How Much Turmeric To Take For Erectile Dysfunction

Toxicol Rep. 2015; 2: 1357–1366.

Dietary supplementation of ginger and turmeric improves reproductive function in hypertensive male rats

Ayodele Jacob Akinyemi,^a, ^b, ^c Isaac A. Adedara,^d, ^eastward Gustavo Roberto Thome,^c Vera Maria Morsch,^c Monique Tomazele Rovani,^f Lady Katerine Serrano Mujica,^f Thiago Duarte,^yard Marta Duarte,^grand Ganiyu Oboh,^a, ^⁎⁎ and Maria Rosa Chitolina Schetinger^c, ^⁎

Ayodele Jacob Akinyemi

^aFunctional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology Akure, Individual Mail Pocketbook 704, Akure 340001, Nigeria

^bDepartment of Biochemistry, Afe Babalola University Ado-Ekiti, Private Mail service Bag 5454, Nigeria

^cPrograma de Pós Graduação em Ciências Biológicas: Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas (CCNE), Universidade Federal de Santa Maria, Campus Universitário, Camobi, CEP 97105-900 Santa Maria, RS, Brazil

Isaac A. Adedara

^dDepartamento de Bioquímica e Biologia Molecular, CCNE, Universidade Federal de Santa Maria, CEP 97105-900 Santa Maria, RS, Brazil

^eDrug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria

Gustavo Roberto Thome

Vera Maria Morsch

Monique Tomazele Rovani

^fLaboratório de Biotecnologia e Reprodução Animal—BioRep, Universidade Federal de Santa Maria, CEP 97105-900, Santa Maria, RS, Brazil

Lady Katerine Serrano Mujica

^fLaboratório de Biotecnologia e Reprodução Brute—BioRep, Universidade Federal de Santa Maria, CEP 97105-900, Santa Maria, RS, Brazil

Thiago Duarte

^yardCentro de Ciências da Saude, Universidade Luterana do Brazil (ULBRA), Campus Santa Maria, Santa Maria, RS, Brazil

Marta Duarte

^gCentro de Ciências da Saude, Universidade Luterana practise Brazil (ULBRA), Campus Santa Maria, Santa Maria, RS, Brazil

Ganiyu Oboh

^aFunctional Foods and Nutraceuticals Unit of measurement, Department of Biochemistry, Federal Academy of Engineering science Akure, Private Mail Pocketbook 704, Akure 340001, Nigeria

Maria Rosa Chitolina Schetinger

Received 2015 Jun xix; Revised 2015 Sep 27; Accepted 2015 October three.

Abstract

Ginger [Zingiber officinale Roscoe (Zingiberaceae)] and turmeric [Curcuma longa Linn (Zingiberaceae)] rhizomes take been reportedly used in folk medicine for the treatment of hypertension. However, the prevention of its complication such every bit male infertility remains unexplored. Hence, the aim of the present study was to investigate the preventive effects of ginger and turmeric rhizomes on some biomarkers of male reproductive role in L-Proper name-induced hypertensive rats. Male Wistar rats were divided into seven groups (n = 10): normotensive control rats; induced (L-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/twenty-four hour period); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. Subsequently xiv days of pre-handling, the animals were induced with hypertension by oral assistants of 50-NAME (40 mg/kg/day). The results revealed significant decrease in serum total testosterone and epididymal sperm progressive motility without affecting sperm viability in hypertensive rats. Moreover, increased oxidative stress in the testes and epididymides of hypertensive rats was evidenced by significant decrease in total and non-poly peptide thiol levels, glutathione S-transferase (GST) activity with concomitant increase in 2′,7′-dichlorofluorescein (DFCH) oxidation and thiobarbituric acid reactive substances (TBARS) production. Similarly, decreased testicular and epididymal NO level with concomitant peak in arginase action was observed in hypertensive rats. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations in biomarkers of reproductive function in hypertensive rats. The inhibition of arginase activity and increase in NO and testosterone levels by both rhizomes could suggest possible machinery of action for the prevention of male infertility in hypertension. Therefore, both rhizomes could be harnessed as functional foods to prevent hypertension-mediated male reproductive dysfunction.

Keywords: Ginger, Turmeric, Hypertension, Male fertility, L-Name

i. Introduction

Hypertension is a chronic medical status in which the blood pressure (BP) in the arteries is elevated [3]. It is considered a major public wellness epidemic and affects more than than 25% of the general population, with its prevalence increasing with age [64]. Evidence suggests that hypertension is associated with an impairment of male sexual role [26] but their pathophysiological pathways are notwithstanding to be clearly elucidated.

The association of hypertension with increased incidence of male sexual dysfunction includes bug related to libido, erection and ejaculation [67], [25]. In addition, information technology has likewise been linked to cause male infertility via a decrease in blood flow to the testis [45], [72]. A reduction of claret period to diverse vital organs of the body as a upshot of vasoconstriction of the arterial vessels is one of the primary manisfestation of hypertension.

