CN113121716B - 一种促凝血紫荆花多糖及其提取分离方法和应用 - Google Patents
一种促凝血紫荆花多糖及其提取分离方法和应用 Download PDFInfo
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Abstract
本发明属于植物提取技术领域,具体涉及一种促凝血紫荆花多糖及其提取分离方法和应用。该提取分离方法是将紫荆花经脱脂、水提醇沉获得粗多糖,然后将除蛋白的粗多糖用DEAE‑52纤维素色谱柱、Sephadex G‑100凝胶色谱柱进一步纯化而得。本发明对该多糖的体外凝血效果进行考察,结果表明该多糖有良好的促凝血作用,可用于制备促凝血药物。
Description
技术领域
本发明属于植物提取技术领域,具体涉及一种促凝血紫荆花多糖及其提取分离方法和应用。
背景技术
紫荆 (Cercis Chinensis Bunge) 属于豆科紫荆属植物,在我国东南部普遍栽培。本种是一美丽的木本花卉植物。树皮可入药,有清热解毒,活血行气,消肿止痛之功效,可治产后血气痛、疗疮肿毒、喉痹;花可治风湿筋骨痛。植物化学研究表明,紫荆中含有黄酮、多酚、酸、甾体、木脂素、挥发油和色素,具有抗氧化、酪氨酸酶、α-葡萄糖苷酶抑制活性等。关于紫荆花多糖,还未见关于紫荆花中分离得到的多糖的结构和活性的研究。
凝血实质上是水溶性的纤维蛋白原转变为水不溶性的固态纤维蛋白的过程,是在内源性或(和)外源性凝血途径下产生凝血酶原激活物,在凝血因子的作用下产生凝血酶,最后在凝血酶的作用下使纤维蛋白原转变为纤维蛋白。血浆凝血酶原时间(PT)主要反映的是外源性凝血途径中凝血因子I、II、V、VII、X的活性;部分活化凝血活酶时间(APTT)主要反映内源性凝血系统状况,与VIII、X、XI、XII等内源性凝血因子活性有关;凝血酶时间(TT)值是主要反映纤维蛋白原转变为纤维蛋白程度的重要指标;血浆纤维蛋白原(FIB)主要反映纤维蛋白原的含量。
能促进血液凝固而使出血停止的药物,称为止血药。临床上因各种病因引起的出血比较常见,如外科和矫形外科:胃肠道、肾、膀胱、前列腺及胸部手术;如创骨折出血、整容外科、颅脑肿痛、挫伤性出血等;内科:肝病出血,肿瘤出血、痔血、肺出血、鼻出血等;妇科手术:子宫及阴道息肉,肌瘤及肿瘤出血,放射治疗后出血;泌尿科:前列腺出血,前列腺切除、肾及膀胱出血等;耳鼻喉科:门诊小手术预防,扁桃体切除、喉部手术;牙科、口腔科:拔牙手术、腭部手术、齿龈炎出血。促凝血药是临床应用范围广泛的药物之一,在创伤中也有重要的应用价值,它主要通过增强体内凝血因素或抑制抗凝血因素,促使凝血,以达到止血目的。
发明内容
本发明目的在于提供一种从紫荆花中提取分离得到具有促凝血作用的紫荆花多糖,同时提供了该紫荆花多糖的提取分离方法及应用。
为实现上述目的,本发明采用以下技术方案:
一种促凝血紫荆花多糖的提取分离方法,其包括以下步骤:
(1)新鲜的紫荆花,阴干,用石油醚回流脱脂,残渣挥干石油醚后,用乙醇浸提,所得残渣挥发至无醇味,再用超纯水加热提取,趁热抽滤,滤液减压浓缩,再向浓缩液中加入乙醇,静置、离心,得到多糖沉淀,冷冻干燥即得紫荆花粗多糖;
(2)采用Sevage法除蛋白;
(3)取除蛋白后的紫荆花粗多糖,用超纯水溶解、离心后加入到DEAE-52纤维素色谱柱,依次用超纯水、0.05 mol/L、0.15 mol/L、0.25 mol/L的NaCl溶液进行洗脱,洗脱液采用苯酚-硫酸法进行检测,测490 nm处的吸光度,以洗脱管数为横坐标,吸光度为纵坐标,绘制洗脱曲线,合并同一洗脱峰的多糖样品,透析、浓缩后冷冻干燥;其中,超纯水的洗脱峰为组分CC-1,0.25 mol/L NaCl溶液的洗脱峰为组分CC-2;
