Co-administration of caffeine and hydromethanolic fraction of Citrullus lanatus seeds improved testicular functions in alloxan-induced diabetic male Wistar rats

Onyeso GI, Nkpaa KW, Omenihu S

1Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, Madonna University Elele Rivers State Nigeria

2Department of Biochemistry (Toxicology Unit), Faculty of Chemical Science, College of Natural and Applied Science, University of Port Harcourt, P.M.B 5323, Choba, Rivers State, Nigeria

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Co-administration of caffeine and hydromethanolic fraction of Citrullus lanatus seeds improved testicular functions in alloxan-induced diabetic male Wistar rats

Onyeso GI1, Nkpaa KW2*, Omenihu S1

1Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, Madonna University Elele Rivers State Nigeria

2Department of Biochemistry (Toxicology Unit), Faculty of Chemical Science, College of Natural and Applied Science, University of Port Harcourt, P.M.B 5323, Choba, Rivers State, Nigeria

ARTICLE INFO

Article history:

Citrillus lanatus

Caffeine

Glybenclamide

Hypoglycemic effect

Diabetes

A BSTRACT

Objective: To investigate the effect of Citrillus lanatus (C. lanatus) seeds and caffeine on blood glucose levels and testicular functions of alloxan-induced diabetic male Wistar rats.. Methods: Alloxan was administered at a single dose of 150 mg/kg BW to induce diabetes. A dose of either 200 mg/kg C. lanatus or 100 mg/kg caffeine or both was administered daily to alloxan-induced diabetic rats for three weeks, after which results were compared with a normal control group and a positive control group that received both alloxan and glybenclamide. Results: C. lanatus seeds extract significantly decreases (P<0.05) blood glucose level and significantly (P<0.05) increased sperm motility, sperm count, normal sperm morphology, sperm viable cells and testosterone in plasma level of alloxan-induced diabetic rats treated with C. lanatus seed extract and caffeine. Conclusions: The present study showed that co-administration of caffeine and hydromethanolic fraction of C. lanatus seed extract have hypoglycemic effect and may consequently ameliorate the impaired testicular general architecture and inhibits sperm death or testicular damage caused by alloxan-induced diabetes.

Document heading doi: 10.1016/j.apjr.2016.01.004

1. Introduction

According to the World Health Organization (WHO), there are approximately 160 000 diabetics worldwide, the number of diabetics has double in the last few years and is expected to double once again in the year 2025 [1]. Due to its high prevalence and potential deleterious effect on a patient’s physical and psychological state, diabetes is a chronic disorder caused by partial or complete insulin deficiency and it’s a major medical concern [2]. The disease remains incurable and can only be controlled with drugs. Despite the availability of medication for management of diabetes, the interest in alternative traditional remedies is increasing [3,4].

C. lanatus (watermelon) seeds contains phytochemical constituents like alkaloids, flavonoids, tannins and Saponin with recognizable hypoglycemic effect as well as the presence of soluble fiber and carbohydrate [4-6]. C. lanatus helps in boosting antioxidant levels because it is exceptionally rich in carotenoids such as lycopene, lutein and 毬 carotene [7]. A regular watermelon juice consumption result in significant increases in blood plasma concentrations of lycopene and 毬 carotene [8-10].

A six weeks study found that treatment with a C. lanatus extract containing six grams of L-citrulline and L-arginine daily on middleaged obese subjects with prehypertension or stage one hypertension experienced reduced ankle blood pressure and altered carotid wave reflection, an indication of improved arterial function of the individuals [10-12]. It is very important to note that all parts of the watermelon have something to offer. For example, the seeds are excellent source of protein. The good nutritional and functional properties of watermelon seed meal proteins suggest their potential use in food formulations and diets [13]. C. lanatus possesses numerous bioactivities from natural source which is of better advantage than conventional therapies [14]. The study was carried out to investigate the effect of hydromethanolic fraction of C. lanatus seed extract and caffeine on blood glucose level and testicular functions of alloxan-induced diabetic male Wistar rats.

