Name: extraction and purification of polyphenols from freeze-dried berry powder for the treatment of vascular smooth muscle cells in vitro
Uploaded: 2017-07-05T14:00:00.000+00:00
Duration: 12 min
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), 1 mL of 5 M NaCl stock solution (50 mM final), 1 mL of 0.5 M EDTA stock solution (5 mM final), 2 mL of 0.5 M NaF stock solution (10 mM final), 286 &#181;L of 700 mM Na3VO4 stock solution (2 mM final), 5 mL of 200 mM Na4P2O7 stock solution (10 mM final), and 5 mL of 20% Triton-X-100 stock solution prepared in water (1% final). Add water to reach a volume of 100 mL. Keep at 4 &#176;C.</li>
		<li>Add 10 &#181;L/mL of protease inhibitor cocktail. Add fresh when the cells are ready for lysis.</li>
	</ol>
	</li>
	<li>Prepare Laemmli sample buffer (4x).
	<ol>
		<li>Add 31.25 mL of 2 M Tris stock solution (pH 6.8), 100 mL of glycerol, 50 mL of a 20% Sodium Dodecyl Sulfate (SDS) solution prepared in water, and 50 mL of &#946;-mercaptoethanol. Add water up to 250 mL. Keep at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare Tris-Buffered Saline (TBS) buffer, weigh 3 g of Tris (25 mM final), 0.18 g of KCl (2.5 mM final), and 8.76 g of NaCl (150 mM final). Adjust the pH to 7.4 and add water up to 1 L. Keep at RT.</li>
	<li>To prepare TBS-T, add 997.5 mL of TBS and 2.5 mL of a 20% Triton-X-100 solution prepared in water. Keep at RT.</li>
</ol>
2. Preparation of Blackerry Extracts
<ol>
	<li>Preparation of crude extract from freeze-dried berry powder.
	<ol>
		<li>Weigh 10 g of freeze-dried blackberry powder (or 5 g of fresh powder). If frozen fruit is used, cut it into very small pieces before starting the extraction.</li>
		<li>Mix the fruit powder with 100 mL of 80% aqueous ethanol in a 1 L round-bottom Erlenmeyer flask.</li>
		<li>Sonicate the mixture for 20 min at 42 kHz, 135 W using an ultrasonic bath at 25 &#176;C, without any time interval in between. Perform the sonication with continuous nitrogen purging in subdued light. For frozen fruit, increase the incubation time to 4 h.</li>
		<li>Filter the mixture through a #2 filter paper using a chilled Buchner funnel with vacuum suction. 
		NOTE: At the end of this step, residuals of the sample appear on the filter paper; this is called filter cake.</li>
		<li>Rinse the filter cake with 50 mL of 100% ethanol.&#160;Save the filtrate and add the filter cake to a 1 L round-bottom flask containing 100 mL of 80% aqueous ethanol.</li>
		<li>Repeat the extraction process for the residue (steps 2.1.2 - 2.1.4).</li>
		<li>Combine the two filtrates into a round-bottom flask with an additional 50 mL of 80% aqueous ethanol. 
		NOTE: The final volume should be approximately 300 mL.</li>
		<li>Use a rotary evaporator at 62 &#176;C and 50 rpm to evaporate the solvent. Continue this process until the ethanol does not evaporate anymore. 
		NOTE: This process takes approximately 45 min.</li>
		<li>Transfer the sample to a 50 mL conical tube. Evaporate the ethanol by injecting nitrogen gas at the top of the tube for 10 min. 
		NOTE: This step is needed to ensure the complete evaporation of the ethanol. The volume of the sample at this step is approximately 20 mL.</li>
		<li>Freeze the sample at -80 &#176;C for at least 24 h. 
		NOTE: This step is needed to allow for a more efficient freeze-drying process.</li>
		<li>Freeze-dry the sample at -50 &#176;C for approximately 8 h. Store the samples at -20 &#176;C. 
		NOTE: The freeze dryer creates a powerful vacuum at -50 &#176;C inside the chamber to dry the samples but preserves most of the compounds, such as polyphenols.</li>
	</ol>
	</li>
	<li>Preparation of purified polyphenol extracts from crude extracts.
	<ol>
		<li>Weigh the freeze-dried extracted samples. 
		NOTE: The volume of the extract at this step is approximately 10 mL.</li>
		<li>Add two volumes (~20 mL) of chloroform to the crude extract and shake the solution for 5 min&#160;in a stir-plate.</li>
		<li>Pour the mixture into a separating funnel. Let the crude extract decant for 1 h at RT to obtain the purified fraction. 
		NOTE: At this step, a two-phase mixture will form.</li>
		<li>Discard the bottom layer (chloroform phase) from the separating funnel and collect the aqueous layer into a clean beaker.</li>
