CN114891130A - 两种何首乌抗炎多糖的制备及其应用 - Google Patents
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Abstract
两种何首乌抗炎多糖的制备及其应用,属于医药技术领域。该多糖由生何首乌经提取、纯化得到,经斑马鱼抗炎模型进行药效评价,具有较好的抗炎活性。何首乌抗炎活性多糖可以应用在保健食品或药品开发中。
Description
技术领域
本发明属于医药保健品技术领域,具体而言,本发明涉及一种何首乌抗炎多糖的制备及其应用。
背景技术
何首乌为蓼科植物何首乌(Polygonum multiflorum Thunb.)的干燥块根。何首乌采挖后,清洗切片,干燥后即为生何首乌饮片,将生何首乌片炮制后称为制何首乌饮片。生何首乌和制何首乌具有不同的功效,生何首乌苦、甘、涩,微温,具有解毒、消痈、截疟及润肠通便的功效,临床上用于疮痈、瘰疬、风疹瘙痒、久疟体虛及肠燥便秘。制何首乌苦、甘、涩,微温,具有补肝肾、益精血、乌须发、强筋骨及化浊降脂的功效。临床上用于血虚萎黄、眩晕耳鸣、须发早白、腰膝酸软、肢体麻木、崩漏带下及高脂血症[2]。
何首乌主要化学成分主要包括二苯乙烯苷、蒽醌、鞣质及磷脂等,我们针对其多糖类成分进行了提取、分离纯化,得到两种均一多糖。应用斑马鱼药理模型,通过孵化率、心跳率、卵黄囊面积、ROS含量、NO 含量等指标对何首乌多糖(WPMP-1、WPMP-2)的抗炎效果进行了评价。结果显示,不同浓度何首乌多糖(10、 50、100μg/mL)对LPS诱导浓度引起的炎症斑马鱼具有保护作用,呈现浓度依赖性,能够恢复卵黄囊正常大小,显著降低心率、ROS生成率以及NO生成率,且当在相同多糖浓度下,WPMP-2高于WPMP-1抗炎活性,说明何首乌多糖具有较好的抗炎活性。该多糖可以进一步应用于保健品和药品的开发,具有很好的市场前景。
发明内容
本发明的第一个目的是提供一种何首乌抗炎多糖的制备方法。
本发明的第二个目的是提供何首乌多糖的应用。
一种何首乌抗炎多糖的制备及其应用,其特征在于,所用原料为蓼科植物何首乌(Polygonum multiflorum Thunb.)的干燥块根,何首乌原料经粉碎、脱脂除杂、提取、分离、精制得到。
两种何首乌抗炎多糖的制备方法,其特征在于:
(1)何首乌饮片适当粉碎,经乙醇浸泡脱脂除杂,其中何首乌与乙醇的质量比=1:(10-20),浸泡次数为 3-5次,浸泡时间为5-10小时;
(2)除杂后的何首乌进行煎煮提取,其中何首乌与水的质量比=1:(10-30),提取次数为1-4次,提取时间为1-4小时,何首乌煎煮提取液减压浓缩至密度为1.12-1.18,再加入无水乙醇至乙醇浓度为75-85%,在4℃冰箱过夜沉淀,收集沉淀,得到粗多糖;
(3)何首乌粗多糖加水溶解至浓度为5-10wt%,经AB-8大孔树脂进行脱色处理,用去离子水2-4BV洗脱,收集过柱液和洗脱液,并减压浓缩5-15wt%,加入1-3wt%的木瓜蛋白酶并置于水浴锅60℃反应2.5h,冷却至室温,然后加入Sevag试剂(Sevag试剂为氯仿:正丁醇体积比=4:1的比例混合均匀),Sevag试剂在混合体系中的体积百分比为,多糖溶液:Sevag试剂=4:1,振摇,离心去除变性蛋白,重复以上操作2-4次,取上清液,得精制多糖溶液。
(4)何首乌精制多糖溶液稀释至浓度为5-10wt%,再经过DEAE-52柱,然后分别用去离子水和0.5M的 NaCl洗脱,收集若干份,每份收集20-100mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1 mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,对应得到两种多糖组分F-1、F-2。多糖组分F-2溶液置于截留分子量为3500Da的透析袋中,在4℃去离子水中透析 48h后得到脱盐多糖组分F-2。
