A. barbadensis vs E. pulcherrima Chromatography Shows Differing Rf Values,
Similar Pigments Verify Like Properties
Vets of the Future (Duhn,
duhn, duhn…)
Jennifer
Beninson
Michele
Fritz
Sarah
Hay
LBS 145 Sec Th1
Abstract:
By:
Michele Fritz; Revised by: Sarah Hay; Revised by: Jennifer Beninson
Many plants have medicinal purposes whereas others have adverse properties such as irritation to the skin and digestive problems when consumed. Our objective was to investigate the structural/functional properties of two plants: healing Aloe barbadensis, and injurious Euphorbia pulcherrima. Three areas were explored including carbohydrates, photosynthesis, and enzyme activity. Benedict’s, Selivanoff’s, Bial’s, and Iodine tests confirmed the presence of various carbohydrates. Photosynthetic properties were examined through paper chromatography to extract and identify various photosynthetic pigments and spectroscopy to detect absorption rates. Litmus paper for pH and polyphenoloxidase (PPO) presence using catechol solution and absorbency were explored. The carbohydrate tests for A. barbadensis indicated aldose, and possibly sucrose. In E. pulcherrima, ketose groups, small traces of starch, and possibly reducing sugars were found. Some tests, however, were inconclusive. E. pulcherrima and A. barbadensis, showed variations on pigments Chlorophyll a, chlorophyll b, carotene and xanthophyll. When compared to the control Rf values, A. barbadensis’s were not similar, while E. pulcherrima’s values were. The light absorbency of E. pulcherrima was 2.94 at 400 nm. At 430 nm A. barbadensis (A) had an absorbency of .506, for A. barbadensis (B) 1.021. From 670-685 nm, A. barbadensis (A) had an absorbency of .251, for Aloe barbadensis (B) .543 (Table 3, Figures 8, 9, 10). Polyphenoloxidase was not present in A. barbadensis, pH of 5.5, but was possibly present in E. pulcherrima, pH of 5.5. According to a chi-squared test (Table 6), E. pulcherrima’s presence of PPO was not large enough to be considered valid.
Discussion:
By:
Jennifer Beninson; Revised by: Michele Fritz; Revised by: Sarah Hay
The differences in carbohydrate, photosynthetic and enzymatic composition of plants that harm, poinsettia, and heal, aloe, were tested to determine how much plants with such opposite effects really differ. To test this curiosity, Aloe barbadensis (Figure 1) and Euphorbia pulcherrima (Figure 2) were chosen as the model plants for harming and healing respectively. To ensure replication and variety, two different plants of each species were used and the plants were labeled A and B accordingly. In all tests performed, either three 11% whole-plant solutions from each plant or 10% aloe gel solutions were tested along with a positive control. The controls were used to ensure the tests themselves were not problematic. It was hypothesized that E. pulcherrima and A. barbadensis would share similar carbohydrate compositions as well as similar photosynthetic properties, but their enzymatic composition would differ greatly due to their opposite properties more specifically A. barbadensis would contain PPO and E. pulcherrima would not.
It was found that Aloe barbadensis and Euphorbia pulcherrima do not share a similar carbohydrate composition according to various performed laboratory tests. When solutions A and B of each plant were put through Benedict’s, Selvanoff’s, and Bial’s tests, observation of color changes and/or precipitate formation (Table 1) indicated which sugars were present in each plant. The Iodine test also indicated which plants contained starch (Table 1). From Benedict’s test, the darkening of solutions from light green to dark emerald showed a slight possibility those plants A and B of E. pulcherrima contained reducing sugars (Figure 3a, 3b). These color changes indicated a possible formation of a red precipitate in both, although the actual ‘redness’ could not be seen by the naked eye due to the solutions’ already dark colors. Since a red precipitate was not clearly detected, Benedict’s tests were deemed technically inconclusive. Spec readings were not taken due to time constraints, although they might have been helpful to detect/indicate any precipitate formation that could not have been seen by the naked eye. Selvanoff’s test showed that disaccharide ketoses were present in both solutions A and B of E. pulcherrima, by turning a light brown within approximately 1 minute, a time that denotes the presence of disaccharide ketoses (Fig 4). Bial’s test yielded information that both solutions of E. pulcherrima contained five-membered rings, called hexose (or higher) furanose rings, because the reaction fluid turned a light olive-brown color (Fig 5a). The Iodine test indicated that both plants of E. pulcherrima contained some starch due to the formation of a black precipitate. Let it be noted, however, that solution A contained a slightly greater amount of precipitate than solution B, which contained only swirls of black precipitate, indicating a smaller amount of starch (Fig 6a, 6b).
