CN106074593B - 附子多糖在制备治疗动脉粥样硬化药物中的应用 - Google Patents
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Abstract
本发明涉及附子多糖在制备治疗动脉粥样硬化药物中的应用，所述应用为附子多糖在制备提高组织抗氧化酶SOD、CAT、GSH‑Px活性的药物中的应用；所述应用还可以为附子多糖在制备降低组织中MDA含量的药物中的应用。本发明首次将附子多糖单体应用在制备治疗动脉粥样硬化药物中，制备得到的药物能够有效降低动脉粥样硬化的发病率。
Description
技术领域
本发明属于药物学和制剂学领域，具体涉及附子多糖在制备治疗动脉粥样硬化药物中的应用。
背景技术
动脉粥样硬化（atherosclerosis，AS）是冠心病、中风等心脑血管疾病的主要病理基础，是危害人类健康的主要疾病。随着生活水平的提高和生活方式的改变，由AS所致的心脑血管疾病已成为严重危害人类健康的疾病之一，在我国动脉粥样硬化的发病率也呈逐年上升的趋势，且发病年龄趋向年轻化。美国、欧洲及日本死于该病者占死亡总数的50%左右。近年来该病的发生率在我国亦有明显增加的趋势。据尸检结果，在40～49岁的人群中冠状动脉和主动脉粥样硬化病变的检出率分别为58.36%和88.31%，并随着年龄的增长而逐渐增加。
因此对于AS发病机制及防治的研究一直是心、脑血管疾病研究的热点。以往虽然人们已经认识到脂质在内皮下的浸润和平滑肌细胞的增殖是AS发生、发展过程中的基本特征，并因此先后提出脂质浸润学说、中层平滑肌细胞增生学说、血栓源学说以及血流动力学学说，但是它们都不能对AS的发生机制提供满意的解释。近年来，“氧化应激损伤机制学说”逐渐为人们所接受，该学说认为体内氧化应激水平增高一方面可以造成血管内皮细胞损伤，这被认为是AS形成的始动因素，内皮损伤不仅导致血细胞的粘附聚集，而且通过激活核因子κB（nuclear factor Kappa B,NF-κB）引发系列炎症细胞因子释放，导致内膜下平滑肌细胞的炎症损伤；另一方面氧化应激水平增高可以氧化修饰体内的低密度脂蛋白（LDL），形成氧化型低密度脂蛋白（ox-LDL），被吞噬细胞吞入导致大量泡沫细胞形成，泡沫细胞凋亡或坏死后将脂质释放出来，形成粥样斑块的主要成分。目前认为，AS既是一种慢性免疫炎症性疾病，又是一种脂质沉积的纤维增生性疾病。
基于AS的机理研究，已开发出多种治疗药物，主要有：（1）调整血脂类抗AS药物，包括他汀类、贝特类、烟酸类药物，其中他汀类药物因为良好的降脂以及抗炎、抗氧化、减少泡沫细胞形成等作用可以稳定/逆转斑块，在临床上得以广泛应用。（2）血管调节类药物，包括抗血小板类药物、扩张血管运动类药物，此类药物对AS斑块中的脂质没有治疗意义。（3）抗氧化剂类药物，包括丙丁酚等，此类药物抑制LDL氧化和改善AS的状况仍处于研究阶段。（4）正在研究的新型抗AS的药物虽然较多，但真正理想的药物并不多，大多数药物价格昂贵，而且都存在一些不良反应。就拿目前应用最广泛、疗效最确切的他汀类药物来说，会引起肝脏转氨酶增高，长期服药肝功能损害的可能性更大，最为严重的副作用还有横纹肌溶解，严重者引起患者死亡。
近年来，随着对AS研究的深入，中药在预防和治疗AS的作用也日益受到重视。目前认为，中药主要从改善脂质代谢，抗脂质过氧化，调节血管内皮功能等方面对基体进行调节从而发挥对AS的治疗作用。中药材中活性多糖成分是近年来药物开发研究的热点，多糖类化合物通常没有细胞毒性，不影响正常细胞功能，具有高效低毒的特点，是开发药物的重要来源。鉴于目前西医药在AS治疗上的局限性，因此发挥中医药优势，利用现代生物技术，研究中药单体在防止AS的作用及其机理，将具有重大的社会和经济价值。
发明内容
本发明的目的在于克服现有技术的不足，提供附子多糖在制备治疗动脉粥样硬化药物中的应用，本发明首次将附子多糖单体应用在制备治疗动脉粥样硬化药物中，制备得到的药物能够有效降低动脉粥样硬化的发病率。
本发明的另一目的在于提供一种用于预防或治疗动脉粥样硬化的药物。
本发明的另一目的在于提供所述附子多糖的提取方法。
为实现上述目的，本发明采用如下技术方案：
附子多糖在制备预防或治疗动脉粥样硬化药物中的应用。
优选地，所述应用为附子多糖在制备提高组织抗氧化酶SOD活性从而预防或治疗动脉粥样硬化药物中的应用。
