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

This work was funded by the American Heart Association (14GRNT20180028) and the Florida State University Council on Research and Creativity (COFRS).

1. Preparation of Reagents
<ol>
	<li>Prepare 80% ethanol (100 mL) by mixing 80 mL of absolute ethanol (molecular biology-grade) and 20 mL of cell culture-grade sterile water.</li>
	<li>To prepare polyphenol extract (10 mg/mL), weigh 10 mg of CE or PE. Add 1 mL of plain Dulbecco&#39;s Modified Eagle Medium (DMEM) under a cell culture hood. Vortex the solution. Aliquot in 200-&#181;L portions and store at -20 &#176;C.</li>
	<li>Prepare lysis buffer.
	<ol>
		<li>Add 5 mL of 1 M HEPES stock solution (pH 7.4; 50 mM final), ...

<ol>
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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. 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Polyphenols isolated from berries contain distinct compositions. The ethanol-based extraction protocol described here allowed for the identification of different levels of phenolic acids and flavonoids present in crude and purified polyphenol extracts of BL (Table 1). CE was enriched in gallic acid, ferulic acid, 4-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid. The purification process did not significantly alter the levels of gallic acid and p-coumaric acid. However, it increased the levels of 3-<e...

Polyphenols are compounds containing at least one phenolic ring in their structure and are abundantly present in the plant kingdom1. Humans have been consuming plants for millennia for medicinal purposes without being aware of the existence of such compounds2. Many fruits and vegetables have some shared polyphenolic compounds, albeit with different quantities, including flavonoids, stilbenes, and phenolic acids3. Although polyphenols are often associated with colorful fruits and vegetables, this is not strictly true. For example, zeaxanthin and xanthine are present in vegetables that are not highly colorful, such as onions and garlic, which are from the family of scallions and are associated with numerous health benefits4. Aside from being associated with several health benefits5, polyphenols also serve plants by protecting them from insects and ultraviolet radiation2. Polyphenols are commonly found in the human diet and are considered powerful antioxidants, as they can scavenge Reactive Oxygen Species (ROS)6,7,8. They also have anti-inflammatory9, antimicrobial10, anti-hypertensive11, and anti-carcinogenic12,13 properties.
Epidemiological studies demonstrate an inverse association between the consumption of flavonoids and cardiovascular disease (CVD) incidence16,17 and mortality14,15. Berries are widely consumed in the US and have high amounts of polyphenols, including flavonoids. For instance, consumption of blackberry (BL) juice (300 mL/d) for eight weeks significantly decreased systolic blood pressure in dyslipidemic patients18. Jeong et al.19 reported that pre-hypertensive men and women consuming 2.5 g of black raspberry (BRB) extract per day had lower 24-h and nighttime blood pressure compared to those consuming a placebo. Raspberries (RB) decreased blood pressure while increasing the expression of superoxide dismutase (SOD) in spontaneously hypertensive rats20. It has recently been shown that BL, RB, and BRB reduce the levels of ROS and senescence induced by angiotensin II (Ang II) in Vascular Smooth Muscle Cells (VSMCs)21. In addition, the anthocyanin fraction from BL extract reduced the expression of inducible nitric oxide synthase (iNOS) and inhibited the activity of Nuclear Factor kappa B (NF-&#954;B) and extracellular signal-regulated kinase (ERK) in lipopolysaccharide (LPS)-stimulated J774 cells22. BRB extracts decreased the NF-&#954;B activation and cyclooxygenase 2 (COX-2) expression in vitro 23, improved the lipid profile, and prevented atherosclerosis lesion formation in mice fed a high-fat diet24. Anthocyanins, which are considered the most abundant flavonoids in berries, modulate the inflammatory response in LPS-stimulated RAW 264.7 macrophages by decreasing Tumor Necrosis Factor alpha (TNF-&#945;) production25 and decrease the proliferation and migration of VSMCs26.
Since there has been growing interest in understanding the role of polyphenols in human health and disease, it is important to optimize the extraction method. Solvent extraction is widely used for that purpose, as it is cost-effective and easily reproducible. In this study, a solvent extraction was used with ethanol, along with an ultrasonic-assisted extraction method, which was adapted from Kim and Lee27. The purification and fractionation of crude extracts (CE) using chloroform and ethyl acetate were performed to obtain the purified extract (PE) fraction that was adapted from Queires et al28. Furthermore, the efficacy of crude versus purified polyphenol extracts from BL at reducing the basal phosphorylation of ERK1/2 were compared, and representative examples of the inhibitory effect of purified BL polyphenol extract on Ang II-induced signaling reductions in VSMCs were provided.

<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

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

It has been previously demonstrated that polyphenol extracts isolated from BL, RB, and BRB reduced the senescence of VSMCs in response to Ang II21. It has been shown that these purified polyphenol extracts modulate Ang II signaling by reducing the phosphorylation of Akt, p38 Mitogen-Activated Protein Kinase (MAPK), and ERK1/2. BL prevents senescence by reducing the expression of the NADPH oxidase (Nox) 1, an enzyme that produces superoxide anions and is strongly up...

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

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

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

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

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