CN114832025B - 一种人参不定根粗提物的制备方法及应用 - Google Patents
一种人参不定根粗提物的制备方法及应用 Download PDFInfo
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- CN114832025B CN114832025B CN202210540063.0A CN202210540063A CN114832025B CN 114832025 B CN114832025 B CN 114832025B CN 202210540063 A CN202210540063 A CN 202210540063A CN 114832025 B CN114832025 B CN 114832025B
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- adventitious root
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Abstract
本发明公开了一种人参不定根粗提物的制备方法及应用,属于医药技术领域。本发明发现人参不定根粗提物在通过异丙肾上腺素诱导的心肌缺血小鼠模型中可以起到心肌缺血预保护的功能。人参不定根粗提物可以通过调控NLRP3炎症小体介导的焦亡通路激活起到抑制心肌缺血导致的心脏损伤从而起到保护心肌缺血的作用。这些体内研究结果为寻找适合的预防心肌缺血措施、改善人类健康具有重要意义,证明人参不定根粗提物补充可以改善心肌缺血的发生发展。拓宽人参不定根粗提物在健康功能食品和药物研制等方面的应用,为心肌缺血的预防和治疗提供新思路。
Description
技术领域
本发明涉及医药技术领域,更具体的说是涉及一种人参不定根粗提物的制备方法及应用。
背景技术
目前,心肌缺血是冠状动脉闭塞引起的心脏局部缺血缺氧,是导致人类死亡的最主要的心血管疾病,心肌缺血后出现心肌细胞凋亡、坏死,心脏重塑,心肌纤维化,心脏功能失调及心律失常等,最终导致充血性心力衰竭甚至猝死。经统计今后10年,我国心血管病患病人数仍将快速增长。目前,心血管病死亡占城乡居民总死亡原因的首位,农村为45.01%,城市为42.61%,心血管病的疾病负担日渐加重,已成为重大的公共卫生问题。因此寻找适合且疗效确切的防治心肌缺血损伤药物一直是心血管领域的研究热点、难点。
焦亡——一种炎症引发的程序性细胞死亡,区别于细胞凋亡、细胞坏死,近年来研究较多。已有研究表明,细胞焦亡参与了心肌缺血病程的发生发展。目前细胞焦亡以NLRP3、Caspase-1、IL-1β为经典信号通路的研究较多。文献指出人参皂甙Rb1通过充当自由基清除剂并减弱由c-Jun N端激酶途径介导的过程中的线粒体ROS生成,从而保护心肌细胞免受氧化损伤同时激活NLRP3炎性小体。NLRP3炎性小体产生时,其作为上游因子可选择性介导Caspase-1(半胱天冬氨酸酶)高分子复合物的成熟和释放,而下游的IL-1β及IL-18等相关前体蛋白的活化成熟则依赖于Caspase-1的刺激,使Caspase-1被切割,导致GSDMD的N端、C端分离,打破其自抑制状态,在细胞膜上形成蜂窝状孔道,释放IL-1β、IL-18炎症因子,从而使细胞发生焦亡。大量文献表明人参皂苷具有抑制心脏炎症反应的作用,而与炎症相关的程序性细胞死亡方式——细胞焦亡存在于心脏疾病中,那么人参皂苷抑制心脏炎症反应的作用机制有可能是通过抑制心脏细胞焦亡实现。因此,人参不定根粗提物对心肌缺血造成的心肌损伤可能具有保护作用。
人参不定根(GAR)是通过发酵培养的人参组织培养物,目前已批准认为与5年以下人参实质等同,可作为新食品原料在医药、食品中应用。毒理学评价表明,人参不定根没有发现急性经口毒性、致突变性和致畸性,90天经口试验表明没有亚慢性毒性。
然而,目前尚未有人参不定根粗提物(GAR)通过缓解焦亡信号通路激活改善/治疗心肌缺血的研究,也没有相关的文献报道。因此,阐明GAR保护心肌缺血的分子机制,发现GAR的心肌缺血保护作用,可以扩大其应用范围,为替代种植人参及健康功能食品的开发提供一定的研究基础。
