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 highl....

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
      ....

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