优选地，所述应用为附子多糖在制备提高组织抗氧化酶CAT活性从而预防或治疗动脉粥样硬化药物中的应用。
优选地，所述应用为附子多糖在制备提高组织抗氧化酶GSH-Px活性从而预防或治疗动脉粥样硬化药物中的应用。
优选地，所述应用为附子多糖在制备降低组织中MDA含量从而预防或治疗动脉粥样硬化药物中的应用。
上述附子多糖的应用，所述附子多糖的制备方法如下：
S1：将附子粉碎，用乙醇进行脱脂处理，然后用水提取并过滤；
S2：将步骤S1过滤所得残渣用水提得残渣水提液，将所述残渣水提液与步骤S1所得水提液合并、减压浓缩并过滤；
S3：将步骤S2所得滤液离心得沉淀物，对所述沉淀物进行脱蛋白处理；
S4：将步骤S3脱蛋白后的提取物置于水中透析，然后醇析、洗涤、干燥，即得粗附子多糖。
优选地，步骤S1中，所述乙醇的体积分数为95%，所述水的温度为90～100℃，所述提取时间为2小时。
优选地，步骤S2中，合并提取液用旋转式蒸发器减压浓缩。
优选地，步骤S3中，选用体积比为5:1的三氯甲烷与正丁醇混合液对所述沉淀物进行脱蛋白处理。
优选地，步骤S4中，将脱蛋白后的提取物置于双蒸馏水中透析三天，再加入3倍体积95%乙醇进行醇析，用无水乙醇洗涤沉淀，然后再40℃进行真空干燥，即得粗附子多糖。
一种用于预防或治疗动脉粥样硬化的药物，所述药物中含有上述附子多糖。
与现有技术相比，本发明具有如下有益效果：
本发明首次将附子多糖单体引入对动脉粥样硬化的防治研究中，先用最接近AS病理的自发性AS动物模型，以AS氧化应激损伤机制为切入点，研究附子多糖抗AS的有效性，研究具有较强的可行性，较高的可信性和较好的研究前景。本研究将现代分子生物学技术与转基因动物平台相结合，研究中药单体-附子多糖的药理学效应，为重要单体防治AS的研究提供新的思路和有效手段。本发明将附子多糖应用在制备治疗动脉粥样硬化药物中，制备得到的药物能够有效降低动脉粥样硬化的发病率。
附图说明
图1为动脉粥样硬化的小鼠主动脉根部进行油红O染色示意图；
图2为动脉粥样硬化的小鼠胸腹主动脉进行油红O染色示意图；
图3为不同剂量的小鼠主动脉根部进行油红O染色的示意图；
图4为不同剂量的小鼠胸腹主动脉进行油红O染色示意图。
具体实施方式
下面结合具体实施例进一步说明本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法：以下各实施例中的apoE基因敲除小鼠购自没过Jackson实验室，所使用原料、助剂等，如无特殊说明，均为可从常规市场购买等商业途径得到的原料和助剂。
实施例1 附子多糖的提取
取干燥的附子100克粉碎成细小颗粒，用95%的乙醇进行脱脂处理，然后用1升90～100℃的双蒸馏水提取2小时并过滤。残渣继续用750ml水提取1小时。合并水提取液用旋转式蒸发器减压（50℃）浓缩并过滤。在4℃的条件下将滤液与4倍体积的95%乙醇混合，离心5000转20分钟。把沉淀物再溶于300ml水中，用60ml体积比为5:1的三氯甲烷和正丁醇的混合液进行脱蛋白处理三次。将脱蛋白后的提取物置于双蒸馏水中透析三天，再加入3倍体积95%乙醇进行醇析。用无水乙醇洗涤沉淀，然后再40℃进行真空干燥，得到粗多糖备用。
实施例2 动脉粥样硬化动物模型的建立
选用apoE基因敲除小鼠，采用高脂动物饲料喂养，该高脂动物饲料由普通小鼠饲料加入脂肪和胆固醇（含21%脂肪和0.15%胆固醇，w/w）制成，辐射消毒。小鼠在学校实验动物中心喂养。
将apoE基因敲除小鼠随机分为对照组、辛伐他汀预防组、辛伐他汀预防高（200mg）、中（100mg）、低（50mg）剂量组和附子多糖预防组、附子多糖治疗高（200mg）、中（100mg）、低（50mg）剂量组9组动物。附子多糖及辛伐他汀各组，动物均选择灌胃给药方式，对照组给以同等剂量的饮用水灌胃，每天1次，12周结束实验。附子多糖预防组、辛伐他汀预防组在实验开始前6周灌胃给药；附子多糖治疗组、辛伐他汀治疗组自实验开始至第7周给以同等剂量饮用水灌胃，从第7周开始灌胃给药。
图1为ApoE-/-小鼠主动脉根部进行油红O染色，肉眼观察可见ApoE-/-小鼠形成严重的动脉粥样硬化斑块。
图2为ApoE-/-小鼠的胸腹主动脉进行油红O染色，肉眼观察可见ApoE-/-小鼠主动脉弓部有明显的粥样斑块形成，呈片状分部，染色较深，且在胸主动脉和腹主动脉部分也存在颗粒状的粥样斑块，染色较浅。