发明内容
有鉴于此,本发明提供了一种人参不定根粗提物的制备方法及应用。
为了实现上述目的,本发明采用如下技术方案:
一种人参不定根粗提物在制备治疗或改善心肌缺血症状的药物中的应用。
进一步的,所述心肌缺血症状包括心肌缺血所致心肌细胞凋亡、心肌缺血所引起的炎症反应、心脏重量提升和心重体重比提升、心肌缺血损伤关键酶提升、抗氧化相关酶降低、心肌缺血导致的心电图变化。
进一步的,人参不定根粗提物通过抑制NLRP3炎症小体介导的焦亡通路激活来起到抑制心肌缺血导致的心脏损伤从而起到保护心肌缺血症状的作用。
一种人参不定根粗提物的制备方法,包括以下步骤:
(1)将人参不定根加入至70%乙醇溶液,加热回流提取2h,将提取液用滤纸过滤,保存滤液备用;
(2)将步骤(1)提取液过滤后的残渣加入至70%乙醇溶液,加热回流提取2h,将提取液用滤纸过滤,将所得滤液与步骤(1)所得滤液合并;
(3)将合并滤液真空干燥,得人参不定根粗提物粉末。
进一步的,步骤(1)和步骤(2)所述回流加热温度为80℃。
进一步的,步骤(1)和步骤(2)所述滤纸孔径为11μm。
进一步的,步骤(1)所述人参不定根为干燥制品。
进一步的,步骤(1)所述人参不定根、步骤(1)所述乙醇溶液与步骤(2)所述乙醇溶液的比例为重量(g):体积(mL):体积(mL)=1:50:50。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种人参不定根粗提物的制备方法及应用,开拓性地提出了人参不定根粗提物在治疗/改善心肌缺血方面的应用,可以拓宽人参不定根粗提物在健康功能食品和药物研制等方面的应用,使其有望开发成为一种健康功能食品或药物,用于预防或治疗心肌缺血,为心肌缺血的预防和治疗提供新思路。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为人参不定根粗提物对心肌缺血小鼠心电图影响;其中(A)对照组;(B)ISO组;(C)500mg/kg GAR+ISO组;(D)1000mg/kg GAR+ISO组;(E)1000mg/kg GAR组;n=10。
图2附图为为人参不定根粗提物心肌缺血小鼠组织病理影响;其中(A)对照组;(B)ISO组;(C)500mg/kg GAR+ISO组;(D)1000mg/kg GAR+ISO组;(E)1000mg/kg GAR组,放大200倍,n=10。
图3附图为人参不定根粗提物对NLRP3炎症通路影响;其中(A)心肌组织中NLRP3mRNA相对水平;(B)心肌组织中Caspase-1mRNA相对水平;(C)心肌组织中IL-1βmRNA相对水平;(D)心肌组织中IL-18mRNA相对水平。*与对照组相比P<0.05,**与对照组相比P<0.01,##与ISO组相比P<0.01,n=4。
图4附图为利用Western Blot检测NLRP3炎症小体信号通路蛋白表达变化;其中(A)NLRP3炎症小体信号通路蛋白表达代表性图片;(B)NLRP3蛋白表达统计图;(C)ASC蛋白表达统计图;(D)Caspase-1蛋白表达统计图;(E)cleaved-Caspase-1蛋白表达统计图;(F)IL-1β蛋白表达统计图。*与对照组相比P<0.05,**与对照组相比P<0.01,***与对照组相比P<0.001,#与ISO组相比P<0.05,##与ISO组相比P<0.01,n=3。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
原料来源:试剂盒购自于南京建成商业试剂盒,人参不定根(GAR)由大连普瑞康生物技术有限公司提供,C57BL/6雄性小鼠由浙江省实验动物中心提供;
本发明未提及材料采购自市售渠道;未提及的实验方法为常规实验方法,在此不再一一赘述。
实施例1
一种人参不定根粗提物的制备方法。
(1)精准称量100g人参不定根加入至5000mL70%乙醇溶液,加热至80℃回流提取2h,将提取液用11μm孔径的whatmanNo.1滤纸过滤,保存滤液备用;