(5)何首乌多糖组分F-1和脱盐多糖组分F-2再分别经过Sephadex G-100柱分离,去离子水洗脱,收集若干份,每份收集20-100mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,得到两种均一多糖组分,分别为WPMP-1和WPMP-2。将WPMP-1和WPMP-2多糖溶液进行冷冻干燥,得到对应的WPMP-1 和WPMP-2何首乌多糖固体粉末。
WPMP-1和WPMP-2何首乌多糖的应用,用于制备抗炎药物或保健品。
进一步将何首乌抗炎多糖与辅料制成口服制剂,进一步制成口服片剂、口服胶囊剂。
本发明方法与现有技术相比具有以下有益效果:
1)本发明应用现代科学原理和技术,对中药何首乌多糖成分进行提取、分离纯化,得到均一多糖组分。
2)本发明得到的何首乌多糖组分具有较好的抗炎效果,可以进一步开发成保健品或药品,具有广阔的市场前景。
附图说明
A 何首乌多糖的DEAE-52柱层析分离;B F-1的Sephadex G-100柱层析分离;C F-2的Sephadex G-100 柱层析分离。
与空白组比较,1)P<0.05。
与空白组比较,1)P<0.05。
A 不同LPS浓度诱导下斑马鱼卵黄囊的形态;B 不同LPS浓度诱导下斑马鱼卵黄囊面积。
与空白组比较,1)P<0.05;与LPS(5μg·mL-1)组比较,2)P<0.05。
A 不同LPS浓度诱导下斑马鱼的荧光图像;B 不同LPS浓度诱导下斑马鱼体内ROS荧光强度定量分析。与空白组比较,1)P<0.01;与LPS(5μg·mL-1)组比较,2)P<0.01。
与空白组比较,1)P<0.05。
A WPMP-1不同浓度下斑马鱼孵化率;B WPMP-2不同浓度下斑马鱼孵化率。
与空白组比较,1)P<0.01;与LPS诱导组比较,2)P<0.05,3)P<0.01。
A 显微镜观察下的斑马鱼形态;B 不同条件下的卵黄囊面积。
与空白组比较,1)P<0.01;与LPS诱导组比较,2)P<0.05,3)P<0.01。
A 荧光倒置显微镜下的斑马鱼荧光图像;B 不同条件下的的斑马鱼荧光强度定量分析。
与空白组比较,1)P<0.01;与LPS诱导组比较,2)P<0.01;与WPMP-1(100μg·mL-1)比较,3)P<0.01。
与空白组比较,1)P<0.01;与LPS诱导组比较,2)P<0.01;与WPMP-1(100μg·mL-1)比较,3)P<0.05。
具体实施方式
下面结合实施例对本发明作进一步说明,但本发明并不限于以下实施例。
实施例1
(1)何首乌饮片1Kg,适当粉碎,经乙醇浸泡脱脂除杂,其中何首乌与乙醇的质量比=1:15,浸泡次数为3次,浸泡时间为8小时;
(2)除杂后的何首乌进行煎煮提取,其中何首乌与水的质量比=1:20,提取次数为2次,提取时间为2小时,何首乌煎煮提取液减压浓缩至密度为1.14,再加入无水乙醇至乙醇浓度为80%,在4℃冰箱过夜沉淀,收集沉淀,得到粗多糖约为180g。
(3)何首乌粗多糖加去离子水3500mL,经AB-8大孔树脂进行脱色处理,用去离子水2BV洗脱,收集过柱液和洗脱液,并减压浓缩约1000mL,加入2wt%的木瓜蛋白酶并置于水浴锅60℃反应2.5h,冷却至室温,然后加入Sevag试剂250mL,振摇,离心去除变性蛋白,重复以上操作2次,取上清液,得精制多糖溶液约800mL;
