小有刺参硫酸软骨素和岩藻聚糖硫酸酯抗血小板聚集活性的比较

目的 从小有刺参中制备纯化硫酸软骨素和岩藻聚糖硫酸酯及其低分子量组分,并比较不同结构硫酸多糖的抗血小板聚集活性,揭示海参硫酸多糖抗血小板聚集活性的构效关系。方法 利用离子交换色谱和自由基降解技术,从小有刺参中制备硫酸软骨素和岩藻聚糖硫酸酯及各自的低分子量组分。采用体外凝血酶诱导血小板聚集模型,比较不同结构的硫酸软骨素和岩藻聚糖硫酸酯对血小板聚集率的影响。结果 4～60 μg/mL硫酸软骨素和低分子量硫酸软骨素具有显著的抑制血小板聚集的活性,在浓度为20 μg/mL时对血小板聚集的抑制率高达94.6%和95.3%,且分子量的降低对其活性无影响。比较不同来源的岩藻聚糖硫酸酯的活性发现,在4～60 μg/mL浓度范围内,海参与海带来源的岩藻聚糖硫酸酯都显著抑制血小板聚集,且活性没有显著差异,但经过自由基降解后,海参和海带来源的岩藻聚糖硫酸酯的抗血小板聚集活性都显著降低。结论 海参硫酸软骨素的抗血小板聚集活性显著高于岩藻聚糖硫酸酯,海参硫酸多糖可以应用于新型抗血小板聚集药物的研究开发。     

不同性质海洋硫酸多糖的制备及其改善大鼠脂肪肝作用的比较研究

目的 探索了不同来源、不同分子量的海洋硫酸多糖对脂肪肝大鼠脂肪代谢的调节作用.方法 SD大鼠随机分为7组,正常对照组、脂肪肝模型对照组、0.1%低分子量海参岩藻聚糖硫酸酯组、0.1%海参岩藻聚糖硫酸酯组、0.2%海参岩藻聚糖硫酸酯组、0.2%海藻岩藻聚糖硫酸酯组、0.2%海参硫酸软骨素组,每组6只.饲料中添加1%乳清酸...     

分子量对海参岩藻聚糖硫酸酯在体内吸收的影响

目的 利用高温高压降解法制备两种不同分子量的岩藻聚糖硫酸酯,探究口服不同分子量的海参岩藻聚糖硫酸酯的吸收特性。方法 采用分子排阻凝胶色谱法、离子高效液相色谱法,检测海参硫酸多糖高温高压降解前后分子量、硫酸根含量的变化,并利用PMP柱前衍生-高效液相色谱法测定岩藻聚糖硫酸酯的单糖组成,以及大鼠血清中单糖的变化。结果 口服低分子量海参岩藻聚糖硫酸酯后,大鼠血清中岩藻糖和半乳糖的吸收速度和最大浓度明显高于中分子量岩藻聚糖组,血清中甘露糖、氨基葡萄糖的含量也显著上升,血清中氨基半乳糖的含量略有上升。而口服中、低分子量岩藻聚糖硫酸酯都能够降低血清中葡萄糖的含量。结论 分子量低于10 kDa的低分子量岩藻聚糖具有很好的体内的吸收率,适合于开发口服岩藻聚糖功能产品。     

不同分子量海参岩藻聚糖硫酸酯的制备及消化吸收特性的初步研究

目的 比较分子量大小对海参岩藻聚糖硫酸酯(SC-FUC)消化吸收特性的影响.方法 采用酶解法制备不同分子量的海参岩藻聚糖硫酸酯.SD大鼠随机分为4组,每组6只,分别灌胃不同分子量的海参岩藻聚糖硫酸酯(1 000 mg·kg-1)后,于0、0.5、2、6、15、24、36和48 h尾静脉取血,采用柱前衍生高效液相色谱法测...     

