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EFFECTS OF WATERMELON POWDER ON RISK FACTORS OF CARDIOVASCULAR DISEASE AND INFLAMMATION IN ATHEROGENIC-DIET FED RATS

Running Head: Effects of watermelon powder on cardiovascular disease and inflammation of rats

Authors:

Katie LechnerNatalie Meltzer

School of Exercise and Nutritional Sciences, San Diego State University, San Diego, CA 92182

Corresponding Author:Mee Young Hong, PhDDepartment of Exercise and Nutritional SciencesSan Diego State University5500 Campanile DriveSan Diego, CA 92182-7251(619) 594 - [email protected]

AbstractObjective

To determine if watermelon powder improves the risk factors of cardiovascular disease and

inflammation in atherogenic diet-fed rats.

Methods

Forty male Sprague-Dawley rats (40-100 g) were equally divided into four groups consuming a

control diet with no inflammation inducing DSS (Dextran Sodium Sulfate), control diet with

DSS, watermelon powder diet with no DSS, and watermelon powder with DSS. Body weight,

food intake and water intake were measured. Designated rats were administered DSS via water

ninety six hours prior to conclusion of study. During the final forty eight hours, water was

restored. The blood, liver, spleen, kidneys and large intestine were collected to assess

triglycerides, serum total cholesterol, HDL-C, LDL-C, CRP, lactate dehydrogenase (LDH) and

total antioxidant levels.

Results

Initial BW, final BW, weight gain before treatment, and food intake before treatment were not

significant. Final BW after treatment, BW on the last day of study, BW gain during treatment,

grand total weight gain, water intake during and after treatment were significant (p<0.05). Spleen

and kidney weights were significant; triglyceride, total cholesterol, HDL-C, and LDL-C, LDH,

total antioxidant capacity, and CRP levels were significant as well (p<0.05). CK, glucose, liver

weight, and epididymal fat weight were not significant.

Conclusion

Watermelon powder reduces the risk of CVD by favorable changes of lipid profile, inflammation

and antioxidant status

Key Words: watermelon; lipid; atherogenic diet; antioxidant; DSS

Introduction

Cardiovascular disease is the leading cause of death for Americans. (1). One of the many

risk factors related to cardiovascular disease is a high fat diet (2). The effects of a high fat diet

include potential chronic diseases, including atherosclerosis (3). By incorporating antioxidants in

the diet, damaged cells can be repaired due to free radicals and may lessen the risk of

cardiovascular disease. The objective of this study was to determine if watermelon powder

improves the risk factors of cardiovascular disease and inflammation in atherogenic-diet fed rats.

Watermelon (Citrullus lanatus or C. lanatus) is a fruit high in lycopene and citrulline

content (2) and (4).In a previous human study, an increased intake of the antioxidant, lycopene,

suggestively amplified levels of antiatherogenic properties (5). In another previous mice study,

the effects of citrulline, a non-essential amino acid, was observed. Citrulline is responsible for

the release of nitric oxide in the body, and is known to improve endothelial dysfunction, reduce

aortic blood pressure and decrease lipid peroxidation in the liver (2). By incorporating

watermelon in the diet, lycopene and citrulline are consumed and will provide beneficial

physiological effects (2) and (4).

The purpose of our study was to directly observe the effects of watermelon powder

consumption within an atherogenic diet with and without DSS treatment, and also amongst an

atherogenic diet without watermelon powder within an atherogenic diet, and to observe its

effects on cardiovascular disease and inflammation. The hypothesis of this study was that

watermelon powder reduces the risk factors of cardiovascular disease and inflammation by

increasing antioxidant and favorable changes of lipid profile and inflammation markers.

Materials and Methods

Animals and diets

A total of forty male Sprague-Dawley rats at twenty-one days old were housed

individually in wire-bottomed cages on a 12-hour light-dark cycle in a research room at San

Diego State University. At the beginning of the experiment the rats were in their weaning stage

and weighed about forty grams. Both temperature and humidity were controlled at

approximately 20-24 degrees Celsius and 40-45% humidity. Water and food were available at all

times for all forty rats. The procedures and training for use of the animals were conducted and

approved by the San Diego State University animal subjects committee.

