J Complement Integr Med. 2015; 12(2): 137–142

Okukwe Obode, Oluwatoyin Okafor, Ochuko Erukainure*, Atinuke Ajayi, Yewande Suberu, Akinyele Ogunji, Teressa Okporua, Oluwatoyin Oluwole, Augusta Ozumba and Gloria Elemo

Protective effect of some selected fruit blends on testicular toxicity in alloxan-induced diabetic rats Introduction

Abstract Background: The protective effect of a developed drink from blends of selected fruits on the sperm quality of alloxan-induced diabetic rats was investigated. Methods: Diabetes was intraperitoneally induced with alloxan; the formulated drink blend was orally administered 2.5 or 5 mL/day. Treatment lasted for 14 days and the rats were humanely sacrificed by cervical dislocation. The antioxidant status via assessment of reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (LPO) was carried out on the testis, while sperm cells were analyzed for sperm motility, counts and abnormality. Results: Induction of diabetes led to a significant (p < 0.05) decrease in GSH level, elevated SOD and CAT activities, significant (p < 0.05) decrease in the sperm quality parameters studied. However, treatment with the formulated drink led to a significant (p < 0.05) reduction of LPO, SOD and CAT activities as well as increase in GSH level. Conclusions: This study shows an improvement in testicular antioxidant activities and sperm qualities by single and double doses of the formulation, suggesting its protective potential against testicular toxicity in diabetic rats. Keywords: catalase (CAT), malondialdehyde (MDA) and reduced glutathione (GSH), sperm cells, superoxide dismutase (SOD) DOI 10.1515/jcim-2014-0032 Received May 18, 2014; accepted February 3, 2015; previously published online March 25, 2015 *Corresponding author: Ochuko Erukainure, Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria, E-mail: [email protected] Okukwe Obode, Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria Oluwatoyin Okafor, Department of Natural Sciences, Albany State University, Albany, GA, USA Atinuke Ajayi, Yewande Suberu, Akinyele Ogunji, Teressa Okporua, Oluwatoyin Oluwole, Augusta Ozumba, Gloria Elemo, Food Technology Division, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria

Diabetes mellitus (DM) is a chronic metabolic disease that affects millions of people all over the world. In the year 2000, the World Health Organization (WHO) reported that 177 million people were affected by diabetes worldwide but by 2025, this figure is projected to rise to over 300 million [1]. Diabetes is characterized by hyperglycemia due to insulin deficiency or insulin resistance. Hyperglycemia occurs when the liver and skeletal muscles cannot store glycogen and/or the cells become unable to utilize glucose. The prevalent treatment of DM besides controlling food intake; treating obesity; proper exercise and changing lifestyle includes administration of oral hypoglycemic drugs and injection of insulin [2]. Factors such as obesity, population growth and aging are thought to be largely responsible [3]. It is a serious metabolic disorder with numerous complications [4]. It is well known that increase of blood glucose levels leads to structural and functional changes in various target tissues and organs [5]. Experimentally induced diabetes in male rats is associated with altered functions of reproductive system [6]. About 90 % of male diabetic patients experienced sexual dysfunction, impotence and infertility caused by testicular failure associated with prolonged hyperglycemia [7]. Infertility is one of the major health problems in life and approximately 30 % of this problem is due to male factors [8]. Several factors can interfere with the process of spermatogenesis and reduce sperm quality and quantity. Some diseases such as coronary heart diseases, DM and chronic liver diseases have been reported to cause deleterious effects on spermatogenesis [9]. On the other hand, intake of antioxidants and vitamins A, B, C and E can increase stability of testicular blood barrier and protect sperm DNA from oxidative stress that caused by active free radicals [10]. The testis contains an elaborate array of antioxidant enzymes and free radical scavengers to protect against oxidative stress. This is of great importance as peroxidative damage is currently regarded as the major cause of impaired testicular functions. Plants have been used as a source of complementary and alternative medicine management of diabetes [11, 12]. This can be attributed to their phytochemicals that have been proven to be therapeutic against DM and other ailments. Of interests are pawpaw (Carica papaya L.) and grape (Citrus

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Obode et al.: Diabetes vs. testicular toxicity

paradisi) fruits. Several studies have reported the antidiabetic properties of these fruits [13, 14]. Blending of two or more fruits has also been shown to have tremendous health benefits as well as ensuring a longer post-harvest shelf life [15, 13, 16]. Blending of juices with other medicinal plants has also been shown to improve the medicinal properties. Erukainure et al. [13] reported the protective role of blends of roselle calyx and selected fruits against diabetic-induced oxidative stress in testicular tissues and sperm cells in albino rats. This study was designed to investigate the effect of blends of some selected fruits on testicular function of diabetic rats.

