CN107056962B - 一种贻贝多糖及其制备方法和应用 - Google Patents
一种贻贝多糖及其制备方法和应用 Download PDFInfo
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- CN107056962B CN107056962B CN201710351379.4A CN201710351379A CN107056962B CN 107056962 B CN107056962 B CN 107056962B CN 201710351379 A CN201710351379 A CN 201710351379A CN 107056962 B CN107056962 B CN 107056962B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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Abstract
本发明提供了一种由贻贝中提取的贻贝多糖及其工业化提取方法和该贻贝多糖制备降血脂、抗动脉粥样硬化的药物和保健食品中的应用。所述贻贝多糖,以(1→4)‑α‑D‑Glc为主链,含有(1→2)‑α‑D‑Glc分枝,构型为α‑吡喃型葡聚糖,平均每12个葡萄糖主链中有1个(1→2)葡萄糖分枝,相对分子量为800‑8000 KDa。所述贻贝多糖通过粗提、酶解、提纯、干燥等步骤提取自贻贝属(Mytilus sp.)煮贝干,提取条件温和、产品纯度高。本发明的贻贝多糖能显著降低甘油三酯、总胆固醇和低密度脂蛋白胆固醇,具有降脂作用,在制备治疗或预防高血脂症、动脉粥样硬化的药物,制备具有降血脂功效的保健品或特殊医学用途配方食品中具有广泛应用。
Description
技术领域
本发明属于天然产物提取及应用领域,涉及一种贻贝多糖及其制备方法和应用。
背景技术
脂质代谢紊乱导致的高脂血症与人类健康的头号杀手心脑血管疾病的发生有着密切的关系,是威胁人类生命健康的最重要的致病因素之一。目前市场上流行的降血脂药物以他汀类药物应用最广、疗效最好,约占调血脂药物销售总额的80%以上。但他汀类药物存在与肝、肾及体内代谢障碍相关的不良反应,另外,还常见有胃肠反应、鼻炎、鼻窦炎、头痛、咽痛、流感综合症、关节炎、胸痛、失眠、肌肉毒性等毒副作用。这使得对他汀类药物替代产品的需求极为迫切。
多糖是所有生物有机体的重要组成部分,具有独特的生物学活性,能提高机体的免疫功能,并参与一切重要的生命活动过程,具有广泛的免疫增强、降血脂、抗肿瘤等作用,同时不良反应又很小。近年来随着开发海洋资源的兴起,人们对海洋生物多糖产生了极大的兴趣 ,发现了许多具有生物活性的多糖类物质,包括具有抗病毒活性的硫酸多糖、促进骨愈合的海洋细菌多糖等。
贻贝多糖来源于海洋动物贻贝(Mytilus sp.)。贻贝是海洋贝类养殖中的重要种类,又名海虹,俗称“海中鸡蛋”,具有明显的补肝肾、降血脂等功效,有很高的药食价值。研究发现贻贝降血脂的功能主要源自于其体内的多糖。目前,贻贝多糖的化学结构尚不明确,其降脂功效和毒性评价也影响着其进一步应用于药物及保健食品。因此,制备高纯度的贻贝多糖并对其进行结构解析、功效研究和毒性评价,对进一步将其开发成天然来源降血脂药物和保健食品具有重要意义。
发明内容
本发明提供了一种由贻贝中提取的贻贝多糖及其工业化提取方法。
本发明另一目的是提供贻贝多糖的在制备降血脂、抗动脉粥样硬化的药物和保健食品中的应用。
为实现上述目的,本发明采用如下技术方案。
一种贻贝多糖,以(1→4)-α-D-Glc为主链,含有(1→2)-α-D-Glc分枝,构型为α-吡喃型葡聚糖,平均每12个葡萄糖主链中有1个(1→2)葡萄糖分枝,其结构式为:
;
