CN111728995B - 空子山核桃提取物在制备抗卵巢癌产品中的应用 - Google Patents
空子山核桃提取物在制备抗卵巢癌产品中的应用 Download PDFInfo
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Abstract
本发明公开一种空子山核桃提取物在制备抗卵巢癌产品中的应用，所述空子山核桃提取物由如下步骤制备得到：步骤(1)、选取竹竿敲落的山核桃，用清水浮选获得空子山核桃，立刻晒干；步骤(2)、将空子山核桃去壳得空子山核桃内容物，将其粉碎；步骤(3)、将空子山核桃内容物粉用甲醇水溶液提取，得到甲醇提取物；步骤(4)、将甲醇提取物过滤、干燥，即得到具有抗卵巢癌活性的空子山核桃提取物。本发明制得的空子山核桃提取物能抑制人卵巢癌细胞OVCAR‑3、A2780/CP70增殖，抑制血管生成，具有良好的抗肿瘤活性，可在制备抗肿瘤药物、保健品和食品中应用，从而为空子山核桃赋予了医用保健价值和经济价值。
Description
技术领域
本发明涉及植物有效成分提取、生物医药技术领域，具体涉及一种空子山核桃提取物在制备抗卵巢癌产品中的应用。
背景技术
与其他妇科恶性肿瘤相比，卵巢癌的发病较为隐匿。有报道，75％以上的卵巢癌患者在确诊时已为晚期(Sinha A,Ignatchenko V,Ignatchenko A,Mejia-Guerrero S,Kislinger T.In-depth proteomic analyses of ovarian cancer cell line exosomesreveals differential enrichment of functional categorise compared to the NCI60proteome.Biochem Biophys Res Commun,2014,445(4):694-701.)。每年全球有239,000例新病例和152,000例死亡(Stephanie L MD,PhD,Marsela B MSc,PhD,Amit M.Oza MD(Lon).Epithelial ovarian cancer:Evolution of management in the era ofprecision medicine[J].CA:A Cancer Journal for Clinicians,2019,69(4):280-304.)。在所有被诊断患有上皮性卵巢癌的妇女中，有三分之二以上的人会死于该病，这一事实在过去的三十年中并没有发生太大变化。目前手术、放射和化疗是最常用的治疗卵巢癌的方法。虽然绝大部分患者使用以铂类药物为主的化学治疗可以取得良好效果，但治疗后仍有60％的复发率(Ferrero A,Ditto A,Giorda G,Gadducci A,Greggi S,Daniele A,Fuso L,Panuccio E,Scaffa C,Raspagliesi F,Sismondi P1,Biglia N.Secondarycytoreductive surgery for isolated lymph node recurrence of epithelialovarian cancer:a multicenter study.Eur J Surg Oncol,2014,40(7):891-898.)。因而探寻新的治疗方法，减轻放、化疗的毒副作用，提高癌症的治疗效果，进而提高患者的生存质量具有重要意义。
山核桃(Carya cathayensis Sarg.)属胡桃科(Juglandaceae)山核桃属(CaryaNutt.)植物，是我国特有的经济林干果，主要分布在天目山等区域。由于山核桃种植在山区，土壤厚度，肥沃程度很不一样(马闪闪，赵科理，丁立忠，黄莎，蔡铃，赵伟明，叶正钱.临安市不同山核桃产区土壤肥力状况的差异性研究[J].浙江农林大学学报，2016，33(6)：953-960.)，因而山核桃成熟程度差异很大。另外，果实成熟期(7-8月)干旱也会造成果实发育迟缓。上述原因造成山核桃在采收过程中产生大量的空子，有的年份空子比率高达30％。目前，山核桃空子缺乏有效的利用手段，收获的空子也主要丢弃在环境中。
