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

Summary

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).

Abstract

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.

Introduction

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-carcinogenic^12,13 properties.

Epidemiological studies demonstrate an inverse association between the consumption of flavonoids and cardiovascular disease (CVD) incidence^16,17 and mortality^14,15. 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-κB) and extracellular signal-regulated kinase (ERK) in lipopolysaccharide (LPS)-stimulated J774 cells²². BRB extracts decreased the NF-κB 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-α) 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.

Protocol

1. Preparation of Reagents

1. Prepare 80% ethanol (100 mL) by mixing 80 mL of absolute ethanol (molecular biology-grade) and 20 mL of cell culture-grade sterile water.
2. To prepare polyphenol extract (10 mg/mL), weigh 10 mg of CE or PE. Add 1 mL of plain Dulbecco's Modified Eagle Medium (DMEM) under a cell culture hood. Vortex the solution. Aliquot in 200-µL portions and store at -20 °C.
3. Prepare lysis buffer.
   1. 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 µL 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 °C.
   2. Add 10 µL/mL of protease inhibitor cocktail. Add fresh when the cells are ready for lysis.
4. Prepare Laemmli sample buffer (4x).
   1. 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 β-mercaptoethanol. Add water up to 250 mL. Keep at 4 °C.
5. 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.
6. 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.

2. Preparation of Blackerry Extracts

1. Preparation of crude extract from freeze-dried berry powder.
   1. 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.
   2. Mix the fruit powder with 100 mL of 80% aqueous ethanol in a 1 L round-bottom Erlenmeyer flask.
   3. Sonicate the mixture for 20 min at 42 kHz, 135 W using an ultrasonic bath at 25 °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.
   4. 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.
   5. Rinse the filter cake with 50 mL of 100% ethanol. Save the filtrate and add the filter cake to a 1 L round-bottom flask containing 100 mL of 80% aqueous ethanol.
   6. Repeat the extraction process for the residue (steps 2.1.2 - 2.1.4).
   7. 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.
   8. Use a rotary evaporator at 62 °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.
   9. 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.
   10. Freeze the sample at -80 °C for at least 24 h.
       NOTE: This step is needed to allow for a more efficient freeze-drying process.
   11. Freeze-dry the sample at -50 °C for approximately 8 h. Store the samples at -20 °C.
       NOTE: The freeze dryer creates a powerful vacuum at -50 °C inside the chamber to dry the samples but preserves most of the compounds, such as polyphenols.
2. Preparation of purified polyphenol extracts from crude extracts.
   1. Weigh the freeze-dried extracted samples.
      NOTE: The volume of the extract at this step is approximately 10 mL.
   2. Add two volumes (~20 mL) of chloroform to the crude extract and shake the solution for 5 min in a stir-plate.
   3. 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.
   4. Discard the bottom layer (chloroform phase) from the separating funnel and collect the aqueous layer into a clean beaker.
   5. 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.
   6. Filter the mixture through #2 filter paper using a chilled Buchner funnel with vacuum suction.
   7. Collect the filtered sample and transfer it to a round-bottom flask to be used with the rotary evaporator.
   8. Set the rotary evaporator to 62 °C and 50 rpm and evaporate the ethyl acetate for about 30 min.
   9. Freeze-dry the sample at -50 °C for approximately 8 h. Transfer the sample to a 50 mL conical tube and store it at -20 °C.

