CN112156120B - 一种帚枝香青正丁醇提取物的用途 - Google Patents
一种帚枝香青正丁醇提取物的用途 Download PDFInfo
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- CN112156120B CN112156120B CN202011238159.9A CN202011238159A CN112156120B CN 112156120 B CN112156120 B CN 112156120B CN 202011238159 A CN202011238159 A CN 202011238159A CN 112156120 B CN112156120 B CN 112156120B
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- butyl alcohol
- tulipa
- alcohol extract
- scoparia
- sugar
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Abstract
本发明涉及一种帚枝香青正丁醇提取物的用途，该帚枝香青正丁醇提取物是从帚枝香青中经过70%乙醇热提取得到，通过体外细胞实验表明该帚枝香青正丁醇提取物具有降血糖及改善高糖损伤引起的肾脏病变，它能显著增加大鼠成肌细胞（L6）对葡萄糖的消耗及高糖诱导的高糖损伤小鼠肾脏细胞（MPC‑5）模型的Na+‑K+‑ATP酶活性，显示出其具有降血糖及调节多元醇通路从而改善高糖损伤引起的肾脏病变的作用。
Description
技术领域
本发明属于中药植物的生物活性领域，涉及一种帚枝香青正丁醇提取物的用途，具体涉及帚枝香青正丁醇提取物在制备治疗糖尿病及高糖损伤引起糖尿病肾脏病变的药物或预防糖尿病及辅助降血糖保健品中的用途。通过细胞实验证实它具有降血糖及改善高糖损伤引起糖尿病肾脏病变的作用。
背景技术
帚枝香青(Anaphalis virgata)，双子叶植物纲菊科香青属植物，生长于海拔3000-4000米处，主要集中在西藏西部、新疆西部、中亚、伊朗等地区。香青属由80种植物组成，主要分布于亚洲热带及亚热带地区；目前在中国发现约有50种，主要集中在我国的南部与西南部。
目前，关于香青属植物的生物活性报道较少：乳白香青(Anaphalis lacteal)作为一种民间藏药，传统上被用作活血化瘀、缓解头痛和止血等，此外，有研究报道，乳白香青挥发油具有治疗慢性支气管炎、抗菌、抗氧化及抗癌活性；香青具有镇咳、祛痰、抗菌，香青挥发油具有抗肿瘤活性；黄腺香青车前叶变种具有清热解毒、驱虫功效，用于治疗牙痛、痢疾、蛔虫病等症，其挥发油具有抗肿瘤活性。而从香青属植物分离的黄酮类、三萜类、甾体类为主要活性成分。
糖尿病是一种由于胰岛素分泌不足(或相对不足)及胰岛素抵抗，引起渐进性糖、脂肪、蛋白质等代谢紊乱的疾病，以高血糖为主要标志。糖尿病现已成为全世界继心血管病和恶性肿瘤之后，第三位威胁人们健康和生命的慢性非传染性疾病。国际糖联盟(IDF)发布的数据显示，2017年全球约4.25亿成年人(20-79岁)患有糖尿病，预计到2045年，这一数字将上升至6.29亿；其中，二型糖尿病(T2DM)患者约占90％。越来越多的糖尿病患者受到由糖尿病引起的并发症的影响，有些并发症影响日常活动和生活质量，而另一些并发症严重到危及生命的程度。
