Echinacea Plant and Supplement Content Analysis

A study to compare organic characteristics of the Echinacea plant to its supplement form, by way of sugar identification, protein quantification, pigments, pH, and glucose levels.

Echinacea is a popular dietary supplement taken by many to fight the common cold. It is believed that in the process of creating the pill form of Echinacea, some of the natural elements that the plant contains are lost. The research performed was to find out if the supplement that people are taking in fact has the same composition as the plant it is made from. Selivanoff's test was used to differentiate between ketoses and aldoses. It was found that both the pill and the Echinacea plant contained ketoses because the solutions in the samples quickly turned to a dark reddish brown color. It was determined whether the Echinacea plant and pill contained monosaccharides as opposed to di- and polysaccharides by using Barfoed’s Test. Both the pill and plant samples became a dark rusty brown color, indicating that they both contained monosaccharides. The Iodine test was used to distinguish whether coiled polysaccharides, or starch was present. The plant and supplement were found to contain starch because the samples turned bluish black. Thin Layer Chromatography (TLC) was performed in order to determine which pigments were present, and was used to quantify Chlorophyll a and b. It was found that the plant contained Xanthophylls, both Chlorophyll a and b, and Beta-carotene, but the supplement results were inconclusive. No pigments were present. The quantity of Chlorophyll a in the plant was 0.140 µg per gram of plant, and Chlorophyll b, 0.238 µg per gram of plant. In order to determine the protein concentration, a Bradford Assay was performed. It was predicted that the Echinacea plant would contain more protein than the supplement perhaps due to processing. The supplement was found to contain more protein than the plant after performing this test. In order to determine the quantity of glucose present in the plant and supplement, a diabetic blood sugar monitor was used. The results of these experiments show that the pill contained more protein, and glucose than the supplement. The pH test performed demonstrated that the pill had a lower pH. The results of the sugar, protein, and glucose tests go against the hypothesis that the pill would be lacking in contents the plant contained.

As the feeling of a cold sets in, one reaches for something to ease the discomfort; some may reach for an herbal supplement such as Echinacea. This can be found in capsule or liquid form at health stores or pharmacies, and is rumored to remarkably strengthen the body's ability to resist infection because of its phytochemical and vitamin content (Anonymous-2, Unknown). In the midst of formulating the plant into a supplement form, many of the organic qualities of the plant are lost. The question at hand is does the pill contain the same types of sugars, starches, and pigments; and does it have the same pH levels and glucose concentration as the pure plant form?

It has been previously found that some Echinacea supplements do not contain all of the proper ingredients or could be contaminated (Anonymous-1, Unknown). In order to pin point the answer to this question the focus will be put on the difference between the pill form of Echinacea and the actual plants macromolecule and pigment content. To test for the presence and types of carbohydrates, proteins, pigments, pH level, and glucose content, various experiments were performed. These experiments included Barfoed’s test, which determined the presence of either monosaccharides or polysaccharides, Selivanoff’s test that determined if the sugar is an aldose or ketose, the Iodine test which detected the presence of starch, a Bradford Assay that quantified proteins in both plant and pill, diabetic strip test for glucose, and pH tests to reveal the acidity of the plant and the supplement. These tests showed whether or not the pill had the same, less, or more of these qualities than the plant. It is already known that various Echinacea pills vary from each other due to the types of processing they undergo (Krochmal et al., 2004). It was therefore hypothesized that the supplement would be missing certain components such as sugars and carbohydrates, starch, proteins, pigments and different pH and glucose levels.

The tests involving carbohydrates are those that test for qualitative information. These tests gave information on the qualities of the samples being testing such as their molecular structure. This gave an idea of what type of carbohydrates each contained, as well as a comparison of the two subjects being tested based on macromolecules alone. Since these tests were qualitative, the results were supported with further research on the plant and pill, such as how much glucose is in the pill compared to the plant and why this may be so. In order to test whether the substances contained monosaccharides, or disaccharides and polysaccharides Barfoed’s test was performed. It was hypothesized that the plant would contain monosaccharides and the pill would contain polysacchrides. This is because only polysccharides are said to be left after drug companies finish producing the pill (Goel, 2003). To determine if starch was present in the plant and pill, the Iodine test was performed. It was hypothesized that the results of this test on the plant would indicate starch presence, and that the supplement may not. This is because all plants contain starch but the supplement may have had the starch removed during processing (Krochmal et al., 2004). To determine whether the supplement and plant contained ketoses or aldoses, Selivanoff’s test was performed. It was hypothesized that both would contain aldoses because all plants make glucose and glucose is an aldose.

