CN116284471A - 黄花倒水莲多糖及其制备方法及应用 - Google Patents
黄花倒水莲多糖及其制备方法及应用 Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
本发明属于医药技术领域,具体涉及黄花倒水莲多糖及其制备方法及应用。该方法包括以下步骤:1、制备黄花倒水莲水提取液;2、经过微滤膜分离,再经过超滤膜截留,收集超滤膜截留液A;3、依次注入阳离子交换树脂和阴离子交换树脂柱中进行吸附,收集离子交换树脂流出液;4、将离子交换树脂流出液再次经过超滤膜纯化、浓缩,收集超滤膜截留液B;5、加入醇至醇浓度达到80‑90%,过滤,收集沉淀;6、将沉淀充分用无水醇洗涤后,用热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,即得。通过本发明可以制备得到黄花倒水莲多糖。且该黄花倒水莲多糖对酒精致肝脏损伤具有明确的保护作用。
Description
技术领域
本发明属于医药技术领域,具体涉及黄花倒水莲多糖及其制备方法及应用,尤其涉及其在制备保肝产品的应用。
背景技术
肝脏是人体代谢和降解毒素的重要器官,但过量的酒精、药物或其他有毒物质会引起肝脏不同程度的损伤,其中酒精是造成肝脏损伤的最主要因素。当机体摄入酒精后,肝脏对酒精进行代谢并产生毒副作用的代谢产物,从而导致大量肝细胞死亡而引发肝脏炎症,随后受损的肝脏长出大量纤维组织逐渐硬化,甚至恶化为肝癌,这是造成死亡的重要原因。而且,近年来中国、欧洲和美国等国家中因过量饮酒引发的肝脏损伤的发病率越来越高,因此,亟需寻找一种安全、有效、价廉的具有预防酒精性肝损伤的产品一直是医药领域研究热点。
众所周知,天然植物成分是发现创新药物的最便捷途径之一,而植物多糖是一类高分子天然产物,因其安全无毒,生物活性广泛受到人们关注。目前在我国上市的植物多糖药物就黄芪多糖、人参多糖、灵芝多糖、猪苓多糖、香茹多糖等。由此可见,植物多糖是创新药物研发的灵感与重要来源。
黄花倒水莲(Polygala fallax Hemsl)为远志科远志属植物,是我国特有物种,该植物以根入药,性平、微苦,具有补益气血、健脾利湿、活血调经之功效,可用于治疗病后体虚、腰膝酸痛、跌打损伤、急慢性肝炎等疾病。黄花倒水莲食用和药用历史悠久,是广西壮、瑶、苗等少数民族常用药食两用滋补药材。由于其良好的疗效,近年来受到人们的广泛关注。研究表明,黄花倒水莲具有调节血脂、抗血凝、抗衰老、抑制乙肝病毒、抗应激、增强免疫力等生理活性,这些功能与其含有丰富的皂苷、多糖、黄酮等功能成分密切相关。然而多糖作为其主要功能成分之一,是否对酒精性肝脏损伤具有保护作用,目前还未清楚。而且目前关于黄花倒水莲多糖的提取工艺存在多次使用有机溶剂,增加安全隐患,不利于规模化生产,而且不同生产工艺制备的多糖成分也不尽相同,功效也有所不同。因此,为了制备一种能够改善酒精性肝损伤的黄花倒水莲多糖,提高黄花倒水莲的利用价值。针对改善酒精性肝损伤功能,建立一种高效、绿色环保、易于规模化生产黄花倒水莲多糖的制备工艺。
发明内容
为解决现有技术的不足,本发明提供了一种黄花倒水莲多糖及其制备方法及应用。
本发明所提供的技术方案如下:
一种黄花倒水莲多糖的制备方法,包括以下步骤:
