CN111990571A - 一种祛炎通脉功能饮料及其制备方法 - Google Patents
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Abstract
本发明提供了一种祛炎通脉功能饮料及其制备方法,制备方法包括以下步骤:将干燥筋骨草、金钱草和三七混合,向其中加入体积浓度为55‑70%的乙醇,于避光条件下浸提15‑20天,收集浸提液;将浸提液减压浓缩至无乙醇残留,得中药原液;将中药原液于75‑85℃避光条件下干燥,得饮料原汁;向饮料原汁中加水,制得。该饮料可有效控制人体内慢性炎症、网状毛细血管循环障碍等问题。
Description
技术领域
本发明属于保健饮料技术领域,具体涉及一种祛炎通脉功能饮料及其制备方法。
背景技术
炎症是导致人体各种慢性疾病或障碍发展的重要因素,研究表明糖尿病、癌症、心脑血管疾病、眼疾、关节炎、肥胖症、自身免疫性疾病等与炎症密切相关。炎症是机体对有害的物理、化学、生物刺激做出的生理反应,是人体组织器官对刺激、损伤、感染产生的一种局部保护的过程,表现为红、肿、热、痛等临床反应,严重时可引起功能障碍。人体组织水平的炎症过程包括一系列小静脉和小动脉扩张、血管通透性增加、血液流动性增强和白细胞渗入组织等步骤。根据炎症过程和细胞机制,炎症主要分为急性炎症和慢性炎症。急性炎症是一个较短的过程,持续数分钟至数天,首先增加血流至发炎区域,随后血管扩张和血管通透性的增强伴随微循环中血浆的渗漏,最终吞噬白细胞等炎症细胞向周围组织迁移。这些细胞和血管反应是由细胞或血浆产生的化学因子介导,是炎症的典型临床症状,例如肿胀,发红,疼痛,发热和功能丧失。慢性炎症的状态与慢性病和疾病风险增加相关,当炎症反应缺乏实际刺激时,通常会发生组织中的慢性炎症。它通常通过内源性保护机制或宿主防御中的其他抵抗机制无法解决的感染而发生,异物的持续存在、持续的化学暴露、反复发作的急性炎症或特定的病原体都是造成慢性炎症的关键原因。
近年,人们对饮料的需求逐渐增大,开发新的迎合消费者需求的饮料产品凸显发展优势,一是人们更倾向于选择洁净的饮水;二是人们对健康饮食意识的提升,蔬菜和水果的成本提高,促使果蔬汁饮料的大力发展;三是固体饮料的保质期长、即冲即饮,符合人们喜欢喝热饮的习惯,有着广阔的发展前景;四是功能饮料能在一定程度上调节人体生理功能,在快节奏、压力大的消费人群广受欢迎,发展迅速。功能饮料是通过调整饮料中天然营养物质的成分和比例,使其具有一定程度上调节人体机能的饮料。目前包括运动饮料、能量饮料和保健饮料,运动饮料、能量饮料以脉动、红牛为代表,而保健功能的天然植物饮料较少。
我国人口众多,糖尿病、心脑血管疾病、肿瘤、关节炎、肥胖、自身免疫性疾病等患者不断增加,这些疾病与人体慢性炎症、网状毛细血管微循环障碍密切相关。目前人体慢性炎症、网状毛细血管微循环障碍临床症状轻微,人们不易觉察,较少引起注意或重视,入院治疗者微乎其微,因此,人们急需一种祛炎通脉保健功能饮料及其制备方法,满足人们预防疾病、延年益寿的需求。
发明内容
针对现有技术中存在的上述问题,本发明提供一种祛炎通脉功能饮料及其制备方法,该饮料可有效控制人体内慢性炎症、网状毛细血管循环障碍等问题。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
一种祛炎通脉功能饮料的制备方法,包括以下步骤:
(1)将干燥的筋骨草、金钱草和三七混合,向其中加入体积浓度为55-70%的乙醇,于避光条件下浸提15-20天,收集浸提液;
(2)将步骤(1)中浸提液减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液于75-85℃避光条件下干燥,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,制得。
