Columbia, Mo USA

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1 Department of Chemistry University of Missouri- Columbia 105 Chemistry Building Taylor Klimt 601 S. College Avenue Columbia, Mo USA April 15, 2016 Dr. Rainer Glaser, Professor of Chemistry Editor, Journal of Organic Chemistry Department of Chemistry, University of Missouri- Columbia Columbia, MO RE: The lowering of cholesterol due to the starch gelatinization of pectin, the soluble dietary fiber found in garbanzo beans By: Taylor Klimt and Dear Dr. Glaser: I would like to submit the manuscript with the above title for the publication as an article in JOC. The submission is made exclusive to the JOC. The paper comes with supporting information. In this article, we report the results of a study done on the effects of adding dietary fiber to an everyday diet, and how this in return lowers cholesterol in the body. The source of the dietary fiber was the garbanzo bean, otherwise known as the chickpea. We specifically tested the soluble fiber pectin. The effects of dietary fiber on cholesterol is important in an age of unhealthy eating, and has not been thoroughly studied before. As possible reviewers we would like to suggest Shelby Herr (Mizzou, sehxbd@mail.missouri.edu), and Joe Schell (Mizzou, jrsg24@mail.missouri.edu) With Best Regards, Grace and Taylor 1

2 The lowering of cholesterol due to the starch gelatinization of pectin, the soluble dietary fiber found in garbanzo beans and Taylor Klimt Department of Chemistry, University of Missouri-Columbia, Missouri

3 Abstract It is been observed that the presence of dietary fiber, both soluble and insoluble, in chickpeas can lower cholesterol levels. In this study, the amount of soluble and insoluble fiber was found for chickpeas along with other legumes. The differences were measured and recorded, with the most amount of soluble fiber found in chickpeas over the others. Soluble fiber, such as pectin, is important in lowering LDL cholesterol levels, which can overall positively affect cardiovascular health. The gelling behavior of pectin is the mechanism that aids in removing cholesterol from the gastrointestinal phase. A gastrointestinal simulation revealed that pectin reacts with bile salts and calcium, which inhibits lipid digestion. 3

4 Introduction There are numerous nutritional and health benefits associated with garbanzo beans. Most health benefits of a garbanzo bean are related to its high dietary fiber content. A typical cup of garbanzo beans is 12.5 grams of fiber, and the recommended daily intake is about 25 to 30 grams of fiber. 1 The two classifications of dietary fiber found in Garbanzo beans are insoluble fiber and soluble fiber. Cellulose is an insoluble fiber that is the major component of cell walls in plants. 2 An unbranched linear chain of several thousand glucose units with β-1,4 glucosidic linkages is the chemical structure of cellulose (Scheme 1). 2 These linkages create a resistance to biological deterioration, as well as resistance to acid hydrolysis, as the result of hydrogen bonding within microfibrils. 2 Scheme 1. Chemical Structure of Cellulose Furthermore, pectin is classified as a soluble dietary fiber. The prominent component of this complex group of polysaccharides is D-galacturonic acid (Scheme 2). 3 Pectin provides structure in most plant cell walls similar to cellulose. Additionally, pectin has the ability to gel when polymer molecules interact over a part of their length to form 4

5 a chain that captures solvent and solute molecules. 4 This gelling characteristic is highly dependent on the degree of methoxylation. This gelling behavior, also known as starch gelatinization, is the function of dietary fiber that helps lower cholesterol. Scheme 2. Chemical Structure of Pectin Here we report the results of a study conducted on 45 living adults. The participants of this study consumed a minimum of 728g of canned, drained chickpeas per week, for 12 weeks. It was hypothesized that including garbanzo beans in an ad libitum diet would significantly increase dietary fiber consumption and with that decrease serum lipids and lipoproteins concentrations in the body. Materials and Methods Fiber Concentrations. The dietary composition, specifically crude fiber, was investigated. Four categories of legumes, peas common beans, chickpeas, and lentils were chosen. One portion of each legume was ground raw into flour and the other portion was washed under water, soaked for 16 hours, cooked, and freeze-dried. Table 1 lists the results of 5

