CN111317751B - 含有硫苷化合物的抗癌组合物及其用途 - Google Patents
含有硫苷化合物的抗癌组合物及其用途 Download PDFInfo
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- CN111317751B CN111317751B CN201910031580.3A CN201910031580A CN111317751B CN 111317751 B CN111317751 B CN 111317751B CN 201910031580 A CN201910031580 A CN 201910031580A CN 111317751 B CN111317751 B CN 111317751B
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Abstract
本发明提供一种利用微生物发酵过程,使硫苷化合物结构改变,并以藻类发酵液包覆硫苷化合物晶体,使所述硫苷化合物的温度耐受性提高,且提升了其生理活性能力,因此减少使用量即可达到抗癌效果。此外,所述发酵过程中可产生的抗氧化酵素,与硫苷化合物具有协同作用,能够达到全面性的保护效果,螯合金属能力则可协助体内排出PM2.5污染物质,避免重金属囤积体内。
Description
技术领域
本发明关于一种含有硫苷化合物的抗癌组合物,特别关于一种含有十字花科蔬菜发酵液及藻类发酵液的抗癌组合物。
背景技术
十字花科蔬菜为双子叶植物十字花目的一科,是植物中最繁盛的科之一,常见十字花科植物如高丽菜、花椰菜、青花菜,特征为叶生长顺序为放射十字状。
硫代化合物具有良好的抗癌功效,在学界中是一项热门的主题,硫代化合物主要存在于前述十字花科蔬菜中,一般提取硫代化合物以合成或萃取方式取得,合成方式皆以化学合成,在食用上具有食用安全上的疑虑;而萃取方式取得的硫代化合物则容易受温度破坏,间接影响功效性。又,硫苷化合物,或称硫代葡萄糖苷,为一类带硫化合物,如萝卜硫素,亦存在于十字花科中,需经由黑芥子酶催化后形成,因此常受到温度破坏,提取困难。目前研究证实,硫苷化合物对于抗癌具有良好的生理活性。
有鉴于此,十字花科所含的硫苷化合物不易提取,容易受到温度破坏影响活性成分,因此如何有效萃取出具有良好抗癌效果的硫苷化合物,如何保护硫苷化合物的抗癌效果,即成为相当重要的课题。
发明内容
有鉴于此,本发明提供一种利用微生物发酵过程,使硫苷化合物结构改变,并以藻类发酵液包覆硫苷化合物,使前述硫苷化合物的温度耐受性提高,且提升了其生理活性能力。减少使用量即可达到抗癌效果。此外,前述发酵过程中可产生的超氧化歧化酶SOD,与硫苷化合物具有协同作用,能够达到全面性的保护效果,螯合金属能力则可协助体内排出PM2.5污染物质,避免重金属囤积体内。
本发明提供一含有硫苷化合物的抗癌组合物,包含:十字花科发酵液和藻类发酵液,所述十字花科发酵液包含硫苷化合物,所述藻类发酵液包含胶体结构,所述胶体结构包覆于所述硫苷化合物,其中所述硫苷化合物含量为200-400mg/L。前述组合物,其中所述十字花科发酵液与所述藻类发酵液的混合比例系为5-10%。
本发明再提供一具有含有硫苷化合物的抗癌组合物的制备方法,其包含以下步骤:
(a)种植十字花科蔬菜,将含硒微生物的肥料施于所述十字花科蔬菜;有机肥料为有机硒含量为100~1000μg/L的液态肥料,以叶面喷洒方式,施肥量为30L/ha,每周1-3次。
(b)待所述十字花科蔬菜长成后收成,以乳酸菌/酵母菌进行发酵处理,产生十字花科蔬菜发酵液;