上述方案中,筋骨草又名白毛夏枯草、金疮小草、散血草、破血丹、青鱼胆草、苦草,性寒、味苦,归肺经,具有止咳化痰、清热凉血、解毒消肿、止痢的功效,可治咳嗽、吐血及妇女血气痛,上呼吸道感染引起的急慢性咽炎、支气管炎、扁桃体炎、肺炎、痢疾、吐血、咳血、疗疮、痈肿及跌打损伤等;金钱草,又名过路黄、镜面草、翠屏草、荷苞草、肉馄饨草、金锁匙、连钱草、对座草、叶金钱草、钱叶草,钱芊金,味甘、微苦、性凉,归肝、胆、肾、膀胱经,有清热利尿、祛风止痛、止血生肌、消炎解毒之功效,可治急慢性肝炎、黄疸型肝炎、胆囊炎、肾炎、泌尿系感染、扁桃腺炎、口腔炎及痈疔疗毒、毒蛇咬伤、乳痈、痢疾、疟疾、肺出血等。三七又名田七、人参三七、参三七、文州三七,味甘、微苦,温,归肝、胃经,具有散瘀止血,消肿定痛功效,用于咯血,吐血,衄血,便血,崩漏,外伤出血,胸腹刺痛,跌扑肿痛等,将筋骨草、金钱草和三七合用,可有效对体内的炎症进行抑制,实现祛炎通脉的作用。
使用一定浓度的乙醇对筋骨草、金钱草和三七避光浸提,可有效将药物中的有效成分提取,避光可避免提取的有效成分见光分解;然后进行减压浓缩,使得浸提液中的乙醇成分蒸发,保留有效成分,再进行避光干燥,制得饮料原汁,将饮料原汁加水后进行密封灌装,即制得祛炎通脉功能饮料,该饮料为纯天然植物提取,其中未添加任何防腐剂等化学物质,具有环保健康的功效。
进一步地,步骤(1)中药材与乙醇的体积比为1:1。
进一步地,步骤(1)中筋骨草、金钱草和三七的质量比为7-10:0.1-1:0.1-2。
进一步地,步骤(1)中筋骨草、金钱草和三七的质量比为8-9:0.1-0.5:0.4-0.8。
进一步地,步骤(1)中筋骨草、金钱草和三七的质量比为9:0.3:0.7。
上述方案中,筋骨草作为主要原料发挥作用,筋骨草和三七作为辅助原料,协助筋骨草发挥作用,三者相互协同,进而提高该功能饮料对体内炎症的抑制效果。
进一步地,步骤(1)中乙醇的体积浓度为60%。
上述方案中,乙醇的体积浓度与极性相关,体积浓度改变后,极性便发挥改变,采用体积浓度为60%的乙醇对药材进行浸提时,乙醇的极性与药材中活性成分的极性相似,利用相似相容的原理,可提高活性成分的浸出量。
进一步地,步骤(2)中减压浓缩时的的压强为9-12KPa。
进一步地,步骤(3)干燥至中药原液的体积减少50%后,停止。
进一步地,步骤(4)中饮料原汁与水的体积占比分别为10-30%和70-90%。
上述方案中,将饮料原汁与水按照上述的体积比混合后,使制得的饮料中营养成分含量适中,实现保健作用。
进一步地,步骤(4)中还加入蔗糖,饮料原汁、蔗糖和水的体积占比为10-30%、8-12%和62-82%。
上述方案中,向其中加入蔗糖,可改善饮料的口感,满足不同人群的口味需求。
本发明所产生的有益效果为:
本发明中的祛炎通脉功能饮料具有良好的抗炎作用,主要是通过促使炎症细胞释放大量的PRDX1和SLPI来改善氧化应激,调节炎症相关蛋白酶催化功能而发挥抗炎作用。
本发明有效抽提了筋骨草、金钱草、三七的活性成分,饮料产品清香、微苦,具有祛炎通脉功效,环保健康,方便消费者携带和饮用,具有较好保健作用,适于在功能饮料行业内推广应用。
附图说明
图1为祛炎通脉功能饮料对小鼠生长的影响情况统计图;
图2为祛炎通脉功能饮料原汁对小鼠生长的影响情况统计图;
图3为小鼠肺组织染色图;
图4为大鼠宫颈组织染色图;
图5为CT组、LPS组和饮料组处理后RAW264.7细胞总RNA电泳检测图;
图6为祛炎通脉功能饮料处理后差异基因统计图;
图7为祛炎通脉功能饮料处理后鞭毛蛋白变化图。
具体实施方式
下面结合附图对本发明的具体实施方式做详细的说明。
实施例1
一种祛炎通脉功能饮料,其制备方法包括以下步骤:
(1)将干燥筋骨草、金钱草和三七混合,筋骨草、金钱草和三七的质量比为7:0.2:0.5,然后向其中加入体积浓度为55%的乙醇,中药材与乙醇的体积比为1:1,于避光条件下常温浸提16天,收集浸提液;
