Name: extraction and purification of polyphenols from freeze-dried berry powder for the treatment of vascular smooth muscle cells in vitro
Uploaded: 2017-07-05T14:00:00
Description: Watch this Scientific Journal Video about Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro at JoVE.com

Dette arbejde blev finansieret af American Heart Association (14GRNT20180028) og Florida State University Council for Research and Creativity (COFRS).

1. Fremstilling af reagenser <ol><li> Tilbered 80% ethanol (100 ml) ved at blande 80 ml absolut ethanol (molekylærbiologi-grade) og 20 ml steriliseret cellekulturklasse. </li><li> For at forberede polyphenol ekstrakt (10 mg / ml), veje 10 mg CE eller PE. Tilsæt 1 ml almindeligt Dulbecco&#39;s Modified Eagle Medium (DMEM) under en cellekulturhætte. Vortex opløsningen. Aliquot i 200 μl portioner og opbevar ved -20 ° C. </li><li> Forbered lysis buffer. <ol><li> Tilsæt 5 ml 1 M HEPES stamopløsning (pH 7,4; 50 mM e...

<ol>
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Polyfenoler isoleret fra bær indeholder tydelige præparater. Den her beskrevne ethanolbaserede ekstraktionsprotokol tillader identifikation af forskellige niveauer af phenol syrer og flavonoider, der findes i rå og rensede polyphenolekstrakter af BL ( tabel 1 ). CE blev beriget i gallinsyre, ferulinsyre, 4-0-caffeoylquininsyre og 5-0-caffeoylquininsyre. Renseprocessen ændrede ikke signifikant niveauerne af gallinsyre og p-coumarsyre. Det øgede imidlertid niveauerne af 3- O- caffeoylquinins...

Polyfenoler er forbindelser indeholdende mindst en phenolisk ring i deres struktur og er rigeligt til stede i planteområdet 1 . Mennesker har brugt planter i årtusinder til medicinske formål uden at være opmærksom på eksistensen af ​​sådanne forbindelser 2 . Mange frugter og grøntsager har nogle delte polyphenoliske forbindelser, om end med forskellige mængder, herunder flavonoider, stilbener og phenol syrer 3 . Selvom polyfenoler ofte er forbundet med farverige frugter og grøntsager, er dette ikke strengt sandt. For eksempel er zeaxanthin og xanthin til stede i grøntsager, der ikke er yderst farverige, såsom løg og hvidløg, der er fra familien af ​​scallions og er forbundet med mange sundhedsmæssige fordele 4 . Bortset fra at være forbundet med flere sundhedsmæssige fordele 5 , tjener polyphenoler også planter ved at beskytte dem mod insekterD ultraviolet stråling 2 . Polyfenoler findes almindeligvis i den menneskelige kost og betragtes som kraftige antioxidanter, da de kan scavenge Reactive Oxygen Species (ROS) 6 , 7 , 8 . De har også antiinflammatoriske 9 , antimikrobielle 10 , antihypertensive 11 og anti-kræftfremkaldende 12 , 13 egenskaber. Epidemiologiske undersøgelser viser en invers forbindelse mellem forbruget af flavonoider og kardiovaskulær sygdom (CVD) incidensen 16 , 17 og dødelighed 14 , 15 . Bær er meget forbrugt i USA og har store mængder polyphenoler, herunder flavonoider. For eksempel forbrug af blackberry (BL) juice (300<html> ML / d) i otte uger signifikant nedsat systolisk blodtryk hos dyslipidemiske patienter 18 . Jeong et al. 19 rapporterede, at prehypertensive mænd og kvinder forbruge 2,5 g sort hindbær (BRB) ekstrakt om dagen havde lavere 24-timers og nattt blodtryk sammenlignet med dem, der indtog en placebo. Hindbær (RB) reducerede blodtrykket, mens ekspression af superoxiddismutase (SOD) blev øget hos spontant hypertensive rotter 20 . Det har for nylig vist sig, at BL, RB og BRB reducerer niveauerne af ROS og senescence induceret af angiotensin II (Ang II) i vaskulære glatte muskelceller (VSMC&#39;er) 21 . Desuden reducerede anthocyaninfraktionen fra BL-ekstrakt ekspressionen af ​​inducerbar nitrogenoxidsyntase (iNOS) og inhiberede aktiviteten af ​​Nuclear Factor kappa B (NF-KB) og ekstracellulær signalreguleret kinase (ERK) i lipopolysaccharid (LPS) -stimuleret J774 cellerAss = &quot;xref&quot;&gt; 22. BRB-ekstrakter reducerede NF-KB-aktiveringen og cyclooxygenase 2 (COX-2) -expressionen in vitro 23 , forbedrede lipidprofilen og forhindrede atheroscleroselæsionsdannelse hos mus, der fodrede en fedtholdig diæt 24 . Anthocyaniner, der betragtes som de mest omfattende flavonoider i bær, modulerer det inflammatoriske respons i LPS-stimulerede RAW 264.7-makrofager ved at reducere Tumor Necrosis Factor alfa (TNF-a) -produktion 25 og reducere proliferationen og migreringen af ​​VSMC&#39;er 26 . Da der har været voksende interesse for at forstå polyphenols rolle i menneskers sundhed og sygdom, er det vigtigt at optimere ekstraktionsmetoden. Opløsningsmiddeludvindingen bruges i vid udstrækning til det formål, da det er omkostningseffektivt og let reproducerbart. I denne undersøgelse blev en opløsningsmiddelekstraktion anvendt med ethanol sammen med en ultralydassisteret ekstraktionN-metode, som blev tilpasset fra Kim og Lee 27 . Rensningen og fraktioneringen af ​​råekstrakter (CE) under anvendelse af chloroform og ethylacetat blev udført for at opnå den oprensede ekstrakt (PE) fraktion, som var tilpasset fra Queires et al. 28 . Endvidere blev effektiviteten af ​​rå versus oprensede polyphenolekstrakter fra BL sammenlignet med BL ved reduktion af den basale phosphorylering af ERK1 / 2 sammenlignet, og repræsentative eksempler på den inhibitoriske virkning af oprenset BL-polyphenolekstrakt på Ang II-inducerede signalreduktioner i VSMC&#39;er blev tilvejebragt.

