601 S. College Avenue Kaitlin L. Siela Columbia, MO Telephone: (816)

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1 Jane M. Jackson Department of Chemistry Telephone: (816) University of Missouri Columbia Chemistry Building 601 S. College Avenue Kaitlin L. Siela Columbia, MO Telephone: (816) USA Dr. Rainer Glaser, Associate Editor April 29, Chemistry Building 601 South College Avenue Columbia, Missouri RE: REVISED Effect of Epigallocatechin Gallate on Micellar Solubility of Cholesterol and Phosphatidylcholine By Jane M. Jackson and Kaitlin L. Siela Dear Dr. Glaser: Thank you for your letter of 23 April regarding the above cited paper. We value the constructive comments by both you and the three reviewers. We have now prepared a revision with the changes described below. Major Changes [M.1] We changed all references to a synthesis of EGCG to sources on extraction. [M.2] In the abstract we included more information regarding the experiment and moved information regarding the background to the introduction. [M.3] Results and Discussion section was expanded. [M.4] We added a scheme about the chemistry of micellar solubility in the Results and Discussion section to improve the explanation of the specific interaction. [M.5] In the Conclusion section we added information about why EGCG is beneficial for human health and why people should include it more in their diet. Response to Reviewer 1 [1.1] Reviewer 1 wrote that we need to shorten our title but we feel that if we were to shorten our original title it would leave out vital information that explains what our research entails. [1.2] The sentence Extraction and isolation using water as the solvent proves to be the best method due to reduced impurities. in the original abstract was deleted. [1.3] We clarified in the abstract that PC is an abbreviation for phosphatidylcholine. [1.4] We revised this sentence using your recommendations and combined with the prior sentence to say by inhibiting oxidation by donating an electron to free radicals.

2 [1.5] We corrected this sentence by saying Omega-3 fatty acids are a specific class of fatty acids, which have [1.6] We clarified the number of hydroxyl groups to say that there are four hydroxyl groups. [1.7] In Scheme 1 we capitalized Omega-3 and Vitamin C. [1.8] We decided that the whole paragraph is about classes of nutraceuticals and there is not enough information to separate into different paragraphs. Also, we thought it was most effective to keep the Scheme as just one instead of three separate. [1.9] Reviewer 1 recommended we change do not need to be approved to are not required to be and this was done. [1.10] We added a scheme and cholesterol is generalized as a lipid and the cholesterol structure is shown in the graphical abstract. [1.11] We deleted the phrase as the solvent in order to reduce impurities. [1.12] We clarified the NMR to be a 13 C-NMR. [1.13] We shortened 37 degrees Celsius to 37ºC and deleted it from one of the sentences. [1.14] We revised the grammatical issues from the reviewers comments. [1.15] We reworded the phrase and changed it to There is no RDA of EGCG due to no found risks. [1.16] We changed the title to Extraction of Epigallocatechin Gallate. [1.17] Reviewer 1 recommended we remove the first paragraph in the appendix. We removed the paragraph. [1.18] Reviewer 1 recommended we changed using a vacuum to in vacuo. We decided that this change was necessary and kept the sentence as it was. [1.19] We included more sources. Response to Reviewer 2 [2.1] We deleted the repeated to act. [2.2] Reviewer 2 recommended that we change the amounts were to the amount, was and this was done. [2.3] We changed the sentence to say that Flavonoids may improve [2.4] Reviewer 2 recommended that we change the sentence beginning with An example of a fatty acid please see [1.5]. [2.5] Reviewer 2 recommended we either change the sentence or remove, we decided to remove the statement starting with Omega-3. [2.6] We changed the sentence to begin with Individual with diets high in [2.7] We bolded the letters a, b, and c under the structures in Scheme 3. [2.8] We decided to delete this section of the results and discussion. [2.9] Reviewer 2 recommended that we change the sentence to say The spectrum supported the conclusion that and this was done. [2.10] We removed the second tab in the first paragraph on pp. S3. 2

3 Response to Reviewer 3 [3.1] We deleted the page number from the letter to the editor and page one starts on the title page. [3.2] We deleted the repeated to act from the abstract and the introduction. [3.3] Reviewer 3 recommended that we shorten information about nutraceuticals in the introduction. We shortened the information given about the three classes of nutraceuticals. [3.4] A scheme was added as recommended by Reviewer 3 to explain galloyl moiety better. [3.5] We decided to delete this section from the results and discussion. [3.6] We expanded upon the antioxidant properties, but did not feel it was necessary to put another experimental data table. I believe we have addressed all the points raised by the reviewer and we are grateful for their thoughtful comments. The revision is more focused and much stronger than the original submission, and we hope that this version will be suitable for publication. Sincerely, Siela and Jackson 3

