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 arbeidet ble finansiert av American Heart Association (14GRNT20180028) og Florida State University Council for forskning og kreativitet (COFRS).

1. Fremstilling av reagenser <ol><li> Tilbered 80% etanol (100 ml) ved å blande 80 ml absolutt etanol (molekylærbiologi-klasse) og 20 ml sterilt vann fra cellekulturer. </li><li> For å forberede polyphenol ekstrakt (10 mg / ml) veier 10 mg CE eller PE. Tilsett 1 ml vanlig Dulbeccos Modified Eagle Medium (DMEM) under en cellekulturhette. Vortex løsningen. Aliquot i 200-l porsjoner og lagre ved -20 ° C. </li><li> Forbered lysis buffer. <ol><li> Tilsett 5 ml 1 M HEPES stamløsning (pH 7,4; 50 mM slutt), 1 ml 5 M NaC...

<ol>
	<li>Morton, L. W., Abu-Amsha Caccetta, R., Puddey, I. B., Croft, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biological+effects+of+dietary+phenolic+compounds:+relevance+to+cardiovascular+disease.">Chemistry and biological effects of dietary phenolic compounds: relevance to cardiovascular disease.</a> Clin Exp Pharmacol Physiol. 27 (3), 152-159 (2000).</li><li>Sekirov, I., Russell, S. L., Antunes, L. C., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiota+in+health+and+disease.">Gut microbiota in health and disease.</a> Physiol Rev. 90 (3), 859-904 (2010).</li><li>Manach, C., Scalbert, A., Morand, C., Remesy, C., Jimenez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenols:+food+sources+and+bioavailability.">Polyphenols: food sources and bioavailability.</a> Am J Clin Nutr. 79 (5), 727-747 (2004).</li><li>Griffiths, G., Trueman, L., Crowther, T., Thomas, B., Smith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onions--a+global+benefit+to+health.">Onions--a global benefit to health.</a> Phytother Res. 16 (7), 603-615 (2002).</li><li>Mazzoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+aspects+of+berries:+from+field+to+health.">The genetic aspects of berries: from field to health.</a> J Sci Food Agric. 96 (2), 365-371 (2016).</li><li>Wang, S. Y., Jiao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenging+capacity+of+berry+crops+on+superoxide+radicals,+hydrogen+peroxide,+hydroxyl+radicals,+and+singlet+oxygen.">Scavenging capacity of berry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen.</a> J Agric Food Chem. 48 (11), 5677-5684 (2000).</li><li>Choi, M. H., Shim, S. M., Kim, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+black+raspberry+seed+containing+anthocyanins+against+oxidative+damage+to+DNA,+protein,+and+lipid.">Protective effect of black raspberry seed containing anthocyanins against oxidative damage to DNA, protein, and lipid.</a> J Food Sci Technol. 53 (2), 1214-1221 (2016).</li><li>Forbes-Hernandez, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Healthy+Effects+of+Strawberry+Polyphenols:+Which+Strategy+behind+Antioxidant+Capacity?.">The Healthy Effects of Strawberry Polyphenols: Which Strategy behind Antioxidant Capacity?.</a> Crit Rev Food Sci Nutr. 56, S46-S59 (2016).</li><li>Figueira, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+a+blueberry+extract+in+acute+inflammation+and+collagen-induced+arthritis+in+the+rat.">Protective effects of a blueberry extract in acute inflammation and collagen-induced arthritis in the rat.</a> Biomed Pharmacother. 83, 1191-1202 (2016).</li><li>Daglia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenols+as+antimicrobial+agents.">Polyphenols as antimicrobial agents.</a> Curr Opin Biotechnol. 23 (2), 174-181 (2012).</li><li>H&#252;gel, H. M., Jackson, N., May, B., Zhang, A. L., Xue, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenol+protection+and+treatment+of+hypertension.">Polyphenol protection and treatment of hypertension.</a> Phytomedicine. 23 (2), 220-231 (2016).</li><li>Niedzwiecki, A., Roomi, M. W., Kalinovsky, T., Rath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+Efficacy+of+Polyphenols+and+Their+Combinations.">Anticancer Efficacy of Polyphenols and Their Combinations.</a> Nutrients. 8 (9), E552 (2016).</li><li>Kresty, L. A., Mallery, S. R., Stoner, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Black+raspberries+in+cancer+clinical+trials:+Past,+present+and+future.">Black raspberries in cancer clinical trials: Past, present and future.</a> J Berry Res. 6 (2), 251-261 (2016).</li><li>Hertog, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavonoid+intake+and+long-term+risk+of+coronary+heart+disease+and+cancer+in+the+seven+countries+study.">Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study.</a> Arch Intern Med. 155 (4), 381-386 (1995).</li><li>Peterson, J. J., Dwyer, J. T., Jacques, P. F., McCullough, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+between+flavonoids+and+cardiovascular+disease+incidence+or+mortality+in+European+and+US+populations.">Associations between flavonoids and cardiovascular disease incidence or mortality in European and US populations.</a> Nutr Rev. 70 (9), 491-508 (2012).</li><li>Cassidy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+anthocyanin+intake+is+associated+with+a+reduced+risk+of+myocardial+infarction+in+young+and+middle-aged+women.">High anthocyanin intake is associated with a reduced risk of myocardial infarction in young and middle-aged women.