2. Materials and methods

2.1. Animal treatment

Thirty albino rats of male sex weighing between 200-250 g were used for this study. The animals were procured and then brought to the animal house of Madonna University Elele, kept in wooden cages and allowed to acclimatize for two weeks after which they were selected into six different groups according to their body weight with five in each groups (n=5). Their health status was closely monitored in a clean wooden cage kept in a clear room. The cages were cleaned regularly to avoid infection of rats. These animals were fed with standard rat feed (Guinea feeds with composition: Protein 14.5%, fat 4.8%, fiber 7.2%, calcium 0.8%, phosphorus 0.62%, sodium 0.15% and metabolizable energy 2 300 kcal/kg, water ad libitum). The experimental protocol was approved by Institutional Animals Ethics Committee (IAEC), for using animals in this experiment. Animals were fasted overnight with free access to water prior to each experiment.

2.2. Extraction of watermelon seed

Ripe watermelon pods were obtained from the local market in Elele, rivers state, Nigeria. The seeds extracted from the pods after allowed rotting manually by washing, only healthy looking seeds[15], (brown in color, not floating on water, without mechanical damage or sign of infection) were collected. The collected seeds were ovendried at 35 曟, until a constant weight was obtained. The dried seeds were reduced into powder using a laboratory grinding mail and frozen until used. The grinded seed was thereafter taken to the laboratory (Biochemistry laboratory of Madonna University), placed in a white bucket and weighed using an electric weighing balance. The weight of the bucket been 320 g, while the watermelon seed weighed as follows; 2 302, 526 and 570 g. Each of the measured watermelon seed was dissolved in 800 mL of methanol and 200 mL of water. These were divided in this manner for a better and accurate result. The buckets used for this processes were properly covered and kept for a 24 hour period. After 24hours the different portions were combined and filtered using Whatman No.1 filter paper and funnel hung on a climb stand. The filtrate was collected in a glass jar as a golden colored liquid. The extract was thereafter placed in a water bath which made it concentrated to a gelatinous substance. After a week the concentrated hydromethanolic extract of C. lanatus seeds was dissolved in 2 litters of water and poured into a white bucket. Thereafter, the extract was then quantitatively transferred into amber colored bottles covered with aluminum foil and stored in a refrigerator at 4 曟 before use. This is to prevent it from losing its potency. A dose of 200 mg/kg was used for high dose and 100 mg/kg for low dose administration as shown on Table 1. Alloxan (hydrate) LR, C4H2N2O4.H2O and glybenclamide (C23H28CIN3O5S) was purchased. Alloxan was dissolved in saline solution (0.9 % sodium chloride, pH 7). The dose of alloxan used was 150 mg/kg as a single dose. This dose was chosen because it was effective to induce diabetes.

Treatments for the 6 groups were listed as following: Group I: Normal feed + water; group II: Normal feed + water + 100 mg/kg caffeine + 150 mg/kg alloxan; group III: Normal feed + water + 150 mg/ kg alloxan + 5 mg/kg glybenclamide; group IV: Normal feed + water + 200 mg/kg C. lanatus + 150 mg/ kg alloxan; group V: Normal feed + water + 150 mg/ kg Alloxan; group VI: Normal feed + water + 100 mg/ kg caffeine + 200 mg/ kg C. lanatus + 150 mg/ kg alloxan.

2.3. Samples collection and analysis

After 30 days of treatment the animals were fasted for 24 hrs prior to sacrifice. The animals were anaesthetized using chloroform and then sacrificed [10]. Thus, blood collected via cardiac punctured and put in a labeled ethylenediamminetetraaetic acid bottle (EDTA) for testosterone hormonal assay and later centrifuged at 7 000 rpm for ten (10) minutes. The serum was then collected and stored at -1 曟. Hormonal assay was carried out on the sample a day after. The animals were then dissected; the testes were removed along with caudal epididymis. The caudal epididymis was separated from testes and lacerated to collect the semen with a microscope glass slide for analysis of sperm characteristics.

