ORIGINAL ARTICLE
Chemotherapeutic efficacy of an ethanolic Moringa oleifera leaf extract against chromium-induced testicular toxicity in rats K. M. Sadek Department of Biochemistry, Faculty of Veterinary Medicine, Damanhur University, Damanhur, Egypt
Keywords Antioxidant—chromium—immunostimulant—Moringa oleifera—sperm parameters Correspondence Kadry M. Sadek, PhD, Department of Biochemistry, Faculty of Veterinary Medicine, Damanhur University, Komhamada, Behera, Egypt. Tel.: (+2)045-3693078; Fax: (+2)045-3591018; E-mail: [email protected]
Accepted: September 30, 2013 doi: 10.1111/and.12196
Summary This study was conducted to determine the mechanism underlying the chemotherapeutic efficacy of an ethanolic Moringa oleifera leaf extract (MOLEE) against chromium-induced impairments of rat testes using biochemical methods. Twenty male Wistar rats were divided into four groups of five animals each. Group I (control), group II injected potassium dichromate (8 mg kg 1) i.p., group III gastrogavaged MOLEE (500 mg kg 1) p.o. and group IV received (potassium dichromate plus MOLEE) by the same doses for 60 days. After the blood samples were collected, the animals were sacrificed to determine the testicular antioxidant status and sperm parameters. The chromiumtreated group exhibited a significant decrease in testicular antioxidant enzymatic activities, local immunity and sperm parameters as well as an increase in inflammatory markers when compared with the control and MOLEE-treated group. However, concurrent administration of chromium and MOLEE significantly ameliorated the chromium effects on the sperm parameters, local immunity, inflammatory markers and antioxidant enzymatic activities compared with rats exposed to chromium alone. This study concludes that chronic exposure to chromium produces clear testicular toxicity, which can either be prevented or at least decreased by concomitant administration of MOLEE. Interestingly, the metal ion chelation could attribute partly the antioxidant activities of MOLEE.
Introduction The pervasiveness of heavy metals in the environment has led to an increase in the incidence of the human organ toxicity. Arsenic, cadmium, lead, mercury, chromium and their inorganic compounds are possibly the most potentially toxic metals in the environment. These metals have many industrial uses, which increase the possibility of human exposure (Leonard et al., 2004). The chromium compounds have been reported to exert toxic effects on body tissues (Sugiyama, 1992). There were at least three forms of chromium compounds have been identified, chromium III, IV and VI with the two latter being more toxic than the former because of their easy permeation at physiological pH through the permease system (DeFlora & Wetterhahn, 1989). The same authors reported that inside the cells, chromium (VI) is reduced to reactive intermediates such as chromium (V), (IV) and finally to the more stable chromium (III) by cellular reductants including © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
glutathione (GSH), cysteine, ascorbic acid and riboflavin as well as NADPH-dependent flavoenzymes such as microsomal cytochrome P450. Chromium contaminates the testes by generating reactive radicals, which results in cellular damage, such as a reduction of enzyme activity and damage to the lipid bilayer and DNA (Stohs & Bagchi, 1995), resulting in amplified oxidative stress damage in the membranes, proteins and DNA in the sperm. The reactive oxygen species (ROS) contribute to cellular ageing, mutagenesis, carcinogenesis and coronary heart disease (Heim et al., 2002). The antioxidants provide protection against degenerative diseases including cancer, coronary heart and Alzheimer’s disease (Iqbal & Bhanger, 2006). Moringa oleifera (Moringaceae) is one of the 14 species from the Moringaceae family, which is native to India, Africa, Arabia, Southeast Asia, South America and the Pacific and Caribbean Islands (Iqbal & Bhanger, 2006). The Moringa oleifera tree (also known as drumstick tree) is a rapidly growing deciduous shrub or small desert tree 1047
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approximately 13 m tall and 35 cm in diameter with an umbrella-shaped open cap (Anjorin et al., 2010). This plant was utilised by the ancient Romans, Greeks and Egyptians. Almost all parts of the plant are used culturally for its nutritional value and supposed medicinal properties and for flavouring as a vegetable and seed (Kasolo et al., 2010). The leaves of Moringa oleifera can be eaten fresh or cooked, and reports have shown that it can be stored as a dried powder for many months without any major loss of its nutritional value (Arabshahi et al., 2007). Moringa oleifera leaves contains specific plant compounds that have been demonstrated to possess the same powerful antioxidant ability as vitamins C, E and A from oranges, pomegranates and carrots, respectively, as well as to contain caffeoylquinic acids, carotenoids-lutein, alpha- and beta carotene, kaempferol, quercetin and rutin (Siddhuraju & Becker, 2003; Aslam et al., 2005; Smolin & Grosvenor, 2007). Moringa oleifera oil and its micronutrients contain antitumor, antioxidant, antiepileptic, antidiuretic, antiinflammatory, hepatoprotective and antidiabetic properties (Hsu et al., 2006; Sreelatha & Padma, 2010). Because of aforementioned medicinal properties of Moringa oleifera, the objective of this study was to investigate the chemotherapeutic efficacy of an ethanolic Moringa oleifera leaf extract (MOLEE) against chromium-induced testicular toxicity and the mechanism responsible for this action, which had not been previously determined.