Next, the Bradford assay to determine protein content was performed. A blue dye was added to both the contents of the pill and the leaf which changed different colors based on the protein concentrations in both specimens. This gave quantitative information and was used to determine which of the two being tested contained more protein. It was hypothesized that the pill form contained more protein because of the way the supplement is created. During processing, Echinacea plants are dried out and important compounds are extracted in different ways from different parts of the plant. They are then combined later to form the supplement (Anonymous 3, Unknown). Because of this research, it is hypothesized that the pill will contain more protein because this process would make the pill more concentrated in nutrients.

Thin Layer Chromatography was used to separate and distinguish the different pigments. The amounts of Chlorophyll a and b were also quantified. It was hypothesized that the pill would contain fewer pigments and less chlorophyll a and b. In previous research it was found that a powder form of Echinacea contained less pigments than the extract when tested using TLC (Krochmal et al., 2004). Echinacea’s phytochemicals are advertised as one of its healing components so it is interesting that in previous research the supplement was found to contain less. The pigment test showed what types of pigments were preserved when the plant form of Echinacea is turned into a pill form. This provided a clue to the type of organic characteristics of the herb being ingested.

Using litmus paper, the pH of the plant and supplement was tested. It was hypothesized that the pH of the supplement would be lower than the plant. After processing the pill all that is left is alkamides, cichoric acid, and polysaccharides (Goel, 2003). The fact that few other components that cichoric acid are left means that cichoric acid makes up a large amount of the pills substance. A larger composition of acid means a lower pH.

Finally, a diabetic blood sugar meter was used to determine the concentration of glucose in the supplement and plant. It was predicted that the plant would contain more glucose because it was actively producing glucose up until the point that the solution for testing was made.

All of these tests were performed in order to answer the question posed as to whether the supplement form of Echinacea contains the same type and amounts of substances as the plant in its natural form. When discussing the methods of extraction of the parts of the plant for the ingredients for a supplement, it is important to mention that most of the natural and organic qualities that the plant possessed will be taken away. All that is left behind is alkamides, cichoric acid, and polysaccharides when drug companies produce the pill (Goel, 2003). This experiment along with research will develop an understanding as to what types of things are being put into a human when one swallows the pill, and to also examine the organic nature of the plant compared to that of the pill on a molecular level.

To create the extract for the various tests, eight Echinacea plants were obtained from Michigan State University’s Horticulture Department. The supplement was obtained form the General Nutrition Companies store in East Lansing, Michigan. The process of creating the extract required a ceramic crucible and grinder. Water, sand, and MgSO4 were ground with the plant leaves and supplement contents in the crucible. A vortex was used on numerous occasions as were centrifuges for large and small test tubes. A pipette was utilized to measure the correct amount of each type of liquid in the various experiments. For Barfoed’s test, Barfoed’s solution was used, and resorcinol in HCl was used for Selivanoff’s Test. As for the Iodine test, I2KI was needed as the staining solution. Beakers of water were needed as was a hot plate to heat the solutions. For the Bradford Assay, Coomassie brilliant blue G-250 dye and a spectrometer were utilized. Test tubes as well as cuvettes were used on many occasions. For the TLC test, TLC strips, a small paint brush, and capillary tubes were used. Also put to use were paper squares and a scalpel used to scrape the pigments to be quantified. Also for this test, 80% acetic acid was used as the running agent. The pH test required litmus paper, an eye dropper, as well as a value legend to compare the colors to. Finally the glucose test made use of a CVS Blood Glucose Monitor and the equipment included.