(c)取藻类,以乳酸菌/酵母菌进行发酵处理,产生藻类发酵液;
(d)将所述十字花科蔬菜发酵液,以及所述藻类发酵液以10:1~30:1比例进行混合,产生前述的组合物;较佳的,以19:1比例进行混合。
较佳地,前述制备具有含有硫苷化合物的抗癌组合物过程中,所述发酵方法,包含:(1)第一阶段发酵:添加乳酸菌/酵母菌、糖类,进行7~14天发酵,(2)第二阶段添加乳酸菌/酵母菌,进行7~14天发酵;添加乳酸菌/酵母菌的含菌量为108CFU/ml,接种量为体积0.1%,发酵温度26-28℃。在某些实施例中,前述发酵时间各为14天。
较佳地,本发明所使用的十字花科蔬菜系包含青花菜、花椰菜、芥菜、小白菜或甘蓝菜。
较佳地,本发明所使用的藻类包含海木耳Sarcodia montagneana、石菜花Gelidium amansii、石莼Ulva lactuca、青丝藻Ulva prolifera、海葡萄Caulerpalentillifera或葛仙米藻Nostoc commune。
较佳地,前述制备具有含有硫苷化合物的抗癌组合物过程中,前述乳酸菌包含Lactobacillus plantarum、Lactobacillus delbrueckii、Lactococcus lactis、Lactococcus acidophillus或Bifidobacterium bifidum,前述酵母菌包含Saccharomycopsis fibufigera、Pichia membramefaciens、Schizosaccharomyes pombe或Saccharomyces cerevisiae。
较佳地,前述十字花科发酵液包含青花菜、花椰菜、芥菜、小白菜或甘蓝的发酵液体。在某些实施例中,前述十字花科发酵液是取等比例质量的青花菜、花椰菜、芥菜、小白菜及甘蓝。
较佳地,前述含硒微生物肥料包含:黄豆液、乳酸菌液、亚硒酸钠。
前述十字花科蔬菜在经过微生物发酵过程中,将十字花科中的硫代葡萄糖苷化合物转换为有机态,并且增加含量;混合后的十字花科发酵液添加藻类发酵液,经高速乳化头乳化后,使藻类发酵液中的胶体包覆于硫苷化合物上,提供保护性与提升功效性。
本发明提供含有硫苷化合物的抗癌组合物,其具有抑制癌细胞生长、抑制肿瘤生长,以及螯合亚铁离子的有利功效,可用于制备治疗癌症的药物或螯合亚铁离子的药物。
附图说明
图1为本发明硫苷结构化合物晶体示意图;
图2A、2B图显示本发明抗癌组合物抑制结肠癌细胞的效果;
图3A、3B图显示本发明抗癌组合物抑制肝癌细胞的效果;
图4A、4B图显示本发明抗癌组合物抑制乳癌细胞的效果;
图5A、5B图显示本发明抗癌组合物抑制肺腺癌细胞的效果;
图6显示本发明抗癌组合物的螯合亚铁离子的效果;
图7显示本发明抗癌组合物抑制小鼠肠癌细胞的效果;
图8显示本发明抗癌组合物的抑制肿瘤体积效果;
图9显示本发明抗癌组合物的对于癌细胞凋亡的作用效果;
图10显示本发明抗癌组合物与他牌酵素液的SOD含量的比较结果。
具体实施方式
为了比较本发明的抗癌组合物的有利功效,本发明实施例以一般种植十字花科,包含青花菜、花椰菜、芥菜、小白菜、甘蓝等,将蔬菜样本洗净后放入发酵桶中,不添加菌种进行30天发酵,各自独立发酵后以等比例混合萃取液,产生十字花科萃取液,作为本发明的抗癌组合物的比较基础。
为了比较本发明的抗癌组合物的有利功效,本发明实施例利用萝卜硫素(Sulforaphane)作为硫代葡糖糖苷标准品,为硫苷化合物之一,依照实验浓度需求配置。