所述贻贝多糖相对分子量为800-8000 KDa。
所述贻贝多糖提取自贻贝属(Mytilus sp.)动物,包括厚壳贻贝(Mytilus coruscus)和紫贻贝(Mytilus edulis)。
一种上述贻贝多糖的提取方法,包括以下步骤:
(1)原料处理:贻贝加热去壳留肉并干燥,粉碎机粉碎,过20-40目筛,得贻贝干粉;
(2)多糖提取:步骤(1)制得的贻贝干粉中加入6倍体积的水,100 ℃提取2 h;过滤去除贻贝肉渣,得多糖提取液;
(3)酶解:将步骤(2)制得的多糖提取液调pH至8.0,加入0.2 %w.t的碱性蛋白酶,50℃酶解2 h,酶解完成后在80℃条件下灭活15-30 min,得酶解液;以硅藻土为助滤剂,抽滤过滤,得到澄清的酶解液;
(4)乙醇沉淀:在步骤(3)制得的多糖溶液中加入1.5倍90-95%(v/v)的乙醇进行沉淀,静置6 h,倾去上清液,得到多糖沉淀;
(5)脱水:在步骤(4)制得的多糖沉淀中加入1倍体积90-95%(v/v)的乙醇进行脱水;
(6)过滤干燥:用400目筛网对脱水后的多糖沉淀进行过滤,收集沉淀,60 ℃真空干燥,得到粗多糖;
(7)重溶:将步骤(6)所得粗多糖溶于水,配制10%贻贝粗多糖溶液,12000 rpm离心1h,将上清液用0.45μm滤膜过滤,得多糖滤液;
(8)离子交换层析:将步骤(7)的多糖滤液用DEAE-琼脂糖(FF)层析柱纯化,以0.05mol/L NaCl溶液为洗脱液,收集洗脱组分;
(9)超滤:用截留分子量为5000的滤膜对步骤(8)所得洗脱组分进行超滤脱盐和浓缩,得浓缩液;
(10)冷冻干燥:将步骤(9)浓缩液冷冻干燥,得贻贝多糖成品。
上述贻贝多糖成品的纯度为99%以上。
所述的贻贝多糖在制备用于治疗或预防心脑血管疾病药物中的应用,所述的心脑血管疾病包括高血脂症、动脉粥样硬化。
所述贻贝多糖在制备具有降血脂功效的保健品或特殊医学用途配方食品中的应用。
本发明具有以下优点:
本发明的贻贝多糖来源于海洋贝类动物贻贝,是海洋贝类养殖中的重要种类,也是最常见的一种食用贝类,可以通过人工养殖大量获得,从而使贻贝多糖来源问题很容易得到解决,又能够拉动海水养殖产业发展。本发明提取贻贝多糖的方法,条件温和、纯度高。本发明的贻贝多糖急性毒性分级属无毒级,安全性高;且贻贝多糖能显著降低甘油三酯、总胆固醇和低密度脂蛋白胆固醇,具有降脂作用。本发明的贻贝多糖在制备治疗或预防高血脂症、动脉粥样硬化的药物,制备具有降血脂功效的保健品或特殊医学用途配方食品中具有广泛应用。
附图说明
图1为贻贝多糖的HPGPC洗脱曲线;
图2为贻贝多糖PMP柱前衍生液相色谱分析谱图:A为单糖标准品的PMP柱前衍生液相色谱图(其中,1为Man;2为GlcN;3为Rha;4为GlcA;5为GalA;6为GalN;7为Glc;8为Gal;9为Xyl;10为Ara;11为Fuc);B为贻贝多糖的PMP柱前衍生液相色谱图。
图3为贻贝多糖光谱分析谱图:A为贻贝多糖的紫外吸收曲线;B为贻贝多糖的红外吸收曲线;
图4为贻贝多糖的1H-NMR图谱;
图5为贻贝多糖的13C-NMR图谱
图6为贻贝多糖的DEPT图谱;
图7为贻贝多糖的HH-COSY图谱;
图8 为贻贝多糖的HSQC图谱;
图9 为贻贝多糖的TOCSY图谱;
图10为贻贝多糖的HMBC图谱;
图11为贻贝多糖甲基化衍生物GC-MS总离子流图;
图12为1,5-二-O-乙酰基-1-脱氧-2,3,4,6-四-O-甲基-D-葡萄糖醇质谱图: a为贻贝多糖所得质谱图;b为CCRC标准质谱图;
图13为1,2,5-三-O-乙酰基-1-脱氧-3,4,6-三-O-甲基-D-葡萄糖醇质谱图: a为贻贝多糖所得质谱图;b为CCRC标准质谱图;
图14为1,4,5-三-O-乙酰基-1-脱氧-2,3,6-三-O-甲基-D-葡萄糖醇质谱图:a为贻贝多糖所得质谱图;b为CCRC标准质谱图;
