CN105744943A - 橄榄的液体植物混杂物的抗血管生成用途 - Google Patents
橄榄的液体植物混杂物的抗血管生成用途 Download PDFInfo
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Abstract
本发明涉及源自含油橄榄的植物水或源自橄榄研磨过程产生的橄榄残渣的富含多酚化合物(例如羟基酪醇和3,4?DHPA?EDA)的植物混杂物或天然浓缩物,其用于治疗和预防血管生成和炎症。具体地,所述血管生成和炎症是病理性血管生成和炎症(例如维持肿瘤发育和肿瘤扩散)或与非肿瘤病理相关的血管生成和炎症。本发明还涉及含所述多酚浓缩物的饮料以及其在治疗和预防血管生成和炎症中的应用。
Description
本发明涉及源自含油橄榄的压制所得的水(通常称为植物水)或源自橄榄研磨过程产生的残余橄榄残渣的富含多酚化合物(具体富含羟基酪醇和3,4-DHPA-EDA)的天然植物混杂物在治疗或预防血管生成和/或炎症中的应用。
具体地,所述血管生成和炎症是病理性类型,例如维持肿瘤发育和肿瘤扩散或与非肿瘤病理相关的血管生成和炎症。
血管生成是个体生长和发育过程期间发生的病理过程,其涉及从已存在的血管腔隙形成新血管。
血管生成过程还表征为各种病理性现象,包括肿瘤。
肿瘤(或瘤)团块可自发生长和发育多达约1-2mm3;然而,为了继续生长,其必须确保自身有必需的营养和氧气供给,所以其需要形成自己的血管腔隙。
肿瘤能分泌血管生成因子,例如VEGF(血管内皮生长因子)、bFGF(碱性成纤维细胞生长因子)和PDGF(血小板衍生生长因子),它们能促进血管发育。肿瘤分泌的血管生成因子激活内皮细胞,其产生响应开始增殖并分泌降解细胞基质和基底膜的物质(例如基质金属蛋白酶-MMP)从而迁移并入侵周围组织。随后,内皮细胞经组织形成由周细胞(即围绕新形成的血管的心肌细胞)稳定的管状结构并能改变血流并调节血管渗透性。
源自肿瘤的血管不规则并且其特征为与“正常”血管不一样的结构元素。例如,它们没有周细胞、大膜孔以及显著的血管扩张。这些特征改变了血管的渗透性和压力水平,并且还因此干扰了抗肿瘤药物的递送,这些药物在隙间液体中分散而不接触肿瘤,因此无法发挥其功能。
考虑到血管生成在肿瘤发育、生长和转移过程中的重要性,进行了目的在于鉴定能阻断肿瘤血管无规则发育并能改善药物向肿瘤位置递送的物质的大量研究。具体地,已在鉴定能阻止血管异常发育的分子中进行了大量尝试;形成了称为“抗血管生成(angioprevention)”的概念(即阻止肿瘤相关的血管生成)。
这些分子中大部分是天然来源(或在一些情况中是合成类似物)。一种非常有趣的示例是源自橄榄油的分子。
事实上,已知心血管疾病和肿瘤的发病率在地中海饮食群体中显著较低,这种饮食基于对橄榄油的消耗。科学证据为研究橄榄油提供了显著的动力,目的在于定义其组成,尤其是鉴定具有医学-药学潜能的物质。
引起特别兴趣的橄榄油的一个特征是其含有高水平的多酚。这些化合物是植物源的天然抗氧化剂,其能抑制自由基的形成。
橄榄油的有益特性引起了橄榄种植和橄榄油生产的大量增长,主要在意大利。因此,我们目睹了橄榄油生产的副产物大量增加,主要是植物水和残渣,它们具有高度污染性,因此对环境产生了显著影响。
这些材料的处置在意大利在国家和地区水平上均受到严格管控,并且法律的实施(11/1996的574号法案)使生产者产生了繁重的成本,它们无法从这些废料中获取任何价值,但其中事实上富含具有高度医药学潜能的分子。
羟基酪醇是植物水中所含的研究最多的多酚。其存在于植物水和残渣中,还通过橄榄苦苷水解形成,后者是主要存在于橄榄树叶子中的物质。
近期研究证实羟基酪醇自身具有针对PC12细胞(嗜铬细胞瘤性细胞系)的细胞保护功能、在给予U937细胞(人骨髓单核细胞系)和C2C12细胞(小鼠成肌细胞系)时抗凋亡、在诱导的骨髓瘤病例中抑制体内乳腺肿瘤增殖、在针对HL60和HL60R肿瘤细胞系(人早幼粒细胞性白血病细胞系和其多药物抗性衍生物)的研究中是化学预防剂、并且预防月经前综合征和骨质疏松症。
此外,已证实体内给予羟基酪醇(亦为高浓度)没有毒性效果。
其他研究证实,当单独给予橄榄苦苷时,其产生抗微生物活性,在结直肠肿瘤细胞系、转移性乳腺肿瘤和ER阴性细胞系中具有抗肿瘤能力,并且具有在细胞骨架水平改变细胞稳定性的能力。
虽然利用植物水进行了许多研究,但仍需要鉴定可以使其废料产生价值的新特征,否则其对生产者来说仅是成本,而对环境则是危害。特别需要鉴定可使这些废料发挥余热的新的营养和药学性质。
在这一方面,申请人惊奇地发现植物水可以在体内和体外阻断/阻止血管生成和炎症,特别是病理血管生成和炎症,例如那些维持肿瘤发育和扩散的血管生成和炎症,或与非肿瘤病理相关的血管生成和炎症。
具体地,申请人发现通过利用反渗透处理而浓缩,植物水的透过物经历微过滤,可获得富含多酚化合物的植物混杂物,其能阻止并阻断血管生成和炎症,尤其是病理性血管生成和炎症,例如与肿瘤相关的那些或与非肿瘤病理相关的那些,其比单独摄取相同化合物(即通过纯化技术从植物水和残渣中分离的相同化合物)所能实现的效果更有效。
