Introduction

Dual burden of malnutrition exists in the Indian society. According to NFHS 3 (2005-2006) diabetes is highly prevalent in urban areas (1.374% women and 1.383% men) than in rural areas (0.641% women and 0.86% men). World Health Report 2002 speculated that cardiovascular diseases (CVDs) will be the largest cause of death and disability by 2020 in India. On the other hand the magnitude of the problem found to be even more serious while giving a glimpse on national nutrition data. According to NFHS 3 (2005-2006) 40.4% children of below 3 years were underweight. Among the adults 36% of women and 34% of men are undernourished, with a BMI less than 18.5. This indicates an alarming situation of nutritional deficiency. In this present scenario recently more attention has been focused to recover valuable components from neglected food parts (food “losses”, “wastes”, “by-products”) and reintroduce them inside the food chain. Watermelon (Citrullus vulgaris) seed, whose cultivar name is Sugar baby is one of such component.

Sample Description

Watermelon seeds are easily available during summer season. Though it is not an oilseed but researches have shown that these seeds contain about 52.6% oil,¹ so these are good source of energy (628k.cal). C. Gopalan has listed watermelon seeds under nuts and oilseeds. C. vulgaris seed kernel contains highest amount of protein (34.1g/100g) compared to that of any other oilseeds. It also contains considerable amount of Arginine (900mg/g of N) and branched chain amino acids (leucine, isoleucine and valine).¹

Inspite of being highly nutritious and having some known medicinal values, these seeds have still not recognized and are less acceptable amongst the people as compared to other oilseeds. Previous work showed that watermelon seed kernel promotes growth due to the presence of good quality protein which was evidenced by significantly increased Protein Efficiency Ratio (PER) among the male albino rats, and therefore may be useful in prevention and treatment of Protein Energy Malnutrition (PEM).² The nutritional importance of the seed kernels of C. vulgaris have been found in some discrete studies, but little work has been done on the effect of the seeds of C. vulgaris on different bodily systems of animals or humans. Hence the objectives of the present research are to:

• prepare a modified diet of male albino rats by adding watermelon seed kernel to the stock diet instead of groundnut oil so that its protein, fat, carbohydrate and calorie content would be same as that of the stock diet.
• study the effects of vulgaris seed kernels on serum glucose and liver glycogen of male albino rats.
• study the effects of C. vulgaris seed kernels on serum lipid profile of male albino rats.

Materials and Methods

Collection of seeds and preparation of sample

Watermelons of specific species (C. vulgaris) were purchased during summer season (April to June) from local markets of Chetla and Rashbehari, Kolkata. The watermelons were cut with a clean, sharp knife, and the seeds were handpicked from them, washed thoroughly with tap water and then dried in shade at room temperature. Then the seeds were screened to remove the bad and immature ones. Finally the seeds were dehusked in a decorticator and the seed kernels were separated from the seed coat. The seed kernels were grounded in a mixer grinder and preserved in a dry, clean container for preparing the experimental diet.

Animal experiment

Eight male albino rats weighing between 105-150g were used for the experiment with due approval of the Institutional Animal Ethical Committee (IAEC). They were assigned into 2 groups, (n = 4), labeled as control and treated group. The control group received normal stock diet (20% protein, 5 % fat, 60 % carbohydrate).³ The experimental group received a modified diet (90g Stock diet excluding groundnut oil + 9g watermelon seed kernel + 1g sugar) and the percentages of protein, fat and carbohydrate were kept almost same as that of the stock diet. All the animals were given water ad libitum. The detail composition of the stock and the modified diet were as follows-

Table 1: Composition and Nutritive value of Stock diet³

Iodised salt- 0.5g/100g,

Vyceneral Vitamin syrup – 0.2ml/100g.

