THE EFFECT OF CAYENNE PEPPER ON CVD RISK FACTORS AND INFLAMMATION IN ATHEROGENIC-DIET FED RATS

Running Head: Effects of cayenne pepper on CVD risk facts in rats Authors: Mee Young Hong, PHD Lauren Doherty School of Exercise and Nutritional Sciences, San Diego State University, San Diego, CA 92182

Corresponding Author: Mee Young Hong, PHD Department of Exercise and Nutritional Sciences San Diego State University 5500 Campanile Drive San Diego, CA 92182-7251 (619)594-2392 Mhong2@mail.sdsu.edu

Abstract
Introduction This study sought to test the effects of cayenne pepper on multiple risk factors for cardiovascular disease. Four groups of Sprague Dawley rats were used to test the effectiveness of cayenne pepper on triglycerides, HDL, LDL, epididymal fat, inflammation marker CRP, liver diseases markers ALT and AP, myocardial infarction marker AST, LDH, leptin, and antioxidant levels. Rats from two groups will also receive DSS treatment for 24 hours to induce inflammation. CVD is considered a chronic inflammatory disease which results in atherosclerosis according to Paul H. Black et. al. Methods For four weeks four groups of ten rats are fed 300 gram atherogenic diets every 48 hours. Two groups (three and four) receive cayenne pepper in addition to the atherogenic diets. Two other groups (two and four) received the DSS treatment for 24 hours. Following the 24 hour DSS treatment, 48 hours later the rats are euthanized and tested for CVD risk factor markers. Results The groups with added cayenne pepper were found to have significantly lower CVD risk factor markers than the control groups with no cayenne pepper. The rats had lower epididymal fat, lower final weight, lower total cholesterol, lower LDL, higher HLD, secreted less leptin, lower ALT, lower AP, and lower AST.

Introduction Cardiovascular Disease is a disease affecting the functionality of the heart. According to the CDC in 2011, 599,413 Americans died of heart disease and 532,304 Americans died of heart disease in 2008[1]. These statistics make CVD the number one killer of Americans followed by Cancer which was roughly 50,000 people less in 2008 and 30,000 less in 2011 [2]. Cardiovascular Disease has many risk factors including high cholesterol levels, smoking, calorie dense diets, high blood pressure, genetic predisposition, and minimal physical activity [3][4]. While genetics cannot be helped, the other risk factors are modifiable however, CVD has remained the number one threat to American lives since at least the 1960s [5] and after 40 years of new medications and millions of deaths it seems the most beneficial solution would likely be an additive to the diet as opposed to more removal of food items from the diet. According to the USDA the average American diet contains an excess of meat, (about double the amount of the DRI) huge amounts of high fat cheese, white refined flour, and about 100 pounds of sugar per year for the average American. As a general guideline according to Bonnie Liebman, a female should consume 6 teaspoons of sugar per year which equates to just under 23 pounds of sugar (22.8) per year [6]. The average American diet contains 5 times the recommended amount of sugar. The average American diet is an atherogenic diet. The high amount of fat, saturated fat, and cholesterol from cheese and meats in particular severely raise lipid profiles and raising the risk factors for CVD. In this study Sprague Dawley rats were fed atherogenic diets to mimic the CVD risk factors and in addition to the atherogenic diet the rats were given cayenne pepper to test the affects it would have on CVD risk factors and inflammation.