图15为1,2,4,5-四-O-乙酰基-1-脱氧-3,6-二-O-甲基-D-葡萄糖醇质谱图:a为贻贝多糖所得质谱图;b为CCRC标准质谱图。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 贻贝多糖的提取制备。
(1)原料处理:将浙江省嵊泗县产厚壳贻贝加热去壳留肉干燥,用粉碎机粉碎,过20目筛,得贻贝干粉;
(2)多糖提取:步骤(1)制得的贻贝干粉中加入6倍体积的水,100℃提取2 h;
(3)除渣:用50目筛网分离贻贝肉渣与多糖提取液;
(4)酶解:将步骤(3)制得的多糖提取液用NaOH调pH至8.0,加入质量比为0.2%的碱性蛋白酶,50 ℃酶解2 h,酶解完成后在80 ℃条件下灭活20 min,得酶解液;
(5)过滤:以硅藻土为助滤剂,抽滤滤过,得到澄清的多糖溶液;
(6)乙醇沉淀:在步骤(5)制得的多糖溶液中加入1.5倍体积质量分数为93%的食用酒精进行多糖沉淀,静置6 h,倾去上清液,得到多糖沉淀;
(7)脱水:在步骤(6)制得的多糖沉淀中加入1倍体积质量分数为93%的食用酒精对多糖沉淀进行脱水;
(8)过滤干燥:用400目筛网对脱水后的多糖沉淀进行过滤,收集沉淀,60 ℃真空干燥,得到粗多糖。
(9)重溶:将步骤(8)所得粗多糖溶于水,配制10%贻贝粗多糖溶液,12000 rpm离心1 h以除去不溶物;
(10)过滤:步骤(9)离心所得上清液用0.45 μm滤膜过滤,得多糖滤液;
(11)离子交换层析:用DEAE-琼脂糖(FF)层析柱纯化多糖,多糖滤液上样后用0.05mol/L NaCl溶液进行洗脱,收集洗脱组分;
(12)超滤:用截留分子量为5000的滤膜对步骤(11)所得洗脱组分进行超滤脱盐和浓缩;
(13)冷冻干燥:浓缩液冷冻干燥,得贻贝多糖样品。
实施例2 贻贝多糖的结构鉴定。
2.1 化学分析
总糖含量采用硫酸/苯酚法测定:吸取1 mL浓度为100 μg/mL的贻贝多糖溶液,加入1 mL 6 %苯酚溶液,迅速加入5 mL浓硫酸,振荡使之混匀,沸水浴20 min后,冷至室温在490 nm波长处测定其吸光值。以葡萄糖浓度-吸光度做标准曲线,线性回归方程为:Y=3.8108X+0.0415(R2=0.9996),计算样品总糖含量。
糖醛酸含量采用硫酸/咔唑法测定:吸取0.5 mL浓度为50 μg/mL的贻贝多糖溶液及空白试剂。在冰水浴中加入3 mL四硼酸钠-硫酸溶液,沸水浴15 min,取出后立即冷却至室温,加0.1%咔唑溶液0.1 mL,摇匀后在530 nm波长处测定其吸光值。以葡萄糖醛酸浓度-吸光度做标准曲线,线性回归方程为:Y=4.1536X+0.0252(R2=0.9990),计算样品糖醛酸含量。
硫酸根含量采用BaCl2-明胶比浊法测定:吸取0.5 mL浓度为10 mg/mL的贻贝多糖溶液及空白试剂,依次加入3%三氟乙酸溶液3.8 mL,氯化钡-明胶溶液1.0 mL,摇匀,室温放置15 min,以1 mol/L HCl为空白对照,在500 nm处测定其吸光值。以硫酸根浓度-吸光度做标准曲线,线性回归方程为::Y=0.4733X-0.0035(R2=0.9977),计算样品硫酸多糖含量。
测定结果结果如下:贻贝多糖样品的总糖含量为99.2%,各批次无明显的糖醛酸组分存在,不含有硫酸根。化学分析结果表明,贻贝多糖是一种具有较高纯度的中性多糖,不含有明显的糖醛酸和硫酸多糖成分。
2.2 相对分子量测定
采用高效液相色谱-分子排阻色谱法对贻贝多糖分子量进行测定。色谱条件如下:岛津LC-20AT高效液相色谱仪;色谱柱为TSK-gel GMPWXL (排阻限500万);柱温:35 ℃;流动相:0.05 mol/mL硝酸钠溶液;流速:0.6 mL/min;检测器:RID-20AT示差折光检测器;上样量20 μL。
