从冷冻干燥的浆果粉中提取和纯化多酚用于治疗血管平滑肌细胞<em>体外</em
Summary
本工作详细介绍了从冷冻干燥的浆果粉中制备富含多酚的提取物的逐步方法。此外，它提供了在使用血管平滑肌细胞（VSMC）的肽激素血管紧张素II（Ang II）存在下如何在细胞培养物中使用这些富含多酚的提取物的详细描述。
Abstract
流行病学研究表明，增加的类黄酮摄入与美国（美国）和欧洲的心血管疾病（CVD）死亡率降低相关。浆果在美国广泛消费，多酚含量高。已经显示多酚与许多分子靶标相互作用并发挥许多积极的生物功能，包括抗氧化剂，抗炎和心脏保护作用。从黑莓（BL），覆盆子（RB）和黑莓（BRB）分离的多酚可以减少血管紧张素II（Ang II）的氧化应激和细胞衰老。这项工作提供了从冻干浆果制备多酚提取物的方案的详细描述。使用80％乙醇水溶液和超声波辅助提取方法，从冷冻干燥的浆果粉中进行多酚提取。粗提物进一步纯化，用氯仿和乙酸乙酯分级分离，分别。在培养物中的血管平滑肌细胞（VSMC）上测试粗制和纯化提取物的作用。
Introduction
多酚是在其结构中含有至少一个酚环的化合物，并且大量存在于植物界1中 。人类已经花了数千年的时间消耗植物用于药用，而没有意识到这种化合物的存在2 。许多水果和蔬菜具有一些共享的多酚化合物，尽管量不同，包括类黄酮，二苯乙烯和酚酸3 。虽然多酚通常与五颜六色的水果和蔬菜有关，但这并不是完全正确的。例如，玉米黄质和黄嘌呤存在于不是高度多彩的蔬菜中，例如洋葱和大蒜，其来自the the家庭并且与许多健康益处相关4 。除了与几种健康益处相关5 ，多酚还通过保护它们免受昆虫的影响而为植物服务d紫外线辐射2 。多酚通常在人类饮食中发现，被认为是强大的抗氧化剂，因为它们可以清除活性氧物质（ROS） 6,7,8 。它们还具有抗炎9 ，抗菌10 ，抗高血压11 ，抗致癌12,13属性。
流行病学研究表明，类黄酮和心血管疾病（CVD）发生率16,17和死亡率14,15之间呈负相关。浆果在美国广泛消费，并含有大量的多酚，包括类黄酮。例如，消费黑莓（BL）果汁（300 mL / d）8周，血脂异常患者的收缩压明显降低18 。 Jeong et al。 19报道，与服用安慰剂的人相比，每天摄入2.5克黑莓（BRB）提取物的高血压男性和女性的24小时和夜间血压较低。覆盆子（RB）降低血压，同时增加自发性高血压大鼠超氧化物歧化酶（SOD）的表达20 。最近已经显示，BL，RB和BRB降低血管平滑肌细胞（VSMC）中血管紧张素II（Ang II）诱导的ROS和衰老水平21 。另外，来自BL提取物的花色素苷分数降低了诱导型一氧化氮合酶（iNOS）的表达，并抑制了脂多糖（LPS）刺激下的核因子κB（NF-κB）和细胞外信号调节激酶（ERK）的活性J774细胞ass =“xref”> 22。 BRB提取物在体外降低NF-κB活化和环氧合酶2（COX-2）表达23 ，改善脂质分布，并预防喂食高脂肪饮食的小鼠动脉粥样硬化病变形成24 。被认为是浆果中最丰富的类黄酮的花青素通过减少肿瘤坏死因子α（TNF-α）产生25并减少VSMC 26的增殖和迁移来调节LPS刺激的RAW 264.7巨噬细胞中的炎症反应。
由于越来越多的兴趣了解多酚在人体健康和疾病中的作用，重要的是优化提取方法。溶剂萃取广泛用于该目的，因为其具有成本效益且易于重现。在本研究中，用乙醇和超声辅助提取物一起进行溶剂萃取n方法，改编自Kim和Lee 27 。使用氯仿和乙酸乙酯对粗提物（CE）进行纯化和分级，得到从Queires 等 28获得的纯化提取物（PE）级分。此外，比较了在降低ERK1 / 2的基础磷酸化时，来自BL的粗制品与纯化多酚提取物的功效，并提供了纯化的BL多酚提取物对血管紧张素II诱导的VSMC信号降低的抑制作用的代表性实例。
Protocol
1.试剂的制备通过混合80 mL无水乙醇（分子生物学级）和20 mL细胞培养级无菌水，制备80％乙醇（100mL）。 为了制备多酚提取物（10mg / mL），称量10mg CE或PE。在细胞培养罩下加入1mL平原Dulbecco's Modified Eagle Medium（DMEM）。涡流解决方案。等分于200μL，储存于-20°C。 准备裂解缓冲液加入5 mL 1M HEPES储备溶液（pH 7.4; 50mM最终），1mL 5M NaCl储备溶液（最终50mM），1mL 0.5M EDTA储备溶…
Representative Results
以前已经证明，从BL，RB和BRB分离的多酚提取物降低VSMC响应Ang II 21的衰老。已经显示这些纯化的多酚提取物通过降低Akt，p38丝裂原活化蛋白激酶（MAPK）和ERK1 / 2的磷酸化来调节Ang II信号传导。 BL通过降低NADPH氧化酶（Nox）1的表达来防止衰老，NADPH氧化酶（Nox）1是产生超氧化物阴离子并被Ang II强烈上调的酶。相比之下，RB和BRB通过Nox1非依赖性机制防止衰老?…
Discussion
从浆果中分离的多酚含有不同的成分。这里描述的基于乙醇的提取方案允许鉴定存在于粗制和纯化的多酚提取物中的不同水平的酚酸和类黄酮（ 表1 ）。 CE富含没食子酸，阿魏酸，4-O-咖啡酰奎宁酸和5-O-咖啡酰奎宁酸。纯化方法没有显着改变没食子酸和对香豆酸的含量。然而，它提高了3-0-椰油酰奎宁酸的水平，从170.5-235.3ppm和槲皮素从24.5-95ppm。相比之下，在EDTA的纯化过程中阿魏酸和?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由美国心脏协会（14GRNT20180028）和佛罗里达州立大学研究和创意理事会（COFRS）资助。