6 the insoluble and soluble dietary fiber concentrations. The concentration values are listed in grams per 100 grams for raw legumes/free-dried cooked legumes. Table 1. Concentration of Insoluble and Soluble Fiber 5 Dietary Fibers Pea (g/100 g) Common Bean (g/100 g) Insoluble Fiber 20.3±0.40 / 19.9±0.19 / Chickpea (g/100 g) 13.9±0.09 / Lentil (g/100 g) 19.0±1.27 / 22.8± ± ± ±2.10 Soluble Fiber 1.73±0.26 / 2.42±0.74 / 5.3±1.67 / 1.44±0.11 / 2.38± ± ± ±0.52 6

7 Dietary Fiber in Chickpeas. In particular, chickpeas provide interest. The dietary fiber content in chickpeas has been found to lower cholesterol. Forty-five adults aged 30 to 70 participated in an eight month study in order to detect how adding chickpeas into an ad libitum diet affected their health. The adults did not take any hypoglycemic or hypolipidemic medication prior to the experiment, but in order to participate, the adults had to have a family history of heart disease, type 2 diabetes, or CVD risk factors. In the first four weeks, participants ate an ad libitum diet routinely to make adjustments. Then, for 12 weeks, participants ate an ad libitum diet with chickpeas, deemed the chickpea phase, and then ate an ad libitum diet without chickpeas, deemed the usual phase, during the following four weeks. Participants logged the food they consumed, weighing and recording the foods during the final week of each phase and halfway through the chickpea phase. The dietary logs were then analyzed using a FoodWorks program, which assigns standard weights to household measures. The usual phase consisted of eating a regular ad libitum diet. During the chickpea phase, however, participants ate a minimum of four 300-gram cans of chickpeas, about 182 grams per week. Blood samples were taken after participants fasted for ten hours during the final week of each dietary phase. The serum total cholesterol, triacylglycerols, high-density lipoprotein (HDL) cholesterol, and plasma glucose levels were reported. There was an increase in fiber during the chickpea phase compared to the usual phase. As seen in Table 2, analysis of the effect of the chickpea phase versus the usual phase was evaluated for total cholesterol and insulin. 7

8 Table 2. Cholesterol and Insulin Total Cholesterol (mg/dl) Insulin (µlu/ml) Mean Difference Chickpea Minus Usual Dietary Fiber (g) (25.89±7.15) Saturated Fatty Acids (17.31±5.21) Polyunsaturated Fatty Acids (17.31±5.21) Protein (17.96±2.85) The amount of fiber available and the serum cholesterol percentage was also measured for two additional foods that contain fiber, wheat bran and whole oats, as seen in Table 3. Table 3. The Effect of Fiber on Serum Cholesterol % 6 Source of Fiber Plant Fiber (g/d) Serum Cholesterol % of Control Wheat Bran Whole Oats Chickpeas

9 The Effect of Pectin in Gastrointestinal Conditions. Pectin, a soluble dietary fiber, is specifically linked to the lowering of cholesterol. Gastrointestinal conditions were simulated and observed to determine how pectin influences lipid digestion. Corn oil, commercial powdered high-methoxyl pectin, lipase from porcine pancreas, Mucin from porcine stomach, and pepsin A from porcine gastric mucose were obtained to make the simulated saliva fluid. The following concentrations of certain chemicals were used, as seen in Table 3. Table 4. Simulated Saliva Fluid in the Oral Phase Compound Concentration (g/l) Sodium Chloride Ammonium Nitrate Potassium Dihydrogen Phosphate Potassium Chloride Potassium Citrate Uric Acid Sodium Salt Urea Lactic Acid Sodium Salt Porcine Gastric Mucin (Type II) 30 These were all mixed in a 1:1 ratio to obtain a mixture containing 1% corn oil and % pectin. The simulated oral phase was shaken for 10 minutes. Simulated gastric fluid was also made from 2 grams of NaCl, 7 ml of concentrated HCl, and 3.2 grams of 9