通过重均分子量分别为180、55500、102000、291600、534000、1185000,2990000Dal的葡聚糖系列对照品绘制以分子量的对数值为纵坐标,以相应色谱峰的保留时间为横坐标的三次标准曲线,得回归方程为:y = -0.00315x3 +0.16469x2 – 3.47013x + 29.24623,R2=0.9988。精密称取贻贝多糖样品50 mg,加流动相溶解并定容至10 ml,HPLC检测,GPC分析。
贻贝多糖的HPGPC洗脱曲线如图1所示,除在11.9 min含有主要多糖组分外,还含有少量低分子杂质。根据标准曲线计算,贻贝多糖主要多糖组分的平均相对分子质量为1212.7 KDa。
2.3 单糖组成分析
采用柱前衍生高效液相色谱法(PC-HPLC)测定贻贝多糖的单糖组成。色谱条件如下:UltiMate 3000高效液相色谱仪;色谱柱为Boston Geen ODS C18色谱柱(250 mm×4.6mm,5 μm);柱温:30 ℃;流动相:磷酸盐缓冲液(pH 6.7)/CH3CN(82:18,V/V);流速:1.0 mL/min;检测器:二极管阵列检测器(DAD,245 nm)。
精密称取贻贝多糖适量,用2 mol/L三氟乙酸(TFA)完全水解。取贻贝多糖降解液100 µL,在碱性条件下与PMP进行衍生反应,经氯仿萃取处理后,HPLC分析。结果如图2所示,结果显示,贻贝多糖中只含有葡萄糖组分。
2.4 紫外和红外光谱分析
以纯水为溶剂配制贻贝多糖溶液使用紫外-可见分光光度计在波长190-500 nm范围内扫描,结果如图3A所示。由图谱可知,贻贝多糖在190-500 nm范围内无明显的紫外吸收,符合多糖的结构特征。
取干燥的贻贝多糖2 mg与干燥溴化钾200 mg研磨后压片,于傅里叶变换红外光谱仪在4000-500 cm-1波段扫描,结果如图3B所示。由图谱可知,贻贝多糖在3391 cm-1范围内存在宽而强的吸收,为O-H的伸缩振动峰;在2928 cm-1的吸收为糖环上次甲基和亚甲基中的C-H伸缩振动,表明样品分子结构中含有亚甲基和次甲基。在1154 cm-1、1081 cm-1、1021 cm-1的吸收峰为糖环C-O-H,C-O-C的特征骨架振动;在1417 cm-1、1368 cm-1、1238 cm-1为吡喃糖环O-H的面内变形振动吸收,577 cm-1为O-H的面外变形振动吸收。848 cm-1处的吸收表明糖环为α-构型;928 cm-1处的吸收表明糖环为D-构型;1646 cm-1处的吸收为多糖类物质常见的微量水分缔合羟基造成。由此可以初步推测,贻贝多糖为α-D-吡喃型葡聚糖。
2.5 核磁共振分析
2.5.1一维核磁共振波谱(1D-NMR)
仪器与试剂:Agilent DD2-500核磁共振波谱仪(500MHz);重水(D2O,99.96%)。
取贻贝多糖20 mg,用1 mL D2O交换3次,冻干后再用D2O溶解,以氘代丙酮为内标,在20 ℃下记录1H-NMR、13C-NMR和DEPT图谱。
在贻贝多糖的1H-NMR图谱(图4)中,在δ5.24 ppm处存在端基H1的信号,且位于较低场(化学位移>δ5.0 ppm),为较宽的单峰,表明贻贝多糖的构型为α-构型。在δ3.0~4.0ppm处的信号为糖环H2~H6信号,因其化学位移重叠严重,需借助H H-COSY、TOCSY和HSQC等图谱进行识别。
在贻贝多糖的13C-NMR图谱(图5)中,在δ99.74 ppm处存在端基C1的信号,且位于较高场(化学位移<δ102 ppm),符合α-构型多糖的特征,与其1H-NMR图谱中和红外光谱中的判断一致;在高场区出现的δ60.48 ppm信号,可能为贻贝多糖糖环C6的信号;在δ69~78 ppm范围内的信号为贻贝多糖糖环C2~C5信号;在δ80~90 ppm范围内未出现C的信号;表明贻贝多糖为吡喃型,且不存在(1→3)糖苷键。