Several studies have reported induction of hypertension in rats by oral administration of N-nitro-fifty-arginine methyl ester hydrochloride (an inhibitor of nitric oxide biosynthesis) [42], [31], [xxx], [22], [21]. Nitric oxide (NO) deficiency has been suggested as a contributory factor in pre-eclampsia [29], [60], while the vasodilatory backdrop of NO are essential for cavernosal smoothen musculus activeness in achieving penile erection [20]. NO tin act as a free radical scavenger, inactivating and even inhibiting production of superoxide anions [10], [46], [24] which cause lipid peroxidation, a process which leads to functional harm of spermatozoa [39]. This propose a benign role for NO in the male person reproductive organization [2].

The inhibition of NO synthesis (by Fifty-Proper noun administration), tin can consequence in a very low concentration of NO-mediated vasodilatation, an increase in vasoconstriction, and subsequently an increment in systemic vascular resistance, which contributes to BP elevation and its complications [19]. The activity of endothelial nitric oxide synthase (eNOS) is competitively inhibited by arginase, which catalyzes the hydrolysis of l-arginine to form l-ornithine and urea, thereby making NO unavailable. Arginase reciprocally regulates eNOS and NO production past competing for l-arginine [66]. In various pathological disorders, arginase has been shown to regulate vascular cell functions primarily through impairment of NO production [66]. Therefore, suppressing high activity of arginase will favor eNOS which in plow leads to increase production of NO. Recent findings take shown phenolic phytochemicals to have promising potential likewise in mitigating this procedure [43].

Zingiber officinale (Ginger, Family Zingiberaceae) roots are ordinarily used as culinary spice and used medicinally for its antioxidant [59]. Information technology is a institute that is used in folk medicine from south-east Asia, and besides in Africa, China, India and Jamaica, the India also cultivate the rhizomes for medicinal purpose [59]. The important active compounds of the ginger root are thought to exist volatile oils and pungent phenol compounds such as gingerols, shogaols, zingerone, and gingerols [59]. Recently, ginger rhizomes are used in traditional medicine as therapy confronting several cardiovascular diseases such as hypertension [33]. It has been reported that ginger lowers claret pressure (BP) through blockade of voltage-dependent calcium channels [32]. Zancan et al. [70] reported the roots and leaves of ginger exhibited antioxidant activity. In add-on, Yang et al. [73] concluded that antioxidants tin can protect sperm DNA and other of import molecules from cell damage induced by oxidation, improve sperm quality and increment reproductive efficiency of men. In rats, Khaki et al. [74] reported that ginger has a protective consequence against Dna harm induced by H₂O₂ and may exist promising in enhancing good for you sperm parameters. Traditionally, ginger rhizome was used in Iran for enhancing male sexuality, regulating female menstrual cycle, and too reducing painful menstrual periods [36].

Other notable member of this plant family unit (Zingiberaceae) is turmeric otherwise called red ginger (Curcuma longa). It is a rhizomatous herbaceous perennial establish, in the ginger family, employed every bit a dye source and food colorant due to its feature yellow color [23]. Turmeric is one of the primary ingredients for curry powder, and used equally an alternative to medicine and tin be made into a drink to treat colds and breadbasket complaints [23]. Like ginger, it is cultivated for used in folk medicine from south-e Asia, and also in Africa, Cathay, India and Jamaica [59]. The curcuminoids chemical compound are the major phytochemicals of the turmeric responsible for the characteristic xanthous color and has been investigated to containing biological activities, such as antioxidant, anti-hypertensive, anti-inflammatory, anticarcinogenic, thrombus suppressive, hypoglycaemic and antiarthritic properties [13], [41], [5], [vii]. In folk medicine, turmeric has been used in lowering claret pressure and every bit tonic and claret purifier [65]. Traditional Indian medicine claims the apply of its powder against biliary disorders, cough, diabetic wounds, hepatic disorder and rheumatism [12]. [56] observed that curcumin is capable of scavenging oxygen costless radicals such as superoxide anions and hydroxyl radicals, which are the initiators of lipid peroxidation.

Both rhizomes are useful in folk medicine against hypertension and are considered safe herbal medicines considering no significant side effect has nevertheless been described using 2–four% dietary supplementation [33], [viii], [9] and their effect on NO product has been published [9].

Thus, because the association of hypertension with male infertility, the ethnopharmacological actions of both rhizomes with express information on their promising potential in improving male fertility in hypertensive individuals, the aim of the present study therefore was to evaluate the preventive event of dietary supplementation of ginger and turmeric, on some biomarkers of reproductive function in L-Proper noun-induced hypertensive male rats.