(2)将步骤(1)中浸提液置于蒸馏罐内,设置蒸馏罐罐底温度为75℃,压强为9KPa,减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液倒入无菌干燥箱的干燥盘内,于75℃避光条件下干燥至中药原液体积减少50%,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,饮料原汁与水的体积比为10-30%和70-90%,然后,于小于30℃、避光条件下低温密封灌装,制得。
实施例2
一种祛炎通脉功能饮料,其制备方法包括以下步骤:
(1)将干燥筋骨草、金钱草和三七混合,筋骨草、金钱草和三七的质量比为9:0.4:1,然后向其中加入体积浓度为65%的乙醇,中药材与乙醇的体积比为1:1,于避光条件下浸提20天,收集浸提液;
(2)将步骤(1)中浸提液置于蒸馏罐内,设置蒸馏罐罐底温度为85℃,压强为12KPa,减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液倒入无菌干燥箱的干燥盘内,于85℃避光条件下干燥至中药原液体积减少50%,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,饮料原汁与水的体积比为15%和85%,然后,于小于30℃、避光条件下低温密封灌装,制得。
实施例3
一种祛炎通脉功能饮料,其制备方法包括以下步骤:
(1)将干燥筋骨草、金钱草和三七混合,筋骨草、金钱草和三七的质量比为9:0.3:0.7,然后向其中加入体积浓度为60%的乙醇,中药材与乙醇的体积比为1:1,于避光条件下浸提18天,收集浸提液;
(2)将步骤(1)中浸提液置于蒸馏罐内,设置蒸馏罐罐底温度为80℃,压强为10KPa,减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液倒入无菌干燥箱的干燥盘内,于80℃避光条件下干燥至中药原液体积减少50%,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,饮料原汁与水的体积比为20%和80%,然后,于小于30℃、避光条件下低温密封灌装,制得。
对比例1
一种祛炎通脉功能饮料,其制备方法包括以下步骤:
(1)将干燥黄连、金钱草和三七混合,黄连、金钱草和三七的质量比为9:0.3:0.7,然后向其中加入体积浓度为60%的乙醇,中药材与乙醇的体积比为1:1,于避光条件下浸提18天,收集浸提液;
(2)将步骤(1)中浸提液置于蒸馏罐内,设置蒸馏罐罐底温度为80℃,压强为10KPa,减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液倒入无菌干燥箱的干燥盘内,于80℃避光条件下干燥至中药原液体积减少50%,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,饮料原汁与水的体积比为20%和80%,然后,于避光条件下低温密封灌装,制得。
对比例2
一种祛炎通脉功能饮料,其制备方法包括以下步骤:
(1)将干燥筋骨草、金钱草和三七混合,筋骨草、金钱草和三七的质量比为11:1.5:2.5,然后向其中加入体积浓度为60%的乙醇,中药材与乙醇的体积比为1:1,于避光条件下浸提18天,收集浸提液;
(2)将步骤(1)中浸提液置于蒸馏罐内,设置蒸馏罐罐底温度为80℃,压强为10KPa,减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液倒入无菌干燥箱的干燥盘内,于80℃避光条件下干燥至中药原液体积减少50%,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,饮料原汁与水的体积比为20%和80%,然后,于小于30℃、避光条件下低温密封灌装,制得。
对比例3