海参硫酸多糖的高温高压降解工艺及其降解机制

目的 分析海参硫酸多糖的高温高压降解工艺和降解产物的结构,探究硫酸多糖的高温高压降解的机理。方法 采用聚丙烯酰胺凝胶电泳法、分子排阻凝胶色谱法、薄层层析法、离子高效液相色谱法和PMP柱前衍生-高效液相色谱法分析多糖的分子量、硫酸根含量和单糖组成的变化,并利用液相色谱-质谱联用技术分析降解产物中硫酸寡糖的结构特征。结果当pH>7时,海参硫酸多糖在高温加压条件下未见明显降解。添加醋酸使溶液的pH降至6以下,海参硫酸多糖在100～121 ℃加热后,pH越低、温度越高、加热时间越长,分子量降低越明显。海参硫酸多糖的硫酸根和单糖组成在降解后变化很小,降解产物中存在一系列奇数和偶数的硫酸岩藻寡糖和硫酸软骨素寡糖,说明高温高压降解为无规断裂型和解聚断裂混合型。相同条件下,岩藻聚糖硫酸酯比硫酸软骨素更易被降解。 结论 本研究建立了高温高压法高效降解海参硫酸多糖的技术,该方法是一种回收率高、易控制、绿色环保的低分子量海参硫酸多糖的制备方法。     

两种颜色仿刺参Apostichopus japonicus中糖胺聚糖表达谱的差异性研究

目的 基于液质联用和糖组学分析技术,分析比较不同颜色仿刺参中的糖胺聚糖,获得不同颜色仿刺参的糖胺聚糖表达谱信息,从而完善海参糖链表达谱信息,促进无脊椎动物的糖链研究。方法 首先对两种颜色仿刺参进行脱脂和蛋白酶酶解处理,然后采用离子交换柱纯化以去除蛋白和高硫酸化聚糖等杂质并获得糖胺聚糖。采用GAGs酶解获得GAGs二糖并对其进行2-AMAC荧光标记,再基于RP-C18-MS/MS-MRM方法进行GAGs表达谱差异分析。结果 在两种颜色仿刺参中CS含量均最高,为2.5~4.5 μg/g,HA含量次之,HS的含量最低。海参中GAGs总量远低于海参中岩藻糖基化硫酸软骨素和岩藻聚糖硫酸酯的含量,白色仿刺参的GAGs总量明显高于青色仿刺参。两种仿刺参中CS二糖以CS-0S、CS-4S、CS-6S为主,HS二糖主要是HS-NS6S和HS-TriS,但是两者在GAGs二糖含量、百分含量、硫酸化模式和硫酸化程度上均存在较大差异。结论 本文首次获得不同颜色仿刺参糖胺聚糖表达谱信息,有助于了解不同颜色仿刺参来源内源性糖链信息,为海参活性成分的研究提供依据。     

壳聚糖-岩藻聚糖硫酸酯纳米粒的制备及性质研究

目的：本文以低分子量岩藻聚糖硫酸酯为交联剂,研究了聚电解质凝聚法制备壳聚糖-岩藻聚糖硫酸酯纳米微粒(Chitosan-fucoidan nanoparticles, CS-Fuc NPs)的制备工艺,并对纳米粒的胃肠道和贮藏稳定性进行研究。方法：采用聚电解质凝聚法制备壳聚糖-岩藻聚糖硫酸酯纳米微粒,采用体外模拟胃液和模拟肠液消化体系,检测纳米粒的胃肠稳定性,常温贮藏实验测定纳米粒的短期贮藏稳定性。结果：壳聚糖-岩藻聚糖硫酸酯纳米微粒的最优制备条件是：壳聚糖(1mg/ml)与岩藻聚糖硫酸酯(1mg/ml)体积比为1.1/1,pH 4.5,温度30℃。所得纳米粒粒径为227.8 nm,Zeta电位为38.4 mV,PDI为0.231,岩藻聚糖硫酸酯复合率(Fuc%)为94.92%。所制备的纳米颗粒在10周内没有显著变化,在模拟胃环境中稳定性良好,在模拟肠道环境中解聚性良好。结论： 壳聚糖-岩藻聚糖硫酸酯纳米粒制备工艺简单,性能良好,有望成为新型的口服药物运送载体。     