(2)将步骤(1)提取液过滤后的残渣加入至5000mL 70%乙醇溶液,加热至80℃回流提取2h,将提取液用11μm孔径的whatmanNo.1滤纸过滤,将所得滤液与步骤(1)所得滤液合并;
(3)将合并滤液用真空旋转蒸发仪蒸干,得20g人参不定根粗提物粉末。
人参皂苷组分检测
取0.2g实施例1制备的人参不定根粗提粉末用10mL甲醇溶解,取1mL溶液经0.24μm滤膜过滤,采用高效液相色谱系统对滤液中的人参皂苷组分进行分析。
高效液相色谱系统:色谱柱:Acclaim 120C18,5μm,250mm X 4.6mm,流速:1mL/min,柱温50℃。洗脱峰在203nm处检测,精密称量人参皂苷标准品Rg1、Rg2、Re、Rb1、Rc、Rb2、Rd进行定量。以水为流动相A,乙腈为流动相B,进行梯度洗脱。
测得人参不定根粗提物中的人参皂苷含量。
表1人参不定根粗提物中的人参皂苷含量
如表1所示,人参不定根中的人参皂苷Rg1含量为1.09mg/g、Rg2含量为0.4mg/g、Re含量为3.3mg/g、Rb1含量为4.72mg/g、Rc含量为0.86mg/g,Rb2含量为10.69mg/g、Rd含量为0.96mg/g,总含量为22.02mg/g。
DBS22/024-2014食品安全地方标准食品原料用人参规定用于食品原料的人参及人参不定根中人参皂苷含量>2%,实施例1选用的人参不定根符合标准。
实验动物与心肌缺血模型设计及预保护方法:
50只C57BL/6雄性小鼠(8w)由浙江省实验动物中心提供,实验动物生产许可证号为SCXK(浙)2019-0002,实验动物使用许可证号为SYXK(浙)2016-0022。实验程序经浙江省医学科学院实验动物福利伦理委员会审查,符合动物保护原则,编号为2018-045。
所有C57BL/6小鼠分为5组,分别为对照组、ISO(异丙肾上腺素)组、500GAR+ISO组、1000GAR+ISO组、1000GAR组,每组10只。人参不定根人体推荐量为3g/d,以生药量计算,根据人与小鼠换算关系,小鼠低剂量组为每天灌胃500mg/kg d人参不定根粗提物(GAR),高剂量组为每天灌胃1000mg/kg d GAR。对照组每天灌胃等体积去离子水,1000GAR组每天灌胃高剂量GAR,均不进行ISO处理。连续灌胃28d,ISO组、ISO+500GAR组、ISO+1000GAR组在第27d和第28d连续两天皮下注射85mg/kg ISO,ISO浓度为17mg/mL,注射间隔24h。
实验场地
实验于杭州医学院黄龙校区6号楼3楼SPF级动物饲养房进行,动物饲养环境为25±2℃,12h明暗交替。小鼠自由进食进水,适应性饲养3d。
动物基本生理状况观察和记录
每天观察小鼠肢体、毛发等生理状况,每天添加适量饲料和饮水,并更换干净垫料。每周测量小鼠体重并记录。
心电图分析
实验期结束时,小鼠腹腔注射200mg/kg 2,2,2-三溴乙醇(Avertin)麻醉,在小鼠左上肢、右上肢、右下肢皮下植入电极,然后记录心电图,观察各组小鼠心电图ST段升高情况。利用iWorx 400系统进行心电数据采集和分析。
实验结果见图1,GAR对心肌缺血小鼠心电图影响。
心肌缺血心电图表现为ST段抬高。对照组和单独GAR组小鼠心电图显示正常,ST段没有抬高,而单独ISO处理小鼠与对照组相比,ST段明显抬高,提示诱导心肌缺血模型成功。GAR提取物预处理改善了心肌缺血小鼠ST段抬高,对心肌缺血具有保护作用。
心重和心脏损伤检测
小鼠记录心电图后采集血样,从每个样本中分离血清进行生化分析。心脏被切除并称重,心重体重比计算方法为心脏重量(mg)除以小鼠体重(g)。小鼠心脏被储存在-80℃低温冰箱,以备进一步使用和分析。
表2 GAR对体重、心重及心重体重比影响(x±s,n=10)
注:**与对照组相比P<0.01,***与对照组相比P<0.001,#与ISO组相比P<0.05,###与ISO组相比P<0.001。