(4)组分CC-1用超纯水溶解、离心后加入到Sephadex G-100凝胶色谱柱,用超纯水洗脱,采用苯酚-硫酸法进行检测,在波长490 nm处测其吸光度,以洗脱管数为横坐标,吸光度为纵坐标,绘制洗脱曲线,得到单一洗脱峰,冷冻干燥得到的多糖命名为CCp-1;
组分CC-2采用上述与组分CC-1相同的方法进行洗脱,得到单一洗脱峰,冷冻干燥得到的多糖命名为CCp-2;
CCp-1和/或CCp-2即为促凝血紫荆花多糖。
本发明提供了利用上述提取分离方法制备得到的促凝血紫荆花多糖。
本发明还提供了上述促凝血紫荆花多糖在制备促凝血药物方面的应用。
其中,DEAE-52纤维素是一种弱酸性阴离子交换剂,主要是其可吸附离子型物质,比如蛋白质、酸性多糖等,而作为大多数的中性多糖则可顺利流出,从而去粗取精、达到分离的目的,常用于多糖的纯化。SephadexG-100凝胶是一种半合成的凝胶,具有一定大小的孔径,不同形状和大小的多糖分子在凝胶层析柱中移动速度不同,从而使大分子多糖与小分子化合物,如盐类、色素、蛋白、小分子多糖等分离开,既可起到纯化多糖的目的,又可以用来鉴定多糖的纯度。
和现有技术相比,本发明的有益效果:
本发明采用特殊的提取分离方法从紫荆花中分离得到多糖CCp-1和CCp-2,并对该多糖的体外凝血效果进行考察,结果表明该多糖有良好的促凝血作用,可用于制备促凝血药物,如促凝血剂。
附图说明
图1为紫荆花粗多糖DEAE-52纤维素柱色谱洗脱曲线;
图2为组分CCp-1和CCp-2的SephadexG-100凝胶柱色谱洗脱曲线;
图3为标准单糖混合物(图上)及CCp-1(图下左)和CCp-2(图下右)水解物的GC色谱图;
图4 为CCp-1和CCp-2对大鼠血浆的凝血时间影响。
具体实施方式
以下通过具体实施方式,对本发明的技术内容再做进一步的详细说明,但是本发明的保护范围并不局限于此。
下述实施例中,所用的材料紫荆花采集于河南省开封市河南大学植物园,为豆科紫荆属植物紫荆Cercis Chinensis Bunge的花。
本文中的乙醇浓度均为体积浓度。
1、一种促凝血紫荆花多糖的提取分离方法,包括以下步骤:
(1)新鲜的紫荆花在阴凉处阴干,用石油醚室温浸泡脱脂3次,每次24 h,残渣挥干石油醚后,用75%乙醇室温浸提2次,每次2 d,回收紫荆花残渣,在通风橱中放置待其挥发至无醇味,然后用超纯水(料液比 1g:15mL)于80℃加热提取2次,每次4 h。趁热抽滤,合并滤液,用旋转蒸发仪浓缩至原体积的1/8左右,向浓缩后的溶液中加入无水乙醇至终浓度为70%,4℃静置12h,离心(4000 r/min,6 min)取其沉淀,冷冻干燥即得紫荆花粗多糖。
(2)将紫荆花粗多糖用适量超纯水溶解获得粗多糖水溶液,按照粗多糖水溶液1/3体积的量加入Sevage试剂(氯仿:正丁醇=4:1,体积比),磁力搅拌震荡30 min,放置使其自然分层,将最下层的溶剂和夹杂在上清液和溶剂之间的变性蛋白质清除,得到上清液,重复上述操作4次基本无变性蛋白层出现。将上清液减压浓缩后加入无水乙醇至终浓度为70%,4℃静置12h,离心后取其沉淀物干燥,即得除蛋白的紫荆花粗多糖。
(3)步骤(2)除蛋白的紫荆花粗多糖,用超纯水溶解、高速离心机离心(12000 r/min,5 min),加入到DEAE-52纤维素柱色谱,依次用超纯水、0.05mol/L、0.15 mol/L、0.25mol/L的NaCl溶液进行梯度洗脱,流速控制在40 rpm,BSZ-100自动部分收集器收集洗脱样品,每8 min收集一管,采取苯酚-硫酸法进行多糖检测,在490 nm处测定其吸光度,以洗脱管数为横坐标,吸光度为纵坐标,绘制洗脱曲线(如图1所示),合并同一洗脱峰的多糖样品,室温下透析24 h(截留分子量8000~14000),浓缩后冷冻干燥,其中,超纯水的洗脱峰为组分CC-1,0.25 mol/L NaCl溶液的洗脱峰为组分CC-2。
