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Abstract Objective To evaluate if watermelon powder improves the risk factors of CVD and inflammation in atherogenic diet-fed rats.

Methods Forty male Sprague-Dawley rats were used and divided in four groups. All groups consumed an atherogenic-diet for four weeks, two groups were fed watermelon powder and two groups were given Dextran Sodium Sulfate for 48 hrs, one group with watermelon and one without. During the study, weight, food, and water intakes were recorded. At the end of the study rats were euthanized and blood was tested for total lipid profiles, CRP, LDH, total antioxidant, createin kinase and serum glucose levels. Results Watermelon groups showed decreased levels in triglycerides, LDL, LDH, CRP and total cholesterol levels compared to the DSS groups which increased these markers (P<.05). Watermelon groups showed to increase total antioxidant levels and kidney weight compared to DSS groups which were lower (P<.05). Conclusion Watermelon powder appears to have positive outcomes on lipid profiles by decreasing inflammation and increasing antioxidant levels, signifying that citrulline in watermelon may decrease the risk of developing cardiovascular disease and inflammation in atherogenic diet-fed rats. Key Words: watermelon, lipid profiles, antioxidants, Inflammation, CVD

Introduction Over the past decades, due to the rise of obesity rates, cardiovascular disease has become the number one cause of death among men and women in the United States. In fact, every year about 1 in 4 deaths or 600,000 deaths are attributed to heart disease (1). About half the population of the United States has at least one of the key risk factors for heart disease which include high blood pressure, high LDL cholesterol, and smoking. However, other risk factors include diabetes, poor diet, physical inactivity, and excessive alcohol use (2). A lot of time and effort has gone into learning more about cardiovascular disease and discovering ways to prevent and or treat heart disease. Watermelon contains many components which would suggest it would be beneficial in helping lower the risks for cardiovascular disease. One of the main components of watermelon is L-Citrulline, which is a naturally occurring amino acid, which is converted in our bodies to L-arginine, another amino acid, which in turn becomes nitric oxide. The formation of the nitric oxide has been found to help vasodilate blood vessels and veins to improve blood flow and lower blood pressure (3). Lower blood pressure means a healthier cardiovascular system. Vasodilation also helps to prevent atherosclerosis, which is the hardening and narrowing of the arteries and the leading cause for heart attacks (4). Watermelon is also a good source for lycopene and beta carotene, both of which contain antioxidant properties. Antioxidants help to eliminate free radicals and reduce oxidative stress, particularly oxidized LDL which contains these free radicals which react with tissues and produce damage to them (5). Damaged tissues, especially around the heart, puts an individual at an extremely high risk for heart disease. The purpose of the

present study was to determine if watermelon powder improves the risk factors for CVD and inflammation in atherogenic diet fed rats. The hypothesis was that watermelon powder reduces the risk of CVD and inflammation by increasing antioxidant and favorable changes of lipid profile and inflammation markers.

Methods and Materials The research model was an in vivo animal study that used male Sprague-Dawley (Harlan, Placentia, CA) rats that were approximately twenty-one days old. They were housed in a room in the life sciences north building at San Diego State University in metal wire cages separately. Temperature and humidity was roughly around 20 to 40C and 40 to 45%, which were controlled strictly. Food and water was accessible for all rats at all times. San Diego State University animal subjects committee approved all procedures and preparation for rats prior to the experiment. There were a total of forty rats, which were divided in four groups of ten. They were grouped using the zig-zag method from least to greatest according to their body weight. There were a total of two diets and two treatments. All of the forty rats diets consisted of an atherogenic components which were sucrose (33%), fat (21%) and cholesterol (3%) by weight (Table 1). The watermelon powder used in our study was provided by Dr. Figueroa from Florida State University (Tallahassee, FL) and originally from Milne fruit products (Prosser, WA). The watermelon product made up 20% of the diet for the first two groups. The other two diets were control groups and contained a placebo made of maltodextrin (20%) instead of the watermelon powders in order to have equal calorie amounts. The treatment consisted of Dextrans Sodium Sulfate, an