本发明的抗癌组合物如图1所示,本发明利用发酵方式将十字花科中硫代化合物,以有机形态方式保留硫代化合物的功效性,并运用海木耳发酵液乳化,形成特殊晶体结构,有助于提升原有功效性,使用量减低,增加生物利用度。
本发明提供一具有含有硫苷化合物的抗癌组合物的制备方法,其包含以下步骤:(a)种植十字花科蔬菜,将含硒微生物的肥料施于该十字花科蔬菜;有机肥料的有机硒含量为100~1000μg/L的液态肥料,以叶面喷洒方式,施肥量为30L/ha,每周1-3次;前述十字花科蔬菜种子包含青花菜、花椰菜、芥菜、小白菜、甘蓝;含硒微生物肥料为100mL黄豆液+1mL乳酸菌液+2.2g亚硒酸钠,30℃培养24小时,硒浓度为10000ppm,施用肥料以喷洒方式,将含硒微生物肥料250ml加水定量至1000mL,每次喷洒2000mL。
(b)待前述十字花科蔬菜长成后,收成,并以整株植株压榨后,添加乳酸菌Lactobacillus plantarum进行14天发酵;之后再添加酵母菌Saccharomyces cerevisiae进行14天发酵;发酵依照发酵情况适度添加醣类供给菌体养分,菌种添加比例为108CFU/ml接种量为体积0.1%,发酵温度26-28℃,产生十字花科蔬菜发酵液;前述青花菜、花椰菜、芥菜、小白菜、甘蓝系各取相同重量(以等比例混合)。
(c)取海木耳,以(b)所述的发酵方法进行发酵,产生海木耳发酵液,所述海木耳为Sarcodia ceylanica;
(d)将前述十字花科发酵液,以及前述海木耳发酵液以10:1~30:1比例进行混合,产生具有硫苷化合物的抗癌组合物;较佳的,以19:1比例进行混合。
在前述发酵过程中,可以依照发酵情况适度添加碳源供给菌体养分,碳源种类可做替换,添加比例占比重为0.1%-5%,碳源种类包含植物或藻类来源的淀粉、多醣、寡糖、双糖或单醣,如藻类多醣体、玉米淀粉、甜菜根糖、蔗糖或糖蜜。
实施例一、硫代化合物含量试验
十字花科萃取液(Cruciferae extract),选自一般种植十字花科(青花菜、花椰菜、芥菜、小白菜、甘蓝),将蔬菜样本洗净后放入发酵桶中,不添加菌种进行30天发酵,各自独立发酵后以等比例混合萃取液,产生十字花科萃取液。
十字花科发酵液(Cruciferae fermentation liquid),选自十字花科(青花菜、花椰菜、芥菜、小白菜、甘蓝)蔬菜,以特殊制程生产,于种植过程中喷洒含硒微生物肥料,收成后经30天,共2株菌(乳酸菌:Lactobacillus plantarum;酵母菌:Saccharomycescerevisiae各发酵两周)发酵生成,各自独立发酵后以等比例混合发酵液,产生十字花科发酵液。
微生物在发酵过程中,将十字花科中硫代葡萄糖苷化合物转换为有机态,并且增加含量;混合后的十字花科发酵液添加5%海木耳发酵液(以同样发酵方式生产发酵液),经高速乳化头乳化后,使海木耳发酵液中的胶体包覆于硫苷化合物上,提供保护性与提升功效性,完成本发明的抗癌组合物。
硫苷化合物的萃取:取20ml十字花科蔬菜发酵液,或20ml十字花科萃取液于均质瓶中,加入100mL含有100%的70℃甲醇,均质处理15秒,于沸水浴中搅拌10分钟(转速为350rpm),再移至70℃水浴进行10分钟,然后离心15分钟(转速为3000rpm),上清液置于35℃烘箱使甲醇挥发,以100mL 0.5M pH 7磷酸缓冲溶液回溶,离心15分钟(转速为3000rpm),以0.45μm的聚偏二氟乙烯过滤膜(PVDF滤膜)过滤,以高效液相色谱法HPLC进行分析。