Approximately 1 kg of fresh tender leaves of Moringa oleifera collected during January–February was used for the study. The plant was identified and authenticated by Dr. Ahmed M Ismail (Faculty of Agriculture, Damanhour University, Egypt). The plant material was air-dried at room temperature. The dried leaves were grounded to a fine powder and stored in an air tight container. Three hundred grams of the dry powder obtained was soaked in 95% ethanol for 24 h in a percolator. After 24 h, it was allowed to percolate slowly and the extract was collected in Petri dishes. The extract was concentrated in vacuum using a rotary flash evaporator. There was a net yield of 30.5 g of the concentrated extract (10.16% w/w). The crude extract was suspended in distilled water before administration to rats on the subsequent day.
Materials and methods
Animal grouping and treatment
Animals Twenty male Wistar rats (Rattus norvegicus) weighing approximately 140–170 g and aged 8 weeks were used for this study and were obtained from the College of Science at Tanta University in Egypt. The animals were housed in stainless steel cages at standard atmospheric temperature (25 5 °C) with a 12-h light/dark cycle and were fed a standard diet and allowed access to water ad libitum. The animals were acclimatised for 2 weeks in their new environment prior to experimentation. All of the animals received humane care in compliance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the study protocol was approved by the local authorities. Chemicals Potassium dichromate (K2Cr2O7), nitric acid, 5, 5-dithiobis(2-nitrobenzoic acid; DTNB, Ellman’s reagent) and 1chloro-2, 4-dinitrobenzene (CDNB) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fresh leaves of the drumstick tree (Moringa oleifera) were obtained 1048
from a local garden at the Badr Centre in the Behera governorate in Egypt. Atomic spectrophotometry standard solutions for iron (Fe in 3% nitric acid) and copper (Cu in 3% nitric acid) were purchased from Ricca Chemical Company (Fenton, MO, USA). Reduced glutathione (GSH) and thiobarbituric acid (TBA) were purchased from Fluka (Buchs, Switzerland). All the other reagents were of analytical, high-performance liquid chromatography (HPLC), or the best available pharmaceutical grade. Preparation of the extract
The rats were randomly divided into the following experimental groups: I (control), II (chromium-treated), III (MOLEE-treated) and IV (chromium + MOLEE combination treated), with each group containing five animals. The chromium-treated, MOLEE-treated and chromium + MOLEE combination group received potassium dichromate (8 mg kg 1, i.p.), MOLEE (500 mg kg 1) p.o. or both daily, respectively, whereas the control group was not given any supplementation. The dose of Potassium dichromate and MOLEE were selected on the basis of previous reports of (Dey & Roy, 2009 and Das & Kanodia, 2012) respectively. The experiment lasted for 60 days. Blood sampling, animal sacrifice and organ harvesting At the end of the experiment and after a night of fasting, blood samples (2 ml) were collected using the retro-orbital plexus method into heparinised tubes. The plasma was separated by centrifugation at 704 g for 15 min, and the samples were frozen at 20 °C until further analysis. The plasma was used to determine the total amount of protein (Peters, 1968), albumin (Doumas et al., 1971), globulin (Coles (1974), C-reactive protein (CRP; Fisher © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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& Nakamura, 1976), Total Antioxidant Capacity (TAC; Koracevic & Koracevic, 2001), IgA (Ojala et al., 1981) and testosterone (TT) (Chen et al., 1991). After the rats were sacrificed by cervical dislocation, the abdominal cavity was opened up through a midline abdominal incision to expose the reproductive organs. The testes were detached, cleared free of the surrounding tissue and weighed with an electronic analytical balance. The testes were used to determine the malonaldehyde (MDA; Placer et al., 1966), superoxide dismutase (SOD; Misra & Fridovich, 1972), catalase (CAT; Sinha, 1971), (GSH; Sedlack & Lindsay, 1968), glutathione S-transferase (GST; Habig et al. (1974) and glutathione peroxidase (GPx; Chiu et al., 1976). A VARIAN spectroAA-20 plus GTA-96 flameless graphite furnace AAS was used to quantify Cu and Fe levels in the testes.
