Comparing the Concentrations of Trans-Resveratrol in Wine Samples To
Resveratrol-Based Supplements Using High Performance Liquid Chromatography
Alex Chan
Spring 2010
Dr. John Fruehauf
University of California Irvine
Abstract
In vivo experiments of mice model’s have shown that trans-resveratrol has been
able to act as an anti-cancer, anti-inflammatory agent, a blood sugar-lowering medium,
delay age-related diseases, mimic affects of dietary restriction, and have other beneficial
effects to the model’s health. Using High Pressure Liquid Chromatography (HPLC), an
experiment was conducted to determine the concentrations levels of trans-resveratrol
based supplements that have been placed on the market to consumers to better their
health. In order to determine the approximate concentration levels of trans-resveratrol in
four unknown resveratrol supplements, an experiment was conducted to create a standard
curve using the known concentrations of stock solutions of resveratrol and compare it
against unknown concentrations of resveratrol supplements. A wine sample was also
measured and compared to the results of the supplements to determine the amount of
resveratrol in a glass of wine would equal a single supplement tablet.
Introduction
In medicine today, many various techniques have been used to cure diseases,
prevent ailments, and help patients. One such technique that shows great promise is the
use of trans-resveratrol (3,5,4-trihydroxystilbene), which was first isolated from the roots
of the plant White Hellabore (Veratrum grandiflorum O. Loes) by M. J. Takaoka in 1940
(Baur, 2006). In 1992, trans-resveratrol was also found to be in the skin of grapes, higher
concentrations being specifically in red grapes (Baur, 2006). In plants, resveratrol acts as
a phytoalexin, a natural antibiotic substance to fight against harmful bacteria and fungi.
Synthesized by an enzyme called stilbene synthase, the synthase has been isolated from
grape vines and transferred to other plants in hopes of having similar affects to plants that
do not naturally produce resveratrol (Kobayashi 2000). These experiments have been
proven successful with the mutant plants showing enhanced resistance against certain
fungi.
According to the well-known French paradox, many people have observed the
French to have a low incidence of coronary heart disease despite having a rich saturated
fat diet. Researchers believe that this low incidence of heart disease is because of red
wines source of resveratrol which have been linked to longevity and cancer prevention in
other species that were tested with trans-resveratrol supplements (Wallenborg, 2009).
Many experiments have been conducted to determine the health benefits of consuming
trans-resveratrol based supplements. When fed to fruit flies, worms, and short-lived fish,
resveratrol treatment extended the life expectancies of these animals as well as reduce the
progression of illnesses such as cancer, cardiovascular disease, and age related diseases.
(Baur, 2006)
When applied to in vivo clinical experiments of mice models, results have come out
both favorable and unfavorable. Resveratrol is believed to be metabolized in the liver and
intestine through glucuronidation and sulphonation. It is thought that trans- Resveratrol
activates the Sirtuin-1 gene in humans inducing a dietary restriction-like environment in
people, which creates a biological stress on the body (Pearson, 2008).
Resveratrol has also been tested to inhibit carcinogenesis at multiple stages of cancer by
inhibiting growth and initiation of tumors (Baur, 2006). The possible beneficial effects of
resveratrol continue to grow as more research is done on the mechanism of this
phytoalexin and further research is conducted.
High Performance Liquid Chromatography is a form of column chromatography
used to separate, identify, and quantify compounds based on their idiosyncratic polarities
and interactions with the column’s stationary phase (Clark, 2007). By forcing a solvent to
go through a column under high pressure, one is able to use a very small particle size for
the column producing a greater area of interaction. With reversed phase HPLC, the
column size is the same, but the silica is modified to make it non-polar by attaching 8 or
18 carbon atoms (Clark, 2007). When combined with a polar solvent, there is a strong
attraction between the polar molecules and polar solvent when passed through the
column. Using High Performance Liquid Chromatography (HPLC) and a research
protocol developed by R.M. Lamuela-Raventos in 1995, we are able to determine the
concentration levels trans-Resveratrol and compare it to an unknown concentration of
resveratrol supplements (Lamuela-Raventos, 1995).