		<li>Add two volumes of ethyl acetate to the aqueous fraction and use a magnetic stir bar to stir the mixture for 5 min at RT.</li>
		<li>Filter the mixture through #2 filter paper using a chilled Buchner funnel with vacuum suction.</li>
		<li>Collect the filtered sample and transfer it to a round-bottom flask to be used with the rotary evaporator.</li>
		<li>Set the rotary evaporator to 62 &#176;C and 50 rpm and evaporate the ethyl acetate for about 30 min.</li>
		<li>Freeze-dry the sample at -50 &#176;C for approximately 8 h. Transfer the sample to a 50 mL conical tube and store it at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Treatment of VSMCs with Berry Extracts
<ol>
	<li>VSMCs culture.
	<ol>
		<li>Isolate VSMCs from the thoracic aortas of Sprague-Dawley rats by performing an enzymatic digestion, as previously described29. Culture the VSMCs as described29.</li>
		<li>Prepare complete medium for the culture of VSMCs using DMEM containing 1 g/L glucose and supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine. Store the complete medium at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Incubation of VSMCs with polyphenol extracts.
	<ol>
		<li>To split the VSMCs, discard the culture medium and wash the cells twice with phosphate-buffered saline (PBS). Add 4 mL of PBS and 2 mL of trypsin EDTA 0.25%. Incubate the cells for 5 min at 37 &#176;C in a CO2 incubator.
		<ol>
			<li>Collect the cells, mix them with 2 mL of complete medium in a 15 mL centrifuge tube, and centrifuge at 1,100 &#215; g for 5 min. Discard the supernatant and resuspend the cell pellet with 1 mL of PBS. Count the cells using a hemocytometer.</li>
			<li>Seed 50,000 VSMCs per well in 6-well culture plates and grow them in complete medium containing 10% FBS until they reach 90% confluency (about 3 d). Maintain the VSMCs at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		</ol>
		</li>
		<li>Prepare the medium for the treatment using DMEM medium containing 1 g/L glucose, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, and 0.5% FBS. Store the treatment medium at 4 &#176;C. 
		NOTE: Polyphenol extracts and Ang II are added directly to cells using this medium.</li>
		<li>Prepare a stock solution of 10 mg/mL Crude Extract (CE) or Purified Extract (PE) of polyphenols by dissolving 10 mg of extract in 1 mL of plain DMEM medium. Keep the stock in 200 &#181;L aliquots at -20 &#176;C.</li>
		<li>Incubate the VSMCs with 2 mL of treatment medium containing 0.5% FBS and add either CE or PE extracts to achieve a concentration of 50 - 500 &#181;g/mL. Change the medium every 24 h by adding fresh polyphenol extracts. Treat the cells for 3 d.</li>
		<li>For treatment with Ang II or other hormones and growth factors, starve the cells for at least 24 h by incubating the cells with treatment medium containing 0.5% FBS before the addition of Ang II (100 nM). 
		NOTE: Incubation with and without CE and PE in 0.5% FBS treatment medium before the addition of Ang II is recommended.
		<ol>
			<li>After 24 h of starvation, add 100 nM Ang II. Replace the treatment medium with fresh CE, PE, or Ang II every 24 h for 3 d.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of cell samples.
	<ol>
		<li>Wash the treated cells twice in cold PBS and lyse them with 200 &#181;L of lysis buffer on ice.</li>
		<li>Collect cell lysates using cell scrapers and transfer the lysates to 1.5 mL microcentrifuge tubes. Incubate the total cell lysates for 20 min on ice and vortex every 5 min.</li>
		<li>Sonicate the cell extracts with three bursts at 125 W for 10 s each, with a 2 s pause in between. Keep the samples on ice throughout the sonication.</li>
		<li>Measure the protein concentration using a protein assay reagent at 595 nm.</li>
		<li>Store the samples at -20 &#176;C for analysis by Western blot.</li>
	</ol>
	</li>
	<li>Western blot.
	<ol>
		<li>Mix equal amounts of protein (about 50 &#181;g) from control non-treated and polyphenol-treated or Ang II-treated samples with lysis buffer to obtain a maximum total volume of 50 &#181;L. Add 16 &#181;L of a 4x Laemmli sample buffer. Heat the samples for 5 min at 75 &#176;C.</li>
		<li>Separate the samples in 10% SDS-PAGE gels and transfer them to PDVF membranes at 10 V for 75 min in a semi-dry transfer system for Western blot analysis with specific antibodies.</li>