Unfortunately, no reactions occurred in A. barbadensis solutions A or B with the Benedict’s, Selivanoff’s, Bial’s, or Iodine tests that were performed (Table 1, Figure 3a,b; 4; 5b; 6c,d). The first 10% solutions used were composed of only aloe gel so new solutions containing 11% whole-plant solutions were made and tested for Bial’s and Iodine tests. This was done to see if there was a greater sugar concentration in the leaves causing the solutions to react with the carbohydrate tests. Again no reactions were detected in Bial’s or Iodine tests. Two other research groups, the Molecules and Pika, also conducted experiments with members of the aloe family. The Molecules prepared and tested several differently concentrated solutions of aloe. Even with a 100% aloe solution, there was no reaction with any of the carbohydrate tests. Pika, however, was able to detect carbohydrates from various different portions of the aloe plant, including the leaf, meat/gel, and the sap. These solutions were 50% concentrated. Color changes were indicated for the leaf in Benedict’s and Barfoed’s tests with only a mild change indicated in Selivanoff’s tests. The meat/gel showed only a slight color change in Selivanoff’s tests, but did not react with any of the other tests. The sap reacted with all tests, Benedict’s, Selivanoff’s, Bial’s, and Iodine, indicated by color changes and/or precipitate formations. The result discrepancies between research groups could be explained by different growth stages. Pika’s plant may have been experiencing a growth phase, therefore storing sugars in its leaves where they could be easily accessible. Due to dormant stages, however, the plants yielding no results may have been storing their sugars in their roots, creating a lower carbohydrate concentration in their leaves. Leaf storage verse root storage could have created a great enough concentration difference to cause the plants to react differently in the various carbohydrate tests.
The abundant sugar found in A. barbadensis could potentially be sucrose since only the Bial’s test showed a possible presence of sucrose by indicating a presence of pyranose, which reflects the glucose portion of sucrose. Sucrose is also a logical source of sugar for the plant since “Sucrose plays a pivotal role in plant growth and development because of its function in translocation and storage…” (Huber and Huber, 1996). In addition, it was concluded that either carbohydrate concentrations were too low to react with the tests or that the properties of the plant, such as latex, interfered with the tests themselves. Logically, A. barbadensis should contain some monosaccharides and starches because glucose, a monosaccharide, results from photosynthesis and is stored in the form of starch (Freeman, 2002). Further research may include determining the plants’ growth stages before examining the presence of sucrose as well as other physical/chemical constituents that may interfere with carbohydrate testing.
To ensure the validity of our findings, positive controls were used with each test. The positive controls used were as follows: Fructose for Benedict’s, Selivanoff’s, and Bial’s tests, starch for Iodine tests, and xylose for Bial’s tests. Each control reacted appropriately indicating that each testing reagent had reacted properly. In Benedict’s tests fructose formed a red precipitate, and in Selivanoff’s tests it formed a rust-brown solution after 45 seconds. The starch solution turned black when iodine was added indicating a positive presence of starch. In Bial’s tests fructose reacted to form a dark olive/brown solution whereas xylose formed an aqua-green solution, both indicating the presence of furanose.