Rats were divided into four groups of ten, consuming two specific diets and two specific

treatments (Table 1). The diet consisted of twenty one percent (21%) fat, thirty-three percent

(33%) sugar and three percent (3%) cholesterol by weight in both the placebo (control), and

watermelon powder diet for a duration of four weeks.The watermelon diet consisted of 0.20 g of

watermelon powder as opposed to the placebo which consisted of maltodextrin used to match

kilocalorie composition of the watermelon powder. Dr. Figueroa of Florida State University

provided the sieved freeze dried watermelon solids obtained from Milne Fruit Products (Prosser,

WA). Fat sources in the diet consisted of such products as dairy butter and corn oil. After two to

three days of acclimation, the experimental diets were put into effect. Within the four weeks of

the experimental duration, prior to euthanasia, the rats in the treatment groups endured a forty

eight hour time period with DSS in their water supply. Immediately after the forty eight hours of

DSS treatment, a normal water supply was restored for the final forty eight hours of the study.

At the conclusion of the study, food and water was removed from the cages, and the

Sprague Dawley rats were euthanized. The blood was collected from the rat carcass with labeled

tests tubes, then allowed to clot at room temperature. The blood was centrifuged for 15 minutes

at 1200 x g at 2-8 degrees Celsius. The serum was stored at -70 degrees Celsius until time of

analysis. The animals were sprayed with an ethanol solution prior to dissection. The epididymal

fat pads, liver, spleen, and kidneys were all individually weighed by one specific student to avoid

error. The large intestine was scraped of feces, measured in length and recorded. A small portion

of the liver and large intestine were administered into two separate cassettes and then placed in

phosphate buffer saline (PBS) solution to maintain physiological integrity. Once each organ was

correctly weighed and recorded, the organs were collected to assess the lipid profile by collecting

serum total cholesterol, triglycerides, high-density lipoproteins (HDL), and serum low-density

lipoprotein (LDL). The serum glucose, C-reactive protein (CRP), lactate dehydrogenase (LDH),

creatine kinase (CK), and total antioxidant levels were also assessed using various kits.The

serum total cholesterol, triglycerides, serum glucose, LDH, CK, and HDL were assessed using a

kit from Stanbio (Boerne, TX). The LDL-cholesterol was calculated by subtracting HDL-

cholesterol from total cholesterol; then the total triglycerides divided by five was subtracted from

the previous number attained. LDH was analyzed by adding a 0.025 mL of serum to its

respective test tube containing a reconstituted agent and allowed to incubate at thirty seven

degrees Celsius for one minute. The sample was read on a spectrometer at 340 nm with distilled

water. The absorbance was recorded at a 60 second interval. Values were derived based on the

absorptivity micro-molar extinction coefficient of NADH.

The CRP was assessed using the ELISA kit from BD Biosciences (San Jose, CA). CRP is

accumulated in the blood during inflammation or tissue trauma. CRP was prepared by

performing 1:2 serial dilutions five times. The final working dilution of serum to wash buffer

was 1:4000. It was incubated at room temperature for 30 minutes. To each microwell, 100 mL of

the detection antibody/enzyme conjugate was added and incubated for 30 minutes at room

temperature. One hundred mL of TMB substrate solution was added to the microwells and

incubated for another five minutes. The reaction was stopped by 100 mL of stop solution. The

absorbance was read at 450 nm within 30 minutes of adding the stop solution. The CRP

concentration was determined by multiplying measured values by the dilution factor used

(1:4000). The ELISA kit utilized a specific antibody and the CRP presented in the rat serum

binded to that specific antibody.

The total antioxidant levels were assessed by the amount of oxidized ABTS in the serum

sample using a kit from Sigma (St. Louis, MO). An ABTS substrate solution was prepared by

adding 25 mL of 3% hydrogen peroxide to 10 mL of ABTS substrate solution. Assays were

prepared in a 96 well plate and 10 uL of trolox standard and 20 mL of myoglobin working

solution were added in wells for the trolox standard curve. One hundred fifty mL of ABTS

working solution were added to each well. The wells were incubated for five minutes at room

temperature until adding 100 ml of stop solution. The absorbance was read at 405 nm using a

plate reader within one hour of incubation time. The antioxidant assay shows the formation of a

ferryl myoglobin molecule, which is a free radical that oxidizes ABTS in the blood, which can be

read on the spectrometer.