		<li>Block the membrane with 2% milk in TBS 0.05% Triton-X100 buffer (TBS-T) for 20 min.</li>
		<li>Remove the milk by washing the membrane at least three times with TBS (5 min each) and incubate with primary antibodies for 1 h at RT or O/N at 4 &#176;C.</li>
		<li>Wash the membrane three times, 10 min each, with TBS-T and incubate with an HRP-linked secondary antibody for 45 min.</li>
		<li>Wash the membrane three times, 10 min each, with TBS-T and develop by enhanced chemiluminescence (ECL).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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-O-caffeoylquinic acid from 170.5 - 235.3 ppm and of quercetin from 24.5 - 95 ppm. In contrast, ferulic acid and rutin were lost during the purification of CE.
The treatment of VSMCs with 50 - 500 &#181;g/mL CE and PE showed that both extracts were effective in reducing the basal phosphorylation of ERK1/2. However, PE showed a stronger downregulation in the activity of this kinase at a lower concentration: 100 &#181;g/mL for PE compared with 400 - 500 &#181;g/mL for CE. These results may reflect the higher concentration of 3-O-caffeoylquinic acid or quercetin found in PE. The use of individual phenolic compounds in cells in culture is needed to identify the specific compound(s) responsible for the downreguation of ERK1/2 phosphorylation.
The decreased activity of signaling kinases, including Akt, ERK1/2, and p38MAPK, caused by purified polyphenols isolated from BL, RB, and BRB21, as well as of ERK1/2 caused by BL CE, shown here, is in agreement with previous reports. For instance, polyphenol extracts isolated from blueberry decreased the tumor growth of mammary cancer cells, in part by reducing the activity of Akt and ERK1/230. As mentioned before, these kinases are also involved in the induction of cellular senescence by Ang II 21, suggesting that polyphenols from BL, RB, and BRB should reduce vascular aging and dysfunction associated with CVD.
As we reported previously21, catalase and SOD2 expression were not upregulated by PE, even at concentrations as high as 500 &#181;g/mL. Since CE was also ineffective at increasing catalase and SOD2 expression, these data suggest that the phenolic compounds lost during the purification protocol are not involved in the regulation of these antioxidant enzymes. These data also suggest that the purification protocol shown here effectively concentrated phenolic compounds relevant to the regulation of Ang II signaling, oxidative stress, and cellular senescence. For example, the Representative Results show that PE strongly downregulated Ang II-induced ERK1/2 phosphorylation. The assessment of the phosphorylation of this kinase is relevant because the inhibition of ERK1/2 activity prevented Ang II-induced cellular senescence21.
In terms of modifications to previous protocols, a sonication was added here to increase the extraction yield for CE. Additionally, for the purification of polyphenols, instead of using a C18 Cartridge, as described by Kim and Lee27, a more traditional method from Queires et al.28 was adopted here. A filtration step, using filter paper to remove impurities, was added to the purification of polyphenol section. In terms of limitations, the sonication and evaporation steps should be carefully monitored according to the type of instrument used, since the duration and temperature of the sonication and evaporation, are the critical steps in this protocol. The modifications added to this method resulted in the highest yield of polyphenols when compared to previous methods27,28 used in our laboratory (data not shown), most likely because of the addition of a sonication step. As mentioned in the protocol section, this protocol can be used for freeze-dried powder from various berries, as well as for frozen fruits. This method has been successfully used to extract and purify polyphenols from raspberries and black raspberries21, as well as from blueberries and strawberries (data not shown). Thus, this method could be also used to extract polyphenols from other types of foods, including vegetables.
In conclusion, this work details a fast, cost-effective, and easily reproducible method to isolate polyphenols from berries, which allows for the retention and concentration of compounds that are protective against oxidative stress in VSMCs.