该效果对人体健康,主要是抗血管生成方面有特别优势。事实上,为此目的的本发明植物水浓缩物可自身使用或与其他抗肿瘤和抗血管生成和抗炎物联用,例如以饮料的形式,用于治疗或预防血管生成和验证,尤其是与肿瘤相关的病理性血管生成和炎症以及与非肿瘤疾病相关的病理性血管生成和炎症。
本发明进一步优势根据下述详细描述而显见,在所附图片的辅助下进行说明,其中:
-图1显示MTT试验的结果,旨在评估在下述逐步稀释物治疗后的HUVE细胞增殖:A)本发明的多酚浓缩物(样品A009);B)空白(样品A012);C)纯化的羟基酪醇(HyT);和D)纯化的羟基酪醇空白(即乙醇-EtOH)。
-图2显示治疗后24和48小时,用下述逐步稀释物治疗后的凋亡和HUVE细胞死亡试验的结果:A,C)本发明的多酚浓缩物(样品A009);B,D空白(样品A012)。
-图3显示治疗后24和48小时,用下述逐步稀释物治疗后的凋亡和HUVE细胞死亡试验的结果:A,C)纯化的羟基酪醇(HyT);B,D纯化的羟基酪醇空白(即乙醇-EtOH)。
-图4显示用下述试剂处理后,基于评估HUVE细胞在基质胶中形成毛状管型结构的能力的形态发生试验:A)无血清培养基(SFM),完全培养基(CTRL),本发明多酚浓缩物(样品A009)的1:500和1:250稀释物,空白(样品A012)的1:500和1:250稀释物;B)无血清培养基(SFM),完全培养基(CTRL),纯化的羟基酪醇(HyT)的1:500和1:250稀释物,纯化的羟基酪醇空白(即乙醇-EtOH)的1:500和1:250稀释物。
-图5显示用下述试剂处理后HUVE细胞的趋化性试验结果:A)完全培养基(CTRL),无血清培养基(SFM),本发明多酚浓缩物(样品A009)的逐步稀释物,空白(样品A012);B)完全培养基(C+),无血清培养基(SFM),纯化的羟基酪醇(HyT)的逐步稀释物,纯化的羟基酪醇空白(即乙醇-EtOH)的逐步稀释物。
-图6显示用下述试剂处理后HUVE细胞的化学侵入(chemoinvasion)试验结果:A)完全培养基(CTRL),无血清培养基(SFM),本发明多酚浓缩物(样品A009)的逐步稀释物,空白(样品A012);B)完全培养基(C+),无血清培养基(C-),纯化的羟基酪醇(HyT)的逐步稀释物,纯化的羟基酪醇空白(即乙醇-EtOH)的逐步稀释物。
-图7显示氧化应激(测定为DCFH-DA+细胞的百分比)对H2O2处理后用下述试剂处理的HUVE细胞的影响的结果:A)本发明多酚浓缩物(样品A009)的逐步稀释物和相应空白(样品A012);B)纯化的羟基酪醇(HyT)的逐步稀释物和相应空白(即乙醇-EtOH)。
-图8显示氧化应激(测定为DCFH-DA+细胞的百分比)对下述试剂预处理后再用H2O2处理的HUVE细胞的影响的结果:A)本发明多酚浓缩物(样品A009)的逐步稀释物和相应空白(样品A012);B)纯化的羟基酪醇(HyT)的逐步稀释物和相应空白(即乙醇-EtOH)。
-图9显示小鼠皮下移植的基质胶接种物的宏观比色测定分析结果,A)无处理(仅基质胶),存在VTH-VEGF、TGF、HGF(正对照C+),存在VTH和本发明多酚浓缩物(样品A009)的1:500稀释物以及相应空白(样品A012);B)无处理(仅基质胶),存在VTH-VEGF、TGF、HGF(正对照C+),存在VTH和纯化的羟基酪醇(HyT)的1:500稀释物。
-图10显示图9的移植的基质胶接种物中血红蛋白的试验。
本发明涉及含多酚化合物(优选羟基酪醇和3,4-DHPA-EDA)的植物水和残渣的植物混杂物或浓缩物,其用于治疗和预防血管生成和炎症。
优选地,所述血管生成和炎症是病理性血管生成和炎症,更优选肿瘤相关血管生成和炎症。或者,血管生成和炎症可不与肿瘤关联而与例如下述的病理关联:风湿性疾病,优选类风湿性关节炎和痛风;结直肠炎症疾病,优选克罗恩氏病、肠易激和肠溃疡性综合症;支气管病理,优选支气管慢性阻塞性肺病和哮喘;肝脏疾病,优选肝硬化和肝纤维化;前列腺疾病,优选前列腺增生和急性/慢性前列腺炎;粘膜炎;皮炎或肿瘤前病变,例如胸、子宫、肺或口腔病变。
事实上,已惊人发现本发明的植物水和/或橄榄残渣的浓缩物在预防肿瘤血管新生(即血管生成)方面特别有效。相比正常血管,这些血管具有不同物理化学结构,并因此具有不同生物学功能。它们大都有孔且通常缺乏周细胞,因而具有不同的血管渗透性。该结构通常导致许多药物治疗的失败或困难,由于这些药物在通过所述血管递送至肿瘤期间损失于间隙空间中,并因此不能到达靶标或不能以有效量到达靶标。抑制此类血管结构的形成明显可使物质递送至肿瘤的效果更好,并因而可改善肿瘤治疗。
植物水优选源自三相(油、植物水和残渣)和两相(油和残渣+植物水)橄榄研磨过程。优选地,研磨产生的植物水可用具有酸性pH(优选pH3-5,优选pH4-5,例如通过添加强酸)、以及具有果胶溶解酶(即水解橄榄表皮的纤维素基质的酶)的溶液处理。