The blood glucose level was checked with a glucose strip and a glucometer. Initial glucose level before inducing alloxan was checked to ensure the rats were not already diabetic, after inducing alloxan the glucose level was checked as well which served as the initial glucose level before inducing and initial glucose level after inducing. The glucose level was checked weekly to see the effect of the extract on the glucose level of the rat.

2.4. Hormonal assay for testosterone

An enzyme- based immunoassay system was used to measure testosterone level in serum samples collected. Blood serum was introduced into micro – plate well for each sample to be measured. Thereafter, an enzyme antigen linked conjugate for testosterone was added to all and rocked for ten seconds there after incubated for one hour at room temperature. The plate was then washed withmicro plate washer to remove all unbound material. After washing, excess fluids were taped off using dry paper towel. Then color was developed by adding color reagent to determine the bound hormone. Quantitative test result was obtained by measuring the absorbance. The color intensity was checked by tasking the ELISA plate to an ELISA reader which is attached to spectrophotometer which read the absorbance.

2.5. Histology of the testes

The testes were fixed in Formalin, after complete fixation the blocks was embedded in paraffin and sections cut at 5 μm (micron) using a microtome and then stained with haematoxylin and eosin and mounted in Canada balsam [16]. Microscopic examination of the sections was then carried out under a light microscope and later the microscopic slides of the testes were photographed at magnification 40伊.

2.6. Statistical analysis

The result obtained from this study was analyzed using the statistical package for social science (SPSS) version 20.0 for windows. Analysis of variance (ANOVA) was used to compare means, and values were considered significant at P<0.05. Post hoc multiple comparisons for differences between groups and within groups were established using least significant difference (LSD) turkey, scheffe and Duncan.

Table 1 Blood glucose level before and after alloxan induction on male wistar rats (mg/dL).

Table 2 Effects of co-administration of caffeine and hydromethanolic fraction of C. lanatus seeds extract on the weekly blood glucose level of alloxan-induced diabetic male wistar rats.

Table 3 Effects of acute administration of caffeine and hydromethanolic fraction of C. lanatus seeds extract on the sperm parameters and testosterone level and of alloxan-induced diabetic male Wistar rats.

3. Results

The results of the effect of co-administration of caffeine and C. lanatus seeds extract on blood glucose level of alloxan-induced diabetic male wistar rats before and after induction are presented on Table 1. The blood glucose level of rats before alloxan induction in group IV (86.30 ± 2.56 mg/dL) significantly increase compared to group I and II (78.90±7.70 and 63.20±4.59 mg/dL ). Although, after alloxan induction, the experimental groups II, III, IV, V and VI (357.50±20.80, 308.70±54.10, 290.80±43.70, 235.80±25.40 and 386.20±25.30 mg/dL) significantly increased (P<0.05) when compared with groups I (78.90±7.70).

The results of the effect of the administration caffeine and C.lanatus seeds extract on weekly blood glucose level of alloxaninduced diabetic male Wistar rats are shown in Table 2. Group I showed no significant different (P>0.05) across week 1, 2 and 3. Group II showed only a marginal increase across the three groups. While the group III significantly (P<0.05) increased blood glucose level after a week (week I) from previous level shown in Table 1. The blood glucose level attained at week 1 in group III significantly (P<0.05) decreased gradually in week 2 and 3. The group IV and VI showed a profound significant (P<0.05) decrease in blood glucose level as shown in Table 2. While the blood glucose level in group V significantly (P<0.05) increase across week 1, 2 and 3 (285.8±38.4, 321.7±35.3 and 389.3±5.42 mg/dL ) respectively (Table 2).

Results of the effects of acute administration of caffeine and hydromethanolic fraction of C. lanatus seeds extract on the sperm parameters and testosterone level and of alloxan-induced diabetic male Wistar rats are shown in Table 3. The result of the study on the sperm mobility between group II and III showed no significant difference, but there was a significant difference (P<0.05) in sperm count between the two groups (P<0.05). The percentage of normal sperm morphology of experimental rats in groups II, III, and V were significantly (P<0.05) different from that of group I. The sperm viable cells in experimental animals groups II, III, and V were also significantly (P<0.05) different from that of group I two. While, the testosterone level in groups III and V were significantly different (P<0.05) when compared with group I.

