CN116947943A - 一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法及其应用 - Google Patents
一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法及其应用,属于生物技术领域。该发明所述蓝莓花色苷采用超高压辅助提取法提取,大孔树脂结合超滤膜纯化,再经减压浓缩、真空冷冻干燥,制备而成。所述蓝莓花色苷包括13种花色苷单体,对肝癌细胞增殖有显著抑制增殖,能够通过上调细胞中Beclin‑1蛋白和LC3 II蛋白表达,下调p62蛋白表达而诱导肝癌细胞自噬,从而发挥抗肿瘤作用。本发明蓝莓花色苷可应用于制备抗肝癌的药物或保健食品,为通过食疗防治肝癌提供新的技术思路。
Description
技术领域
本发明属于生物技术领域,涉及一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法及其应用。
背景技术
肝癌是危害人类健康的重要疾病之一,发病率居恶性肿瘤第4位、病死率居第3位。目前手术切除结合化疗手段是治疗肝癌的首选方案。化疗药物毒副作用大,在杀伤肿瘤细胞的同时也会对正常细胞产生较大的杀伤性,严重影响患者生命安全和生存质量,致使术后复发率高达60%以上。而通过食疗抑制肝癌细胞增殖,从而达到防治肝癌的良好效果已引起国内外学者的广泛关注。自噬是癌细胞程序性自主死亡的主要途径之一,因此,开发出疗效好,安全性高、具有高自噬诱导率的肝癌辅助治疗药物具有很重要的意义。
蓝莓属杜鹃花科越橘属,富含花青素、黄烷醇、酚酸、维生素C等生物活性物质,具有很高的营养保健功能,被联合国粮农组织(FAO)列为人类五大健康食品之一,开发前景极其广阔。蓝莓渣是蓝莓加工副产物,目前只有20%的利用率,资源浪费非常严重。因此,蓝莓渣花色苷的高效制备及其在营养健康产业中的应用,有利于实现蓝莓的高值化利用,推进蓝莓产业发展、改善国民身体素质。
已有研究表明,蓝莓花青素是迄今为止所发现的最有效的天然水溶性自由基清除剂,是蓝莓生物保健功能的主要物质基础,长期食用具有美容养颜、抗氧化、抗炎、抗衰老、保护视网膜、保护心血管、延缓衰老、增强免疫力等多种药理保健功效,在国内外市场非常畅销。但是迄今为止,市场上尚无蓝莓花色苷抗肝癌药物或保健食品,有关蓝莓花色苷通过诱导肝癌细胞自噬发挥抗癌作用的文献报道也未见报道。
发明内容
针对现有技术的不足,本发明目的在于提供一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,该方法制备的蓝莓花色苷能诱导肝癌细胞自噬。
本发明另一个目的在于提供一种诱导肝癌细胞自噬的蓝莓花色苷在制备抗肝癌的药物或保健食品中的应用。
为了实现上述目的,本发明的技术方案如下:
一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,包括如下步骤:
(1)将蓝莓渣干粉与适当体积的酸化乙醇溶液混合,料液比为1∶20~1∶30(g/mL),装入聚乙烯袋中,封口后置于超高压设备中处理,然后抽滤,收集滤液,滤渣再重复处理一次;合并两次滤液,通过旋转蒸发仪减压浓缩去除乙醇,浓缩至总体积的0.1~0.3倍,得蓝莓花色苷提取液。
(2)用大孔树脂吸附蓝莓花色苷提取液,然后采用一定浓度乙醇进行洗脱,收集洗脱液,得蓝莓花色苷初纯液。
(3)将蓝莓花色苷初纯液通过超滤设备进一步纯化,采用的超滤膜的截留分子量为30KD,超滤压力为1.5MPa,得超滤液。
(4)将超滤液通过旋转蒸发仪减压浓缩,再经冷冻干燥后即得蓝莓花色苷。
步骤(1)中,所述酸化乙醇的浓度为50~60%,pH为3,处理压力300~400MPa,保压时间6~12min。
步骤(2)中,所述大孔树脂为AB-8树脂或NKA-9树脂;所述大孔树脂吸附的具体过程为:将蓝莓花色苷提取液以1.5mL/min的流速注入大孔树脂中,树脂吸附平衡后,用去离子水清洗至溶液无色澄清,然后用80%乙醇溶液以2.0mL/min的流速洗脱1h。