残渣优选经捶打、稀释和预过滤。残渣优选具有0.5-1毫米(mm)的尺寸或截止值,更优选约0.7mm。截止值的示例是振动筛选所获得的那些。
若需要,捶打的残渣可溶解或分散于pH3-5,优选pH3.5-4的水性基质中。
溶解步骤目的在于溶解多酚,否则其仍困于橄榄表皮的固体基质中。
本发明优选实施方式中,植物水和/或橄榄残渣的浓缩物(下述“浓缩物”)还包括:至少一种优选自酪醇、氯原酸、β-羟基毛蕊花甙、芸香苷、毛蕊花甙和藤黄菌素的其他酚类化合物;和至少一种优选自钠、钙、镁和钾的金属;和至少一种优选自氯化物、硫酸盐、磷酸盐和硝酸盐的阴离子;和至少一种选自葡萄糖、果糖、甘露醇和蔗糖的糖。
本发明其他实施方式中,浓缩物包括含氮物(蛋白质、氨基酸),优选含量为15-60mg/kg,更优选20-40mg/kg(mg氮/L活性溶液)。任何情况中,浓缩物中存在的最大量的酚类化合物是羟基酪醇和3,4-DHPA-EDA。
优选地,羟基酪醇的含量范围为1-10克/L植物水(g/L),更优选1.5-5g/L,更优选2-3g/L。
优选地,4-DHPA-EDA的含量为0.5-8g/L,更优选1-6g/L,更优选1.5-2.5g/L。
优选地,酪醇的含量为0.1-0.4g/L,更优选0.15-0.25g/L。
优选地,氯原酸的含量为0.06-0.24g/L,更优选0.8-0.16g/L。
优选地,β-羟基毛蕊花甙的含量为0.3-1.5g/L,更优选0.5-1g/L。
优选地,芸香苷的含量为0.05-0.2g/L,更优选0.08-0.15g/L。
优选地,毛蕊花甙的含量为0.4-1.7g/L,更优选0.6-1g/L。
优选地,藤黄菌素的含量为0.1-0.5g/L,更优选0.15-0.28g/L。
优选地,钠的含量为75-300mg/L,更优选120-180mg/L。
优选地,钙的含量为5-10g/L,更优选2-5g/L。
优选地,镁的含量为220-900mg/L,更优选400-500mg/L。
优选地,钾的含量为3-15g/L,更优选6-9g/L。
优选地,氯化物的含量为1.5-7g/L,更优选2.5-4.5g/L。
优选地,硫酸盐的含量为12-45g/L,更优选18-28g/L。
优选地,磷酸盐的含量为1.5-7g/L,更优选2.5-5g/L。
优选地,硝酸盐的含量为12-50mg/L,更优选18-30mg/L。
优选地,葡萄糖的含量为15-60g/L,更优选25-35g/L。
优选地,果糖的含量为3.5-15g/L,更优选5-9g/L。
优选地,甘露醇的含量为1-4g/L,更优选1.5-3g/L。
优选地,蔗糖的含量为4-16g/L,更优选6-10g/L。
在本发明的优选实施方式中,浓缩物通过/可通过包含下述步骤的方法获得:(i)微过滤植物水和/或橄榄油渣的样品,获得微流化的透过物和浓缩物;和(ii)通过反渗透处理步骤(i)的微过滤透过物来进行浓缩。
优选地,微过滤在前述溶解步骤之后进行。
微过滤目的在于分离浓缩物,即植物水/残渣的悬液中内含物的浓缩组分,例如微片段、纤维和微粒材料例如细胞和细菌。其在此类基质的标准条件下进行。
除了浓缩物以外,通过微过滤步骤可获得透过物,即清澈组分,其特征为与起始材料不同的颜色以及含有植物水/残渣中的溶解组分,例如蛋白质、糖、盐、多酚、有机酸和各种可溶有机分子。
优选地,用至少一种且优选两种陶瓷膜进行微过滤。所述膜优选特征为管状。
在优选实施方式中,所述膜由氧化铝和氧化锆制成。
优选地,所述膜具有下述特征:外直径约30-约40mm、优选约25mm;长度约500-约1500mm、优选约1200mm;系列通道直径(优选水压直径)约2.5-约5mm、优选约3.5mm;过滤表面约0.15-约0.7m2、优选约0.35m2;分子截留值为约0.1微米-约300kDa。
浓缩获自前述植物水/残渣的微过滤的透过物的反渗透步骤在此类基质的标准条件下进行,优选通过使用多聚膜,更优选使用多胺。
具体地,所述膜具有卷绕螺旋形状以及排斥高盐的分子截留值,即能排斥99.9%的氯化钠分子。这意味着渗透膜捕获感兴趣的生物医药分子并仅允许水分子通过。
优选地,多聚膜的过滤面积为约5-约15m2,更优选约7m2。
反渗透步骤用作浓缩获自微过滤的透过物,优选浓缩约4倍;这表示100L微流化透过物会得到25L浓缩物。
在这种情况中,体积浓度比例(VCR)为4,即100/25。
基于起始基质(植物水)以及主要基于其盐含量,VCR可发生变化,这是由于反渗透过程必须抗衡发生浓缩的基质的渗透压。
本发明还涉及获自/可获自上述过程的植物水/残渣的浓缩物(或植物混杂物)。
浓缩物优选具有前述组成,涉及酚类化合物、金属、糖、阴离子和氮的含量。其可单独使用或与其他物质、分子或抗肿瘤和抗血管生成和抗炎治疗组合以治疗并防止血管生成和炎症,优选病理性血管生成和炎症,具体为与肿瘤相关的血管生成和炎症,或与例如下述病症的非肿瘤病理相关的血管生成和炎症:风湿性疾病,优选类风湿性关节炎和痛风;结直肠炎症疾病,优选克罗恩氏病、肠易激和肠溃疡性综合症;支气管病理,优选支气管慢性阻塞性肺病和哮喘;肝脏疾病,优选肝硬化和肝纤维化;前列腺疾病,优选前列腺增生和急性/慢性前列腺炎;粘膜炎;皮炎或肿瘤前病变,例如胸、子宫、肺或口腔病变。优选地,本发明的浓缩物可单独使用或与其他物质/分子组合使用,用于抑制(优选以预防方式)肿瘤血管形成。