Results of the histological examination of the testis are shown in Figures 1-6. The testis of experimental rats in group I exhibited a normal testicular architecture and presence of several normal spermatocytes showing apparent normal outline of the seminferous tubules, interstitium and spermatogenic cells (arrows). The spermatozoa are arranged in rows between and around the cells of sertoli. There was no thickening of the basement membrane. Tubules appeared healthy and no areas of fibrosis were found. Healthy quantities of Sertoli and Leydig cells were present as shown in Figure 1. Group II exhibited deeply stained microstructure and slight reduction in the population of spermatogenic cells. The interstitial spaces were wider than those of group I as shown in Figure 2. While testis in group III showed loss of spermatogenic cells, reduced density of mature spermatozoa within the lumens (L) of the seminiferous tubules, scanty supporting cells (arrows) and some degrees of disintegration and degeneration of the cells (Figure 3). However, histological sections of testis in group IV showed apparent normal testicular architecture as shown in Figure 4, while testis in group VI showed reduced staining intensity, wider luminal diameter, with reduced population of mature sperm cells in the lumen (Figure 6). Moreso, testis in group V showed atrophy and degeneration of seminiferous tubules and loss of spermatogenesis, decrease in spermatogenic cell layer and germinal epithelium (Figure 5).

Figure 1. Photomicrograph of testis of experimental rats administered water (伊200 H&E).

Figure 2. Photomicrograph of testis of experimental rats administered 100 mg/kg caffeine (伊200 H&E).

Figure 3. Photomicrograph of testis of experimental rats administered with 150 mg/kg alloxan and 5 mg/kg glybenclamide (伊200 H&E).

Figure 4. Photomicrograph of testis of experimental rats administered 200 mg/kg hydromethanolic fraction of C. lanatus seeds extract and 150 mg/kg alloxan (伊200 H&E).

Figure 5. Photomicrograph of testis of experimental rats administered 150 mg/kg alloxan (伊200 H&E).

Figure 6. Photomicrograph of testis of rats administered 100 mg/kg caffeine, 200 mg/kg hydromethanolic fraction of C. lanatus seeds extract and 150 mg/kg alloxan (伊200 H&E).

4. Discussion

Alloxan has been observed to cause a massive reduction of the B-cells of the islets of Langerhans and induce hyperglycaemia[17,18]. In our study we have found that co-administration of caffeine and hydromethanolic fraction of C. lanatus seeds extract decreases blood glucose in alloxan diabetic rats, which is consistent with study done by Omigie and Agoreyo [4]. Also, administration of glybenclamide (a known hypoglycemic drug) significant decrease blood glucose level. However administration of C. lanatus seeds extract only exhibited far more hypoglycaemic effect when compared with co-administration of caffeine and hydromethanolic fraction of C. lanatus seeds extract and glybenclamide alone. Nasiri et al [19] also reported that C. lanatus seed have hypoglycaemic effect. The possible mechanism by which administration of hydromethanolic fraction of C. lanatus seeds extract brings about its hypoglycaemic action may be by potentiating the insulin effect by increasing either the pancreatic secretion of insulin from the B -cells of islets of Langerhans or its release from bound insulin. This result is in consistent with Mahesh and Menon, [20] study which reported that the effect of flavonoid which is one phytochemical of C. lanatus on pancreatic B-cells may lead to proliferation and secretion of more insulin. It could also be that the extract caused a hypoglycemic effect by inhibiting the process of glycogenolysis (break down of glycogen to form glucose) or it inhibited the process of glycogenesis by the liver.