In order to determine whether the Echinacea plant and supplement contain monosaccharide or di- and polysaccharides, Barfoed’s Test was used. The positive control for this experiment was a 1% glucose solution as its already known to contain monosaccharides, and the negetive control was water. Separately, 1.5g of supplement contents and plant leaves were weighed. They were ground up in crucibles containing 12ml of distilled water, 2.5g of sand, and 2.5g of magnesium sulfate. The samples were put in test tubes, sealed, and centrifuged for three minutes at 5900rpm to separate the solid material from the liquid. Five hundred µl of the extract from the pill were placed in each of three test-tubes. The same thing was done for the plant, water, and the 1% glucose solution. Three replications for each of the four samples were performed in order to have more accurate results. Three ml of Barfoed’s solution was then pipetted into each of the 12 test-tubes. Each test-tube was then placed in a boiling water bath for two minutes and the color was recorded. A rusty brownish-red color indicated monosaccharides whereas no color change indicated di- or polysaccharides. This addressed the hypothesis by testing whether the sugars in the plant and supplement were composed of monosaccharides as opposed to di- or polysaccharides.

Selivanoff’s test was used to determine if the Echinacea plant and supplement containd ketoses or aldoses. The control for this experiment was galactose as it is a known aldose, and the negetive control was water. Separately, 1.5g of supplement contents and plant leaves were weighed. They were ground up in crucibles containing 12ml of distilled water, 2.5g of sand, and 2.5g of magnesium sulfate. The samples were put in a centrifuge for three minutes at a speed of 5900rpm to separate the solid material from the liquid. Three hundred fifty µl of the extract from the pill where placed in each of three test-tubes. The same thing was done for the plant, water, and the galactose solution resulting in 12 test tubes. Three milliliters of Selivanoff’s solution was then pipetted into each of the 12 test-tubes. Each test-tube was then placed in a boiling water bath for two minutes and the color and time were recorded as soon as color change was visible. A quick reaction with a dark brownish red color change indicated ketoses are in the sample. If it reacted slowly over several minutes, the sample contained aldoses. This addressed the question of whether or not the plant contents are similar to the supplement, by testing whether the sugars in the plant and supplement are of similar molecular structure. More information about the location of the Carbonyl group in the carbohydrates was obtained by Selivanoff’s test.

To test for coiled polysaccharides, or starch, the Iodine test was performed. For this experiment, starch was used as the positive control as it is known to contain starch, and the negative control was water. Separately, 1.5g of supplement contents and plant leaves were weighed. They were ground up in crucibles containing 12ml of distilled water, 2.5g of sand, and 2.5g of magnesium sulfate. The samples were put in a centrifuge for three minutes at a speed of 5900rpm to separate the solid material from the liquid. Three hundred fifty µl of the extract from the pill were placed in each of three test-tubes. The same thing was done for the plant, water, and the starch solution. Three ml of I2KI reagent was then pipetted into each of the 12 test-tubes. The tubes were kept at 22 degrees Celsius, or room temperature, and the color change was observed. If the solution became bluish black, it was an indication that starch was present. This addressed the hypothesis by testing whether or not starch was present in the extract of supplement, then comparing that to the results of the test on the plant leaves.

In order to determine the protein concentration in the Echinacea plant and supplement, a Bradford Assay was performed on each. A standard curve dilution series was first created using bovine serum albumin (BSA). This was done by creating a test tube with 2ml of 2 mg/ml bovine serum albumin and labeling the tube 2. Six test tubes were labeled 1, 0.5, 0.25, 0.125, 0.0625, and 0. One ml of distilled water was added to each tube. One ml of the solution in test tube 2 was added to test tube 1 and vortexed.

For the other five tubes, one ml from the previous tube was placed into the next tube until each was left with 1 mL of solution. Fifty µL of each tube was put in 7 new test tubes, and then mixed with 50 µL of 0.1 M NaOH. Following this, 3ml of Bradford reagent was added to each, vortexed, and left to sit for five minutes. Coomassie brilliant blue G-250 dye (CBBG) was used, which yielded different color responses based on various protein concentrations in the samples. In order to determine the amount of protein in the supplement and Echinacea plant, extracts had to be prepared. Separately, 1.5g of supplement contents and plant leaves were weighed. They were ground up in different crucibles containing 2.5g of sand. The samples were put in a centrifuge for three minutes at 5900rpm to separate the solid material from the liquid. One and a half milliliters of each extract was put in separate cuvettes. The cuvettes were placed in a spectrometer and the absorbances of each solution were read at 595 nm. The results were plotted against the standard curve created from the dilution series. Three replications were done for both the supplement and the plant. Performing this experiment and its replications helped provide more information on whether or not the plant contained more protein than the supplement as predicted by our hypothesis.

