CN111759883B - 山核桃内果皮提取物在制备抗卵巢癌产品中的应用 - Google Patents
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Abstract
本发明公开了一种山核桃内果皮提取物在制备抗卵巢癌产品中的应用，山核桃内果皮提取物由如下步骤制备得到：步骤(1)、选取充分成熟、外表光滑无裂缝且无异味的山核桃，将山核桃去壳、剥离山核桃内果皮；步骤(2)、将山核桃内果皮粉碎，得到山核桃内果皮粉；步骤(3)、将山核桃内果皮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山核桃内果皮提取物。本发明提取物表现出了对人卵巢癌细胞：OVCAR‑3更高的选择毒性，具有良好的抗癌活性。可以预期发展为抗卵巢癌产品，为山核桃赋予了新的开发和利用价值，对推动山核桃产业和医药工业的发展具有积极作用。
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，具体涉及一种山核桃内果皮提取物在制备抗卵巢癌产品中的应用。
背景技术
卵巢癌是发病率位于第二的女性生殖恶性肿瘤，其病死率在妇科肿瘤中居于首位。在全球范围内，每年约有23万人被确诊为卵巢癌，每年造成15万人死亡(S.Lheureux,M.Braunstein,A.M.OzaEpithelial ovarian cancer:evolution of management in theera of precision medicine，CA Cancer J.Clin.,69(2019),pp.280-304.)。目前，卵巢癌治疗主要采用手术治疗和化疗为主，效果有限，患者5年和10年生存率仅为32％和15％(KimJ,Park EY,Kim O,Schilder JM,Coffey DM,Cho CH,et al.Cell origins of high-gradeserous ovarian cancer.Cancers 2018；10(11):pii:E433.)。原因在于虽然绝大部分卵巢癌患者初期使用以铂类药物为主的化学治疗可以取得良好效果，但后期往往会由于耐药性而复发(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.)。因而筛选新的具有活性的化疗药物，尤其是低毒且疗效好的抗肿瘤生物活性物质，对保护和提高肿瘤患者免疫力，减轻放化疗的毒副作用，具有重要意义。
山核桃(Carya cathayensis Sarg.)属胡桃科(Juglandaceae)山核桃属(CaryaNutt.)植物。全国每年产量2万余吨，浙、皖两省分布较广，浙江山核桃主产于浙皖交界天目山区。山核桃果皮由外果皮、中果皮和内果皮三层结构组成。山核桃仁的果肉上会覆盖一层红褐色的薄皮，这层薄皮就是山核桃的内果皮。目前对山核桃外果皮、中果皮功能性研究较多，而内果皮涉及较少。至今尚未见到利用山核桃内果皮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山核桃内果皮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：
一种山核桃内果皮提取物在制备抗卵巢癌产品中的应用，所述山核桃内果皮提取物由如下步骤制备得到：
步骤(1)、选取充分成熟、外表光滑无裂缝且无异味的山核桃，将山核桃去壳、剥离山核桃内果皮；
步骤(2)、将步骤(1)得到的山核桃内果皮粉碎，得到山核桃内果皮粉；
步骤(3)、将步骤(2)得到的山核桃内果皮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山核桃内果皮提取物。
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)选取的山核桃使用20～50℃的温水浸泡30-40min后再去壳。
进一步地，步骤(2)山核桃内果皮粉碎后过60～80目筛。
进一步地，步骤(3)甲醇水溶液为80～85v/v％的甲醇水溶液。
进一步地，步骤(3)山核桃内果皮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：
本发明制得的山核桃内果皮提取物表现出了对人卵巢癌细胞OVCAR-3更高的选择毒性，具有良好的抗癌活性，可以预期发展为抗卵巢癌产品，为山核桃赋予了新的开发和利用价值，对推动山核桃产业和医药工业的发展具有积极作用。
附图说明
图1是山核桃内果皮提取物对卵巢癌细胞OVCAR-3及卵巢正常细胞IOSE364存活率的影响。
图2是山核桃内果皮提取物对卵巢癌细胞OVCAR-3及卵巢正常细胞IOSE364凋亡率的影响。
图3是山核桃内果皮提取物对卵巢癌细胞OVCAR-3凋亡流式细胞仪分析结果图。
图4是山核桃内果皮提取物对卵巢正常细胞IOSE364凋亡流式细胞仪分析结果图。
图5是山核桃内果皮提取物对OVCAR-3细胞caspase-3/7酶活性的影响。