10 pepsin A. The mixture was diluted with distilled water, and 1 M HCl was added to lower the ph to 1.2. Samples from the oral phase and gastric fluid were mixed in a 1:1 ratio so that the final mixture had 0.5% corn oil and % pectin. 1 M NaOH was added and shaken for two hours. This mixture was used in the intestinal phase. The interaction of pectin and gastrointestinal components was analyzed. Results and Discussion Fiber Concentrations. Table 1 displays the results of soluble dietary fiber and insoluble dietary fiber. The amount of soluble fiber increased from raw legumes to freeze-dried cooked legumes for both the pea and common bean. The amount of soluble fiber was the greatest for chickpeas, thus indicating that chickpeas are an important source of dietary fiber that can reap health benefits. The amount of insoluble fiber increased from raw legumes to the freeze-dried cooked legumes for all samples. Although chickpeas had the lowest number of insoluble fiber, fiber concentrations still remain an important aspect of chickpeas due to its ability to lower cholesterol. Dietary Fiber in Chickpeas. The participants typically consumed about 119 g/day, or 4.6 cans per week, of chickpeas. There were significantly higher amounts of fiber content after adding chickpeas to their diet. The total fiber in the chickpea phase was 28.6 ± 7.0, and the total fiber in the usual phase was 21.8 ± 8.2. Thus, there was a 6.77-gram difference between the two phases, with an increase of fiber in the chickpea phase. There were significant decreases in mean serum total cholesterol during the chickpea phase compared with the 10

11 usual phase. As seen in Table 2, total cholesterol decreased by 7.7 mg/dl, LDL cholesterol decreased by 7.3 mg/dl, and fasting insulin decreased by 0.89 µlu/ml. High fiber diets such as during the chickpea phase was observed to lower the serum total cholesterol levels. As seen in Table 3, chickpeas contain the highest amount of plant fiber and the lowest amount of serum cholesterol percentage of the control. On average, diets with added beans lowered serum cholesterol levels by 19%. Chickpeas are rich in soluble fibers, such as pectin, which work to lower serum total cholesterol concentrations. The lower number of serum cholesterol percentage of the control for chickpeas than wheat bran and whole oats indicates that it is more effective at lowering cholesterol levels. The mechanism for the lowering of these levels can be explained by the increase of bile salts found in fecal excretion, which diverts cholesterol synthesized in the liver. Another possible mechanism includes that soluble fibers are short chains of fatty acids that inhibit cholesterol synthesis. The Effect of Pectin in Gastrointestinal Conditions. Pectin reduced cholesterol levels by interfering with the reabsorption of bile salts in the small intestine. In addition, pectin interacted with certain gastrointestinal components and other components in the gastrointestinal tract, like CaCl 2, NaCl, and other digestive enzymes in order to reduce lipid digestion. Pectin was able to decrease lipid digestion due to a gelling behavior that it exhibits. Pectin interacted particularly strongly with the calcium ions and bile salts, which formed microgels that decreased the ability of emulsified liquids to be accessed by lipases in the gastrointestinal tract and 11

12 inhibited digestion. Lipid digestion is inhibited if enough of the calcium and bile salts are trapped within the microgels and cannot remove the long-chain fatty acids from liquid droplet surfaces. Lipid digestion can again be delayed if liquid droplets are trapped within pectin microgels and ligase cannot access the surfaces of the lipid. Thus, foods that are high in fiber are able to inhibit lipid digestion and lower cholesterol levels. Conclusion In this experiment, chickpeas had the highest amount of soluble fiber compared to other legumes. In addition, chickpeas showed to lower the serum cholesterol levels the most compared to other foods rich in fiber. This suggests that the soluble fiber found in chickpeas significantly lowers the cholesterol levels in humans. Pectin is an example of soluble fiber that interferes with bile salts and calcium levels in the gastrointestinal tract, thus lowering lipid digestion. Pectin exhibits a gelling behavior that lowers the surface area of a lipid and prevents ligase from reaching the lipid. This inhibits the digestion of the lipid and thus lowers the cholesterol levels. The main finding of this experiment is that garbanzo beans can aide in lowering cholesterol. Thus suggesting that adding chickpeas into your daily diet brings many health benefits including the lowering of one s cholesterol. To further this study, we will be experimenting with adults that suffer from abnormally high cholesterol. The adding of chickpeas to their diet may or may not have an impact on his or her health. 12