Materials and methods Plant materials Unripe pawpaw fruits (Carica papaya), grape fruits (Citrus paradisi) as well as guava leaves (Psidium guajava) were identified and obtained from National Horticultural Research Institute (NIHORT), Ibadan, Nigeria (Table 1). Table 1: Product formulation. Extracts

Percentage, %

Pawpaw Grape fruit Guava leaves Sweetener (aspartame)

    tablets/L

Development of blend: Extracts of pawpaw fruit, guava leaf and grape fruit were produced using hot extraction methods as described by Okafor et al. [17]. Unripe pawpaw fruits were washed and peeled, seeds were removed and the fruits were cut into smaller sizes. This was boiled for 30 min at 60 °C. The boiled fruits were then milled in a warring blender with the water in which they were boiled to get slurry, which was then filtered with a muslin cloth to get the extract. Grape fruits were washed, peeled, cut into smaller pieces; seeds were removed and milled in a warring blender into slurry. The slurry was then filtered using a fine sieve. The guava leaves were sorted, washed and boiled in water separately for 20 min, and the extract was decanted. Varying proportion of the extracts (pawpaw fruit 60 %, grape fruit 20 % and guava leaves 20 %) obtained were mixed to get a fruit blend and was pasteurized at 80 °C.

Animals Twenty male albino rats of Wister strain weighing between 150 and 200 g were used for the study. They were fed on standard rat pellet diet and acclimatized for one week. They were provided water ad libitum and maintained under standard laboratory conditions of natural photo period of 12-h light–dark cycle.

Induction of diabetes Diabetic state was induced by a single-dose intraperitoneal injection of 150 mg/kg of alloxan monohydrate in normal saline in a volume of about 3 mL. After 72 h, the diabetic rats (glucose level > 150 mg/dL) were separated and used for the study. The formulated drink blend was administered orally, 2.5 or 5 mL/day for 14 consecutive days after induction of diabetes. The animals used in the present study were maintained in accordance with the approval of the Animal Ethical Committee, Federal Institute of Industrial Research, Oshodi, Lagos, Nigeria. The rats were divided into four groups, each consisting of five animals. Group 1 – Pelletized mouse chows Group 2 – Diabetic (untreated) Group 3 – Diabetic þ 2.5 mL of blend Group 4 – Diabetic þ 5 mL of blend The rats were monitored daily for food intake, water intake and body weight. Blood glucose levels of the rats were monitored on weekly basis using a glucometer. Treatment lasted for 14 days after induction. At the end of the treatment trials, the rats were fasted overnight and humanely sacrificed by cervical dislocation.

Preparation of tissue homogenates The testes were harvested, rinsed in ice-cold 1.15% KCl solution to wash off excess blood, blotted dry with filter paper and weighed. The organs were homogenized in four parts of homogenizing buffer and centrifuged at 10,000 rpm for 15 min in an ultracentrifuge at a temperature of –2 °C. The post-mitochondrial fraction was decanted and stored at –4 °C for subsequent analysis.

Determination of oxidative stress parameters Lipid peroxidation (LPO) was determined by measuring malondialdehyde (MDA) formed by thiobarbituric acid reaction (TBAR) [18]. Catalase (CAT) activity was estimated by measuring the rate of decomposition of H2O2 [19]. Superoxide dismutase (SOD) activity was determined by the method of Kakkar et al. [20], while the method of Ellman [21] was adopted in estimating the level of reduced glutathione (GSH) activity.

Evaluation of sperm quality The tail of the epididymis was cut into small pieces in 2 mL of normal saline and sperm collected by squeezing it gently on clean slide [22]. Sperm motility and count were evaluated using conventional methods [23]. Sperm abnormality was carried out by microscopical examination of the seminal smears stained with eosin and nigrosin stain. Presence of epithelial cells, triple phosphate crystals and oil droplets was also investigated.

Statistical analysis To address the biological variability, each set of experiments was repeated at least three times. Statistical significance was established using one-way analysis of variance (ANOVA), and data were reported

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Obode et al.: Diabetes vs. testicular toxicity

as mean  standard deviation. Significant difference was established at p < 0.05. Statistical analyses were carried out using SPSS for Windows, version 15.0 (SPSS Inc., Chicago, IL).