</a> Circulation. 127 (2), 188-196 (2013).</li><li>Jacques, P. F., Cassidy, A., Rogers, G., Peterson, J. J., Dwyer, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+flavonoid+intakes+and+CVD+incidence+in+the+Framingham+Offspring+Cohort.">Dietary flavonoid intakes and CVD incidence in the Framingham Offspring Cohort.</a> Br J Nutr. 114 (9), 1496-1503 (2015).</li><li>Aghababaee, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+blackberry+(Morus+nigra+L.)+consumption+on+serum+concentration+of+lipoproteins,+apo+A-I,+apo+B,+and+high-sensitivity-C-reactive+protein+and+blood+pressure+in+dyslipidemic+patients.">Effects of blackberry (Morus nigra L.) consumption on serum concentration of lipoproteins, apo A-I, apo B, and high-sensitivity-C-reactive protein and blood pressure in dyslipidemic patients.</a> J Res Med Sci. 20 (7), 684-691 (2015).</li><li>Jeong, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Rubus+occidentalis+extract+on+blood+pressure+in+patients+with+prehypertension:+Randomized,+double-blinded,+placebo-controlled+clinical+trial.">Effects of Rubus occidentalis extract on blood pressure in patients with prehypertension: Randomized, double-blinded, placebo-controlled clinical trial.</a> Nutrition. 32 (4), 461-467 (2016).</li><li>Jia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antihypertensive+effect+of+ethyl+acetate+extract+from+red+raspberry+fruit+in+hypertensive+rats.">The antihypertensive effect of ethyl acetate extract from red raspberry fruit in hypertensive rats.</a> Pharmacogn Mag. 7 (25), 19-24 (2011).</li><li>Feresin, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blackberry,+raspberry+and+black+raspberry+polyphenol+extracts+attenuate+angiotensin+II-induced+senescence+in+vascular+smooth+muscle+cells.">Blackberry, raspberry and black raspberry polyphenol extracts attenuate angiotensin II-induced senescence in vascular smooth muscle cells.</a> Food Funct. 7 (10), 4175-4187 (2016).</li><li>Pergola, C., Rossi, A., Dugo, P., Cuzzocrea, S., Sautebin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+nitric+oxide+biosynthesis+by+anthocyanin+fraction+of+blackberry+extract.">Inhibition of nitric oxide biosynthesis by anthocyanin fraction of blackberry extract.</a> Nitric Oxide. 15 (1), 30-39 (2006).</li><li>Lu, H., Li, J., Zhang, D., Stoner, G. D., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+involved+in+chemoprevention+of+black+raspberry+extracts:+from+transcription+factors+to+their+target+genes.">Molecular mechanisms involved in chemoprevention of black raspberry extracts: from transcription factors to their target genes.</a> Nutr Cancer. 54 (1), 69-78 (2006).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+extract+of+unripe+Rubus+coreanus+fruit+attenuates+atherosclerosis+by+improving+blood+lipid+profile+and+inhibiting+NF-&#954;B+activation+via+phase+II+gene+expression.">Aqueous extract of unripe Rubus coreanus fruit attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-&#954;B activation via phase II gene expression.</a> J Ethnopharmacol. 146 (2), 515-524 (2013).</li><li>Wang, J., Mazza, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anthocyanins+and+other+phenolic+compounds+on+the+production+of+tumor+necrosis+factor+alpha+in+LPS/IFN-gamma-activated+RAW+264.7+macrophages.">Effects of anthocyanins and other phenolic compounds on the production of tumor necrosis factor alpha in LPS/IFN-gamma-activated RAW 264.7 macrophages.</a> J Agric Food Chem. 50 (15), 4183-4189 (2002).</li><li>Pascual-Teresa, S., Moreno, D. A., Garcia-Viguera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavanols+and+anthocyanins+in+cardiovascular+health:+a+review+of+current+evidence.">Flavanols and anthocyanins in cardiovascular health: a review of current evidence.</a> Int J Mol Sci. 11 (4), 1679-1703 (2010).</li><li>Kim, D. O., Lee, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+Isolation+of+Polyphenolics.">Extraction and Isolation of Polyphenolics.</a> Curr Protoc Food Analyt Chem. 1, 2.1-2.12 (2002).</li><li>Queires, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenols+purified+from+the+Brazilian+aroeira+plant+(Schinus+terebinthifolius,+Raddi)+induce+apoptotic+and+autophagic+cell+death+of+DU145+cells.">Polyphenols purified from the Brazilian aroeira plant (Schinus terebinthifolius, Raddi) induce apoptotic and autophagic cell death of DU145 cells.</a> Anticancer Res. 26 (1A), 379-387 (2006).</li><li>Griendling, K. K., Taubman, M. B., Akers, M., Mendlowitz, M., Alexander, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+phosphatidylinositol-specific+phospholipase+C+from+cultured+vascular+smooth+muscle+cells.">Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells.</a> J Biol Chem. 266 (23), 15498-15504 (1991).</li><li>Vuong, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+a+polyphenol-enriched+preparation+on+chemoprevention+of+mammary+carcinoma+through+cancer+stem+cells+and+inflammatory+pathways+modulation.">Role of a polyphenol-enriched preparation on chemoprevention of mammary carcinoma through cancer stem cells and inflammatory pathways modulation.</a> J Transl Med. 14, (2016).</li></ol>