Results Testicular oxidative stress The activities of the testicular antioxidant enzymes SOD, CAT and TAC and GSH in the chromium-treated group decreased significantly (P < 0.05) when compared with the control and MOLEE-treated group. However, administration of chromium induced no significant changes in the activities of GPx and GST when compared with the control group. Following co-administration with MOLEE, there was an improvement in the testicular antioxidant status when compared with the chromium-treated group (Tables 1 and 2). The testicular content of MDA in the chromium-treated group was significantly elevated when compared with the control and MOLEE-treated group (P < 0.05). There was a significant decrease in the MDA levels of the chromium + MOLEE combination group when compared with the chromium-treated group (P < 0.05; Table 2).
Sperm characteristics As described by Saalu et al. (2007), sperm motility, concentration and progressive motility were determined by removing the caudal part of epididymis and placing it in a beaker containing 1 ml of physiological saline solution, after which, each section was quickly incised with a pair of sharp scissors and left for a few minutes to release its spermatozoa into the saline solution. Semen drops were placed on a slide, and two drops of warm 2.9% sodium citrate were added. The slide was covered with a cover slip and examined with a light microscope using a 409 objective to determine sperm motility. The sperm concentration was performed using an enhanced Neubauer hemocytometer. The sperm morphology was carried out by staining technique.
Trace mineral levels Compared with the control animals, the testicular Fe and Cu contents were significantly higher (P < 0.05; Table 2) in chromium-treated rats. The MOLEE + chromium-treated rats had significantly decreased levels of Fe and Cu when compared with the group treated with chromium alone. Body weight changes Table 3 reported that rats in the chromium-treated group lost body weight when compared with rats in both the control and MOLEE-treated group. The rats in the chromium + MOLEE combination group had a significant increase in body weight when compared with the chromium-treated group (P < 0.05), it was just portrayed in the Table 3.
Statistical analysis The results are expressed as the mean SE. The analysis of variance for the obtained data was performed using SAS (2002). Values of P < 0.05 were considered to be statistically significant.
Table 1 Effects of chromium and/or MOLEE on TAC, SOD, CAT, GPx and GST in rats
Groups Group I (control) Group II (chromium) Group III (MOLEE) Group IV (chromium/ MOLEE)
1
TAC (mM l ) 0.98 0.38 2.79 1.85
c
0.13 0.08d 0.24a 0.16b
SOD (U mg protein) 89.65 67.65 119.91 90.37
1
CAT (K s protein) b
5.43 6.56c 6.77a 4.49b
96.47 52.34 95.63 89.46
1
mg
a
3.35 3.87cd 4.42a 4.54ab
1
GPX Activity IU g Wet tissue 69.42 63.38 69.74 67.86
a
1.72 3.19ab 2.42a 2.23a
1
GST Activity mol CDNB per min 1 g 396.1 397.5 479.7 433.7
1
Wet tissue
c
7.42 6.61c 9.87a 6.37b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract; TAC, total antioxidant capacity; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferase.