2. Materials and methods

2.1. Chemicals

The substrate l-arginine, likewise as urea, N-(l-naphthyl)ethylene-diamine dihydrochloride, Tris–HCl buffer, HEPES, Fifty-NAME, and Coomassie bright blue Grand were obtained from Sigma Chemical Co. (St. Louis, MO, U.s.) and bovine serum albumin, nitrate, vanadium chloride (VCl_three) from Reagen (Colombo, PR, Brazil). All the other chemicals used in this experiment were of the analytical grade, while the water was glass distilled. All the kits used for the bioassay were sourced from Randox Laboratories Ltd. (Crumlin, Northern Ireland, Uk).

2.two. Sample drove

The fresh samples of ginger (Z. officinale) and turmeric (C. longa) rhizomes were obtained from a farmland at Akure metropolis, Nigeria. Authentication of the plants was carried out at the Department of Biology, Federal University of Engineering science, Akure, Nigeria.

2.iii. Animals

Adult male Wistar rats (200–300 g) from the Central Animal House of the Federal University of Santa Maria were used in this experiment. The animals were maintained at a constant temperature (22 ± 2 °C) on a 12 h calorie-free/dark wheel with free access to food and water. The animals were used according to the guidelines of the National Council for Animal Experiments Control (CONCEA) and are in accord with international guidelines. The research projection was approved by the ethics committee of the Federal University of Santa Maria—Brazil by the number 23/2011.

2.four. Experimental protocol

The rats were acclimatized for two weeks and randomly divided into 7 groups of ten animals each (n = 10). Grouping 1: (Control) serve every bit the normotensive control group placed on a basal nutrition; Grouping two: (Induced) serve as the hypertensive (L-Proper noun) group placed on a basal nutrition plus L-Proper name; Group 3: (L-Name + AT) serve as the positive command placed on a basal diet plus L-NAME plus atenolol (x mg/kg/mean solar day); Grouping 4: (RG Normal) serve equally the normotensive nutrition grouping placed on a diet supplemented with (4%); Group 5: (RG + L-NAME) serve as the hypertensive diet group placed on a diet supplemented with (4%) plus L-NAME; Grouping half-dozen: (WG Normal) serve as the normotensive diet group placed on a diet supplemented with ginger (four%); and Group seven: (WG + L-Proper name) serve as the hypertensive diet grouping placed on a diet supplemented with ginger (4%) plus L-Proper noun. The rats were placed on their respective diet for 2 weeks before induction of hypertension (Table 1). Daily feed intake was monitored and body weight was taken both at the beginning and at stop of the experiment. In the hypertensive groups, hypertension was induced past the oral assistants of the nitric oxide synthase (NOS) inhibitor L-Name (40 mg/kg/24-hour interval) by gavage for 10 days [31]. In the normotensive groups, the animals received h2o past gavage throughout the experiment. All the animals were sacrificed 24 h afterwards the terminal handling under ketamine and xylazine anesthesia.

Table 1

Nutrition formulation for basal and supplemented diets for control and examination groups.

Treatment	Group i	Group ii	Group 3	Group 4	Grouping 5	Gourp vi	Group 7
Skimmed milk	39.4	39.4	39.4	39.4	39.iv	39.4	39.4
Oil	10.0	x.0	x.0	10.0	10.0	x.0	10.0
Vitamin premix	4.0	iv.0	four.0	iv.0	iv.0	iv.0	four.0
Corn starch	46.vi	46.6	46.half dozen	42.6	42.six	42.6	42.vi
Ginger	–	–	–	4.0	four.0	four.0	4.0
Total	100 g	100 g	100 1000	100 g	100 g	100 g	100 g

2.5. Diet conception

The diets were freshly formulated according to a modified method of Akinyemi et al. [seven] (Table 1).

2.6. Hemodynamic parameter determination

In all rats, systolic claret pressure (SBP) was measured in awaken animals, by tail-cuff plethysmography (Kent Scientific; RTBP1001 Rat Tail Blood Pressure Organization for rats and mice, Litchfield, USA). Rats were conditioned with the apparatus earlier measurements were taken. SBP was recorded at the end of experiment (final treatment week).

2.7. Sperm motion and viability assays

The sperm were nerveless immediately subsequently a rat was sacrificed. Briefly, epididymal sperm was obtained past cutting the epididymis with surgical blades and released onto a sterile clean drinking glass slide. The sperm was subsequently diluted with 2.ix% sodium citrate dehydrate solution, mixed thoroughly and covered with a 24 × 24 mm coverslip before examination under a stage contrast microscope at 200× magnification to evaluate the sperm progressive motility. The data were expressed as percentage of sperm progressive motion. Sperm viability was determined past staining with 1% eosin and 5% nigrosine in three% sodium citrate dehydrate solution co-ordinate to established procedures [68], [iv].

two.8. Serum testosterone

The serum total testosterone level was measured past ELISA method using DRG Elisa testosterone kit (ELISA Environmental impact assessment-1559, 96 Wells kit, DRG Instruments, GmbH, Marburg, Germany) according to the standard protocol supplied by the kit manufacturer.