Polyfenoler isolert fra bær inneholder forskjellige sammensetninger. Den etanolbaserte ekstraksjonsprotokollen beskrevet her er tillatt for identifisering av forskjellige nivåer av fenoliske syrer og flavonoider som finnes i rå og rensede polyfenol ekstrakter av BL ( tabell 1 ). CE ble anriket i gallinsyre, ferulinsyre, 4-0-koffeokylsyre og 5-0-koffeokylsyre. Rensingsprosessen endret ikke signifikant nivåene av gallinsyre og p-kumarinsyre. Det økte imidlertid nivåene av 3- O- koeoylkinsyr...

Polyfenoler er forbindelser som inneholder minst en fenolisk ring i deres struktur og er rikelig tilstede i planterike 1 . Mennesker har spist planter i årtusener til medisinske formål uten å være klar over eksistensen av slike forbindelser 2 . Mange frukter og grønnsaker har noen delte polyphenoliske forbindelser, om enn med forskjellige mengder, inkludert flavonoider, stilbener og fenol syrer 3 . Selv om polyfenoler ofte er forbundet med fargerike frukter og grønnsaker, er dette ikke helt sant. For eksempel er zeaxanthin og xantin til stede i grønnsaker som ikke er svært fargerike, som løk og hvitløk, som er fra familien av scallions og er forbundet med mange helsemessige fordeler 4 . Bortsett fra å være assosiert med flere helsemessige fordeler, tjener polyfenoler også planter ved å beskytte dem mot insekter ogD ultrafiolett stråling 2 . Polyfenoler finnes ofte i det menneskelige kostholdet og betraktes som kraftige antioksidanter, da de kan scavenge Reactive Oxygen Species (ROS) 6 , 7 , 8 . De har også antiinflammatorisk 9 , antimikrobiell 10 , antihypertensiv 11 og anti-kreftfremkallende 12 , 13 egenskaper. Epidemiologiske studier viser en invers forbindelse mellom forbruket av flavonoider og kardiovaskulær sykdom (CVD) incidens 16 , 17 og dødelighet 14 , 15 . Bær er mye konsumert i USA og har høye mengder polyfenoler, inkludert flavonoider. For eksempel forbruk av blackberry (BL) juice (300<html> ML / d) i åtte uker signifikant redusert systolisk blodtrykk hos dyslipidemiske pasienter 18 . Jeong et al. 19 rapporterte at prehypertensive menn og kvinner som bruker 2,5 g svart bringebær (BRB) ekstrakt per dag, hadde lavere 24-timers og nattt blodtrykk sammenlignet med de som brukte en placebo. Hindbær (RB) reduserte blodtrykket mens du økte ekspresjonen av superoksiddismutase (SOD) i spontant hypertensive rotter 20 . Det har nylig blitt vist at BL, RB og BRB reduserer nivåene av ROS og senescence indusert av angiotensin II (Ang II) i vaskulære glatte muskelceller (VSMCs) 21 . I tillegg reduserte anthocyaninfraksjonen fra BL-ekstrakt ekspresjonen av inducerbar nitrogenoksydsyntase (iNOS) og hemmet aktiviteten av Nuclear Factor kappa B (NF-KB) og ekstracellulær signalregulert kinase (ERK) i lipopolysakkarid (LPS) -stimulert J774 cellerAss = &quot;xref&quot;&gt; 22. BRB-ekstrakter reduserte NF-KB-aktiveringen og cyklooksygenase 2 (COX-2) -ekspresjonen in vitro 23 , forbedret lipidprofilen og forhindret ateroskleroselesjonsdannelse hos mus med et fettfattig kosthold 24 . Anthocyaniner, som betraktes som de mest omfattende flavonoider i bær, modulerer den inflammatoriske responsen i LPS-stimulerte RAW 264.7-makrofager ved å redusere Tumor Necrosis Factor alfa (TNF-a) -produksjonen 25 og redusere proliferasjonen og migrasjonen av VSMCs 26 . Siden det har vært økende interesse for å forstå rollen av polyfenoler i menneskers helse og sykdom, er det viktig å optimalisere ekstraksjonsmetoden. Løsningsmiddelekstraksjon er mye brukt til det formålet, da det er kostnadseffektivt og enkelt reproduserbart. I denne studien ble en løsningsmiddelekstraksjon brukt med etanol, sammen med et ultralydassistert ekstraktN-metode, som ble tilpasset fra Kim og Lee 27 . Rensingen og fraksjoneringen av råekstrakter (CE) ved anvendelse av kloroform og etylacetat ble utført for å oppnå den rensede ekstrakt (PE) fraksjon som ble tilpasset fra Queires et al., 28 . Videre ble effekten av rå versus rensede polyfenolekstrakter fra BL sammenlignet med å redusere basal fosforylering av ERK1 / 2, og representative eksempler på den hemmeffekten av renset BL-polyfenol-ekstrakt på Ang II-induserte signalreduksjoner i VSMCs ble tilveiebrakt.