inflammation inducing agent. Two groups from the study were treated with DSS, one that contained watermelon powder and another group that contained a placebo, while the other two groups were not treated with DSS (Table 2). When the rats first arrived to the lab they were first given two to three days to get acclimated to their new environment. The study began immediately, where two of the groups were given watermelon powder diets for a span of 4 weeks in length. Body weight, food intake, and water intake were measured before and after the start of the four weeks. Before euthanasia, two groups, one with a watermelon diet and one group without were given 48 hours with the 3% DSS treatment added to their drinking water and then following the 48 hours none of the groups were given the DSS treatment in their drinking water so treatment could take effect. Final body weights, food intake and water intake were measured after this procedure. After euthanization, rats bodies were dissected so that livers, spleens, epididymal fat and kidneys were removed to evaluate and compare the weight of each organ. Blood samples were also drawn in order to measure serum glucose, total lipid profile, lactate dehydrogenase (LDH), C-reactive protein (CRP), creatine kinase (CK) and total antioxidant levels. Testing kits used to measure serum glucose, triglycerides, HDL, LDH, and Creatine Kinase were manufactured by StanBio (Boerne, TX). Total antioxidant capacity kits were manufactured by Sigma (St. Louis, MO) and the CRP kit was manufactured by Biosciences (San Jose, CA). Serum glucose was assessed to determine the effect of the diet and treatment on glucose levels. Glucose assay from stanBio uses a single reagent; glucose oxidase oxidizes glucose when hydrogen peroxide bonds with phenol and 4-aminoantipyrine,

where a red-violet quinone complex is formed. The color brightness of the red-violet quinone complex is an indicator of high glucose content. Total lipid profiles were conducted to determine the total cholesterol, total triglycerides and HDL cholesterol levels in effects to their diet and treatment. These three markers are also very important because they are used to determine total LDL cholesterol in effect from the diets and treatments as well. LDL cholesterol was calculated using a specific equation that goes as follows, LDL (mg/dL) = total cholesterol HDL TG/5. Lactate dehydrogenase (LDH) was also measured in order to determine cardiac muscle damage in effects to the diet and treatment given to each rat, but it is also an indicator of liver disease, pernicious and megaloblastic anemias, pulmonary emboli, malignancies, and muscular dystrophy. LDH easily speeds the oxidation of lactate to pyruvate with the reduction of NAD to NADH; this redox reaction shows LDH activity respectively. The spectrometer measures the absorbance of NADH at 340 nm, which increases after 60 seconds which gives us the results of LDH activity in U/L. C-reactive protein (CRP) is a sensitive phase where the protein made by the liver during a state of inflammation by using the DSS treatment. The ELISA kit used is intended to find and quantize rat CRP in rat serum. CRP from diluted unhemolyzed blood serum from rats will bind to the antibodies, horseradish peroxidase conjugated anti-rat CRP is added to create an antibody-antigen-antibody sandwich, which is indicated with a blue color. A substrate was added which would produce the color which is measured by spectrometer at 450 nm.

Serum creatine kinase (CK) levels have shown to be important in evaluation of cardiac and skeletal muscle diseases, including heart attacks and muscular dystrophy. CK speeds up the reaction with the phosphorylation of ADP to ATP, with these series of reactions NADPH is produced at a rate that corresponds to the CK levels. The assay determines NADPH absorbance increasing every 60 sec at 340 nm using the spectrometer. Total antioxidant levels were measured to find the concentration of antioxidants capacity in the body, an important indicator for reduced risk of cardiovascular disease, cancer, liver damage, and proper nerve function. Antioxidants stop cell damage from the formation of reactive oxygen species (ROS) formed from environmental pollutants, UV-light, radiation, and cigarette smoke. Sigma-Aldrich assay shows that the formation of ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, which oxidizes ABTS to produces ABST+, a cation radical. The production of the radical cation produces a green color, which is read at 405 nm using the spectrometer. Statistical Analysis All results that were gathered from this study were analyzed by ANOVA procedure using SPSS (IBM, Armonk, New York) in order to determine the effects of both treatments and diets on body weight, food intake, water intake, triglycerides, HDL, lactate dehydrogenase, C-reactive protein, serum glucose, creatine kinase, and total antioxidant levels. All values were analyzed and present as mean+/-SE with an alpha value of p<0.05, which is considered significant.