在贻贝多糖的DEPT(图6)图谱中,δ60.4 ppm的信号为负峰,表明对应的C信号为亚甲基(-CH2),可能为贻贝多糖糖环结构中的C6信号,这与其在13C-NMR图谱的推测一致。因在贻贝多糖的DEPT图谱中,其它位置均无负峰信号,表明贻贝多糖结构中不存在(1→6)糖苷键。
2.5.2二维核磁共振波谱(2D-NMR)
仪器与试剂:Agilent DD2-500核磁共振波谱仪(500MHz);重水(D2O,99.96%)。
试验方法:取贻贝多糖样品20 mg,用1 mL D2O交换3次,冻干后再用D2O溶解,以氘代丙酮为内标,在20 ℃下记录HSQC、HH-COSY、TOCSY和HMBC图谱。
根据贻贝多糖的HH-COSY图谱(图7),结合HSQC图谱(图8)、TOCSY图谱(图9)、HMBC图谱(图10),可以对贻贝多糖C和H的化学位移归属进行确认(表1)。
表1贻贝多糖的1H-NMR和13C-NMR信号归属
在贻贝多糖的HMBC图谱中,可以分别看到H1(δ5.24 ppm)与C4(δ76.74 ppm)的偶合相关信号,C1(δ99.74 ppm)与H4(δ3.51 ppm)的偶合相关信号;H1(δ5.24 ppm)与C2(δ77.26 ppm)的偶合相关信号,C1(δ99.74 ppm)与H2(δ3.47 ppm)的偶合相关信号;表明贻贝多糖中存在(1→4)糖苷键和(1→2)糖苷键。
综合对贻贝多糖的1D-NMR和2D-NMR分析,可以初步确定贻贝多糖是以(1→4)-α-D-Glc为主链,含有(1→2)-α-D-Glc分枝的α-吡喃型葡聚糖。
2.6 单糖连接方式分析
将贻贝多糖进行甲基化反应结合GC-MS分析测定单糖连接方式。
采用改良的Hakomori法对贻贝多糖进行甲基化反应。称取经五氧化二磷充分干燥48 h后的多糖组分2 mg,加入无水DMSO,在N2氛中磁力搅拌1 h使样品充分溶解。快速加入干燥的NaH粉末,继续搅拌反应1 h。逐滴加入碘甲烷1 mL,在N2氛中避光搅拌反应1.5 h,然后加纯水1 mL终止反应。用氯仿多次萃取反应液,合并氯仿层,减压干燥即得甲基化多糖。
将贻贝多糖甲基化样品采用2 mol/L TFA完全酸水解,经硼氘化钠还原,用吡啶和乙酸酐反应生成乙酰化衍生物后进行GC-MS分析。质谱分析条件为:气相色谱柱:AgilentDB-225(0.25 mm×30 m×0.25 µm);载气:氦气;载气流速:1.0 mL/min;进样口温度:250℃;检测器:质谱检测器(MSD);进样量:1 µL。
贻贝多糖甲基化衍生物进行GC-MS分析的总离子流图如图11所示。从图中可以看出,贻贝多糖主要在21.48 min、24.42 min、25.13 min和27.82 min处出峰。参照美国佐治亚大学糖复合物研究中心的(Complex Carbohydrate Structure Database,CCSD)的GC/MS数据库,对贻贝多糖甲基化样品GC-MS分析中的色谱峰进行质谱解析归属如下。
在21.48 min处的色谱峰,为1,5-二-O-乙酰基-1-脱氧-2,3,4,6-四-O-甲基-D-葡萄糖醇(图12),由末端Glc-(1→产生。
在24.42 min处的色谱峰,为1,2,5-三-O-乙酰基-1-脱氧-3,4,6-三-O-甲基-D-葡萄糖醇(图13),是由→2)-Glcp-(1→产生。
在25.13 min处的色谱峰,为1,4,5-三-O-乙酰基-1-脱氧-2,3,6-三-O-甲基-D-葡萄糖醇(图14),是由→4)-Glcp-(1→产生。
在27.82 min处的色谱峰,为1,2,4,5-四-O-乙酰基-1-脱氧-3,6-二-O-甲基-D-葡萄糖醇(图15),是由→2,4)-Glc-(1→产生。