13 Supplemental Material Available: The appendix contains a more detailed explanation of how the macronutrient was prepared. In addition, there is further information provided about pectin, including an 1 H NMR spectra and a graph detailing the effect that pectin has on free fatty acids. References (1) Chickpeas Nutrition Facts. Self Web. 07 March 2016 < (2) Dhingra, D.; Michael, M.; Rajput, H.; Patil, R. T. Dietary Fibre in Foods: a Review. J. Food Sci. Technol. 2012, 49, (3) BeMiller, J. An Introduction to Pectins. Chapter 1 in Chemistry and Functions of Pectins. Fishman, M.; Jen, J., Eds. American Chemical Society, 1986, 9, (4) Urias-Orona, V.; Rascón-Chu, A.; Lizardi-Mendoza, J.; Carvajal-Millán, E.; Gardea, A. A.; Ramírez-Wong, B. A Novel Pectin Material: Extraction, Characterization and Gelling Properties. Int. J. Mol. Sci. 2010, 11, (5) Vasishtha, H.; Srivastava, R. P.; Verma, P. Effect of dehusking and cooking on protein and dietary fibre of different genotypes of desi, kabuli and green type chickpeas (Cicer arietinum). J Food Sci. 2014, 51, (6) Anderson, James; Lin Chen, Wen-Ju. Legumes and Their Soluble Fiber: Effect on Cholesterol-Rich Liporopteins. ACS Symp Series, 1983, 214,

14 Supporting Information The lowering of cholesterol due to the starch gelatinization of pectin, the soluble dietary fiber found in garbanzo beans and Taylor Klimt Department of Chemistry, University of Missouri-Columbia, Missouri S1

15 Table of Contents Dietary Fiber in Chickpeas... S3 NMR Spectroscopy for Methylated Pectin... S4 The Influence of Pectin on FFA... S5 Bibliography... S6 S2

16 Dietary Fiber in Chickpeas. Soluble and insoluble fibers can specifically be extracted from pulses through the following method. A mixture of dry, ground pulse flour, 80 ml of 0.5 M MES/Tris buffer, and 300 U α-amylase was heated for 35 minutes at degrees Celsius. After 20 ml of deionized water was added and the mixture cooled to 69 degrees Celsius, 60 U of protease was added and stirred for 30 minutes. In order to adjust the ph to about , HCl and 3260 U of amyloglucosidase was added, followed by more stirring for 30 minutes. The solution was boiled for 10 minutes to deactivate enzymes and then centrifuged for 15 minutes. In order to collect the insoluble residue, it was washed with 10 ml of 95% ethanol two times and 10 ml of acetone once. The remaining liquid was decanted, mixed with four times its volume of 95% ethanol to precipitate the soluble fiber, and then centrifuged for 15 minutes. The soluble residue was also rinsed with 10 ml of 95% ethanol and acetone. The soluble and insoluble fibers collected were dried in an oven at 60 degrees Celsius, milled to a powder, and dried in a vacuum oven at 80 degrees Celsius. S3

17 NMR Spectroscopy for Methylated Pectin The NMR Spectroscopy is used to show the analysis of free galaturonic acid and methyl esters in tri- and tetrameric pectin fragments. Figure 1 shows this analysis with a degree of methylation at 84%. Figure S1. 1 HNMR Spectrum (D 2 O) of a methylated Pectin S4

18 The Influence of Pectin on FFA Figure S2 shows that the more pectin available in the intestine the less free fatty acids released in the intestinal phase. Figure S2. The influence of pectin concentration on free floating molecules. S5

19 Bibliography De Almeida Costa, G.; da Silva Quieroz-Monici, K.; Reis, S.; Oliviera, A. Chemical composition, dietary fibre, and resistant starch contents of raw and cooked pea, common bean, chickpea, and lentil legumes. Food Chem. 2004, 94, Pittaway, J.; Robertson, I.; Ball, Madeleine. Chickpeas May Influence Fatty Acid and Fiber Intake in an Ad Libitum Diet, Leading to Small Improvements in Serum Lipid Profile and Glycemic Control. J Am Diet Assoc. 2008, 108, Christensen, T.; Lundt, I.; Rosenbohm, C.; Young, N. Chemically methylated and reduced pectins; preparation, characterization by 1 H NMR spectroscopy, enzymatic degradation, and gelling properties. Carbohydr. Res. 2006, 93, (NMR) Espinal-Ruiz, M.; Parada-Alfonso, P.; Restrepo-Sánchez, L.; Narváez-Cuenca, C.; McClements, D. Interaction of a Dietary Fiber (Pectin) with Gastrointestinal Components (Bile Salts, Calcium, and Lipase): A Calorimetry, Electrophoresis, and Turbidity Study. J. Agric. Food Chem. 2014, 62, (Pectin Bar Graph) S6