(4)组分CC-1用超纯水溶解、高速离心机离心(12000 r/min,5 min)后加入到Sephadex G-100凝胶色谱柱进一步分离纯化,用超纯水洗脱,流速控制在3 rpm,BSZ-100自动部分收集器收集洗脱样品,每35 min收集一管,采用苯酚-硫酸法进行多糖检测,在波长490 nm处测其吸光度,以洗脱管数为横坐标,吸光度为纵坐标,绘制多糖洗脱曲线(如图2所示),得到单一洗脱峰,冷冻干燥得到的多糖命名为CCp-1;
组分CC-2采用上述与组分CC-1相同的方法进行洗脱,得到单一洗脱峰,冷冻干燥得到的多糖命名为CCp-2;
CCp-1和/或CCp-2即为促凝血紫荆花多糖。
2、促凝血紫荆花多糖CCp-1和CCp-2的分子量测定和单糖组成成分分析
2.1 分子量测定
按照《中国药典》(2015年版,总则0514)的方法,采用高效体积排阻色谱法(HPSEC) 测定CCp-1和CCp-2的均一性和分子量。结果显示:CCp-1和CCp-2为均一的多糖。根据标准右旋糖苷的校正,估CCp-1和CCp-2的平均分子量分别为17060和8303 Da,详见表1。
表1 CCp-1 和CCp-2的分子量
2.3 单糖组成成分分析
2.3.1多糖的水解
准确称取多糖CCp-1和CCp-2各10 mg,加入5 mL安瓿瓶中,加入 2 mL 4 mol/L的三氟乙酸,氮气封管。在 110℃下水解3 h,旋转蒸发除去三氟乙酸溶液,加入少量甲醇溶解残渣,旋转蒸发至干燥,如此重复 4次,得到的水解物备用。
2.3.2单糖的衍生化
向水解产物中加入盐酸羟胺10 mg和吡啶0.5 mL,振荡混匀,放入90℃油浴中加热反应30 min。取出冷却至室温,加入醋酸酐0.5 mL,在 90℃下继续反应30 min以进行乙酰化,反应产物经0.22 μm滤膜过滤后注入气相色谱进行分析;用相同方法处理标准单糖,并制成标准单糖衍生物混合液。
2.3.3气相色谱条件
色谱柱:HP(30 m×0.35 mm,0.25 μm);进样温度:250 ℃;检测器温度:280 ℃;色谱柱升温程序:初始温度110 ℃保持 5 min,然后以 5℃/min 的速度由 110 ℃升至 190℃,保持4 min,以 3℃/min 的速度由190℃升至 210℃,保持8 min;载气:高纯氮气,流速2mL/min;进样量为2 µL。
2.3.4单糖组成分析结果
标准单糖混合物和CCp-1和CCp-2经水解后的单糖GC色谱图见图3所示,通过和标准单糖图谱中保留时间比较可以确定样品的单糖构成。由图3和表2可知,CCp-1和CCp-2均为杂多糖。在CCp-1和CCp-2中均检测到6种单糖:鼠李糖(Rha)、阿拉伯糖(Ara)、木糖(Xyl)、甘露糖(Man)、葡萄糖(Glc)和半乳糖(Gal),其主要单糖虽然均为鼠李糖和半乳糖,但单糖的摩尔比不同。
表2 CCp-1和CCp-2的单糖组成成分
3促凝血紫荆花多糖CCp-1和CCp-2的活性分析
方法:采用大鼠体外血浆凝血三项对紫荆花花多糖进行凝血活性检测
3.1仪器和试剂
TGL-16gR高速台式冷冻离心机(上海安亭科学仪器厂);
RAC-030全自动凝血分析仪(深圳雷杜生命科学股份有限公司);
氯化钠注射液(辰欣药业股份有限公司,1910012706);
云南白药(云南白药集团股份有限公司,ZJA1708);
注射用灯盏花素(昆明龙津药业股份有限公司,20190813-1);
凝血酶原时间(PT)测定试剂盒(20191209M);活化部分凝血酶时间(APTT)测定试剂盒(20200319M);凝血酶时间(TT)测定试剂盒(20190821M)均由深圳雷杜生命科学股份有限公司生产。
实验动物:SD大鼠,SPF级,雄性,体重180~200 g,由河南省实验动物中心提供(SCXK(豫)2017-0001)。
3.3样品溶液配制
3 mg的样品LLp-1b用1000 μL溶剂溶解制3 mg/mL溶液。取灯盏花素8 mg用600 μL溶剂制得13.33 mg/mL,取云南白药1 mg用25 μL溶剂制得40 mg/mL。溶剂(也作为空白溶剂)为:无水乙醇:1,2-丙二醇:生理盐水=1:1:3(体积比)。