2.nine. Measurement of nitric oxide (NO)

NO content in testes and epididymis supernatant was estimated in a medium containing 400 mL of 2% vanadium chloride (VCl₃) in 5% HCl, 200 mL of 0.1% North-(50-naphthyl)ethylene-diamine dihydrochloride, 200 mL of 2% sulfanilamide (in v% HCl). Afterward incubating at 37 °C for sixty min, nitrite levels, which corresponds to an estimative of levels of NO, were adamant spectrophotometrically at 540 nm, based on the reduction of nitrate to nitrite by VCl₃ [48]. Testes and epididymis nitrite and nitrate levels were expressed as nanomole of NO/milligram of protein.

2.10. Arginase activeness assay

The arginase activity of testes and epididymis from normotensive and hypertensive rats were assay every bit described past Mendez et al. [47]. Briefly, tissue lysate (fifty μL) was added into 75 μL of Tris–HCl (50 mmol/L, pH 7.5) containing 10 mmol/Fifty MnCl_ii. Heating the lysate at 55–60 °C for 10 min activated arginase. The hydrolysis reaction of 50-arginine by arginase was performed by incubating the mixture containing activated arginase with fifty μL of l-arginine (0.v mol/L, pH 9.7) at 37 °C for 1 h and was stopped by adding 400 μL of the acid solution mixture (H_iiSO₄:H_iiiPO₄:H_twoO = 1:3:seven). For calorimetric determination of urea, α-isonitrosopropiophenone (25 μL, 9% in absolute ethanol) was then added and the mixture was heated at 100 °C for 45 min. After placing the sample in the dark for x min at room temperature, the urea concentration was adamant spectrophotometrically past the absorbance at 550 nm measured with a microplate reader. The amount of urea produced, after normalization with protein, was used every bit an index for arginase activity.

two.eleven. Interpretation of antioxidant status

The correct testes and epididymis of each rat were homogenized in 50 mM Tris–HCl buffer (pH vii.four). The resulting homogenate was centrifuged at 10,000 ×chiliad for 15 min at four °C and the supernatant was subsequently collected for estimation of antioxidant status. Protein was measured by the Coomassie blue method according to [xviii] using serum albumin equally standard.

2.11.1. Determination of glutathione-S-transferase activity

Glutathione-S-transferase activity was determined co-ordinate to the method of [35] using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The assay reaction mixture consisted of 270 μL of a solution containing (20 mL of 0.25 M potassium phosphate buffer, pH 7.0, 10.5 mL of distilled water, and 500 μL of 0.1 Chiliad GSH at 25 °C), 20 μL of sample (i:l dilution), and 10 μL of 25 mM CDNB. The reaction was monitored for 5 min (30 s intervals) at 340 nm in a SpectraMax plate reader (Molecular Devices, CA, The states) and the data were expressed as μmol/min/mg protein using the molar extinction coefficient (ϵ) of nine.vi mM⁻¹ cm⁻¹ for CDNB cohabit.

2.11.2. Full thiol (T-SH) conclusion

Full thiol content was determined according to the method previously described by [27]. Briefly, the reaction mixture consisted 40 μL of testicular or epididymal homogenate, 10 μL of 10 mM DTNB and 0.1 M potassium phosphate buffer (pH 7.four) in a final volume of 200 μL. The mixture was incubated for 30 min at ambience temperature and so read the absorbance at 412 nm using a SpectraMax plate reader (Molecular Devices, CA, USA). A standard curve was plotted for each measurement using cysteine every bit a standard and the results expressed equally μmol/mg protein.

2.11.iii. Decision of non-protein thiols (NPSH)

NPSH levels were adamant by the method of [27]. Briefly, an aliquot of testicular or epididymal homogenate was mixed (ane:1) with 10% trichloroacetic acid. Subsequent to precipitation of protein, the resulting solution was centrifuged at 10,000 ×k for 5 min at 4 °C and the free —SH groups were adamant in the supernatant. The reaction mixture consisting 50 μL of sample, 450 μL phosphate buffer and 1.v mL of 0.ane mM of v′,5′-dithiobis 2-nitro benzoic acrid was incubated for 10 min at 37 °C. The absorbance was measured at 412 nm using a SpectraMax plate reader (Molecular Devices, CA, USA). NPSH levels were expressed equally μmol/mg of protein.