(4)何首乌精制多糖溶液800mL,再经过DEAE-52柱,然后分别用去离子水和0.5M的NaCl洗脱,收集若干份,每份收集50mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,对应得到两种多糖F-1、F-2 分别各约400mL。多糖组分F-2溶液置于截留分子量为3500Da的透析袋中,在4℃去离子水中透析48h后得到脱盐多糖组分F-2。
(5)何首乌多糖组分F-1和脱盐多糖组分F-2再分别经过Sephadex G-100柱分离,去离子水洗脱,收集若干份,每份收集20mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,分别得到WPMP-1和 WPMP-2两种均一多糖组分各约200mL。将WPMP-1和WPMP-2多糖溶液进行冷冻干燥,得到WPMP-1 何首乌多糖固体粉末90g和WPMP-2何首乌多糖固体粉末65g。
实施例2
(1)何首乌饮片500g适当粉碎,经乙醇浸泡脱脂除杂,其中何首乌与乙醇的质量比=1:20,浸泡次数为4次,浸泡时间为5小时;
(2)除杂后的何首乌进行煎煮提取,其中何首乌与水的质量比=1:15,提取次数为3次,提取时间为1小时,何首乌煎煮提取液减压浓缩至密度为1.16,再加入无水乙醇至乙醇浓度为80%,在4℃冰箱过夜沉淀,收集沉淀,得到粗多糖约85g。
(3)何首乌粗多糖加去离子水1500mL,经AB-8大孔树脂进行脱色处理,用去离子水2BV洗脱,收集过柱液和洗脱液,并减压浓缩约500mL,加入3wt%的木瓜蛋白酶并置于水浴锅60℃反应2.5h,冷却至室温,然后加入Sevag试剂150mL,振摇,离心去除变性蛋白,重复以上操作3次,取上清液,得精制多糖溶液约400mL;
(4)何首乌精制多糖溶液400mL,再经过DEAE-52柱,然后分别用去离子水和0.5M的NaCl洗脱,收集若干份,每份收集30mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,对应得到两种多糖F-1、F-2 分别各约200mL。多糖组分F-2溶液置于截留分子量为3500Da的透析袋中,在4℃去离子水中透析48h后得到脱盐多糖组分F-2。
(5)何首乌多糖组分F-1和脱盐多糖组分F-2再分别经过Sephadex G-100柱分离,去离子水洗脱,收集若干份,每份收集20mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,分别得到WPMP-1和 WPMP-2两种均一多糖组分各约150mL。将WPMP-1和WPMP-2多糖溶液进行冷冻干燥,得到WPMP-1 何首乌多糖固体粉末40g和WPMP-2何首乌多糖固体粉末30g。
下面试验进一步说明本发明:
1材料与方法
1.1实验动物
AB野生型斑马鱼亲本由北京中医药大学惠赠,实验所用幼鱼由实验室养殖孵育,养殖及繁殖条件参照《Zebrafifish Book》[15],雌雄鱼分开养殖,养殖水质电导率为450~550μs·cm-1,水温为28℃,采用自然光照,使用循环水。
1.2材料与试剂
生何首乌购于北京同仁堂;氯化钠(货号:ylh429)、乙醇(货号:ylh145)、(货号:yly014)、碳酸氢钠(货号:ylh096)、氯化钾(货号:M155394)购自北京益利精细化工公司;氯化钙(货号:C804986)、正丁醇(货号:B802835)皆为分析纯、AB-8大孔吸附树脂(货号:A875381)、木瓜蛋白酶(货号:M047338)、葡聚糖凝胶G-100(货号:M047983)购自北京迈瑞达科技有限公司;DEAE-52离子交换树脂(货号:K100335) 购自上海笛柏生物科技有限公司;脂多糖(LPS)(货号:S11060)购自源叶生物公司;活性氧测定试剂盒 (货号:S0033S)、一氧化氮测定试剂盒(货号:S0021S),Western及IP细胞裂解液(货号:P0013)、PMSF (货号:ST506)购自上海碧云天生物技术有限公司,BCA测定试剂盒(货号:PC0020)购于北京索莱宝公司。