<table border="0" cellpadding="0" cellspacing="0" width="971">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Angiotensin II</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">A9525-10MG</td>
			<td style="width:343px;">Treatment of VSMCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">&#946;-actin</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">A2228</td>
			<td>Primary antibody (1:5000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Blackberry fruit</td>
			<td style="width:157px;">Mercer Foods</td>
			<td style="width:168px;"></td>
			<td>Freeze-dried blackberry powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Catalase&#160;</td>
			<td style="width:157px;">Calbiochem</td>
			<td style="width:168px;">219010</td>
			<td>Primary antibody (1:1000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td style="width:157px;">Biotech Grd, Inc.</td>
			<td style="width:168px;">97064-678</td>
			<td>Preparation of purified polyphenol extracts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:157px;">Mediatech, Inc.</td>
			<td style="width:168px;">10-014-CV</td>
			<td>Culture of VSMCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol (absolute molecular biology grade)</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">E7023-500ML</td>
			<td>Preparation of polyphenol extracts&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylacetate</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">439169</td>
			<td>Preparation of purified polyphenol extracts</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:303px;">ERK1/2</td>
			<td style="width:157px;">Cell Signaling Technology, Inc.</td>
			<td style="width:168px;">9102S</td>
			<td>Primary antibody (1:500)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA, 500 mM, pH 8.0</td>
			<td style="width:157px;">Teknova, Inc.</td>
			<td style="width:168px;">E0306</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Freeze-Dryer</td>
			<td style="width:157px;">Labconco</td>
			<td style="width:168px;">VirTis Benchtop K</td>
			<td>Preparation of polyphenol extracts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">FBS</td>
			<td style="width:157px;">Seradigm</td>
			<td style="width:168px;">1400-500</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">HEPES</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">H3375</td>
			<td>Lysis buffer&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaCl</td>
			<td style="width:157px;">EMD Millipore, Inc.</td>
			<td style="width:168px;">7760</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaF</td>
			<td style="width:157px;">J.T.Baker, Inc.</td>
			<td style="width:168px;">3688-01&#160;</td>
			<td>Lysis buffer</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:303px;">Na3VO4</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">450243</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:303px;">Na4P2O7 , decahydrate</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">S-9515</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:303px;">phospho ERK1/2&#160;</td>
			<td style="width:157px;">Cell Signaling Technology, Inc.</td>
			<td style="width:168px;">9101S</td>
			<td>Primary antibody (1:1000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor cocktail</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">P8340-5ml</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:303px;">Protein assay dye reagent</td>
			<td style="width:157px;">Bio-Rad Laboratories, Inc.</td>
			<td style="width:168px;">500-0006</td>
			<td>Protein concentration Measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PVDF transfer membrane</td>
			<td style="width:157px;">Thermo Scientific, Inc.</td>
			<td style="width:168px;">88518</td>
			<td>Western blots</td>
		</tr>
		<tr height="20">
			<td height="41" style="height:41px;width:303px;">Rotatory Evaporator</td>
			<td style="width:157px;">Buchi Labortechnik</td>
			<td style="width:168px;">Rotavapor 
			R3000</td>
			<td>Preparation of polyphenol extracts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile water</td>
			<td style="width:157px;">Mediatech, Inc.</td>
			<td style="width:168px;">25-055-CV</td>
			<td>Preparation of polyphenol extracts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sonicator</td>
			<td style="width:157px;">QSonica, LLC</td>
			<td style="width:168px;">Q125</td>
			<td>Preparation of cell extracts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SOD2</td>
			<td style="width:157px;">Enzo Life Sciences, Inc.</td>
			<td style="width:168px;">ADI-SOD-110-F</td>
			<td>Primary antibody (1:1000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton-X-100</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">X100</td>
			<td>Western blots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Whatman #2 filter paper</td>
			<td style="width:157px;">GE Healthcare, Inc.</td>
			<td style="width:168px;">28317-241</td>
			<td>Preparation of polyphenol extracts</td>
		</tr>
	</tbody>
</table>