Results There were significant differences (p < 0.05) in all the studied antioxidant parameters. Induction of diabetes led to a significant (p < 0.05) reduction of GSH activity in the testes of the experimental rats (Figure 1); however, treatment with the blend at both single and double doses significantly (p < 0.05) increased the activity. There was an increase in the activity of SOD in the untreated diabetic rats. This was reduced significantly in the treated groups, with the double dose (5 mL) having greater significant decrease as shown in Figure 2. There was an

increase in the CAT activity in the testes of the untreated diabetic rats as shown in Figure 3. However, treatment with both doses reduced its activity. Induction of diabetes led to increase in LPO (Figure 4). This was, however, significantly reduced by both single and double doses of the blends. The effects of the blend at both single and double doses are shown in Table 2. The sperm cells were observed to be grayish, opaque and watery. Sperm count and motility were significantly (p < 0.05) decreased in the untreated diabetic group as compared with the control group. These were observed to increase significantly in the treated groups (in a dose-dependent manner). There was an increase in sperm abnormality for the untreated group. Treatment with single dose (2.5 mL) reduced the abnormality of the cells. The pus cells were not significantly affected by treatment, but

700

25

600

b,d

b,d

20 15

400

a,c

a,c

10

µ/mol

µ/mol

500

300 200

5

100

0

0

Group 1

Group 2

Group 3

Group 4

Figure 1: Effect of blends on GSH activities of testes in diabetic rats. Notes: Values ¼ mean þ SD; n ¼ 5. a ¼ statistically significant (p < 0.05) as compared with group 1; b ¼ statistically significant (p < 0.05) as compared with group 2; c ¼ statistically significant (p < 0.05) as compared with group 3; d ¼ statistically significant (p < 0.05) as compared with group 4.

Group 1

Group 2

µ/mol

7 a,c,d

6

µ/mol

120

4

b b b

3

100

2

80

1

60

0 Group 1

40 20 0 Group 1

Group 2

Group 3

Group 4

Figure 2: Effect of blends on SOD activities of testes in diabetic rats. Note: Value ¼ mean  SD; n ¼ 5.

Group 4

Figure 3: Effect of blends on CAT activities of testes in diabetic rats. Note: Value ¼ mean  SD; n ¼ 5.

5 140

Group 3

Group 2

Group 3

Group 4

Figure 4: Effect of blends on LPO in testes in diabetic rats. Notes: Values ¼ mean þ SD; n ¼ 5. a ¼ statistically significant (p < 0.05) as compared with group 1; b ¼ statistically significant (p < 0.05) as compared with group 2; c ¼ statistically significant (p < 0.05) as compared with group 3; d ¼ statistically significant (p < 0.05) as compared with group 4.

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Obode et al.: Diabetes vs. testicular toxicity

Table 2: Effect of blend on sperm quality of diabetic rats. Parameters Motility Sperm count Abnormal count Epithelial cells Triple phosphate crystal Oil droplet

Group 

Group 

Group 

Group 

%    mL % þ Nil þþ

Nil    mL % þþ þþ Nil

%    mL % þ Nil Nil

%    mL % þ Nil þ

the epithelial cells were reduced compared to the untreated diabetic group. There were no triple phosphate crystals in the treatment groups compared with the untreated diabetic.

Discussion Hyperglycemia leads to the increased production of reactive oxygen species (ROS) via at least four different routes: increased glycolysis; intercellular activation of the sorbitol pathway; auto-oxidation of glucose and non-enzymatic protein glycation [24], which in turn, causes an increase in oxidative stress to the body. The lipids in sperm are the main substrates for peroxidation and researches have shown that excess amounts of ROS and free radicals have adverse effects on sperm quality and function. The testes are susceptible to oxidative stress despite the low oxygen tensions that typify the testicular cells. This is attributed to abundance of highly unsaturated fatty acids and the presence of potential ROS-generating systems [25]. The testes have developed an array of antioxidant systems comprising both enzymatic and non-enzymatic constituents to counteract this effect [25]. However, in diabetes these antioxidant systems are compromised, subjecting the testes to free radical damage and testicular oxidative stress. The effect of blends of selected fruits on testicular antioxidant activities and sperm quality in diabetic rats was investigated and reported in this paper. The significant increase in testicular LPO and decrease in testicular antioxidants results from the induction of diabetes by alloxan, reflecting the inhibitory action of alloxan on both enzymatic and non-enzymatic antioxidants in testes [26]. Alloxan exhibits its action by generating ROS resulting to destruction of the pancreatic β cells, thereby leading to diabetes [27]. Reduced cellular level of GSH has been reported to be a marker of oxidative stress [28]. The reduction in the