1.3仪器与设备
R-1002N旋转蒸发仪、WB-2002水浴锅(郑州长城科工贸有限公司);ES225SM-DR电子天平(瑞士Precisa 公司);DHP-9052电热恒温培养箱(上海一恒科技有限公司);Centrifuge5430R离心机(德国Eppendorf 公司);LCD数字显微镜(米欧特公司);AxioObserverA1荧光倒置显微镜(德国Zeiss公司);FM70制冰机(北京长流科学仪器有限公司);Enspire2300-001A酶标仪(美国PerkinElmer公司)。
1.4多糖的提取
1.4.1多糖采用水提醇沉法进行提取何首乌饮片适当粉碎,经乙醇脱脂除杂,以料液比1:10的比例, 80℃加热提取4h,收集水提液,弃去残渣。将水提液减压浓缩至适当体积,加入无水乙醇至乙醇浓度为 80%,在4℃冰箱过夜沉淀,收集沉淀,得到粗多糖。
1.4.2多糖的纯化与分离在粗多糖溶液中加入2%的木瓜蛋白酶并置于水浴锅60℃反应2.5h,冷却至室温,然后加入Sevag试剂(氯仿:正丁醇=4:1的比例混合均匀),振摇20min,离心去除变性蛋白,取上清液,用AB-8大孔树脂进行脱色处理,收集洗脱液并减压浓缩至一定体积,得精制多糖溶液。精制多糖溶液再经过DEAE-52柱,用去离子水和0.5M的NaCl洗脱,得到两种多糖组分F-1、F-2,每种多糖组分再经过Sephadex G-100柱分离,得到两种均一多糖组分,分别为WPMP-1和WPMP-2。
1.5 LPS诱导斑马鱼炎症模型
1.5.1斑马鱼喂养及胚胎收集成年野生AB系斑马鱼置于室温(28℃)自然光照周期下饲养,每天喂食3次,斑马鱼胚胎由健康雌雄鱼(1:2)自然交配获得。收集胚胎,及时移除水中排泄物及杂质等,更换为胚胎培养液(3.5g·L-1NaCl、0.05g·L-1KCl、0.1g·L-1CaCl2、0.025g·L-1NaHCO3)并转移至干净培养皿中,于28.5℃恒温培养箱中待后续实验。
1.5.2 LPS浓度确定将收集的斑马鱼胚胎移入6孔培养板中,随机分为每孔50枚胚胎,除去残留培养液后,每组每孔分别加入LPS使终浓度为0、5、10、20、30、40μg·mL-1,实验重复三次,胚胎于28.5℃培养箱中培养,每隔一段时间除去死亡胚胎,48h脱膜后及时除去悬浮膜外壳,计算斑马鱼胚胎孵化率。
1.5.3 LPS致斑马鱼胚胎炎症模型优化将收集的斑马鱼胚胎移入6孔培养板中,随机分为每孔50枚胚胎,除去残留培养液后,将斑马鱼胚胎浸润于LPS诱导溶液中,28.5℃培养箱中培养,48hpf(hours post fertilization)时期测定卵黄囊面积,72hpf时期测定心率、ROS生成率、NO生成率。
1.6何首乌多糖抗炎活性的评价
1.6.1何首乌多糖对孵化率的影响将收集的斑马鱼胚胎移入6孔培养板中,随机分为每孔50枚胚胎,除去残留培养液后,每组每孔分别加入多糖使终浓度为0、10、50、100μg·mL-1,实验重复三次,胚胎于28.5℃培养箱中培养,每隔一段时间除去死亡胚胎,48h脱膜后及时除去悬浮膜外壳,计算斑马鱼胚胎孵化率。