According to Brittany Risher, Capsaicin, the compound that gives hot peppers their kick, raises your core body temperature, causing you to burn more calories, capsaicin is fund in cayenne pepper [7]. Typically when hot peppers are used in studies capsaicin is used as the active ingredient as opposed to the entire pepper. This study uses ground cayenne pepper as an alternative to the usual capsaicin. Ground cayenne pepper can be purchased at any grocery store in contrast to capsaicin which is more expensive and requires a less convenient trip to another store such as a specialty health store. This study sought to test the effects of cayenne pepper on multiple risk factors for cardiovascular disease. Four groups of Sprague Dawley rats were used to test the effectiveness of cayenne pepper on triglycerides, HDL, LDL, epididymal fat, inflammation marker CRP, liver diseases markers ALT and AP, myocardial infarction marker AST, LDH, leptin, and antioxidant levels. Rats from two groups will also receive DSS treatment for 24 hours to induce inflammation. CVD is considered a chronic inflammatory disease which results in atherosclerosis according to Paul H. Black et. al [8]. Methods In the San Diego State University research lab forty, twenty-one day old Sprague Dawley rats were divided into four groups via the zigzag method. The rats were striated by weight to even weights of each rat within each group. Each group contained ten rats and received different diets and treatments with or without cayenne pepper (from TradeWinds, Amerifoods trading Co. Los Angeles, CA) and with or without Dextran sodium sulfate, an inflammation inducing agent (Table 1.) The four groups included two diets and two treatments. Group one (control) received

no Dextran sodium sulfate (DSS) with atherogenic diet (33% sucrose, 21% fat, 3% cholesterol.) Group two received no DSS along with a slightly modified atherogenic diet with 2.90 grams cayenne pepper. Group three received an atherogenic diet with no cayenne pepper and no DSS. Group four received an atherogenic diet with 2.90 grams of cayenne pepper and the DSS treatment (Table 2.) Each rat is given 300 grams of food and every forty-eight hours the spillage is measured to account for the food not consumed by the rats. The amount of water drank is also measured. The rats were given two to three days of acclimation time before the new diets and treatments ensued. For four weeks the rats were given their assigned diets and treatments. Following the four weeks groups which received the DSS treatment were given water containing DSS for twenty-four hours. Following the twenty-four hours the DSS containing water was removed and for the following forty-eight hours and rats were given pure water. After forty-eight hours the rats were euthanized. Following euthanasia the rats are weighed to record their final weights. The rats were then dissected. The spleen, kidney, and liver are all extracted and weighed for comparison. Blood samples were collected from each rat and multiple tests were run. The tests included total cholesterol, HDL, LDL by calculation, CRP, LDH, total antioxidant levels, ALT, AST, AP, and Leptin. Serum total cholesterol (triglyceride and HDL), LDH, AST, ALT, and AP test kits came from StanBio in Boerne, TX. LDL by calculation, CRP from Biosciences in San Jose, CA, total antioxidant levels test from Simga, St. Louis Missouri, and Leptin EMD from Millipore, Billerica, MA. Using the test kit from StanBio and serum samples from the rats, total cholesterol is

measured using HDL and triglycerides. LDL is determined by calculation using the calculation of LDL (mg/dL) = total cholesterol HLD TG/5. Serum Lactate Dehydrogenase (LDH) is a measure for multiple things including but not limited to, myocardial infarction, megaloblastic anemia, pernicious anemia, liver damage, and muscular dystrophy. LDH reagent was reconstituted amd 7.5 uL of the rats unhemolyzed serum was added to the 300 uL of reagent. The spectrometer was zeroed at 340nm and the results were read after 60 seconds. C-Reactive Protein (CRP) is used to test for inflammation. CRP from 100 micro liters of diluted unhemolyzed serum from the rats will bind to the antibody, horseradish peroxidase conjugated anti-rat CRP is added and used to create a sandwich between the antibodies and antigens. A substrate was added which would produce a color for the spectrometer to read at 450nm. Antioxidant assay (total antioxidant levels) test is used to find the concentration of antioxidants in the body. Trolox standard solution, reconstituted Myoglobin, ABTS substrate working solution, and rat samples are placed in the well and incubated for five minutes. Following incubation a stop solution was added to the wells and results were read at 405nm from the spectrometer. Alanine aminotransferase (ALT) test is used to test for hepatic diseases such as Cirrhosis. Reconstituted ALT reagent was added to each well along with serum samples. The samples were read at 340nm from the spectrometer. Results we recorded after 60 seconds. Aspartate aminotransferase (AST) is used to test for liver diseases or myocardial infarctions which have occurred within two days. Reconstituted AST reagent was added to

serum samples from the rats. The spectrometer was zeroed at 340nm and the results were recorded after 60 seconds. Alkaline phosphatase (AP) test is a measure if liver functionality. AP working reagent is created by adding buffer and substrate. The AP working reagent was added to serum samples and the results were read at 405nm from the spectrometer every minute for 15-20 minutes. Leptin is a reflection of epididymal fat which regulates kcal intake. Leptin is an anorexagenic hormone secreted when there is an abundance of epididymalfat. Multiple diluted buffers were added to each well with periodic incubations after each. Substrate was added, incubated for 20 minutes, and then a stop solution was added. The results were read from the spectrometer at an absorbance of 450nm.