4 Effect of Epigallocatechin Gallate on Micellar Solubility of Cholesterol and Phosphatidylcholine Kaitlin Siela and Jane Jackson Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri

5 Abstract. Epigallocatechin gallate (EGCG), the most abundant type of catechin, displays antioxidant properties and is thought to act as an antioxidant in biological systems. Unfermented green tea is the most prominent source of catechins in human nutrition with over 30% of its content being catechin. EGCG can be a major cause of the inhibition of cholesterol absorption. Green tea catechins, like EGCG, that contain a galloyl moiety have a higher affinity for hydrophobic bilayers than free catechins. It was found that green tea catechins with a galloyl moiety decrease micellar solubility of cholesterol via specific interaction of phosphatidylcholine (PC). It was found that analogues of galloyl moiety without the catechin skeleton such as propyl gallate also showed obvious slow-binding inhibition, whereas the green tea ungallated catechin did not. In the absence of green tea catechins, the amount of cholesterol incorporated into the bile micelles was 100%. EGCG and GCG effectively decreased the micellar solubilities of cholesterol and PC in a dose-dependent manner. 2

6 Introduction Nutraceuticals are food, or parts of food, that provide medical or health benefits including the prevention and treatment of disease. 1 There are three major classes of nutraceuticals: antioxidants, oils and fatty acids, and vitamins. Antioxidants are compounds in our food that prevent and/or repair damage to our cells by inhibiting oxidation by donating an electron to free radicals. 2 Flavonoids, which all have a three-ring structure, are an example of an antioxidant (Scheme 1a). 3 Flavonoids may improve memory, learning and general cognitive function, including reasoning skills, decision-making, verbal comprehension and numerical ability. 3 The second class of nutraceuticals is oils and fatty acids. Fatty acids influence physiological processes, such as, calcium transport across the cell membrane, cell proliferation, and immune cell function. 4 Omega-3 fatty acids are a specific class of fatty acids which have a carboxylic acid and a long polyunsaturated fatty acid tail (Scheme 1b). Individuals with diets high in omega-3, either through food or supplements, can experience lower chance of cancer. The third class of nutraceuticals is vitamins. Vitamins are organic compounds that our bodies need to grow and develop normally. These vitamins can be produced from our bodies or obtained through diet. An example of a vitamin is vitamin C, which is a single ring structure with four hydroxyl groups (Scheme 1c). If vitamin C is obtained through one s diet, it can help lower risk of heart disease and certain cancers. 5 Scheme 1. Chemical Structures of A. Flavonoid B. Omega-3 C. Vitamin C. A. B. C. 3

7 Whether it is to help with muscle gain, shed a few extra pounds, or to simply promote wellness many Americans have taken up using nutraceuticals in their everyday life. By eating a diet of improved foods and nutraceuticals one can greatly improve their health and see an increase in their performance. Unlike over-the-counter drugs, nutraceuticals are not required to be approved by the Federal Drug Administration before being released to the market. The manufacturers of the supplements must have evidence that their product is safe and the label does not mislead the public, but due to the Dietary Supplement Health and Education Act, the FDA is prevented from regulating the supplements. 1 Despite the lack of regulation and testing, nutraceuticals have shown a rapid emergence due to the growing dissatisfaction with drug costs and conventional healthcare. 1 This growing trend focuses on preventive medicine and the public perception that natural is good. Here we report the results of an experiment aimed to decrease micellar solubility of cholesterol, focusing on epigallocatechin gallate via specific interaction with phosphatidylcholine (PC). 7 Epigallocatechin gallate (EGCG), a catechin, was extracted and isolated from green tea leaves. An expanding body of preclinical evidence suggests EGCG has the potential to impact a variety of human diseases. 8 EGCG displays antioxidant properties and is thought to act as an antioxidant in biological systems. There are two classes of catechins: free catechins and catechins with a galloyl moiety. EGCG is a catechin with a galloyl moiety along with a comparison compound, gallocatechin gallate (GCG). A galloyl moiety has inhibition characteristics. 9 The other comparison compound used in this experiment is a free catechin, epigallocatechin (EGC). Catechins containing a gallate ester moiety have the highest activity of antioxidants and are the most effective inhibitors of cholesterol absorption. EGCG acts as a chain-breaking antioxidant, which traps peroxyl radicals and thus suppresses radical chain 4