糖尿病肾病(Diabetic nephropathy，DN)是由糖尿病引起的肾脏病变，其主要表现是从早期肾小球高滤过开始，随后是微量白蛋白到大量白蛋白尿的发展，随后肾小球滤过率(glomerular filtration rate,GFR)下降。糖尿病肾病约占糖尿病患者的40％，是全世界慢性肾脏病(chronic kidney disease,CKD)的主要原因，也是终末期肾病(end stagerenal disease,ESRD)的主要原因。糖尿病患者全身脏器出现糖代谢障碍，而约50％的糖在肾脏代谢，加重肾脏的糖负荷导致肾脏损伤。近年来，对肾小球滤过屏障结构和功能的研究主要集中于足细胞在DN发生发展中的作用。足细胞是附着在肾小球基底膜上的终末分化上皮细胞，相邻足细胞之间交替的足突构成间裂孔隔膜，在维持肾小球滤过屏障结构和功能的完整性方面起着重要作用。高糖诱导足细胞凋亡，导致足细胞减少，引起蛋白尿，加速足突消失。
多元醇通路，又称山梨醇通路，是组织细胞葡萄糖代谢的重要途径，其通路异常是糖尿病肾脏病变重要发病机制之一。由醛糖还原酶(aldose reductase,AR)及山梨醇脱氢酶(sorbitol dehydrogenase,SDH)共同构成。醛糖还原酶主要存在于肾脏、晶状体、视网膜、及神经等组织中，多元醇通路的异常会导致醛糖还原酶活性增强，胞内Na+-K+-ATP酶活性下降。现有上市醛糖还原酶抑制剂依帕司他，但有时会伴随红斑、水疱、呕吐等过敏症状及胃部不适等副作用，且患者不能长期服用。
此外，有研究证实，二型糖尿病患者的脂肪细胞，骨骼肌和肝脏中蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase1B，PTP1B)活性增强或过度表达，当蛋白酪氨酸磷酸酶被剔除后，不仅胰岛素敏感性增高，而且糖尿病症状也有所改善，PTP1B在胰岛素信号的传导中起负调控作用,PTP1B活性增强或过度表达使激活的胰岛素受体去磷酸化而终止胰岛素信号的传导。因此PTP1B是引起胰岛素抵抗的重要原因，抑制PTP1B是防治糖尿病的有效途径。PTP1B通过对胰岛素受体激酶(insulin receptor kinase，IRK)或IRK活性片段的磷酸化酪氨酸残基去磷酸化作用对胰岛素信号传导进行负调节。
现目前关于帚枝香青的生物活性研究还未见报道。
发明内容
本发明的目的在于，提供一种帚枝香青正丁醇提取物的用途，该帚枝香青正丁醇提取物是从帚枝香青中经过70％乙醇热提取得到，通过体外细胞实验表明该帚枝香青正丁醇提取物具有降血糖及改善高糖损伤引起的肾脏病变，它能显著增加大鼠成肌细胞(L6)对葡萄糖的消耗及高糖诱导的高糖损伤小鼠肾脏细胞(MPC-5)模型的Na+-K+-ATP酶活性，显示出其具有降血糖及调节多元醇通路从而改善高糖损伤引起的肾脏病变的作用。
本发明所述的一种帚枝香青正丁醇提取物的用途，所述帚枝香青正丁醇提取物在制备治疗糖尿病及高糖损伤引起肾脏病变的药物中的用途。
一种帚枝香青正丁醇提取物的用途，所述帚枝香青正丁醇提取物在制备预防糖尿病及辅助降血糖保健品中的用途。
本发明所述的一种帚枝香青正丁醇提取物的用途，其中所涉及的帚枝香青正丁醇提取物的提取方法：
a.取30g帚枝香青，粉碎后，采用浓度为70％乙醇按质量体积比20倍量水浴加热回流，水浴温度为60℃，提取2次，，每次提取1.5小时，收集提取液；
b.将提取液进行减压蒸馏除去其中的乙醇，浓缩至糖浆状，温度45℃干燥，即得到帚枝香青粗提物浸膏；
c.将帚枝香青粗提物浸膏以1:15-1:20的料液比，用水分散后，依次用石油醚、二氯甲烷、乙酸乙酯和正丁醇萃取，将正丁醇萃取液减压蒸至糖浆状，温度45℃干燥，得到帚枝香青正丁醇提取物。
本发明所述的一种帚枝香青正丁醇提取物的用途，通过体外酶学水平筛选，测定帚枝香青正丁醇提取物对醛糖还原酶(Aldose Reductase,AR)的抑制活性；并采用高糖诱导，构建小鼠肾足细胞(MPC-5)高糖损伤模型，检测细胞内Na+-K+-ATP酶活性，结合统计分析手段，根据对照组、模型(高糖损伤)组及给药组MPC-5细胞内Na+-K+-ATP酶活性变化，深入分析帚枝香青正丁醇提取物对糖尿病肾病的改善作用，结果表明：帚枝香青正丁醇提取物对小鼠肾足细胞(MPC-5)作用后，高糖损伤的小鼠肾足细胞(MPC-5)，其多元醇通路中的Na+-K+-ATP酶活性显著升高，表明帚枝香青正丁醇提取物对于糖尿病肾脏病变具有明显的改善作用，可以在防治糖尿病肾病的药物中应用。