本发明的所述肿瘤优选为结直肠癌、乳腺癌和前列腺癌、皮肤癌(黑素瘤和其他)、胰腺癌、肺癌、卵巢癌、膀胱癌、肾癌和肝癌。
本发明所述的与血管新生关联的炎症症状为:风湿性疾病,优选类风湿性关节炎和痛风;结直肠炎症疾病,优选克罗恩氏病、肠易激和肠溃疡性综合症;支气管病理,优选支气管慢性阻塞性肺病和哮喘;肝脏疾病,优选肝硬化和肝纤维化;前列腺疾病,优选前列腺增生和急性/慢性前列腺炎;粘膜炎;皮炎或肿瘤前病变,例如胸、子宫、肺或口腔病变。
本发明的其他方面涉及含前述植物水/残渣的浓缩物的饮料,以及可能包含生产各种饮料所通常添加的赋形剂。
所述饮料可基于水和果汁和奶。本发明特别优选的实施方式中,所述饮料基于果汁,优选基于葡萄汁。特别优选葡萄汁和葡萄液,优选源自有机葡萄。所述饮料可任选经冻干。
或者,前述植物水/残渣的浓缩物可配制成片剂、锭剂或药片用于口服。
实际中,饮料或口服制剂可作为食物补充,尤其是用于防止血管生成和炎症,优选是病理性血管生成和炎症,尤其是与肿瘤相关的那些或与例如下述疾病的非肿瘤病理相关的那些:风湿性疾病,优选类风湿性关节炎和痛风;结直肠炎症疾病,优选克罗恩氏病、肠易激和肠溃疡性综合症;支气管病理,优选支气管慢性阻塞性肺病和哮喘;肝脏疾病,优选肝硬化和肝纤维化;前列腺疾病,优选前列腺增生和急性/慢性前列腺炎;粘膜炎;皮炎或肿瘤前病变,例如胸、子宫、肺或口腔病变。
饮料或口服制剂优选可作为食物补充用于抑制(优选以预防方式)肿瘤血管形成。
任选地,所述饮料可与抗肿瘤和抗血管生成和抗炎物质、分子、药物或治疗联用。
本发明的其他方面涉及含前述植物水/残渣的浓缩物和可能的赋形剂的乳膏、油、油膏、雾、香波或凝胶。
所述乳膏、油、油膏或凝胶可用于治疗(优选局部)和/或防止由血管生成增加和/或改变和炎症所引起的生理病理性症状。
实施例
从橄榄植物水/残渣生产浓缩的多酚
整个生产过程主要使用基于膜的切向流过滤和分离技术。
从橄榄植物水和/或残渣生产多酚浓缩物所进行的膜处理仅使用两步过滤:微过滤和反渗透。然而,基于所需产品,可能用超滤和纳米过滤完成所述过程。
微过滤能分离悬浮固体、细菌和脂肪,同时反渗透捕获所有存在的物质,包括离子(还有单价离子)并仅允许水穿透。
所述过程从植物水开始进行,所述植物水源自三相橄榄研磨过程,但其还可用于源自预处理后的二相过程的湿残渣。
湿残渣可进行捶打、稀释和以截止值约0.7mm进行预过滤(例如通过振动筛),然后用膜系统处理,或者其可在三相倾滗器中可用稀释液处理并在膜过程处理之前在三相倾滗器中再处理。
所进行的微过滤过程目的在于分离植物水和/或残渣中存在的悬浮液中全部内含物的浓缩组分(微片段、纤维和微粒子材料例如细胞和细菌)。
微过滤透过物是清澈组分,其颜色根据处理的橄榄的品种而不同,含有植物水和/或残渣的所有可溶成分,例如蛋白质、糖、盐、多酚、有机酸和各种可溶有机分子。
为了进行微过滤,使用具有氧化锆选择层的两种管状氧化铝陶瓷膜,其具有下述特征:外径25mm、长度1178mm、通道水力直径为3.5mm的23个通道、过滤表面0.35m2并且分子截止值为0.14微米-300kDa。
微过滤中,通过使用具有通道水力直径为3.5mm的23个通道的膜,可以浓缩植物水/残渣直到获得总固体含量约12%的浓缩组分。
考虑总固体含量约7%的经处理的植物水,其可浓缩4倍,从而获得总固体含量为5.5%的透过物和固体含量12.1%的浓缩物。
本发明的多酚浓缩物通过浓缩透过物制成,所述透过物获自通过反渗透对植物水进行微过滤。
使用具有卷绕螺旋形状、高盐排斥并且过滤表面为7m2的聚酰胺制成的聚合膜,还可使用低盐排斥并且透过物中多酚损失较小的膜。
反渗透中,处理的植物的微过滤透过物可浓缩约3.6倍。
很明显,体积浓度比可基于起始基质、主要基于其盐含量、因此基于渗透压而不同。
测试的植物水的组成
多酚浓缩物的组成示于下表I:
表I
表1所示物质包含在识别码为A009的样品中,而空白(即阴性对照)用码A012识别。
空白获自用XAD7树脂的多酚浓缩物的分批色谱分离。
详细地,约50cc的XAD7树脂用蒸馏水漂洗,在乙醇中再生,然后再用蒸馏水漂洗。
用0.45微米滤器真空过滤来回收树脂,并在烧杯中加入约75ml的植物水浓缩物。树脂与浓缩物室温震荡接触约30分钟。
真空过滤可回收用XAD7树脂处理的植物水的浓缩物,电导率23.4mS/cm。
用XAD7树脂处理后回收的植物水浓缩物再用XAD7树脂处理,在乙醇中重生并用蒸馏水漂洗。
真空过滤后,回收用XAD7树脂处理两次的植物水浓缩物的空白的第三样品,电导率16.92mS/cm。
HUVEC(人脐带静脉内皮细胞)用作本发明多酚浓缩物的靶细胞模型,考虑到内皮细胞构成血管生成过程的基础单元。