8 autoxidations. Catechins react with peroxyl radicals via a single electron transfer followed by deprotonation (Scheme 2). 7 Beneficial effects of antioxidants are to revoke the deleterious consequences of elevated free radicals are implicated in disease prevention and cost-effective strategy. 10 Scheme 2: Formation of Catechin Phenoxyl Radical by One-Electron Oxidation We expect that green tea catechins with a galloyl moiety have a higher affinity for hydrophobic lipid bilayers than free catechins (Scheme 3). Therefore, it is possible that green tea catechins with a galloyl moiety can interact directly with cholesterol through their hydrophobicity since cholesterol is also a hydrophobic molecule. Scheme 3: Structure of Galloyl Moiety Materials and Methods Catechins are most easily extracted and isolated from green tea leaves using water. The tea leaves are extracted in 80 degrees Celsius water for sixty minutes using a 10:1 solvent to tea 5

9 ratio (ml/g). 6 A more detailed description of the synthesis as well as spectroscopic characterization can be found in the supporting information. Scheme 4: Chemical Structures of A. EGCG B. GCG C. EGC The catechins, EGCG, GCG, and EGC (Scheme 4), were purified and were confirmed to be >98% by 13 C-NMR analysis. The purified catechins were tested to see their effects on the micellar solubility of cholesterol and PC. A bile salt solution containing 6.6 mmol/l sodium taurocholate, 0.6 mmol/l PC, 0.5 mmol/l cholesterol, 132 mmol/l NaCl, and 15 mmol/l sodium phosphate was prepared by sonication and stored for 24 hours. Various amounts of EGCG, EGC, and GCG were added to deionized water and stored. Both mixtures were stored at 37ºC. The mixtures were added to 3 ml micellar solutions and incubated for 1 hour at 37ºC. The supernatant was passed through a 0.2 micrometer syringe filter, and the cholesterol and fatty acid contents originating from the phospholipids were analyzed by gas chromatography using an SPB-1 column and DB-WAX column, respectively (Table 1). 6

10 Table 1: Effect of Green Tea Catechins on Micellar Solubility of Cholesterol and Phosphatidylcholine Catechin Concentration (mmol/l) Cholesterol Concentration in micelles (%) Phosphatidylcholine concentration in micelles (%) EGCG EGC GCG Results and Discussion First we extracted and isolated the new nutraceutical, epigallocatechin gallate, from the green tea leaves. After purification of catechin from reversed-phase high-performance liquid chromatography (RP-HPLC), 13 C-NMR, IR, and a UV-Vis spectrum were taken and analyzed. The spectrum supported out conclusion that we had successfully isolated the nutraceuticals EGCG. In the absence of green tea catechins, the amounts of cholesterol incorporated into the bile micelles were 100%. EGCG and GCG effectively decreased the micellar solubilities of cholesterol and PC in a dose-dependent manner (Scheme 5). EGC slightly decreased the micellar solubilities of cholesterol and PC. The concentrations of micellar concentration and PC following the addition of 2 mmol/l of GCG were lower than the concentration following 2 mmol/l of EGCG. 7

11 Scheme 5: Micellar Solubility of Lipids The availability of EGCG is very high. EGCG is the most abundant catechin in green tea. There is no RDA of EGCG due to no found risks. EGCG is water-soluble, so it is unlikely to build up in your body. The therapeutic range is 100 mg to 1,000 mg, approximately 1 to 8 cups to tea per day. This range depends on how the tea is brewed. Green tea catechins (EGCG, GCG, and EGC) are synergistic with vitamin E and vitamin C. EGCG can be 20 to 200 times more powerful than vitamin E in neutralizing pro-oxidants and free radicals. While EGCG is the most abundant catechin, GCG has a greater effect on cholesterol and phosphatidylcholine concentration in micelles after 1 mmol/l concentration. EGCG and GCG are more effective than EGC whose dose did not effect a change in the amount of cholesterol and phosphatidylcholine concentration in micelles. This difference in results between the catechins is most likely because of the structural differences. EGCG and GCG are structurally similar while EGC does not have the ester group and third benzene ring. It was found that because EGC did not contain a galloyl moiety that it did not inhibit as well as EGCG and GCG. The galloyl moiety also increased the affinity for hydrophobic lipid bilayers. In our experiment we also found that EGCG eliminated cholesterol as well as PC in bile salt micelles. 8

