CN112274576A - 香蕉伪茎嫩心萃取物于抗乳癌的用途 - Google Patents
香蕉伪茎嫩心萃取物于抗乳癌的用途 Download PDFInfo
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Abstract
本发明是有关于一种香蕉伪茎嫩心萃取物于抗乳癌的用途,其是施予有效剂量的香蕉伪茎嫩心萃取物于所需个体以达到抑制乳癌细胞生长的功效。
Description
技术领域
本发明是有关于一种香蕉伪茎嫩心萃取物于抗乳癌的用途,尤其是指经由特定处理条件萃取的香蕉伪茎(pseudo-stem)嫩心(tender core)具有抑制乳癌细胞生长与促进乳癌细胞凋亡等抗乳癌功效,藉此可增加香蕉伪茎的经济价值。
背景技术
香蕉为中国台湾重要经济作物之一,生产香蕉所产生的废弃物量非常的庞大,对环境也造成很大的问题;根据Bello et al.(2014)指出生产一吨的香蕉时,所产生废弃物包含:香蕉伪茎(pseudo-stem)三吨、蕉梗160公斤、蕉叶480公斤及440公斤的香蕉皮。在香蕉废弃物中,显然香蕉伪茎所占比例最高,仅少数在东南亚地区有利用伪茎配合其他食材做为汤料。
目前有学者研究将香蕉伪茎制成生物质酒精,亦有学者发现香蕉假茎其组织汁液含量常达80%以上,且具较高有机酸及电解质含量,因而尝试将香蕉假茎汁液用做植物电池的材料。
另,CN105394778A公开了一种香蕉茎干粉的制备方法,其是将香蕉收获后的树去掉叶片并剥去外皮得到香蕉茎干,将所得到的茎干进行压榨脱水成片状物料,厚度为5~30mm,利用纤维切断机将其切成0.3~2mm长度的段,进行热风干燥,使含水率控制在3%~8%,再利用粉碎机将干燥好的物料进行粉碎过筛后得到香蕉茎干粉末。藉此,经由物理加工法把香蕉茎干加工成粉末状态,可作为膳食纤维的原料加以利用。
CN104814356A公开了一种用香蕉假茎为主要原料制备肉猪饲料的方法,其是将香蕉采收后的废弃物香蕉假茎加工生产肉猪饲料,其方法包括(1)将香蕉假茎一片片剥出,去除腐烂、变质的叶鞘及其他杂质;(2)将香蕉假茎打碎放入发酵罐中,调整pH6.5-7.0得到发酵液,接入培养成熟的乳酸菌液体菌种,再加入纤维素酶,搅拌,静置,发酵结束后,取出沥干得到香蕉假茎粉;(3)将狼尾草打碎后,加入玉米粉、麦皮和香蕉假茎粉,按比例混合;最后(4)干燥。藉此使香蕉假茎变废为宝,并可降低饲养成本。
CN103826817A公开了用香蕉茎制造板的方法以及用如此方法生产的板,使用香蕉废弃物尤其是香蕉假茎来简单地和尽可能生态地生产可用于装饰目的的新材料,具体而言,所述方法包括先从香蕉假茎的中心部分切出茎秆,将茎秆切成条带(或薄片),在辊轧机中层压所获得的条带,通过组装条带使交叠在预定的宽度上,以形成板,最后再加压和干燥所获得的板;尤其通过加压彼此抵靠的条带的交叠部分,使条带连接在一起,无需借助于外部添加粘结剂。
有些农民会将香蕉伪茎拿来当作种植的基底,然而,大部分仍将香蕉伪茎视为无活性部分而丢弃。
发明内容
本发明主要目的为提供一种香蕉伪茎嫩心萃取物于抗乳癌的用途,其是指藉由特定处理条件萃取而得的香蕉伪茎(pseudo-stem)嫩心(tender core)萃取物具有抑制乳癌细胞生长与促进乳癌细胞凋亡等抗乳癌功效,藉此可增加香蕉伪茎的经济价值。
于本发明的一实施例中,香蕉伪茎嫩心萃取物是以80℃热水进行杀菁1-20分钟且榨汁,并于干燥与磨粉后利用乙醇溶液萃取所得;较佳而言,乙醇溶液是95%(V/V)乙醇,干燥条件是以100℃进行干燥至少36小时。