硫苷化合物分析条件:以去离子水配制0.02M四丁基硫酸氢胺tetrabutylammonium hydrogensulfate(TBA)水溶液,再以磷酸氢二钠·二水(Na2HPO4·2H2O)调整pH值至7.0,以0.45μm滤膜过滤并去气后作为移动向A液,100%乙晴以0.45μm滤膜过滤并去气后作为移动向B液,以梯度洗脱进行分析0min:B 5%,25min:B 25%,35min:B25%,35.1min:B 5%,40min:Stop,流速1mL/min,UV侦测器波长设定227nm,取10μL样品注入高效液相色谱法HPLC进行分析。
实施例一的结果如表一所示,十字花科萃取液的硫苷化合物为25±0.01μmole/ml,十字花科蔬菜发酵液的硫苷化合物为70±0.02μmole/ml。
表一、硫苷化合物的HPLC分析结果
实施例二、本发明抗癌组合物用以抑制癌细胞之效果
本实施例针对抑制癌细胞的效果进行测试,设计四个不同组别,分别为:控制组(control)、萝卜硫素(Sulforaphane)、十字花科萃取液(Cruciferae extract)、十字花科蔬菜发酵液(Cruciferae femented liquid)。
(1)抑制结肠癌细胞:
以WiDr细胞(结肠癌细胞)作为模型,WiDr(细胞数为5×104cell/well,样品浓度为0.01、0.025、0.05、0.075、0.1μM(此为换算含有硫代葡萄糖苷/萝卜硫素浓度),分别培养24与48小时,与样品共培养后以MTT assay检验抑制癌细胞生长能力。
其结果如图2A和图2B所示,结果显示,以十字花科蔬菜发酵液处理的细胞,在抑制癌细胞生长能力上,无论何种浓度及时间处理,都与以萝卜硫素或十字花科萃取液处理的癌细胞有显著的差异。
(2)抑制肝癌细胞:
以HepG2(肝癌细胞)作为模型,HepG2细胞数为5×104cell/well,样品浓度为0.01、0.025、0.05、0.075、0.1μM(此为换算含有硫代葡萄糖苷或萝卜硫素之浓度,分别培养24与48小时,与样品共培养后以MTT assay检验抑制癌细胞生长能力。
其结果如图3A和图3B图所示,结果显示,以十字花科蔬菜发酵液处理的细胞,在抑制癌细胞生长能力上,无论何种浓度及时间处理,都与以萝卜硫素或十字花科萃取液处理的癌细胞有显著的差异。
(3)抑制乳癌细胞:
以MCF-7(乳癌细胞)作为模型,MCF-7细胞数为5×104cell/well,样品浓度为0.1、0.5、1、1.5、2μM(此为换算含有硫代葡萄糖苷或萝卜硫素浓度),分别培养24与48小时,与样品共培养后以MTT assay检验抑制癌细胞生长能力。
其结果如图4A和图4B图所示,结果显示,以十字花科蔬菜发酵液处理的细胞,在抑制癌细胞生长能力上,无论何种浓度及时间处理,都与以萝卜硫素或十字花科萃取液处理的癌细胞有显著的差异。
(4)抑制肺腺癌细胞:
以LCC-1(肺腺癌细胞)作为模型,LCC-1细胞数为5×104cell/well,样品浓度为0.1、0.5、1、1.5、2μM(此为换算含有硫代葡萄糖苷或萝卜硫素浓度),分别培养24与48小时,与样品共培养后以MTT assay检验抑制癌细胞生长能力。
其结果如图5A和图5B图所示,结果显示,以十字花科蔬菜发酵液处理的细胞,在抑制癌细胞生长能力上,无论何种浓度及时间处理,都与以萝卜硫素或十字花科萃取液处理的癌细胞有显著的差异。
实施例三、本发明抗癌组合物用以螯合亚铁离子能力
螯合亚铁离子能力实验设计:
0.02mL的FeCl2·4H2O(2mM)于1.5ml离心管中,加入0.94mL待测样品及0.04mL的Ferrozine(5mM)混合均匀,在室温下静置反应10分钟,使用光谱仪检测562nm吸光值,吸光值越低表示样品螯合亚铁离子能力越强,EDTA为标准品。
其结果如图6所示,结果显示,以十字花科蔬菜发酵液处理的细胞,在螯合亚铁离子能力上,无论何种浓度处理,都与以萝卜硫素或十字花科萃取液处理的癌细胞有显著的差异。
实施例四、本发明抗癌组合物的抗肿瘤效果
本试验分为四组:第一组为控制组(control);第二组为5-FU组,5-FU为已知癌症化疗药物5-氟尿嘧啶;第三组为十字花科蔬菜发酵液(Cruciferae femented liquid)(本发明抗癌组合物);第四组为5-FU和十字花科蔬菜发酵液,以十字花科蔬菜发酵液作为辅助治疗(5-FU+Cruciferae femented liquid)。
(1)抑制小鼠大肠癌细胞:
以小鼠BALB/c为模型,CT-26细胞株经过继代为106细胞数量的细胞液,于4周龄时在左后肢皮下注射CT-26细胞液100μL,诱导大肠癌后晶由样本治疗,以腹腔注射食盐水/5-FU,发酵液为每日进行管喂,使用量为人体建议使用量转换,0.5ml/kg,实验持续4周后牺牲,于每周进行肿瘤大小测量。其结果如图7所示,结果显示,十字花科发酵液抑制肿瘤大小可比拟化疗药物5-FU,作为化疗药物辅助实更能降低肿瘤的大小,可发展为抗癌药物或化疗辅助药物。
(2)小鼠牺牲后肿瘤体积评估:
治疗28天后小鼠牺牲,将肿瘤取出比对肿瘤大小。其结果如图8所示,结果显示十字花科发酵液能有效缩小动物模型中肿瘤的大小,作为辅助药物实效果最佳。
(3)肿瘤组织切片:
将牺牲小鼠肿瘤进行切片后镜检,判断细胞凋亡程度。其结果如图9所示,结果显示,使用十字花科发酵液的肿瘤组织,细胞凋亡状况皆有显著影响,以凋亡型态判断,化疗药物5-FU可减少肿瘤的生长,但对于癌细胞无明显诱导凋亡作用;经十字花科发酵液治疗,能显著提升癌细胞凋亡作用,此结果证实十字花科发酵液中的硫代化合物具有靶向特性,可针对癌细胞诱导其凋亡作用。
实施例五、本发明抗癌组合物之SOD含量检验
本发明抗癌组合物有经过SOD含量检验。有关于SOD与硫代化合物协同作用为目前文献研究中已知结果。
本实施例以商业分析套组(Superoxide dismutase assay kit,Cayman,AnnArbor,MI,USA)分析本发明抗癌组合物的SOD的活性。可于波长450nm下测定其吸光值,依据SOD的标准活性曲线计算出发酵液中超氧歧化酵素的含量,单位以Uint/mL表示。其结果如图10所示A、B、C发酵液分别含890、280、950Uints(单位)/mL的SOD,综合发酵液含3500Units/mL的SOD。
前述A、B和C为不同菌种的发酵液,蔬果种类、数量相同,蔬果种类为西红柿、青梅、红甜椒、番石榴、甘蓝、西兰花、葡萄、苹果、花椰菜、苦瓜、甘薯、梨子、红茶、菠菜、丝瓜、橙子、香蕉、莴苣、西瓜、西番莲、芥菜、葡萄柚、萝卜、冬瓜、芥蓝、空心菜、黄瓜、小白菜、菠萝、黑木耳、香菇、柠檬、李子、猕猴桃、芹菜、茄子、甜瓜、苋菜、金橘、胡萝卜、哈密瓜、莲雾、大白菜、荔枝、芦笋、火龙果、番荔枝、莲藕、柿子、茼蒿、油菜、杨桃、木瓜、豆瓣菜、菜豆、芜菁、马铃薯、山药、芋头和南瓜。
前述A发酵液的制程以乳酸菌(ATCC 14917)菌株进行60天发酵;前述B发酵液的制程以乳酸菌(ATCC 8014)菌株+Coopers市售酵母菌进行60天发酵;前述C发酵液的制程以乳酸菌(ATCC 14917)菌株+Coopers市售酵母菌进行60天发酵。