步骤1,在干燥的黄花倒水莲粗粉中加入8~10倍重量的纯水,90~100℃加热回流提取30min~60min,过滤,滤渣再次加入3~5倍量的纯水,90~100℃加热回流提取25~35min,过滤,合并2次提取液,得到黄花倒水莲水提取液;
步骤2,将步骤1得到的所述的黄花倒水莲水提取液经过微滤膜分离,微滤膜透过液再经过超滤膜截留,收集超滤膜截留液A;
步骤3,将步骤2得到的所述的超滤膜截留液A依次注入阳离子交换树脂和阴离子交换树脂柱中进行吸附,收集离子交换树脂流出液;
步骤4,将步骤3得到的所述的离子交换树脂流出液再次经过超滤膜纯化、浓缩,收集超滤膜截留液B;
步骤5,将步骤4所述的超滤膜截留液B中加入醇至醇浓度达到80-90%,过滤,收集沉淀;
步骤6,将步骤5所述的沉淀充分用无水醇洗涤后,用热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,得到本发明所述的黄花倒水莲多糖。
上述技术方案的有益效果如下:
黄花倒水莲中不仅含有丰富的多糖成分,而且还含有丰富的皂苷、黄酮、单糖、低聚糖等植物小分子次生代谢产物,因此,常规的多糖提取分离方法对黄花倒水莲多糖成分的分离纯化带来巨大困难。因此,本发明的发明原理如下:
(1)基于多糖分子的亲水性特点,以水作为提取溶媒,即安全,又便宜,同时能够有效地将黄花倒水莲多糖成分提取出来,但不可避免将大量水溶性的次生代谢产物、氨基酸、蛋白质等成分一同提取出来。
(2)为了更好去除提取液中其他非多糖成分,本发明基于多糖分子量大的特性,应用膜分离技术与离子交换树脂分离技术相结合,选择适宜截留分子量的分离膜设备,通过分子量的控制分离杂质,并通过离子交换树脂吸附除去具有带电基团的杂质,从而定向制备多糖成分,并获得特定分子量的活性多糖。
具体的,步骤2所述的微滤膜的截留分子量为200KD~400KD。步骤2所述的超滤膜的截留分子量为8kD~10kD。
选择上述截留分子量的微滤膜和超滤膜,不仅可以有效地去除胶质、蛋白质或更大分子量的多糖(纤维素等)等非活性成分,而且还可以去除皂苷、黄酮、单糖等小分子植物代谢产物,从而根据需求,定向制备所需分子量的多糖。
具体的,步骤3所述的阳离子交换树脂为001×7、001×8或D001中任一种。步骤3所述的阴离子交换树脂为201×7、M511或D301中任一种。
采用上述型号的离子交换树脂,能够有效的从超滤膜截留液A中吸附多糖溶液中的色素分子、可溶性氨基酸或蛋白质等带电荷杂质,进一步去除杂质。
具体的,步骤4所述的超滤膜截留分子量为4kD~6kD。基于该超滤膜不仅可以进一步去除残余的小分子成分,而且还可以对多糖溶液进行浓缩,避免常规高温浓缩造成多糖结构变性,影响多糖活性。
具体的,步骤5所述的醇为80-90%乙醇。本发明采用醇沉法的原因在于,乙醇可降低水溶性多糖的溶解度而沉淀,而且不同浓度的乙醇沉淀的多糖组分也是不同的。因此,本发明在80-90%乙醇条件下获得多糖具有较好的纯度与活性。
具体的,步骤6所述的无水醇为无水乙醇。本发明采用无水乙醇作为洗涤剂,其对许多植物色素或其他植物次生代谢产物具有较好的溶解性,能够进一步提高多糖纯度。
本发明的目的之二是提供了根据上述制备方法制备得到的黄花倒水莲多糖。
本发明的目的之三是提供上述黄花倒水莲多糖的应用,用于制备保肝或护肝产品。
具体的,用于制备改善酒精引发肝脏损伤的保肝或护肝产品。