藉此,相较于冻干、杀菌冻干、榨汁等萃取方法,本发明藉由榨汁且杀菁得到的香蕉伪茎嫩心萃取物具有较高的抗乳癌能力,可将香蕉伪茎嫩心开发为健康保健食品。
附图说明
图1:本发明其一具体实施例的步骤流程图。
图2:不同香蕉伪茎嫩心萃取物对于乳癌细胞凋亡调控蛋白表达的分析图。
图3:杀菁榨汁香蕉伪茎嫩心萃取物(香蕉心萃取物)对于乳癌细胞凋亡调控蛋白表达的分析图。
图4:为图3的定量分析图。
图5:杀菁榨汁香蕉伪茎嫩心萃取物(香蕉心萃取物)对于乳癌细胞凋亡情形的分析图。
图6:为图5的定量分析图。
图7:杀菁榨汁香蕉伪茎嫩心萃取物(香蕉心萃取物)对于乳癌细胞存活率影响的分析图。
图8:杀菁榨汁香蕉伪茎嫩心萃取物(香蕉心萃取物)对于乳癌细胞Cleaved PARP表达情形的分析图。
图9:杀菁榨汁香蕉伪茎嫩心萃取物(香蕉心萃取物)对于乳癌细胞Cleavedcaspase-7表达情形的分析图。
图10:为图9的定量分析图。
具体实施方式
本发明的目的及其结构功能上的优点,将依据附图所示的结构,配合具体实施例予以说明,俾使阅读者能对本发明有更深入且具体的了解。
本发明提供一种香蕉伪茎嫩心(pseudo-stem tender core)萃取物于抗乳癌的用途,其可促进乳癌细胞凋亡(apoptosis)达到抗乳癌效果,其中香蕉伪茎嫩心萃取物是以80℃热水进行杀菁1-20分钟且榨汁,并于75℃-100℃进行干燥至少24小时(可例如为以100℃进行干燥至少36小时)与磨粉后利用乙醇溶液(可例如为95%(V/V)乙醇)萃取所得,最后亦可进一步进行减压浓缩。
此外,藉由下述具体实施例,可进一步证明本发明可实际应用的范围,但不意欲以任何形式限制本发明的范围。
实施例一:制备香蕉伪茎嫩心萃取物
香蕉品种为目前中国台湾最主要的香蕉栽种品种,学名为Musa sapientum L.,俗名北蕉,产地来自中国台湾台中太平,收成季节为每年七、八月份。
请参阅图1,为本发明其一具体实施例的步骤流程图。香蕉伪茎嫩心的萃取方式有以下四种,包括(1)冻干(Freeze-dry)、(2)杀菁冻干(Blanch and Freeze-dry)、(3)榨汁(Squeeze)、(4)杀菁榨汁(Blanch and Squeeze),详细萃取步骤如下:
(1)冻干组:将新鲜的香蕉伪茎嫩心经清洗、分切后,直接将香蕉伪茎嫩心置于–20℃冰箱冷冻,取出放置于铁盘上并铺平,放入冷冻干燥机中干燥。干燥完成的样品用粉碎机粉碎、均质,分装于真空袋中真空包装,并置于–20℃冰箱保存备用。
(2)杀菁冻干组:将新鲜的香蕉伪茎嫩心经清洗、分切后,以80℃热水杀菁1分钟,并将杀菁后的香蕉伪茎嫩心切片,放于–20℃冰箱冷冻,取出放置于铁盘上并铺平,放入冷冻干燥机中干燥。干燥完成的样品用粉碎机粉碎、均质,分装于真空袋中真空包装,并置于–20℃冰箱保存备用。
(3)榨汁组:将新鲜的香蕉伪茎嫩心经清洗、分切后,放入榨汁机榨汁,并将残渣与汁液分开存放于–20℃冰箱冷冻,分别取出残渣与汁液放置于铁盘上并铺平,放入冷冻干燥机中干燥。干燥完成的样品用粉碎机粉碎、均质,分装于真空袋中真空包装,并置于–20℃冰箱保存备用。
(4)杀菁榨汁组:将新鲜的香蕉伪茎嫩心经清洗、分切后,以80℃热水杀菁1分钟,并将杀菁后的香蕉伪茎嫩心切块,放入榨汁机榨汁,并将残渣与汁液分开放在–20℃冰箱冷冻,分别取出残渣与汁液放置于铁盘上并铺平,放入冷冻干燥机中干燥。干燥完成的样品用粉碎机粉碎、均质,分装于真空袋中真空包装,并置于–20℃冰箱保存备用。
实施例二:分析香蕉伪茎嫩心萃取物对于乳癌细胞凋亡(apoptosis)调控蛋白表达的影响
1.细胞培养