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><tbody><tr><td>Angiotensin II</td><td>Sigma-Aldrich, Inc.</td><td>A9525-10MG</td><td>Treatment of VSMCs</td></tr><tr><td>&amp;beta;-actin</td><td>Sigma-Aldrich, Inc.</td><td>A2228</td><td>Primary antibody (1:5000)</td></tr><tr><td>Blackberry fruit</td><td>Mercer Foods</td><td></td><td>Freeze-dried blackberry powder</td></tr><tr><td>Catalase&amp;nbsp;</td><td>Calbiochem</td><td>219010</td><td>Primary antibody (1:1000)</td></tr><tr><td>Chloroform</td><td>Biotech Grd, Inc.</td><td>97064-678</td><td>Preparation of purified polyphenol extracts</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM )</td><td>Mediatech, Inc.</td><td>10-014-CV</td><td>Culture of VSMCs</td></tr><tr><td>Ethanol (absolute molecular biology grade)</td><td>Sigma-Aldrich, Inc.</td><td>E7023-500ML</td><td>Preparation of polyphenol extracts&amp;nbsp;</td></tr><tr><td>Ethylacetate</td><td>Sigma-Aldrich, Inc.</td><td>439169</td><td>Preparation of purified polyphenol extracts</td></tr><tr><td>ERK1/2</td><td>Cell Signaling Technology, Inc.</td><td>9102S</td><td>Primary antibody (1:500)</td></tr><tr><td>EDTA, 500 mM, pH 8.0</td><td>Teknova, Inc.</td><td>E0306</td><td>Lysis buffer</td></tr><tr><td>Freeze-Dryer</td><td>Labconco</td><td>VirTis Benchtop K</td><td>Preparation of polyphenol extracts</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1400-500</td><td>Cell culture</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, Inc.</td><td>H3375</td><td>Lysis buffer&amp;nbsp;</td></tr><tr><td>NaCl</td><td>EMD Millipore, Inc.</td><td>7760</td><td>Lysis buffer</td></tr><tr><td>NaF</td><td>J.T.Baker, Inc.</td><td>3688-01&amp;nbsp;</td><td>Lysis buffer</td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, Inc.</td><td>450243</td><td>Lysis buffer</td></tr><tr><td>Na&lt;sub&gt;4&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt; , decahydrate</td><td>Sigma-Aldrich, Inc.</td><td>S-9515</td><td>Lysis buffer</td></tr><tr><td>phospho ERK1/2&amp;nbsp;</td><td>Cell Signaling Technology, Inc.</td><td>9101S</td><td>Primary antibody (1:1000)</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich, Inc.</td><td>P8340-5ml</td><td>Lysis buffer</td></tr><tr><td>Protein assay dye reagent</td><td>Bio-Rad Laboratories, Inc.</td><td>500-0006</td><td>Protein concentration Measurement</td></tr><tr><td>PVDF transfer membrane</td><td>Thermo Scientific, Inc.</td><td>88518</td><td>Western blots</td></tr><tr><td>Rotatory Evaporator</td><td>Buchi Labortechnik</td><td>Rotavapor&lt;br/&gt; R3000</td><td>Preparation of polyphenol extracts</td></tr><tr><td>Sterile water</td><td>Mediatech, Inc.</td><td>25-055-CV</td><td>Preparation of polyphenol extracts</td></tr><tr><td>Sonicator</td><td>QSonica, LLC</td><td>Q125</td><td>Preparation of cell extracts</td></tr><tr><td>SOD2</td><td>Enzo Life Sciences, Inc.</td><td>ADI-SOD-110-F</td><td>Primary antibody (1:1000)</td></tr><tr><td>Triton-X-100</td><td>Sigma-Aldrich, Inc.</td><td>X100</td><td>Western blots</td></tr><tr><td>Whatman #2 filter paper</td><td>GE Healthcare, Inc.</td><td>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 upregulated by Ang II. In contrast, RB and BRB prevent senescence by a Nox1-independent mechanism, as they increase the expression of the antioxidant enzymes SOD1, SOD2, and glutathione peroxidase 1 (GPx-1). BL fails to increase SOD2 expression, while none of the extracts attenuate the downregulation of catalase by Ang II21. BL was focused on to determine whether the lack of effect of BL on SOD2 and catalase expression could be explained by a loss of polyphenol compounds during the purification process or by an inadequate concentration of a certain polyphenolic compound in the extract. VSMCs were incubated