Separation of pigments from the Echinacea plant and supplement was possible through the use of thin-layer chromatography (TLC). These pigments were separated for observation. Spinach extract was used as the control in the experiment because it contained all four of the pigments that were being tested for. Separately, 2.5g of supplement contents and plant leaves were weighed. They were ground up in crucibles containing 12ml of distilled water, 5g of sand, and 2.5g of magnesium sulfate. The samples were put in a centrifuge for five minutes at a speed of 5000rpm to separate the solid material from the liquid. Eight ml of 100% acetone was added to the extract. A spatula was used to mix the acetone with the slurry, and the solution was left to settle for 10 minutes. Another two minutes in the centrifuge at 5000rpm separated the solids from the liquid once again. Two TLC strips were prepared by drawing a light pencil line 2cm from the bottom. Using a paint brush, the liquid extract was brushed onto the pencil line. A new coating was added 10 more times with each addition following the drying of the previous one. The strips were placed in large test tubes with 80% acetone filled only enough to where the liquid was still below the pencil line. The test tubes were corked immediately. After 15 minutes of allowing the acetone run up the strip, the colors were observed and recorded. This experiment separated the pigments from our samples and shed light on the presence or lack of Chlorophyll a, Chlorophyll b, Xanthophyll, and Beta-carotene in the Echinacea plant and supplements extract. Once the separation was completed, the strips were removed from the test tubes. Chlorophyll a and b were then scraped off the strips onto separate paper squares and put into capillary tubes. The tubes were filled with 1.5ml 80% acetone. Using a small centrifuge, the two capillary tubes were centrifuged at 5900rpm for three minutes. The Chlorophyll a and b solutions were then transferred to cuvettes and placed in the spectrometer.

An independent assay was performed to determine the pH of the plant and pill samples. Separately, 1.5g of supplement contents and plant leaves were weighed. They were ground up in crucibles containing 12ml of distilled water, and 2.5g of sand. The samples were put in test tubes, sealed, and centrifuged for three minutes at 5900rpm to separate the solid material from the liquid. Five hundred µl of the extract from the pill were placed in each of three test-tubes. The same thing was repeated for the plant. Nine strips of pH paper were obtained, three for the plant extract, three for the pill extract, and three for the control, water, which is known to have a neutral pH of 7. An Eye dropper was used to fully wet the litmus paper for all three replications of both extracts, and water. Results in the blue and green range indicated a base while red and orange results indicated an acid. If there was a difference in acidity between the supplement and the plant, then questions could be raised about possible differences in components of the Echinacea plant and supplement.

A glucose concentration test was utilized as a second independent assay to acquire more information on the potential differences between the Echinacea plant and supplement. Separately, 1.5g of supplement contents and plant leaves were weighed. They were ground up in crucibles containing 12ml of distilled water, and 2.5g of sand. The samples were put in test tubes, sealed, and centrifuged for three minutes at 5900rpm to separate the solid material from the liquid. Five hundred µl of the extract from the pill were placed in each of three test-tubes. The same thing was done for the plant. Once the extract was created, a glucose strip was obtained from the kit and a drop of the sample solution which came with the kit was put on the strip. This glucose sample solution was used to set the machine to the default 15mg/dl. After this, another strip was obtained and a drop of the plant extract was placed on it. The machine then measured the results which were recorded. This was repeated two more times for the plant, and three replications were done on the supplement extract. The measurement of Glucose concentration was a very fitting way to compare the Echinacea plant to the supplement, and to measure quantitatively, the difference between the two.

After completing the procedures for the sugar tests, the results obtained from the supplement and plant extracts were similar (Table 1). For Barfoed’s Test on the plant extract, all three replications yielded a dark precipitate (Figure 1). The 1% glucose solution used as the positive control also yielded a dark precipitate indicating monosaccharides (Figure 4). No precipitate was observed for the water, which was used as the negative control (Figure 3). As for the supplement extract, a dark precipitate was indeed observed in all three replications (Figure 2), similar in appearance to the plant extract.

For Selivanoff’s Test, the positive control galactose turned into a brownish color in under two minutes (Figure 7). Galactose is a known aldose, making the results suitable for comparison to the results of the plant and pill tests. The negative control, water, showed no color change (Figure 8). For the test on the supplement, all three replications turned into a dark brownish red color very quickly (Figure 5). The three trials for the plant extract also quickly turned into a dark brownish red color (Figure 6).