本发明开展了黄花倒水莲多糖对酒精致小鼠肝脏损伤的保护作用研究。研究结果表明,摄食本发明制备的黄花倒水莲多糖能够降低小鼠血清谷草转氨酶、谷丙转氨酶和丙二醛的含量;提高超氧化物歧化酶活性,减少氧化损伤;抑制肝脏中炎症介质肿瘤坏死因子α和白细胞介素1β分泌,降低炎症反应,从而改善酒精对小鼠肝脏损伤,验证了本发明制备的黄花倒水莲多糖对酒精致肝脏损伤具有保护作用。
具体的,所述的产品剂型为固体、半固体或液体制剂。
具体的,所述的产品为药品或食品、或者功能食品。
本发明的有益效果:
通过本发明可以制备得到花倒水莲多糖。且该黄花倒水莲多糖对酒精致肝脏损伤具有明确的保护作用,可为酒精性肝损伤患者提供新的选择。
具体实施方式
以下对本发明的原理和特征进行描述,所举实施例只用于解释本发明,并非用于限定本发明的范围。
实施例1
步骤1,取干燥的黄花倒水莲根粗粉5kg,加入9倍量的纯水,95℃加热回流提取45min,过滤,滤渣再次加入4倍量的纯水,95℃加热回流提取30min,过滤,合并2次提取液,得到黄花倒水莲水提取液。
步骤2,将步骤1所述的黄花倒水莲水提取液,先经过300KD的微滤膜分离,微滤膜流出液再经过9KD超滤膜分离,收集超滤膜截留液A。
步骤3,将步骤2所述的超滤膜截留液A依次注入001×7阳离子交换树脂和201×7阴离子交换树脂柱中进行吸附,收集离子交换树脂流出液。
步骤4,将步骤3所述的离子交换树脂流出液经过5KD超滤膜纯化,浓缩,收集超滤膜截留液B。
步骤5,将步骤4所述的超滤膜截留液B中加入乙醇至乙醇浓度达到85%,过滤,收集沉淀。
步骤6,将步骤5所述的沉淀充分用无水乙醇洗涤后,热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,得到本发明所述的黄花倒水莲多糖112g,硫酸苯酚法检测,多糖含量为68.5%。
实施例2
步骤1,取干燥的黄花倒水莲根粗粉5kg,加入10倍量的纯水,100℃加热回流提取30min,过滤,滤渣再次加入4倍量的纯水,100℃加热回流提取25min,过滤,合并2次提取液,得到黄花倒水莲水提取液。
步骤2,将步骤1所述的黄花倒水莲水提取液,先经过400KD的微滤膜分离,微滤膜流出液再经过10KD超滤膜分离,收集超滤膜截留液A。
步骤3,将步骤2所述的超滤膜截留液A依次注入001×8阳离子交换树脂和M511阴离子交换树脂柱中进行吸附,收集离子交换树脂流出液。
步骤4,将步骤3所述的离子交换树脂流出液经过6KD超滤膜纯化,浓缩,收集超滤膜截留液B。
步骤5,将步骤4所述的超滤膜截留液B中加入乙醇至乙醇浓度达到90%,过滤,收集沉淀。
步骤6,将步骤5所述的沉淀充分用无水乙醇洗涤后,热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,得到本发明所述的黄花倒水莲多糖108g,硫酸苯酚法检测,多糖含量为66.5%。
实施例3
步骤1,取干燥的黄花倒水莲根粗粉5kg,加入8倍量的纯水,90℃加热回流提取60min,过滤,滤渣再次加入4倍量的纯水,90℃加热回流提取35min,过滤,合并2次提取液,得到黄花倒水莲水提取液。
步骤2,将步骤1所述的黄花倒水莲水提取液,先经过200KD的微滤膜分离,微滤膜流出液再经过8KD超滤膜分离,收集超滤膜截留液A。
步骤3,将步骤2所述的超滤膜截留液A依次注入D001阳离子交换树脂和D301阴离子交换树脂柱中进行吸附,收集离子交换树脂流出液。