untreated diabetic group corresponds to previous studies which reported reduced GSH level in diabetic patients and in experimental models [29, 30]. The decrease in testicular GSH was reversed by the blend at both doses, correlating with its antioxidant potentials. Increase in SOD and CAT activities due to oxidative stress has been reported [31]. Their observed increase in the testes of untreated diabetic rats in the present study could be attributed to hyperglycemia-induced oxidative stress. The importance of SOD in controlling O2– leakage from testicular mitochondria has been reported [32]. Gu and Hecht further reported that its mRNA is higher in the testes than the liver. CAT catalyzes the decomposition of hydrogen peroxide (H2O2) into less reactive gaseous oxygen and water molecules [33]. This study demonstrated significant decreases in SOD and CAT activities in testes of rats treated with the blends. Induction of LPO in diabetes has been observed in numerous tissues both in vitro and in vivo [34]. The testes are potent target owing to its high concentration of polyunsaturated fatty acids. In the present study, induction of diabetes led to an increase in the level of testicular LPO in accordance to previous studies. Reduction of the LPO level by both doses of the blend indicates the protective potentials of the blend against hyperglycemia-induced peroxidation of testicular membrane lipids. Increasing evidence suggests that diabetes has an adverse effect on male reproduction function and oxidative stress may be involved [35]. This is seen in the quality of the sperm of the untreated diabetic rats. Their low sperm motility and count as well as percentage abnormality corresponds to previous studies on the sperm quality of diabetic rats [25, 7, 35]. At single dose of the blend, the motility was restored significantly. The observed improvement of the sperm count by the blends suggests its potency in the management of the diabetes-induced testicular toxicity. The reduced concentration of epithelial cells and absence of triple phosphate crystals in the testes of the treated group further indicates the potency of the blend [13].

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Obode et al.: Diabetes vs. testicular toxicity

Conclusions This study shows improved testicular antioxidant activities and sperm qualities by single and double doses of juice blends from selected fruits, thus suggesting the protective potential of the formulation against testicular toxicity in diabetic male rats. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: The authors are sincerely grateful to the management of the Federal Institute of Industrial Research, Oshodi (FIIRO), Lagos, Nigeria for funding of this project. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References 1. World Health Organization (WHO). Diabetes: the cost of diabetes. Fact sheet No. 236, 2002. 2. Rang HP, Dale MM. Pharmacology, 2nd ed., Chap. 20. London: Churchill Livingstone, 1991: 506. 3. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047–53. 4. Yanardag R, Ozsoy-Sacan O, Bolkent S, Orak H, Karabulut-Bulan O. Protective effects of metformin treatment on the liver injury of streptozotocin-diabetic rats. Hum Exp Toxicol 2005;24:129–35. 5. Cai L, Chen S, Evans T, Deng DX, Mukherjee K, Chakrabarti S. Apoptotic germ-cell death and testicular damage in experimental diabetes: prevention by endothelin antagonism. Urol Res 2000;28:342–7. 6. Orth JM, Murray FT, Bardin CW. Ultrastructural changes in Leydig cells of streptozotocin-induced diabetic rats. Anat Rec 1979;195:415–30. 7. Amaral S, Moreno AJ, Santos MS, Seiça R, Ramalho- Santos J. Effects of hyperglycemia on sperm and testicular cells of GotoKakizaki and streptozotocin-treated rat models for diabetes. J Theriogenol 2005;66:2056–67. 8. Isidori AM, Pozza CD, Gianfrilli D, Isidori A. Medical treatment to improve sperm quality. J Reprod Biomed 2006;12:704–14. 9. Mosher WD, Pratt WF. Fecundity and infertility in the United States: incidence and trends. J Fertil Steril 1991;56:192–3. 10. Jedlinska MG, Bomba K, Jakubowski T, Rotkiewicz BJ, Penkowski A. Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in rats. J Reprod 2006;52:203–9.