The iodine test for starch required a positive control of starch. The bluish black color observed after doing this test indicated the presence of starch (Figure 12). The three replications of the iodine test on the Echinacea plant extract turned bluish black (Figure 9). The same bluish black results were observed in all three trials when testing the supplement (Figure 10). The negative control used for this test was glucose because it is known to contain no starch (Figure 11)

The TLC test aided in the identification and quantification of pigments in the Echinacea samples. For this test, spinach leaf extract was used as the control as it contains Chlorophyll a, Chlorophyll b, Xanthophyll, as well as Beta-carotene. The Echinacea plant was found to contain Chlorophyll a, Chlorophyll b, Xanthophyll, as well as Beta-carotene after doing the TLC test (Figure 13). The supplement yielded no distinct results. None of the four pigments were visible on the TLC strip (Figure 14), therefore no quantification was possible. As for the plant, Chlorophyll a was quantified to be 0.140ml/g of plant, and Chlorophyll b was quantified to be 0.238ml/g of plant (Figure 15).

The Bradford Assay for protein concentration revealed differing absorbance results. The control or blank used to create the standard curve was water. After running the plant extract through the spectrometer, the absorbance was observed to be 0.880. The absorbance of the supplement extract was 0.958. The equation of Y=0.0167x-0.0204 was found to be the best fit line to the standard curve (Figure 16). Using the equation and the absorbance, the protein concentration for the plant extract was determined to be 53.93mg/ml, while the supplement extract yielded 58.60mg/ml.

The glucose test on the pill and plant yielded differing results (Table 1). The three trials on the plant yielded 56mg/dl, 59,mg/dl and 59mg/dl. These values are much lower when compared to the supplement glucose test results of 138mg/dl, 126mg/dl, and 129mg/dl

After testing for pH of the extract of the supplement (Figure 17) and plant (Figure 18), the litmus paper colors were compared to the values associated with those colors. It was found that the pH of the plant was 6.5 and the pH of the supplement was 5.5.

Multiple tests were done on the popular herb, Echinacea, which is taken by many people for a variety of claimed health benefits. The tests on the plant leaves and supplement quantitatively and qualitatively compared the contents found in each. Barfoed’s Test, Selivanoff’s Test, and the Iodine test for coiled polysaccharides compared the types of sugars found in each sample. A thin-layer chromatography (TLC) test was done to identify the presence of Chlorophyll a, Chlorophyll b, Xanthophyll, and Beta-carotene. A Bradford Assay was performed to quantitatively compared protein concentration. Also, pH levels were assessed as well as glucose concentration. It was predicted that the Echinacea plant would contain more natural elements including glucose, protein, carbohydrates, and pigments due to processing.

The first sugar test performed was Barfoed’s test. Results from the experiment showed that both the pill and plant extracts contained monosaccharides as opposed to di- or polysaccharides. This qualitative test suggests that molecular structures of the sugars are single rings rather than two or more rings linked together. It was hypothesized that the plant would contain monosaccharides due to the presence of glucose used by the plant for energy. The Supplement was thought to contain polysaccharides due to the manner in which pills are processed by drug companies (Goel 2003). From the results obtained, the hypothesis was supported for the plant. As for the supplement, the indication of monosaccharides does not follow the previous research as well as the hypothesis.

There was potential for error in making the solutions that were tested. There may have been leftover particles in the crucible from previous experiments by other scientists who did not properly clean the equipment. These particles may have been ground in with the mixtures being created for the extract, and could have produced a positive test result for monosaccharides. Also, Barfoed’s reagent may have become contaminated due to its long time under the hood, and there may have been neglect on the part of others in changing the pipette tips.

The results of Selivanoff’s test showed that both the plant and the pill extract contained ketoses. It was previously hypothesized that both the pill and plant would contain aldoses because plants contain glucose, which is an aldose. While the results show the presence of ketoses, it does not necessarily imply the absence of aldoses. This is because due to the nature of Selivanoff’s test, ketoses react quicker than aldoses, and the presence of ketoses would have been covered up by the indication that aldoses were present.