Statistics Data from treatment and diet were analyzed by a two way ANOVA procedure with SPSS (IBM, Armork, New York.) Using SPSS food intake, water intake, body weight, total cholesterol, LDL, HDL, CRP, LDH, AST, ALT, AP, leptin, and antioxidant levels were analyzed and presented Mean SE with a alpha levels P less than 0.05 being significant. Results Final body weights of groups one and two varied significantly from groups three and four. The groups given cayenne pepper weighed significantly less than groups not given cayenne pepper with a P-value of 0.045 (Table 3.) There were no statistical differences between the food and water consumed in groups one through four with the exception of during the DSS treatment. However, while receiving the DSS treatment, groups two and four drank significantly

less water with a P-value of 0.016 and ate significantly less food with a P-value of 0.004. Spleen, liver, and kidney weights did not differ significantly. Epididymal fat varied significantly. Groups one and two had higher amounts of epididymal fat than groups three and four with a significant P-value of 0.037. Total cholesterol, LDL, and HDL were significantly altered. Total cholesterol was lowered in groups three and four in comparison to groups one and two with a P-value of 0.038. LDL cholesterol was also lowered in groups thee and four with a significant P-value of 0.031. HDL cholesterol was raised in groups three and four with a significant P-value of 0.045. The groups given cayenne pepper had higher HDL, lower LDL, and lower total cholesterol (Table 4.) LDH results were a measure of the amount NADH produced from the reduction of NAD to NADH following the oxidation of lactate to pyruvate. The results of this test were not significant. The DSS treatment did not have an effect on LDH. For a positive result CRP testing samples would turn a blue to yellow color while a negative reaction would remain colorless. The amount of absorbance from the spectrometer reflected a significant P-value of 0.036. The amount of CRP differed significantly from the control group one with no DSS and control group two with DSS from group three with cayenne pepper and four with cayenne pepper and DSS (Table 5.) Calculations of the antioxidant concentrations were done using the linear regression of the standard curve. Averages of the absorbance of each well were taken and plotted on a standard curve. The calculations produced a significant P-value of <0.001 for those receiving the DSS treatment and 0.011for the different diets. The control diet with no DSS had a significantly different concentration of antioxidant levels then the diet containing cayenne pepper and no

DSS. Control group three with cayenne pepper and no DSS had significantly higher amounts of antioxidants than the group four which received cayenne pepper and DSS and the control groups two which received DSS but no cayenne pepper (Table 6.) Elevated ALT suggests evidence of a hepatic disease. A significant P-value result of 0.041 showed that the control diet with DSS had a higher ALT than all other groups and group three, the group which received cayenne pepper with no DSS had significantly lower ALT than group one with no DSS and no cayenne pepper and group two which received DSS but no cayenne pepper (Table 5.) Elevated AST for a short period of time can be indicative of a myocardial infarction or long term elevated AST can be indicative of a liver disease. The amount of AST found in groups one and groups two, the control groups both without cayenne pepper and group two with DSS were significantly different with a P-value of 0.040 from groups three and four both with cayenne pepper added to their diet (Table 5.) Results for AP were calculated using "absorptivity micromolar extinction coefficient" of 4-nitrophenol at 405 nm (0.01845). The AP activity in groups one and two were significantly different from groups three and four with a P-value of 0.016. The diets with no cayenne pepper had significantly different AP activity from the groups with cayenne pepper with no variation in DSS (Table 5.) Groups one and two varied significantly from groups three and four with leptin. Groups two and three had significantly lower levels of leptin secretions with a P-value of 0.045. The groups with cayenne pepper to their diets had lower levels of leptin than those with no cayenne pepper (Table 7.)