Statistical analysis

All data was analyzed to evaluate the effects of the diets on cardiovascular disease and

inflammation using the ANOVA procedure using SPSS (IBM, Armonk, New York) to evaluate

the effects of diets and treatment on weight, food intake, water intake, lipid profiles, CRP, LDH,

and total antioxidant. Data will be presented Mean±SE. An alpha level of p<0.05 will be

considered significant.

Results

The initial and final body weight before DSS treatment among the four groups was not

significant, as evidenced by similar mean body weights (Table 2). Lower mean body weights

were recorded in DSS treated animals in their final body weights (p=0.003). Decreased final

body weight prior to euthanasia showed the DSS treatment was significant (p=0.007). Weight

gain before DSS treatment was similar throughout the four groups, although weight gain during

treatment (p=0.001) and weight gain in grand total (p=0.003) were significantly different. The

DSS treatment lowered mean weight gain in the Sprague-Dawley rats. Watermelon powder did

not show to reduce final body weight.

Food intake before and after treatment did not alter between groups (Table 3). Food

intake was lowered during DSS treatment and was determined significant (p<0.001). After the

treatment, the animals returned to their previous food pattern and consumed the same amount as

the nontreated groups. Water intake before DSS treatment was insignificant because all four

groups consumed a similar amount of water. Water intake during DSS treatment decreased

(p<0.001) and increased after treatment (p=0.020), (Table 3). Decreased water intake during

treatment may have been from the ability of the animals to taste the DSS solution in their water

supply, therefore causing them to drink less fluid.

Liver and epididymal fat weight were not significant due to similar mean values between

groups. The spleen weights in the DSS treated rats were lighter than the non DSS treated rats and

determined significant (p=0.049), (table 4). The kidney weights among the watermelon diet

groups were heavier (p=0.024) and the DSS treated groups indicated lower kidney weights

(p=0.019).

While glucose levels were insignificant among the groups, triglyceride levels were

decreased and showed significance in the watermelon diet (p=0.031), (Table 5). According to the

data, watermelon reduces total triglyceride levels. Total cholesterol was significant as indicated

by decreased levels in the watermelon diet (p<0.001) as well as in the DSS treatment, as

indicated by increased levels (p=0.011). Watermelon proved to reduce total cholesterol while

inflammation continued to increase it. DSS treatment reduced HDL-cholesterol and was

statistically significant (p=0.041). LDL-cholesterol was significantly lowered in the watermelon

diet (p<0.001) yet was raised in the DSS treatment (p=0.008), (Figure 1). Watermelon decreased

LDL-cholesterol and had no effect on HDL-cholesterol, but inflammation increased LDL-

cholesterol while also lowering HDL-cholesterol (Figure 1).

There was a significant decrease in LDH levels in the watermelon diet (p<0.001), (Table

5). The purpose of LDH is to show cardiac tissue breakdown and can also be an indicator of

myocardial infarction. Therefore, lower values helped to support the potential of watermelon to

decrease risk factors of cardiovascular disease. CK proved to be insignificant in either of the four

groups. The watermelon diet improved total antioxidant capacity (p=0.049), (Figure 2). The DSS

treated groups had lowered total antioxidant capacity which showed inflammation lowers total

capacity (p=0.015), (Figure 2). CRP levels were significant; CRP levels were lowered in the

watermelon diet (p<0.001) and raised in the DSS treated groups (p=0.001), (Table 5). CRP is

most commonly used to screen for inflammatory diseases. Thus, proved that inflammation

increased the number of free radicals in the animals. Also, these values indicated that watermelon

powder decreases inflammation.

Discussion

Observational studies have discovered a link between an increased intake of dietary

antioxidants and a reduction of cardiovascular morbidity (5). For instance, antioxidant and anti-

inflammatory effects due to lycopene, a component of watermelon, have aided in heart protective

properties (4). Thus, the benefits of incorporating watermelon into the diet is evident based on

the data collected during this study.This is relevant because we know lycopene is an abundant

antioxidant present in watermelon. Values from this study prove that watermelon powder

incorporated into the diet increased the total antioxidant capacity.

Citrulline, a component of watermelon, is responsible for the release of Nitric oxide in

the body (2). Nitric oxide has shown to regulate mammalian metabolism of energy substrates (3).