Error was possible during this test in a few areas. Selivanoff’s reagent could have been contaminated from its use by others, thus affecting the outcome of the experiment. Neglect in changing pipette tips, as well as leftover substances in the crucible during the grinding stage, also may have negatively affected the test.

The iodine test results did not support the hypothesis that only the plant would contain starch. It was predicted that the process of making the supplement would eliminate the starch present in the plant. When the plants are processed, they are stripped of their moisture in a dehydration process. Warm air is blown over the plant material to remove the water content. To avoid nutritional loss, the heat is applied in stages using low temperatures, which prevents cells from rupturing (Murray 2006). This careful method of creating the supplement may be why there was still starch present in the supplement after the Iodine test.

Error may have occurred from contaminated I2KI reagent. Once again there may have been substances left over in the crucible from previous use, which may have had an effect on the outcome of the experiment.

For all of the sugar tests performed (Barfoed’s, Selivanoff’s, and Iodine), the same results where observed when comparing the pill to the plant for each respective test. The qualitative tests on the pill and plant extract showed that they both contained monosaccharides, starch, and ketoses. These sugar test results reveal that the processing of the plant into a supplement does not change the molecular structure for the sugar tests to say otherwise. Starch, monosaccharides, and ketoses were all still present in the supplement after processing. Although the tests were not quantitative and could not indicate if amounts of these carbohydrates were lost due to processing, they still in fact indicate their presence.

In regards to the TLC test, the lack of the four pigments being tested for in the supplement suggests that these pigments were lost in the processing of the pill. The TLC findings support the hypothesis that the pill will lack pigments found in the plant. Finding pigments such as Chlorophyll a and b, Xanthophyll and Beta-carotene means that the plant contains photosynthetic properties that the pill does not. This helps prove that the processing of the Echinacea pill ends the production and eliminates the presence of these pigments. The Beta-carotene found in the plant has distinct antioxidant properties. As previously mentioned antioxidants are very beneficial to the body and protect body cells from the damaging effects of oxidation (DeEll 2005). The lack of Beta-carotene in the supplement should be noted as being a potential missed benefit of taking the Echinacea supplement instead of the plant.

There was chance for error when performing this test. The 80% acetone may not have been corked soon enough causing some to be dispersed into the air. This could have effected the separation of the pigments on the TLC strip. When scraping Chlorophyll a and Chlorophyll b from the plant TLC strip, the edges of the other pigments may have been scraped as well, causing the absorbances and quantification to be incorrect. There may have been fingerprints on the cuvettes before each reading. This may have affected the absorbances. The spectrometers in the lab vary from day to day and do not always correctly zero before each reading, adding another potential source of error.

The results of the Bradford Test for protein concentration went against the hypothesis that the plant would contain more protein then the pill. It could be that the mass of plant leaves measured in the methods of the Bradford test was partly composed of water. In other words, two equivalent masses of plant leaves and supplement would contain a great difference in the water content. The leaves clearly contained more water, which represented non-protein mass. The water mass of the leaves must have been replaced by other compounds (possibly protein) in the supplement in order to obtain the same mass as the leaves. There was an attempt to remove the excess water from the leaves before making the extract, but the centrifuge was unable to separate the water from the rest of the plant. When creating the supplement, Echinacea plants are dried out and important compounds are extracted in different ways from different parts of the plant. They are then combined later to form the supplement. One of these compounds is protein (Murray 2006). Therefore gram for gram, the supplement will contain a greater concentration of protein than the plant leaves. Knowing this, it was proved that the Echinacea supplement contained more protein than the plants they were made from.

Potential sources of error could have occurred while creating the extract used to quantify the protein. While grinding the plant leaves with the other materials, all the slurry may not have been collected from the crucible, and would thus lower the amount of possible protein present. The spectrometers in the lab vary in their consistency and do not always correctly zero before each reading, adding another potential source of error. In addition, there may have been fingerprints on the cuvettes before each reading. This may have affected the absorbencies.

It was hypothesized for the pH test that the plant would have a lower pH than the supplement because plants were shown to be acidic in previous studies (Hanstein, 1999). The test results demonstrated that the supplement in fact was more acidic. Although acidity had not been mentioned in previous research on this subject, the fact that the other study shows plant leaves to be acidic lead to the hypothesis that the leaves would be more acidic. When reviewing the methods of supplement creation, there is no mention of the addition of any acids that would affect the pH.