extraction and purification of polyphenols from freeze-dried berry powder for the treatment of vascular smooth muscle cells in vitro

Epidemiologiske undersøgelser tyder på, at øget flavonoid indtagelse korrelerer med nedsat dødelighed på grund af hjerte-kar-sygdomme (CVD) i USA og USA. Bær er meget forbrugt i USA og har et højt polyphenolisk indhold. Polyfenoler har vist sig at interagere med mange molekylære mål og udøve adskillige positive biologiske funktioner, herunder antioxidant, antiinflammatoriske og kardioprotektive virkninger. Polyfenoler isoleret fra blackberry (BL), hindbær (RB) og sort hindbær (BRB) reducerer oxidativ stress og cellulær senescence som svar på angiotensin II (Ang II). Dette arbejde giver en detaljeret beskrivelse af protokollen, der anvendes til fremstilling af polyphenolekstrakterne fra frysetørrede bær. Polyphenolekstraktioner fra frysetørret bærpulver blev udført under anvendelse af 80% vandig ethanol og en ultralydassisteret ekstraktionsmetode. Det rå ekstrakt blev yderligere oprenset og fraktioneret under anvendelse af chloroform og ethylacetat,henholdsvis. Virkningerne af både rå og oprensede ekstrakter blev testet på vaskulære glatte muskelceller (VSMC&#39;er) i kultur.

Det er tidligere blevet påvist, at polyphenolekstrakter isoleret fra BL, RB og BRB reducerede senescensen af ​​VSMC&#39;er som svar på Ang II 21 . Det har vist sig, at disse oprensede polyphenolekstrakter modulerer Ang II-signalering ved at reducere phosphoryleringen af ​​Akt, p38-mitogenaktiveret proteinkinase (MAPK) og ERK1 / 2. BL forhindrer senescens ved at reducere ekspressionen af ​​NADPH oxidasen (Nox) 1, et enzym der producerer superoxidanion...

Watch this Scientific Journal Video about Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro at JoVE.com

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Dette arbejde beskriver en trin-for-trin metode til fremstilling af polyphenol-rige ekstrakter fra frysetørret bærpulver. Derudover giver den en grundig beskrivelse af, hvordan man bruger disse polyphenolrige ekstrakter i cellekultur i nærvær af peptidhormon angiotensin II (Ang II) ved hjælp af vaskulære glatte muskelceller (VSMC&#39;er).

Ekstraktion og oprensning af polyphenoler fra frysetørret bærpulver til behandling af vaskulære glatte muskelceller<em&gt; In Vitro</em

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Research

JoVE Journal

Chemistry

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

Rapid High Throughput Amylose Bestemmelse i frysetørret kartoffelknolden Prøver

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

Syntese og oprensning af Iodoaziridines Inddragelse kvantitativ udvælgelse af det optimale stationære fase til kromatografi

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Supercritical Nitrogen Processing for the Purification of Reactive Porous Materials

Superkritisk Nitrogen Behandling til rensning af reaktive porøse materialer

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

X-ray af pulver i Conservation Science: Mod Rutinemæssig Krystalstrukturbestemmelse af korrosionsprodukter på Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Syntese af Protein biokonjugater<em&gt; via</em&gt; Cystein-maleimid Kemi

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

En praktisk metode til udvinding og analyse med højtryks væskekromatografi catecholamin neurotransmittere og deres metabolitter

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Nanothermite with Meringue-like Morphology: From Loose Powder to Ultra-porous Objects

Nanothermite med marengs-lignende morfologi: fra løs pulver til ultra-porøse objekter

The goal of the protocol described in this article is to prepare aluminothermic compositions (nanothermites) in the form of porous, monolithic objects. ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

Adskillelse af aldehyder og reaktive ketoner fra blandinger ved hjælp af en Bisulfite udvinding protokol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Extraction of Lignin with High ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Ekstraktion af Lignin med højt indhold af β-O-4 ved Mild Ethanol udvinding og dens virkning på Depolymerization udbyttet

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Syntese og Bioconjugation af Thiol-reaktiv reagenser til oprettelse af hjemmeside-selektivt modificerede Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...