2.11.4. Reactive oxygen species (ROS) detection

ROS production was quantified by the 2′,vii′-dichlorodihydrofluorescin diacetate (DCFH-DA) method based on the ROS-dependent oxidation of DCFH-DA to DCF [71], [44]. Briefly, l μL of testes and epididymides homogenates, Tris–HCl buffer (10 mM; pH 7.iv) and DCFH-DA solution at final concentration of 50 μM were incubated in the dark for 30 min to allow the probe to exist incorporated into any membrane-leap vesicles, and the diacetate groups cleaved past esterases. Fluorescence of the samples was monitored at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a SpectraMax plate reader (Molecular Devices, CA, The states). Groundwork fluorescence was corrected by inclusion of parallel blanks. DCF levels were expressed as percentage of command.

ii.eleven.5. Lipid peroxidation

Lipid peroxidation was determined as the formation of thiobarbituric acid reactive substances (TBARS) during an acid-heating reaction co-ordinate to previously published written report [54]. Briefly, the reaction mixture consisting 200 μL of testes and epididymides homogenates or standard (0.03 mM MDA), 200 μL of 8.i% sodium dodecyl sulfate (SDS), 500 μL of 0.8% TBA and 500 μL of acetic acrid solution (2.five K HCl, pH iii.four) was heated at 95 °C for one h. The absorbance was measured at 532 nm using a SpectraMax plate reader (Molecular Devices, CA, U.s.a.). TBARS tissue levels were expressed equally μmol MDA/mg of protein.

2.12. Statistical assay

The statistical analysis used was one-way ANOVA, followed by Duncan'due south multiple range tests, p < 0.05 was considered to represent a meaning difference in both analyses used. All data were expressed as mean ± S.E.Grand.

iii. Results

3.i. Systolic blood pressure

In this study, the oral administration of 50-NAME by gavage was associated with a significant rise in the final systolic blood force per unit area (SBP) when compared with the normotensive control rats, validating the hypertensive model. However, we could discover that dietary supplementation of both turmeric and ginger as well as anti-hypertensive drug (atenolol) clearly possesses hypotensive consequence, causing a significant reduction of SBP in the L-NAME-induced hypertensive rats (Fig. i).

An external file that holds a picture, illustration, etc. Object name is gr1.jpg

Effects of dietary supplementation of turmeric and ginger on the final systolic blood pressure (SBP) measurements in control and L-Proper name-induced hypertensive rats. Data are presented as mean + SEM (n = 10). Bars with different letters are statistically different (p < 0.05). Command: Normotensive control rats placed on basal diet; Induced: Hypertensive rats placed on basal diet; Fifty-Proper noun + AT: Hypertensive rats placed on basal diet + atenolol (x mg/kg/solar day); RG Command: Normotensive rats placed on basal diet supplemented with 4% turmeric; RG + 50-Proper name: Hypertensive rats placed on basal diet supplemented with four% turmeric; WG Control: Normotensive rats placed on basal diet supplemented with four% ginger; WG + L-Proper noun: Hypertensive rats placed on basal nutrition supplemented with 4% ginger.

3.2. Body weight, absolute and relative organ weights

The body weight, absolute and relative organ weights of command and 50-NAME exposed rats are presented in Table 2. Following induction of hypertension with L-NAME, there was a significant decrease in the body weight of induced group (hypertensive rats) whereas no significant different was observed in testes and epididymides weights when compare to the control. Withal, pre-treatment with dietary supplementation of both rhizomes equally well as anti-hypertensive drug (atenolol) prevented a significant subtract in the trunk weight and did not alter testes and epididymides weights.

Table 2

Trunk weight, absolute and relative organ weights of L-NAME induced hypertensive rats treated with dietary supplementation with red and white ginger.

Treatment groups	Final torso weight (g)	Accented testis weight (g)	Absolute epididymis weight (g)	Relative testis weight (g)	Relative epididymis weight (g)
Control	365.0 ± 31.six^a	ane.68 ± 0.thirteen^a	0.27 ± 0.02^a	0.50 ± 0.06^a	0.08 ± 0.006^a
L-NAME	345.six ± 32.nine^b	1.51 ± 0.12^a	0.26 ± 0.02^a	0.45 ± 0.06^a	0.07 ± 0.01^a
L-Name + AT	373.7 ± eleven.9^a	ane.56 ± 0.09^a	0.26 ± 0.02^a	0.42 ± 0.03^a	0.07 ± 0.007^a
RG Normal	363.3 ± sixteen.9^a	1.76 ± 0.05^a	0.30 ± 0.03^a	0.48 ± 0.03^a	0.08 ± 0.008^c
RG + L-NAME	352.0 ± 20.9^a	1.88 ± 0.10^a	0.32 ± 0.02^a	0.52 ± 0.05^a	0.09 ± 0.006^a
WG Normal	355.iii ± 23.3^a	1.74 ± 0.17^a	0.25 ± 0.03^a	0.49 ± 0.04^a	0.07 ± 0.007^a
WG + 50-Proper noun	363.half-dozen ± 19.7^a	1.69 ± 0.xi^a	0.27 ± 0.03^a	0.46 ± 0.04^a	0.07 ± 0.01^a

three.three. Sperm progressive movement and viability

The motility of epididymal sperm in the hypertensive rats decreased significantly as compared to the control (normotensive rats). However, pre-treatment with dietary supplementation of both turmeric and ginger rhizomes and atenolol caused a significant recovery in the sperm motility when compared with the L-NAME-induced hypertensive rats (Table 3). However, sperm viability was not afflicted in all handling groups.