Materials and Methods
Sample stock solutions were created using 99.9% concentrated trans-Resveratrol
from Chromadex, in a solid form and mixed with HPLC-grade methanol. Instead of using
the original samples of resveratrol from Sigma-Aldrich, it was decided to use a different
brand of resveratrol for these tests. Elution ratios of 1:32 (0.03125 mg/mL), 1:16 (0.0625
mg/mL), 1:8 (0.125 mg/mL), 1:4 (0.25 mg/mL), and 1:2 (0.50 mg/mL), were created and
run through the HPLC system. Concentrations were stopped at above 1:2 (0.50 mg/mL)
because the HPLC system was unable to read concentrations of samples that were so
highly concentrated and readings capped at around 200,000 mAU’s. Each sample was
first mixed with half of the amount of methanol on a plastic weigh tray in which it was
measured in, to avoid any loss of mass from the supplement when transferring. Once the
supplement/methanol mixture was placed into the container, the second half of methanol
was finally added.
An HP/Agilent 1050 series DAD HPLC system consisting of three major parts was
used for the experiment. An HP 1050 autosampler was used to introduce a liquid sample
automatically into the system at a constant measurement. Approximately 100µL of each
sample was injected into the HPLC system. Once each sample was run through the
system, the HP 1050 quaternary pump moved the sample through a densely packed
column that separated particles by size and solvent elution.
The solvent elution profile used was the same as the previous tester who used two
solvents for separation. Solvent A consisted of glacial acetic acid that was diluted with
HPLC-grade water to get the pH to 2.40. Solvent B consisted of 20% of Solvent A and
80% HPLC-grade acetonitrile. Both were combined in the HPLC system at a flow rate of
1.5 mL/min. The time interval profile it followed was: 0 min, 82% of A, 18% of B; 15
min, 82% of A, 18% of B; 25 min, 0% of A, 100% of B.
Figure 1. Elution gradient plot following the protocal by R.M. LamuelaRaventos. Shows what percentage of each solvent was run during the time interval.
Solvent A consisted of glacial acetic acid that was diluted with HPLC-grade water
till it had a pH of 2.4. Solvent B consisted of 80% of HPLC-grade acetonitrile and
20% of Solvent A
The wavelengths were observed at two different levels, 285 nm and 306 nm. To
focus on the trans-resveratrol isomers, our tests focused mainly on the 306 nm
wavelength. Once the sample was run through the HP 1050 DAD detector, which
provided characteristic information of the retention time, the data from each test was
consolidated to a single computer. The surface area of each peak at its highest retention
time was measured and plotted onto a graph. A linear regression line was then calculated
in order to find a standard curve to compare to other experiments.
Four supplements of unknown concentrations of trans-resveratrol supplements were
then conducted using the same HPLC protocol. Supplement A was described as a white
capsule with an off-white powder substance inside. When taken apart supplement A
weighed 448.47mg. 1mg of Supplement A was massed out and diluted with 2mL of
methanol and tested. Supplement B was a dark green capsule with a fine white powder in
the inside. The mass of the powder measured to be 481.4mg. Supplement C was a darker
brown capsule with ground brown substance consisting of other granules of unknown
content and weighed 594.4mg. Supplement D was a lighter brown capsule and contained
a more ground brown substance that weighed 880.8mg. Supplements B, C, and D were
diluted with 4mL of methanol. The HPLC machine had difficulties reading a 0.25mg/mL
concentration of Supplement A, so a 0.5mg/mL was used instead.
Once all supplements dissolved into a completely liquid form, each sample was
placed into an individual container and run through the HPLC system where all results
were recorded. It is also important to note that after every sample was run through the
system, it was necessary to run 100µL of HPLC-grade acetonitrile for 20 minutes. This
was done in order to clean the system so that no data was skewed by leftover traces of
residue from the previous sample. Because each sample took so long to run, the data
collected from this experiment was conducted over a period of 10 weeks. Tests were
done Monday, Wednesday, and Thursday in the afternoon and multiple samples were run
to best optimize the experiment.
Following the supplement samples, a wine sample was also run through the HPLC
system through similar procedures. 300L of Black Swan Australian red wine was
measured out and filtered through an 0.2m inorganic anopore filter. 100L was injected
into the HPLC system and results were compared to the results from the standard to
determine the amount of trans-resveratrol in the wine sample. Concentration of
resveratrol was then compared to the results of the supplement tests.
Results
Using the results from the stock solutions, a linear regression line was created to
determine the average relationship between the surface areas of the peaks and the
concentration of each solution. The equation of the linear regression line was determined
to be Surface Area of Peak = 74.26 x (trans-Resveratrol concentration mg/mL) - 12167
where R2 is equal to 0.971. The closer the R2 value is to 1 represents the data following a
perfectly linear regression.