1.6.2不同何首乌多糖组分抗炎效果的评价将收集的斑马鱼胚胎移入6孔培养板中,随机分为每孔50 枚胚胎,除去残留培养液后,每组每孔加入多糖使终浓度分别为0、0、10、50、100μg·mL-1,后于每孔幼鱼中添加LPS使其达到诱导浓度,其中已设置阴性对照组和阳性对照组,胚胎于28.5℃培养箱中培养,每隔一段时间除去死亡胚胎,48h脱膜后及时除去悬浮膜外壳,以备后续实验,48hpf时期进行卵黄囊面积的检测,72hpf时期测定心率、ROS生成率和NO生成率。
1.7炎症评价指标
1.7.1心率根据文献[16],实验幼鱼发育至72hpf时期,每组选择3条斑马鱼幼鱼,显微镜下对各组幼鱼心跳进行人工计数(30s),并计算各组与空白组心率之比。
心率(%)=给药组心率/空白组心率x100%
1.7.2卵黄囊面积根据文献[16],实验幼鱼发育至48hpf时期,每组选择3条斑马鱼幼鱼,显微镜下对各组幼鱼卵黄囊进行观察拍照,并计算各组与空白组卵黄囊面积之比。
卵黄囊面积(%)=给药组卵黄囊面积/空白组卵黄囊面积x100%
1.7.3活性氧(ROS)的分析使用荧光探针染料2,7-二氯二氢荧光素二酸酯(DCF-DA),检测斑马鱼幼虫中ROS的生成。每组选择10条斑马鱼幼鱼转移到6孔培养板中,除去残留培养液,以胚胎培养基稀释 DCF-DA为10mM添加到培养板中,28.5℃暗箱孵育1h,每隔一段时间轻轻摇匀,使探针和幼鱼充分接触,孵育结束后用胚胎培养液冲洗3-5次,0.02%三卡因麻醉后,置于荧光倒置显微镜下观察活性氧的生成情况,并利用ImageJ软件进行定量分析。
1.7.4 NO的分析参考文献[17],使用NO测定试剂盒对斑马鱼幼鱼中的NO水平进行测定。每组选择30 条斑马鱼转移到6孔培养板,除去残留培养液,0.02%三卡因冰上麻醉,加入含1%PMSF的WB/IP裂解液,充分研磨,所有裂解样品的步骤均在冰上进行,13000rpm离心5min取上清,上清液作为样品,BCA蛋白试剂盒测定样品蛋白浓度,NO测定试剂盒测定样品NO含量,最终NO含量比较以NO含量/蛋白浓度表示。
1.8数据处理
所有试验独立重复3次,数据均以表示,运用OriginLab OriginPro 9.0.0软件绘制图表,用 SPSS 17.0软件进行统计学分析,组间比较采用方差分析和t检验,P<0.05表示差异有统计学意义,P<0.05 表示差异显著,P<0.01表示差异极显著。
2结果与分析
2.1何首乌多糖的分离和纯化
何首乌粗多糖经DEAE-52阴离子交换层析纯化,得到两种组分F-1、F-2(图1中A),两种多糖组再分别通过Sephadex G-100凝胶过滤层析进一步纯化,收集含量最大组分,得到均一多糖WPMP-1和WPMP-2(图 1中B、图1中C)。
2.2 LPS诱导斑马鱼炎症模型
2.2.1 LPS浓度确定如图2所示,以不同浓度LPS诱导斑马鱼胚胎后,均造成了一定数量的胚胎死亡现象,且LPS浓度越大,孵化率越低。当LPS诱导浓度分别在5、10μg·mL-1时,LPS诱导组孵化率均与空白组孵化率无显著性差异(P>0.05);而当浓度高于10μg·mL-1时,LPS诱导组孵化率与空白组孵化率表现出显著性差异(P<0.05),因此选择LPS终浓度为5、10μg·mL-1。
2.2.2 LPS诱导浓度对斑马鱼心率的影响心率升高通常发生在心血管受到损害时,是斑马鱼评估药物毒性一种典型标志物[17]。实验以人工计数方式对分别浸润于5、10μg·mL-1LPS诱导浓度的斑马鱼幼鱼进行心率测定。结果如图3所示,当LPS诱导浓度为5、10μg·mL-1时,两组心率均与空白组心率具有显著性差异(P<0.05),而两组之间心率无显著性差异(P>0.05)。当LPS诱导浓度为10μg·mL-1时,斑马鱼心率显著提高至117.85%。表明在10μg·mL-1浓度LPS的刺激下,幼鱼心跳加快,表现出对LPS刺激的毒性反应。