Euphorbia pulcherrima and Aloe barbadensis share similar pigmentation. The chromatography strip yielded four colors for each plant (Figure 7). E. pulcherrima’s and A. barbadensis strips contained orange, which denotes the presence of carotene or similar pigments, xanthophylls which are an orange/yellow color, as well as blue-green, chlorophyll a and similar pigments, and pale green, chlorophyll b and similar pigments. Rf values were obtained from the pigments on the chromatography strip. The control Rf values were carotene 1, xanthophylls .8, chlorophyll a .71, and chlorophyll b .53 (Table 2). The averaged Rf values obtained from A. barbadensis (A) were carotene 1, xanthophylls .51, chlorophyll a .29, and chlorophyll b .15 (Table 2). The averaged Rf values we obtained from A. barbadensis (B) were carotene 1, xanthophylls .57, chlorophyll a .29, and chlorophyll b .17 (Table 2). The Rf values for E. pulcherrima were carotene 1, xanthophylls .72, chlorophyll a .61, and chlorophyll b .4 (Table 2a). The Rf values in A. barbadensis do not correlate well with the control Rf values indicating differing pigments from the ones mentioned. The Rf values for E. pulcherrima are closer to the control Rf values meaning that the pigments are very similar to the ones mentioned even thought they may not be exact.
Carotene and xanthophylls belong to pigments called carotenoids, which absorb wavelengths that chlorophyll are unable to absorb. Carotenoids, therefore, broaden the absorption spectrum of any plant that contains these pigments (Freeman, 2002). The light absorbency of E. pulcherrima was 2.94 at 400 nm whereas A. barbadensis (A) had an absorbency of .506 and for A. barbadensis (B) 1.021 at 430 nm. A. barbadensis (A) had an absorbency of .251 and for A. barbadensis (B) .543 from 670 nm to 685 nm (Table 3, Figures 8, 9, 10). E. pulcherrima (B) was not able to be tested due to the fact that all the leaves had been used up in earlier experiments and the leaves didn’t have enough time to regenerate. E. pulcherrima absorbed the most light at the 400nm end which means that it absorbs blue/violet light and reflects red pigments. This makes perfect sense because most of the leaves had a reddish tint to them. It also reflects green pigments because a portion of the leaves used for the solutions also contained green leaves. In the aloe plants, there were two peaks in the absorption vs. wavelength graph (Figure 8,9). The peaks were located at either end of the visible light spectrum; 430nm and between 670-685nm. This also makes sense because the aloe leaves appeared green to the naked eye. Green pigments absorb blue light and red light, which would account for the peaks in the graph. The lowest readings on the aloe graph appear in the green region meaning that aloe leaves reflect green pigments. The range of light that each plant could absorb paralleled the absorption ranges of the pigments found on their respective chromatography strips.
Next, pH was tested for in E. pulcherrima and A. barbadensis by adding a small strip of litmus paper directly to the inner part of the plant. E. pulcherrima reflected a pH of 5.5 and A. barbadensis reflected a pH of 5.5. Unlike carbohydrate and photosynthetic properties, these plants’ enzymatic compositions are similar: both E. pulcherrima and A. barbadensis do not show evidence of polyphenoloxidase. Polyphenoloxidase (PPO) catalyzes the oxidation of certain organic compounds and is responsible for the browning of fruits and vegetables (Maleszewski et al., 2003). PPO was tested for by first taking readings of just the E. pulcherrima solutions without catechol and then by adding catechol to both solutions of E. pulcherrima and A. barbadensis. The spectrometer was used for E. pulcherrima solutions and readings were taken at 445nm, however, readings were supposed to be taken at 480nm. Since the absorbencies were all taken at the same wavelength they should be proportional to what would have been recorded at 480nm. The absorbencies from the solutions without catechol were compared to the ones with catechol. Catechol is a colorless substrate that when oxidized by PPO, turns a brownish color indicating the presence of PPO. This brown color results from the chemical o-benzoquinone, the product of the catechol-PPO reaction (Maleszewski et al., 2003). Results showed that there was a possible presence of PPO in E. pulcherrima, however, a chi-squared analysis showed that the data (Table 5) was not significant enough to declare presence of PPO (Table 6). PPO was tested for in A. barbadensis by adding 9 drops of catechol directly to 2ml of the solution to see if a color change could be detected with the naked eye, but no change was noticed. Due to the clear nature of the aloe solutions, it seemed appropriate to document any changes visually as was done in the control experiment Lab 3, pg 79-82 (Maleszewski et al., 2003).