Table three

Sperm progressive motility, sperm viability and testosterone level in L-Name induced hypertensive rats treated with dietary supplementation with blood-red and white ginger.

Treatment groups	Sperm motion (%)	Sperm viability (%)	Testosterone (ng/dl)
Control	70.0 ± 6.3^a	82.iii ± 18.2^a	103.vii ± x.iv^a
L-NAME	43.3 ± 8.2^b	65.three ± two.9^a	34.8 ± 6.2^b
L-Proper noun + AT	60.0 ± sixteen.2^a	75.0 ± xvi.6^a	123.ane ± half dozen.1^a
RG Normal	56.7 ± viii.half dozen^a	75.0 ± xvi.7^a	232.nine ± 13.0^c
RG + Fifty-Proper noun	60.0 ± xi.9^a	66.seven ± 16.7^a	73.eight ± v.4^a
WG Normal	56.7 ± 11.vii^a	75.0 ± xvi.7^a	135.iv ± 6.7^a
WG + L-Name	57.v ± 4.3^a	eighty.0 ± 16.7^a	126.8 ± 9.two^a

iii.4. Testosterone concentration, Nitric oxide (NO) level and arginase activeness

Serum testosterone concentration significantly decreased in L-Name hypertensive animals. Nevertheless, dietary supplementation with turmeric and ginger significantly increased the testosterone level when compared with the hypertensive group (Tabular array 2). Nitric oxide (NO) level in the testes and epididymis were decreased in induced group (hypertensive rats) when compared with the control (normotensive) grouping. In the diet-supplemented hypertensive group the levels of NO were clearly elevated compared to the induced group (hypertensive rats) but were not significantly dissimilar from the command (normotensive animals) as presented in Fig. two. Testiscular and epididymal arginase activeness of hypertensive rats increased significantly when compared to the normotensive command rats (Fig. 3). Nevertheless, pre-treatment with dietary supplementation of ginger and turmeric rhizomes as well as positive command drug acquired a pregnant subtract in the arginase activity when compared with induced grouping (hypertensive rats).

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Effects of dietary supplementation of turmeric and ginger on the testicular and epididymal nitric oxide (NO) level in control and L-NAME-induced hypertensive rats. Data are presented every bit mean + SEM (n = 10). Bars with different letters are statistically different (p < 0.05). Command: Normotensive control rats placed on basal diet; Induced: Hypertensive rats placed on basal diet; L-Proper name + AT: Hypertensive rats placed on basal diet + atenolol (10 mg/kg/solar day); RG Control: Normotensive rats placed on basal diet supplemented with iv% turmeric; RG + L-Proper name: Hypertensive rats placed on basal nutrition supplemented with 4% turmeric; WG Control: Normotensive rats placed on basal diet supplemented with four% ginger; WG + 50-Name: Hypertensive rats placed on basal diet supplemented with iv% ginger.

An external file that holds a picture, illustration, etc. Object name is gr3.jpg

Effects of dietary supplementation of turmeric and ginger on the testicular and epididymal arginase activity in control and L-Proper noun-induced hypertensive rats. Data are presented as mean + SEM (n = 10). Bars with different letters are statistically unlike (p < 0.05). Control: Normotensive command rats placed on basal nutrition; Induced: Hypertensive rats placed on basal diet; 50-NAME + AT: Hypertensive rats placed on basal diet + atenolol (10 mg/kg/day); RG Control: Normotensive rats placed on basal diet supplemented with 4% turmeric; RG + 50-NAME: Hypertensive rats placed on basal diet supplemented with 4% turmeric; WG Control: Normotensive rats placed on basal diet supplemented with 4% ginger; WG + 50-NAME: Hypertensive rats placed on basal nutrition supplemented with 4% ginger.

iii.5. Testicular and epipidymal antioxidant condition

The antioxidant status of the testes and epididymis in normotensive and Fifty-Proper name induced hypertensive rats is presented in Fig. 4, Fig. v. Oral administration of Fifty-Proper noun drug resulted in a pregnant subtract in the GST activities every bit well as in GSH and T-SHs levels with concomitant summit in the ROS and TBARS production in the testes and epididymis of L-Proper name-induced hypertensive rats when compared with control (normotensive rats). However, pre-treatment with dietary supplementation of turmeric and ginger as well equally positive control drug acquired a significant increase in the GST activities also as in GSH and T-SHs levels with concomitant decrease in the ROS and TBARS production when compared with hypertensive rats.