2.2.3 LPS诱导浓度对斑马鱼卵黄囊面积的影响胚胎到幼鱼时期的斑马鱼可仅利用卵黄囊中的营养存活,但当受到有害刺激时,斑马鱼幼鱼则会表现出卵黄囊吸收延迟,发育迟缓,严重影响了幼鱼的发育[18]。由图4可知,在显微镜观察下,LPS诱导组的胚胎发育至48h后出现卵黄囊水肿的现象(图4中A),当 LPS浓度为10μg·mL-1时,该组卵黄囊面积与空白组卵黄囊面积、与5μg·mL-1LPS诱导组卵黄囊面积均表现出显著性差异(P<0.05),表明10μg·mL-1LPS严重影响了斑马鱼的正常发育,卵黄囊面积约为空白组卵黄囊面积的1.580倍(图4中B)。
2.2.4 LPS诱导浓度对斑马鱼ROS生成率的影响活性氧(ROS)的过度也称为氧化应激,与人类疾病以及衰老相关。受到有害刺激的细胞产生过量ROS是炎症反应的一个指标[19]。由图5可知,空白组中斑马鱼体内的荧光强度较弱,反映了幼鱼体内ROS含量较低,当斑马鱼幼鱼受到一定诱导浓度LPS刺激后,在荧光倒置显微镜下可以观察到其体内的荧光强度增强,ROS含量增高(图5中A)。当LPS浓度为10μg·mL-1时,该组ROS含量与空白组ROS含量、与5μg·mL-1LPS诱导组NO含量均表现出极显著性差异(P<0.01),表明10μg·mL-1LPS诱导浓度可显著提高斑马鱼体内的ROS含量,约为空白组ROS含量的439.00%(图 5中B)。
2.2.5 LPS诱导浓度对斑马鱼NO生成率的影响当免疫细胞被炎症刺激物激活时,细胞诱导产生炎症介质和炎症细胞因子,包括一氧化氮(NO)和前列腺素E2(PGE2)以及TNF-α和IL-1β,所以NO含量也是炎症标志[16]。图6可知,空白组斑马鱼体内的NO含量较低,当斑马鱼幼鱼受到一定浓度LPS刺激后,NO 含量增高。当LPS诱导浓度为5、10μg·mL-1时,两组NO含量均与空白组NO含量表现出显著性差异(P <0.05),而两组之间NO含量无显著性差异(P>0.05)。且当LPS浓度为10μg·mL-1时,该组NO含量与空白组NO含量表现出显著性差异(P<0.05),约为空白组NO含量的168.70%,表现出斑马鱼炎症反应。
2.2.6 LPS最优浓度确定斑马鱼炎症模型建立在以不对斑马鱼造成生理性伤害的基础上,仍需表现出较强的炎症反应。在此研究中,10μg·mL-1LPS诱导浓度可显著提高斑马鱼心率,导致斑马鱼卵黄囊水肿,延缓了斑马鱼的发育,同时显著提高斑马鱼体内的ROS生成率及NO生成率,全面表现出炎症反应,确定了10μg·mL-1LPS诱导斑马鱼炎症是本研究中炎症造模的最优浓度,与文献类似[17]。因此,后续实验以 10μg·mL-1LPS诱导造模并评价何首乌多糖的抗炎保护作用。
2.3多糖抗炎活性评价
2.3.1不同组分不同浓度何首乌多糖对斑马鱼胚胎发育的影响健康斑马鱼自然交配后,收集胚胎,加入何首乌多糖的不同组分WPMP-1和WPMP-2后在28.5℃下孵育,并观察斑马鱼胚胎0-96hpf的发育情况。结果如图7所示,实验结果表明,在实验的多糖浓度下,斑马鱼胚胎孵化率正常,且无畸形、个体弯曲等异常形态(图7中A、B),说明多糖对斑马鱼胚胎发育阶段没有任何毒性作用,且对胚胎发育期促进作用。
2.3.2不同组分不同浓度何首乌多糖对LPS诱导的斑马鱼心率的影响由图8可知,在10μg·mL-1LPS 刺激下,斑马鱼幼鱼的心率显著提高(P<0.01),在多糖的处理下,斑马鱼幼鱼心率减慢,10μg·mL-1多糖即可使斑马鱼幼鱼心率与LPS诱导组心率表现出显著差异(P<0.05),100μg·mL-1多糖使斑马鱼幼鱼心率与空白组心率无显著性差异(P>0.05),恢复到正常水平,可知多糖在一定浓度下避免了LPS对斑马鱼幼鱼的毒性损伤。但在同一浓度下不同组分WPMP-1、WPMP-2对心率的恢复不明显(P>0.05)。
