Original Article

INTERNATIONAL JOURNAL OF
WOMEN'S HEALTH AND REPRODUCTION SCIENCES
http://www.ijwhr.net
doi: 10.15296/ijwhr.2013.16

Effects of Watermelon Seed Extract (Citrullus Vulgaris) on

Spermatogenesis in Rat
Arash Khaki1*, Fatemeh Fathiazad2, Mohammad Nouri1

Abstract:
Article History: Objective: Citrullus vulgaris is an antioxidant that has been
Received July 2013 shown to reduce oxidative stress. Worldwide studies have been
Accepted September 2013 done in order to use as herbal medicine in different fields of
Available online September 2013 medicine. Based on ancient Persians traditional books use of
herbal medicine has positive effect on treatment of different
Keywords: diseases. Previous studies confirmed antioxidants have
Citrullus vulgaris, significant effect on infertility by their role on reactive oxygen
Rat species. The aim of this study was to see effect of this herb on
Sperm spermatogenesis.
Material and Methods: Wistar male rats (n=20) were allocated
Corresponding Author: into two groups, control group (n=10) and citrullus vulgaris
Arash Khaki, Women’s (C.V) group (n=10) that received 55mg.kg-1.day-1 C.V by
Reproductive Health Research gavage method for 4 weeks; however, the control group just
Center, Tabriz University of received an equal volume of distilled water daily. Animals were
Medical Sciences, Tabriz, Iran. kept in standard condition. In 28th day, testicle tissues were
Tel: +989143138399 collected then prepared for sperm analysis.
Email: arashkhaki@yahoo.com Results: sperm parameters significantly increased in
experimental group in comparison to control group (P<0.05).
Conclusion: Since in our study 55mg.kg-1 (C.V) has significantly
increased sperm population, motility and viability,it seems that
using it in infertile patients has beneficial effects.

1- Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
2- Department of Pharmacognosy, Tabriz University of medical sciences, Tabriz, Iran

Int J Women’s Health Reproduction Sci Vol. 1, No. 3, Autumn 2013 ISSN 2330-4456
 A Khaki et al 100