Judgment error may have occurred when comparing the color of the litmus paper to the given shades and the pH numbers associated with them. Skin oils may have been absorbed by the paper during the setup of the experiment.

The quantitative test on glucose was hypothesized to show that the plant would contain more glucose than the supplement. However, it was found that the supplement in fact contained more glucose. As with the protein example previously mentioned, the supplement is more concentrated in its nutrients. The plant leaves contained more water and thus two equivalent masses demonstrated that the leaves contained less glucose than the concentrated pill. The method of drying out the plants and extracting useful compounds is related to glucose as it could be included as one of the compounds being extracted (Murray 2006).

As with the other tests, a source of error could have occurred by not collecting all of the ground up leaves from the crucible. The glucose meter may not have been totally accurate as each reading was slightly different for replications of the same liquid.

The tests performed on the Echinacea supplement to the plant itself were helpful in qualitatively and quantitatively comparing the contents of the two. There is plenty of opportunity for further research on this subject.

The same macromolecule experiments could be repeated on different brands of Echinacea supplement, and the results compared among each other. It is known that different types of Echinacea pills vary in their contents due to the types of processing they undergo (Krochmal 2004). Also, experiments could be done on people who are infected with the common cold. Some would be given placebo pills, some a placebo plant, others would receive Echinacea pills and the fourth group would consume the Echinacea plant. The length and severity of their colds could then be observed. In order to understand exactly what the plant loses and/or gains from processing, the composition of an Echinacea plant could be determined before and after it is made into supplement form. By testing the exact same plant in supplement and natural form, it would eliminate the possibility of difference due to natural variation between plants. These are just a few of a variety of experiments that could be done to further the research on Echinacea.

Figure 1. The results of Barfoed’s test on the Echinacea plant extract. Barfoed’s test was used to determine if monosaccharides or di- and polysaccharides were present. All three trials yielded a dark precipitate indicating the presence of monosaccharides. Each tube contained 3ml of Barfoed’s solution and 500µl of the plant extract. The tubes were put in boiling water and observations were made at two minutes.

Figure 2. Results of Barfoed’s test on glucose, the positive control. A precipitate is visible indicating the presence of monosaccharides. Each tube contained 3ml of Barfoed’s solution and 500µl of the supplement solution. The tubes were put in boiling water and observations were made at two minutes.

Figure 3. Result of Barfoed’s test on water, the negative control. No precipitate formed in any of the three trials. Each tube contained 3ml of Barfoed’s solution and 500µl of water. The tubes were put in boiling water and observations were made at two minutes. No precipitate formed indicating no monosaccharides.

Figure 4. The results of Barfoed’s test on the Echinacea supplement. Barfoed’s test was used to determine if monosaccharides or di- and polysaccharides were present. All trials yielded a dark precipitate indicating the presence of monosaccharides. Each tube contained 3ml of Barfoed’s solution and 500µl of the supplement solution. The tubes were put in boiling water and observations were made at two minutes.

Figure 5. The results of Selivanoff’s test on the positive control galactose. Galactose is a known aldose therefore the test turned the solutions slightly orange in color slowly (after two minutes). Each tube contained 3ml of Selivanoff’s reagent and 350µl of galactose. The tubes were placed in boiling water and observations began immediately.

Figure 6. The results of Selivanoff’s test on water which was the negative control. The solutions mixed with 3ml of Selivanoff’s reagent and 350µl of water yielded no color change, and therefore contained no aldoses or ketoses. The yellow tint to the test tube on the left was due to impurities in the Selivanoff’s reagent used for the test.

Figure 7. Selivanoff‘s Test results after performing the procedures on the supplement extract. Selivanoff’s test was used to determine the presence of ketoses and aldoses. All trials quickly became dark red in color indicating the presence of ketoses. Each tube contained 3ml of Selivanoff’s reagent and 350µl of plant extract. The tubes were placed in boiling water and observations began immediately. A reaction after two minutes would suggest the samples contained aldoses.

Figure 8. The results of Selivanoff’s test on the Echinacea plant. Selivanoff’s test was used to determine the presence or ketoses and aldoses. All trials quickly became dark red in color indicating the presence of ketoses. If the color change happened after two minutes, the test would suggest aldoses were present. Each tube contained 3ml of Selivanoff’s reagent and 350µl of plant extract. The tubes were placed in boiling water and observations began immediately.