一种祛炎通脉功能饮料,其制备方法包括以下步骤:
(1)将干燥筋骨草、金钱草和三七混合,筋骨草、金钱草和三七的质量比为9:0.3:0.7,然后向其中加入体积浓度为80%的乙醇,中药材与乙醇的体积比为1:1,于避光条件下浸提10天,收集浸提液;
(2)将步骤(1)中浸提液置于蒸馏罐内,设置蒸馏罐罐底温度为80℃,压强为10KPa,减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中药原液倒入无菌干燥箱的干燥盘内,于80℃避光条件下干燥至中药原液体积减少50%,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,饮料原汁与水的体积比为20%和80%,然后,于小于30℃、避光条件下低温密封灌装,制得。
试验例
一、祛炎通脉功能饮料安全性实验
(1)祛炎通脉功能饮料对小鼠饮用安全性实验
取五周龄KM小鼠(川北医学院实验动物中心提供,合格证号:SYXK(川)2014-076)雌雄各40只分开,分别分4组(每组10只)用基础饲料(川北医学院实验动物中心提供)喂养,其中一组给洁净水对照、另一组给祛炎通脉功能饮料(实施例3)作为小鼠饮用水,每日称量基础饲料重量,每周称量小鼠体重计算生长量,观察祛炎通脉功能饮料对小鼠的影响情况,具体结果见图1。
通过图1结果表明,祛炎通脉功能饮料组雌雄小鼠生长情况良好,发育状态和精神状态等生物学特性与对照组雌雄小鼠相比没有发现统计学差异。
(2)祛炎通脉原汁对大鼠灌胃安全性实验
取清洁级SD大鼠(川北医学院实验动物中心提供,合格证号:SYXK(川)2014-078)雌雄各40只分开,分别分4组(每组10只)用基础饲料(川北医学院实验动物中心提供)喂养,其中两组灌胃洁净水作对照、两组每日每只灌胃祛炎通脉原汁(实施例3)1ml,每周称量大鼠体重计算生长量,观察祛炎通脉原汁对大鼠生长情况的影响,具体结果见图2。
通过图2结果表明,祛炎通脉原汁组雌雄大鼠生长情况良好,发育状态和精神状态等生物学特性与对照组雌雄大鼠相比没有发现统计学差异。
二、祛炎通脉饮料功能性实验
(1)祛炎通脉功能饮料对小鼠急性肺炎保护作用实验
取五周龄KM小鼠(由川北医学院实验动物中心提供,合格证号:SYXK(川)2014-076)雌雄各半,建立急性肺炎模型,小鼠垂直固定,用移液枪吸取5mg/ml LPS溶液80ul,分3次滴鼻,待液体全部经鼻吸入后,保持垂直数秒,使其能均匀感染肺部,放于笼中让其自然苏醒,同时设置空白对照组,以等量的PH7.4 10mmol/L PBS 80ul分3次滴鼻。将KM小鼠72只随机分为6组:①空白对照组(12只)②模型组(12只)③实施例3中祛炎通脉功能饮料低计量组(4g/kg,12只)、中(8g/kg,12只)、高剂量(12g/kg,12只)组和④地塞米松组(2mg/kg,12只)。于造模前一周起,祛炎通脉功能饮料组小鼠每天定时灌胃给饮料一次,模型组和空白对照组灌服生理盐水(10ml/kg);地塞米松组于造模前1h腹腔注射,具体实验结果见表1。
表1实验各组小鼠血清TNF-α、IL-1β(pg/ml)差异
注:与空白对照组比较:*P<0.05,有显著差异,**P<0.01,有极显著差异;与模型组比较:△P<0.05,有显著差异,△△P<0.01,有极显著差异;与祛炎通脉功能饮料高剂量组比较□P<0.05,有显著差异□□P<0.01,有极显著差异。
通过表1实验结果表明,祛炎通脉功能饮料对小鼠血清炎症因子表达具有统计学意义,随着祛炎通脉功能饮料剂量的增加,小鼠血清炎症因子数量逐渐减少。
分别对空白对照组、模型组、祛炎通脉功能饮料组和地塞米松组的小鼠肺泡组织进行染色,具体染色结果见图3。
通过图3可以得知,空白对照组(K)小鼠肺泡结构完整,肺泡腔清晰,肺泡壁无增厚、无炎性细胞浸润,未见明显病理改变;模型组(M)小鼠肺泡腔内可见大量的炎性细胞浸润,肺泡壁明显增厚;地塞米松组(X)上述变化明显减轻,仅见少量炎性细胞浸润和局灶性肺泡间隔增厚;祛炎通脉功能饮料组局灶性肺泡壁增厚,可见炎细胞浸润,但较模型组有所改善。