3. Treatment of VSMCs with Berry Extracts

1. VSMCs culture.
   1. Isolate VSMCs from the thoracic aortas of Sprague-Dawley rats by performing an enzymatic digestion, as previously described²⁹. Culture the VSMCs as described²⁹.
   2. 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 °C.
2. Incubation of VSMCs with polyphenol extracts.
   1. 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 °C in a CO₂ incubator.
      1. Collect the cells, mix them with 2 mL of complete medium in a 15 mL centrifuge tube, and centrifuge at 1,100 × g for 5 min. Discard the supernatant and resuspend the cell pellet with 1 mL of PBS. Count the cells using a hemocytometer.
      2. 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 °C in a humidified 5% CO₂ incubator.
   2. 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 °C.
      NOTE: Polyphenol extracts and Ang II are added directly to cells using this medium.
   3. 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 µL aliquots at -20 °C.
   4. 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 µg/mL. Change the medium every 24 h by adding fresh polyphenol extracts. Treat the cells for 3 d.
   5. 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.
      1. 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.
3. Preparation of cell samples.
   1. Wash the treated cells twice in cold PBS and lyse them with 200 µL of lysis buffer on ice.
   2. 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.
   3. 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.
   4. Measure the protein concentration using a protein assay reagent at 595 nm.
   5. Store the samples at -20 °C for analysis by Western blot.
4. Western blot.
   1. Mix equal amounts of protein (about 50 µg) from control non-treated and polyphenol-treated or Ang II-treated samples with lysis buffer to obtain a maximum total volume of 50 µL. Add 16 µL of a 4x Laemmli sample buffer. Heat the samples for 5 min at 75 °C.
   2. 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.
   3. Block the membrane with 2% milk in TBS 0.05% Triton-X100 buffer (TBS-T) for 20 min.
   4. 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 °C.
   5. Wash the membrane three times, 10 min each, with TBS-T and incubate with an HRP-linked secondary antibody for 45 min.
   6. Wash the membrane three times, 10 min each, with TBS-T and develop by enhanced chemiluminescence (ECL).

Results

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...

Discussion

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-

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Angiotensin II	Sigma-Aldrich, Inc.	A9525-10MG	Treatment of VSMCs
β-actin	Sigma-Aldrich, Inc.	A2228	Primary antibody (1:5000)
Blackberry fruit	Mercer Foods		Freeze-dried blackberry powder
Catalase	Calbiochem	219010	Primary antibody (1:1000)
Chloroform	Biotech Grd, Inc.	97064-678	Preparation of purified polyphenol extracts
Dulbecco's Modified Eagle Medium (DMEM )	Mediatech, Inc.	10-014-CV	Culture of VSMCs
Ethanol (absolute molecular biology grade)	Sigma-Aldrich, Inc.	E7023-500ML	Preparation of polyphenol extracts
Ethylacetate	Sigma-Aldrich, Inc.	439169	Preparation of purified polyphenol extracts
ERK1/2	Cell Signaling Technology, Inc.	9102S	Primary antibody (1:500)
EDTA, 500 mM, pH 8.0	Teknova, Inc.	E0306	Lysis buffer
Freeze-Dryer	Labconco	VirTis Benchtop K	Preparation of polyphenol extracts
Fetal Bovine Serum (FBS)	Seradigm	1400-500	Cell culture
HEPES	Sigma-Aldrich, Inc.	H3375	Lysis buffer
NaCl	EMD Millipore, Inc.	7760	Lysis buffer
NaF	J.T.Baker, Inc.	3688-01	Lysis buffer
Na3VO4	Sigma-Aldrich, Inc.	450243	Lysis buffer
Na4P2O7 , decahydrate	Sigma-Aldrich, Inc.	S-9515	Lysis buffer
phospho ERK1/2	Cell Signaling Technology, Inc.	9101S	Primary antibody (1:1000)
Protease inhibitor cocktail	Sigma-Aldrich, Inc.	P8340-5ml	Lysis buffer
Protein assay dye reagent	Bio-Rad Laboratories, Inc.	500-0006	Protein concentration Measurement
PVDF transfer membrane	Thermo Scientific, Inc.	88518	Western blots
Rotatory Evaporator	Buchi Labortechnik	Rotavapor R3000	Preparation of polyphenol extracts
Sterile water	Mediatech, Inc.	25-055-CV	Preparation of polyphenol extracts
Sonicator	QSonica, LLC	Q125	Preparation of cell extracts
SOD2	Enzo Life Sciences, Inc.	ADI-SOD-110-F	Primary antibody (1:1000)
Triton-X-100	Sigma-Aldrich, Inc.	X100	Western blots
Whatman #2 filter paper	GE Healthcare, Inc.	28317-241	Preparation of polyphenol extracts