Benedict’s, Barfoed’s, Selivanoff’s, Bial’s tests show different sugars exist in ginkgo biloba extract and leaves
http://www.msu.edu/~john1670/biologyblues.html
February 27, 2003
Abstract
By: Nurit
Golenberg, Nik Spiro, Philip Johnson
In this investigation the makeup of a ginkgo leaf was compared to an herbal supplement made primarily with ginkgo extract. It is hypothesized that the sugar makeup of the leaf differs from that of the extract, that there will be more pigments in the leaf than in the extract, that the enzyme PPO will be present in the extract, but not in the leaf, and that ginkgo extract would enhance memory. Different properties of the sugars were tested in the ginkgo leaf and the ginkgo extract using Benedict’s test, Barfoed’s test, Selivanoff’s test, Bial’s test and iodine test. Bial’s and Selivanoff’s tests showed the leaves to have different properties than the extract such as a monosaccharide ketose and hexose (or higher) furanose, which supports the hypothesis. Pigment presence and absorbance was also evaluated. Separating the pigments using paper chromatography should have allowed for the identification of the pigments present, but the results were inconclusive. They showed the pigment chlorophyll b could be present in the leaf, but the extract did not contain any pigments at all. Another test was done to detect the presence of PPO. Contrary to the hypothesis, the results were the same for both in that neither the extract nor the leaf had any PPO present. Finally, a memory test was given to test the validity of memory-enhancement claims for the extract. The results support that it did indeed provide a slight increase in memory ability.
Discussion
The focus of this investigation was to determine what changes were made to the Ginkgo leaf in order to transform it into a supplement. It was hypothesized that the sugar makeup of the leaf would indeed be different than that of the supplement. It was also predicted that the leaf form of Ginkgo would contain more chlorophyll pigments than that of the supplement because the extraction process would remove some of them. It was hypothesized that the enzyme PPO would be present in the leaf but not the extract. Finally, it was hypothesized that the ginkgo extract would increase memory capabilities
Unknown sugar analysis
The several different Carbohydrate Tests performed on the Ginkgo leaf and the 50mg Herbal Plus Standardized Ginkgo Biloba herbal supplement showed many things. Most importantly, every test for the ginkgo extract did not produce a change. Most likely, this is because the extract does not contain any sugar at all. While there is a chance that this could happen with sugar present, probably all sugar was removed in the extraction process.
However, there were results for the leaf solution. With the use of tests like Benedict’s, Selivanoff’s, Barfoed’s, Bial’s, and Iodine, it was possible to get an idea of the structure of the sugars (Table 1). Benedict’s test showed no change for any of the three leaf trials. This reveals that the sugar is not a reducing sugar (it does not contain a free or potentially free aldehyde group). Barfoed’s test was used to find if the sugars in the extract and leaf were mono- or polysaccharide. The leaf and extract solutions once again did not produce any red precipitate, which means the subjects do not contain monosaccharides. Instead, it must only contain di- and/or polysaccharides if any sugars at all. The solutions made from Ginkgo leaf turned red after less than a minute of heating in Selivanoff’s test. This shows that the leaves contain sugars that are ketoses. Ketose sugars have their carbonyl group in the middle of the carbon chain. Bial’s test enabled us to determine if the sugars contained furan rings. The leaf solutions turned a muddy brown. Like the fructose that was used as a control, the leaf must contain sugars that contain hexose, or perhaps higher furanose rings. Finally, the absence of starch was discovered by performing the Iodine test. The test performed on the Ginkgo leaf remained a dark yellow color meaning there was no starch present.
The hypothesis predicted that the sugars in the extract and leaf of the Ginkgo would differ. This prediction was somewhat true: the sugar content was indeed different, but this is because all sugar was removed during the extraction process. The sugar contained has a hexose furanose and is a ketone. This could very likely be fructose. This fructose must be part of a larger sugar, however, because tests indicated only di- or polysaccharides. It could perhaps be from the disaccharide sucrose.
Photosynthesis
Our data supports the hypothesis that the ginkgo leaf contains more pigments than the extract does. Unfortunately, the separations in all the trials did not produce good results, even though they were all redone in an attempt of a better outcome (Figure 6, Table 2). The first effort (3 trials each of both the extract and the leaf) produced no pigments whatsoever on the strip. However, the second attempt did show something – a smear of pale green color rising up through the center of the paper strip. The problem was that the pigment, presumably chlorophyll b, did not manifest itself in a line as we were expecting. Instead, it covered an area that began at the original sample dot and faded out.
Still, one strip of the leaf did indeed show a definitive front (Figure 7). The strip had an Rf value similar to that of chlorophyll b, which supports the color seen (Table 2). On the other hand, the extract does not contain any pigments at all, as can be seen on all the strips done (Figure 6). This was surprising, as some pigment was thought to have remained in the extract even after it was removed from the leaf. Also, it was expected that the leaf would also contain more pigments than was separated on the strips. Perhaps the fact that they are somewhat old and dry caused this, because they were already turning more of a brown color. A remedy to this for future experiments would be to find a source for fresh, newly cut ginkgo leaves. However, these could not be found anywhere when this experiment was being conducted. Also, since all the strips contained less pigment than was expected, it is possible something did not work correctly with the procedure, which might also explain the lack of pigment fronts for the leaves.