用多酚浓缩物进行的分析所得结果与相同条件下仅用羟基酪醇处理HUVEC所获结果进行比较。选择羟基酪醇作为比较物质是因为其是样品A009中最多的多酚(2.70g/L)。
目的在于证明植物水(以所述浓缩物的形式)相较单一物质而言,除了具有抗血管生成效果之外还表现出更好的抑制能力。
为了评估所述效果是否确实是由于羟基酪醇而非其溶于的乙醇溶液,用相同浓度的含乙醇培养基进行分析,其中羟基酪醇作为空白样品溶解。
评估植物水的抗血管生成性能
样品的稀释物对应于表II中的mg/ml和μM值:
表II
稀释物 | mg/ml羟基酪醇 | μM羟基酪醇 |
1:100 | 0.0270 | 174.96 |
1:250 | 0.0108 | 69.984 |
1:500 | 0.0054 | 34.992 |
1:1000 | 0.0027 | 17.496 |
1:2500 | 0.00108 | 6.9984 |
1:5000 | 0.00054 | 3.4992 |
1:10000 | 0.00027 | 1.7496 |
评估植物水对内皮细胞的抗增殖效果
样品A009和A012对人内皮细胞(HUVEC)的影响通过MTT活性试验(四唑盐、[3-(4,5-二甲基噻唑-2-基)]-2,5-二苯基溴化四唑)进行评估。所述试验基于MTT化合物被线粒体酶、琥珀酸脱氢酶代谢的能力。盐的还原产生蓝色晶体产物甲瓒,其不溶于水。活力细胞(不同于无活力细胞)还原盐并且所产生的甲瓒的量与存在的细胞数量成正比。形成的晶体可溶,并通过分光光度仪读取570nm波长处的吸光度。
5000HUVE细胞接种于96孔板的各孔。具体地,所述孔用1%明胶包被,并在不同处理时间进行试验(24h,48h,72h,96h),各时间测试浓缩物的不同稀释物(1:10000-1:100间隔)。
对内皮细胞增殖的影响用下述浓缩物稀释物评估:1:10000-1:5000-1:2500-1:1000-1:500-1:250-1:100。
图1A可看出,用样品A009处理的内皮细胞的活力显著下降,具体地从1:1000稀释物处理24小时后开始。
样品A012(空白)对HUVEC活力没有影响(图1B)。
羟基酪醇还引起HUVE细胞的活力下降,具体从1:250稀释物处理24小时后开始(图1C)。含乙醇的培养基用作对照,对细胞活力没有任何影响(图1D)。实验重复进行2次。
因此可得出结论,样品A009对细胞活力的影响大于羟基酪醇。
细胞凋亡评估
用膜联蛋白-V和7-氨基-放线菌素D(7-AAD)标记来评估诱导浓缩物处理的内皮细胞凋亡(程序性细胞死亡)。这些标记的细胞荧光检测分析可区分在相同细胞群体中所述处理诱导不同阶段细胞死亡(早期、后期、晚期)的能力。膜联蛋白-V是能结合磷脂酰丝氨酸的分子,一种细胞膜的甘油磷脂,通常与内部胞浆层关联,其作为细胞损伤的后果而暴露于细胞外侧。7-AAD是仅能在损伤细胞或凋亡细胞中穿过细胞膜的化合物;对该标记物阳性表示细胞死亡并产物具有毒性。
150,000HUVE细胞接种在用1%明胶包被的6孔板的孔中。然后细胞用不同样品稀释液处理(24h和48h),然后通过胰蛋白酶处理从板上分离并用7-AAD和膜联蛋白V染色以评估凋亡水平。
以样品A009、A012和HyT-EtOH的不同稀释物(1:2500–1:250的稀释范围)处理内皮细胞24和48小时,并分析对两种标记物的阳性。
从图2A和2C可看出,样品A009的1:250稀释物在24小时和48小时处理后分别诱导50%和75%的内皮细胞发生后期阶段的凋亡。样品A012的相同稀释物不产生任何凋亡前效应(约95%细胞具有活力)。
相反,用羟基酪醇和用含乙醇培养物的处理不增加细胞坏死(图3)。
试验重复进行2次。
因此,羟基酪醇比本发明浓缩物的活性低。
评估植物水对内皮细胞的形态的影响
当在胞外基质中体外培养并暴露于合适的活化刺激时,内皮细胞能组装成管状结构,模拟血管的内腔结构。
通过基质胶中的形态试验(即由层粘连蛋白、胶原蛋白IV、副层连蛋白、硫酸乙酰肝素蛋白多糖、生长因子(例如PDGF,EGF,TGF-β和MMP)组成的聚合物)可评估所选化合物的抗血管生成能力。为了评估本发明浓缩物体外抑制管的形成的能力,24孔板的各孔包被300μL基质胶(10mg/mL),然后基质多聚化后,1mL完全培养基中的50,000HUVE细胞接种在所述多聚化基质中。用样品A009、A012、HyT和EtOH(1:500和1:250)的不同稀释物预处理细胞24小时。
阳性和阴性对照分别使用由补充有下述物质的M199培养基组成的完全培养基(CM):10ng/mlaFGF(酸性成纤维细胞生长因子)、10ng/mlbFGF(碱性成纤维细胞生长因子)、10ng/mlEGF(表皮生长因子)、0.1mg/ml肝素、0.10μg/ml氢化可的松、10%FBS(胎牛血清)、1%谷氨酰胺(Gln)、1%氨苄青霉素/链霉素(P/S)和生长因子以及无血清培养基M199(SFM)。
样品的抑制影响在37℃和5%CO2下孵育6小时后通过在显微镜下观察管状结构的形成评估。