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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Table 2 Effects of chromium and/or MOLEE on MDA, GSH, testicular Fe and Cu levels in rats 1
Groups
MDA (nmol g
Group Group Group Group
119.5 5.26 158.44 4.67a 87.3 5.75c 121.5 6.71b
I (control) II (chromium) III (MOLEE) IV (chromium/MOLEE)
wet tissue)
GSH (lmol g
b
68.34 43.43 89.28 73.36
1
wet tissue)
Fe (lg g
bc
2.68 3.78de 3.69a 4.64b
106.68 215.77 83.68 177.78
1
wet tissue) c
1.39 2.33a 1.39d 1.81b
Cu (lg g 33.16 67.55 31.96 52.89
1
wet tissue)
1.21c 0.96a 1.15c 1.37b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract; MDA, malonaldehyde; GSH, reduced glutathione; Fe, iron; Cu, copper.
Table 3 Effects of chromium and/or MOLEE on body weight, body weight difference, testes weight, testes weight/body weight ratio and plasma testosterone level in rats
Groups
Initial body weight (g)
Group Group Group Group
160.8 172.5 165.3 170.5
I (control) II (chromium) III (MOLEE) IV (chromium/MOLEE)
4.7cd 5.4a 4.9bc 5.2ab
Final body weight (g) 169.8 143.3 162.7 161.9
4.3a 2.0cd 3.9b 5.6b
Body weight difference (g)
Testis weight (g)
9.0b 29.2a 2.6c 8.6b
1.61 0.53 1.78 1.27
0.5ab 0.3de 0.5a 0.2c
Testis weight per Body weight Ratio
Testosterone (ng ml 1)
0.0095ab 0.0037de 0.010a 0.0078c
5.63 1.39 5.59 3.45
1.1a 0.2c 1.7a 0.6b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract. Table 4 Effects of chromium and/or MOLEE on total protein, albumin and globulin, IgA and CRP in rats Groups
Total protein (g dl 1)
Group Group Group Group
7.92 7.69 8.27 8.38
I (control) II (chromium) III (MOLEE) IV (chromium/MOLEE)
0.33ab 0.21bc 0.35a 0.29a
Albumin (g dl 1) 4.60 3.97 4.57 4.63
0.33a 0.31b 0.36a 0.28a
Globulin (g dl 1) 3.32 3.72 3.70 3.75
0.08ab 0.06a 0.13a 0.05a
IgA (mg dl 1) 98.66 87.45 104.83 102.58
5.43ab 4.56c 4.52a 5.18a
CRP (mg l 1) 5.82 41.91 6.23 31.46
2.51cd 3.78a 1.21cd 2.69b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract; IgA, immunoglobulin A; CRP, C-reactive protein.
Changes in testis weight Table 3 revealed that the testicle weight and testicle weight to body weight ratios of the rats in the chromium-treated group were significantly lower than those of the control, MOLEE-treated and chromium + MOLEE combination group (P < 0.05). Testosterone level Table 3 showed a significant decrease in the TT levels of the chromium-treated group when compared with the control and MOLEE-treated group (P < 0.05). Following co-treatment with MOLEE, the TT levels significantly increased when compared with the chromium-treated group (P < 0.05).
MOLEE-treated group (P < 0.05). However, the chromium + MOLEE combination group showed a significant increase in the total protein and albumin levels when compared with the chromium-treated group (P < 0.05). Immunoglobulin A and C-reactive protein concentration Table 4 showed a significant decrease in the IgA levels and increase in the CRP levels of the chromium-treated group when compared with the control and MOLEE-treated group (P < 0.05). When co-treated with MOLEE, the level of IgA increased and the level of CRP decreased significantly (P < 0.05) when compared with the group treated with chromium alone.