(2)祛炎通脉功能饮料对大鼠慢性宫颈炎保护作用实验
取清洁级SD大鼠(川北医学院实验动物中心提供,合格证号:SYXK(川)2014-078),喂养适应一周后,将0.5ml玻璃注射器(用灌胃针头)轻轻插入大鼠阴道内约3~4mm处,向其中缓慢注入苯酚胶浆0.1ml,注入同时往外撤针,而后再向阴道穹隆部腔内注入胶浆0.2ml,并予自制医用无菌棉堵塞阴道口,接着将动物头低尾高体位倾斜放置5min后放回笼内,每隔2天注射1次,连续5次。造模成功表现为大鼠阴道有白色分泌物,阴道红肿,阴道内充血。将大鼠随机分为5组:模型组(M),祛炎通脉功能饮料低(4g/kg,12只)、中(8g/kg,12只)、高剂量(12g/kg,12只)组,地塞米松组(X)(4mg/kg,12只)。灌胃给药从第一次注射造模后第3天开始,模型组和空白对照组灌服生理盐水(10ml/kg),连续10天不断,统计酶联免疫检测前列腺素2(PGE2),具体结果见表2。
表2实验各组大鼠宫颈组织PGE2(pg/ml)差异
注:与空白对照组比较:*P<0.05,有显著差异,**P<0.01,有极显著差异;与模型组比较:△P<0.05,有显著差异,△△P<0.01,有极显著差异;与祛炎通脉功能饮料高剂量组比较□P<0.05,有显著差异□□P<0.01,有极显著差异。
通过表2结果表明,酶联免疫检测前列腺素2(PGE2)具有统计学意义,随着祛炎通脉功能饮料的剂量增加,宫颈组织内酶联免疫检测前列腺素2(PGE2)数量逐渐减少。
分别对空白对照组、模型组和地塞米松组的大鼠宫颈组织进行染色,具体染色结果见图4。
通过图4可以得知,与空白组比较,模型组宫颈组织鳞状上皮明显增生,黏膜下层毛细血管扩张充血,炎性细胞浸润明显,说明宫颈炎模型复制成功。与模型组比较,祛炎通脉功能饮料组病变有所减轻(J1、J2、J3),其中高剂量组炎细胞浸润程度最低。
(3)祛炎通脉功能饮料对LPS诱导的RAW264.7细胞炎症介质的影响实验RT-PCR法测定细胞中TNF-α、IL-6、IL-1β、iNOS和COX-2的mRNA表达水平,具体为:将对数生长的RAW264.7细胞消化收集起来,1000rpm,5min离心后,弃上清,加入适量培养基重悬细胞,计数调整细胞密度为3×105cells/mL,每孔2mL铺入6孔板中,每组三个复孔,放入37℃、5%二氧化碳培养箱中继续培养,培养适当时间待细胞贴壁后,弃去原培养基,空白孔和模型组每孔加入2mL完全培养基,实验孔加入50μg/mL中药提取液,避光加样,加样完成后继续放入37℃、5%二氧化碳培养箱中继续培养2h。药物保护作用2h后,取出孔板,模型组和各浓度药物组每孔加入终浓度为1μg/mL的LPS,空白组加入等量培养基,加样完成后继续放入37℃、5%二氧化碳培养箱中继续培养28h。
总RNA提取:
a.作用28h后,弃去原培养基,用PBS洗两遍细胞,每孔避光加入1mL Trizol,静置3-5min后用无酶枪头将细胞裂解液收集于专用1.5mL EP管中;
b.在上述EP管中,每管加入0.2mL氯仿,用漩涡混合器振荡混匀30s,室温静置3min;
c.4℃、12000g×20min离心后,转移500μL上层水相到新的无酶EP管中;
d.在上述EP管中,加入500μL异丙醇上下颠倒混匀,室温放置10min;
e.4℃、12000g×10min离心后,去掉上清,保留胶状沉淀,轻轻加入1mL 75%乙醇(RNase free水配制),缓缓颠倒清洗;
f.4℃、7500g×5min离心后,用枪头去掉上清,尽可能去除液体,保留胶状沉淀,室温干燥5-10min后,加入30μL RNase free水溶解。
g.若不立即操作,可置于-80℃冰箱中保存。
RNA质量检测:
a.RNA浓度和纯度检测:
取1μL体积的RNA待测样品,使用分光光度计测定RNA的A260、A280及A260/A280(Ratio,R)。根据A260的值能够计算出RNA的浓度,即一个单位等于40μg/mL的单链RNA。根据R值可判断RNA的纯度与质量,如果R值在1.8-2.2之间,则RNA质量较好;若R值<1.8说明有蛋白污染;R值>2.2说明RNA水解成核苷酸。
b.RNA电泳检测:
取8μL体积的RNA待测样品,加入2μL的5×RNA loading buffer,混合均匀,用浓度为1.2%的琼脂糖凝胶对RNA样品进行电泳,电泳条件为120V、30min,具体结果见图5。
通过图5结果看出,肉眼可见清晰的28S、18S、5S三条带,且28S的条带亮度约为18S的2倍,原始点样孔处没有条带残留,说明RNA的提取效果比较好。
逆转录反应:
a.将1μg上述总RNA为模板进行逆转录反应,合成cDNA,根据RNA浓度换算得到1μgRNA大致体积约为1μL,反应体系为20μL;
b.在0.5mL的PCR专用管中配制20μL反应体系;
c.先将RNA模板和RNase-free dH2O加在一起后,进行热变性(条件:65℃,5min,冰上1min),再加入其他组分在漩涡混合器上充分混匀,4000rpm,15s离心;
d.然后放入PCR扩增仪中,设置反应程序:30℃10min,42℃20min,99℃5min,4℃进行逆转录合成cDNA,若不立即操作,可放置于-80℃冰箱保存。
荧光定量PCR反应:
a.引物设计
b.RT-PCR反应体系
①将逆转录得到的cDNA模板稀释20倍(5μL cDNA+95μL RNase-free dH2O),备用;
②在RT-PCR专用八联管中依次加入如下体系:
③按照上述体系加样,内参孔需只将目标基因引物替换成内参基因引物,每个标本都要对应测内参基因,每组三个复孔,加样完成后在漩涡混合器上充分混匀,4000rpm,15s离心后上机;
④扩增条件:95℃预变性10min,95℃变性15s,60℃退火40s,共进行40个循环;反应完成后从60℃到95℃缓慢升温,每个循环上升0.5℃,整个过程连续采集荧光信号,产生熔解曲线。
熔解曲线分析:SYBR GreenⅠ染料法实时荧光定量PCR反应的特异性可通过对其产物的熔解曲线的分析来评价。PCR产物在熔解过程中,其荧光信号会发生变化,而PCR仪能够捕捉到这些变化并自动绘制出熔解曲线。在熔解反应过程中,DNA双链随着温度的升高而逐渐解开,与此同时荧光信号逐渐变低。当温度达到DNA溶熔温度(Tm)值作用时双链数量会发生骤降,荧光信号量也急速下降,由此可知Tm值,从而确定扩增产物的特异性。
⑤对小鼠RAW264.7细胞中TNF-α、IL-6、IL-1β、iNOS、COX-2基因的mRNA相对表达量的测定是通过相对定量分析得到,相对定量分析采用的是Ct值比较法,具体结果见表3-表7。
表3祛炎通脉功能饮料对LPS诱导RAW264.7细胞TNF-αmRNA表达(X±S,n=3)
表4祛炎通脉功能饮料对LPS诱导RAW264.7细胞IL-6mRNA表达(X±S,n=3)
表5祛炎通脉功能饮料对LPS诱导RAW264.7细胞中IL-1βmRNA表达(X±S,n=3)
表6祛炎通脉功能饮料对LPS诱导RAW264.7细胞中iNOS mRNA表达(X±S,n=3)
表7祛炎通脉功能饮料对LPS诱导RAW264.7细胞COX-2mRNA表达(X±S,n=3)
通过表3-表7的RT-PCR实验结果表明,与LPS对照组相比,原汁饮料能够显著降低RAW264.7细胞中炎症细胞因子TNF-α、IL-6、IL-1β和炎症介质iNOS、COX-2基因的表达水平。
四、祛炎通脉功能饮料祛炎机制实验
本实验中以Mus_musculus.GRCm38.p6为参考基因组,采用Illumina测序平台对空白对照组、LPS模型对照组、乙酰哈巴苷组和祛炎通脉功能饮料组进行测序,并对测序结果进行差异基因的筛选,利用差异基因功能富集等生物信息学方法,探讨祛炎通脉功能饮料对巨噬细胞产生的影响,再进一步实验验证效应靶点的调控方向,具体结果见图6。
