LBS 145 Molecules

Effect of pH displays greater enzymatic activity in
Carnegia gigantea (Aloe Vera) possibly causing medicinal benefits.

The Molecules

LBS 145 section M1 - Dr. Douglas Luckie

February 17th, 2003

Jonathan Backos

Maureen O'Brien

Adam Jablonowski

Stephen Gottschalk

Abstract

The Carnegiea gigantean (Aloe Vera) was compared to the Cereus giganteus (Saguaro Cactus) to see if there were any major differences in if reducing sugars were present, the carbohydrates produced, the photosynthesis absorption and action spectrum, and the enzyme activity in the two plants. The reason for our investigation was to see if the differences in the Aloe Vera compared to the Saguaro Cactus could produce evidence about why the Aloe Vera has medical benefits. We suspected that the Aloe Vera plant would contain a greater quantity of carbohydrates, photosynthetic absorbency, and enzymatic activity. The tests used in the investigation were the dehydration of carbohydrates test, the iodine test for coiled polysaccharides, the photosynthesis test, the enzyme activity tests, and the effect of pH test (Maleszewski et al., 2003). After conducting the five carbohydrate tests with three different concentrated solutions our results displayed no difference in carbohydrates present in Aloe Vera and the Saguaro Cactus. The cactus had a better photosynthetic absorbency overall, but both plants followed the same trend. There was a slight presence of the PPO enzyme in the Aloe Vera plant which led to a greater enzymatic activity than in the Saguaro Cactus which lacked PPO. The greater enzymatic activity in the Aloe Vera plant, compared to the Saguaro Cactus, could be the trait that distinguishes why the Aloe Vera plant has medicinal benefits.

Table 1: The carbohydrates present in the Aloe Vera. The results after conducting Benedict's test, Barfoed's test, Selivanoff's test, Bial's test, and iodine test for coiled polysaccharides the results were found to be inconclusive. In our research we later found that water makes up 99% of the Aloe Vera plant which suggests a possible explanation why the tests did not reveal any carbohydrates presence even though we know that they exist.

Carbohydrates	Benedict's	Selivanoff's	Bial's	Iodine
Aloe	No Red Precipitate	No Color Change	No Color Change	No Color Change
Cactus	No Red Precipitate	No Color Change	No Color Change	No Color Change
Water (neg.control)	No Red Precipitate	No Color Change	No Color Change	No Color Change
Glucose	Red Precipitate	Aldose, reacted over 1 minute	Yellow, Pyranos	n/a
Fructose	n/a	Ketose, reacted under 1 minute.	Light Brown, Hexos Furanos	n/a
Xylose	n/a	n/a	Green, Furnos	n/a
Starch	n/a	n/a	n/a	Purple, Starch in mixture

Figure 16: Absorbance spectrum of the Aloe Vera and the Saguaro Cactus between 400 and 700 nm. This absorbance spectrum shows that the Saguaro Cactus has a better absorbance from 400 nm to 520 nm but then the Aloe Cera has better absorbance between 545nm and 700nm. Overall, the cactus absorbs more light than the Aloe Vera plant in all regions of the action spectrum, but both plants absorbance follow the same decreasing trend.

