硫酸软骨素快速提取法研究

采用先高温蒸煮后加稀碱与酶解相结合 ,并用氯仿反萃取的方法提取硫酸软骨素 ,与其它提取方法相比较 ,缩短了一半工艺流程 ,同时提高了纯度和提取率 ,减轻了碱法提取所带来的环境污染。     

硫酸软骨素研究现状

硫酸软骨素是从动物软骨中提取的黏性多糖,在医药、化妆品和食品工业上有广泛的用途,市场前景广阔。本对硫酸软骨素的性质和用途做了简要的介绍,并阐述了近几年硫酸软骨素生产工艺的发展状况。     

硫酸软骨素快速提取法研究

硫酸软骨素是用于治疗冠心病、神经系统疼痛及链霉素引起的肝脏障碍和肝炎辅助治疗的药物,来源于动物的软骨。本文采用先高温蒸煮后加稀碱与酶解相结合的方法提取药物,并用氯仿反萃取,较其他方法缩短了原工艺一半流程,提高了纯度,减轻了碱盐提取所带来的环境污染。     

硫酸软骨素蛋白聚糖在脑发育中的作用

蛋白聚糖 (ＰＧ)是一种或多种糖胺聚糖链 (ＧＡＧ)和核心蛋白共价结合形成的复合物 ,硫酸软骨素蛋白聚糖 (ＣＳＰＧ)即核心蛋白与硫酸软骨素 (ＣＳ)链共价交连的一类蛋白聚糖 ,不同的核心蛋白与ＣＳ链相连形成不同的ＣＳＰＧ。聚集蛋白聚糖家族 (ａｇｇｒｅｃａｎｆａｍｉ ｌｙ) ,磷酸蛋白聚糖 (ｐｈｏｓｐｈａｃａｎ) ,神经蛋白聚糖Ｃ(ｎｅｕｒｏｇｌｙＣ)是哺乳动物脑发育中的 3种经典的ＣＳＰＧ。其它如星形软骨素蛋白聚糖(ａｓｔｒｏｃｈｏｎｄｒｉｎ) ,饰胶蛋白聚糖 (ｄｅｃｏｒｉｎ) ,睾丸蛋白聚糖 (ｔｅｓｔｉｃａｎ) ,细胞蛋白聚糖 …     

硫酸软骨素提取工艺的研究

硫酸软骨素是生物界广泛存在的酸性粘多糖,广泛存在于动物的器官软骨。以猪器官软骨为原料,采用碱提取和酶解相结合的方法提取硫酸软骨素,利用正交实验对碱提取过程中的温度,碱浓度以及提取时间等三个关键因素进行优化,并对硫酸软骨素成品的相关指标进行检测。正交实验结果表明,最佳的碱提取条件为：温度40℃、提取时间8h、碱提取浓度为0．15g／mL。在此条件下获得的碱提取液,通过酶解、脱色、醇沉、干燥,得到白色硫酸软骨素成品,总得率为20％,各项指标均符合国家部颁标准,达到出口要求。     

膜分离技术在硫酸软骨素分离方面的应用

介绍了硫酸软骨素的生产工艺 ,应用膜分离技术对现有工艺进行了改进 ,使硫酸软骨素纯度达 95 .2 % ,收率提高3% ,并且简化了操作     

鹿茸硫酸软骨素蛋白聚糖的分离纯化研究

报道了用DEAE-纤维素(DE-23)离子交换柱层析从鹿茸二杠中分离、纯化及鉴定硫酸软骨素的方法.首先用适量蒸馏水浸泡鹿茸二杠并将其捣碎,离心取沉淀用盐酸胍浸提,浸提液对尿素液透析后经DEAE-纤维素(DE-23)离子交换柱层析,吸附大量的硫酸软骨素;再用含盐尿素溶液梯度洗脱、分离后,经软骨素酶消化及琼脂糖凝胶电泳, 与硫酸软骨素标准品比较,证实得到的物质为纯的硫酸软骨素蛋白聚糖,其得率约为48.77%.该方法使硫酸软骨素分离纯化一步完成,大大简化了纯化步骤.     

硫酸软骨素酶产生菌的筛选及酶的分离纯化

利用平板快速筛选法,从鱼腹中筛选到一株产生硫酸软骨素酶的细菌YH311,通过生物特性和生化反应试验考察,初步鉴定为温和气单胞菌（Aeromonas sobria）。培养至36h时,该菌株产酶达到高峰期,培养液的酶活力达09U/mL。利用超声波破碎菌体细胞,分别测定培养液和菌体细胞的酶活力,发现培养液的酶活力要远远大于菌体细胞的酶活力,显示该酶为分泌型表达,属于胞外酶。发酵液经离心后,上清液用硫酸铵分级沉淀,再分别经过CMCellulose、QAEsephadex A50和Sephadex G150层析柱进行逐级分纯,并跟踪酶活,最后获得硫酸软骨素酶。SDSPAGE检测为单一条带,其分子量约为80kD。     