Sperm parameters Changes in total protein, albumin and globulin Table 4 revealed a significant decrease in the total protein and albumin of the chromium-treated group with no effect on globulin when compared with the control and 1050
Morphology As shown in Table 5, the sperm morphology of the control, MOLEE-treated and chromium + MOLEE combination group was normal when compared with the © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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Okpuzor J, Ogbunugafor H, Kareem GK (2009) Antioxidative properties of ethyl acetate fraction of Globimetula branuii in normal rats. J Biol Sci 9:470–475. Pakade V, Cukrowska E, Chimuka L (2013) Comparison of antioxidant activity of Moringa oleifera and selected vegetables in South Africa. S Afr J Sci 109:1–5. Peters TJ (1968) Proposals for standardization of total protein assays. Clin Chem 14:1147. Placer ZA, Crushman L, Son BC (1966) Estimation of product of lipid peroxidation (malondialdehyde) in biochemical system. Anal Biochem 16:359–364. Pourmorad F, Hosseinimehr SJ, Shahabimajd N (2006) Antioxidant activity, phenol and flavonoids content of some selected Iranian plants. Afr J Biotechnol 5:1142–1145. Rakesh S, Singh VJ (2010) In vivo antioxidant activity of Moringa oleifera leaf and pod extracts against carbon tetra chloride induced liver damage in albino mice. J Chem Pharm Res 2:275–283. Saalu LC, Adesanya AO, Oyewopo AO, Raji Y (2007) An evaluation of the deleterious effect of unilateral cryptorchidism on the contralateral normally descended testis. Sci Res Essays 2:74–78. Saalu LC, Osinubi AA, Akinbami AA, Yama OE, Oyewopo AO, Enaibe BU (2011) Moringa oleifera Lamarck (drumstick) leaf extract modulates the evidences of
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hydroxyurea–induced testicular derangement. Int J Appl Res Nat Prod 4:32–45. SAS (2002) User’s Guide Statistical Analysis system, Version 6, 4th edn. SAS Institute, Cary, NC, USA. Sedlack J, Lindsay RH (1968) Estimation of total protein bound and non protein sulfhydryl groups in tissues with Ellman reagent. Anal Biochem 86:271–278. Siddhuraju P, Becker K (2003) Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam) leaves. J Agric Food Chem 51:2144– 2155. Sinha KA (1971) Calorimetric assay of catalase. Anal Biochem 47:389–394. Smolin LA, Grosvenor MB (2007) Nutrition Science and Applications, 4th edn. John Wiley & Sons Inc., New York, pp 123–125. Sreelatha S, Padma PR (2010) Antioxidant activity and total phenolic content in Moringa oleifera leaves in two stages of maturity. Plant Foods Hum Nutr 64:303–311. Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18:321–336. Sugiyama M (1992) Role of physiological antioxidant in chromium (VI)-induced cellular injury. Free Radic Biol Med 12:397–407.
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induced disturbances in Cu/Fe homeostasis reflected in increased their levels in the testes. The mechanism related to these disturbances has not been clearly defined. Simultaneous treatment with MOLEE, which has been shown to contain bioflavonoids and other effective antioxidants (Saalu et al., 2011), resulted in a notable amelioration of the unbalanced sperm parameters of the testes in the chromium + MOLEE combination group. Pre-treatment with the MOLEE has been shown to shield the testes from a variety of toxic substances (Stohs & Bagchi, 1995). Siddhuraju & Becker (2003) and Saalu et al. (2011) reported that Moringa oleifera contains fundamental antioxidants and phenolic compounds that help to protect against oxidative changes brought about by toxic materials and certain antineoplastic agents. Saalu et al. (2011) showed the antioxidative properties of Moringa oleifera and its ability to increase the antioxidant capability. Similarly, the phenolic compounds in Moringa oleifera are a class of antioxidant agents that act as free radical terminators and are involved in the impedance of oxidative degradation of lipids (Pourmorad et al., 2006). Pre-treatment with Moringa oleifera hydro-alcoholic leaf and aqueous pod extracts improved the SOD, CAT, glutathione and peroxidase levels significantly and reduced lipid peroxidation in CCl4-induced hepatocellular damage and high-fat diet-induced nonalcoholic fatty liver disease respectively (Rakesh & Singh, 2010; Nilanjan et al., 2012). The total phenolic content and the total flavonoid content of the Moringa leaves were almost two and three times that of the vegetables, respectively (Pakade et al., 2013). It is known that phenolic and flavonoid contents are directly linked to antioxidant properties (Siddhuraju & Becker, 2003). Flavonoids can exert their antioxidant activities by various mechanisms, such as scavenging or quenching free radicals and inhibiting enzymatic systems responsible for free radical generation (Lukacinova et al., 2008). Unexpectedly, the decreased testicular Fe++ and Cu+ by MOLEE in this study added another mechanism of chelating metal ions. Thereby, chemotherapeutic