本发明所述蓝莓花色苷是由13种不同的花色苷单体组成的混合产物,具体包括:飞燕草色素-3-半乳糖苷、飞燕草色素-3-葡萄糖苷、矢车菊素-3-半乳糖苷、飞燕草色素-3-阿拉伯糖苷、矢车菊素-3-葡萄糖苷、牵牛花色素-3-半乳糖苷、牵牛花色素-3-葡萄糖苷、芍药素-3-半乳糖苷、牵牛花色素-3-阿拉伯糖苷、芍药素-3-葡萄糖苷、锦葵色素-3-半乳糖苷、锦葵色素-3-葡萄糖苷、锦葵色素-3-阿拉伯糖苷。
本发明所述的蓝莓花色苷的纯度≥52.93%。
一种诱导肝癌细胞自噬的蓝莓花色苷的应用,可应用于制备抗肝癌的药物或保健食品中。
一种诱导肝癌细胞自噬的蓝莓花色苷的应用,所述蓝莓花色苷通过上调Beclin-1蛋白和LC3 II蛋白表达,下调p62蛋白表达而诱导肝癌细胞自噬发挥抗肿瘤作用。
相对于现有技术,本发明的优点如下:
1)本发明采用蓝莓渣为原料制备一种诱导肝癌细胞自噬的蓝莓花色苷,属于废弃物利用,极大的提高了蓝莓资源的综合利用率。
2)本发明蓝莓花色苷在制备时,采用的超压力辅助提取技术属于低温提取技术,不仅显著提高了花色苷提取效率、还能有效解决普通溶剂提取、微波提取、超声提取等技术造成的花色苷降解、功能与活性丧失等技术缺陷,具有极大的商业开发价值。
3)本发明制备的蓝莓花色苷可显著抑制肝癌细胞增殖,诱导肝癌细胞自噬,将此作为药物或保健食品,在取代或部分取代化疗药物具有潜在价值,可显著降低化学药物对肝癌患者造成的毒副作用,提高术后病人的生存质量。
附图说明
图1为提取压力对蓝莓花色苷得率的影响
图2为提取时间对蓝莓花色苷得率的影响
图3为乙醇浓度对蓝莓花色苷得率的影响
图4为蓝莓花色苷HPLC分析图
图5为蓝莓花色苷对肝癌细胞HepG2生长的抑制作用
图6为蓝莓花色苷对肝癌细胞自噬泡形成的影响。其中A为对照组,B为50μg/mL蓝莓花色苷处理组,C为100μg/mL蓝莓花色苷处理组,C为200μg/mL蓝莓花色苷处理组。
图7为蓝莓花色苷对细胞自噬相关蛋白的影响
具体实施方式
下面详细说明本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。
以下实施例用于说明本发明,但并不限定于本发明。若无特别说明,下述实施例中所用技术方法均为常规方法;若无特别说明,下述实施例中所用的实验材料,均为常规实验材料。
实施例1蓝莓花色苷的制备方法
本发明所述蓝莓花色苷的制备方法,包括如下步骤:
(1)将蓝莓渣干粉与pH为3、浓度为50~60%的酸化乙醇溶液按照为1∶20~1∶30(g/mL)料液比混合,装入聚乙烯袋中,封口后置于超高压设备中(HPP600MPa,包头科发高压科技公司)处理,抽滤,收集滤液,滤渣再重复处理一次;合并两次滤液,通过旋转蒸发仪(D-RE-600A型,上海亚荣仪器厂)于50℃条件下减压浓缩去除乙醇,浓缩至总体积的0.1~0.3倍,得蓝莓花色苷提取液。
以花色苷提取率为指标,设计单因素试验,研究不同酸化乙醇浓度(40%、50%、60%、70%、80%)、不同处理压力(100、200、300、400、500MPa)、不同提取时间(3、6、9、12、15min)对花色苷得率的影响,确定适宜的提取条件。
根据图1~图3单因素试验结果,再结合经济成本,确定蓝莓花色苷的超高压提取条件为:酸化乙醇的浓度为50~60%,pH为3,处理压力为300~400MPa,保压时间为6~12min。
(2)采用AB-8树脂或NKA-9大孔树脂,将蓝莓花色苷提取液以1.5mL/min的流速注入大孔树脂中,树脂吸附平衡后,用去离子水清洗至溶液无色澄清,然后用80%乙醇溶液以2.0mL/min的流速洗脱1h。收集洗脱液,得蓝莓花色苷初纯液。
为了确定适宜的大孔树脂,以树脂的静态吸附率和解析率为指标,分别考察NDS-17、HPD-300、NKA-9、HPD-400、AB-8大孔树脂对蓝莓花色苷纯化的影响。根据表1试验结果,确定吸附率和解析率都比较高的AB-8树脂或NKA-9大孔树脂用于蓝莓花色苷的初步纯化。
表1五种树脂的静态吸附率和解吸率
注:a-d表示不同树脂之间显著性差异(p<0.05)。
(3)将蓝莓花色苷初纯液通过超滤设备(有机膜分离试验机BONA-GM-18,山东博纳集团)进一步纯化,采用的超滤膜的截留分子量为30KD,超滤压力为1.5MPa,得超滤液。