海带低分子量岩藻聚糖硫酸酯的制备和抗血小板聚集活性研究

目的：研究低分子量海带岩藻聚糖硫酸酯的制备和纯化技术,筛选抗血小板聚集活性的低分子量海带岩藻聚糖硫酸酯。方法：以海带硫酸多糖为原料,采用自由基降解和强阴离子交换色谱技术,制备纯化出高硫酸根和高岩藻糖的低分子量岩藻聚糖硫酸酯;采用体外凝血酶诱导血小板聚集模型,筛选出抗血小板聚集活性的低分子量岩藻聚糖硫酸酯。结果：强阴离子交换色谱能够有效地将复杂的低分子量海带岩藻聚糖硫酸酯分离纯化,得到4个纯度较高的高硫酸根、高岩藻糖的低分子量多糖,其岩藻糖含量超过85% ~ 95%,硫酸根含量35%以上,分子量在20 ku ~ 30 ku。体外抗血小板聚集试验结果证明,这4个高硫酸根高岩藻糖组分的抗血小板聚集活性最高,而低硫酸根组分含有较高的半乳糖、甘露糖和糖醛酸,其抗血小板活性非常低。结论：采用自由基氧化降解和强阴离子交换色谱法,可获得抗血小板活性高的高硫酸根高岩藻糖的低分子量岩藻聚糖硫酸酯。     

子安辐肛参皂苷lecanoroside A和C的抗真菌和抗肿瘤活性

子安辐肛参(Actinopyga lecanora)为海参纲楯手目海参科棘皮动物,在我国主要分布于西沙群岛、海南岛海域,资源十分丰富.通过对其体内的皂苷类成分进行研究,从中分离并鉴定了其中5种三萜皂苷:holothurin A、B,lecanoroside A、B和C,其中后3种皂苷为新化合物[1].     

海参中总皂苷含量测定方法的研究

目的 建立一种海参中总皂苷含量的测定方法.方法 以海参三萜皂苷Echinoside A作为标准品,采用香草醛-高氯酸作为显色体系,以60%乙醇提取,正丁醇萃取得海参样品中总皂苷,利用标准曲线测定了11种市售海参样品的总皂苷含量,并采用.皂蛋比(皂苷/蛋白质)作为指标讨论了海参品种、生长环境、加工方法等因素对皂苷含量的影响.结果 确定测定波长为560nm,标准曲线为y=1.3414x-0.0077,该方法在0～0.5 mg范围内呈现良好的线性关系(R2=0.9994).本测定方法的加样回收率为(99.01±2.82)%,RSD为4.68%.结论 方法简便,重现性好,可作为海参中总皂苷含量测定的方法.     

Effect of marine glycosides on adenosinetriphosphatase activity

Marine glycosides from the sea cucumbers Actinopyga agassizi, Holothuria atra, Bohadschia argus, Cucumaria fraudatrix, Astichopus multifidus and Thelenota ananas inhibit both Na+-K+ ATPase and Mg2+-ATPase of rat brain in vitro. The glycoside-cholesterol complex of these compounds does not influence ATPase activity. Asterosaponins from starfishes Linckia guildingi and Linckia laevigata possess a slight inhibiting effect. The triterpene glycosides from sea cucumbers are more powerful inhibitors than steroidal glycosides from starfishes.     

Antischistosomal effect of holothurin extracted from some Egyptian sea cucumbers

Context: Holothuria polii (H. polii) Linnaeus (Holothuriidae), Actinopyga mauritiana (A. mauritiana) Quoy & Gaimard (Holothuriidae) andBohadschia vitiensis(B. vitiensis) Semper (Holothuriidae) are sea cucumbers inhabiting the coasts of Egypt. Their tegument and the cuvierian gland contained a substance called holothurin that was used in traditional medicine. These three species are abundant in the Egyptian coast, however there are no reports about their efficacy as antiparasitic agent.

Objective: The antischistosomal effect of the holothurin extracted from the three species of sea cucumber is investigated.