Because the lines of all but one trial were not very definitive, the data is inconclusive. Although, as discussed in the previous paragraph, the pigment could have been chlorophyll b, perhaps it was not. It is a possibility that it could be chlorophyll a instead, or perhaps not even a pigment at all. It is impossible to tell from these results and different procedures would need to be used to further study the pigments involved.
As a result of the lack of pigments, the absorbance did not turn out very well. The absorbance spectrum of the leaf does resemble that of chlorophyll a or b (Figure 8) (Freeman, 2002). However, the same could be said for the extract’s spectrum, which the previous test showed contained no pigments. One explanation is that the paper chromatography results were flawed and both the extract and the leaf contained more pigments than was shown. This is a possibility since those results were inconclusive. Another possible explanation is that the settling of larger particles in the solutions to the bottom of the container created this spectrum. The data shows the absorbance gradually decreasing as time increases on both graphs. This could be explained by the settling of larger particles sinking to the bottom of the container – quickly at first and then more slowly as the amount decreased. This could possibly produce the results shown in the absorbance spectrums. The leaf showed higher absorbencies than that of the extract on average (Figure 8). This difference could be explained if the leaf contains more pigments and they absorb some of the light that is passed through it. However, once again, the lack of good results in the previous test is a problem even after multiple trials, and would have to be fixed in future experiments.
Enzymes
The enzyme tests that were outlined in the methods were chosen in order to determine the presence of PPO, and to observe the effects of heat and inhibitors, differences in pH, and varied concentrations of substrate on the activity of the enzyme. It was predicted that the ginkgo supplement would be less inhibited in function than the enzymes contained in the leaf. Countering our hypothesis, no PPO was found in either of the two test subjects. Due to the inability to test for other enzymes, the enzyme portion of the experiment was scrapped.
Memory
Test
It is believed that ginkgo and its herbal supplements are able to improve many functions in the human body. One such function, memory enhancement by increased blood flow to the brain, proved to be an interesting subject. Using sheets with random numbers and timed tests, it seemed possible to observe the pills effects on a person. After performing the tests on several volunteers, our hypothesis was shown to be correct in that there was an overall increase in memory. Possible factors that could have brought some of the scores down between tests would be the individual volunteer’s daily activities such as class studying or sleeping. After all tests were completed percent increase for each person's score was calculated then an average of all the percentages was made. The ginkgo biloba supplement showed an average of +5.74% increase in one’s recall abilities (Table 3).
Many of the results found were unexpected. The sugar content looked to be indeed different, but it was discovered that the extract probably did not contain any sugar at all. The data showed there were minimal pigments in the leaf, and apparently none in the extract, which was not predicted. PPO was not found to be present in either the leaf nor the extract, which was unexpected. However, the extract did tend to increase memory a small amount, which supports both the hypothesis and claims about the supplement.
Table 1. The results of the carbohydrate tests. Benedict’s tests for reducing sugars and is indicated by a red/brown color. Barfoed’s tests for monosaccharides which are indicated by a change of color to red. Selivanoff’s differentiates between ketoses and aldoses. Ketoses turn red in less than a minute, aldoses in greater than a minute. Bial’s tests for furanose rings, and a brown color indicates a hexose (or higher)-furanose. Finally, the iodine tests for starch, which is indicated by a blue/black color.
|
Sample |
Benedict's |
Barfoed's |
Selivanoff's |
Bial's |
Iodine |
|
Ginkgo
Leaf (1) |
No change (Blue) |
No change
(Blue) |
Red<1min |
Muddy Brown |
No change (Yellow) |
|
Ginkgo
Leaf (2) |
No change (Blue) |
No change
(Blue) |
Red<1min |
Muddy Brown |
No change (Yellow) |
|
Ginkgo
Leaf (3) |
No change (Blue) |
No change
(Blue) |
Red<1min |
Muddy Brown |
No change (Yellow) |
|
Ginkgo Extract
(1) |
No change (Blue) |
No change
(Blue) |
No change
(in 2 min) |
No Change |
No change (Yellow) |
|
Ginkgo Extract
(2) |
No change (Blue) |
No change
(Blue) |
No change
(in 2 min) |
No Change |
No change (Yellow) |
|
Ginkgo Extract
(3) |
No change (Blue) |
No change
(Blue) |
No change
(in 2 min) |
No Change |
No change (Yellow) |
|
Positive Control |
(Glucose) Red/Brown |
(Fructose) Red |
(Fructose) Red<1min |
(Fructose) Brown |
(Starch) Blue/Black |
References
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Newton,
MA: Integrative Medicine Communications, 2000.
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medicine,
Tokyo; New York: Springer, 1997.
3. Ward, Christopher P., et al. Ginkgo biloba extract: Cognitive enhancer or anti-stress buffer. Pharmacology, Biochemistry and Behavior. 2002; 72:913-922
4. LeBars, Pierre L., et al. A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. JAMA. Oct 22, 1997 v278 n16 1327-1333
5. Maleszewski, Wilterding, Sayed, Luckie. LBS-145: Cell and Molecular Biology Lecture/Lab Spring 2003 Course Packet. East Lansing: MSU, 2003.
6. Freeman, Scott. Biological Science.