综上所述,本发明经乳酸菌和酵母菌发酵过的十字花科蔬菜萃取物,再混以海木耳发酵液,可将抗癌成分硫苷化合物形成一硫苷化合物晶体,提升了有效成分的抗癌效果,并增加螯合亚铁离子能力。
本发明所揭露的所有特征应可以任何结合方式实现。本发明所揭露的每一特征应可以相同、均等或相似目的的取代物所取代。因此,除非有明确的指定,否则所揭露的每一个特征仅仅只是均等物或相似特征的一个种类的一实施例。
Claims (9)
1.一种发酵液组合物,其特征是,包含十字花科蔬菜发酵液和藻类发酵液,所述十字花科蔬菜发酵液包含硫苷化合物,所述藻类发酵液包含胶体结构,所述胶体结构包覆于该硫苷化合物,所述发酵液组合物透过一特殊发酵步骤制成,所述特殊发酵步骤包含以下步骤:
(a)种植十字花科蔬菜,将含硒微生物的肥料施于所述十字花科蔬菜,所述十字花科蔬菜为青花菜、花椰菜、芥菜、小白菜和甘蓝;
(b)待所述十字花科蔬菜长成后收成,进行发酵处理,产生十字花科蔬菜发酵液,所述十字花科蔬菜发酵液是取等比例的青花菜、花椰菜、芥菜、小白菜和甘蓝;
(c)取藻类,进行发酵处理,产生藻类发酵液,所述藻类为海木耳;
(d)将所述十字花科蔬菜发酵液,以及所述藻类发酵液以10:1~30:1比例进行混合,产生所述的发酵液组合物,其中:
所述特殊发酵的步骤(b)和(c)中,所述发酵处理包含: (1) 第一阶段发酵:添加A菌,进行7~14天发酵;(2) 第二阶段添加B菌,进行7~14天发酵;添加的A菌或B菌含菌量为108CFU/ml,接种量为体积0.1%,发酵温度26-28℃,所述A菌为Lactobacillus plantarum,所述B菌为Saccharomyces cerevisiae。
2.如权利要求1所述的发酵液组合物,其特征是,所述硫苷化合物含量为40~100 μmole/ml。
3.一种如权利要求1所述的发酵液组合物的制备方法,包含以下步骤:
(a)种植十字花科蔬菜,将含硒微生物的肥料施于所述十字花科蔬菜,所述十字花科蔬菜为青花菜、花椰菜、芥菜、小白菜和甘蓝;
(b)待所述十字花科蔬菜长成后收成,进行发酵处理,产生十字花科蔬菜发酵液,所述十字花科蔬菜发酵液是取等比例的青花菜、花椰菜、芥菜、小白菜和甘蓝;
(c)取藻类,进行发酵处理,产生藻类发酵液,所述藻类为海木耳;
(d)将所述十字花科蔬菜发酵液,以及所述藻类发酵液以10:1~30:1比例进行混合,产生如权利要求1所述的发酵液组合物;
所述步骤(b)和(c)中,所述发酵处理包含: (1) 第一阶段发酵:添加A菌,进行7~14天发酵;(2) 第二阶段添加B菌,进行7~14天发酵;添加的A菌或B菌含菌量为108 CFU/ml,接种量为体积0.1%,发酵温度26-28℃,所述A菌为Lactobacillus plantarum,所述B菌为Saccharomyces cerevisiae。
4.如权利要求3所述的制备方法,其特征是,所述含硒微生物肥料包含:黄豆液、乳酸菌液或亚硒酸钠。
5.一种如权利要求1所述的发酵液组合物在用于制备治疗癌症的药物中的用途。
6.如权利要求5所述的用途,其特征是,所述癌症为大肠癌、肝癌、乳癌或肺腺癌。
7.如权利要求5所述的用途,其特征是,所述癌症为结肠癌。
8.一种如权利要求1所述的发酵液组合物在用于制备螯合亚铁离子的药物中的用途。
9.如权利要求8所述的用途,其特征是,所述用途包含协助体内排出PM2.5污染物质,避免重金属囤积体内。
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