The diabetic hyperglycemia induces reduction of sperm parameters and testosterone level which are considered to be significant biomarkers of male fertility. This study showed significant decrease in sperm motility, sperm count, normal sperm morphology, sperm viable cells and testosterone in diabetic groups. These results indicated that diabetes could lead to impotence in male. However, after treatment of with caffeine and hydromethanolic fraction of C. lanatus seeds extract, the level of sperm motility, sperm count; normal sperm morphology, sperm viable cells and testosterone in alloxan-diabetic groups significantly increased. This could be due to the presence of a phytonutrient citrulline, found in water melon seed. When it is consumed citrulline has an added benefit, it is converted to arginine which has been known to boost sperm count in men [21]. Also, arginine is an important amino acid also found in caffeine. Nasir [5] reported that C. lanatus contains tannins and saponin with recognizable hypoglycemic effect as well as the presence of soluble fiber and carbohydrate which may contribute to the hypoglycemic effect.

The health benefit of C. lanatus may also be attributed to the presence of phenol and flavonoids in water melon seed and caffeine[22]. The property of almost every flavonoid is their capacity to act as an antioxidant [22]. Therefore the high phenolic and flavonoid content of water melon seed and caffeine suggest possible high antioxidant potential that helps to return the sperm motility towards normal. Also, the significant increase seen in group VI on Table 4 may be due to the presence of ascorbic acid and flavonoid in caffeine and water melon seed reported by Edward et al, [23] which is known to influence the synthesis of luteinizing hormone from the anterior pituitary gonadal axis. Luteinizing hormone stimulates the leydig cells in the production of testosterone [24]

The loss of spermatogenic cells, reduced density of mature spermatozoa within the lumens (L) of the seminiferous tubules, scanty supporting cells (arrows), disintegration and degeneration of the cells indicated that diabetes may induce sperm death. Also, alloxan-induced diabetes lead to atrophy and degeneration of seminiferous tubules and loss of spermatogenesis decrease in spermatogenic cell layer and germinal epithelium. This also resulted in shrunken seminiferous tubules and reduced cellularity. The blood vessels were also noted to be dilated and congested. All these suggested that the testis was heavily damaged by alloxan administration as shown in Figure 5. On the other hand, treatment of the diabetic rats with caffeine and C. lanatus normalized the general architecture of the testis. From the results above it could be concluded that co-administration of caffeine and hydromethanolic fraction of C. lanatus seeds extract are able to normalize the blood glucose levels and could ameliorate the impaired testicular general architecture and inhibit sperm death.

Conflict of interests statment

The authors declare that there is no conflict of interests

References

[1] Etuk EU. Animals models for studying diabetes mellitus. Agric Biol J N Am 2010;1(2): 130-134

[2] Sharma R, Dave V, Sharma S, Jain P, Yadav S. Experimental models on diabetes: A comprehensive review. Inter J Advances in Pharm Sci 2013; 4:1-8.

[3] Pari L, Umamaheswari J. Antihyperglycaemic activity of Musa sapientum flowers: effect on lipid peroxidation in alloxan diabetic rats. Phytother Res 2002; 14: 1-3.

[4] Omigie IO, Agoreyo FO. Effects of watermelon (Citrullus lanatus) seed on blood glucose and electrolyte parameters in diabetic wistar rats. J Appl Sci Environ Manage 2014; 18 (2):231-233.

[5] Nasir,M, Khaki A, Gharachurlu S, Ashteani A. Effect of ginger on spermatogenesis in alloxan induced diabetic rat. Iran J Med Plants 2009; 8(31):1118-1125.

[6] Johnson IT. Antioxidants and antitumour properties. In: Pokorny J, Yanishlieva N, Gordon M (eds). Antioxidants in food: practical applications. Cambridge: Woodhead Publishing; 2001,p. 100-123.

[7] Chandrika UG, Fernando KSSP, Ranaweera KKDS. Carotenoid content and in vitro bioaccessibility of lycopene from guava (Psidium guajava) and watermelon (Citrullus lanatus) by high-performance liquid chromatography diode array detection. Int J Food Sci Nutr 2009; 60(7):558-66.