Results Initial body weight and final body weight before DSS treatment of the 40 Sprague-Dawley rats were not significantly different between groups (Table 3). However, final body weight after the DSS treatment was slightly lower for the groups given the DSS treatment (p< 0.003) and final body weight on the last day of the study was also slightly lower for the group given the DSS treatment (p< 0.007). There was no significance between the groups for weight gain before the DSS treatment, but the groups who did not receive the DSS treatment had more of a weight gain during the treatment (p<0.001) and after the treatment (p<0.003) than those groups with the treatment. The groups on the DSS treatment had had less food intake during the treatment than those groups who did not have the treatment (P<0.001). On the other hand, food intake before the treatment and after the treatment had no significant difference. Water intake before the treatment had no significant differences between groups. Water intake during the treatment and after the treatment was slightly lower for the DSS groups (p<0.001 and p=0.02). Liver weight and epididymal fat weight were not statistically different between groups, whereas spleen weight was slightly lower for those groups with the DSS treatment (p=0.049) and kidney weight was higher for the watermelon group (p=0.024) and lower for the group treated with DSS (p=0.019) as demonstrated in table 5. There were no statistical differences between groups for glucose levels. Figure 1 demonstrates the effects of the diets on the serum lipid profile. Triglyceride levels were lower for the groups given the watermelon powder (p=0.031). Non-DSS treatment groups had a higher HDL than DSS groups (p=0.041). Total

cholesterol was lower for the groups with watermelon in the diet (p <0.001) and higher for groups treated with the DSS treatment (p=0.011).The same results were found for LDL; the watermelon group had a lower LDL than the control groups (p< 0.001) and the DSS treated group had a higher LDL than the non- DSS treated group (p=0.008). Figure 2, 3, and 4 demonstrates the effects of the diets on cardiac muscle damage and lowering the risk factors for CVD. LDH, which measures cardiac muscle damage, was lower for the groups with the watermelon in the diet (p<0.001). On the other hand, CK, which also measures cardiac muscle damage, had no significant differences between the groups. The antioxidant capacity was higher for groups on the watermelon diet (p=0.049) and lower for the groups with the DSS treatment (p=0.015). CRP, which measures inflammation, was higher for the groups on the DSS treatment (p<0.001) and lower for the groups on the watermelon diet (p=0.001).

Discussion Cardiovascular disease is influenced by many different biological markers, such as inflammation, high blood lipid profiles, cardiac muscle damage, and low antioxidant capacity. Studies have shown that an increase in inflammation has greatly affected the risks for cardiovascular disease (6). Inflammation is one of the key biological markers for cardiovascular disease due to the fact that it elevates blood lipid levels and causes atherosclerosis. When atherosclerosis, or a buildup of plaque in the blood vessels causing inhibition of blood flow, occurs, damage is ultimately done to the heart. However, citrulline, which is one of the main components of watermelon, may help prevent inflammation thus reducing the risk for cardiovascular disease.

In the results we found that the DSS treatment influenced final body weight of the rats by decreasing the weight as compared to the rats without the DSS treatment. The groups that received the DSS treatment also did not gain as much weight as compared to the groups without the treatment. The reasons for this could be due to the fact that the DSS treatment caused dehydration and loss of appetite in the rats, which was also shown in the results because the groups with the DSS treatment had a lower food and water intake during the treatment. However, since the study was only four weeks long there wasnt enough time to determine if the DSS treatment essentially affects weight gain. In a study done by Trivedi et al, evidence shows that DSS induces ulcerative colitis, which inflames the colon causing loss of fluids through the feces (7). This was evident in our study as the groups on the DSS treatment had lower weight gain, lower food and water intake, and lower final body weight. The diet and the DSS treatment did not play a significant role in the difference of both the epididymal fat and the liver weight due to the fact that the study only lasted four weeks in length. However, the DSS treatment did affect both the spleen and the kidney weights by producing a lower final weight than the organs of those groups without the treatment due to the severe inflammation it produced. The groups on the watermelon diets had higher kidney weights, which helps regulate blood pressure and body fluid balance(8). The results of this study also revealed that inflammation caused by the DSS treatment influenced many of the biochemical markers for cardiovascular disease in a negative way. For example, the main marker for inflammation, which is the C-reactive protein, had higher values for those groups with the DSS treatment. Also, the higher

