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, lipid presence, protein quantification, pigments, pH and glucose levels.

 By: Annette Agemak, Phillip Ajlouny, Bethan Roberts, and Phong Vu

LBS 145
Tuesday 3-6 pm
James Hardie & Lauren Feinberg
February 27, 2006

Abstract

    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 Barfoedâs 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. 

Discussion

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.    Barfoedâs Test, Selivanoffâs 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 Barfoedâs 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.   

The results of Barfoedâs test on the Echinacea plant extract. Barfoedâs 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 Barfoedâs solution and 500µl  of the plant extract. The tubes were put in boiling water and observations were made at two minutes.

 The results of Barfoedâs test on the Echinacea supplement.  Barfoedâs 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 Barfoedâs solution and 500µl of the supplement solution. The tubes were put in boiling water and observations were made at two minutes.

    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, Barfoedâs 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 Selivanoffâs 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 Selivanoffâs 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.  Selivanoffâs 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 (Barfoedâs, Selivanoffâs, 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). 

Glucose Test Results- The table shows the results of the three replications on the plant and the supplement extracts.

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.

References 

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 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): 305ö313. Published online 2004 October 6

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