山核桃中起到抗氧化作用的部位绝大部分是山核桃的种皮，空子山核桃因为发育受阻或者因为受到病理性损害，而无法生成正常的山核桃果实，但空子山核桃中同样具有红褐色种皮。利用这些空子山核桃资源开发出应用潜力较高的天然抗氧化产物，变废为宝，提高山核桃产业的经济效益具有深远意义。目前对山核桃功能性研究较多，而空子山核桃涉及较少。至今尚未见到利用空子山核桃抗癌特别是卵巢癌的相关报道。
发明内容
本发明的目的是提供一种空子山核桃提取物在制备抗卵巢癌产品中的应用，以解决现有技术的不足。
本发明采用以下技术方案：
一种空子山核桃提取物在制备抗卵巢癌产品中的应用，所述空子山核桃提取物由如下步骤制备得到：
步骤(1)、选取竹竿敲落的山核桃，用清水浮选获得空子山核桃，立刻晒干；
步骤(2)、将步骤(1)得到的空子山核桃去壳得空子山核桃内容物，将其粉碎；
步骤(3)、将步骤(2)得到的空子山核桃内容物粉用甲醇水溶液提取，得到甲醇提取物；
步骤(4)、将步骤(3)得到的甲醇提取物过滤、干燥，即得到具有抗卵巢癌活性的空子山核桃提取物。
进一步地，所述产品为药物或保健品或食品。
进一步地，步骤(1)选取的空子山核桃晾晒1～2天。
进一步地，步骤(2)空子山核桃内容物粉碎后过60～80目筛。
进一步地，步骤(3)甲醇水溶液为70～85v/v％的甲醇水溶液。
进一步地，步骤(3)空子山核桃内容物粉和甲醇水溶液料液比为1g：10～12ml。
进一步地，步骤(3)提取的方式为超声波辅助提取，提取功率为200～240W，提取温度为20～25℃，提取时间为2～2.5h，提取次数为1～2次。
进一步地，步骤(4)采用离心的方式过滤杂质，转速为5000～6000r/min，时间为8～10min。
进一步地，步骤(4)离心过滤得到的滤液旋转蒸发之后再真空干燥。
进一步地，步骤(4)离心过滤得到的滤液于40～45℃旋转蒸发，蒸发至溶液即将蒸干，再于40～45℃真空干燥12～24h。
本发明的有益效果：
空子山核桃是山核桃种植过程中的副产物之一，由于果实发育不饱满而被山核桃种植农户废弃。本发明制得的空子山核桃提取物能抑制人卵巢癌细胞OVCAR-3、A2780/CP70增殖，抑制血管生成，具有良好的抗肿瘤活性，可在制备抗肿瘤药物、保健品和食品中应用，从而为空子山核桃赋予了医用保健价值和经济价值。
附图说明
图1是空子山核桃提取物对卵巢癌细胞OVCAR-3、A2780/CP70存活率的影响。
图2是空子山核桃提取物对卵巢癌细胞OVCAR-3、A2780/CP70凋亡率的影响。
图3是空子山核桃提取物对凋亡途径相关蛋白表达影响的蛋白印记图。
图4是空子山核桃提取物对凋亡途径相关蛋白：Bad表达的影响。
图5是空子山核桃提取物对凋亡途径相关蛋白：Bax表达的影响。
图6是空子山核桃提取物对凋亡途径相关蛋白：Puma表达的影响。
图7是空子山核桃提取物对细胞周期相关蛋白表达影响的蛋白印记图。
图8是空子山核桃提取物对细胞周期蛋白：P21表达的影响。
图9是空子山核桃提取物对细胞周期蛋白：cyclinB1表达的影响。
图10是空子山核桃提取物对OVCAR-3细胞中VEGF的影响。
具体实施方式
下面结合实施例和附图对本发明做更进一步地解释。下列实施例仅用于说明本发明，但并不用来限定本发明的实施范围。
实施例1：空子山核桃提取物的制备
(1)原料的选择和处理
选取用竹竿敲落的山核桃，去除外果皮，用清水浮选获得空子山核桃，立刻晒干，去壳(中果皮)后，人工挑选出正常未变质内容物，将内容物于40℃真空干燥48h后，用液氮处理研磨至粉碎，过60目筛，得到空子山核桃内容物粉，用密封袋密封，-80℃保存备用。
(2)空子山核桃提取物的制备