图6是山核桃内果皮提取物对内源性途径相关蛋白表达影响的蛋白印记图。
图7是山核桃内果皮提取物对内源性途径相关蛋白：Puma表达的影响。
图8是山核桃内果皮提取物对内源性途径相关蛋白：Bax表达的影响。
图9是山核桃内果皮提取物对内源性途径相关蛋白：Bad表达的影响。
图10是山核桃内果皮提取物对内源性途径相关蛋白：cleaved PARP表达的影响。
图11是山核桃内果皮提取物对p53蛋白表达的影响。
图12是山核桃内果皮提取物对OVCAR-3细胞中VEGF的影响。
图13是山核桃内果皮提取物对HIF-1α蛋白表达影响的蛋白印记图。
图14是山核桃内果皮提取物对HIF-1α蛋白表达的影响。
图15是山核桃内果皮提取物对HIF-1α-VEGF通路抑制VEGF分泌的影响。
图16是山核桃内果皮提取物对PTEN途径相关蛋白表达影响的蛋白印记图。
图17是山核桃内果皮提取物对PTEN途径相关蛋白：PTEN表达的影响。
图18是山核桃内果皮提取物对PTEN途径相关蛋白：NFKB表达的影响。
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：山核桃内果皮提取物的制备
(1)原料的选择和处理
选取充分成熟、外表光滑无裂缝且无异味的临安本地山核桃，将山核桃用40℃左右的温水浸泡30min，目的是使种子和种皮能够更方便的剥离。去壳，使果肉与果壳(外果皮和中果皮)分离，得到山核桃仁后，再人工剥离种子与种皮，得到的种皮即为山核桃的内果皮，经液氮粉碎，过60目筛，得到山核桃内果皮粉，用密封袋密封，-80℃保存备用。
(2)山核桃内果皮提取物的制备
将山核桃内果皮粉用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，得到山核桃内果皮提取物。
实施例2：山核桃内果皮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和卵巢正常细胞IOSE 364，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。用含有不同浓度山核桃内果皮提取物的培养基(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方法测定山核桃内果皮提取物对人卵巢癌细胞OVCAR-3和卵巢正常细胞IOSE 364抑制效果。如图1所示，在OVCAR-3细胞中，在提取物(5μg/mL-40μg/mL)处理24h后，细胞存活率从87.6％下降到4.7％(p＜0.05)，IC50值为10.33±0.166μg/mL；而在IOSE 364细胞中，在40μg/mL时，卵巢正常细胞IOSE 364存活率为62.4％，远大于OVCAR-3细胞的存活率。这说明山核桃内果皮提取物对卵巢癌细胞具有良好的抑制效果，而对卵巢正常细胞具有较小的毒性，表明山核桃内果皮提取物对癌细胞的抑制作用具有较好的选择性。
实施例3：山核桃内果皮提取物通过p53依赖的内源性途径诱导卵巢癌细胞凋亡
使用Alexa Flour 488膜联蛋白(AnnexinV)细胞凋亡试剂盒(Invitrogen)检测山核桃内果皮提取物对卵巢癌细胞凋亡的影响。将OVCAR-3和IOSE 364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山核桃内果皮提取物的培养基(培养基为RPMI-1640+10v/v％FBS)在37℃、5％CO2下培养24h，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-图4所示，山核桃内果皮提取物治疗后OVCAR-3细胞凋亡的增加呈剂量依赖性。当用20μg/mL的山核桃内果皮提取物处理后，细胞凋亡率为44.21％。与上述细胞活力结果相似，卵巢正常细胞IOSE 364也没有明显的凋亡。这进一步证实了山核桃内果皮提取物对卵巢癌细胞的选择性抑制作用。
为了验证山核桃内果皮提取物对卵巢癌细胞的促凋亡作用，对凋亡执行蛋白Caspase-3/7酶活进行检测。将OVCAR-3细胞(104)接种在96孔板中(培养基为RPMI-1640+10v/v％FBS)，在37℃、5％CO2下培养过夜。然后更换含不同浓度山核桃内果皮提取物的培养基(培养基为RPMI-1640+10v/v％FBS)，在37℃、5％CO2下孵育4h。然后使用Caspase-Glo3/7试剂盒测定细胞的Capase-3/7酶活性。酶活性通过蛋白质浓度校准，并表示为空白组的百分比。
将卵巢癌细胞OVCAR-3(106)接种在60mm培养皿中(培养基为RPMI-1640+10v/v％FBS)，在37℃、5％CO2下过夜孵育使细胞贴壁，去除培养基。然后加入含不同浓度山核桃内果皮提取物的培养基(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SuperSignal West PicoSubstrate(Pierce)显色剂进行显色。