(4)将超滤液通过旋转蒸发仪(D-RE-600A型,上海亚荣仪器厂),于50℃条件下减压浓缩,再经冷冻干燥后即得蓝莓花色苷。
采用Agilent 1200高效液相色谱仪,配用反相色谱柱Eclipse XDB-C18色谱柱(250mm×4.6mm,5μm)检测本发明蓝莓花色苷的组成成分。流动相包括A(含1%磷酸的超纯水)和B(100%乙腈)。梯度洗脱如下:5%流动相B(0~5min),5%~10%流动相B(5~15min),10%流动相B(15~25min),10%~12%流动相B(25~35min),12%~15%流动相B(35~50min),15%~18%流动相B(50~60min),18%~25%流动相B(60~80min),25%~30%流动相B(80~90min)。流速为0.6mL/min;检测波长520nm;进样量为10μL;柱温25℃。结果见图4和表2。
表2蓝莓花色苷HPLC分析结果
由表1和图4可知,制备的蓝莓花色苷是由13种不同的花色苷单体组成的混合产物。分别为飞燕草色素-3-半乳糖苷、飞燕草色素-3-葡萄糖苷、矢车菊素-3-半乳糖苷、飞燕草色素-3-阿拉伯糖苷、矢车菊素-3-葡萄糖苷、牵牛花色素-3-半乳糖苷、牵牛花色素-3-葡萄糖苷、芍药素-3-半乳糖苷、牵牛花色素-3-阿拉伯糖苷、芍药素-3-葡萄糖苷、锦葵色素-3-半乳糖苷、锦葵色素-3-葡萄糖苷、锦葵色素-3-阿拉伯糖苷。其中锦葵色素含量最高。
蓝莓花色苷的纯度≥52.93%。
实施例2蓝莓花色苷对肝癌细胞(HepG2)增值的抑制作用
取实施例1制备的蓝莓花色苷,分别将其溶于10%胎牛血清的DMEM培养基(Gbico公司产品)中,使蓝莓花色苷在培养基中的浓度分别为50、100、150、200、250、300μg/mL,经水系0.22μm滤器过滤后,备用。
肝癌细胞HepG2细胞株(购自美国Solarbio公司),用含10%胎牛血清的DMEM培养基于37℃、5%CO2、95%饱和湿度条件的培养箱中培养至对数生长期,胰酶消化后制成单细胞悬液,调整其密度为5×104/mL,接种于96孔板,置于培养箱中培养24h,待细胞贴壁后换无血清培养基继续孵育12h后,弃去上清;分别加入配制好的含不同浓度的蓝莓花色苷溶液的培养基200μL,并设置等体积只加入含10%胎牛血清的DMEM培养基的空白对照组和调零组(无细胞,只加入含10%胎牛血清的培养基),分别培养24h、48h、72h。结束前4h每孔加入5mg/mL MTT溶液20uL,继续培养4h后吸去上清,每孔加入150uL二甲基亚砜(DMSO),避光振荡10min,使结晶物充分溶解,用酶标仪在490nm下测定各孔吸光值(OD值)。并按下列公式计算抑制率:抑制率(%)=[1-(实验OD值-调零OD值)/(对照组OD值-调零OD值)]×100%。实验重复3次。实验结果见图5。
根据图5所示MTT检测结果显示:用含50~300μg/mL蓝莓花色苷的DMEM培养基溶液培养细胞24h时,细胞的生长抑制率达13.98%~71.26%,培养48h时细胞的生长抑制率达14.05%~81.27%,培养72h时细胞的生长抑制率达21.91%~87.49%。表明蓝莓花色苷能明显抑制肝癌细胞生长,其抑制作用随蓝莓花色苷浓度的提高和时间的延长而增强。
实施例3蓝莓花色苷通过诱导肝癌细胞自噬发挥抗癌活性
1.吖啶橙染色检测肝癌细胞的自噬
吖啶橙渗透入细胞内,结合胞核中双链DNA和胞质呈现出绿色荧光。自噬过程中形成的酸性膜泡,使得吖啶橙在低pH环境下与自噬溶酶体结合呈现出红色荧光,通过红色荧光的数量,可判断细胞自噬发生的程度。在6孔板中的放置细胞爬片,用培养液对肝癌细胞HepG2进行适当稀释,加入6孔板中使铺板细胞密度为1×105个/孔。分别用含不同浓度(终浓度为0、50、100、200μg/mL)的花色苷培养液培养细胞。培养24h后,加入1mL吖啶橙试剂(2μg/mL),于暗处反应15min,在荧光显微镜(IX53,日本Olympus公司)下观察HepG2细胞。结果见图6。