with 50-500 &#181;g/mL CE (Figure 1A) or PE (Figure 1B) in medium containing 0.5% FBS for three days. Neither CE nor PE increased SOD2 or catalase levels at any of the concentrations tested. As a positive control, ERK1/2 phosphorylation was measured, and it was found that CE reduced the phosphorylation of this kinase at concentrations higher than 300 &#181;g/mL. In contrast, PE was effective at about 100 &#181;g/mL (Figure 1B). Low phosphorylation levels were observed at concentrations of 200-500 &#181;g/mL. These data support the previous observation showing that 200 &#181;g/mL BL PE is sufficient to reduce Ang II signaling21. These results suggest that higher concentrations of polyphenol compounds in PE, compared with CE, could explain the higher efficiency of this extract at reducing ERK1/2 phosphorylation. To test this idea, the polyphenol compositions of both extracts were compared. The identification and quantification of polyphenol compounds in CE was performed by HPLC, as previously described21 (Table 1). 3-O-caffeoylquinic acid and quercetin were present at higher levels in PE compared to CE, while ferulic acid and rutin were found only in CE. Next, VSMCs were treated with 200 &#181;g/mL BL PE for 24 h before the addition of 100 nM Ang II (Figure 1C). As previously reported21, the BL polyphenol extract reduced Ang II-induced ERK1/2 phosphorylation but demonstrated no effect on catalase and SOD2 expression.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55605/55605fig1.jpg" /> 
Figure 1: Blackberry Polyphenols Reduce Basal and Ang II-induced ERK1/2 Phosphorylation in VSMCs. VSMCs cultured in 10% FBS at about 90% confluency were incubated with 50 - 500 &#181;g/mL BL Crude Extract (CE) (A), Purified Extract (PE) (B), or 200 &#181;g/mL PE (C) in 0.5% FBS DMEM medium for 3 d. C) After 24 h of incubation with BL PE, Ang II (100 nM) was added and the cells were incubated for 3 d. The medium, with fresh extracts and Ang II, was changed every day. The cells were then washed and lysed, and the total cell extracts were separated in 10% PAGE-SDS gels. Western blots were tested with rabbit antibodies against phosphorylated ERK1/2 (Thr 202/Tyr 204), ERK1/2, catalase, and SOD2 and mouse antibody against &#946;-actin. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55605/55605fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analytes (ppm)</td>
			<td>PE</td>
			<td>CE</td>
		</tr>
		<tr>
			<td colspan="3">Phenolic acids</td>
		</tr>
		<tr>
			<td>gallic acid</td>
			<td>243.5</td>
			<td>321.9</td>
		</tr>
		<tr>
			<td>p-coumaric acid</td>
			<td>32.9</td>
			<td>46.5</td>
		</tr>
		<tr>
			<td>Ferulic acid</td>
			<td>-</td>
			<td>236.5</td>
		</tr>
		<tr>
			<td>Chlorogenic acids</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-O-caffeoylquinic acid</td>
			<td>235.3</td>
			<td>170.5</td>
		</tr>
		<tr>
			<td>4-O-caffeoylquinic acid</td>
			<td>13</td>
			<td>76.9</td>
		</tr>
		<tr>
			<td>5-O-caffeoylquinic acid</td>
			<td>14.1</td>
			<td>49.9</td>
		</tr>
		<tr>
			<td colspan="3">FLAVONOIDS</td>
		</tr>
		<tr>
			<td colspan="3">Flavonols</td>
		</tr>
		<tr>
			<td>Quercetin</td>
			<td>95</td>
			<td>24.5</td>
		</tr>
		<tr>
			<td colspan="3">Flavanones</td>
		</tr>
		<tr>
			<td>Rutin</td>
			<td>-</td>
			<td>37.8</td>
		</tr>
	</tbody>
</table>
Table 1: Analysis of the Polyphenol Composition of Blackberry in Crude and Polyphenol-purified Extracts. Phenolic acids and flavonoids in Crude Extract (CE) and Purified Extract (PE) were analyzed using High Performance Liquid Chromatography (HPLC). The concentration of analytes is expressed as ppm. The composition of polyphenols in BL PE was recently published21 and added to the table to be compared to CE.