本发明利用帚枝香青正丁醇提取物对大鼠成肌细胞(L6)葡萄糖消耗情况进行测定，结合统计学分析手段，根据对照组、给药组L6细胞对培养基葡萄糖消耗情况，深入分析帚枝香青正丁醇提取物对葡萄糖消耗的影响；结果表明：帚枝香青正丁醇提取物给药浓度为3.125μg/mL、6.25μg/mL、12.5μg/mL、25μg/mL均促进了葡萄糖消耗，给药浓度为3.125μg/mL、6.25μg/mL、12.5μg/mL、25μg/mL时，显著增强了葡萄糖的摄取；且给药+低浓度胰岛素(100nM)组刺激葡萄糖消耗，产生加和作用，葡萄糖消耗量增多。结果表明帚枝香青正丁醇提取物具有显著增强葡萄糖消耗作用，可以在降糖药物中有所应用。
本发明通过帚枝香青正丁醇提取物的干预治疗作用，表明帚枝香青正丁醇提取物对改善糖尿病高血糖及高糖损伤诱导肾脏病变方面具有显著保护作用，为降血糖及高糖损伤诱导肾脏病变提供了新途径，对于帚枝香青进一步深入研究具有重要意义和价值，有很广阔的潜在市场价值和应用。
附图说明
图1为本发明不同浓度帚枝香青正丁醇提取物对小鼠肾足细胞(MPC-5)活力的影响；
图2为本发明不同浓度帚枝香青正丁醇提取物对小鼠肾足细胞(MPC-5)Na+-K+-ATP酶活力的影响图；
图3为本发明不同浓度帚枝香青正丁醇提取物对大鼠成肌细胞(L6)活力的影响；
图4为本发明不同浓度帚枝香青正丁醇提取物对大鼠成肌细胞(L6)葡萄糖消耗的影响图。
具体实施方式
实施例1
a.取30g帚枝香青，粉碎后，采用浓度为70％乙醇按质量体积比20倍量水浴加热回流，水浴温度为60℃，提取2次，每次提取1.5小时，收集提取液；
b.将提取液进行减压蒸馏除去其中的乙醇，浓缩至糖浆状，温度45℃干燥，即得到帚枝香青粗提物浸膏；
c.将帚枝香青粗提物浸膏以1:15-1:20的料液比，用水分散后，依次用石油醚、二氯甲烷、乙酸乙酯和正丁醇萃取，将正丁醇萃取液减压蒸至糖浆状，温度45℃干燥，得到帚枝香青正丁醇提取物。
实施例2
将实施例1获得的帚枝香青正丁醇提取物用于生物活性筛选：
用还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，DL-甘油醛混合液作为底物，以在阳性药槲皮素为对照，利用酶标仪进醛糖还原酶的高通量筛选，根据定量且过量的烟酰胺腺嘌呤二核苷酸磷酸(NADPH)存在时，以DL-甘油醛为底物，以检测烟酰胺腺嘌呤二核苷酸磷酸(NADPH)的减少来反映醛糖还原酶(Aldose Reductase,AR)的活性，酶反应体系组成如下，磷酸缓冲盐溶液(pH7.4)，醛糖还原酶，烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，DL-甘油醛，醛糖还原酶特异抑制剂槲皮素(3.125μg/mL)，反应体系混匀后闭光放置30min，置比色仪上测定340波长条件下的吸收值(A)，测定结果减去本底值后计算酶活性；
抑制率(I％)＝[(OD340空白-OD340样品)/OD340空白]×100％
筛选结果见表1：
结论：从实验结果表明，帚枝香青正丁醇提取物具有抑制醛糖还原酶活性的功效。
实施例3