表2为GAR预处理对小鼠体重、心脏重量、心重体重比的影响见表3。各组间的体重没有显著差异,ISO处理组小鼠体重略有下降,差异没有统计学意义(P>0.05)。ISO诱导的心肌缺血小鼠中,心脏重量和心重体重比显著增加。1000mg/kg d GAR预处理显著改善心肌缺血小鼠心重和心重体重比,差异具有统计学意义(P<0.05,P<0.001)。
心肌缺血小鼠血清中相关酶检测
提取小鼠血清10μL,利用南京建成商业试剂盒,根据说明书检测血清中心肌缺血损伤的特异性标记酶包括乳酸脱氢酶(LDH)、肌酸激酶-MB(CK-MB)、丙氨酸转氨酶(ALT)和cTn-T。
表3 GAR对心肌缺血小鼠血清中相关酶影响(x±s,n=5)
注:*与对照组相比P<0.05,**与对照组相比P<0.01,#与ISO组相比P<0.05,##与ISO组相比P<0.01,###与ISO组相比P<0.001。
表3为LDH、CK-MB、ALT和cTn-T为心肌损伤的标志性酶,心肌缺血发生时血清中含量均升高。如表3所示,与正常对照组相比,ISO处理组血清LDH(232.31±36.33vs.106.15±46.39)、CK-MB(8.51±1.71vs.6.51±0.53)、ALT(142.35±67.62vs.45.20±11.61)和cTn-T(90.57±23.60vs.53.22±22.46)含量明显增加,具有统计学意义。而高剂量组GAR预处理明显减少心肌缺血小鼠血清中LDH、CK-MB、ALT和cTn-T水平(P<0.05;P<0.01;P<0.001),差异具有统计学意义。结果表明GAR对心肌缺血损伤具有保护作用。
氧化指标检测
心脏中过氧化物酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)、过氧化氢酶(CAT)水平降低被认为是抗氧化能力下降和氧化应激的指标。准确称量0.1g心肌组织,按组织重量(g):体积(mL)=1:9比例加入9倍体积的生理盐水,冰浴条件下,制备成10%组织匀浆,2500r/min离心10min,取上清。利用南京建成商业试剂盒根据说明书检测上清液中SOD、GSH-px和CAT抗氧化酶活性。
表4GAR对心肌缺血小鼠心肌组织抗氧化酶影响(x±s,n=4)
注:*与对照组相比P<0.05,#与ISO组相比P<0.05
表4GAR对氧化应激影响
表4所示,ISO处理明显减少抗氧化酶SOD、GSH-px及CAT活性(P<0.05),缺血心脏抗氧化能力减弱,将引发氧化应激损伤。低剂量和高剂量GAR预处理明显恢复ISO导致的SOD和GSH-px活性降低(P<0.05),提高心肌组织抗氧化能力,但对CAT抗氧化活性有升高的趋势,没有统计学意义。
心脏组织HE染色
全心组织在室温下用4%多聚甲醛固定24h,用石蜡包埋,切成5μm厚切片。依次用无水乙醇作用5min,90%乙醇作用2min,80%乙醇作用2min,70%乙醇作用2min,蒸馏水作用2min。苏木素染色液染色10min,浸自来水约10min洗去多余的染色液,再用蒸馏水洗涤数秒钟。之后,用分化液分化约30s,自来水冲洗10min。伊红染色液染色2min。再依次用70%乙醇作用10s,80%乙醇作用10s,90%乙醇作用10s,无水乙醇作用10s,再用二甲苯透明5min。换用新鲜的二甲苯,再透明5min。封片剂封片。显微镜下观察,细胞核呈蓝色,而细胞浆呈粉红色或红色。
实验结果见图2,GAR心肌缺血小鼠组织病理影响。
正常组心肌细胞排列整齐、形态正常。ISO处理明显导致心肌损伤,心肌细胞排列紊乱,心肌间质炎症浸润,有心肌纤维化发生。高剂量组GAR明显改善ISO导致的心肌组织病理形态学改变,对心脏组织具有一定的保护作用。
心肌缺血组织NLRP3炎症小体信号mRNA检测
提取RNA