Materials and methods: The ethanol extract was made from the tegument of bothH. poliiandA. mauritianawhile it was made from the cuvierian gland ofB. vitiensis. The body wall (or cuvierian gland) of the sea cucumber was blended with 95% ethanol in a volume = 4 × tissue weight. Extraction was done at room temperature for one day then filtered. The ethanol was removed by evaporation using Rotavapour (BÜCHI 461 water bath REIII) at 40°C. Later the aqueous residue was placed in a vacuum oven at 20°C for about 48 h to remove water. The resulting dried mass was then stored at ?4°C until use. The percentage yield and the LD₅₀ were calculated for each extract. Each extract was administered orally toShistosoma mansoni infected mice in acute and chronic phases of infection. The dose of one-tenth of LD₅₀ of each extract was administrated to mice (5.4, 62.2, and 10 mg/kg body weight/mouse forH. polii extract (HPE),A. mauritianaextract (AME), and cuvierian gland ofB. vitiensis, respectively) for 24 h. The effects of each extract on the worm burden and total egg count was studied. The effects of each extract on the worm tegument using scanning electron microscope (SEM) were investigatedin vivo andin vitro.

Results: The percentage yield of cuvierian gland extract (CGE) was higher (70%) than the tegument AME (33.4%) and HPE (9.3%). The 24 h LD₅₀ of investigated sea cucumber ethanol extracts were 54.46, 627, and 100 mg/kg body weight/mouse for HPE, AME, and CGE. Oral administration of HPE caused decrease in male and female worm burden of 30-day infected mice to reach 60 and 90%, respectively. HPE decreased the egg count significantly in those mice with 30-day (1.75 egg counts/g tissue,p < 0.05) and 45-day (3.25 egg counts/g tissue,p < 0.05) infections. SEM studies of recovered worms from treated mice with all extracts showed different tegumental changes like formation of blebs, wrinkling, formation of numerous pores, and rupturing of some tubercles. These effects were more pronounced in those worms treatedin vitro represented by severe shrinkage of the tegument, deformation of spines, rupturing, and collapsing of tubercles.

Discussion and conclusion: Results support the hypothesis that holothurin is a promising antischistosomal agent.     

重组人红细胞生成素胶原蛋白双层微条的制备及含量测定

目的:制备重组人红细胞生成素(rhEPO)胶原蛋白双层微条并建立其含量测定方法。方法:以胶原蛋白为包裹材料,以硫酸软骨素钠为释放调节剂,采用凝胶加压成型的方法制备双层微条;采用反相高效液相色谱法测定双层微条中rhEPO的含量。结果:rhEPO检测浓度线性范围为0 .25～20μg/ml(r=0 9998) ,平均回收率为96 .2% ,日内、日间精密度分别为0. 9%、2 .6%(n=3)。结论:rhEPO胶原蛋白双层微条制备方法简单可行,含量测定方法准确、可靠。     

Screening of marine extracts for schistosomicidal activity in vitro. Isolation of the triterpene glycosides echinosides A and B with potential activity from the Sea Cucumbers Actinopyga echinites and Holothuria polii

Context: Praziquantel (PZQ) is the drug available for the treatment of schistosomiasis. The reported reduced cure rates, the failure of treatment after PZQ administration in patients and the existence of resistant parasite strains, reinforce the need to rapidly discover new effective molecules against Schistosoma parasite.

Objective: To screen the methanol extracts of 79 marine organisms for their schistosomicidal activities against Schistosoma mansoni adult worms in vitro and perform bio-assay directed chromatography for the most active extracts to isolate the active compounds.

Materials and methods: Screening of the marine organisms and bio-assay directed chromatography of the most active extracts together with identification of the active isolates using 1D and 2D NMR analysis, were investigated.

Results: Results indicated that the isolates echinosides A and B from the sea cucumbers Actinopyga echinites Jaeger and Holothuria polii Delle Chiaie (Holothuriidae) were highly active. Their LC₅₀ values were equal to 0.19 μg/ml and 0.27 μg/ml, respectively. Detailed ¹HNMR data for echinosides A and B are reported here for the first time.

Discussion and conclusion: These findings demonstrate that the isolated echinosides possess potential in vitro schistosomicidal activity against S. mansoni adult worms. Therefore, echinosides are promising as lead compounds for the development of new schistosomicidal agents.     

