The Antioxidant Properties of Green Coffee 'Coffea arabica' Beans
Dr. Marco (UNIBE), Dr. Eduardo Agueras (UNIBE), Ana Julia Gonzalez
(UNIBE),Vanessa Okafor (SUNY Oswego), Maame Hayfron (SUNY Oswego)
Introduction
Objective
Environmental stresses such as pollutants, toxins, radiation, smoking and unhealthy diets introduce
molecules known as free radicals into our bodies. Free radicals are molecules with at least one unpaired
electron (Halliwel, 1999). In humans, the most common form of free radicals is oxygen. When an oxygen
molecule (O2) becomes electrically charged, it tries to steal electrons from other molecules, causing
damage to the DNA and other molecules. (National Cancer Institute- NCI, 2004). High levels of free
radicals begin to form reactive chains which proceed to wreak havoc on important cellular components
of the body. Molecules known as antioxidants have been shown to combat the actions of free radicals
(Goldfarb, 1993). Antioxidants defend our bodies by terminating the chain of reaction of free radicals
before vital molecules are destroyed. By providing the free radicals with the electron they are seeking,
antioxidants succeed in stabilizing the unpaired electrons.
Cancer results from exposure to environmental carcinogen agents such as chemicals, radiation and
viruses. Along with the double bond in the carbon chain of caffeic acid, (a component of Coffea arabica)
the length of the carbon chain between the aromatic ring and the terminal carboxylic group has been
shown to affect cancerous cells (Gomez et al., 2003). Elimination of, or avoiding exposure to these
carcinogens is the key to elementary cancer prevention (Surh., J 1999).
The objective of this study was to extract caffeic acid from Coffea arabica and
further investigate its antioxidant properties by comparing its chemical structure
to that of other compounds know to be antioxidant in nature.
Theory and Methods
200.02 g of ground green coffee bean was weighed into a beaker and mixed with 100 mL of distilled water. The
mixture was then blended for one minute to create an even mixture. In order to make the caffeic acid soluble in
water, 1.51 g of NaOH was dissolved in 100 mL of distilled water. This solution was mixed with the coffee puree and
stirred constantly while being heated for an hour. The resulting mixture was then sieved twice. Hydrochloric acid was
introduced drop by drop into the filtrate until the pH of the solution decreased from 6 to approximately 2. The total
mixture, which was measured and found to be 430 mL, was poured into a 500 mL separatory funnel and filled with 60
mL of chloroform. For a homogenous solution free of bubbles, the mixture was carefully stirred. A few drops of
isopropyl alcohol were then pipetted into the fluid to form a distinct separation. About 60 mL of the desired extract,
which was settled at the bottom of the funnel, was drained out. An additional 60 mL of chloroform was added to the
remaining puree left in the funnel. Upon separation, the process was repeated four more times to extract as much
caffeic acid from the puree as possible. A small amount of anhydrous MgSO4 was added in order to pair up with the
extra water molecules in the final extract. The extract was then filtered to get rid of any traces of the puree in it. The
filtrate was purified in a rotary evaporator at about 65oC. The resulting glossy and gelatinous sample was diluted with
minute drops of chloroform. With a pasteur pipette, the sample was carefully transferred into a jar, covered with filter
paper, and secured with a rubber band. Finally, several holes were punctured into the filter paper in order to allow the
chloroform to evaporate, leaving the caffeic acid to crystallize. The crystallized caffeic acid was extracted from the jar
with a few drops of chloroform to carry out the thin layer chromatography. The mobile phase for the separation was
prepared using a mixture of benzene, methanol and acetic acid in the ratio 90:16:8 respectively. The thin layer
chromatography was then conducted on a TLC silica gel aluminium sheet. However, the result gotten
was
a
undesirable so the process was repeated, but this time sodium sulphate was added to the solvent (the mobile phase)
in order to absorb the water from the acetic acid. This produced a clearer separation of the compound. Another thin
layer chromatography was carried out using a mixture of acetone, chloroform, butanol and ammonium hydroxide as
the mobile phase in the ratio 30:30:40:10. After the process was completed, the sheet used was sprayed with a
substance that was prepared in order to properly highlight the separation of the compound. This spray consisted of
5g of ferric chloride, 2g of iodine dissolved in 50mL of acetone, and 20% aqueous tartaric acid.The sample was then
taken to the lab for Infrared and ultraviolet tests to be carried out on it.
Figure 1
depicts the
set-up used
to extract
the caffeic
acid from
the coffee
bean puree.
Figure 2 above shows
the crystallized caffeic
acid after it had gone
through the rotary
evaporator.
Figure 3 above is the infrared
machine used to analyze the
sample.
Results
3,00
75,0
2,8
70
2,6
Functional group
65
205,95
60
Wavelength (cm -1) Bhatt B., Wavelength (figure
2011
5)
2,4
55
2,2
2,0
45
1,8
40
RO-H (alcohol)
2358,24
50
861,28
927,65
3110,74
1,6
A
%T 30
1,4
25
1,2
20
2852,66
15
1,0
272,87
10
0,8
2922,63
1599,48
744,69
5
1184,58
1549,27
1358,92
1459,85
0,6
0
0,4
-10,0
4000,0
0,00
200,0
1237,78
1691,17
1663,89
-5
0,2
972,42
1023,71
1286,57
250
300
350
400
450
nm
500
550
600
650
700,0
Figure 4 shows the UV-Vis absorption spectra of
caffeic acid in the wavelength regions of 200 - 700
nm. The graph consists of two maximum points
where the first maximum point is at 205.95 nm. The
second peak rests at 272.87 nm. These values
correspond well with previous findings at 217 nm and
324 nm respectively.
Conclusion
It was concluded that caffeic acid had been extracted and
it in fact possesses qualities which can be said to be
antioxidant in nature. This is because the -OH functional
groups present in caffeic acid are capable of providing
free radicals with the extra electron they desire, and in
essence, destroying them. Caffeic acid, which is a
polyphenol can therefore act as an inhibitor and play a
major role in the prevention of diseases stemming from
free radical activity in the body; specifically cancer. This
goes to show that an increased intake of foods rich in this
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
3284.58
COO-H (carboxylic
acid)
3284,58
35
3368.42
800
2650.2
2852.66
C=O
1680.87
1691.17
C=C
1575-1650
1663.58-1599.48
sp3 C-O
1123.18
1184.58
Table 1 compares the IR values gotten with the theoretical ones
for caffeic acid.
690,0
Figure 5 represents several functional groups present
in caffeic acid. The exact values (cm-1) are given and
compared with generalized positions and relative
intensities of absorption bands in Table I. It is evident
that the right side of the diagram contains a very
complicated series of absorptions. This area, known as
the finger print region, is mainly due to a manner of
bending vibrations within the molecule.
Upon completion of the investigation, results not only show that
the ground coffee bean contains caffeic acid but also reveal the
double bonds exhibited in a phenolic compound. In comparison
with earlier observations (Belay, 2009), a consistent correlation is
observed between the UV-Vis values. Considerable attention has
recently focused on the possibility that polyphenols bind with
specific kinases and act as inhibitors, contributing to their
biological effects in preventing disease. In accordance with the
obtained results, this suggests that this compound (caffeic acid)
could act as a potent chemo preventive agent against cancer.
References
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(2006)
Surh, Y.J. “Molecular mechanisms of chemopreventive effects of
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Chemical and Pharmaceutical Research. 3(3): 176-181 (2011)
Belay, A.; Gholap, A.V. “Characterization and determination of
chlorogenic acids (CGA) in coffee beans by UV-Vis spectroscopy.”
Acknowledgements