如图4A所示,样品A009能以剂量依赖的方式干扰内皮细胞形成稳定的管状结构。样品A012(空白)的相同稀释物不产生任何抑制效果。
羟基酪醇也能以剂量依赖的方式干扰内皮细胞形成稳定的管状结构,而乙醇稀释物(空白)仅产生轻微抑制效果(图4B)。试验重复进行2次。
因此,羟基酪醇抑制内皮形态的效果低于本发明浓缩物,样品A009。
评估植物水对内皮细胞迁移活性的抑制能力
内皮细胞的特征为能沿着趋化梯度向特定位置迁移。为了评估HUVEC的迁移能力,用Boyden腔室和截止值12μm并用胶原(50μg/mL)浸没的多孔滤器设置迁移试验。
各腔室的低级隔室装载210μLCM,含有10ng/mLαFGF、10ng/mlbFGF、10ng/mLEGF、0.1mg/mL肝素、0.10μg/mL氢化可的松、10%FBS、1%Gln、1%P/S,或生长因子和无血清培养基M199(SFM)。
用浓缩物、空白、羟基酪醇或含乙醇培养基的多样稀释物(1:2500、1:1000、1:500和1:250)预处理24小时的50000HUVE接种在腔室的上部隔室的500μL无血清培养基中。
将细胞在37℃,5%CO2下孵育6小时。
试验末期,机械法清洁滤器从而消除上侧未迁移细胞;然后将其在纯乙醇中固定5分钟,随后再水化并用活力染色DAPI标记。各滤器上存在的细胞在荧光显微镜下计数。具体地,各滤器随机分析5个光学区域。
如图5A所示,本发明浓缩物(样品A009)的1:500和1:250稀释物能以统计上显著的程度(分别为p值=0.0057和p值=0.0003)干扰内皮细胞的迁移能力,而样品A012(空白)不干扰其迁移能力。
相比乙醇培养基,1:250稀释的羟基酪醇降低内皮细胞的迁移能力,p值=0.0302(图5B)。还可观察到在羟基酪醇的高稀释物中,内皮细胞迁移能力提高,而本发明的浓缩物对其抑制。试验重复进行2次。
因此,用单一物质的羟基酪醇干扰内皮细胞迁移的能力较低,即其并不有效。
评估植物水对内皮细胞侵入活性的抑制能力
原位形成后,内皮细胞能侵入周围组织,产生能降解胞外基质的因子,所述胞外基质构成对其实际迁移至趋化位置的物理屏障。
用Boyden腔室和截止值12μm并用基质胶(1mg/mL)包被的多孔滤器设置侵入试验。
各腔室的低级隔室装载210μL完全培养基CM,含有10ng/mLaFGF、10ng/mlbFGF、10ng/mLEGF、0.1mg/mL肝素,0.10μg/mL氢化可的松、10%FBS、1%Gln、1%P/S,或210μL生长因子和无血清培养基M199(SFM)。
用500μLSFM重悬并用浓缩物、空白、羟基酪醇或无羟基酪醇的溶液的不同稀释物(1:2500、1:1000、1:500和1:250)预处理24小时的50000HUVE细胞37℃,5%CO2孵育24小时。
试验末期,机械法清洁滤器从而消除上侧未穿透基质的细胞。含以侵入基质胶的细胞的滤器在纯乙醇中固定5分钟,随后再水化并用活力染色DAPI标记。各滤器上的细胞在荧光显微镜下计数;各滤器随机选取5个光学区域进行分析。
图6A的结果表明样品A009(本发明浓缩物)的1:500和1:250稀释物能以统计上显著的程度(分别为p值=0.0335和p值=0.0011)干扰内皮细胞的侵入能力,而样品A012(空白)不影响其侵入能力。
如图6B所示,相比乙醇培养基,1:500和1:250稀释的羟基酪醇降低内皮细胞的侵入能力,p值=0.0016和p值=0.0159。
羟基酪醇在较长的时间内具有更好的效果,因其抑制细胞侵入而非迁移。
本发明的浓缩物针对迁移和侵入均有效。
评估对用H
2
O
2
处理后的植物水的氧化应激引起的损伤的保护作用
活性氧物质(ROS)代表细胞损伤的主要机制之一,其在炎症过程中有基础作用,因此在炎症相关的血管生成中也有基础作用。因此评估面对HUVE细胞的抗氧化剂能力,所述细胞用H2O2处理后用植物水浓缩物处理,通过DCFH-DA(2’,7’-二氯荧光素-二乙酸酯)标记进行评估。
DCFH-DA是能显示胞内H2O2浓度的物质,可用细胞荧光仪检测。
150,000HUVE细胞接种在6孔板的包被有1%明胶的各孔中的完全培养基中。然后细胞用胰蛋白酶消化并在完全M199培养基中重悬,H2O2浓度250μM。然后将细胞置于孵育器中,37℃和5%CO2暗培养15分钟。然后细胞用DPBS清洗,然后通过5分钟1200rpm离心消除上清。最后,在含10μMDCFH-DA和H2O2(阳性对照)或浓缩物(A009和A012)、羟基酪醇或无羟基酪醇的溶剂的稀释液的DPBS中重悬细胞。在含5%CO2的大气中于37℃暗孵育细胞45分钟。最后,用FACSCanto读取结果。
图7A的结果表明样品A009(本发明的浓缩物)能以剂量依赖方式产生高度显著(p-值1:2500=0.0310,p-值1:1000=0.0001,p-值1:500=0.0001,p-值1:250<0.0001)的保护效果。
羟基酪醇产生抗氧化效果(图7B),而溶解乙醇的培养基没有抗氧化效果。