Physiological levels of this signaling molecule stimulate uptake and oxidation of glucose and

fatty acids by skeletal muscle, heart, liver, and adipose tissue (3) and (6). Nitric oxide inhibits the

synthesis of glucose, glycogen, and fat within the insulin sensitive tissues, and enhance lipolysis

in white adipocytes (3). It is crucial for proper body functions and benefits blood circulation, the

immune system, and intra-cellular communication (3). Our data shows significantly lowered

CRP levels, proving watermelon has positive effects, such as decreased inflammation and a

lowered risk of cardiovascular disease. The enhancement of nitric oxide, due to the incorporation

of watermelon, has shown to reduce blood pressure by vasodilation and reduce the risk of

atherosclerosis.

A similar study was conducted in LDL receptor-deficient mice (2). The study conducted

by Poduri et al provided one group of mice with a C. lanatus extract on hypercholesterolemia-

induced atherosclerosis (2). The mice consuming the C. lanatus extract had significantly

increased plasma citrulline concentrations, when compared to their control group (2). Other

results in Poduri et al study included lowered body weight, lowered fat mass, decreased plasma

cholesterol concentrations, and attenuated development of atherosclerosis when C. lanatus was

incorporated into the diet (2). The results of this study show significant decreases in harmful

lipid profile levels within the watermelon diet. Data supported a decrease in total cholesterol,

triglycerides and LDL-cholesterol. Therefore, watermelon powder helped to decrease the risk

factors of cardiovascular disease.

The results of Poduri et al study concluded evidence similarly to the study conducted at

San Diego State University. Similar results included an increased total antioxidant capacity,

which mirrored the increased plasma citrulline concentrations. A difference amongst the studies,

however, included an effect in the HDL-cholesterol based upon the DSS treatment, which was

not relevant to the Poduri et al study. The data in this study proved that watermelon powder did

not have an effect on HDL-cholesterol. Another major difference between the Poduri et al and

this study included the lowered body weight and lowered fat mass. This study did not find

significant data to base effects of watermelon or DSS treatment on fat mass and body weight.

In summary, this study demonstrated that watermelon does reduce, to some extent, the

risk factors of cardiovascular disease and inflammation by increasing antioxidant capacity and

favorable changes in lipid profiles. These effects might be attributed to the antioxidant lycopene

or amino acid citrulline properties present in watermelon. The improved lipid profile and

antioxidant capacity proved watermelon to be beneficial. This may suggest that watermelon does

in fact improve the risk factors of cardiovascular disease and inflammation in an atherogenic-fed

diet. Further research should be conducted to learn effects of the incorporation of watermelon in

the diet, as well as fat mass and body weight. Limitations of this study may be reflected by the

short duration of a four week period. Future studies should conduct this experiment over a longer

period of time to determine potential long term physiological effects of watermelon.

In conclusion, watermelon powder appeared to have a beneficial effect on favorable

changes in lipid profile and antioxidant capacity, suggesting that watermelon may contribute to

decreasing one’s risk for developing cardiovascular disease or inflammatory diseases.

Acknowledgments:

The authors wish to acknowledge the contributions of the Spring 2013 Advanced Nutrition

Laboratory students and professor at San Diego State University who assisted in conducting and

evaluating this research.

References

1. Center for Disease Control and Prevention. Available at: http://www.cdc.gov/nchs/fastats/lcod.htm. Published: January 11, 2013. Accessed April 29, 2013.

2. Poduri A, Rateri DL, Saha SK, Saha S, Daugherty A. Citrullus lanatus 'sentinel' (watermelon) extract reduces atherosclerosis in LDL receptor-deficient mice. J Nutr Biochem. 2013;24(5):882-886.

3. Dai Z, Wu Z, Yang Y, Wang J, Satterfield MC, Meininger CJ, Bazer FW, Wu G. Nitric oxide and energy metabolism in mammals. biofactors. 2013;10(1):1002-1099

4. McEneny J, Wade L, Young IS, Masson L, Duthie G, McGinty A, McMaster C, Thies F.Lycopene intervention reduces inflammation and improves HDL functionality in moderately overweight middle-aged individuals. J Nutr Biochem. 2013;24(1):163-168

5. Bohm V. Lycopene and heart health. Mol Nutr Food Res. 2012;56(2):296-303

6. Jabecka A, Ast J, Bogdaski P, Drozdowski M, Pawlak-Lemaska K, Cielewicz AR, Pupek-Musialik D. Oral L-arginine supplementation in patients with mild arterial hypertension and its effect on plasma level of asymmetric dimethylarginine, L-citruline, L-arginine and antioxidant status. Eur Rev Med Pharmocol Sci. 2012;16(12):1665-1674

Figure legend

Figure 1: Lipid profile concentrations of rats during atherogenic-diet with and without

watermelon powder and during atherogenic-diet with and without DSS treatment. TG:

triglyceride; TC: total cholesterol; HDL: high density lipoprotein cholesterol; LDL: low density

lipoprotein cholesterol

Figure 2: Effects of watermelon on total antioxidant capacity (mM). Bars represent means±SE.