生物安全柜紫外照射15min,再用DEPC水配制的75%酒精擦拭操作台面及用具。动物:将心脏组织从-80℃冰箱中取出,剪取约0.03g心脏左心室组织置于1.5mL EP管中。每管加入1mL TRNzol试剂,研磨棒将肾脏组织研碎。向管中加入200μL氯仿,剧烈摇晃15s,静置3min后,13500rpm离心15min。离心后溶液分层,小心吸取上层水相溶液到另一个新的1.5mLEP管中,再向其中加入500μL异丙醇。静置10min,13500rpm离心10min。离心后将上清液弃去,保留RNA沉淀。加入1mL 75%乙醇洗涤沉淀,13500rpm离心1min后弃去液体,将管子置于通风口挥干乙醇。最后用50μL DEPC水溶解沉淀。用Nanodrop核酸蛋白测定仪测RNA浓度及纯度。
RNA逆转录
逆转录体系如下:RNA 1000ng、2×RT BufferMix 10μL、20×Enzyme mix1μL,不足部分用无RNA酶水补齐,配制成20μL反应体系。反应程序为:25℃10min→→85℃5min→→37℃120min→→4℃∞。
荧光定量Real-time PCR
反应混合物体系如下:按10μL SYBR Green染料、各基因上下游引物各1μL,ddH2O7μL的比例配制反应混合物,轻轻混匀后取19μL/孔加入0.2mL PCR反应板中,每孔再加入各基因1μL cDNA。PCR 96孔板1000rpm离心1min,使液体沉在孔底。置于荧光定量PCR仪中,95℃5min预变性后,按下列程序完成反应:95℃15s→65℃30s→72℃30s,40循环。实验所需引物如下所示。
实验结果见图3,GAR对心肌缺血组织NLRP3炎症小体信号mRNA影响。
(A)心肌组织中NLRP3 mRNA相对水平;(B)心肌组织中Caspase-1mRNA相对水平;(C)心肌组织中IL-1βmRNA相对水平;(D)心肌组织中IL-18mRNA相对水平。*与对照组相比P<0.05,**与对照组相比P<0.01,##与ISO组相比P<0.01,n=4。
心肌缺血30min以上既可导致心肌细胞不可逆损伤,在缺血过程中,氧化应激反应可激活NLRP3炎症小体,促使Caspase-1裂解为有活性的cleaved-Caspase-1(p20),进一步促进IL-1β和IL-18前体变为有活性的IL-1β和IL-18,导致炎症风暴,触发心肌炎性损伤。利用荧光定量PCR检测NLRP3炎症小体信号通路中NLRP3、Caspase-1、IL-1β、IL-18mRNA水平,如图3所示,GAR明显抑制ISO导致的NLRP3、Caspase-1、IL-1β、IL-18mRNA水平上调,差异具有统计学意义(P<0.01,P<0.001)。
心肌缺血小鼠NLRP3炎症小体信号通路蛋白检测
蛋白质提取
取小鼠心脏左心室约0.02g于1.5mL离心管中,配制蛋白裂解液(RIPA:10%SDS:蛋白酶抑制剂:磷酸酶抑制剂=60:40:1:1),每管加入200-300μL蛋白裂解液。在冰上将肾脏组织充分研磨成匀浆,用超声充分裂解。4℃,13500rpm离心30min,取上清液保存于-80℃冰箱,用于后续实验。
蛋白质处理
用BCA法测定蛋白浓度。BCA试剂盒中A液和B液按50:1比例混合,96孔板每孔中加入200μL BCA混合液、19μL PBS缓冲液,1μL待测蛋白样,轻轻震荡混匀后置于37℃培养箱中孵育30min。30min后利用酶标仪测定蛋白标准曲线及蛋白样吸光度值。根据标准曲线及蛋白样的吸光度值计算出蛋白的浓度,蛋白质上样量为80μg/孔,用RIPA裂解液将所有样品调整至相同体积,加入5×loading buffer后充分混匀,干式恒温器加热5min(100℃),使蛋白质变性。
WesternBlotting检测蛋白质表达水平
电泳:将胶板固定在胶架上,放入电泳槽中,倒入电泳液至总体积的1/3,排除气泡后,再将电泳液加至刻度线。首先,按分组在孔内加入一定体积的蛋白处理样,然后在蛋白样本两边分别加入一孔3μL marker(左边)和一孔1μL marker(右边)。扣上电泳槽盖子,接通电源,设置电压70V,电泳时间持续20-30min,直到marker分开后再将电压调至110V,根据目的蛋白分子量的大小调整电泳时间,约为1-1.5h。