对贻贝多糖的GC-MS结果进行归属分析,发现贻贝多糖中主要含有末端→(Glc、→4)-Glc-(1→、→2)-Glc-(1→和→2,4)-Glc-(1→等连接方式。对各信号进行积分,可以计算贻贝多糖中不同糖苷键连接方式的摩尔组成(表2)。
表2 贻贝多糖的甲基化分析结果
根据贻贝多糖甲基化样品经硼氘化钠还原和乙酰化衍生后的GC-MS分析结果,可以确定贻贝多糖的连接方式主要为→4)-Glcp-(1→,还含有→2)-Glcp-(1→和→2,4)-Glcp-(1→等连接方式,这与其2D-NMR的分析结果相吻合。三种糖苷键连接方式的摩尔比依次约为11:1:1。
2.7 贻贝多糖的化学结构
综合贻贝多糖的化学分析和紫外光谱(UV)、红外光谱(IR)、核磁共振波谱(NMR)和气质联用分析(GC-MS)等现代仪器分析结果,可以确定贻贝多糖是一种纯度较高的,以(1→4)-α-D-Glc为主链,含有(1→2)-α-D-Glc分枝的α-吡喃型葡聚糖,且平均每12个葡萄糖中有1个末端的葡萄糖分枝。
实施例3 贻贝多糖降血脂功效研究。
3.1 试验动物
选雄性SD大鼠,由济南朋悦实验动物繁育有限公司提供,动物生产许可证号:SCXK(鲁)20140007。动物合格证号:No.37009200002065,体重170±10 g。
3.2 试验方法
随机分为6组,分别为空白组、模型组、L1组(100 mg/kg)、L2组(200 mg/kg)、L3组(400 mg/kg)、L4组(800 mg /kg)。全部大鼠喂养基础饲料观察1周,禁食12 h后称重,眼内眦取血,室温25 ℃静置1 h,3000 rpm离心10 min,取血清,用自动生化分析仪测定TC,TG,HDL-C和 LDL-C水平。空白组始终基础饲料喂养,其余各组大鼠均用高脂饲料喂养(高脂饲料配方为胆固醇1%、胆盐0.1%、猪油10%、蛋黄粉与全脂奶粉分别为5%,购于北京华阜康生物科技有限公司)。4周后眼内眦取血(取血前禁食12 h),测定血清TC(总胆固醇),TG(甘油三酯),HDL-C(高密度脂蛋白胆固醇)和LDL-C(低密度脂蛋白胆固醇)水平。用高脂饲料喂养4周后各组大鼠血清总胆固醇水平和甘油三酯水平与空白组比较有显著性差异(p<0.05),表明脂代谢紊乱模型成功建立。
脂代谢紊乱模型建立成功后,空白组与模型组每日灌胃蒸馏水1 mL,L1、L2、L3、L4组分别按照相应的剂量每天灌胃给药1次,连续8周。给药8周后大鼠隔夜禁食12 h,取血和肝脏,测定血清TC,TG,HDL-C和 LDL-C水平。
3.3 统计分析
计量资料以x±SD表示,均使用SPSS 13.0软件统计分析。若计量资料的方差齐,采用单因素方差分析LSD检验进行组间比较;若计量资料的方差不齐,采用两独立样本非参数检验。P<0.05认为有统计学意义。
3.4 试验结果
在高脂模型建立后的给药过程中,连续用药8周,与模型组比较,贻贝多糖所有剂量组均能显著降低大鼠血清甘油三酯水平(P<0.01),其中400 mg/kg·d、800 mg/kg·d剂量组可明显降低血清总胆固醇和低密度脂蛋白胆固醇含量(P<0.05)(表3)。提示贻贝多糖有显著降血脂功能,在低剂量条件下只降低血清甘油三酯水平,高剂量条件下可降低血清甘油三酯、总胆固醇和低密度脂蛋白胆固醇水平。
表3 给药前后各组大鼠的血清TC、TG、HDL-C、LDL-C比较(mmol/L)
注:n=10,# #表示P<0.01,与正常组比较;*表示P<0.05,**表示P<0.01与模型组比较
实施例4 贻贝多糖急性毒性试验。
4.1 试验动物
昆明种(KM)小鼠,SPF级,40只,雌雄各半,由济南朋悦实验动物繁育有限公司生产,合格证号:37009200000705,实验动物生产许可证号:SCXK(鲁)2014-0007。体重范围为18.4-22.0 g。