人类三阴性乳癌细胞株(triple negative breast cancer cells)Hs578T购自American Type Culture Collection(ATCC;Manassas,USA)。将Hs578T细胞以培养液DMEM(含10%FBS、1.5g/L NaHCO3、100unit/mL penicillin、100μg/mL streptomycin,pH=7.2)培养于培养皿中(BD Biosciences,San Jose,CA,USA)培养于直径10厘米培养皿中,细胞置于5%CO2、恒温37℃培养箱中,每1-2天更换培养液。待细胞繁殖至九分满,去除培养液,并以37℃的PBS(磷酸盐缓冲液,含0.01M Na2HPO4、0.01M NaH2PO4.H2O、0.14M NaCI,pH=7.3)清洗细胞2次,再加入1ml 0.05%Trypsin-EDTA(0.25%Trypsin、1mM EDTA)使细胞脱离培养皿底盘。待细胞脱离底盘后,加入1mL DMEM培养液(含10%FBS)至培养皿中使Trypsin-EDTA终止反应,并将细胞悬浮液移至含有DMEM培养液(含10%FBS)的15mL离心管,以1500rpm离心5分钟,随后去除上清液,并加入5mL DMEM培养液(含10%FBS)将细胞冲散使其悬浮。再于10厘米培养皿中加入10mL DMEM培养液(含10%FBS),再加入实验需要量的细胞液,水平摇晃使细胞均匀分散于培养皿中后培养于含5%CO2、恒温37℃培养箱中。
计数细胞时是将细胞悬浮液以DMEM培养液(含10%FBS)稀释5倍(10μL细胞悬浮液与40μL DMEM培养液)并均匀冲散,从中取出10μL置入细胞计数盘中以进行细胞计数。
2.细胞处理
人类乳癌细胞Hs578T培养于6厘米培养皿中至八分满,分别加入四种制法所得的400μg/ml香蕉伪茎嫩心萃取物(含10%或1%FBS的DMEM培养液)培养24小时,或加入不同浓度为0、100、200、400μg/ml杀菁榨汁的香蕉伪茎嫩心萃取物培养24小时。接续,利用西方墨点法分析香蕉心萃取物对于乳癌细胞凋亡调控蛋白Bax和Bcl-2相较于对照组(控制组)的表达情形。所使用的抗体信息分别为Bax抗体购自Santa Cruz Biotechnology,Inc.(sc-526;稀释比例1:500),Bcl-2抗体购自Santa Cruz Biotechnology,Inc.(sc-7382;稀释比例1:500),β-actin抗体购自GeneTex Inc.(稀释比例1:1000)。各组实验分析数据以平均值加减标准偏差(Mean±SD)方式表示。实验统计数据采用SPSS统计软件中单因子变异数分析(One-way analysis of variance,ANOVA)以及Tukey’s test(Tukey’s multiplecomparison test)进行分析,当p值<0.05时,以不同英文字母代表各组间有显著差异。
结果请参阅图2,相较于控制组为未加入香蕉伪茎嫩心萃取物,四种不同萃取方式所得的香蕉伪茎嫩心萃取物中,以杀菌冻干、榨汁及杀菁榨汁萃取物(400μg/ml)处理,可显著诱发Hs578T乳癌细胞促凋亡蛋白Bax表达,同时抑制抗凋亡蛋白Bcl-2表达,其中又以榨汁和杀菁榨汁的香蕉伪茎嫩心萃取物效果较为显著,杀菁榨汁组最佳,上述结果说明此具有抗乳癌生长潜力。
请再参阅图3与图4,将不同浓度0、100、200、400μg/ml杀菁榨汁的香蕉伪茎嫩心萃取物(香蕉心萃取物)处理乳癌细胞24小时,结果显示香蕉伪茎嫩心萃取物处可显著诱发乳癌细胞促凋亡蛋白Bax表达,同时抑制抗凋亡蛋白Bcl-2表达,呈剂量关系。上述结果说明此香蕉伪茎嫩心萃取物(杀菁榨汁萃取方式)确实具有促乳癌细胞凋亡潜力。
