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.     

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.     

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.     

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.     

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。     

小球藻糖蛋白的分离纯化与抗氧化活性评价

小球藻经热水抽提、脱蛋白、醇析得粗糖蛋白,再经葡聚糖凝胶层析得到两种糖蛋白组分(CGPⅠ和CGPⅡ),经SDS-聚丙烯酰胺凝胶电泳,证明为电泳纯物质,分子量分别为63.7,21.0kDa。利用α-脱氧核糖法、连苯三酚自氧化法进行抗氧化实验,结果显示:CGPⅠ和CGPⅡ均有一定抗氧化能力,且CGPⅡ高于CGPⅠ。     

Characterization of water soluble polysaccharides from organs of Chinese Jujube (<Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. cv. Dongzao)

Water-soluble polysaccharides (WSPs) in Chinese jujube leaves, fruits and flowers were extracted with hot water from the alcohol insoluble residues (AIRs) and precipitated with ethanol allowed to concentrate the fraction as 7.8, 5.1, 18.3%. Pectic polysaccharides were the major components in all WSPs since all extractions were very rich in sugars such as uronic acid, arabinose and galactose. Polymers extracted with hot water from different organs were different in the degree of branching, degree of esterification. All WSPs revealed immunobiological activities especially in fruits and flowers.     

植物生长调节剂对城市污水中小球藻生长和油脂积累的影响

研究了不同质量浓度植物生长调节剂(IAA、TRIA、6-BA、2,4-D)对蛋白核小球藻(Chlorella pyrenoidosa)生长和油脂积累的影响,并从藻细胞形态,脂肪酸组成及元素含量等方面对机理进行了探讨。结果表明:2,4-D对城市污水中小球藻生长的促进作用最为显著,当2,4-D的质量浓度为1.5 mg/L时小球藻干重和油脂产量分别达到最大值,比空白对照组分别提高了30.77%和51.43%。利用扫描电镜观察可以发现处理组小球藻个体大小略大于空白对照组。通过对生成的脂肪酸甲酯进行气相色谱分析,结果显示植物生长调节剂的投加对小球藻脂肪酸组成影响不大,但提升了单不饱和脂肪酸含量,有利于提高所得生物柴油的品质。     

Antioxidant properties of polysaccharides from Ganoderma tsugae

Ganoderma tsugae Murrill (Ganodermataceae) were available in the form of mature and baby Ling chih, mycelia and fermentation filtrate. From these four forms, hot water extracted and hot alkali extracted polysaccharides were prepared and their antioxidant properties were studied. Polysaccharides showed good antioxidant activity as evidenced by their particularly low EC₅₀ values (<0.1 mg/ml). At 20 mg/ml, both extracted polysaccharides from mycelia showed reducing powers of 0.41–0.52 whereas reducing powers of other polysaccharides were in the range of 0.87 to 1.14. At 20 mg/ml, scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl radicals increased to 93.7–100%, except for that of the hot water extracted polysaccharide, from filtrate, being 74.9%. At 20 mg/ml, scavenging abilities of both extracted polysaccharides from mycelia on hydroxyl radicals were 13.9 and 24.4%, respectively whereas scavenging abilities of the other polysaccharides were in the range of 39.0–55.2%. At 10 mg/ml, the chelating abilities of polysaccharides from mature and baby Ling chih, mycelia and filtrate were 93.9–100%, 97.6–100%, 85.1–88.0% and 51.2%, respectively. Overall, both extracts of polysaccharides possessed good antioxidant properties except for scavenging ability on hydroxyl radicals and can be developed as a new dietary supplement and functional food.     

Antioxidation and antiglycation of polysaccharides from Misgurnus anguillicaudatus

Misgurnus anguillicaudatus is a fish widely inhabiting rivers, lakes, ponds, swamps and rice fields in China, Japan, Korea and other Asian countries. It is edible and can be used to treat xiao ke (namely diabetic) in traditional Chinese medicine. Polysaccharides from M. anguillicaudatus (MAPs) were extracted and separated into four parts which were named MAP I, MAP II MAP III and MAP IV, respectively. The percentage of polysaccharide in dry weight is about 3.06%. Hydroxyl radical and superoxide radical anion scavenging activities together with glycation inhibition of these polysaccharides were investigated. The results showed that all the MAPs had an inhibitory effect on oxidation and glycation and exhibited a dose-dependent response. Especially MAP IV displayed a stronger antioxidation effect than glucosamine and better antiglycation activities than aminoguanidine. As oxidation and glycation are relevant to diabetic complications, the result of this work suggests that MAPs may be effective in preventing and treating diabetic complications.     