Introduction: effect of Citrullus vulgaris seeds as an

Phytotherapy has recently gained popularity antioxidant on sperm parameters.
in Europe and the United States. The Material & Methods:
watermelon fruit has deep green or yellow Animals
colored smooth thick exterior rind with gray Twenty adult wistar albino male rats which
or light green vertical stripes. Inside the were 8 weeks old and weighing 250±10 g,
fruit is pink, red or even yellow in color with were obtained from animal facility of
small black seeds embedded in the middle pasture institute of Iran. Male rats were
third of the flesh. Generally, watermelon housed in temperature controlled rooms
flesh is the main consumable portion; (25C) with constant humidity (40-70%)
however, outer rind is also used in some and 12h/12h light/ dark cycle before using
parts of the world (1, 2). Watermelon with in experimental protocols. All animals were
red flesh is a significant source of lycopene. treated in accordance with the Principles of
Preliminary researches indicate that the Laboratory Animal Care. The experimental
consumption of watermelon may have protocol was approved by the Animal Ethical
antihypertensive effects (3). water . Committee in accordance with the guide for
Watermelon like many other fruits is a the care and use of laboratory animals
source of vitamin C. The amino-acid prepared by Tabriz medical University. All
citrulline was first extracted from Rats were fed a standard diet and water. The
watermelon and analyzed. Watermelon daily intake of animal water was monitored
contains a significant amount of citrulline at least one week before starting treatment
and after consumption of several kilograms, in order to determine the amount of water
an elevated concentration is measured in the needed for each experimental animal.
blood plasma; this could be mistaken for Thereafter, the wistar male rats (n=20)
citrullinaemia or other urea cycle disorders were randomly allocated into two groups,
(4, 5). In human nutrition, watermelon control group (n=10) and experimental
contributes to the integrity of the blood group (n=10) that received 55mg.kg-1.day-
vessels. Various preliminary studies reveal 1 Citrullus vulgaris by gavage method for 4
novel pharmaceutical properties of it. weeks; however, the control group just
Flavonoids are products of plant metabolism received an equal volume of distilled water
and have different phenolic structures (6, 7). daily.
Enhanced oxidative stress and changes in Preparation of extract:
antioxidant capacity are considered to play Citrullus vulgaris seeds were bought from
an important role in the pathogenesis of local market in Tabriz City of Iran. Seeds
chronic diabetes mellitus (8, 9). Although were washed and dried in room
the mechanisms underlying the alterations temperature. This hydro alcoholic extract
associated with diabetes mellitus are was kept in refrigerator for all experiments.
presently not well understood, 2.3. Surgical procedure
hyperglycemia leads patients to increased In 28th day, the Pentobarbital sodium (40
oxidative stress because the production of mg/kg) was administered intra peritoneal
several reducing sugars (through glycolysis for anesthesia, and the peritoneal cavity was
and the polyol pathway) is enhanced. These opened through a lower transverse
reducing sugars can easily react with lipids abdominal incision. Thereafter, epididymis
and proteins (nonenzymatic glycation in control and experimental groups were
reaction) and increase the production of immediately removed. The weights of
reactive oxygen species (ROS) (10, 11, 12). epididymis in both groups were registered.
The balance of ROS and antioxidant is a The animals were decapitated between 9:00
major mechanism in preventing damage by AM and 11:00 AM.
oxidative stress. Therefore, the dietary 2.4. Epididymis weight, Sperm count,
supplement of antioxidants such as vitamins, viability and motility
flavonoids has been used to prevent the Sperms from the cauda epididymis were
occurrence of many chronic diseases (13, released by cutting into 2 ml of medium
14). Therefore, we decided to study the (Hams F10) containing 0.5% bovine serum

Int J Women’s Health Reproduction Sci Vol. 1, No. 3, Autumn 2013 ISSN 2330-4456
 A Khaki et al 101

albumin (15).After 5 min incubation at 37°C with the oxidation process by reacting with
(with 5% CO2), the cauda epididymis sperm free radicals, chelating catalytic metals,
reserves were determined using the acting as oxygen scavengers (20, 21) and
standard hemocytometric method and preventing lipid auto oxidation (22, 23).
sperm motility was analyzed with Worldwide studies have been done in order
microscope (Olympus IX70) at 10 field and to use of herbal medicine in different fields
reported as mean of motile sperm according of medicine. Based on ancient Persians
to WHO method (16). traditional books use of herbal medicine has
Statistical analysis positive effect on treatment of different
Statistical analysis was done using the diseases especially infertility. Several
independent sample T-test for comparison studies have reported that antioxidants and
of data in the control group with the vitamin A, B, C, and E in diet can protect
experimental groups. The results were sperm DNA from free radicals and increase
expressed as Mean ± S.E.M (standard error blood-testis barrier stability (24, 25).
of means). P-value less than 0.05 were Evidences suggest that certain
considered significant and are written in the phytochemicals found in citrus sources, such
parentheses. as flavonoids and limonoids, have major role
Results : in treating or retarding chronic diseases.
Weight of Epididymis: Positive properties of these phytochemicals
The obtained results in this study were include anti-oxidative, anti-carcinogenic,
illustrated in table 1. There was no cardiovascular protective, neuro-protective,
significant difference in epididymis weights bone health promotion and anti-
between two groups. inflammatory protection. Antioxidants
Sperm motility, viability and count : protect DNA and other important molecules
Administration of 55 mg/kg Citrullus from oxidation and can improve sperm
vulgaris extract for 28 consecutive days quality and consequently increase fertility
significantly increased Sperm rate in men (26). Therefore, it is suggested
concentrations, motility and viability in that, increased use of herbal medicine, fruit,
experimental group as compared with the vegetables, onion, tea and black burgundy
control group (P<0.05) (Table 1). grape which are full of flavonoids and
citrullus vulgaris seeds such as herbal
Discussion: antioxidant can improve sperm parameters
In this study, male rats were subdivided into
and increase chances in infertile couple to
two groups, control and experimental group.
get new life.
Experimental group receiving watermelon
seed extract (citrullus vulgaris) showed Conclusion:
increased sperm motility, viability and This study shows increase in sperm viability,
concentration in comparison to control motility and population so it seems it as
group. This increase could be due to the positive effects on male infertility.
protective effect of citrullus vulgaris seed Acknowledgments:
extract administration. Phytonutrients with This research was supported by Women’s
significant role in human health and disease Reproductive Health Research Center,
prevention decline incidences of several Tabriz University of Medical Sciences,
physiological threats (17). These Tabriz, Iran. The code of this research
phytonutrients have anti-oxidative activity protocol was 9018.
due to having phenolics and carotenoid
compounds (18). Watermelon has
numerous antioxidants (e.g. lycopene, beta-
carotene) and some specific amino acids
(e.g. arginine, citrulline). Fresh watermelon
consumption ¬is considered a healthy
addition to diet owing to the presence of
lycopene (19). Antioxidants can interfere