Discussion

The interest in the healing properties of the Aloe Vera plant was the driving force behind this research project. To find these healing properties, we investigated characteristics of carbohydrates, photosynthetic processes, and different enzyme activities occur in the Aloe Vera. By looking at these three characteristics compared to a similar arid dwelling species like the cactus, we hypothesized that differences between the two plants would show why the Aloe Vera possesses healing properties. Our hypothesis includes that we suspected that Aloe Vera would contain the PPO enzyme (polyphenoloxidase), and more carbohydrates that the cactus would lack. The different enzymes and enzyme activity of the Aloe Vera compared to the cactus could be characteristics that could potentially explain the absence of healing properties in the cactus. Our hypothesis included that the Aloe Vera plant would have a greater absorbency than the cactus when testing the photosynthetic absorbency. We also anticipated that there would be more pigments in Aloe Vera and they would have a greater Rf value. We expected that the deficiency of these enzymes, photosynthetic properties and carbohydrates caused the cactus to lack the same benefits.
The first experiments that were done were the carbohydrate tests. The tests that were used to conclude what different types of carbohydrates were present in each of the plant's structure were Benedict's test, Bial's test, Selivanoff's test, and the Iodine test for coiled polysaccharides. The first set of experiments were done with a pair of stock solutions that had 2.5 grams of plant tissue and 250ml of water that were blended together for five minutes. With this first stock solution no substantial results were seen or recorded with the three experimental test tubes, but the negative and positive controls did produce the correct results. Another pair of stock solutions were tried which contained 3.5g of plant tissue and 50ml of water that were blended for five minutes. Again the five experiments were performed with three test tubes with the experimental stock solution, and one test tube for the negative control and one test tube for the positive control. The second pair of stock solutions did not produce any significant results in the experimental test tubes but the negative and positive controls did produce the correct results.
Finally, we changed our approach and prepared another pair of stock solutions by only taking the internal components of the plant, adding 20ml of water, and mixing the solutions for 5 minutes. Again, the five experiments were performed with three test tubes containing the experimental stock solution, one test tube for the negative control and one test tube for the positive control. The third pair of stock solutions did not produce any significant results in the experimental test tubes but the negative and positive controls did produce the correct results.
It is apparent that our tests were not producing accurate data because all plants, including cactus and Aloe Vera contain starch. The Future Vets were also working with aloe and testing for carbohydrates in their research. Three more concentrations were performed. They used a 10% concentration made of just the gel of the Aloe Vera plant. This was done by taking 480ml of the aloe gel and swirled it with 4,320ml of water. This concentration was used for Selivanoff's, Bial's, and the Iodine test. All of which gave no results. Another concentration was made with 10g of the whole aloe plant and 90ml of water. This was blended in a Waring Commercial Blender at medium speed for 1 minute. The mixture was then strained through six layers of cheesecloth yielding a 11% concentration.(Per. Comm. with Future Vets).
From the information that was gathered from the experiments, it was concluded that the way our stock solutions were created needed to be re-evaluated. The major reason why our experiment stock solutions did not produce any results could possibly be explained because the stock solution were not concentrated enough with carbohydrates that should be present in the two plants. Even though we altered the concentrations significantly, we later found that Aloe Vera is composed of 99% water, which could make the carbohydrates difficult to isolate (Robson et.al., 1982: 157-163). From our research it is known that sucrose and starch should be the primary carbohydrate present (Freeman, 2002:151) but other carbohydrates like glucose, galactose and xylose should also be present in the plant tissue. Another major reason why no carbohydrates were found was because plants usually make starch during the day but at night turn starch is turn into sucrose molecules (Freeman, 2002: 151). Since the experiments were done at 7 to 10 PM most of the starch was probably turned into sucrose. Also, once the sucrose is made in plants it usually is broken down to power other plant cells or sent to the roots for storage (Freeman, 2002: 151). Only the plant's leave tissue was tested for carbohydrates and not the root systems of the plants so this could have also affect out results. Taking all these facts into consideration it has been learn that other places on the plant should be tested for carbohydrates and when making test solutions less water should be used to so that the solutions do not become too diluted. With this knowledge other techniques to test for carbohydrates will be done in the future.
In the second part of the lab both the saguaro cactuses and the Aloe Vera were tested for which pigments were present in each plants chloroplast. A technique called paper chromatography was used to test for the pigments in the chloroplast of each of the plants. It was found that the Aloe Vera and the saguaro cactuses both contained pigments of carotene, chlorophyll a, xanthophylls, and chlorophyll b, but chlorophyll b and carotene were more abundant in the Aloe plant. From this data is can be concluded that Aloe Vera can absorb red orange and yellow light better then saguaro cactuses.
Another photosynthesis test that was done was the absorption spectrum test. The results of the test showed that the Aloe Vera absorbed energy in the red and blue areas of the absorbance spectrum fairly well and reflected energy in the green areas of the absorbance spectrum. The saguaro cactuses absorbed less energy then the Aloe Vera in the red areas but absorbed much more energy then the Aloe Vera in the blue areas of the absorbance spectrum. To explain this Aloe Vera must grow in an environment where it has less access to light so the Aloe Vera must be better adapted to absorb any light energy that reaches its leaves. The saguaro cactuses lives in an environment that has an abundant source of light so it is highly adapted to efficiently absorbing blue light energy. The cactus had a better photosynthetic absorbency overall, but both plants followed the same trend.
The test for the presents of the PPO enzyme in the Aloe Vera showed a slight positive result for the PPO enzyme. One reason why there was not a greater color change in the PPO enzyme test was because the concentration of PPO directly in the Aloe Vera was diluted because 99% of the plant solution is water. The test for the presents of the PPO enzyme in the Saguaro Cactus showed a negative result for the PPO enzyme. The lack of the PPO enzyme in the Saguaro Cactus could have been caused by different evolutionary paths between the Aloe Vera and the Saguaro Cactus.
There was a slight presence of the PPO enzyme in the Aloe Vera plant which led to a greater enzymatic activity than in the Saguaro Cactus which lacked PPO. In the Aloe Vera plant the range of absorbance for the enzyme activity was between a high of 0.747 absorbance and a low of 0.390 absorbance. In the Saguaro Cactus the range of absorbance for the enzyme activity was between a high 0.166 absorbance and a low of 0.390 absorbance. The data for the Saguaro Cactus was mostly constant but the small variation in the absorbance data was caused by other factors then enzyme activity, which could have been small particles in the cactus solution or the variation in the color of the solution. Since the Aloe Vera showed a large amount of PPO enzyme activity compared to the Saguaro Cactus it was concluded that this enzyme activity was a characteristic that cause healing properties in the Aloe Vera. The greatest PPO enzyme activity was in the pH range of 5.5 and 6.0 which is close to the pH of the skin of humans. Since the Aloe Vera healing properties worked best when the enzyme can work in a favorable pH environments like human skin it can be concluded that this enzyme activity data is a strong connection to the healing properties of Aloe Vera. These findings support our initial hypothesis that the Aloe Vera plant's healing properties are connected to its enzyme activity.
Since the test for PPO enzyme on the Aloe Vera only showed a slight positive result, it was thought that there were other enzymes that might have a large impact on the healing properties of the plant. So our research group asked: "What other enzymes are active in the Aloe Vera?" After doing some more research it was found that the major enzyme that has been tested in the Aloe Vera plant was the enzyme Bradykinase (Fujita, 1976: 205). In the medical world this enzyme is also referred to as the Angiotensin-Converting Enzyme (ACE) (Ghannam et al., 1986: 288-294). Our new findings about this enzyme led to many more questions. We were unable to attain the procedures and supplies to test for the presence of Bradykinase in Aloe Vera and the cactus due to financial and time restrictions of the lab. However, with more time and more resources, we could further pursue this study. For example, we want to test if bradykinase is found in Saguaro Cactus. We researched it extensively but were not able to find any tests done by other scientists on cactus and bradykinase. However, the lack of a significant amount data suggesting that the carbohydrate and absorbency properties are responsible for the biological and chemical differences between the two plants, leads us to believe that further research of the enzymes involved may answer more questions.