如图5-图10所示，与对照组相比，用20μg/mL山核桃内果皮提取物处理24h后，OVCAR-3的caspase-3/7酶活性最大增加到3.57倍，而且发现BAX(1.73倍)、BAD(1.53倍)、cleaved PARP(1.5倍)、Puma(2.9倍)等促凋亡蛋白表达上调。这些促凋亡蛋白与线粒体凋亡途径有关。因此，线粒体凋亡途径可能是山核桃内果皮提取物诱导细胞凋亡的主要原因。另外，我们还发现山核桃内果皮提取物会导致p53基因表达水平上调(如图11所示)，因而我们推测山核桃内果皮提取物通过p53介导的内源性凋亡通路来诱导卵巢癌细胞的凋亡。
实施例4：山核桃内果皮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RPMI-1640+10v/v％FBS)并过夜孵育使细胞贴壁。除去培养基，采用不同浓度山核桃内果皮提取物(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12所示，在山核桃内果皮提取物(5μg/mL-40μg/mL)处理后，OVCAR-3癌细胞分泌的VEGF水平分别被抑制到57.12、36.66、22.55和7.87％。结果表明山核桃内果皮提取物能够明显降低OVCAR-3细胞中VEGF的表达。
为明确山核桃内果皮提取物抑制VEGF分泌的分子机理，我们采用Western Blot法对影响VEGF分泌的关键转录因子HIF-1α进行分析，如图13和图14所示，结果发现山核桃内果皮提取物处理显著抑制HIF-1α的表达。与对照组相比，20μg/mL山核桃内果皮提取物时HIF-1α的表达降低了65.8％。为对上述分子通路进行确认，将OVCAR-3癌细胞(1×104个细胞/孔)接种到96孔板上(培养基为RPMI-1640+10v/v％FBS)，过夜孵育。用0.6μL的jetPRIME试剂(VWR，West Chester，Pennsylvania，USA)转染VEGF荧光素酶报告基因(0.05μg)和HIF-1α(0、0.0625和0.125μg)或SR-a(作为载体)质粒，培养细胞4h。除去培养基，采用10μg/mL山核桃内果皮提取物处理16h(以DMSO作为对照)，采用荧光酶标仪分析荧光强度。如图15所示，山核桃内果皮提取物导致的VEGF分泌水平下调能够被转录的HIF-1α质粒逆转，这说明山核桃内果皮提取物导致的卵巢癌细胞分泌VEGF水平下降与降低HIF-1α转录因子的表达直接相关。
如图16-图18所示，山核桃内果皮提取物可以引起PTEN基因表达上调和NFKB的表达下调。与对照组相比，10μg/mL山核桃内果皮提取物时PTEN的表达增加了75.1％，20μg/mL山核桃内果皮提取物时NFKB的表达降低了65.8％。因为PTEN与NFKB基因均与肿瘤细胞凋亡和抑制血管生成作用相关，因此，我们推断山核桃内果皮提取物诱导细胞凋亡和抗血管生成作用可能部分归因于提取物对PTEN的上调和NFKB的表达下调。
Claims (9)
1.一种山核桃内果皮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；所述山核桃内果皮提取物由如下步骤制备得到：
步骤（1）、选取充分成熟、外表光滑无裂缝且无异味的山核桃，将山核桃去壳、剥离山核桃内果皮；
步骤（2）、将步骤（1）得到的山核桃内果皮粉碎，得到山核桃内果皮粉；
步骤（3）、将步骤（2）得到的山核桃内果皮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山核桃内果皮提取物。
2.根据权利要求1所述的山核桃内果皮提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（1）选取的山核桃使用20～50℃的温水浸泡30～40min后再去壳。
3.根据权利要求1所述的山核桃内果皮提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（2）山核桃内果皮粉碎后过60～80目筛。
4.根据权利要求1所述的山核桃内果皮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80～85v/v%的甲醇水溶液。
5.根据权利要求1所述的山核桃内果皮提取物在制备抗卵巢癌产品中的应用，其特征在于，步骤（3）山核桃内果皮粉和甲醇水溶液料液比为1g：10～12ml。
6.根据权利要求1所述的山核桃内果皮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所述的山核桃内果皮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所述的山核桃内果皮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所述的山核桃内果皮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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