Int J Women’s Health Reproduction Sci Vol. 1, No. 3, Autumn 2013 ISSN 2330-4456
 A Khaki et al 102

Table 1. Sperm parameters and epididymis weight of control and experimental groups.

Variables Control group (n=10) Citrullus vulgaris group (n=10)

epididymis (gr) 1 ±0.33 1±0.55

Sperm concentration (total count)
38.40±1.29 63.20±2.35*
(No of sperm/rat ´106)
Motility (℅) 33±3 70±1. 79*
Viability(℅) 58±2.55 89.60±1.47*

Int J Women’s Health Reproduction Sci Vol. 1, No. 3, Autumn 2013 ISSN 2330-4456
 A Khaki et al 103

References:

1. Touhami M, Laroubi A, Elhabazi K, Loubna F, Zrara I, Eljahiri, et al. Lemon juice has
protective activity in a rat urolithiasis model. BMC urology. 2007; 7(1): 18.

2. Levi A, Thomas CE, Keinath AP, Wehner TC. Genetic diversity among watermelon
(Citrullus lanatus and Citrullus colocynthis) accessions. Genet. Resour. Crop Evol. 2001; 48:
559-566.

3. Figueroa A, Sanchez-Gonzalez MA, Wong A, Arjmandi BH. Watermelon extract

supplementation reduces ankle blood pressure and carotid augmentation index in obese adults
with prehypertension or hypertension". American journal of hypertension. 2012; 25 (6): 640-
3.

4. Tlili I, Hdider C, Lenucci MS, Ilahy R, Jebari H, Dalessandro G. Bioactive compounds and
antioxidant activities during fruit ripening of watermelon cultivars. J Food Compost Anal.
2013; 24(7), 923-928.

5. Mandel H, Levy N, Izkovitch S, Korman, S H.Elevated plasma citrulline and arginine due
to consumption of Citrullus vulgaris (watermelon). Berichte der deutschen chemischen
Gesellschaft. 2005; 28 (4): 467-472.

6. Guzmán-Gutiérrez SL, Navarrete A . Pharmacological exploration of the sedative

mechanism of hesperidin identified as the active principle of Citrus sinensis flowers. Planta.
Med. 2009; 75(4): 295-301.

7. Ohtsuki K, Abe A, Mitsuzumi H. Glucosyl hesperidin improves serum cholesterol

composition and inhibits hypertrophy in vasculature. J Nutr Sci Vitaminol. 2003; 49(6): 447-
450.

8. Baynes JW, Thorpe SR.Role of oxidative stress in diabetic complications: a new perspective
on an old paradigm. J of Diabetes.1999; 48: 1-9.