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 ...

extraction-purification-polyphenols-from-freeze-dried-berry-powder

Nanyang Technological University

Research

JoVE Journal

Chemistry

19.0K Views.  University of Arkansas for Medical Sciences.  In dit werk wordt een stap-voor-stap methode beschreven om polyphenolrijke extracten uit gevriesdroogd bessenpoeder te bereiden. Daarnaast biedt het een grondige beschrijving van hoe deze polyphenol-rijke extracten in celkweek kunnen worden gebruikt in aanwezigheid van vasculaire gladde spiercellen (VSMC's) van het peptidehormoon-angiotensine II (Ang II). 

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

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

Environment

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

Biochemistry

Polyol-Based Microwave-Assisted Extraction for Phenolic and Antioxidant Yields

Microwave-Assisted Extraction of Phenolic Compounds and Antioxidants for Cosmetic Applications Using Polyol-Based Technology

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert behavior with plant bioactive chemicals. This study explores the sustainable extraction of phenolic compounds and natural antioxidants from coffee silverskin using the microwave-assisted extraction (MAE) method with polyol-based solvents: glycerin, propylene glycol (PG), butylene glycol (BG), methylpropanediol (MPD), isopentyldiol (IPD), pentylene glycol, 1,2-hexanediol, and hexylene glycol (HG). A comparative analysis was conducted on conventional and non-conventional solvent extractions, focusing on their impact on the bioactive compounds of MAE, encompassing parameters such as total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities like the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), the 2,2&#8242;-azino-bis(-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay (ABTS), and the ferric reducing antioxidant power assay (FRAP). The highest values were observed for TPC with aqueous-1,2-hexanediol extraction (52.0 &#177; 3.0 mg GAE/g sample), TFC with aqueous-1,2-hexanediol extraction (20.0 &#177; 1.7 mg QE/g sample), DPPH with aqueous-HG&#160;extraction (13.6 &#177; 0.3 mg TE/g sample), ABTS with aqueous-pentylene glycol extraction (8.2 &#177; 0.1 mg TE/g sample), and FRAP with aqueous-HG extraction (21.1 &#177; 1.3 mg Fe (II) E/g sample). This research aims to advance eco-friendly extraction technology through natural plant components, promoting sustainability by minimizing hazardous chemical use while reducing time and energy consumption, with potential applications in cosmetics.

Author Spotlight: Eco-Friendly Extraction of Bioactive Compounds Using Polyol-Based Microwave-Assisted Techniques

This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use extracts.

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Biology

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

Medicine

Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug against hyperlipidemia in Traditional Chinese Medicine (TCM) and Indian Medicine theories, however the potential mechanism requires further exploration. The present research aims to reveal the mechanism of FP against hyperlipidemia based on an integrated strategy combining network pharmacology prediction with metabolomics validation. A high-fat diet (HFD)-induced mice model was established by evaluating the plasma lipid levels, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Network pharmacology was applied to find out the active ingredients of FP and potential targets against hyperlipidemia. Metabolomics of plasma and liver were performed to identify differential metabolites and their corresponding pathways among the normal group, model group, and intervention group. The relationship between network pharmacology and metabolomics was further constructed to obtain a comprehensive view of the process of FP against hyperlipidemia. The obtained key target proteins were verified by molecular docking. These results reflected that FP improved the plasma lipid levels and liver injury of hyperlipidemia induced by a HFD. Gallic acid, quercetin, and beta-sitosterol in FP were demonstrated as the key active compounds. A total of 16 and six potential differential metabolites in plasma and liver, respectively, were found to be involved in the therapeutic effects of FP against hyperlipidemia by metabolomics. Further, integration analysis indicated that the intervention effects were associated with CYP1A1, AChE, and MGAM, as well as the adjustment of L-kynurenine, corticosterone, acetylcholine, and raffinose, mainly involving tryptophan metabolism pathway. Molecular docking ensured that the above ingredients acting on hyperlipidemia-related protein targets played a key role in lowering lipids. In summary, this research provided a new possibility for preventing and treating hyperlipidemia.

The present protocol describes an integrated strategy for exploring the key targets and mechanisms of Fructus Phyllanthi against hyperlipidemia based on network pharmacology prediction and metabolomics verification.

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug ...

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact on the effectiveness and quality of the final product. However, existing stability evaluation methods are often time-consuming and labor-intensive, requiring long-term observation and the operation of complex equipment (such as high-performance liquid chromatography), and it is difficult to obtain more physical information about the instability of the system. Therefore, there is an urgent need to establish a fast and accurate stability analysis technology for traditional Chinese medicine. Multiple light scattering is a cutting-edge analytical method that can accurately and rapidly evaluate the stability of traditional Chinese medicines in an environment-friendly manner without changing the nature or state of the sample or using organic reagents.
In this work, using the precise scanning data of multiple light scattering, the present protocol rapidly acquired the variation curves for layer thickness, particle migration speed, and average particle size over time. This enabled the precise identification of the mechanism and crucial characteristics causing the system&#39;s instability in its early stages. Of note, the research period for the extraction process can be considerably shortened by the detailed quantification of the system stability, which also allows for a quick, accurate, and in-depth analysis of the effects of various extraction processes on the stability of Phyllanthus emblica L.

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Here, we introduce a stability evaluation method based on multiple light scattering technology to evaluate the stability of traditional Chinese medicine extracts.

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

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

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