CN108309991A - 富含dha的磷脂在改善急性肾损伤制品中的应用 - Google Patents
富含dha的磷脂在改善急性肾损伤制品中的应用 Download PDFInfo
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Abstract
本发明公开了一种富含DHA的磷脂在改善急性肾损伤制品中的应用。本发明以鱿鱼卵为原料制备DHA磷脂，进一步分离和转化，制得的磷脂的纯度均在90%以上。本发明提供的鱿鱼卵磷脂提取物的制备方法成本低，步骤简单，使用的有机溶剂量少，对环境产生的有机溶剂污染小。通过动物实验验证，急性肾损伤小鼠仅补充干预200‑400mg/kg富含DHA卵磷脂，就明显提高了肾损伤小鼠的生存时间，以及降低了小鼠的肾脏组织损伤程度，即证明了富含DHA的磷脂能够应用于改善急性肾损伤制品中。
Description
技术领域
本发明属于活性物质筛选技术领域中，尤其涉及富含DHA的磷脂的提取及制备方法，以及其在预防、改善或治疗急性肾损伤制品中的应用。
背景技术
急性肾损伤(acute kidney injury, AKI)，主要是由肾缺血或肾毒性药物而导致的一类重症疾病，临床主要表现为血肌酐和尿素氮水平的急剧增高，其中绝大分的患者肾脏病理表现为急性肾小管坏死(acute tubular necrosis, ATN)。目前已有的研究表明，AKI 的主要发病机制包括线粒体破坏、炎症反应、氧化应激反应及肾小管上皮细胞坏死和凋亡等。由于 AKI有着十分复杂的发病机制，且目前仍缺乏有效的治疗手段，故关于 AKI的具体发病机制及防治的研究已经成为肾脏病学领域亟待解决的问题之一。
目前，临床上针对急性肾损伤的治疗，目前尚无有效的治疗手段，因此开发安全有效的具有预防、减缓或治疗急性肾损伤的膳食补充剂或者功能性食品成为迫切要求。
近年来研究发现，w-3多不饱和脂肪酸（n3多不饱和脂肪酸）， 如二十二碳六烯酸（DHA）可以保护多种疾病，包括动脉粥样硬化、糖尿病、癌症、炎症，然而富含多不饱和脂肪酸的鱼油对于急性肾损伤的研究尚未有报道。目前市场上的DHA产品主要为乙酯型和甘油酯型，存在于磷脂中的不饱和脂肪酸相对于甘油酯型和乙酯型不饱和脂肪酸，不仅氧化稳定性更好，而且消化吸收速度更快，再加上磷脂本身具有改善脂肪代谢、增强免疫力等活性，到目前为止，对于富含DHA的磷脂改善急性肾损伤的功效，尚未有相关的研究报道，因此富含DHA的磷脂可能作为一种改善急性肾损伤的新型脂质保健产品。
发明内容
本发明的目的是提供一种富含DHA的磷脂的提取及制备方法，并研究其在改善急性肾损伤方面的应用，以弥补现有技术的不足。
为达到上述目的，本发明提供的具体技术方案如下：
一种富含DHA的磷脂在改善急性肾损伤制品中的应用。
进一步的，所述富含DHA磷脂，其DHA磷脂含量为30-40%。
进一步的，所述富含DHA的磷脂为所述富含DHA磷脂为DHA磷脂酰胆碱、DHA磷脂酰乙醇胺、DHA磷脂酰丝氨酸和DHA磷脂酰肌醇中的一种或多种混合。
本发明还提供了一种富含DHA的磷脂的制备方法，包括以下步骤：
1）鱿鱼卵经真空冷冻干燥粉碎后，制成粉末；