将实施例1获得的帚枝香青正丁醇提取物对小鼠肾足细胞(MPC-5)活力影响检测：小鼠肾足细胞接种于96孔板，8000个/孔，培养16小时使细胞贴壁后，更换培养基建立高糖损伤模型,对照组采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+11mmol/L D-葡萄糖1640培养基培养24小时，模型(高糖)组采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+30mmol/L D-葡萄糖1640培养基刺激24小时，给药组采用高糖刺激同时加入不同浓度帚枝香青正丁醇提取物培养24小时，24小时后，3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法(MTT)检测细胞活力；用Graphpad Prism统计分析，检验对照组与给药组统计学差异，研究表明：帚枝香青正丁醇提取物处理高糖损伤条件下MPC-5细胞24小时后，不同浓度与对照组相比，细胞活力均在100％附近，结果证明浓度为0-25μg/mL帚枝香青正丁醇提取物对MPC-5细胞没有毒性作用。
MPC-5细胞活力检测见表2：
研究表明：帚枝香青正丁醇提取物处理高糖损伤条件下小鼠肾足细胞(MPC-5)24小时后，不同浓度与对照组相比，细胞活力均在100％附近，结果证明浓度为0-25μg/mL帚枝香青正丁醇提取物对小鼠肾足细胞(MPC-5)没有毒性作用(图1)。
实施例4
细胞培养及构建小鼠肾足细胞(MPC-5)高糖损伤模型：
小鼠肾足细胞(MPC-5)细胞用含有10％胎牛血清、青霉素100U/mL、链霉素100U/mL的1640培养基(D-葡萄糖浓度：11mmol/L)，在温度37℃、浓度5％％CO2条件下培养，取小鼠肾足细胞(MPC-5)用于实验，其高糖损伤模型条件：正常组采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+11mmol/L D-葡萄糖1640培养基，高糖组采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+30mmol/L D-葡萄糖1640培养基放置培养箱培养48小时；
将实施例1获得的帚枝香青正丁醇提取物用于生物活性筛选：
小鼠肾足细胞(MPC-5)接种于6孔板中培养16小时使其贴壁，更换培养基，条件：对照组采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+11mmol/L D-葡萄糖1640培养基培养48小时，模型(高糖)组采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+30mmol/LD-葡萄糖1640培养基刺激48小时，给药组采用高糖刺激24小时后加入不同浓度帚枝香青正丁醇提取物继续培养24小时，收集细胞，根据试剂盒方法检测细胞内Na+-K+-ATP酶活力，不同浓度帚枝香青正丁醇提取物对细胞Na+-K+-ATP酶活力结果见表4：
结果显示：小鼠肾足细胞(MPC-5)高糖损伤后Na+-K+-ATP酶活力显著降低，Na+-K+-ATP酶活力正常组vs.造模组具有显著性差(P<0.05)；不同浓度帚枝香青正丁醇提取物给药24小时后，Na+-K+-ATP酶活力显著增强，12.5μg/mL及25μg/mL剂量组vs.模型组均具有极其显著性差异(***P<0.001)；结果表明：不同浓度帚枝香青正丁醇提取物在高糖损伤造成多元醇通路异常方面具有改善作用，并具有保护肾足细胞抗高糖损伤作用(图2)。
实施例5
将实施例1获得的帚枝香青正丁醇提取物作为糖尿病降糖用途的生物活性筛选：
以阳性药3-(3,5-二溴-4-羟基-苯甲酰基)-2-乙基-苯并呋喃-6-磺酸-(4-(噻唑-2-基磺酰胺基)-苯基)-酰胺为阳性对照，利用酶标仪进行PTP1B酶抑制剂的高通量筛选，根据PTP1B水解pNPP的磷酸基团而产生颜色反应来测定PTP1B的活性；酶反应体系组成如下：PBS缓冲液(pH7.4)，PTP1B融合蛋白，pNPP，PTP1B特异抑制剂3-(3,5-二溴-4-羟基-苯甲酰基)-2-乙基-苯并呋喃-6-磺酸-(4-(噻唑-2-基磺酰胺基)-苯基)-酰胺(2.5μM)，反应体系混匀后闭光放置30min，置比色仪上测定405波长条件下的吸收值(A)，测定结果减去本底值后计算酶活性；
