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

The overall goal of this protocol is to provide a simple and reproducible method for the extraction and purification of plant polyphenols that could be used to treat cells in the future. This method can help answer key questions regarding the role of function of foods in human health and disease. This ethanol-based and ultrasonic assisted polyphenol extraction is an easy and reproducible method that allows for a higher polyphenol yield.

The use of chloroform allows for the removal of sugars, fatty acids, proteins and other molecules while ethyl acetate allows for polyphenol fractionation. This concentrated polyphenol extracts can then be used in vitro and in vivo to study the effects of polyphenols as well as their mechanisms of action on various tissues, including the cardiovascular system, as described here. To begin this procedure, prepare the reagents as outlined in the text protocol.

Next, weigh out 10 grams of freeze-dried blackberry powder. Add the berry powder to a one liter round bottom flask containing 100 milliliters of 80%aqueous ethanol. Place the flask in an ultrasonic bath at 25 degrees Celsius.

Under continuous nitrogen purging in subdued light, sonicate the mixture for 20 minutes at 42 kilohertz and 135 watts. After this, use a chilled Buchner funnel with vacuum suction to filter the mixture through a number two filter paper. Rinse the filter cake with 50 milliliters of 100%ethanol.

Save the filtrate and add the filter cake to a one liter round bottom flask containing 100 milliliters of 80%aqueous ethanol. Then, repeat the extraction process by sonicating, filtering and washing the mixture. Combine the two filtrates in a round bottom flask containing 50 milliliters of 80%aqueous ethanol.

Using a rotary evaporator at 62 degrees Celsius and 50 rpm, evaporate the solvent. Continue this until the ethanol no longer evaporates. Next, transfer the sample to a 50 milliliter conical tube.

Inject nitrogen gas at the top of the tube for 10 minutes to evaporate any remaining ethanol. Freeze the sample at negative 80 degrees Celsius for at least 24 hours. Then, freeze dry the sample at negative 50 degrees Celsius for approximately eight hours.

Store the samples at negative 20 degrees Celsius until ready to use. To begin, weigh the freeze-dried extracted samples. Add approximately 20 milliliters of chloroform to a 100 milliliter beaker with the sample.

Using a stir plate, mix the solution for five minutes. Then, pour the mixture into a separating funnel. Let the crude extract decant for one hour at room temperature.

After this, discard the bottom layer and collect the aqueous layer into a clean beaker. Add two volumes of ethyl acetate to the beaker. Using a magnetic stir bar, stir the mixture for five minutes at room temperature.

Next, use a chilled Buchner funnel with vacuum suction to filter the mixture through number two filter paper. Transfer the filtered sample to a round bottom flask. Using a rotary evaporator at 62 degrees Celsius and 50 rpm, evaporate the ethyl acetate for 30 minutes.

After this, freeze dry the sample at negative 50 degrees Celsius for eight hours. Transfer the sample to a 50 milliliter conical tube and store at negative 20 degrees Celsius until ready to use. After culturing the vascular smooth muscle cells, discard the culture medium and wash the cells twice with phosphate buffered saline.

Then, add four milliliters of PBS and two milliliters of 0.25%Trypsin-EDTA. Incubate in a carbon dioxide incubator at 37 degrees Celsius for five minutes. After this, transfer the cells to a 15 milliliter centrifuge tube containing two milliliters of complete medium.

Centrifuge at 1, 100 times g for five minutes. Discard the supernatant and re-suspend the cell pellet with one milliliter of PBS. Using a hemocytometer, count the cells.

Next, in six-well culture plates, seed 50, 000 cells per well. Grow the cells to 90%confluency in complete medium containing 10%fetal bovine serum. Prepare the treatment medium as outlined in the text protocol and store at four degrees Celsius.

Then, add 10 milligrams of either crude or purified extract to one milliliter of plain DMEM to prepare a 10 milligram per milliliter stock solution. Store the stock solution in 200 microliter aliquots at negative 20 degrees Celsius. After this, add two milliliters of treatment medium, containing 0.5%fetal bovine serum and the desired concentration of stock solution to the cells.

Incubate for three days at 37 degrees Celsius in a humidified 5%CO2 incubator. Then, if treating with hormones or growth factors, starve the cells for at least 24 hours by incubating with treatment medium containing 0.5%fetal bovine serum. After starvation, add the desired hormone, replacing the treatment medium every 24 hours for three days, as detailed in the text protocol.

Next, wash the treated cells twice in cold PBS. Lyse the cells using 200 microliters of lysis buffer on ice. Using cell scrapers, collect the cell lysate and transfer to a 1.5 milliliter microcentrifuge tube.

Incubate the collected lysate for 20 minutes on ice, vortexing every five minutes. After this, sonicate the cell extracts with three bursts at 125 watts for 10 seconds each, with a two second pause in between each burst. Using a protein assay reagent at 595 nanometers, measure the protein concentration.

Mix 50 micrograms of protein with lysis buffer to obtain a maximum total volume of 50 microliters. Add 16 microliters of a 4x Laemmli sample buffer. Then, heat the samples for five minutes at 75 degrees Celsius.

Separate samples in 10%SDS-PAGE gels and transfer to PVDF membranes for Western blot analysis with specific antibodies. In this study, an ethanol based extraction is used to identify the different levels of phenolic acids and flavonoids present in crude and purified polyphenol blackberry extracts. While the purification process did not significantly alter the levels of gallic acid or p-coumaric acid, it did increase the levels of 3-O-caffeoylquinic acid from 170.5 to 235.3 ppm and of Quercetin from 24.5 to 95 ppm.

In contrast, Ferulic acid and Rutin were lost during the purification of the crude extract. After the extraction, VSMCs were incubated in 50 to 500 micrograms per milliliter of either crude or purified blackberry extract in medium containing 0.5%fetal bovine serum for three days. Both extracts are seen to be effective at reducing the basal phosphorylation of ERK.

However, the purified extract is seen to down regulate strongly at 100 micrograms per milliliter, while 400 to 500 micrograms per milliliter of crude extract is required. This suggests that the purified extract's greater efficiency at inhibiting ERK phosphorylation is due to its higher concentrations of polyphenol compounds. After this, VSMCs are treated for 24 hours with 200 micrograms per milliliter of purified blackberry extract before the addition of 100 nanomoles of angiotensin II.As previously reported, the blackberry polyphenol extract reduces angiotensin II induced ERK phosphorylation, but demonstrates no effect on catalase or SOD2 expression.

Once mastered, polyphenol extraction can be prepared in a few hours. This technique can aid researchers in the field of functional foods to explore the role of polyphenols in vitro and in animal models. After watching this video, you should be able to extract and purify polyphenols from fruits and vegetables and use the polyphenol extracts to treat cells in culture.

We have used this method to demonstrate the role of blackberry polyphenols in preventing aging of aortic vascular smooth muscle cells. This is significant, considering that aging of the vascular system is a major risk factor for the development of cardiovascular diseases.