对硫酸软骨素传统提取方法的改进

软骨中硫酸软骨素与蛋白质结合成蛋白多糖,并与胶原蛋白结合在一起。本文采用先高温蒸煮,后加稀碱与酶解相结合提取该药物,ＴＣＡ沉淀蛋白质后,高岭土吸附,再用氯仿连续反萃取,使产品质量达到优级纯,较其他方法缩短了原工艺流程,提高了纯度,减轻了碱－盐提取所带来的环境污染。     

发酵法生产硫酸软骨素的研究进展

硫酸软骨素是一种典型的硫酸化糖胺聚糖,具有多种药物活性,广泛应用于药品、保健品及化妆品行业。硫酸软骨素是动物软骨中蛋白聚糖的主要成分和少数几种细菌的荚膜多糖,因此可利用动物提取法和发酵法进行生产。以下综述了硫酸软骨素的发酵生产及其合成机制的研究进展,并对其发展趋势进行了展望。     

Tissue specific distribution of sulfated mucopolysaccharides in mammals

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied.The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

Glycosaminoglycan structures required for strong binding to midkine,a heparin-binding growth factor

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.     

Changes in sulfated mucopolysaccharide composition of mammalian tissues during growth and in cancer tissues

The distribution of sulfated mucopolysaccharides in different tissues during growth and in cancer tissues is reported. It is shown that most of the tissues of 1 day-old rats and rabbits contain chondroitin sulfate A/C, chonroitin sulfate B and heparan sulfate in about the same proportions, whereas in adult animals chondroitin sulfate A/C decreases in concentration or disappears. Changes in the relative proportions of chondroitin sulfate B and heparan sulfate were also observed in most of the tissues. In rats, these changes occur in the first 25 days of extrauterine development. A great increase of chondoitin sulfate A/C was observed in human tumors of different origins when compared with the normal adjacent tissues. Changes in the relative proportions of chondroitin sulfate B and heparan sulfate were also observed in most of the tumors analysed. The possible role of chondroitin sulfate A/C in cell division is discussed in view of the present findings.     

Molecular cloning of squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase and synthesis of a unique chondroitin sulfate containing E-D hybrid tetrasaccharide structure by the recombinant enzyme

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO(4)) residues in chondroitin sulfate (CS). We previously purified squid GalNAc4S-6ST and cloned a cDNA encoding the partial sequence of squid GalNAc4S-6ST. In this paper, we cloned squid GalNAc4S-6ST cDNA containing a full open reading frame and characterized the recombinant squid GalNAc4S-6ST. The cDNA predicts a Type II transmembrane protein composed of 425 amino acid residues. The recombinant squid GalNAc4S-6ST transferred sulfate preferentially to the internal GalNAc(4SO(4)) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO(4)) could be sulfated efficiently when the GalNAc(4SO(4)) residue was included in the unique nonreducing terminal structure, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), which was previously found in CS-A. Shark cartilage chondroitin sulfate C (CS-C) and chondroitin sulfate D (CS-D), poor acceptors for human GalNAc4S-6ST, served as the good acceptors for the recombinant squid GalNAc4S-6ST. Analysis of the sulfated products formed from CS-C and CS-D revealed that GalNAc(4SO(4)) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) was generated in the resulting sulfated glycosaminoglycans. These observations indicate that the recombinant squid GalNAc4S-6ST is a useful enzyme for preparing a unique chondroitin sulfate containing the E-D hybrid tetrasaccharide structure.     

Chlorophyll fluorescence performance of sweet almond [Prunus dulcis (Miller) D. Webb] in response to salinity stress induced by NaCl

One-year old sweet almond (Prunus dulcis) seedlings were submitted to four levels of salt stress induced by NaCl, namely 0.3, 0.5, 0.7, and 1.0 S m⁻¹. Effects of salt stress on a range of chlorophyll (Chl) fluorescence parameters (Chl FPs) and Chl contents were investigated in order to establish an eco-physiological characterization of P. dulcis to salinity. Salt stress promoted an increase in F₀, F_s, and F₀/F_m and a decrease in F_m, F′_m, F_v/F_m, q_P, ΔF/F′_m, F_v/F₀, and UQF_(rel), in almost all Chl fluorescence yields (FY) and FPs due to its adverse effect on activity of photosystem 2. No significant changes were observed for quenchings q_N, NPQ, and q_N(rel). The contents of Chl a and b and their ratio were also significantly reduced at increased salt stress. In general, adverse salinity effects became significant when the electric conductivity of the nutrient solution (EC_n) exceeded 0.3 S m⁻¹. The most sensitive salt stress indicators were F_v/F₀ and Chl a content, and they are thus best used for early salt detection in P. dulcis. Monitoring of a simple Chl FY, such as F₀, also gave a good indication of induced salt stress due to the significant correlations observed between the different Chl FYs and FPs. Even essential Chl FYs, like F₀, F_m, F′_m, and F_s, and mutually independent Chl FPs, like F_v/F₀ and q_P, were strongly correlated with each other.