9. Wolff SP, Jiang ZY, Hunt JV.Protein glycation and oxidative stress in diabetes mellitus and
ageing. J. Free Radic Biol. Med. 1991; 10: 339 -52.

10. Mrowicka M. The role of disorders of the prooxidant-antioxidant system in diabetes

etiopathology. Postepy Hig Med Dosw. 2011;65:534–541.

11. Yokozawa T, Cho EJ, Park CH, Kim JH. Protective Effect of Proanthocyanidin against
Diabetic Oxidative Stress. Evid Based Complement Alternat Med. 2012:623879

12. Moussa SA. Oxidative stress in diabetes mellitus. Romanian J Biophys. 2008; 18(3): 225-
236.

13. García-Lafuente A, Guillamón E, Villares A, Rostagno MA, Martínez JA. Flavonoids as

anti-inflammatory agents: implications in cancer and cardiovascular disease. Inflamm Res.
2009; 58(9): 537-552.

Int J Women’s Health Reproduction Sci Vol. 1, No. 3, Autumn 2013 ISSN 2330-4456
 A Khaki et al 104

14. Khaki A, Fathiazad F, Nouri M, Khaki AA, Jabbari-kh H, Hammadeh M. Evaluation of

androgenic activity of Allium cepa on spermatogenesis in Rat. Folia Morphologica. 2009;
68(1): 45-51.

15. Feng R, He W, Ochi H. A new murine oxidative stress model associated with Senescence.
Mech Ageing Dev. 2001; 122: 547-559.

16. World Health Organization (1999.). WHO Laboratory Manual for the Examination of
Human Semen and Semen-Cervical Mucus Interaction. 4th ed. Cambridge University Press,
New York.

17. Engelhard YN, Gazer B, Paren E. Natural antioxidants from tomato extract reduce blood
pressure in patients with grade-1 hypertension: A double blind, placebo-controlled pilot study.
Am Heart J. 2006; 151: 1-6.

18. Shahidi, F. Nutraceuticals and functional foods: Whole versus processed foods. Trends
Food Sci. Technol. 2009; 20: 376-387.

19. Kamil MM, Gamal MF, Shaheen MS. Fourier Transformer Infrared Spectroscopy for
Quality Assurance of Tomato Products. J Am Sci.2011; 7: 558-572.

20. Shahidi, F. and P.K.J.P.D. Wanasundara. Phenolic antioxidant. Crit. Rev. Food Sci Nutr.
1992; 32: 67-103.

21. Sánchez-Moreno C, Larrauri J, Saura-Calixto F. Free radical scavenging capacity and

inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents. Food
Research International. Food Res Int. 1999; 32(6), 407-412.

22. Bondet V, Brad-William W, Berset C. Kinetics and mechanisms of anti-oxidant activity

using DPPH free radical method. LWT Food Sci. Technol. 1997; 31: 609-615.

23. Brand-Williams W, Cuvelier ME and Berset C. Use of a free radical method to evaluate
antioxidant activity. LWT Food Sci. Technol. 1997; 28: 25-30.

24. Jedlinska-Krakowska M, Bomba G, Jakubowski K, Rotkiewicz T, Jana B, Penkowski A .

Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in
rats. J Reprod Dev. 2006; 52: 203-209.

25. Fathiazad F, khak A, Nouri M, Khaki A.A. Effect of Cinnamon Zeylanicum on serum
Testosterone and anti-oxidants levels in Rats. IJWHR. 2013; 1(1): 29-35.

26. Yang HS, Han DK, Kim JR, Sim JC. Effects of alpha-tocopherol on cadmium-induced
toxicity in rat testis and spermatogenesis. J Korean Med Sci. 2006; 21:445-451.

Int J Women’s Health Reproduction Sci Vol. 1, No. 3, Autumn 2013 ISSN 2330-4456