Discussion Based on the results of this study there is a clear correlation between the addition of cayenne pepper to an atherogenic diet and lowering CVD risk factors. In this study the rats that consumed cayenne pepper along with being fed an atherogenic diet had significantly less epididymal fat, lower cholesterol levels, specifically LDL cholesterol, and lower triglycerides. Excess fat, triglycerides, and high LDL cholesterol are related to the risk of CVD. Lowering LDL lowers the risk of atherosclerosis related to heart attacks and strokes. In addition to the atherogenic diets, the rats in groups three and four which received cayenne pepper had significantly higher HDL. HDL cholesterol removes LDL cholesterol from the arteries and helps to prevent atherosclerosis which also lowers CVD risk factors. The control groups also had significantly higher amounts of CRP suggesting the control groups with atherogenic diets alone had elevated inflammation levels when compared to the diets with cayenne pepper. Like other studies have stated before, cayenne pepper has been shown to be a weight loss helper by boosting metabolism by up to 25% after eating a spicy meal according to Kimberly Daly [9]. In addition to metabolism boosting, according to Mark Tallon a 2007 study from Japan showed that the addition of capsaicin to a non-specific diet improved the mobilization of fat from adipose tissues to the blood stream and used for energy [10]. The study used active males as subjects. The subjects were given capsaicin to use before their usual workouts. The lower epididymal fat shows a correlation with this study based on the lower levels of leptin released in groups three and four in comparison to groups one and two. Also according to Mark Tallon, a different study published in 2007 showed that rats fed

hypercholestrolemic diets and non- hypercholestrolemic diets lowered their triglycerides and liver cholesterol significantly with the addition of cayenne pepper to their diet [9]. Triglycerides and cholesterol were lowered less in the hypercholestrolemic diet which suggests that while the addition of cayenne pepper to a diet may be effective, it is likely more effective in an already healthy diet. This study produced results which coincide with both of the previously mentioned studies. Triglycerides, epididymal fat, and cholesterol were all lowered in addition to AP, ALT, AST, CRP, and leptin. Leptin secretion was likely reduced in reaction to the lowered epididymal fat which is what triggers the secretion of leptin. In another study, capsaicin was used to test hepatoprotective activity. Capsaicin was shown to keep liver damage minimal after a period of 8 weeks of testing. According to Memy H. Hassan et al this study produced similar results using similar markers of lower AST and ALT [11]. Although this study acknowledges that the antioxidant properties of capsaicin may be partially responsible. The lower AST and ALT levels in the rats given cayenne pepper suggest the same findings that Hassan found. Both studies produced lower amounts of AST and ALT. According to a Health and Wellness article, Some research suggests that items like green tea and cayenne pepper can increase your metabolism, but according to de Mille, they seem to elevate your metabolism only slightly for a brief time--not enough to result in significant weight loss [12]. This leaves the door open for more research on the matter. At the end of the study the rats given cayenne pepper weighed significantly less despite consuming the same amount of water and food for the entirety of the study. This study also showed a significant decrease in triglycerides, epididymal fat, LDL, leptin, and other liver disease and

myocardial infarction markers such as AST, ALT, CRP, and AP however, this study only lasted for four weeks. A longer research study would be recommended to refute previous and contradictory research findings.