[8] Edwards AJ, Vinyard BT, Wiley ER, Brown ED, Collins JK. Penelope Perkins-Veazie, et al. Consumption of watermelon juice increases plasma concentrations of lycopene and beta-carotene in humans. J Nutr 2003; 133(4):1043-1050.

[9] Zhang C, Ho SC, Chen Y, Fu J, Cheng S, Lin F. Greater vegetable and fruit intake is associated with a lower risk of breast cancer among Chinese women. Int J Cancer 2009; 125(1):181-188.

[10] Onyeso G, Nkpaa KW, Nwaka E. Ameliorative potential of methanolic extract of citrullus lanatus (watermelon) seeds on the sperm parameters, testosterone level and testicular cytoarchitecture of male albino rats induced with lead-acetate. Bri J Pharm Res 2015; 6 (1): 35-43.

[11] Figueroa A, Sanchez-Gonzalez MA, Wong A, Bahram H, Arjmandi BH. Watermelon extract supplementation reduces ankle blood pressure and carotid augmentation index in obese adults with prehypertension or hypertension. Am J Hypertens 2012; 12: 342-353.

[12] Christensen AS, Viggers L, Hasselstr?m K, Gregersen S. Effect of fruit restriction on glycemic control in patients with type 2 diabetes--a randomized trial. Nutr J 2013; 12:29.

[13] Langowski HC, Wani AA. A warm welcome to food packaging and shelf life. Food Packaging & Shelf Life 2014;1(1):1-2

[14] Erhirhie EO, Ekene NE. Medicinal values of Citrullus lanatus. Inter J Res Pharm Biom Sci 2013;4(4): 1303-1312.

[15] Okunrobo OL, Uwaya OJ, Imafidon EK, Osarumwense OP, Omorodion EJ. Quantitative determination, metal analysis and antiulcer evaluation of methanol seeds extract of Citrullus lanatus Thunb (Cucurbitaceae) in rats. Asian Pacific J Trop Dis 2012;3(supplement):1261-1265.

[16] Galigher AE, Kayloff EN. Essentials of practical microtechniques. Philadelphia: Lea and Febiger;1971,p.77.

[17] Goldner M, Gomori G. Alloxan induced diabetes. Endocrinology 1943; 33:297–299.

[18] Stanely P, Mainzen P, Venugopal PM. Hypoglycaemic and other related actions of Tinospora cordifolia roots in alloxan-induced diabetic rats. J Ethnopharmacol 2000; 70; 9–15.

[19] Nassiri M, Khaki A, Gharachurlu S, Ashteani A, Rastegarnea K, Rezazadeh S. Effects of Ginger on spermatogenesis in streptozotocin induced Diabetic Rat. Iran J Med Plants 2009; 8(31):118-125.

[20] Mahesh T, Menon VP. Quercetin allievates oxidative stress in streptozotocin-induced diabetic rats. Phytother Res 2004; 18:123–127.

[21] Andersen AG, Jensen TK, Carlsen E, J?rgensen N, Andersson AM, Krarup T, et al. High frequency of sub-optimal semen quality in an unselected population of young men. Hum Reprod 2000;15:366–372.

[22] Rimando AM, Perkins-Veazie PM. Determination of citrulline in watermelon rind. J Chromatogr 2005; 1078(1-2):196-200.

[23] Edwards AJ, Vinyard BT, Wiley ER, Brown ED, Collins JK, Penelope Perkins-Veazie, et al. Consumption of watermelon juice increases plasma concentrations of lycopene and beta-carotene in humans. J Nutr 2003; 133(4):1043-1050.

[24] Sonmez M, Turk G, Yuce A. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male Wistar rats. Theriogenology 2005; 63: 2063-2072.

15 September 2015

Nkpaa KW, Department of Biochemistry (Toxicology Unit), Faculty of Chemical Science, College of Natural and Applied Science, University of Port Harcourt, P.M.B 5323, Choba, Rivers State, Nigeria.

E-mail: nkwilly@gmail.com

Tel: +2348066626323

Received in revised form 27 October 2015 Accepted 2 November 2015

Available online 1 March 2016