Table 2: Composition and Nutritive value of modified diet

Iodised salt- 0.5g/100g,

Vyceneral Vitamin syrup – 0.2ml/100g

The total experimental period was 28 days during which the rats were weighed at weekly intervals. After 28 days of feeding with the modified diet, the animals were anesthetized using chloroform anesthesia following ethical procedures. Blood samples were then collected via cardiac puncture and serum was separated for the analysis of glucose and lipid profile. Serum glucose, liver glycogen and lipid profile were analyzed by the following methods-

Serum glucose by GOD – POD Method

Serum glucose level of all the rats were assessed by Glucose-Oxidase Peroxidase kit in protein free supernatant^4,5 provided by Sigma Aldrich Corporation, USA.

Liver glycogen was estimated by the standard method described in the book named “A Manual of Laboratory Techniques” of ICMR³

The liver was taken out rapidly from the animal & the excess blood was removed by blotting between folds of filter paper. Immediately 1g of liver was cut and weighed in digital balance and put into test tubes containing 2ml of 30% KOH with a tissue concentration 2ml/g of liver. After that the tissue of different rats were digested in a boiling water bath for 1hour and then cooled in ice cold water. 4 ml of 95% ethanol were then added & the mixture was heated just to boiling, taking care so that no spurting occurs during the process. It was then left to stand for overnight in the refrigerator. Next day the tubes were centrifuged and the supernatant was discarded. The precipitate was dissolved in 10 ml of warm water. 2 volumes of 95% ethanol were added to reprecipitate the glycogen. 60% ethanol was used for several washings of the centrifuged precipitate. 2 ml of 2N H₂SO₄ was added to it and was placed in boiling water bath for 3-4 hours for hydrolysis. Neutralization was done by adding NaOH. The solution was filtered and Glucose was estimated from the aliquot. The conversion factor 0.93 was used to obtain the value of glycogen from glucose.

Total cholesterol and HDL-C by CHOD-POD / Phosphotungstate method ^[6]

For Total Cholesterol

Free Cholesterol and Fatty acids are obtained by hydrolysis of Cholesterol esters using Cholesterol Esterase (CE). In the next step, Cholest-4-en-3-one and Hydrogen Peroxide are liberated through the oxidation of the 3-OH group of free Cholesterol by Cholesterol Oxidase (CHOD). Red Quinoneimine dye is produced by the coupling of Hydrogen Peroxide with 4-Aminoantipyrine (4-AAP) and Phenol in presence of Peroxidase (POD). The absorbance of the produced coloured dye is measured at 505nm in the spectrophotometer.

For Hdl-Cholesterol

Polyethylene glycol 6000 (PEG) is added to precipitate the LDL, VLDL and chylomicron fractions. After centrifugation, the supernatant is used for HDL estimation by CHOD-PAP method.

Serum triglyceride (TG) by GPO-POD method with TBHA chromogen⁷

Glycerol and free fatty acids are obtained by the catabolism of serum triglycerides with the help of the enzyme Lipoprotein lipase. Liberated Glycerol is converted to Glycerol – 3 phosphate in presence of glycerol kinase and ATP. Hydrogen peroxide is formed by the action of Glycerol– 3 phosphate oxidase on Glycerol – 3 phosphate. This together with phenolic compound TBHA and 4 Aminoantipyrine in presence of peroxidase gives the red colour complex. The intensity of the colour is measured at 505 nm (500 – 546 nm) in the spectrophotometer.

• VLDL = TG/5 mg/dl.⁸
• LDL = Total cholesterol – (HDL + VLDL).⁸
• Atherogenic Index (AI) = LDL-C/HDL-C.⁸

Statistical analysis

Demographic characteristics of Control and Treated groups were similar. All statistical tests were two-tailed and were conducted at 5% significance level. For comparison of means Student’s t-test was applied. Statistical analysis was done by using SPSS software version 16.

Results and Discussions

Table 3: Effect of the modified diet on the diabetes detecting parameters of male albino rats

n = number of animals in each group. df = 6, NS = Not-significant, ** = p < 0.01.