将空子山核桃内容粉用80v/v％的甲醇水溶液按料液比1g:10ml溶解，经超声波辅助提取，提取功率为240W，提取温度为25℃，提取时间为2h。在5000r/min下离心10min，重复提取1次，合并滤液，滤液于40℃下经旋转蒸发器浓缩至溶液即将蒸干，再于45℃下经真空干燥箱真空干燥12h，得到空子山核桃提取物。
实施例2：空子山核桃提取物对卵巢癌细胞增殖的抑制效果
各取100uL人卵巢癌细胞OVCAR-3和A2780/CP70，以10,000个细胞/孔的浓度接种于96孔板(培养基为RPMI-1640+10v/v％FBS)，并在37℃、5％CO2下培养16h以附着细胞，除去培养基，用PBS缓冲液(0.01M，pH 7.4)洗涤一次。用含有不同浓度空子山核桃提取物的培养基(培养基为RPMI-1640+10v/v％FBS)处理24h。然后除去培养基，用PBS缓冲液(0.01M，pH7.4)洗涤两次，加入100μL新鲜配置的AQueous One检测溶液，在37℃孵育1h，采用酶标仪在490nm下进行细胞活力分析。
实验采用AQueous One方法测定空子山核桃提取物对人卵巢癌细胞OVCAR-3和A2780/CP70抑制效果。如图1所示，随着提取物浓度的增加，卵巢癌细胞OVCAR-3和A2780/CP70的存活率明显下降；在OVCAR-3细胞中，在提取物(5μg/mL-40μg/mL)处理24h后，细胞存活率由88.3％下降到37.9％(p＜0.05)；同样的，在A2780/CP70细胞中，提取物(5μg/mL-40μg/mL)使细胞存活率从91.7％下降到36.7％(p＜0.05)。表明该提取物能显著性抑制人卵巢癌细胞OVCAR-3和A2780/CP70的增殖。
实施例3：空子山核桃提取物诱导卵巢癌细胞凋亡
空子山核桃提取物对细胞凋亡的影响使用Alexa Flour 488膜联蛋白(AnnexinV)细胞凋亡试剂盒(Invitrogen)测定。OVCAR-3和A2780/CP70细胞以106/皿的浓度接种在60mm培养皿中(培养基为RPMI-1640+10v/v％FBS)，在37℃、5％CO2下培养16h，去除培养基。然后加入含有不同浓度空子山核桃提取物的培养基(培养基为RPMI-1640+10v/v％FBS)在37℃、5％CO2下培养24h后，收集细胞，采用Annexin V/PI双染色法进行染色，染色后采用流式细胞仪进行分析。如图2所示，在OVCAR-3和A2780/CP70细胞中，当无空子山核桃提取物添加时，凋亡率分别为6.6％和8.1％；当用20μg/mL的空子山核桃提取物处理后，凋亡率分别增加到了10.0％和22.5％。结果表明：空子山核桃提取物能显著性促进卵巢癌细胞OVCAR-3和A2780/CP70的凋亡。
实施例4：空子山核桃提取物对卵巢癌细胞凋亡途径以及细胞周期蛋白的作用
将卵巢癌细胞OVCAR-3(106)接种在60mm的培养皿中(培养基为RPMI-1640+10v/v％FBS)，在37℃、5％CO2下培养过夜使细胞贴壁，去除培养基。然后加入含不同浓度空子山核桃提取物的培养基(培养基为RPMI-1640+10v/v％FBS)处理24h，去除培养基，细胞用PBS缓冲液(0.01M，pH 7.4)洗涤一次，然后在100μL哺乳动物蛋白提取试剂中裂解，该试剂包括1μL Halt蛋白酶，1μL磷酸酶抑制剂和2μL乙二胺四乙酸(EDTA)。细胞裂解物通过10％SDS-PAGE凝胶电泳分离，并使用Mini-Protean 3System(Bio-Rad)将蛋白质转移到硝酸纤维素膜上。将膜用含0.1v/v％Tween-20的5w/v％脱脂牛奶Tris缓冲液封闭1h(室温)。然后按照说明书进行一抗、二抗孵育。用TBST缓冲液洗涤后，添加Super Signal West PicoSubstrate(Pierce)显色剂进行显色。
本实验测定了几种与细胞凋亡途径相关蛋白(Puma、Bad、Bax)的表达。如图3-6所示，在提取物(5μg/mL-20μg/mL)处理后，蛋白Bad，Bax和Puma表达量均上升。Bad和Bax蛋白是细胞主要的促凋亡蛋白。因此从结果可以看出该提取物能够通过上调Bad，Bax，Puma蛋白的表达来诱导卵巢癌细胞OVCAR-3凋亡。