血浆的制备:大鼠,采用腹主动脉脉取血,置于一次性抗凝负压真空管,轻轻颠倒混匀,3000 rpm离心15 min,取上层清液备用。
3.4实验方法
(1)对APTT影响的检测方法
分别在不同测试杯中加入25 μL各个样品溶液,再加入50 μL血浆,放入凝血仪器中,自动加入50 μL APTT试剂,37 ℃孵育后加入37 ℃预温的50 μL CaCl2溶液,记录凝固时间,即为APTT值。
(2)对PT影响的检测方法
分别在不同测试杯中加入25 μL各个样品溶液,再加入50 μL血浆,放入凝血仪器中,自动加入100 μL PT试剂,记录凝固时间,即为PT值。
(3)对TT影响的检测方法
分别在不同测试杯中加入25 μL各个样品溶液,再加入100 μL血浆,放入凝血仪器中,自动加入100 μL TT试剂,记录凝固时间,即为TT值。
3.5 数据处理
结果采用算术平均值和标准差表示,数值统计采用SPSS19.0软件单因素方差分析法(One-Way ANOVA)比较其显著性差异。测定结果见表3和图4。
表3 CCp-1和CCp-2对大鼠血浆的凝血时间影响
注:与空白比较, *** p<0.001< ** p<0.01< * p<0.05;
与云南白药比较,▲▲▲ p < 0.001<▲▲ p< 0.01<▲p< 0.05.
CCp-1和CCp-2对APTT、PT和TT的影响如表3和图4所示。结果显示:与对照组相比,云南白药作为促凝剂阳性对照可显著缩短APTT、PT和TT (p<0.05或p<0.01),灯盏花素作为抗凝剂阳性对照可显著延长APTT、PT和TT (p<0.05或p<0.01或p<0.01)。与云南白药相比,CCp-1和CCp-2能显著缩短APTT、PT和TT (p<0.001),说明CCP-1和CCP-2有良好的促凝作用。
Claims (3)
1.促凝血紫荆花多糖的提取分离方法,其特征在于,包括以下步骤:
(1)新鲜的紫荆花,阴干,用石油醚室温浸泡脱脂3次,每次24 h,残渣挥干石油醚后,用乙醇浸提,所得残渣挥发至无醇味,再按料液比 1g:15mL用超纯水于80℃加热提取2次,每次4 h,趁热抽滤,滤液减压浓缩,再向浓缩液中加入乙醇,静置、离心,得到多糖沉淀,冷冻干燥即得紫荆花粗多糖;
(2)采用Sevage法除蛋白;将紫荆花粗多糖用适量超纯水溶解获得粗多糖水溶液,按照粗多糖水溶液1/3体积的量加入Sevage试剂进行除蛋白,Sevage试剂为体积比4:1的氯仿和正丁醇;
(3)取除蛋白后的紫荆花粗多糖,用超纯水溶解、离心后加入到DEAE-52纤维素色谱柱,依次用超纯水、0.05 mol/L、0.15 mol/L、0.25 mol/L的NaCl溶液进行洗脱,洗脱液采用苯酚-硫酸法进行检测,测490 nm处的吸光度,以洗脱管数为横坐标,吸光度为纵坐标,绘制洗脱曲线,合并同一洗脱峰的多糖样品,透析、浓缩后冷冻干燥;其中,超纯水的洗脱峰为组分CC-1,0.25 mol/L NaCl溶液的洗脱峰为组分CC-2;
(4)组分CC-1用超纯水溶解、离心后加入到Sephadex G-100凝胶色谱柱,用超纯水洗脱,采用苯酚-硫酸法进行检测,在波长490 nm处测其吸光度,以洗脱管数为横坐标,吸光度为纵坐标,绘制洗脱曲线,得到单一洗脱峰,冷冻干燥得到的多糖命名为CCp-1;
组分CC-2采用上述与组分CC-1相同的方法进行洗脱,得到单一洗脱峰,冷冻干燥得到的多糖命名为CCp-2;
CCp-1和/或CCp-2即为促凝血紫荆花多糖。
2.利用权利要求1所述的提取分离方法制备得到的促凝血紫荆花多糖。
3.权利要求2所述促凝血紫荆花多糖在制备促凝血药物方面的应用。
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