2.3.3不同组分不同浓度何首乌多糖对LPS诱导的斑马鱼胚胎卵黄囊面积的影响由图9可知,多糖处理组的卵黄囊面积逐渐变小(图9中A),50μg·mL-1多糖处理组斑马鱼卵黄囊面积与LPS诱导组卵黄囊面积表现出显著性差异(P<0.05),至100μg·mL-1多糖浓度时斑马鱼卵黄囊面积与LPS诱导组卵黄囊面积表现出极显著性差异(P<0.01),与空白组卵黄囊面积无显著差异(P>0.05)(图9中B),说明多糖能抑制LPS刺激造成的斑马鱼卵黄囊水肿,发育迟缓的炎症反应,使其恢复正常发育。但在同一浓度下不同组分WPMP-1、WPMP-2对减小卵黄囊的作用不明显(P>0.05)。
2.3.4不同组分不同浓度何首乌多糖对LPS诱导的斑马鱼体内ROS生成率的影响由图9可知,LPS诱导后提高了斑马鱼体内ROS含量,而多糖处理后表现出荧光强度的下降(图10中A),10μg·mL-1多糖处理组ROS含量即可表现出与LPS诱导组ROS含量的极显著性抑制(P<0.01)(图10中B),减轻了氧化损伤。且当多糖浓度均为100μg·mL-1时,不同组分WPMP-1、WPMP-2对抑制LPS诱导ROS生成率表现出显著性差异(P<0.01),分别降低了209.17%和236.87%。可见WPMP-2的抑制活性要大于WPMP-1。
2.3.5不同组分不同浓度何首乌多糖对LPS诱导的斑马鱼体内NO生成率的影响由图11可知,LPS诱导后斑马鱼幼鱼体内产生了大量的NO。经多糖处理后,幼鱼体内的NO含量逐渐下降,50μg·mL-1多糖处理组NO含量即与LPS诱导组NO含量表现出极显著性差异(P<0.01),表明50μg·mL-1多糖处理多糖能够显著抑制NO产生,从而减轻炎症反应。当何首乌多糖浓度为100μg·mL-1,不同组分WPMP-1、WPMP-2 对抑制LPS诱导NO生成率表现出显著性差异(P<0.05),分别降低了48.17%和57.80%,可见WPMP-2的抑制活性要大于WPMP-1。
3结论
研究表明,10μg·mL-1LPS处理斑马鱼胚胎可造成如心率升高以及卵黄囊水肿、尾巴弯曲等形态异常毒性作用,也可引起斑马鱼幼鱼体内ROS和NO含量显著增加,分别为空白组的439.00%及168.70%,表现出炎症反应。
经过100μg·mL-1何首乌多糖处理的炎症斑马鱼模型,可以使斑马鱼幼鱼心率减慢,恢复到正常心率,缓解了LPS刺激引起的毒性反应。同样经过100μg·mL-1何首乌多糖处理的炎症斑马鱼模型,可以使斑马鱼幼鱼的卵黄囊面积减小,与空白组卵黄囊面积无显著差异,使斑马鱼发育正常化,证明了何首乌多糖可以保护斑马鱼幼鱼免受LPS引起的表型变化。并且当何首乌多糖浓度为50μg·mL-1以上时,均极显著抑制了斑马鱼体内NO和ROS的生成,表明何首乌多糖能够减轻LPS对斑马鱼的氧化损伤,显示出良好的抗炎活性。该实验结果与Sheng等报道食用褐藻中多糖对斑马鱼胚胎的保护作用相似[19]。
通过抗炎活性指标的比较可以发现,当何首乌多糖浓度为100μg·mL-1时,WPMP-1、WPMP-2多糖组分也表现出活性显著差异性,将LPS诱导的ROS生成率分别降低了209.17%和236.87%,将LPS诱导的NO 生成率分别降低了48.17%和57.80%,结果显示WPMP-2多糖组分的抗炎抑制活性要大于WPMP-1多糖组分。根据已有的对何首乌多糖结构的研究表明,WPMP-1与WPMP-2在分子量、单糖组成、糖醛酸含量及支链结构方面存在显著差异,WPMP-2具有更大的分子量、更高的糖醛酸含量以及分支化度,所以在一般情况下 WPMP-2比WPMP-1具有更高的生物活性[20],通过抗炎活性实验,进一步表明了这一规律不仅适用于何首乌多糖的抗氧化、免疫调节活性[9-10],也同样适用于何首乌多糖的抗炎活性。