Bars with different superscripts differ significantly at P <0.05

Table 1. Composition of diet mixture

Ingredient (g) Control (placebo) Watermelon Powder

Cornstarch 12.30 g 12.30 g

Sucrose 33.00 g 33. 00 g

Cellulose 5.00 g 5.00 g

Casein 20.00 g 20.00 g

Corn oil 5.00 g 5.00 g

Dairy Butter 16.00 g 16.00 g

Cholesterol 3.00 g 3.00 g

Salt mix, AIN-93G 3.50 g 3.50 g

Vitamin mix, AIN-93G 1.00 g 1.00 g

Methionine 0.30 g 0.30 g

Sodium cholate 0.50 g 0.50 g

Choline chloride 0.40 g 0.40 g

Placebo 0.20 g 0.00 g

Watermelon powder 0.00 g 0.20 g

Total 100.20 g 100.20 g

● 33% sugar, 21% fat by weight, 3% cholesterol by weight

Table 2. Initial body weights, final body weights and weight gain

Cont-noDSS Cont-DSS Watermelon-noDSS

Watermelon-DSS

Initial Body Weight (g)

59.83 ± 1.05 59.80 ± 0.93 59.79 ± 0.88 59.80 ± 0.84

Final Body Weight (g)

254.80± 5.08a 240.80± 4.09b 254.80± 5.83a 238.64± 11.39b

Weight gain(g, before DSS treatment)

179.12± 3.69 179.35± 3.03 178.86± 5.19 180.13± 3.41

Weight gain (g, 195.22± 41.7a 181.00± 3.48b 195.01± 5.40a 178.84± 3.31b

during treatment)

Weight gain(g, grand)

198.98± 4.48a 182.61± 3.61b 198.64± 5.56a 185.61± 4.22b

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

Table 3. Food intake and water intake


Watermelon-DSS

Food intake - before treatment (g/d)

16.10± 0.26 16.04± 0.28 16.46± 0.42 16.44± 0.24

Food intake - during treatment (g/d)

17.61± 0.36a 14.21± 0.36b 17.67± 0.42a 13.30± 0.42b

Food intake - after treatment (g/d)

18.22± 0.46 15.44± 0.70 17.49± 0.45 17.57± 1.38

Water intake - before treatment (g/d)

23.75± 0.86 24.12± 1.43 26.28± 1.70 26.14± 1.94

Water intake - during treatment (g/d)

26.66± 0.56a 21.81± 1.08b 28.39± 1.79a 19.55± 2.08b

Water intake - after treatment (g/d)

29.71± 1.04a 36.97± 3.58b 31.55± 1.71a 46.65± 8.24b


Table 4. Liver, spleen, epididymal fat and kidney weight


Watermelon-DSS

Liver weight (g) 19.31± 0.78 17.82± 0.46 18.51± 1.29 17.30± 0.52

Spleen weight (g)

1.15± 0.07a 1.13± 0.04b 1.24± 0.06a 1.03± 0.05b

Epididymal fat weight (g)

1.89± 0.13 1.75± 0.14 1.83± 0.11 1.73± 0.16

Kidney weight (g)

2.02± 0.06a,b 1.90± 0.02a 2.18± 0.08a,b 2.02± 0.06


Table 5. Glucose, LDH, CK, and CRP concentration


Watermelon-DSS

Glucose (mg/dl) 113.59± 18.62 138.17± 8.48 121.55± 7.55 133.25± 11.50

LDH (mg/dl) 235.86± 8.25a 215.89± 14.09a 183.64± 15.36b 154.16± 15.32b

CK (U/L) 465.04± 66.60 438.98± 19.47 438.54± 53.96 455.76± 50.26

CRP (ug/mL) 132.36± 11.05a 217.46± 13.87b 87.86± 9.91c 169.02± 18.65a


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