实施例三:分析香蕉伪茎嫩心萃取物对于乳癌细胞凋亡(apoptosis)的影响
细胞培养方式如实施例二所示,在此实施例中使用流式细胞仪(flow cytometer)检测乳癌细胞凋亡情形。
人类乳癌细胞Hs578T培养于6厘米培养皿中至8分满,加入0、100、200、400μg/ml香蕉伪茎嫩心萃取物(含10%FBS的DMEM培养液)培养24小时。将处理完毕的细胞,收集培养皿中条件培养基(Conditioned medium)置于15mL离心管中,培养皿以PBS清洗两次后,加入500μL 0.05%Trypsin-EDTA(0.25%Trypsin、1mM EDTA)等待细胞脱离底盘,随后加入500μL DMEM培养液(含10%FBS)至培养皿中使Trypsin-EDTA终止反应,并将细胞悬浮液移至含有DMEM培养液(含10%FBS)的15mL离心管,以1500rpm离心5分钟后去除上清液,加入1mLPBS将细胞冲散使其悬浮并移至1.5mL离心管,于4℃、700g离心5分钟去除上清液,加入500μL的1X Annexin-V binding buffer(将10X Annexin-V binding buffer以灭菌水稀释成1X)并置于冰上。于避光环境下,加入Propidium Iodide Solution及Annexin-V至1.5mL离心管中并将细胞冲散,于冰中静置5分钟。利用Filter过滤后置于冰中,即可利用流式细胞仪进行细胞凋亡检测分析。所使用的染剂FITC Anexin V与碘化丙啶(Propidium Iodide,PI)皆购自BD Biosciences(San Jose,CA,USA)。
请参阅图5和图6,结果显示杀菁榨汁萃取方式的香蕉伪茎嫩心萃取物(香蕉心萃取物)可诱发Hs578T乳癌细胞凋亡,凋亡效果呈剂量关系,上述结果说明高剂量香蕉心萃取物(杀菁榨汁萃取方式)显著促乳癌细胞凋亡。
实施例四:分析香蕉伪茎嫩心萃取物对于乳癌细胞存活率的影响
人类乳癌细胞Hs578T培养于直径3厘米培养皿中,待生长至7分满后,分别加入0、25、50、100、200、400μg/ml香蕉伪茎嫩心萃取物(含10%或1%FBS之DMEM培养液)培养24小时。处理完毕后,以37℃ PBS清洗2次,加入1mL含0.5%mg/mL MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,购自Sigma-Aldrich Ins.)的DMEM培养液,放于5%CO2、温度37℃的培养箱内,暗反应3小时。随后移除含有MTT培养液并加入1ml异丙醇(isopropanol,购自Merck Chemical Company),于平摇式震荡器摇晃10分钟,利用活细胞粒线体琥珀酸去氢酶作用下所产生的蓝紫色非水溶性formazan(1-[4,5-dime-thythi-azol-2-yl]-2,5-dpheny-formazan)产物使之溶出,吸取Isopropanol溶液并放至1.5mL微量离心管中,以7000rpm离心5分钟使其分层,取上清液200μL置于96孔盘中,利用微量盘酶免疫分析仪(Multiskan FC,Thermo scientific Inc.,Japan)以波长570nm读取吸光值,比较对照组(控制组)与处理组吸光值后,即可求得细胞相对存活率。各组实验分析数据以平均值加减标准偏差(Mean±SD)方式表示。实验统计数据采用SPSS统计软件中单因子变异数分析(One-way analysis of variance,ANOVA)以及Tukey’s test(Tukey’smultiple comparison test)进行分析,当p值<0.05时,以不同英文字母代表各组间有显著差异。