抑制率(I％)＝[(OD405空白-OD405样品)/OD405空白]×100％
筛选结果见表5：
结论：从实验结果表明，帚枝香青正丁醇提取物具有蛋白酪氨酸磷酸酶1B抑制剂功效。
实施例6
实施例1获得帚枝香青正丁醇提取物对大鼠成肌细胞(L6)活力影响：
大鼠成肌细胞以5×103个/孔的密度均匀的接种于96孔板中，培养基采用10％胎牛血清+100U/mL青霉素+100U/mL链霉素+25mM D-葡萄糖DMEM培养基，培养16小时使细胞贴壁，更换培养基，设置调零孔、对照组、实验组，条件：调零孔为DMEM全培养基不含细胞，对照组为DMEM全培养基+细胞，实验组为不同药物浓度的DMEM全培养基+细胞，24小时后，3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐比色法(MTT)检测细胞活力；用Graphpad Prism统计分析，检验对照组与实验组统计学差异，研究表明：帚枝香青正丁醇提取物处理L6细胞24小时后，不同浓度实验组与对照组相比，细胞活力均在90％以上，无显著性差异，结果证明0-25μg/mL帚枝香青正丁醇提取物对L6细胞没有毒性作用；L6细胞活力结果见表6：
结论：帚枝香青正丁醇提取物处理高糖(25mM D-Glucose)条件下大鼠成肌细胞(L6)24小时后，不同浓度与对照组相比，细胞活力均在90％以上，结果证明帚枝香青正丁醇提取物对大鼠成肌细胞(L6)无显著毒性(图3)。
实施例7
将实施例1获得的帚枝香青正丁醇提取物作为糖尿病降糖用途的生物活性筛选：
L6细胞以8×103个/孔的密度均匀的接种于96孔板中，置于温度37℃、浓度5％CO2恒温培养箱中培养；待细胞长至80％左右，将细胞的DMEM完全培养基更换为含有0.25％BSA的无血清DMEM培养基，饥饿培养3.5h；分别加入不同浓度梯度的药物：0μg/mL，0μg/mL+胰岛素(100nM)，3.125μg/mL，3.125μg/mL+胰岛素(100nM)，6.25μg/mL，6.25μg/mL+胰岛素(100nM)，12.5μg/mL，12.5μg/mL+胰岛素(100nM)，25μg/mL，25μg/mL+胰岛素(100nM)，另设置空白对照组，每组设3个复孔，在温度37℃、浓度5％CO2恒温培养箱中继续培养；培养24h后，取出培养基上清，按葡萄糖氧化酶试剂盒说明书进行葡萄糖消耗测定；
结论：帚枝香青正丁醇提取物给药浓度为3.125μg/mL、6.25μg/mL、12.5μg/mL、25μg/mL均促进了葡萄糖消耗，给药浓度为3.125μg/mL、6.25μg/mL、12.5μg/mL、25μg/mL时，显著增强了葡萄糖的摄取；且给药+低浓度胰岛素(100nM)组刺激葡萄糖消耗，产生加和作用，葡萄糖消耗量增多。结果表明：不同浓度帚枝香青正丁醇提取物具有显著增强葡萄糖消耗的作用(图4)。
Claims (1)
1.一种帚枝香青正丁醇提取物的用途，其特征在于，所述帚枝香青正丁醇提取物在制备治疗糖尿病及高糖损伤引起肾脏病变的药物中的用途，所述帚枝香青正丁醇提取物的提取方法为：
a.取30g帚枝香青，粉碎后，采用浓度为70%乙醇按质量体积比20倍量水浴加热回流，水浴温度为60℃，提取2次，每次提取1.5小时，收集提取液；
b.将提取液进行减压蒸馏除去其中的乙醇，浓缩至糖浆状，温度45℃干燥，即得到帚枝香青粗提物浸膏；
c.将帚枝香青粗提物浸膏以1:15-1:20的料液比，用水分散后，依次用石油醚、二氯甲烷、乙酸乙酯和正丁醇萃取，将正丁醇萃取液减压蒸至糖浆状，温度45℃干燥，得到帚枝香青正丁醇提取物。
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