2）取鱿鱼卵干粉，加入乙醇，用匀浆机匀浆50min，过滤后将乙醇浸提液用真空旋转蒸发旋干，得到的提取物即为乙醇浸提物，将乙醇浸提物溶于氯仿甲醇混合液,然后转移至分液漏斗中，加入氯化钠溶液，摇匀并静止过夜，收集下层氯仿层，经减压浓缩得到鱿鱼卵脂质粗提取物；
3）将硅胶经活化装柱后，用氯仿平衡硅胶柱；再将步骤 2）得到的鱿鱼卵的脂质粗提物用氯仿溶解后，缓慢倒入硅胶柱中，依次用氯仿、氯仿-甲醇混合液、丙酮和甲醇进行洗脱，收集洗脱液，洗脱液进行薄层硅胶板层析，并用碘和Dittmer试剂显色确认后减压浓缩去除残留有机溶剂即得到鱿鱼卵磷脂；其中, 收集氯仿-甲醇的洗脱液为DHA磷脂酰乙醇胺（DHA-PE）；收集甲醇的洗脱液为DHA磷脂酰胆碱（DHA-PC）；
4）将醋酸/醋酸钠溶液缓冲液放入集热式恒温加热磁力搅拌器中预热，将 L-丝氨酸溶于其中，加入上述步骤3）中的DHA-PC，最后加入磷脂酶D，在 40 ℃下搅拌反应48h，加入盐酸，灭活酶，反应终止，用正己烷-异丙醇提取反应产物，即得到DHA磷脂酰丝氨酸（DHA-PS）；
5）将步骤 2）中鱿鱼卵脂质粗提取物，加入丙酮、三甲胺和乙酸酐，冷却过滤后，用丙酮洗脱，得到DHA磷脂酰肌醇（DHA-PI）。
进一步的，所述制品为改善急性肾损伤的组合物：包括功能性食品或药品，及相关制品，如乳剂等。
本发明的有益效果：
本发明以鱿鱼卵为原料制备DHA磷脂，进一步分离和转化，制得的磷脂的纯度均在90%以上。本发明提供的鱿鱼卵磷脂提取物的制备方法成本低，步骤简单，使用的有机溶剂量少，对环境产生的有机溶剂污染小。
通过动物实验验证，急性肾损伤小鼠仅补充干预200-400mg/kg富含DHA卵磷脂，就明显提高了肾损伤小鼠的生存时间，以及降低了小鼠的肾脏组织损伤程度。本发明制得的各个组分的DHA磷脂能够极大地延长了急性肾损伤小鼠的生存时间，并且能明显减缓小鼠肾脏组织病变速度，即DHA磷脂均能明显改善急性肾损伤小鼠的肾损伤程度，说明了DHA磷脂能有效预防、改善或治疗急性肾损伤。
附图说明
图 1 为本发明实施例2中富含DHA的磷脂对急性肾损伤小鼠的生存时间影响的结果图。
图 2 为本发明实施例2中富含DHA的磷脂对急性肾损伤小鼠肾脏组织病理学影响的结果图。
图3为本发明实施例2中富含DHA的磷脂对急性肾损伤小鼠血清肾功能指标影响的结果图。
具体实施方式
以下采用实施例并结合附图来详细说明本发明。
实施例1：富含DHA磷脂的制备
（1）将市售的乌贼卵经真空冷冻干燥粉碎后，磨成粉末（200目）。
（2）取鱿鱼卵干粉，加入乙醇，用匀浆机匀浆50min，过滤后将乙醇浸提液用真空旋转蒸发旋干，得到的提取物即为乙醇浸提物，将乙醇浸提物溶于氯仿甲醇混合液,然后转移至分液漏斗中，加入氯化钠溶液，摇匀并静止过夜，收集下层氯仿层，经减压浓缩得到鱿鱼卵脂质粗提取物；
（3）将硅胶经活化装柱后，用氯仿平衡硅胶柱 ；再将步骤（2）得到的鱿鱼卵的脂质粗提物用氯仿溶解后，缓慢倒入硅胶柱中，依次用氯仿、氯仿-甲醇混合液、丙酮和甲醇进行洗脱，收集洗脱液，洗脱液进行薄层硅胶板层析，并用碘和Dittmer试剂显色确认后减压浓缩去除残留有机溶剂即得到鱿鱼卵磷脂；其中, 收集氯仿-甲醇的洗脱液为DHA磷脂酰乙醇胺， 收集甲醇的洗脱液为DHA磷脂酰胆碱；