References

Heron, M. (2012). Retrieved from National Vital Statistics Reports website: http://www.cdc.gov/nchs/data/nvsr/nvsr60/nvsr60_06.pdf Hoyert Ph.D., D., & Xu M.D., J. (2011). Retrieved from CDC website: http://www.cdc.gov/nchs/data/nvsr/nvsr61/nvsr61_06.pdf Peracino, A. , & Paoletti, R. (2009). Appendix a: Diet as a risk factor for cvd. Atherosclerosis (Supplements) (Component), 10(1), 29. Kaplan, G., Strawbridge, W., Cohen, R., & Hungerford, L. (1996). Retrieved from http://aje.oxfordjournals.org/content/144/8/793.full.pdf html Luepker, R. (2011). Cardiovascular disease: Rise, fall, and future prospects. Annual Review of Public Health, 32(1), 1-3. Liebman, B. (2010). The changing american diet. Nutrition Action Health Letter, 37(2), 10-11. Risher, B. (2010). Metabolism booster!: Hot peppers. Prevention,62(9), 42-83. Black, P. , & Garbutt, L. (2002). Stress, inflammation and cardiovascular disease. Journal of Psychosomatic Research, 52(1), 1-23. Daly, K. (2011). 5 metabolism boosters. Shape, 30(7), 124. Tallon, M. (2008). Spicing up health with capsaicin. Natural Foods Merchandiser, 29(7), 38. Hassan, M. , Edfawy, M. , Mansour, A. , & Hamed, A. (2012). Antioxidant and antiapoptotic effects of capsaicin against carbon tetrachloride-induced hepatotoxicity in rats. Toxicology and Industrial Health, 28(5), 428-438. Rev up your metabolism to burn off pounds. (2009). Food & Fitness Advisor, 12(2), 1-2.

Appendix A
Table 1. Number of rats in each group; treatment and diet from each group. Control Group 1 (10 rats) Group 2 (10 rats) Cayenne Pepper Group 3 (10 rats) Group 4 (10 rats)

No Dss DSS

Table 2. Group diet composition Ingredient (g) Cornstarch Sucrose Cellulose Casein Corn Oil Anhydrous Milkfat Cholesterol Salt Mix, AIN-76 Vitamin Mix, AIN-76 Methionine Sodium Cholate Choline Chloride Cayenne Pepper Total Diet 1 (control) 12.30 33.00 5.00 20.00 5.00 16.00 33.00 3.50 1.00 0.30 0.50 0.40 0.00 100.00 Diet 2 (cayenne pepper) 11.60 32.69 4.00 19.60 4.61 15.90 33.00 3.50 1.00 0.30 0.50 0.40 2.90 100.00

Table 3. Final body weights, epididymal fat and triglyceride. Control + no DSS 230.53 6.01 1.7100 .15226 49.05 4.65 Control + DSS 229.18 4.80 1.6622 .11025 45.71 4.47 Cayenne + no DSS 217.60 6.83 1.4700 .07881 39.68 3.30 Cayenne + DSS 218.33 5.01 1.4760 .10081 38.73 4.10

Final Body Weight (g) Epididymal Fat (g) Triglyceride (mg/dL)

* Presented Meat SE p<0.05 Table 4. Total cholesterol, HDL, LDL. Control + no DSS Total Cholesterol (mg/dL) 136.45 9.05 HDL (mg/dL) 31.12 7.73 LDL (mg/dL) 101.68 10.98 * Presented Mean SE p<0.05 Control + DSS 140.15 14.59 29.86 4.31 103.06 13.24 Cayenne + no DSS 116.32 5.68 47.37 12.94 74.86 15.34 Cayenne + DSS 119.37 7.27 49.46 13.79 78.82 11.78

Table 5. CRP, AST, ALT, AP levels in each group. Control + no DSS 94.47 8.86 107.95 9.50 44.29 2.05 151.55 9.85 Control + DSS 113.60 11.66 112.17 6.66 56.56 3.75 167.86 14.83 Cayenne Pepper + no DSS 88.07 4.84 93.51 4.09 39.76 1.38 136.60 3.78 Cayenne Pepper + DSS 79.20 6.57 94.45 5.79 41.81 2.62 134.94 5.45

CRP (ug/ml) AST (U/L) ALT (U/L) AP (U/L)

* Presented Meat SE p<0.05 Table 6. Total antioxidant levels.

0.6 0.5 0.4 0.3 0.2 0.1 0 Antioxidant (mM)

A B A

Control + no DSS Control + DSS

* Presented Mean SE p<0.05. Different letters represent statistically significant difference. Table 7. Total leptin levels.
8 7 6 5 4 3 2 1 0 Leptin (ng/ml) Control + no DSS Control + DSS

A A

Cayenne + no DSS Cayenne + DSS

* Presented Mean SE p<0.05. Different letters represent statistically significant difference.