步骤4,将步骤3所述的离子交换树脂流出液经过4KD超滤膜纯化,浓缩,收集超滤膜截留液B。
步骤5,将步骤4所述的超滤膜截留液B中加入乙醇至乙醇浓度达到80%,过滤,收集沉淀。
步骤6,将步骤5所述的沉淀充分用无水乙醇洗涤后,热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,得到本发明所述的黄花倒水莲多糖105g,硫酸苯酚法检测,多糖含量为65.8%。
对比例
步骤1,取干燥的黄花倒水莲根粗粉5kg,加入9倍量的纯水,95℃加热回流提取45min,过滤,滤渣再次加入4倍量的纯水,95℃加热回流提取30min,过滤,合并2次提取液,得到黄花倒水莲水提取液。
步骤2,将步骤1所述的黄花倒水莲提取液60℃减压浓缩后,加入乙醇至乙醇浓度达到85%,过滤,收集沉淀。
步骤3,将步骤5所述的沉淀充分用无水乙醇洗涤后,热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,得到对比例黄花倒水莲多糖256g,硫酸苯酚法检测,多糖含量为23.8%。
功能评价实验:黄花倒水莲多糖改善酒精致小鼠肝脏损伤的药效评价
1.实验动物
SPF级雄性昆明小鼠42只,6-8周龄,适应性饲养1周。
2.分组、给药及处理
将小鼠随机分为7组,即正常组、模型组、阳性对照组(联苯双酯组,100mg/kg/d)、实施例1-3组(200mg/kg/d)和对比例组(200mg/kg/d),每组8只,按上述分组方案连续灌胃给药7d,每天1次,正常组和模型组给予等量的生理盐水。
从给药第6d起,给药结束后,除正常组外,其余各组立即灌胃50%食用酒精,灌胃3次,两次间隔12h,单次给予酒精剂量为5g/kg,建立小鼠酒精性肝损伤模型。小鼠最后一次给药4h后,通过眼底静脉采血制备血清,并处理死小鼠后,取肝脏。
3.实验结果
3.1黄花倒水莲多糖对酒精致肝脏损伤小鼠血清中谷草转氨酶、谷丙转氨酶的影响
血液中的谷草转氨酶(AST)和谷丙转氨酶(ALT)活性是反映肝脏损伤的重要指标。结果如表1所示,与正常组相比较,模型组小鼠的血清AST和ALT显著升高,表明酒精致小鼠肝脏损伤模型建立成功。当给予本发明制备的黄花倒水莲多糖后,小鼠血清中AST和ALT活性显著降低,表明黄花倒水莲多糖能够改善酒精致小鼠肝脏损伤。
表1黄花倒水莲多糖对酒精性肝损伤小鼠血清AST和ALT活性的影响
注:与正常对照组比较,##p<0.01;与模型组比较,**p<0.01
3.2黄花倒水莲多糖对酒精致肝脏损伤小鼠血清中超氧化物歧化酶和丙二醛的影响
酒精在肝脏代谢过程中会产生大量的活性氧和丙二醛等代谢产物,破坏机体氧化还原平衡和线粒体功能,导致氧化应激和脂质代谢异常,进而造成肝脏损伤。结果如表2所示,与正常组相比较,模型组小鼠血清超氧化物歧化酶(SOD)显著降低,丙二醛(MDA)显著上升,表明酒精代谢造成SOD大量消耗并产生大量有害物MDA。当给予本发明的黄花倒水莲多糖后,能够显著提升SOD含量,并减少MDA产生,表明黄花倒水莲多糖能够改善酒精代谢造成的机体氧化还原失调,减少MDA产生,达到保护肝脏作用。
表2黄花倒水莲多糖对酒精致肝脏损伤小鼠血清中SOD和MDA的影响
注:与正常对照组比较,##p<0.01;与模型组比较,**p<0.01