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Effects of dietary supplementation of turmeric and ginger on the testicular and epididymal glutathione Due south-transferase (GST) activity, total thiol (T-SHs) and not-protein thiol (NPSH) or reduced glutathione (GSH) level in control and 50-Name-induced hypertensive rats. Data are presented as mean + SEM (n = 10). Confined with different letters are statistically different (p < 0.05). Command: Normotensive control rats placed on basal diet; Induced: Hypertensive rats placed on basal nutrition; Fifty-NAME + AT: Hypertensive rats placed on basal diet + atenolol (x mg/kg/day); RG Control: Normotensive rats placed on basal diet supplemented with 4% turmeric; RG + Fifty-Name: Hypertensive rats placed on basal diet supplemented with 4% turmeric; WG Control: Normotensive rats placed on basal diet supplemented with 4% ginger; WG + L-NAME: Hypertensive rats placed on basal diet supplemented with 4% ginger.

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Furnishings of dietary supplementation of turmeric and ginger on the testicular and epididymal reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) level in control and 50-NAME-induced hypertensive rats. Data are presented every bit hateful + SEM (n = ten). Bars with unlike letters are statistically different (p < 0.05). Control: Normotensive control rats placed on basal diet; Induced: Hypertensive rats placed on basal diet; L-NAME + AT: Hypertensive rats placed on basal nutrition + atenolol (x mg/kg/day); RG Command: Normotensive rats placed on basal nutrition supplemented with iv% turmeric; RG + L-NAME: Hypertensive rats placed on basal diet supplemented with four% turmeric; WG Control: Normotensive rats placed on basal nutrition supplemented with 4% ginger; WG + L-Proper noun: Hypertensive rats placed on basal diet supplemented with four% ginger.

4. Discussion

In the present study, we observed a meaning ascension in systolic blood pressure level after treatment with L-NAME (xl mg/kg bwt/day) past oral gavage. This outcome is in understanding with previously described studied past Balbinott et al. [16]. Still, dietary supplementation with ginger and turmeric also as treatment with a positive control drug (atenolol) caused a significant reduction of SBP in the hypertensive rats. This clearly indicates that both ginger varieties possess hypotensive effect. This is in agreement with [32], where they reported hypotensive effect of aqueous extract of ginger in normotensive rats. The clan of hypertension with increased incidence of male person infertility has been reported [26], [45], [72], [fifteen] merely their pathophysiological pathways are even so to exist clearly elucidated. The marked subtract in sperm motility and sperm count with concomitant elevated sperm abnormalities observed in the nowadays study indicates an adverse consequence of oxidative damage on male reproductive role in vivo as a consequence of oral assistants of L-Name drug to induced hypertension. The chronic inhibition of NO can bear upon sperm function and hence, availability of NO is an essential mediator in the male reproductive tracts [2]. Also, NO can act every bit a gratis radical scavenger, inactivating and fifty-fifty inhibiting production of superoxide anions [ten], [46], [24] which cause lipid peroxidation, a procedure which leads to functional impairment of spermatozoa [39].

The rest between antioxidant defense organization and ROS generation in the male reproductive system is required to maintain the regulation of normal sperm role/fertility [6], [xiv]. Moreover, we observed an imbalance between antioxidant defense system and ROS generation in testes and epididymis of hypertensive male person rats. This clearly is an indication of oxidative stress which has been linked to cause male infertility [14]. There was a significant elevation in the testicular and epididymal ROS and TBARS production in the hypertensive rats with a concomitant decrease in GST activeness, GSH and TSH levels. These observations could result in the inadequacy of the testes and epididymis antioxidant status to effectively mitigate induction of oxidative stress in hypertensive rats. The harm due to oxidative stress in testes and epididymis in hypertensive rats was evident by the elevated production of ROS and TBARS in the induced rats. Excessive generation of TBARS from lipid peroxidation may crusade over utilization of GSH. The level of GSH and T-SHs besides as GST action was decreased in the testes and epididymis in the nowadays study. The decrease in the GSH level suggests overutilization in the detoxification procedure in other to cope with oxidative stress while the decrease in GST activity may result from decrease substrate GSH or inhibition by increased free radicals in the testes and sperm of L-NAME treated rats. However, dietary supplementation with both ginger rhizomes finer prevented the decrease in GST activeness, GSH and TSH levels thereby resulting in a significant reduction in ROS and TBARS levels in testes and epididymides of L-NAME hypertensive rats. This observation may exist due to the protective role of phenolic acids and flavonoid compounds on testicular androgenesis and spermatogenesis [1], [38] which have already been characterized in the present constitute study as reported by Akinyemi et al. [9]. Also, the antioxidant effect and ability to forbid lipid peroxidation by the rhizomes [52] could be a possibility to prevent the induction of oxidative stress in the testis and epididymis of rats treated with L-NAME compound.