请参阅图7,结果显示杀菁榨汁萃取方式的香蕉伪茎嫩心萃取物(香蕉心萃取物)可诱发Hs578T乳癌细胞死亡,死亡效果呈剂量关系。上述结果说明高剂量香蕉心萃取物(杀菁榨汁萃取方式)显著促乳癌细胞死亡。
实施例五:分析香蕉伪茎嫩心萃取物对于乳癌细胞Cleaved PARP(poly(ADP-ribose)polymerase)及Cleaved caspase-7表达的影响
裂解态的PARP(cleaved PARP)及裂解态的凋亡蛋白酶-7(cleaved caspase-7)为启动细胞凋亡的核内标示蛋白(Marker),若cleaved PARP或cleaved caspase-7蛋白表达量增加代表细胞凋亡情形增加。
细胞培养方式如实施例二所示,利用西方墨点法分析香蕉心萃取物对于启动乳癌细胞凋亡的核内标示蛋白cleaved PARP和cleaved caspase-7相较于对照组(控制组)的表达情形。本实施例所使用的抗体信息分别为PARP抗体购自Roche(11835238001;稀释比例1:1000),caspase-7抗体购自Cell signaling technology(稀释比例1:1000),β-actin抗体购自GeneTex Inc.(稀释比例1:1000)。各组实验分析数据以平均值加减标准偏差(Mean±SD)方式表示。实验统计数据采用SPSS统计软件中单因子变异数分析(One-way analysisof variance,ANOVA)以及Tukey’s test(Tukey’s multiple comparison test)进行分析,当p值<0.05时,以不同英文字母代表各组间有显著差异。
请参阅图8,结果显示杀菁榨汁萃取方式的香蕉伪茎嫩心萃取物(香蕉心萃取物)可诱发Hs578T乳癌细胞Cleaved PARP蛋白表达,效果呈剂量关系,说明高剂量香蕉心萃取物(杀菁榨汁萃取方式)确实促乳癌细胞死亡。
请再参阅图9与图10,结果显示杀菁榨汁萃取方式的香蕉伪茎嫩心萃取物(香蕉心萃取物)亦可诱发Hs578T乳癌细胞Cleaved caspase-7蛋白表达,效果呈剂量关系。上述结果说明高剂量香蕉心萃取物(杀菁榨汁萃取方式)确实促乳癌细胞死亡。
由上述的实施说明可知,本发明藉由特定萃取条件步骤,证实香蕉伪茎嫩心萃取物具有抗乳癌的新用途,因此常被视为废弃物丢弃的香蕉伪茎嫩心可被开发制成具有抗乳癌能力的保健营养食品,大幅增加香蕉伪茎的经济价值和产业利用性。
综上所述,本发明的香蕉伪茎嫩心萃取物于抗乳癌的用途,的确能藉由上述所揭露的实施例,达到所预期的使用功效。
Claims (6)
1.一种香蕉伪茎嫩心(pseudo-stem tender core)萃取物用于制备抗乳癌组合物的用途。
2.如权利要求1所述的用途,其中所述香蕉伪茎嫩心萃取物是促进乳癌细胞凋亡(apoptosis)。
3.如权利要求1所述的用途,其中所述香蕉伪茎嫩心萃取物是以80℃热水进行杀菁1-20分钟且榨汁,并于干燥与磨粉后利用乙醇溶液萃取所得。
4.如权利要求3所述的用途,其中所述乙醇溶液是95%(V/V)乙醇。
5.如权利要求4所述的用途,其中所述干燥条件是以75℃-100℃进行干燥至少24小时。
6.如权利要求5所述的用途,其中所述干燥条件是以100℃进行干燥至少36小时。
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