total cholesterol and LDL levels in the DSS treated groups help to justify the negative effects of inflammation on blood lipid profiles. In a study done by Choy et al, interpreting lipid levels in the context of high-grade inflammatory states, it was found that the higher the inflammatory states the higher the lipid levels would be and the greater the risk for cardiovascular disease(9). Not only did the DSS affect the lipid profiles but it also had a negative effect on total antioxidant capacity, lowering the levels and therefore, affecting the immune system and the ability for the body to fight disease, especially any diseases affecting the heart. On the other hand, the results indicated that the watermelon groups had a positive effect on lipid profiles, total antioxidant capacity, and CRP. In Gil et als study, testing free radicals and the effects of antioxidants, Gil found that antioxidants such as Vitamin C, E, carotenoids, lycopenes, and flavonoids prevent damage caused by free radicals and reduce markers for inflammation(5). Watermelon contains the antioxidant lycopene and b-carotene and so therefore total antioxidant capacity was found to be higher in groups given the watermelon. By lowering the TG, TC, and LDL levels it can be concluded that the components of watermelon, especially the citrulline which inhibits oxidative damage in the arteries, help improve blood lipid profiles. The citrulline also improves the endothelial function, therefore reversing any cardiac muscle damage that has occurred, which was evident in the study by the lower levels of LDH (3). The results also indicated that the watermelon fed helped reduce inflammation by lowering the CRP levels. In summary, this study demonstrated that the effects of inflammation affect the body in a negative way and greatly increase the risk factors for cardiovascular disease.

However, it was also demonstrated that many key components of watermelon can help prevent some of the negative aspects of inflammation and ultimately help lower the risks for cardiovascular disease. Nevertheless, future research examining watermelon and its effects on inflammation and cardiovascular disease over a longer duration with a DSS treatment given for a longer time, should be conducted in order to determine how much of an effect watermelon can have on the risk factors of cardiovascular disease and whether or not it is worth it to use watermelon as a major component of the diet to treat and prevent inflammation.

In conclusion, watermelon powder appears to have reduced the risk factors for cardiovascular disease and inflammation by increasing antioxidant and favorable changes of lipid profiles and inflammation markers.

References [1] http://www.cdc.gov/heartdisease/facts.htm Accessed on May 2, 2013. [2] Jarret BD, Alan D, et al. Lifetime risks of cardiovascular disease. N Engl J Med. 2012; 366(4):321-329. [3] Wileman SM, Mann GE, et al. Role of L-citrulline transport in nitric oxide synthesis in rat aortic smooth muscle cells activated with LPS and interferon-. Br J Pharmacol. 2003; 140(1): 179185. [4] http://www.webmd.com/heart-disease/what-is-atherosclerosis Accessed on May 2, 2013. [5] Gil NS, Dhiman BJ, et al. Evaluation of free radical scavaging, anti-inflammatory and analgesic potential of benincasa hispida seed extract. International Jour. of Pharmacology 2010; 6(5):652-657. [6] Pearson A, Thomas, Mensah A, George et al. Markers of inflammation and cardiovascular disease. Circulation 2003; 107:499. [7] Trivedi PP. Ulcerative colitis-induced hepatic damage in mice: studies on inflammation, fibrosis, oxidative DNA damage and GST-P expression. Chem Biol Interact. 2013; 201:19-30. [8] http://science.howstuffworks.com/life/human-biology/kidney.htm Accessed on May 6, 2013. [9] Choy E, Sattar N. Interpreting lipid levels in the context of high-grade inflammatory states with a focus on rheumatoid arthritis: a challenge to conventional cardiovascular risk actions. Ann Rheum Dis.2009;68:460-469.

Table 1: Composition of Experimental Diets


Ingredients (g) Cornstarch Sucrose Cellulose Casein Corn Oil Dairy Butter Cholesterol Salt Mix Vitamin Mix Methionine Sodium cholate Choline chloride Placebo Watermelon Powder Control (Placebo) 12.30 33.00 5.00 20.00 5.00 16.00 3.00 3.50 1.00 0.30 0.50 0.40 0.20 0.00 100.20 Watermelon Powder 12.30 33.00 5.00 20.00 5.00 16.00 3.00 3.50 1.00 0.30 0.50 0.40 0.00 0.20 100.20

* 33% sugar, 21% fat by wt, 3% cholesterol by wt

Table 2: Groups Diets Treatments NO DSS DSS (3%) Control 10 10 Watermelon Powder 10 10

Table 3. Initial body weight, final body weight (before, during, and after DSS treatment, and weight gain (before, during, and after DSS treatment) for control and watermelon groups with and without DSS treatments *

Cont- no DSS Initial Body Weight (g) Final Body Weight, before DSS Treatment(g) Final Body Weight, right after DSS Treatment (g) Final Body Weight, last day (g) Weight gain, before DSS treatment (g) Weight gain, during DSS treatment (g) Weight gain, last day (g) 59.83 1.05 238.70 4.30