<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>
		</tr>
		<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>
		</tr>
		<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 studier indikerer at økt flavonoidinntak korrelerer med redusert dødelighet på grunn av hjerte-og karsykdommer (CVD) i USA og Europa. Bærene er mye konsumert i USA og har et høyt polyphenolisk innhold. Polyfenoler har vist seg å interagere med mange molekylære mål og utøve mange positive biologiske funksjoner, inkludert antioksidanter, antiinflammatoriske og kardioprotektive effekter. Polyfenoler isolert fra brombær (BL), bringebær (RB) og svart bringebær (BRB) reduserer oksidativt stress og cellulær senescens som svar på angiotensin II (Ang II). Dette arbeidet gir en detaljert beskrivelse av protokollen som brukes til å fremstille polyphenol-ekstraktene fra frysetørkede bær. Polyfenol ekstraksjoner fra frysetørket bærpulver ble utført ved anvendelse av 80% vandig etanol og en ultralydassistert ekstraksjonsmetode. Råekstraktet ble ytterligere renset og fraksjonert ved anvendelse av kloroform og etylacetat,henholdsvis. Effektene av både rå og rensede ekstrakter ble testet på Vascular Smooth Muscle Cells (VSMCs) i kultur.

Det har tidligere blitt påvist at polyfenolekstrakter isolert fra BL, RB og BRB reduserte senescensen av VSMCs som svar på Ang II 21 . Det har blitt vist at disse rensede polyfenolekstrakter modulerer Ang II-signalering ved å redusere fosforyleringen av Akt, p38-mitogen-aktivert proteinkinase (MAPK) og ERK1 / 2. BL forhindrer senescens ved å redusere ekspresjonen av NADPH-oksidasen (Nox) 1, et enzym som produserer superoksydanioner og er sterkt oppregulert av ...

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 arbeidet beskriver en trinnvis metode for å fremstille polyphenolrike ekstrakter fra frysetørket bærpulver. I tillegg gir den en grundig beskrivelse av hvordan man bruker disse polyfenolrike ekstraktene i cellekultur i nærvær av peptidhormon angiotensin II (Ang II) ved bruk av vaskulære glatte muskelceller (VSMCs).

Ekstraksjon og rensing av polyfenoler fra frysetørket bærpulver til behandling av vaskulære glatte muskelceller<em&gt; I 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 høy gjennomstrømning Amylose Bestemmelse i Freeze Tørket potet Tuber Samples

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 rensing av Iodoaziridines Involvering Kvantitativ Valg av Optimal stasjonær fase for 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

Kritisk Nitrogen Processing for rensing av reaktiv 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 Powder Diffraksjon i Conservation Science: Mot Rutine Crystal Structure Bestemmelse av korrosjonsprodukter på Heritage Kunstverk

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 av Protein Bioconjugates<em&gt; via</em&gt; Cystein-maleimide kjemi

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 for utvinning og analyse med høyt trykk væske kromatografi katekolaminer nevrotransmittere 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 svært porøs 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

Separasjon av aldehyder og reaktive ketoner fra blandinger bruker en Bisulfite utvinning protokoll

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

Utvinning av Lignin med høy β-O-4 innhold av Mild etanol utvinning og dens effekt på Depolymerization avkastningen

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 Thiol-reaktive reagenser for etableringen av nettstedet-selektivt endret Immunoconjugates

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