Materials
| Angiotensin II | Sigma-Aldrich, Inc. | A9525-10MG | Treatment of VSMCs |
| β-actin | Sigma-Aldrich, Inc. | A2228 | Primary antibody (1:5000) |
| Blackberry fruit | Mercer Foods | Freeze-dried blackberry powder | |
| Catalase | Calbiochem | 219010 | Primary antibody (1:1000) |
| Chloroform | Biotech Grd, Inc. | 97064-678 | Preparation of purified polyphenol extracts |
| DMEM | Mediatech, Inc. | 10-014-CV | Culture of VSMCs |
| Ethanol (absolute molecular biology grade) | Sigma-Aldrich, Inc. | E7023-500ML | Preparation of polyphenol extracts |
| Ethylacetate | Sigma-Aldrich, Inc. | 439169 | Preparation of purified polyphenol extracts |
| ERK1/2 | Cell Signaling Technology, Inc. | 9102S | Primary antibody (1:500) |
| EDTA, 500 mM, pH 8.0 | Teknova, Inc. | E0306 | Lysis buffer |
| Freeze-Dryer | Labconco | VirTis Benchtop K | Preparation of polyphenol extracts |
| FBS | Seradigm | 1400-500 | Cell culture |
| HEPES | Sigma-Aldrich, Inc. | H3375 | Lysis buffer |
| NaCl | EMD Millipore, Inc. | 7760 | Lysis buffer |
| NaF | J.T.Baker, Inc. | 3688-01 | Lysis buffer |
| Na3VO4 | Sigma-Aldrich, Inc. | 450243 | Lysis buffer |
| Na4P2O7 , decahydrate | Sigma-Aldrich, Inc. | S-9515 | Lysis buffer |
| phospho ERK1/2 | Cell Signaling Technology, Inc. | 9101S | Primary antibody (1:1000) |
| Protease inhibitor cocktail | Sigma-Aldrich, Inc. | P8340-5ml | Lysis buffer |
| Protein assay dye reagent | Bio-Rad Laboratories, Inc. | 500-0006 | Protein concentration Measurement |
| PVDF transfer membrane | Thermo Scientific, Inc. | 88518 | Western blots |
| Rotatory Evaporator | Buchi Labortechnik | Rotavapor R3000 |
Preparation of polyphenol extracts |
| Sterile water | Mediatech, Inc. | 25-055-CV | Preparation of polyphenol extracts |
| Sonicator | QSonica, LLC | Q125 | Preparation of cell extracts |
| SOD2 | Enzo Life Sciences, Inc. | ADI-SOD-110-F | Primary antibody (1:1000) |
| Triton-X-100 | Sigma-Aldrich, Inc. | X100 | Western blots |
| Whatman #2 filter paper | GE Healthcare, Inc. | 28317-241 | Preparation of polyphenol extracts |
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Cite This Article
Feresin, R. G., Pourafshar, S., Huang, J., Zhao, Y., Arjmandi, B. H., Salazar, G. Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro. J. Vis. Exp. (125), e55605, doi:10.3791/55605 (2017).