The original hypothesis that enzymes differ between A. barbadensis and E. pulcherrima could not be supported by the data
that was obtained, however, according to Constabel, polyphenoloxidase (PPO) is everywhere in the
plant kingdom. In the plant kingdom,
there are many different kinds of polyphenoloxidase (Constabel et al., 2000). Research has only focused on a small
percentage of plants, and of the plants that were studied, only one type of PPO
was tested – the PPO that causes food discoloration. It is possible, that the plants studied in this experiment had
PPO, but that the tests used were not sufficient in finding it. It would be beneficial to try other tests to
determine the presence of PPO in E.
pulcherrima and A. barbadensis. Research group Pika also concluded no
presence of PPO within the aloe family.
Using a 7mL aloe solution to 1mL of distilled water (7:1 solution) they
zeroed the spectrometer at 480nm and used the 7:1 solution as a blank. One mL catechol was added to the 7:1
solution and an immediate reading was taken using the spectrometer. After ten minutes, another reading was
taken. Their results showed changes no
greater than .003 differences between absorbency readings. From this they concluded that no PPO was
present in aloe, which is consistent with this experiment’s findings.
In conclusion, these experiments
were successful. The collected data
could not support the hypothesis that E.
pulcherrima and A. barbadensis
share similar carbohydrate properties, however the data did support the
hypothesis that the species contain similar photosynthetic properties but
contain different enzymatic compositions.
As with any experiment, there were some possible sources of error, one
being the plants themselves. It was
unknown whether or not the plants had been previously exposed to pesticides or
air born particles that could have changed the composition of the plants, thus
creating false test reactions. Due to limited plant sources, it was not
possible to explore different solution concentrations. Another constraint would
be time, which restricted the ability to perform more trials and additional
experiments. Also, human error could
account for any other inaccuracies found within the preformed experiments, as
humans are not infallible.
Table 2. Paper Chromatography:
The Rf values for Aloe
barbadensis plants A and B and Euphorbia
pulcherrima plants A are given; two trials were done for each. Euphorbia
pulcherrima (B) was not
tested due to lack of subject material.
The Rf value reflects the rate of flow, a ratio of the
distance traveled by the dissolved substances to the distance the solvent
moves. Similar Rf values
indicate similar pigments: A. barbadensis
contain pigments that vary from
the controlled pigments of carotene, xanthopyll, chlorophyll a, and chlorophyll
b whereas E. pulcherrima contain pigments more closely related to
the control.
|
|
Rate of flow = Rf values |
|||||||
|
Color bands → |
Carotene |
Avg |
Xanthophyll |
Avg |
Chlorophyll A |
Avg |
Chlorophyll B |
Avg |
|
Aloe barbadensis A1 |
1 |
1 |
.533 |
.506 |
.320 |
.289 |
.160 |
.152 |
|
Aloe barbadensis A2 |
1 |
.486 |
.257 |
.143 |
||||
|
Aloe barbadensis B1 |
1 |
1 |
.600 |
.569 |
.333 |
.290 |
.187 |
.171 |
|
Aloe barbadensis B2 |
1 |
.538 |
.246 |
.154 |
||||
|
Euphorbia pulcherrima A1 |
1 |
1 |
.771 |
.719 |
.629 |
.608 |
.400 |
.400 |
|
Euphorbia pulcherrima A2 |
1 |
.667 |
.587 |
.400 |
||||
Control
|
1 |
|
.847 |
|
.780 |
|
.568 |
|