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 (O
2
) 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
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) .
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
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. 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 3 above is the infrared machine used to analyze the sample.
3,00
2,8
2,6
205,95
75,0
70
65 Functional group
Wavelength (cm -1) Bhatt B.,
2011
Wavelength (figure
5)
60
2,4
55
2,2
2358,24
RO-H (alcohol) 3368.42
3284.58
2,0
1,8
1,6
A
1,4
1,2
1,0
0,8
0,6
0,4
0,2
272,87
0,00
200,0 250 300 350 400 500 550 600 650 700,0 450 nm
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.
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 substance may prevent cancer in such people. However,
50
45
40
35
%T
30
25
20
15
10
5
0
-5
3284,58
3110,74
2852,66
2922,63
1286,57
1599,48
1184,58
1691,17
1663,89
1549,27
1459,85
1358,92
1237,78
972,42
1023,71
861,28
927,65
744,69
COO-H (carboxylic acid)
C=O
2650.2
1680.87
2852.66
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.
-10,0
4000,0 3600 3200 2800 2400 2000 1600 1400 1200 1000 800 690,0 1800 cm-1
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.
Barry Halliwell. “Reactive Species and Antioxidants. Redox Biology is a
Fundamental Theme of Aerobic Life.” Plant Physiology, 141: 312-322
(2006)
Surh, Y.J. “Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances.” Mutation
428(1-2): 305-327 (1999)
Bhatt, B. “Chemical constituents of Solanum zanthocarpum. Journal of
Belay, A.; Gholap, A.V. “Characterization and determination of chlorogenic acids (CGA) in coffee beans by UV-Vis spectroscopy.”
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.