结果表明本发明浓缩物能产生更显著的抗氧化效果。羟基酪醇的p值实际上更低:p-值1:2500=0.0115,p-值1:1000=0.0062,p-值1:500=0.0082和p-值1:250=0.0223。
试验重复进行2次。
评估对用H
2
O
2
处理前的植物水的氧化应激引起的损伤的保护作用
150,000HUVE细胞接种在6孔板的包被有1%明胶的各孔的完全培养基中。然后用胰蛋白酶消化细胞并重悬在标准稀释的完全M199培养基和植物水浓缩物(A009和A012)、羟基酪醇或含乙醇培养基中。
然后将细胞置于孵育器中,37℃和5%CO2暗培养30分钟。DPBS清洗后,对阳性对照和分析的样品均将细胞在含10μMDCFH-DA和H2O2的DPBS中重悬。细胞置于在5%CO2、37℃的暗孵育器中45分钟。最后,用FACSCanto读取结果。
图8中可知样品A009以剂量依赖方式产生保护效果,同时可观察到ROS的产生在空白(A012)处理的样品中依然很高。ROS生产的降低在样品A009的情况中非常显著:p-值1:2500=0.0056,p-值1:1000=0.0015,p-值1:500<0.0001和p-值1:250<0.0001。
图8B的结果表明若在H2O2的诱导氧化后使用羟基酪醇没有显著抗氧化效果。此外,可观察到没有羟基酪醇的溶剂对细胞没有显著效果,如用H2O2预处理的情况所示。
试验重复进行2次。
综上,除了比本发明浓缩物具有更低的抗氧化能力之外,羟基酪醇若在H2O2的诱导后的预处理中使用则不能保持其能力。
在小鼠模型中评估植物水的抗血管生成能力
植物水浓缩物体内抑制血管形成的能力通过基质胶海绵(sponge)试验进行评估,如Albini等“AngiogenicpotentialinvivobyKaposi'ssarcomacell-freesupernatantsandHIV-1tatproduct:inhibitionofKS-likelesionsbytissueinhibitorofmetalloproteinase-2(Kaposi's瘤无细胞上清和HIV-1tat产物的体内抗血管生成能力:通过金属蛋白酶-2组织抑制剂抑制KS样损伤)(AIDS,1994)”所述。
具体地,c57/BL6品系的8周雄性小鼠用作动物模型。基质胶粒料与不同稀释的样品A009或对应空白(A012)的组合皮下注射入小鼠。基质胶4℃时表现为液体形式,室温快速聚合;该特性开发用于刺激皮下肿瘤,分泌促血管生成物质。实验中,液体形式的基质胶与含血管生成所需所有因子(由VEGF(100ng/μl),TNFα(1.2ng/μl)和肝素(25U/ml)组成)的称为VTH的混合物关联。
VTH作为阳性对照,由于VEGF代表内皮细胞的主要生长因子,TNFα构成招募炎症细胞组分的必须细胞因子,肝素用于维持内皮招募和活化所在粒料中形成的血液。一旦皮下注射入动物胁腹,基质胶快速聚合并释放其中所含因子。其作为内皮和炎症细胞的化学引诱物,一旦其浸润基质胶,其保持被捕获的状态并在基质形成的“微环境”中被活化。为了所研究的试验,各条件使用三只小鼠。所采用的条件是:1)仅基质胶(阴性对照)、2)基质胶和VTH(阳性对照)、3)含VTH与2种稀释的植物水浓缩物(1:500和1:250)的基质胶,对待分析的样品和空白样品均是如此。此外,针对羟基酪醇测试相同的稀释物。
第0天注射基质胶粒料;第4天处死小鼠移出粒料。移植的粒料经称重并置于300μlPBS中,然后分为两部分:一半用于血红蛋白试验,其为血管生成的标志物(内皮细胞招募和活化的结果是粒料中存在血液),而另一半包埋于OCT中用于进行免疫组化染色以评估内皮细胞和炎症组分。
为了定量血红蛋白,粒料经机械崩解,4℃13000g离心12分钟,移出上清。
然后200μl上清置于Eppendorf管中,其中加入800μlDrabkin溶液。该物质结合血红蛋白,形成沉淀晶体,因而能产生分光光度仪读取的吸光度(540nm):血红蛋白浓度与形成的晶体数量和记录的吸光度直接成正比。血红蛋白定量根据下述数学模型:
HB=(540nm吸光度/粒料重量mg)x100
图9A中可观察到染色与内皮浸润和因此存在的血液成正比。
图9B可观察到用1:500羟基酪醇处理的粒料的染色比阳性对照强。
图10显示关于粒料的血红蛋白含量的定量相关的数据。可观察到相比与样品A012(空白)和羟基酪醇关联的粒料,与本发明浓缩物(A009)关联的血红蛋白浓度的降低在统计上显著(p-值=0.0058)。
综上,上述实验结果清楚地表明获自植物水的多酚浓缩物(样品A009)具有抗血管生成活性。事实上,本发明浓缩物抑制内皮细胞的活力并增加其凋亡。此外,其还阻止用于刺激胞外基质的合成基质的迁移和侵入。最后,通过用本发明浓缩物处理HUVEC,还可观察到对血管系统的管状结构形成的抑制。
除了体外结果,本发明浓缩物的抗血管生成能力还在生理系统中清楚地得以证明。
所有实验通过比较浓缩物和相同的多酚纯化浓缩物(空白样品)来进行,从而能确定所观察到的效果是由于感兴趣的分子而非可能干扰实验的其他因子。
此外,还单独测试羟基酪醇,所有测试表明其效果和活性比本发明的浓缩物差。