In male reproductive system, leydig cells are predominantly responsible for the biosynthesis and secretion of testosterone which is vital in the initiation and maintenance of spermatogenesis by affecting Sertoli prison cell androgen receptors [37], [62]. The reduction in the serum concentration of testosterone in the present study may result from oxidative damage in the testes of the 50-NAME hypertensive rats. Low levels of testosterone adversely affect spermatogenesis and tin pb to Sertoli cells dysfunction [69]. However, pre-treatment with dietary ginger and turmeric rhizomes respectively caused an increase in testosterone hormone. The increment in serum testosterone level, reported in this study, were in agreement with those obtained by Morakino et al. [49] and Moselhy et al. [50] where extract of Z. officinale possess pro-fertility backdrop in male rats which might be a product of both its potent antioxidant properties and androgenic activities. Moreover, Khaki et al. [74] reported that assistants of 100 mg/kg/day of ginger significantly increased sperm concentration, viability, movement and serum full testosterone in H_iiO₂ induced male infertility. This suggested that ginger varieties may be promising in enhancing male infertility induced by hypertensive condition.

Previous studies take demonstrated that reductions in blood flow to the testis could play an important role in the pathogenesis of male infertility via formation of hypo-spermatogenesis with consequent compromise in reproductive capability. The NO-cGMP pathway has been implicated to plays a cardinal office in the male sexual function via production of NO, a strong vasodilator [40]. Endothelium nitric oxide synthase (eNOS) utilizes L-arginine and oxygen as substrates to produce nitric oxide (NO) and citrulline. l-Arginine is besides utilized by another enzyme arginase, a metalloenzyme that catalyzes the hydrolysis of 50-arginine to produce 50-ornithine and urea. The primal role of arginine as a substrate for both nitric oxide synthase and arginase serves equally a potential signal of regulation for the NO/cGMP pathway such that an upwards-regulation of 1 enzyme leads to the downward-regulation of the other. In the present report, in that location was a meaning increase in the arginase activity in the testes and epididymides of L-NAME-treated rats when compared with command without L-Name administration (Fig. 3). This outcome is contrary to what has been previously published by Reisser et al. [57], where L-Proper noun inhibited arginase activity in vivo. The departure in arginase activeness could be due to experimental model or organ differences. L-Name was previously demonstrated to inhibit the activity of arginase in lysates from rat colon cancer cells and liver in vitro which was confirmed by in vivo in tumor nodules and liver [57]. Notwithstanding, the increase in arginase activeness could be due to the inhibition of eNOS activity equally a event of Fifty-Name treatment (strong inhibitor of NO production) thereby favouring arginase pathway. Furthermore, our issue was accompanied by significant subtract in the NO level in both the testes and epididymides of hypertensive rats. The reduction in the NO level has been shown to be associated with elevated vascular superoxide anion product and consequently, harm of vasodilation [55]. However, dietary ginger rhizomes treatment were able to cause an inhibition of arginase action leading to an increment in NO product in genital tissues in hypertensive rats, thus affecting male sexual function. The ability of the rhizomes to inhibit arginase activity in the present study is in line with Akinyemi et al. [eight], where dietary supplementation of two ginger varieties inhibits arginase activity in hypercholesterolemic rats showing that both rhizomes have inhibitory effects on arginase activity under pathological country. Nitric oxide (NO) is a potent vasodilator that plays a vital physiological/pharmacological touch in several diseases associated with vasoconstriction. Hence, the implication of the pharmacological benefits of ginger and turmeric rhizomes in the prevention of male infertility in hypertensive rats.

Thus, in mechanistic term, ginger rhizomes supplementation clearly ameliorated hypertension-mediated reproductive dysfunction via enhancement of NO bioavailability and macerated ROS formation which are associated with vascular endothelial dysfunction and hypertension. The significant reduction in oxidative stress has been shown to prevent the activation of diverse molecular mechanisms involved in vascular remodelling associated with hypertension, particularly NOXs in angiotensin signaling [58].

five. Decision

In conclusion, this study demonstrated that L-NAME-induced hypertension resulted in male reproductive dysfunction via alterations in the anti-oxidant status in the testes and epididymides, testosterone level and sperm motility. Still, dietary supplementation with turmeric or ginger rhizome was associated with restoration of systolic blood pressure, sperm motility, testosterone level and improvement of antioxidant status in the epididymides and testes of L-NAME-induced hypertensive rats. Therefore, nosotros can suggest that both rhizomes could be harnessed as functional foods to foreclose hypertension-mediated male reproductive dysfunction.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

Ayodele Jacob Akinyemi is a beneficiary of 2014CNPq/TWAS sandwich postgraduate fellowship. The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Rio Grande practise Sul (FAPERGS), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Academy of Sciences for the Developing World (TWAS) for their back up towards this report.

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