（4）将醋酸/醋酸钠溶液缓冲液放入集热式恒温加热磁力搅拌器中预热，将 L-丝氨酸溶于其中，加入上述步骤（3）中的DHA-PC，最后加入磷脂酶D，在 40 ℃下搅拌反应48h，加入盐酸，灭活酶，反应终止，用正己烷-异丙醇提取反应产物，即得到DHA磷脂酰丝氨酸。
（5）将步骤（2）中鱿鱼卵脂质粗提取物，加入丙酮、三甲胺和乙酸酐，冷却过滤后，用丙酮洗脱，得到DHA磷脂酰肌醇。
实施例2：动物实验
1.材料和方法
（1）动物喂养和行为学分析
实验动物采用 Balb/c小鼠，雄性，体重 20-22g，购自北京维通利华实验动物技术有限公司。动物房湿度65±15%，室温23±2℃，12h:12 h明暗交替，小鼠可自由摄食和饮水。小鼠在适应性喂养一周后，将小鼠分为8组 ：正常对照组、模型对照组、PC、DHA、DHA-PC、DHA-PS、DHA-PE及DHA-PI组，每组 19只，分别喂食相应的饲料。饲料参照 AIN-93G 啮齿动物饲料配方并加以改良，记录和统计摄食量，每隔一天称量记录体重变化，连续喂养1周。除正常组外，其余五组每日腹腔注射盐酸万古霉素（(400 mg /kg.bw/day），DHA的灌胃剂量为300 mg/kg.bw/day，受试物组的DHA和PC含量基本相同，模型对照组组灌胃等量的生理盐水。干预一周后，每组取9只小鼠禁食过夜处死，收集血液和肾组织样本，用于生化指标检测。剩余小鼠继续进行万古霉素注射和受试物干预，每隔6h观察小鼠死亡状况，并进行记录，最后绘制小鼠死亡曲线，计算实验组小鼠和对照组小鼠的死亡率。
（2）统计学处理
实验数据以`x ± SEM表示，采用 SPSS 18.0 软件进行 Student’s t test andTukey’s test 分析， 以 P ＜ 0.05 为有显著差异。
实验结果；
富含DHA的磷脂对万古霉素诱导的急性肾损伤小鼠生存时间的影响如图1所示，模型组相对于正常对照组而言，小鼠的生存时间明显的减短了，正如我们期望的，在富含DHA的磷脂干预后，与模型组相比，小鼠的生存时间明显的延长了，这提示富含DHA的磷脂能明显的改善万古霉素诱导的急性肾损伤小鼠的生存时间。
富含DHA的磷脂对急性肾损伤小鼠的肾脏组织的病理学HE结果如图2所示，与正常组小鼠相比，模型组小鼠注射万古霉素会引起肾脏组织发生明显的变化：大面积坏死区域，肾小管出现明显的萎缩、 扩张，坏死的肾小管上皮细胞明显增多，肾近曲小管上皮细胞有空泡变性、坏死，管腔可见脱落上皮细胞，肾小管出现蛋白管型，管腔狭窄，出现典型的急性肾损伤病理表现，而富含DHA的磷脂明显的减轻肾损伤程度，细胞排列逐渐规则，肾小管萎缩程度逐渐减轻，坏死的肾小管上皮细胞明显减少，空泡变性明显减少，肾小球和肾小管结构逐渐趋于完整。 这提示富含DHA的磷脂能明显的改善万古霉素诱导的急性肾损伤小鼠的肾脏组织的病理改变。
图3为小鼠血清中急性肾损伤标记物水平变化，如图所示，与对照组相比，模型组小鼠血清中BUN、Cr、KIM-1和CysC水平显著增加，说明造模成功。而在富含DHA的磷脂干预后，小鼠的肾损伤标记物水平均明显降低了，这提示富含DHA的磷脂能明显的保护小鼠的肾损伤。
以类似急性肾损伤作为实验对象，其病症产生机理较复杂，包括线粒体功能障碍介导的细胞凋亡，MAPK信号通路，炎症，肾脏内氧化应激和内质网应激等，实施例2中主要机制可能为肾脏内氧化应激导致的细胞凋亡造成的肾小管损伤，即说明了富含DHA的磷脂能显著的改善万古霉素诱导的急性肾损伤。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
