimethylbiguanide treatment (P > 0.05). The mechanism of POMC's hypoglycemic action might be similar to that of dimethylbiguanide.

Industrial relevance

Opuntia cladode has been used traditionally as a herbal medicine for treating diabetes, burns, bronchial asthma and indigestion in many countries over the world. Polysaccharides in it might be responsible for these beneficial properties. In the present study, polysaccharides were prepared from Opuntia cladode and their hypoglycemic effects were determined. The results indicated that this polysaccharides could improve the control in blood glucose and serum lipid levels of streptozotocin-induced diabetic rats. This conclusion would be helpful for further developing this traditional medicinal herb.     

Isolation, partial characterisation and immunomodulatory activities of polysaccharide from Morchella esculenta

BACKGROUND: Polysaccharides extracted from fungi or algae have shown a variety of medical activities, and the exploitation of polysaccharides for application in pharmacy has been very promising. In this study, the immunomodulatory properties of polysaccharides from Morchella esculenta were investigated to identify immunostimulatory activity and potentially contribute to the therapeutic potential of Morchella esculenta. RESULTS: A water‐soluble polysaccharide, MEP, was obtained from the fermentation broth of Morchella esculenta. Two fractions of this polysaccharide (MEP‐I and MEP‐II) were extracted and purified. High‐performance gel permeation chromatography analysis showed the average molecular weights of two polysaccharides. Structural properties and compositions of these two fractions were examined by Fourier transform infrared and a high‐performance liquid chromatography with an evaporative light scattering detector. Active experiments suggested that the MEP had typical immunostimulatory activity. CONCLUSION: These bioactivity tests showed that the polysaccharides from Morchella esculenta presented significant immune modulating activity, and this finding may offer the basis for the popular use of polysaccharides in functional foods or medicine. Copyright © 2011 Society of Chemical Industry     

高效液相色谱法测定蛋白核小球藻中的叶黄素

采用反相高效液相色谱法(RP-HPLC)分离测定了蛋白核小球藻中的叶黄素。样品制备采用甲醇-二氯甲烷(体积比为2：1)混合溶剂为叶黄素提取剂，所用色谱柱为Nova-Pak~(?)C_(18)不锈钢柱，以甲醇-乙腈-水(体积比为80：10：10)和甲醇-乙腈(体积比为40：60)为流动相，线性梯度洗脱，流速0.8mL／min，在447nm下用光电二极管阵列检测器(DAD)检测。测定结果显示，在0～60mg／L范围内，标准曲线呈良好的线性关系(r=0.9999)，叶黄素的保留时间为11.25min，浓度的相对标准偏差为0.49％(n=6)，平均加样回收率为99.7％。该法简单、快速、准确。     

烘焙温度对小球藻脂肪酸、色素及乙醇提取物抗氧化活性的影响

探讨烘焙温度对小球藻脂肪酸组成、色素成分及乙醇提取物抗氧化活性的影响。结果表明:小球藻的脂肪酸以多不饱和脂肪酸为主,其含量达到细胞总脂的76.69%。其中,亚油酸(C_(18:2))含量最高,亚麻酸(C_(18:3,n3))次之。温度不高于150℃时,烘焙处理对小球藻的脂肪酸含量及组成无明显影响;但当温度达到200℃时,烘焙处理会显著降低多不饱和脂肪酸的含量。小球藻的色素成分及乙醇提取物的抗氧化活性对烘焙温度很敏感。当烘焙温度高于100℃时,小球藻的叶绿素和类胡萝卜素降解严重;当烘焙温度高于150℃时,小球藻的抗氧化活性也明显减弱。