Table 4: Effect of the modified diet on the lipid profile parameters of male albino rats

n = number of animals in each group. df = 6, NS = Not-significant, * = p < 0.05.

Serum glucose level decreased significantly (p < 0.01) among the treated group of animals than that of the control group but there was no significant difference in the liver glycogen content of the two groups of rats (Table-3). Type of fat intake, may be linked to insulin resistance, which can increase risk of developing type 2 diabetes and impaired postprandial lipid metabolism.^9,10 Previous studies showed that C. vulgaris seed kernel contained branched chain amino acids (BCAAs) i.e.  leucine (400mg/g of N), isoleucine (310mg/g of N) and valine (260mg/g of N)¹ and 16.1 ± 0.88mg Vitamin E¹¹ per 100g of seed kernels. There are reports that glucose metabolism can be modulated by BCAAs. In skeletal muscle of non-diabetic rats glucose is augmented by BCAAs through enhancement of translocation of glucose transporter 4 to the plasma membrane via phosphatidylinositol 3-kinase and protein kinase C pathways. The same study additionally reported that mammalian target rapamycin (mTOR) in the hepatocytes are upregulated by BCAAs. During long term insulin treatment mTOR in the skeletal muscles degrades insulin receptor substrates by insulin-mediated PI3K activity.¹² It has also been showed that the post-prandial blood sugar levels were decreased in the vitamin E supplemented patients of the type I DM patients over 24 months and also in the type II DM patients. This decline was statistically significant only at the end of 24 months.¹³ Therefore, presence of substantial amounts of BCAAs and Vitamin E are probably the causative agents of hypoglycemic function of this seed.

The results obtained from this study reveal that after consuming the modified diet containing watermelon seed, serum triglyceride (TG) and VLDL-C of the treated group were significantly decreased (p < 0.05 and p < 0.05 respectively) in comparison to that of the control group. Serum total cholesterol, LDL and AI (Atherogenic Index) were decreased whereas HDL (good cholesterol) increased in the treated group compared to that of the control group (Table-4). Arginine (900mg/ g of N) present in watermelon seeds reported to have positive effect on serum lipid profile parameters. Endothelial nitric oxide synthase (eNOS) helps in the production of Nitric Oxide (NO) from l-arginine. NO plays an important role in regulation of endothelium-dependent vasodilatation.¹⁴ 59.64% of Linoleic acid is present in watermelon seed kernels¹ which can be metabolized into to gamma-linolenic acid (GLA) in the body. According to some preliminary evidence GLA, either alone or in combination with omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may help to reduce high blood pressure.¹⁵ In mammalian metabolism Arachidonic acid (AA) is formed from Linoleic acid (LA) by subsequent Δ⁶ desaturation, chain elongation and Δ⁵ desaturation. In this metabolic pathway Δ⁶ desaturation step is rate-limiting and the step can be by-passed by dietary  -linolenic acid (GLA). Most of the GLA is elongated to dihomo- -linolenic acid (DGLA) by the action of elongase and a small amount of GLA is desaturated by Δ⁵ desaturase into AA. The DGLA can enter into cycloxygenase pathway to form prostaglandins of the 1-series (PGE1) additionally or alternately it can enter into 15-lipoxygenase pathway to produce eicosatrienoic acid (15-HETrE). The PGE1 and 15HETrE have been proved to have suppressive effects on chronic inflammation, hypotensive effect through vasodilation and anti-atherosclerotic effect by retarding the vascular smooth muscle cell proliferation.¹⁶ So, the presence of high amount of linoleic acid in watermelon seed kernels may be one of the possible reasons of improving lipid profile parameters.

Conclusion

Worldwide studies have been done to make use of herbal medicine. The proposed research work showed that consumption of watermelon seed kernel would improve lipid profile parameters, therefore may be useful in prevention and treatment of cardiovascular diseases. Presence of BCAAs and Vitamin-E in watermelon seed kernels are probably responsible for its hypoglycemic property. Thus, watermelon seed kernel may be used for the treatment of various ailments without any undesirable side effect, especially in those countries in which more classical therapeutic approach is not easily available. Still there is an ample scope of research to expose hitherto unknown bioactive ingredients and their mechanism of action responsible for the positive health benefits of these seeds.