塔尔油对蛋白核小球藻抑藻效应及机理研究

以塔尔油为化感物质,研究了塔尔油对蛋白核小球藻的抑藻效果,并对其机理进行了初步探究。研究结果表明,150和100 mg/L的塔尔油可有效控制蛋白核小球藻的生长繁殖,抑藻率超过80%,最高可达88%。在塔尔油的作用下,蛋白核小球藻的过氧化氢酶和超氧化物歧化酶活力均出现升高现象,高于对照组4倍以上,藻细胞的叶绿素A含量直线下降。塔尔油的作用可使藻细胞外观出现凹陷,甚至破裂。与对照组相比,加入塔尔油的实验组Zeta电位绝对值最高下降56%,藻细胞发生絮聚趋势增大。塔尔油可使藻液颗粒粒径分布范围变大,小于正常细胞的碎片含量增加,大于正常细胞粒径分布的藻细胞絮聚团增加,使藻液颗粒平均粒径比对照组增大了25%。加入塔尔油的实验组中,死亡细胞的比例显著增大,从对照组的9.7%分别增至62.8%(100 mg/L实验组)和75.7%(150 mg/L实验组)。     

Effect of ultrasonic treatment on the recovery and DPPH radical scavenging activity of polysaccharides from longan fruit pericarp

Ultrasonic technique was employed to extract polysaccharides from longan fruit pericarp (PLFP). The optimal conditions for ultrasonic extraction of PLFP were determined by response surface methodology. Box–Behnken design was applied to evaluate the effects of three independent variables (ultrasonic power, time and temperature) on the recovery and 1,1′-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of PLFP. The correlation analysis of two mathematical-regression models indicated that quadratic polynomial model could be employed to optimize the ultrasonic extraction of PLFP. From response surface plots, ultrasonic power, time and temperature exhibited independent and interactive effects on the extraction of PLFP. The DPPH radical scavenging activity of PLFP could be improved by application of various ultrasonic power, time and temperature, which was possible due to the degradation of polysaccharides to different extent. The optimal conditions to obtain the highest recovery and the strongest DPPH radical scavenging activity of PLFP were 120 W, 22 min and 60 °C, as well as 241 W, 18 min and 51 °C, respectively. Under these optimal conditions, the experimental values agreed with the predicted ones by analysis of variance. It indicated high fitness of two models used and the success of response surface methodology for optimizing PLFP extraction.     

Effect of methylation on the structure and radical scavenging activity of polysaccharides from longan (Dimocarpus longan Lour.) fruit pericarp

Polysaccharides of longan fruit pericarp (PLFP) were purified by gel filtration chromatography and methylated by methyl iodide. The structure of methylated PLFP was identified by gas chromatography/mass spectrometry. The results indicated that the percentages of methylated Ara, Glc and Gal increased gradually to a maximal value with increasing volume of methyl iodide. Methylation resulted in a decrease in the DPPH radical scavenging activity of PLFP, while the superoxide anion radical scavenging activity of PLFP decreased with increasing the degree of methylation. When the degree of methylation reached up to 47.4% or a higher value, a promoted effect on the generation of superoxide anion was observed. Furthermore, a high correlation coefficient between degree of methylation and superoxide anion radical scavenging activity of PLFP was determined, which indicated the important role of hydroxyl groups of monosaccharide units in the radical scavenging activity of PLFP.     

Immunomodulatory and anticancer activities of phenolics from Garcinia mangostana fruit pericarp

The methanolic extract of Garciniamangostana fruit pericarp was partitioned into butanol and water fractions in this work. Three major phenolics were purified and identified as P₁ [1,3,6,7-tetrahydroxy-2,8-(3-methyl-2-butenyl) xanthone], P₂ [1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl-2-butenyl) xanthone] and P₃ (epicatechin). Strong antioxidant activities were detected for P₁–P₃. In vitro cell proliferation trials indicated that P₁ and P₃ exhibited good immunomodulatory activities when 7.5 μg/ml was used. Furthermore, P₁ and P₃ showed good cytotoxicities against human breast cancer cells (MCF-7) and human colon cancer cells (LOVO). P₁ exhibited the maximal cytotoxicity of 73.06% against MCF-7 cells and of 46.27% against LOVO cells when 62.5 μg/ml was used. The cytotoxicities of P₁, P₂, P₃ and paclitaxel against normal embryonic lung fibroblast cells (HELF) were in a decreasing order: paclitaxel > P₃ > P₁ > P_2. These results suggested that P₁ and P₃ could be used as a potential anticancer agent.