Cont- DSS 59.80 0.93 239.15 3.48

Watermelonno DSS 59.79 0.88 238.65 5.64

WatermelonDSS 59.80 0.84 239.93 3.69

254.80 5.08a

240.80 4.09b

254.80 5.83 a

238.64 3.80 b

258.56 5.34 a

242.41 4.19 b

258.43 5.97 a

245.41 4.49 b

179.12 3.69 195.22 4.17 a 198.98 4.48 a

179.35 3.03 181.00 3.48 b 182.61 3.61 b

178.86 5.19 195.01 5.40 a 198.64 5.56 a

180.13 3.41 178.84 3.31 b 185.61 4.22 b

* Data presented as means SE (standard error). Data in rows with different subscript letters are statistically different ( p<0.05).

Table 4. Food intake and water intake before, during, and after the DSS treatment for control and watermelon groups with and without DSS treatment *

Cont-no DSS Food Intake, before the DSS treatment (g) Food intake, during the DSS treatment (g) Food intake, after the DSS treatment (g) Water Intake, before the DSS treatment (g) Water Intake, during the DSS treatment (g) Water Intake, after the DSS treatment (g) 16.10 0.26 17.61 0.39a

Cont-DSS 16.04 0.28 14.21 0.36b

Watermelon-no DSS 16.46 0.42 17.67 0.42 a

WatermelonDSS 16.44 0.24 13.30 0.42 b

18.22 0.46

15.44 0.70

17.49 0.45

17.57 1.38

23.75 0.86 26.66 0.56 a 29.71 1.04 a

24.12 1.43 21.811.08 b 36.97 3.58 b

26.28 1.70 28.39 1.79 a 31.55 1.71 a

26.14 1.94 19.55 2.08 b 46.65 8.24 b

* Data presented as means SE (standard error). Data in rows with different subscript letters are statistically different ( p<0.05).

Table 5. Liver, spleen, kidney, and epididymal fat weights for control and watermelon groups with and without DSS treatment *

Cont- no DSS Liver Weight (g) Spleen Weight (g) Kidney Weight (g) Epididymal Fat Weight (g) 19.31 0.78 1.15 0.07 a 2.02 0.06a,b 1.89 0.13

Cont- DSS 17.82 0.46 1.13 0.04b 1.90 0.02 a 1.75 0.14

Watermelonno DSS 18.51 1.29 1.24 0.06 a 2.18 0.08 b 1.83 0.11

WatermelonDSS 17.30 0.52 1.03 0.05 b 2.02 0.06a,b 1.73 0.16

* Data presented as means SE (standard error). Data in rows with different subscript letters are statistically different ( p<0.05).

250

200

a a,b b a a,b b c
Control-no DSS Control-DSS Watermelon-no DSS Watermelon-DSS

Concentration mg/dl)

150

a a b b

100

50

a b a b

0 TG TC HDL LDL

Figure 1. Serum lipid concentrations of rats fed watermelon powder and given DSS treatment versus those without watermelon powder and DSS treatment. Data are presented as mean SE (standard error). TG: triglyceride; TC: total cholesterol; HDL: high density lipoprotein cholesterol; LDL: low density lipoprotein cholesterol. Bars with different superscripts differ significantly at P <0.05.

300 LDH (lactate dehydrogenase, U/L) 250 200 150 100 50 0 Control-no DSS Control-DSS Watermelon- no DSS Watermelon-DSS a a b b

Figure 2. Effects of watermelon and DSS treatment on LDH (lactate dehydrogenase). Bars represent meansSE. Bars with different superscripts differ significantly at P <0.05.
0.9 b a a a

Total Antioxidant Capacity (mM)

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Control-no DSS Control-DSS Watermelon -no DSS Watermelon-DSS

Figure 3. Effects of watermelon and DSS treatment on antioxidant capacity. Bars represent meansSE. Bars with different superscripts differ significantly at P <0.05.

250

b a

200 C-reactive protein (g/ml) a

150

c 100

50

0 Control-no DSS Control-DSS Watermelon-no DSS Watermelon-DSS

Figure 4. Effects of watermelon and DSS treatment on C-reactive protein levels. Bars represent meansSE. Bars with different superscripts differ significantly at P <0.05.