实施例3 附子多糖对动脉粥样硬化的防治作用
油红0染色检测粥样斑块面积：
将动物麻醉后断头处死，显微镜下解剖出主动脉，10%甲醛固定24小时后，用0.3%油红0染色液染色，60%异丙醇脱去未染色的油红0，苏木素复染，1%HCl分色及返蓝。
显微镜观察主动脉根部和主动脉弓处红色动脉粥样硬化斑块，应用全自动图像分析系统分析统计主动脉粥样斑块面积占主动脉内膜总面积的百分比。
预实验选用apoE基因敲除小鼠，置于安静清洁环境下分笼喂养，室内温度控制在24±2，定时给予清洁饮水和专用饲料。适应性喂养1周后，无不良反应，饮食、饮水正常，将小鼠分别称重后随机分为5组：对照组、附子多糖低剂量预防组、附子多糖高剂量预防组，每组6只。空白对照组给予常规饮食，其它各族给予高脂饲料。
说明书附图3为各组ApoE-/-小鼠主动脉根部进行油红O染色，图3中左图为对照组，中图为附子多糖低剂量组，右图为附子多糖高剂量组。由图可知，肉眼观察可见附子多糖低剂量组和高剂量组可减轻主动脉粥样硬化斑块的形成，以高剂量组更为明显。
说明书附图4为各组ApoE-/-小鼠的胸腹主动脉进行油红O染色，图3中左图为对照组，中图为附子多糖低剂量组，右图为附子多糖高剂量组。由图可知，肉眼观察可见附子多糖低剂量组和高剂量组能减轻ApoE-/-小鼠主动脉弓部粥样斑块形成及胸主动脉和腹主动脉部分粥样斑块形成，以高剂量组更为明显。
实施例4 附子多糖对动脉粥样硬化防治作用的机制研究
（1）附子多糖对小鼠氧化应激损伤的影响
ELISA检测各组小鼠SOD、CAT、GSH-PX活力。
（2）附子多糖对小鼠腹腔游离巨噬细胞中ox-LDL的影响
DiL荧光标记检测ox-LDL：
将小鼠腹腔提取的巨噬细胞经前处理后接种于激光扫描共聚焦显微镜检测专用培养皿，培养2小时后，加入终浓度为1mg/ml的DiL-ox-LDL，37℃孵育4小时。
用激光扫描共聚焦显微镜照相，激发波和发射波分别置于520nm和580nm。运用IPP软件测定细胞内平均荧光强度，反映巨噬细胞内ox-LDL浓度。
（3）附子多糖对NF-κB通路的炎症因子的影响
DNA结合活性检测检测各组NF-κB的活性，ELISA、RT-PCR、Western blot检测TNF-α、IL-2、IL-6、IL-8、单核细胞趋化蛋白-1和细胞间粘附分子-1的表达。
（4）附子多糖对小鼠血脂代谢的影响
ELISA检测各组小鼠血脂水平的差异，包括TC、TL、LDL、HDL；肝脏LDL-R的表达及定量研究等。
实施例5 附子多糖安全性评价
小鼠急性毒性（LD50）试验。小鼠给药前12小时内禁食不禁水，随机分为7组，每组10只小鼠。实验中以灌胃方式对小鼠进行给药，根据预防试验测得的LD100和LD0剂量，按照寇氏法将相邻两组剂量之比（γ）设定为1.246，第7组为空白对照组，给以等体积溶媒溶液。给药后24小时内观察小鼠摄食、行为、中毒症状，统计每组死亡动物数。
Claims (1)
1.附子多糖在制备预防或治疗动脉粥样硬化药物中的应用，所述附子多糖的制备方法如下：
取干燥的附子100克粉碎成细小颗粒，用95%的乙醇进行脱脂处理，然后用1升90～100℃的双蒸馏水提取2小时并过滤；残渣继续用750ml水提取1小时；合并水提取液用旋转式蒸发器减压50℃浓缩并过滤；在4℃的条件下将滤液与4倍体积的95%乙醇混合，离心5000转20分钟；把沉淀物再溶于300ml水中，用60ml体积比为5:1的三氯甲烷和正丁醇的混合液进行脱蛋白处理三次；将脱蛋白后的提取物置于双蒸馏水中透析三天，再加入3倍体积95%乙醇进行醇析；用无水乙醇洗涤沉淀，然后再40℃进行真空干燥，得到粗多糖备用。
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| 附子多糖对力竭运动小鼠心肌过氧化损伤的保护作用;刘古锋等;《海南医学》;20081203;第19卷(第7期);67-69 * |
| 附子多糖对大鼠食诱性高胆固醇血症的预防作用及机制研究;周芹等;《中国药理学通报》;20110430;第27卷(第4期);492-496 * |
| 附子多糖提取纯化工艺研究;叶强等;《中药与临床》;20131231;第4卷(第2期);29-31 * |
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