3.3黄花倒水莲多糖对酒精致肝脏损伤小鼠肝脏中肿瘤坏死因子α和白介素1β的影响
肝脏在酒精代谢过程中产生的内毒素会引发肝脏炎症,分泌大量的炎症介质,进一步加重肝脏损伤。结果如表3所示,与正常组相比较,模型组肝脏组织中肿瘤坏死因子α(TNF-α)和白介素1β(IL-1β)含量显著升高,表明酒精代谢引发肝脏炎症。当给予本发明的黄花倒水莲多糖后,小鼠肝脏中TNF-α和IL-1β均显著下降,表明黄花倒水莲多糖能够抑制酒精致小鼠肝脏炎症反应,进而保护肝脏。
表3黄花倒水莲多糖对酒精致肝脏损伤小鼠肝脏中TNF-α和IL-1β的影响
注:与正常对照组比较,##p<0.01;与模型组比较,**p<0.01
可见,本发明制备的黄花倒水莲多糖能够显著降低小鼠血清AST、ALT和MDA的含量;提高SOD活性,减少氧化损伤,;抑制肝脏中炎症介质TNF-α和IL-1β分泌,降低炎症反应,进而改善酒精对小鼠肝细胞损伤。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种黄花倒水莲多糖的制备方法,其特征在于,包括以下步骤:
步骤1,在干燥的黄花倒水莲粗粉中加入8~10倍重量的纯水,90~100℃加热回流提取30min~60min,过滤,滤渣再次加入3~5倍量的纯水,90~100℃加热回流提取25~35min,过滤,合并2次提取液,得到黄花倒水莲水提取液;
步骤2,将步骤1得到的所述的黄花倒水莲水提取液经过微滤膜分离,微滤膜透过液再经过超滤膜截留,收集超滤膜截留液A;
步骤3,将步骤2得到的所述的超滤膜截留液A依次注入阳离子交换树脂和阴离子交换树脂柱中进行吸附,收集离子交换树脂流出液;
步骤4,将步骤3得到的所述的离子交换树脂流出液再次经过超滤膜纯化、浓缩,收集超滤膜截留液B;
步骤5,将步骤4所述的超滤膜截留液B中加入醇至醇浓度达到80-90%,过滤,收集沉淀;
步骤6,将步骤5所述的沉淀充分用无水醇洗涤后,用热水溶解至饱和,冷却至室温,离心,取上清液冷冻干燥,得到本发明所述的黄花倒水莲多糖。
2.根据权利要求1所述的黄花倒水莲多糖的制备方法,其特征在于:步骤2所述的微滤膜的截留分子量为200KD~400KD,超滤膜的截留分子量为8kD~10kD。
3.根据权利要求1所述的黄花倒水莲多糖的制备方法,其特征在于:步骤3中:所述的阳离子交换树脂为001×7、001×8或D001中任一种;阴离子交换树脂为201×7、M511或D301中任一种。
4.根据权利要求1所述的黄花倒水莲多糖的制备方法,其特征在于:步骤4所述的超滤膜截留分子量为4kD~6kD。
5.根据权利要求1所述的黄花倒水莲多糖的制备方法,其特征在于:步骤5所述的醇为乙醇。
6.根据权利要求1所述的黄花倒水莲多糖的制备方法,其特征在于:步骤6所述的的无水醇为无水乙醇。
7.一种根据权利要求1至6任一所述的制备方法制备得到的黄花倒水莲多糖。
8.根据权利要求7所述的黄花倒水莲多糖的应用,其特征在于:用于制备保肝或护肝产品。
9.根据权利要求8所述的黄花倒水莲多糖的应用,其特征在于:用于制备改善酒精引发肝脏损伤的保肝或护肝产品。
10.根据权利要求8所述的黄花倒水莲多糖的应用,其特征在于:
所述的产品剂型为固体、半固体或液体制剂;
所述的产品为药品或食品、或者功能食品。
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