转膜:提前准备好冰槽,转膜槽置于冰槽中。将所需滤纸和NC膜用冰冷的转膜液浸泡数分钟。将胶板从胶架上取下,浸泡在转膜液中。从玻璃胶板上割下胶,然后按海绵垫1张、滤纸2张、胶、NC膜、滤纸2张、海绵垫1张的顺序放入转膜夹中,将每一层的气泡排干净且保持湿润。将夹好的转膜夹放入转膜槽中,加转膜液至刻度,盖上盖子,接通电源,将电流调节至300mA,根据蛋白分子量大小调整转膜时间,约75-105min。
封闭:转膜完成后将NC膜取出,浸泡在配制好的封闭液中,室温下摇床55rpm摇晃封闭2h。
孵育一抗:根据抗体说明书,用一抗二抗稀释液将一抗原液稀释到合适倍数后倒入抗体盒中,将2张剪出有目的蛋白的NC膜背靠背放入相应抗体盒中,于4℃冰箱的摇床上55rpm摇晃孵育过夜。NLRP3(1:500),ASC(1:1000),Caspase-1(1:1000),IL-1β(1:1000)、IL-18(1:1000),GAPDH(1:4000)。
孵育二抗:根据抗体说明书,用一抗二抗稀释液将二抗原液1:10000稀释。从4℃冰箱中取出NC膜,置于室温摇床上55rpm摇晃1h。之后用PBST在摇床上摇晃洗膜3次,每次10min。之后,将NC膜平铺于桌面上,根据目的蛋白一抗特异性加入700nm兔源或荧光二抗,室温避光孵育1h。孵育完成后PBST洗膜3次,每次8min。
扫膜:洗膜后使用红外荧光扫描系统Odyssey扫描检测,GAPDH为内参,根据灰度值对蛋白条带进行分析。
实验结果见图4,GAR对心肌缺血小鼠NLRP3炎症小体信号通路蛋白表达影响。
其中(A)NLRP3炎症小体信号通路蛋白表达代表性图片;(B)NLRP3蛋白表达统计图;(C)ASC蛋白表达统计图;(D)Caspase-1蛋白表达统计图;(E)cleaved-Caspase-1蛋白表达统计图;(F)IL-1β蛋白表达统计图。
*与对照组相比P<0.05,**与对照组相比P<0.01,***与对照组相比P<0.001,#与ISO组相比P<0.05,##与ISO组相比P<0.01,n=3。
ISO诱导的缺血心肌组织中,NLRP3、ASC、Caspase-1、cleaved-Caspase-1和IL-1β表达明显升高(P<0.01,P<0.001),差异具有统计学意义。高剂量GAR预处理对NLRP3/Caspase-1通路相关蛋白异常表达有明显的恢复作用,表明GAR可能对心肌缺血炎症损伤有一定的抑制作用。结果表明GAR可能通过抑制NLRP3炎症小体信号改善ISO导致的心肌缺血损伤。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (3)
1.一种人参不定根粗提物在制备治疗或改善心肌缺血药物中的应用,其特征在于,所述人参不定根粗提物的制备方法,包括以下步骤:
(1)将人参不定根加入至70%乙醇溶液,加热至80℃回流提取2h,将提取液用滤纸过滤,保存滤液备用;
(2)将步骤(1)提取液过滤后的残渣加入至70%乙醇溶液,加热至80℃回流提取2h,将提取液用滤纸过滤,将所得滤液与步骤(1)所得滤液合并;
(3)将合并滤液真空干燥,得人参不定根粗提物粉末;
步骤(1)所述人参不定根、步骤(1)所述70%乙醇溶液与步骤(2)所述70%乙醇溶液的比例为重量:体积:体积=1g:50mL:50mL。
2.根据权利要求1所述的一种人参不定根粗提物在制备治疗或改善心肌缺血药物中的应用,其特征在于,步骤(1)和步骤(2)所述滤纸孔径为11μm。
3.根据权利要求1所述的一种人参不定根粗提物在制备治疗或改善心肌缺血药物中的应用,其特征在于,步骤(1)所述人参不定根为干燥制品。
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KR20100050192A (ko) * | 2008-11-05 | 2010-05-13 | 성신여자대학교 산학협력단 | 프로토파낙사디올과 프로토파낙사트리올 비율을 조절하여 생리효과를 나타내는 인삼 추출 조성물 |
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