因此,上述实验结果表明通过使植物水的微过滤透过物经历反渗透所获得的多酚浓缩物具有抗血管生成性质。
具体地,所述浓缩物相比仅羟基酪醇具有改善的抗血管生成效果,后者是植物水中含有的主要多酚化合物。
Claims (14)
1.含羟基酪醇和3,4-DHPA-EDA的植物水和/或橄榄残渣的浓缩物,其用于治疗和/或预防血管生成。
2.如权利要求1所述的浓缩物,其还包括:至少一种优选自酪醇、氯原酸、β-羟基毛蕊花甙、芸香苷、毛蕊花甙和藤黄菌素的酚类化合物;和/或至少一种优选自钠、钙、镁和钾的金属;和/或至少一种优选自氯化物、硫酸盐、磷酸盐和硝酸盐的阴离子;和/或至少一种选自葡萄糖、果糖、甘露醇和蔗糖的糖;和/或氮。
3.生产植物水和/或橄榄残渣的浓缩物的方法,所述方法包括:
(i)微过滤植物水和/或橄榄残渣的样品,获得微过滤的透过物和浓缩物;和
(ii)通过反渗透处理步骤(i)的微过滤透过物来进行浓缩。
4.如权利要求3所述的方法,其中所述微过滤步骤涉及使用至少一种陶瓷膜,其优选管状。
5.如权利要求4所述的方法,其中所述膜由氧化铝和氧化锆制造。
6.如权利要求3-5中任一项所述的方法,其中所述反渗透通过使用聚合膜进行,所述聚合膜优选由聚酰胺制成,所述膜优选为螺旋形。
7.用权利要求3-6所述的方法可获得的植物水和/或橄榄残渣的浓缩物,其用于治疗和/或预防血管生成。
8.如权利要求1或2或7所述的浓缩物,其中所述血管生成是病理性血管生成,优选所述血管生成是肿瘤或非肿瘤血管生成。
9.如权利要求8所述的浓缩物,其中所述非肿瘤血管生成与下述关联:风湿性疾病,优选类风湿性关节炎和痛风;结直肠炎症疾病,优选克罗恩氏病、肠易激和肠溃疡性综合症;支气管病理,优选支气管慢性阻塞性肺病和哮喘;肝脏疾病,优选肝纤维化和肝硬化;前列腺疾病,优选良性前列腺增生和急性/慢性前列腺炎;粘膜炎;皮炎或肿瘤前病变,优选乳腺、子宫、肺或口腔病变。
10.如权利要求1或2或7中任一项所述的植物水和/或橄榄残渣的浓缩物,其用于抑制肿瘤血管形成。
11.含权利要求1或2或7中任一项所述的植物水和/或橄榄残渣的浓缩物的饮料。
12.如权利要求11所述的饮料,其用于治疗和/或预防血管生成。
13.如权利要求12所述的饮料,其中所述血管生成是病理性血管生成,优选所述血管生成是肿瘤或非肿瘤血管生成。
14.如权利要求11-13中任一项所述的饮料,其中所述饮料基于水和/或水果和/或奶;优选基于葡萄汁和/或葡萄液。
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US11918007B2 (en) | 2017-12-14 | 2024-03-05 | Universidade Do Porto | Foodstuff composition comprising a derivate of olive pomace |
IT201800002266A1 (it) * | 2018-01-31 | 2019-07-31 | Fattoria La Vialla Di Gianni Antonio E Bandino Lo Franco Soc Agricola Semplice | Uso cosmetico di acque di vegetazione |
WO2020014610A1 (en) * | 2018-07-12 | 2020-01-16 | International Flavors & Fragrances Inc. | Verbascoside and related compounds for sweetness enhancement |
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Also Published As
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HK1221903A1 (zh) | 2017-06-16 |
BR112016009606A2 (pt) | 2017-09-19 |
ITMI20131814A1 (it) | 2015-05-01 |
US20160250272A1 (en) | 2016-09-01 |
JP2017501209A (ja) | 2017-01-12 |
US10226498B2 (en) | 2019-03-12 |
WO2015063736A1 (en) | 2015-05-07 |
CN105744943B (zh) | 2020-10-09 |
CA2928913A1 (en) | 2015-05-07 |
EP3062805A1 (en) | 2016-09-07 |
JP6687527B2 (ja) | 2020-04-22 |
EP3062805B1 (en) | 2019-08-28 |
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