Figure 2. The Standard Regression Curve of known concentrations of transResveratrol. The known concentrations were plotted again the peak surface area
and a linear regression line was placed with an equation of y=74.26x -12167. The
R2 was equal to 0.971 and showed to be very well fit to all the possible point
Mass (mg)
Ratio (mg/mL)
Expected Peek Surface Area (maU's)
Concentration of Resveratrol (M)
Resveratrol/1mg Supplement
Expected Resveratrol (mg)
Amount of Wine Necessary (L)
Amount of Wine Necessary
(1 Glass=240mL)
Supplement A
Supplement B
Supplement C
Supplement D Wine Sample
448.47
481.40
594.40
880.80
(1:2)
(1:4)
(1:4)
(1:4)
(1:1)
96496.65
102516.50
37599.31
99697.22
33.74
1463.27
1544.33
670.15
1506.37
164.28
0.4650166
0.9938
0.306095
0.9639
0.03746
208.54
478.41
181.94
849.04
26.695
7.81
17.92
6.82
31.81
32.55
74.67
28.40
132.52
Table 1. Experiment showing individual supplements tested. Results are recorded in
mass, elution gradient mg/mL, the expected peek surface when the surface area is
entered into linear regression equation, amount of resveratrol/1mg of supplement,
the expected amount of total resveratrol in each supplement in mg, the amount of
wine necessary for each supplement in L, and the amount of wine necessary for each
supplement in 1 glass of wine (240mL).
The values of the stock solutions followed a general trend of 1:2 mg/mL having a
surface area of around 158,000 mAU’s, the 1:4 mg/mL having a surface area of 50,000
mAU’s, the 1:8 raio having a surface area of around 33,000 mAUs, and so forth all the
way to the 1:32 mg/mL. Peaks were noticed to spike the highest at 22.5 minutes into the
system and were consistently on time. Samples above a ratio of 1:2 mg/mL were too
concentrated for the HPLC system to read and had to be taken out of the equation
because the system capped at 200,000 mAU’s and were not accurate results.
Taking the area under the curve calculated by the HPLC system from each
supplement and substituting that number into the Y component of the linear regression
equation, we were able to get a measurement of how much resveratrol was in each
sample. That number was then multiplied by the sequence of numbers calculated to get
the molarity of each sample to get an expected amount of resveratrol per 1mg of each
supplement. Once that number was received, it was then multiplied by the total mass of
the supplement to determine how much of the supplement really consisted of transresveratrol.
Chromatogram of Wine Sample
Figure 3. Chromatogram of the Wine Sample at 306nm wavelength with an
injection of 100L. The first set of peaks is from bubbles and excess substance that
was left from the previous sample. Small dots can be seen at 22.5 minutes which
represent the very little peaks from the wine sample. The peaks height only reached
a maximum of 27 mAU’s, thus the peaks are very small.
Table 1. Experimental results of wine sample test at 306nm wavelength. Peak 11 and
12 show where the small amount of resveratrol peaked at with an area of 25.89 and
7.85 mAU’s respectfully.
The wine sample followed the same procedure as the supplement samples. The
sample was substituted into the linear regression equation and the amount of resveratrol
was determined by solving the equation backwards. The number resolved was the amount
of trans-resveratrol in mg/L of wine. The amount of wine per 1 liter was then converted
into per 240mL, which equaled 1 glass of wine, and the results were compared to each
supplement sample.
Discussion
Optimizing a linear regression line from the known concentrations of transresveratrol samples allowed us to compare the results from unknown samples to the
known linear regression line. Because the linear regression line showed a coefficient of
determination of 0.971, statistically we can say there is a direct correlation between
peak surface area and concentration. Diluting each supplement sample and testing it with
HPLC gave us a surface area where the resveratrol would peak within the column.
The peak formed at 22.5 minutes at the 306 nm wavelength and instead of using the
maximum height of the peak, we used the total surface area underneath the curve that
formed. This number showed to be more accurate because it gave us a larger number with
more information about the curve.
Each sample supplement was tested independently from one another and ran
through the HPLC machine on different days to avoid the possibility of mixing samples
in the HPLC machine. Some samples were run multiple times because of inaccurate
readings from the HPLC machine. The average mean was used as results for samples that
were run more then once. The results from each chromatogram were stable and consistent
which allowed us to get more accurate results from each test. According to the
experiment, Supplement A, which weighed 448.47mg contained 208.54mg of transresveratrol. Supplement B had a mass of 481.4mg and contained 181.94mg of resveratrol.