Figure 9. Iodine test on starch, the positive control. Each tube contained 350µl of the supplement solution and 35µl or IKI reagent. The tubes were kept at room temperature until a color change was observed. The starch solution turned bluish black indicating the presence of starch.

Figure 10. Iodine test results on the Echinacea plant extract. This test was used to indicate the presence of coiled polysaccharides. The three trials became bluish black in color indicating the presence of coiled polysaccharides. Each tube contained 350µl of the plant solution and 35µl of IKI reagent. The tubes were kept at room temperature and the color change was observed.

Figure 11. The results of the Iodine test on the Echinacea supplement. The iodine test is used to indicate the presence of coiled polysaccharides. All trials became bluish black in color indicating the presence of coiled polysaccharides. Each tube contained 350 µl of the supplement solution and 35µl or IKI reagent. The tubes were kept at room temperature and the color change was observed.

Figure 12. The results of the iodine test for coiled polysaccharides on glucose, the negative control. The yellowish brown color indicates no starch present. Each tube contained 350µl of the supplement solution and 35µl or IKI reagent. The tubes were kept at room temperature until a color change was observed.

Figure 13. Results from Thin Layer Chromatography of the Echinacea plant. The colors separated into four distinct pigments; Chlorophyll a (A), Chlorophyll b (B), Carotene (C), and Xanthophylls (D).

Figure 14. The results from Thin Layer Chromatography of the Echinacea Supplement. The tests were inconclusive for the four different trials. There were no distinct pigments present in the supplement TLC.

Figure 15. From left to right, cuvettes of Xanthophyll, Beta-carotene, Chlorophyll b, and Chlorophyll a before being analyzed by the spectrophotometer. The pigments were scraped off the TLC strip and used to make the solutions. After being analyzed, Chlorophyll a was quantified to be 0.140ml/g and Chlorophyll b was quantified to be 0.238ml/g.

Figure 16. Graph of the Standard Curve for the Bradford Assay. The points were obtained by creating a standard curve dilution series. The absorbencies were plotted, and a best fit curve with its equation is shown. The absorptions of both the plant and pill extract were 0.880 and 0.958 respectively. Using the equation of the curve, the protein concentrations were determined to be 53.93 for the plant and 58.60 for the suppleme

Figure 17. Results of the pH test on the Echinacea supplement. The Yellow tint indicates a more acidic pH then the neutral 7. The pH of three replications (A, B, and C) yielded identical results of about 5.5.

Figure 18. Results of the pH test on the Echinacea plant. The Yellow/Orange tint indicates a slightly more acidic pH then the neutral 7. All three replications (A, B, and C) yielded a pH of about 6.5

The table shows the results of the three replications on the plant and the supplement extracts.

	Trial 1	Trial 2	Trial 3
Supplement Extract	138mg/dl	126mg/dl	129mg/dl
Pill Extract	56mg/dl	59mg/dl	59mg/dl

Anonymous-2. Unknown. Echinacea: Can the 'prairie doctor' give your immune system a boost?

Anonymous-3. Unknown. To end with the very best Dietary Supplements you must begin with the very best practices.

http://www.omafra.gov.on.ca/english/crops/hort/news/orchnews/2005/on_0305a10.htm

Goel V., Lovlin R., Barton R., Lyon M. R., Bauer R., T.D.G. Lee, Basu T.K. 2003. Efficacy of a standardized Echinacea preparation for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial. Journal of Clinical Pharmacy & Therepeutics. Accessed January 17, 2006 from Blackwell Synergy Web site: http://www.blackwell-synergy.com

Hanstein, S., Felle, H. H. (1999). The influence of atmospheric NH₃ on the apoplastic pH of green leaves: a non-invasive approach with pH-sensitive microelectrodes .New Phytologist 143 (2), 333-338.

Krochmal, R., Hardy, M., Bowerman S., Lu, Q., Wang, H-J., Elashoff, RM., and Heber, DH. 2004 December. Phytochemical Assays of Commercial Botanical Dietary Supplements Based Complement Alternate Medicine.1(3): 305–313. Published online 2004 October 6