由图6可见,吖啶橙染色细胞后,可见点状橙红色荧光颗粒散布于细胞核周围,与对照组相比,蓝莓花色苷浓度越高,点状橙红色荧光颗粒越多,提示蓝莓花色苷能显著诱导肝癌细胞自噬。
2.Western Blot法检测自噬相关蛋白的表达
用含有不同浓度(0、50、100、200μg/mL)蓝莓花色苷的培养基培养HepG2细胞24h后,在冰上裂解细胞,收集裂解液,按照BCA蛋白定量试剂盒检测步骤对蛋白质进行定量。通过电泳仪(Mini protean 3 cell电泳仪,美国Bio-Rad公司)用12%SDS-PAGE分离蛋白样品,每孔上样量为15μg蛋白,将SDS-PAGE胶上的蛋白质通过电转仪(TE77XP电泳仪,美国Hoefer公司)转移到PVDF膜上,5%脱脂奶粉封闭1h,用TBST洗涤3次,每次洗涤时间为5min,然后将PVDF膜分别浸泡在稀释好的兔抗Beclin-1、LC3I、LC3II(abcam生物科技有限公司)、p62(博奥森生物科技有限公司)中,并4℃孵育过夜。次日,用TBST洗涤PVDF膜3次,然后用羊抗兔二抗(jackson公司)孵育1h,再用TBST洗涤3次。用ECL化学发光液显色,用化学发光凝胶成像系统(Tanon-5200型化学发光成像系统,上海天能科技有限公司)拍照。结果见图7。
如图7所示,蓝莓花色苷对HepG2细胞自噬水平均存在显著影响。与对照组相比,蓝莓花色苷处理组的Beclin-1和LC3-II蛋白表达水平显著提高,p62蛋白显著降低(p<0.05)。提示蓝莓花色苷能通过自噬杀伤肝癌细胞。
综上所述,实验结果证实了蓝莓花色苷上调Beclin-1蛋白和LC3 II蛋白表达,下调p62蛋白表达,能通过诱导肝癌细胞自噬发挥抗肿瘤作用,可应用于制备抗肝癌的药物或保健食品中。
Claims (8)
1.一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,其特征在于:包括如下步骤:
(1)将蓝莓渣干粉与适当体积的酸化乙醇溶液混合,料液比为1∶20~1∶30(g/mL),装入聚乙烯袋中,封口后置于超高压设备中处理,抽滤,收集滤液,滤渣再重复处理一次;合并两次滤液,通过减压浓缩去除乙醇,浓缩至总体积的0.1~0.3倍,得蓝莓花色苷提取液。
(2)用大孔树脂吸附蓝莓花色苷提取液,然后采用一定浓度乙醇进行洗脱,收集洗脱液,得蓝莓花色苷初纯液。
(3)将蓝莓花色苷初纯液通过超滤设备进一步纯化,采用的超滤膜的截留分子量为30KD,超滤压力为1.5MPa,得超滤液;
(4)将超滤液减压浓缩,再经冷冻干燥后即得蓝莓花色苷。
2.根据权利要求1所述的一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,其特征在于,步骤(1)中,所述酸化乙醇的浓度为50~60%,pH为3,处理压力300~400MPa,保压时间6~12min。
3.根据权利要求1所述的一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,其特征在于,步骤(2)中,所述大孔树脂为AB-8树脂或NKA-9树脂;所述大孔树脂吸附的具体过程为:将蓝莓花色苷提取液以1.5mL/min的流速注入大孔树脂中,树脂吸附平衡后,用去离子水清洗,然后用80%乙醇溶液以2.0mL/min的流速洗脱1h。
4.根据权利要求1所述的一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,其特征在于,所述蓝莓花色苷是由13种不同的花色苷单体组成的混合产物。
5.根据权利要求1所述的一种诱导肝癌细胞自噬的蓝莓花色苷的制备方法,其特征在于:所述蓝莓花色苷的纯度≥52.93%。
6.权利要求1-5所述的一种诱导肝癌细胞自噬的蓝莓花色苷的应用。
7.根据权利要求6所述的应用,其特征在于,所述蓝莓花色苷在制备抗肝癌的药物或保健食品中的应用。
8.根据权利要求7所述的应用,其特征在于,所述蓝莓花色苷通过上调Beclin-1蛋白和LC3 II蛋白表达,下调p62蛋白表达而诱导肝癌细胞自噬发挥抗肿瘤作用。
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