咔唑法测定鲨鱼硫酸软骨素含量的方法学研究

目的建立用咔唑法测定鲨鱼硫酸软骨素含量的方法。方法采用咔唑法,以鲨鱼硫酸软骨素为对照品,在浓硫酸介质中经咔唑显色反应后,于515nm波长处分别测定对照品溶液和供试品溶液的吸收度,按标准曲线法计算含量。结果硫酸软骨素量在29~145μg范围内与吸收度呈良好的线性关系,相关系数r=0.999 8,加样回收率为100.5%,RSD=1.3%(n=6)。结论此法简单、快速,结果准确,可用作鲨鱼硫酸软骨素含量的测定方法。     

酶解高效液相色谱法测定硫酸软骨素的含量

目的建立酶解高效液相色谱法测定硫酸软骨素含量的方法。方法采用硫酸软骨素ABC酶制备样品,高效液相色谱法测定含量。以Ultimate XB-NH2柱(4.6 mm×25 cm,5μm)分离样品,醋酸钠缓冲液(pH 5.6)-乙腈(950∶50)为流动相,流速1.0 mL/min,检测波长232 nm,进样量10μL,柱温30℃。结果在此色谱条件下,硫酸软骨素A、B、C三组分分离良好;硫酸软骨素浓度在300～3 000μg/mL之间与峰面积呈良好线性关系;高、中、低三个浓度平均加样回收率为102.1%,100.4%和97.5%。结论本法简便、准确、可靠,可用于硫酸软骨素的含量测定。     

四种多糖抗溃疡作用的研究

糊精、人参果胶、肝素和硫酸软骨素A对四种大鼠实验性胃溃疡(消炎痛型、应激型、幽门结扎型及醋酸型)均有不同程度的抑制作用,前两种植物多糖抗溃疡作用比后两种酸性粘多糖强。其中以糊精的抗溃疡作用最显著。多糖的抗溃疡作用可能与其降低胃酸和抑制胃蛋白酶活性有关。     

鲨鱼硫酸软骨素中蛋白质比色法定量的方法学研究

目的建立用比色法测定鲨鱼硫酸软骨素原料中蛋白质含量的方法。方法采用福林-酚试剂法对蛋白质进行显色反应,以牛血清白蛋白为标准品,在760 nm波长处测定蛋白质标准品溶液和供试品溶液的吸收度,按标准曲线法计算供试品中蛋白质的含量。结果标准曲线蛋白质量在21.1~105.5μg范围内与吸收度呈良好的线性关系,相关系数r=0.999 0,加样回收率为99.8%,RSD=0.91%(n=6)。结论此法简单、快速,结果准确,可用作鲨鱼硫酸软骨素原料中蛋白质含量的测定方法。     

Frondoside A inhibits human breast cancer cell survival, migration, invasion and the growth of breast tumor xenografts

Breast cancer is a major challenge for pharmacologists to develop new drugs to improve the survival of cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A inhibited the growth of pancreatic cancer cells in vitro and in vivo. We investigated the impact of Frondoside A on human breast cancer cell survival, migration and invasion in vitro, and on tumor growth in nude mice, using the human estrogen receptor-negative breast cancer cell line MDA-MB-231. The non-tumorigenic MCF10-A cell line derived from normal human mammary epithelium was used as control. Frondoside A (0.01-5 μM) decreased the viability of breast cancer cells in a concentration- and time-dependent manner, with 50%-effective concentration (EC50) of 2.5 μM at 24h. MCF10-A cells were more resistant to the cytotoxic effect of Frondoside A (EC50 superior to 5 μM at 24 h). In the MDA-MB-231 cells, Frondoside A effectively increased the sub-G1 (apoptotic) cell fraction through the activation of p53, and subsequently the caspases 9 and 3/7 cell death pathways. In addition, Frondoside A induced a concentration-dependent inhibition of MDA-MB-231 cell migration and invasion. In vivo, Frondoside A (100 μg/kg/dayi.p. for 24 days) strongly decreased the growth of MDA-MB-231 tumor xenografts in athymic mice, without manifest toxic side-effects. Moreover, we found that Frondoside A could enhance the killing of breast cancer cells induced by the chemotherapeutic agent paclitaxel. These findings identify Frondoside A as a promising novel therapeutic agent for breast cancer.