Claims (5)
1.一种富含DHA的磷脂在改善急性肾损伤制品中的应用。
2.如权利要求1所述的应用，其特征在于，所述富含DHA磷脂中，其DHA磷脂含量为30-40%。
3.如权利要求1所述的应用，其特征在于，所述富含DHA的磷脂为所述富含DHA磷脂为DHA磷脂酰胆碱、DHA磷脂酰乙醇胺、DHA磷脂酰丝氨酸和DHA磷脂酰肌醇中的一种或多种混合。
4.一种富含DHA的磷脂的制备方法，其特征在于，该方法包括以下步骤：
1）鱿鱼卵经真空冷冻干燥粉碎后，制成粉末；
2）取鱿鱼卵干粉，加入乙醇，用匀浆机匀浆50min，过滤后将乙醇浸提液用真空旋转蒸发旋干，得到的提取物即为乙醇浸提物，将乙醇浸提物溶于氯仿甲醇混合液,然后转移至分液漏斗中，加入氯化钠溶液，摇匀并静止过夜，收集下层氯仿层，经减压浓缩得到鱿鱼卵脂质粗提取物；
3）将硅胶经活化装柱后，用氯仿平衡硅胶柱 ；再将步骤 2）得到的鱿鱼卵的脂质粗提物用氯仿溶解后，缓慢倒入硅胶柱中，依次用氯仿、氯仿-甲醇混合液、丙酮和甲醇进行洗脱，收集洗脱液，洗脱液进行薄层硅胶板层析，并用碘和Dittmer试剂显色确认后减压浓缩去除残留有机溶剂即得到鱿鱼卵磷脂；其中, 收集氯仿-甲醇的洗脱液为DHA磷脂酰乙醇胺，收集甲醇的洗脱液为DHA磷脂酰胆碱；
4）将醋酸/醋酸钠溶液缓冲液放入集热式恒温加热磁力搅拌器中预热，将 L-丝氨酸溶于其中，加入上述步骤3）中的DHA-PC，最后加入磷脂酶D，在 40 ℃下搅拌反应48h，加入盐酸，灭活酶，反应终止，用正己烷-异丙醇提取反应产物，即得到DHA磷脂酰丝氨酸；
5）将步骤 2）中鱿鱼卵脂质粗提取物，加入丙酮、三甲胺和乙酸酐，冷却过滤后，用丙酮洗脱，得到DHA磷脂酰肌醇。
5.如权利要求1所述的应用，其特征在于，所述改善急性肾损伤制品为改善急性肾损伤的组合物：包括功能性食品或药品，及相关制品。
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| CN112315984A (zh) * | 2020-09-09 | 2021-02-05 | 山东省科学院生物研究所 | 海洋来源磷脂在促进血管生成方面的应用 |
| CN112315984B (zh) * | 2020-09-09 | 2023-02-14 | 山东省科学院生物研究所 | 海洋来源磷脂在促进血管生成方面的应用 |
| CN112315953A (zh) * | 2020-11-06 | 2021-02-05 | 中国海洋大学 | 一种姜黄素酯在改善急性肾损伤制品中的应用 |
| CN112315953B (zh) * | 2020-11-06 | 2023-03-07 | 中国海洋大学 | 一种姜黄素酯在改善急性肾损伤制品中的应用 |
| CN113662948A (zh) * | 2021-09-10 | 2021-11-19 | 中国海洋大学 | 磷脂及其衍生物在制备改善系统性红斑狼疮制品中的应用 |
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