Acknowledgements

The authors acknowledge financial help from the University Grants Commission (UGC) of India.

References

1. Gopalan C., Ramasastri B.V. and Balasubramaniam S.C. “Nutritive value of indian foods”. Hyderabad: National Institute of Nutrition. 1971.
2. Biswas R., Chattopadhyay A. and Datta (De) S. Effect of watermelon (Citrullus vulgaris) seed kernels on serum lipid profile and body weight of male albino rats. IOSR Journal of Environmental Science, Toxicology and Food Technology. 2015;9(10):13-16.
3. Raghuramulu N., Madhuvan Nair K., Kalyan Sundaram S. “A manual of laboratory techniques”. Hyderabad: National Institute of Nutrition. 1983.
4. Huggett A.S., Nixon D.A. Use of giucose oxidase, peroxidase and O-dianisidine in determination of blood and urinary glucose. Lancet. 1957;273(6991):368-370.
   CrossRef
5. John B.H., Gerald K. An automated determination of glucose utilizing a glucose oxidase-peroxidase system. Journal of Laboratory and Clinical Medicine. 1961;57:970-980.
6. Allain C.C., Poon L.S., Chan C.S., Richmond W., Fu P.C. Enzymatic determination of total serum cholesterol. Clinical Chemistry. 1974;20(4):470-475.
7. Jacobs N.J., Vandemark P.J. The purification and properties of the alpha-glycerophosphate-oxidizing enzyme of Streptococcus faecalis 10C1. Archives of Biochemistry and Biophysics. 1960;88: 250-255.
   CrossRef
8. Nku C. O., Ikpi, D.E., Nna, V.U., Agiande, G.Altered serum lipid profile in albino wistar rats following the consumption of Cola nitida rubra (kola nut). Australian Journal of Basic and Applied Sciences. 2014;8(13):82-89.
9. Hu F.B., van Dam R.M., Liu S. Diet and risk of type II diabetes: the role of types of fat and carbohydrate. Diabetologia. 2001;44(7):805–817.
   CrossRef
10. Thomsen C., Rasmussen O., Lousen T., Holst J.J., Fenselau S., Schrezenmeir J., Hermansen K. Differential effects of saturated and monounsaturated fatty acids on postprandial lipemia and incretin responses in healthy subjects. The American Journal of Clinical Nutrition. 1999;69(6):1135–1143.
11. Ejoh S.I., Ketiku O.A., Vitamin E content of traditionally processed products of two commonly consumed oilseeds – groundnut (Arachis Hypogea) and melon seed (Citullus Vulgaris) in Nigeria. Nutrition and Food Sciences. 2013;3(2):187.
12. Rajendram, Rajkumar, Victor R. Preedy, and Vinood B. Patel. Branched chain amino acids in clinical nutrition. Springer. 2015;1.
13. Jain B., and  Jain V. A. Vitamin E, its beneficial role in diabetes mellitus (DM) and its complications. Journal of Clinical & Diagnostic Research. 2012;6:1624–1628.
    CrossRef
14. Ignarro L.J, Napoli C. Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis. Current Atherosclerosis Reports. 2004;6(4):281–287.
    CrossRef
15. Frenoux J.M.R., Prost E. D., Belleville J. L. and Prost J. L. A polyunsaturated fatty acid diet lowers blood pressure and improves antioxidant status in spontaneously hypertensive rats. The Journal of Nutrition. 2001;131(1):39-45.
16. Xiaoping Wang, Huanping Lin, and Yan Gu. Multiple roles of dihomo-γ-linolenic acid against proliferation diseases. Lipids in health and disease. 2012;11(25):1-9.
    CrossRef