通过图6表明,祛炎通脉功能饮料引起67个基因上调,95个基因下调。结合四组样本分析,取所有组别与LPS对照组的交集基因,共同的差异基因最终筛选出5个,在共同差异基因中,按照基因改变调控方向一致与统计意义最佳最终确定的目标分子为Prdx1(过氧化物还原酶1)与Slpi(分泌型白细胞肽酶抑制因子)。靶分子具有的生物功能有催化活性、抗氧化活性、分子伴侣、酶调节活性以及充当分子功能调节剂的作用。在基因与蛋白水平,Prdx1与Slpi表达趋势一致,在LPS诱导后均会引起两个分子含量的轻度升高,在祛炎通脉功能饮料处理后,两个分子含量大量增多,参与炎症反应调节。
用祛炎通脉功能饮料原汁处理后检测PRDX1、SLPI分子蛋白质水平的变化,以验证祛炎通脉功能饮料对PRDX1、SLPI蛋白水平的影响。具体结果见图7。
通过图7表明,蛋白质免疫印迹条带图和对应的基因表达匹配,说明蛋白质变化水平与基因变化水平一致。
综上所述,祛炎通脉功能饮料具有良好抗炎作用,PRDX1和SLPI是祛炎通脉功能饮料作用的共同效应分子。祛炎通脉功能饮料是通过促使炎症细胞大量释放PRDX1和SLPI来改善氧化应激,调节炎症相关蛋白酶催化功能而发挥抗炎作用。
本发明有效抽提了筋骨草、金钱草、三七的活性成分,饮料产品清香、微苦,具有祛炎通脉功效,环保健康,方便消费者携带和饮用,具有较好保健作用,适于在功能饮料行业内推广应用。
Claims (10)
1.一种祛炎通脉功能饮料的制备方法,其特征在于,包括以下步骤:
(1)将筋骨草、金钱草和三七混合,向其中加入体积浓度为55-70%的乙醇,于避光条件下浸提15-20天,收集浸提液;
(2)将步骤(1)中浸提液减压浓缩至无乙醇残留,得中药原液;
(3)将步骤(2)中的中药原液于75-85℃避光条件下干燥,得饮料原汁;
(4)向步骤(3)的饮料原汁中加水,制得。
2.如权利要求1所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(1)中筋骨草、金钱草和三七的质量比为7-10:0.1-1:0.1-2。
3.如权利要求1或2所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(1)中筋骨草、金钱草和三七的质量比为8-9:0.1-0.5:0.4-0.8。
4.如权利要求3所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(1)中筋骨草、金钱草和三七的质量比为9:0.3:0.7。
5.如权利要求1所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(1)中乙醇的体积浓度为60%。
6.如权利要求1所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(2)中减压浓缩时的压强为9-12KPa。
7.如权利要求1所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(3)中干燥至中药原液的体积减少50%后,停止。
8.如权利要求1所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(4)中饮料原汁与水的体积占比分别为10-30%和70-90%。
9.如权利要求1所述的祛炎通脉功能饮料的制备方法,其特征在于,步骤(4)中还加入蔗糖,饮料原汁、蔗糖和水的体积占比为10-30%、8-12%和62-82%。
10.权利要求1-9中任一项所述的方法制备得到的祛炎通脉功能饮料。
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