Testing Supplement C, it had a weight of 594.4mg, and in that, contained 181.94mg of
resveratrol. Finally supplement D massed at 880.8mg of resveratrol and of that,
849.04mg was expected to be resveratrol. From the results, some of the samples seem
like a plausible amount of trans-resveratrol to have in a supplement. Supplements sold
can contain anywhere from 5mg to 500mg of trans-resveratrol. Supplement A and C
seem like the most feasible results of resveratrol containing supplements because both
have a significantly lower mass of resveratrol in the total weight of the supplement. It is
expected that supplements also have excess chemicals, vitamins, and nutrients and very
rarely contain 100% pure resveratrol. Results of supplements B and D show that both
have almost a 99% concentrated supplement of resveratrol, which seems very unlikely.
According to the data, it shows that each of these supplements had a very high
concentration of trans-resveratrol. Having done these experiments for two quarters now,
I’m happy to se all the results come out clean and uniform. However that does not mean
that any experimental errors could have occurred while conducting this experiment.
Because HPLC works with such small numbers that are very precise, any sort of
miscalculation in massing or diluting could have easily affected the results. There also
could have easily been trace amounts of resveratrol from past experiments stuck in the
column also. To try to avoid this problem, not only did we run the system with
acetonitrile after each sample, we switched the column upside down and ran it with
methanol to clean the column. However this method of cleaning was only done a couple
of times and trace amounts of resveratrol still could have skewed our results.
The wine sample had an expected concentration of 164.28M, which was then
converted into mg of trans-resveratrol. The expected amount of trans-resveratrol in 1 liter
of wine was determined to be 26.74mg. The amount of trans-resveratrol in mg was then
divided by the amount of trans-resveratrol in each supplement to determine how much
resveratrol is needed for each supplement. Answers were then converted to 240mL,
which was determined to be the amount of wine that was contained in a single glass of
wine. It was determined that one would need to drink 32.55 glasses of red wine to match
the amount of resveratrol in supplement A, 74.67 glasses of wine to match supplement B,
28.40 glasses of wine to match supplement C, and 132.52 glasses of red wine to match
supplement C. Because red wine contains only a very little amount of grape skin itself,
and isn’t very concentrated, wine only has a small amount of resveratrol which matches
the results of the data. A large amount of wine would need to be consumed in order to
match the amount of trans-resveratrol in a single resveratrol based supplement. Better
results could have been determined if we had used an even smaller amount of sample
solution to create a more accurate linear regression line for small amounts of resveratrol.
The method used to determine the concentration of resveratrol in resveratrol-based
supplements can also be easily applied to other chemicals. While it is understood that the
concentration of these resveratrol based products can be determined by HPLC, further
experimentation is necessary to more accurately determine the exact concentration of
these substances, how they are formed, and what we can do to better improve our results.
Work Cited
Barger, Jamie L., Tsuyoshi Kayo, and James M. Van. "A Low Dose of Dietary
Resveratrol Partially Mimics Caloric Restriction and Retards Aging
Parameters in Mice ." PLoS ONE 3.6 (2008): 264-74. Print.
Baur, Joseph A., and Sinclair A. David. "Therapeutic potential of resveratrol: the in
vivo evidence." Nature 5 (2006): 493-506. Print.
Clark, Jim. High Performance Liquid Chromatography - HPLC Direct HPLC Analysis of
Cis- and trans-Resveratrol and Piceid Isomers in Spanish Red Vitis vinifera
Wines . N.p., 2007. Web. 5 Dec. 2009.
<http://www.chemguide.co.uk/analysis/chromatography/hplc.html>.
Kobayashi, Shozo. "Advances of Resveratrol Research." National Institute of Fruit Tree
Science 1.15 (2000): 2-20. Print.
Lamuela-Raventos, Rosa M., Ana I. Romero-Perez, Andrew L. Waterhouse, and M.
Carmen Torre-Boronat. "Direct HPLC Analysis of cis- and trans-Resveratrol
and Piceid Isomers in Spanish Red Vitis vinifera Wines ." Agricultural Food
Chemistry 43 (1995): 281-83. Print.
Pearson, Kevin J., and Joseph A. Baur. "Resveratrol Delays Age-Related Deterioration
and Mimics Transcriptional Aspects of Dietary Restriction without Extending
Life Span." Cell Metabolism 8 (2008): 157-68. Print.
Wallenborg, Karolina, Pinelopi Vlachos, Sofi Eriksson, Lukas Huijbregts et al. "Red
wine triggers cell death and thioredoxin reductase inhibition: Effects beyond
resveratrol and SIRT1 ." Experimental Cell Research 315 (2009): 1360-71.