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11. Cheryl A. Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus. J Ethnobiol Ethnomed 2006;2:45. 12. Gupta RK, Kesari AN, Murthy PS, Chandra R, Tandan V, Watal G. Hypoglycemic and anti-diabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals. J Ethnopharm 2005;99:75–81. 13. Erukainure OL, Okafor OY, Obode OC, Ajayi A, Oluwole OB, Oke OV, et al. Blend of roselle calyx and selected fruit modulates testicular redox status and sperm quality of diabetic rats. J Diab Metab 2012;3:214. 14. Lans CA. Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus. J Ethnobiol Ethnomed 2006;2:45. 15. Bhardwaj RL, Pandey S. Juice blends-a way of utilization of underutilized fruits, vegetables, and spices: a review. Crit Rev Food Sci Nutr 2011;51:563–70. 16. FAO. Prevention of post-harvest food losses fruits, vegetables and root crops a training manual. Rome: FAO, 1989. 17. Okafor O, Daramola K, Pikuda Y, Oke O, Omosebi B, Elemo G, et al. Chemical profile of selected fruit extracts used for diabetes control. In: Martirosyan DM, Abate N, editors. Functional foods for chronic diseases, vol. 5: Diabetes and related diseases. Richardson, TX: Food Science Publisher, 2010:194–205. 18. Chowdhury P, Soulsby M. Lipid peroxidation in rat brain is increased by simulated weightlessness and decreased by a soy-protein diet. Ann Clin Lab Sci 2002;32:188–92. 19. Chance B, Maehly AC. Assay of catalase and peroxidase. Methods Enzymol 1955;2:764–75. 20. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem Biophys 1984;21:130–2. 21. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. 22. Shalaby MA, Mouneir SM. Effect of Zingiber officinale roots and Cinnamon zeylanicum bark on fertility of male diabetic rats. Glob Vet 2010;5:341–7. 23. Raji Y, Udoh US, Mewoyeka OO, Ononye FC, Bolarinwa AF. Implication of endocrine malfunction in male infertility efficacy of Azadiraktha indica extract in rats. Afr Med Med Sci 2003;32:159–65. 24. Wrighten SA, Piroli GG, Grillo CA, Reagan LP. A look inside the diabetic brain: contributors to diabetes-induced brain aging. Biochem Biophys Acta 2009;1792:444–53. 25. Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. Oxid Med Cell Longev 2008;1(1): 15–24. 26. El-Missiry MA. Enhanced testicular antioxidant system by ascorbic acid in alloxan diabetic rats. Comp Biochem Physiol C 1999;124:233–7. 27. Lenzen S. The mechanisms of alloxan- and streptozotocininduced diabetes. Diabetologia 2008;51:216–26. 28. Jorge AP, Horst H, de Sousa E, Pizzolatti MG, Silva FR. Insulinomimetic effects of kaempferitrin on glycaemia and on 14C-glucose uptake in rat soleus muscle. Chem Biol Interact 2004;149:89–96. 29. Dincer Y, Akcay T, Alademir Z, Ilkova H. Effects of oxidative stress on glutathione pathway in red blood cells from patients with insulin-dependent diabetes mellitus. Metabolism 2002;51:1360–2.

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Obode et al.: Diabetes vs. testicular toxicity

30. Obrosova I, Fathallah L, Liu E, Nourooz-Zadeh J. Early oxidative stress in diabetic kidney: effect of DL-alpha lipoic acid. Free Radical Biol Med 2003;34:186–95. 31. Onyema OO, Farombi EO, Emerole GO, Ukoha AI, Onyeze GO. Effect of vitamin E on monosodium glutamate induced hepatoxicity and oxidative stress in rats. Ind J Biochem Biophys 2005;43:20–4. 32. Gu W, Hecht NB. Developmental expression of glutathione peroxidase, catalase, and manganese superoxide dismutase mRNAs during spermatogenesis in the mouse. J Androl 1996;17:256–62.

33. Chelikani P, Fita I, Loewen PC. Diversity of structures and properties among catalase. Cell Mol Life Sci 2004;61:192–208. 34. Akondi RB, Kumar P, Annapurna A, Pujari M. Protective effect of rutin and naringin on sperm quality in streptozotocin (stz) induced type 1 diabetic rats. Iran J Pharm Res 2011;10:585–96. 35. Hakim P, Sani HA, Noor MM. Effects of Gynura procumbens extract and glibenclamide on sperm quality and specific activity of testicular lactate dehydrogenase in streptozotocin-induced diabetic rats. Mal J Biochem Mol Biol 2008;16:10–14.

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Protective effect of some selected fruit blends on testicular toxicity in alloxan-induced diabetic rats.

The protective effect of a developed drink from blends of selected fruits on the sperm quality of alloxan-induced diabetic rats was investigated...
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