12 Conclusion A novel antioxidant, epigallocatechin gallate, has been extracted, isolated, and characterized. In an in vitro assay, the effects of EGCG on the micellar solubility of cholesterol and phosphatidylcholine were measured. GCG and EGC were used as comparative compounds. It was found that the three catechins lowered concentration of cholesterol and PC in micelles. EGCG had the greatest effect at 1 mmol/l and EGC had the least effect. The discovery, successful extraction, and natural availability of EGCG are promising for the development of lowering cholesterol concentration in micelles. Lowering your cholesterol is important; increased cholesterol levels lead to a higher risk of heart attacks and strokes. EGCG can be a major cause of the inhibition of cholesterol absorption. Other green tea catechins that contain a galloyl moiety have a higher affinity for hydrophobic bilayers than free catechins. It was found that green tea catechins with a galloyl moiety decrease micellar solubility of cholesterol via specific interaction of PC. More detailed studies are necessary to understand how green tea catechins with a galloyl moiety cause the precipitation of cholesterol from bile salt micelles. Supplemental Material Available: Description of the preparation of epigallocatechin gallate and of its spectroscopic characterization ( 13 C-NMR, IR, UV-Vis). References (1) What are Nutraceuticals. National Nutraceutical Center, (accessed 5/8/2014). 9

13 (2) Nutrition Spotlight: The Super Power of Antioxidants. National Center on Health, Physical Activity, and Disability, (accessed 5/8/2014). (3) Franz, M. "Your Brain on Blueberries." Scientific American Mind. 2011, 26, (4) Effects of Omega-3 Fatty Acids on Cancer Risk. American Medical Association (5) Vitamins. MedlinePlus, (accessed 5/8/2014). (6) Vuong, Q. V., Golding, J. B., Nguyen, M. and Roach, P. D. J. Sep. Science 2010, 33, (7) Valcic, S., Muders, A., Jacobsen N., Liebler, D., Timmermann, B. Chem. Res. Toxicol. 1999, 12, (8) Singh, B.; Shankar S.; Srivastava, R. Biochemical Pharmacology 2011, 82, (9) Wang, X.; Song, K.-S.; Guo, Q.-X.; Tian, W.-X. Biochemical Pharmacology 2003, 66, (10) Brown, M. K.; Evans, J. L.; Luo, Y. Pharmacology Biochemical Behavior 2006, 85,

14 Supporting Information Effect of Epigallocatechin Gallate on Micellar Solubility of Cholesterol and Phosphatidylcholine Kaitlin Siela and Jane Jackson Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri S1

15 Table of Contents Extraction of EGCG S3 13 C-NMR Spectrum of EGCG S4 IR Spectrum of EGCG... S5 UV-Vis Spectrum of EGCG S6 Bibliography.. S7 S2

16 Extraction of Epigallocatechin Gallate The most prominent source of catechins in human nutrition is through unfermented green tea, with over 30% of its content being catechin. After harvest, the tea leaves must be quickly treated to prevent any oxidation of the catechins. In a 10:1 ratio of solvent to tea (ml/g) the tea constituents are extracted in 80 degrees Celsius water for sixty minutes. The tea constituents are then put through reversed-phase high-performance liquid chromatography (RP-HPLC) in order to isolate and purify catechins from the other tea components. In a semi-preparative RP-HPLC column (10x250mm, 7µm) the tea constituents are first extracted using chloroform to remove caffeine form the tea infusion. The catechins in the infusion were then separated into six fractions using silica gel column chromatography and chloroform/methanol/water (65:35:10 ratio) to elute the six fractions from the column. To isolate epigallocatechin gallate (EGCG) individually, the fifth and sixth fractions were put through the RP-HPLC column with an isocratic water/acetonitrile/methanol/acetic acid (159:36:4:1) solvent system. A flow rate of 3mL/min is used for all of the column chromatography process. The extracts were then dried using a vacuum in order to obtain a powder form. S3

17 Figure S1. 13 C-NMR of Epigallocatechin Gallate. S4

18 Figure S2. IR spectrum of EGCG. S5

19 Figure S3. UV-Vis spectrum of EGCG. S6

20 Bibliography Kobayashi, M.; Nishizawa, M.; Inoue, Nao; H., Takahiro; Y., Masahito; Ukawa, Y.; Sagesaka, Y.; Doi, T.; Nakayama, T.; Kumazawa, S.; Ikeda, I. J. Agric. Food Chem. 2014, 62, S7