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Claims (6)
1.两种何首乌抗炎多糖的制备方法,其特征在于,包括以下步骤:
(1)何首乌饮片适当粉碎,经乙醇浸泡脱脂除杂,其中何首乌与乙醇的质量比=1:(10-20),浸泡次数为3-5次,浸泡时间为5-10小时;
(2)除杂后的何首乌进行煎煮提取,其中何首乌与水的质量比=1:(10-30),提取次数为1-4次,提取时间为1-4小时,何首乌煎煮提取液减压浓缩至密度为1.12-1.18,再加入无水乙醇至乙醇浓度为75-85%,在4℃冰箱过夜沉淀,收集沉淀,得到粗多糖;
(3)何首乌粗多糖加水溶解至浓度为5-10wt%,经AB-8大孔树脂进行脱色处理,用去离子水2-4BV洗脱,收集过柱液和洗脱液,并减压浓缩5-15wt%,加入1-3wt%的木瓜蛋白酶并置于水浴锅60℃反应2.5h,冷却至室温,然后加入Sevag试剂(Sevag试剂为氯仿:正丁醇体积比=4:1的比例混合均匀),Sevag试剂在混合体系中的体积百分比为,多糖溶液:Sevag试剂=4:1,振摇,离心去除变性蛋白,重复以上操作2-4次,取上清液,得精制多糖溶液;
(4)何首乌精制多糖溶液稀释至浓度为5-10wt%,再经过DEAE-52柱,然后分别用去离子水和0.5M的NaCl洗脱,收集若干份,每份收集20-100mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,对应得到两种多糖组分F-1、F-2。多糖组分F-2溶液置于截留分子量为3500Da的透析袋中,在4℃去离子水中透析48h后得到脱盐多糖组分F-2。
(5)何首乌多糖组分F-1和脱盐多糖组分F-2再分别经过Sephadex G-100柱分离,去离子水洗脱,收集若干份,每份收集20-100mL,每份取其中1mL样品溶液,加用去离子水至2mL,加入1mL的5%的苯酚溶液和5mL的浓硫酸。混匀后静置5min,沸水浴中加热10min,取出试管放入冷水中冷却至室温,应用紫外-可见分光光度计在490nm处测定吸光值,合并对490nm有吸收组分,得到两种均一多糖组分,分别为WPMP-1和WPMP-2。将WPMP-1和WPMP-2多糖溶液进行冷冻干燥,得到对应的WPMP-1和WPMP-2何首乌多糖固体粉末。
2.按照权利要求1所述的两种何首乌抗炎多糖的制备方法,其特征在于,何首乌为蓼科植物何首乌(Polygonum multiflorum Thunb.)的干燥块根。
3.按照权利要求1所述的方法制备得到的两种何首乌抗炎多糖WPMP-1和WPMP-2。
4.按照权利要求1所述的方法制备得到的两种何首乌抗炎多糖的应用,用于制备抗炎药物或保健品。
5.按照权利要求4所述的应用,何首乌抗炎多糖与辅料制成口服制剂。
6.按照权利要求5所述的应用,制成口服片剂、口服胶囊剂等。
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CN116693711A (zh) * | 2023-06-09 | 2023-09-05 | 中国食品药品检定研究院 | 一种何首乌多糖及其提取方法和应用 |
CN116693711B (zh) * | 2023-06-09 | 2024-02-02 | 中国食品药品检定研究院 | 一种何首乌多糖及其提取方法和应用 |
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