既往的研究表明CyclinB1是G2/M周期检测点的关键调控蛋白，而p21是CyclinB1-CDK1复合物的抑制因子。在研究中，从图7-9可以看出，该提取物能够上调p21蛋白的表达并抑制CyclinB1蛋白的表达。表明该提取物可能能够通过上调p21蛋白的表达并抑制CyclinB1蛋白的表达和活性来阻滞卵巢癌细胞OVCAR-3的细胞周期。
实施例5：空子山核桃提取物对卵巢癌细胞血管生成的影响
细胞培养上清中的VEGF水平通过Quantikine人VEGF免疫测定试剂盒(R&DSystemems)进行分析。将OVCAR-3细胞(2×104/孔)接种到96孔板中(培养基为PRMI-1640+10v/v％FBS)并过夜孵育使细胞贴壁。除去培养基，采用不同浓度空子山核桃提取物(培养基为RPMI-1640+10v/v％FBS)，继续培养24h。收集培养基上清液。按照制造商的说明测量VEGF的量，将其标准化为总蛋白水平，并表示为未处理对照的百分比。如图10所示，在空子山核桃提取物(5μg/mL-40μg/mL)处理后，OVCAR-3细胞中的VEGF的蛋白水平为空白组的57.9％和11.7％，这说明空子山核桃提取物能够明显降低OVCAR-3细胞中VEGF的表达，从而显示出抑制卵巢癌细胞血管形成的能力(体外)。
Claims (9)
1.一种空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，所述产品为药物；
所述空子山核桃提取物由如下步骤制备得到：
步骤（1）、选取竹竿敲落的山核桃，用清水浮选获得空子山核桃，立刻晒干；
步骤（2）、将步骤（1）得到的空子山核桃去壳得空子山核桃内容物，将其粉碎；
步骤（3）、将步骤（2）得到的空子山核桃内容物粉用甲醇水溶液提取，得到甲醇提取物；
步骤（4）、将步骤（3）得到的甲醇提取物过滤、干燥，即得到具有抗卵巢癌活性的空子山核桃提取物。
2.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（1）选取的空子山核桃晾晒1~2天。
3.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（2）空子山核桃内容物粉碎后过60～80目筛。
4.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（3）甲醇水溶液为70～85v/v%的甲醇水溶液。
5.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（3）空子山核桃内容物粉和甲醇水溶液料液比为1g：10～12ml。
6.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（3）提取的方式为超声波辅助提取，提取功率为200～240W，提取温度为20～25℃，提取时间为2～2.5h，提取次数为1～2次。
7.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（4）采用离心的方式过滤杂质，转速为5000～6000r/min，时间为8～10min。
8.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（4）离心过滤得到的滤液旋转蒸发之后再真空干燥。
9.根据权利要求1所述的空子山核桃提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（4）离心过滤得到的滤液于40～45℃旋转蒸发，蒸发至溶液即将蒸干，再于40～45℃真空干燥12～24h。
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