4.2 试验方法
小鼠分性别、按体重随机分为实验组和对照组,每组20只,雌雄各半。配制0.6 g/mL贻贝多糖溶液,实验组一日内灌胃给药2次,每次给药体积40 mL/kg,间隔约10 h,总给药剂量为48 g/kg·d。对照组同法灌胃给予纯水。
给药后即刻观察动物的反应情况,记录动物的中毒症状、外观体征以及中毒症状出现时间、持续时间和恢复情况等。给药当日应密切观察,然后每日上午观察一次,连续观察14天,药前、药后第3、7、10、14天各称一次体重。体重值用x±SD表示,以DAS1.0软件进行组间t检验比较,以观察药物对动物体重增长的影响。
4.3 试验结果
药后第3、7、10、14天实验组与对照组体重基本一致(表4)。小鼠主要症状表现为药后约2 min内出现小鼠活动次数减少,约20 min内基本恢复,其它观察未见明显异常。观察结束后处死动物剖检,脑、心、肝、脾、肺、肾等主要脏器未有肉眼可见异常。
贻贝多糖小鼠最大给药量为48 g/kg·d,约相当于人每日拟用剂量(16 mg/kg·d)的3000倍,急性毒性分级属无毒级。
表4 贻贝多糖灌胃给药对小鼠体重的影响(x±SD,n=20)
综上所述,一定剂量的贻贝多糖能够显著降低大鼠血清中甘油三酯和低密度脂蛋白胆固醇的含量;能显著降低大鼠血清甘油三酯水平,显著降低血清总胆固醇和低密度脂蛋白胆固醇含量;贻贝多糖急性毒性分级属无毒级。
Claims (5)
1.一种贻贝多糖,其特征在于,以(1→4)-α-D-Glc为主链,含有(1→2)-α-D-Glc分枝,构型为α-吡喃型葡聚糖,平均每12个葡萄糖主链中有1个(1→2)葡萄糖分枝,其结构式为:
;
所述贻贝多糖相对分子量为800-8000 KDa。
2.根据权利要求1所述的贻贝多糖,其特征在于,提取自贻贝属动物,包括厚壳贻贝和紫贻贝。
3.一种如权利要求1所述的贻贝多糖的提取方法,其特征在于,包括以下步骤:
(1)原料处理:贻贝加热去壳留肉并干燥,粉碎机粉碎,过20-40目筛,得贻贝干粉;
(2)多糖提取:步骤(1)制得的贻贝干粉中加入6倍体积的水,100 ℃下提取2 h;过滤去除贻贝肉渣,得多糖提取液;
(3)酶解:将步骤(2)制得的多糖提取液调pH至8.0,加入0.2 %w.t的碱性蛋白酶,50 ℃酶解2 h,酶解完成后在80 ℃条件下灭活15-30 min,得酶解液;以硅藻土为助滤剂,抽滤过滤,得到澄清的酶解液;
(4)乙醇沉淀:在步骤(3)制得的多糖溶液中加入1.5倍90-95%v/v的乙醇进行沉淀,静置6 h,倾去上清液,得到多糖沉淀;
(5)脱水:在步骤(4)制得的多糖沉淀中加入1倍体积90-95%v/v的乙醇进行脱水;
(6)过滤干燥:用400目筛网对脱水后的多糖沉淀进行过滤,收集沉淀,60 ℃真空干燥,得到粗多糖;
(7)重溶:将步骤(6)所得粗多糖溶于水,配制10%贻贝粗多糖溶液,12000 rpm离心1 h,将上清液用0.45μm滤膜过滤,得多糖滤液;
(8)离子交换层析:将步骤(7)的多糖滤液用DEAE-琼脂糖FF层析柱纯化,以0.05 mol/LNaCl溶液为洗脱液,收集洗脱组分;
(9)超滤:用截留分子量为5000的滤膜对步骤(8)所得洗脱组分进行超滤脱盐和浓缩,得浓缩液;
(10)冷冻干燥:将步骤(9)所得浓缩液冷冻干燥,得贻贝多糖成品。
4.一种如权利要求1所述的贻贝多糖在制备用于治疗或预防心脑血管疾病药物中的应用,其特征在于,所述心脑血管疾病包括高血脂症、动脉粥样硬化。
5.一种如权利要求1所述的贻贝多糖在制备具有降血脂功效的保健品或特殊医学用途配方食品中的应用。
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