action of MOLEE against chromium-induced testicular toxicity could be attained partly by mechanism related to mineral homeostasis or binding the redox-active metals such as Fe and Cu. Antioxidant properties also can be due to the presence of carotenoids, alkaloids, and proanthocyanidins in this plant. The increased antioxidant enzymatic activity after administration of MOLEE could partially be due to the plant’s ability to prevent activity impairment. MOLEE effectively scavenges the free radicals generated and subsequently reduces the destructive effects of oxidative stress as well as decreases the need of antioxidant enzymes to counteract the increased free radicals (Arabshahi et al., 2007). The elevated activity of SOD and CAT may suggest an induction of the enzymes by MOLEE in the rats. SOD 1052
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and CAT are both inducible enzymes whose production can be stimulated. Previous reports support this finding; including applications with black rice extract (Chiang et al., 2006) and the ethyl acetate fraction of the Globimetula branuii leaf extract (Okpuzor et al., 2009). Increased activity of the enzymes could mean enhanced antioxidative capacity of the animals (Lobo et al., 2010). However, high vitamin C content in the leaves of Moringa oleifera has been reported (Siddhuraju & Becker, 2003; Aslam et al., 2005). Vitamin C can act as a pro-oxidant as well as an antioxidant. It could be suggested that vitamin C may play a pro-oxidant role in the observed induction of antioxidant enzymes. The decreased lipid peroxidation observed in this study correlates with the induction of antioxidant enzymes above basal levels by MOLEE. Regarding the anti-inflammatory effect of Moringa oleifera, Ezeamuzie et al. (1996) demonstrated that the roots of Moringa oleifera contain anti-inflammatory properties that may be useful in the treatment of the acute inflammatory conditions such as carrageenaninduced oedema. Amelioration of airway inflammation associated with ovalbumin treatment in guinea pigs could be possible with the potent anti-inflammatory activity of Moringa oleifera bioactive compounds (Mahajan et al., 2009). The ethanolic extract of the Moringa oleifera leaves showed significant reduction of experimentally induced colitis, which may be attributed to its anti-inflammatory property (Das & Kanodia, 2012). This result could provide a rationalisation for the observations in this study as to why the group treated with chromium and MOLEE combination showed enhanced sperm parameters and increases in antioxidant enzymes activity and IgA as well as decreased testicular MDA and CRP inflammatory marker levels. In this regard, the results of this study are consistent with another report (D’cruz, 2005) which showed that the sperm cytoplasm contained low concentrations of free radical scavenging enzymes and, as a result, that an increase in the antioxidant enzyme system levels after Moringa oleifera treatment can favour reproductive potential. Regarding the effects of Moringa oleifera on total protein and albumin levels, this study showed that MOLEE increased the total amount of protein and albumin levels in chromium-treated rats, which further indicated the nutritional properties of Moringa oleifera and its hepatoprotection efficacy. Herein, the globulin level in Chromium- and/or MOLEE-treated group remained unchanged compared with control group. There was shortage in literature which discussed the effects of chromium and moringa oleifera on serum globulin level. The increased IgA in MOLEE-treated group and decreased IgA in chromium-treated group might be associated with decreased or increased in other subclasses of globulin respectively. © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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Conclusion The increased oxidative stress and inflammation and decreased local immunity resulting from chromium intoxication in testicular tissue might be a cause of abnormal sperm parameters. However, ethanolic MOLEE exerted a therapeutic effect against the toxicity, which was shown by the improvement in the sperm parameters. The antioxidant, anti-inflammatory and immunostimulatory potential of Moringa oleifera was reflected by decreases in the oxidative stress and CRP levels and increased IgA levels in the chromium + MOLEE-treated combination group. The metal ion chelation could partly attribute the antioxidant activities of MOLEE. Thus, Moringa oleifera could provide multiple health benefits to humans by having various biological activities. Acknowledgements The author gratefully acknowledges to Mr. Abdel Rahman Saleh for providing the plant leaves and to Dr. Ahmed M. Ismail for plant identification and authentication. References Anjorin ST, Ikokoh PS, Okolo A (2010) Mineral composition of Moringa oleifera leaves pods and seeds from two regions in Abuja. Nigeria. Int J Agric Biol 12:1560–1569. Arabshahi DS, Devi V, Urooj A (2007) Evaluation of antioxidant activity of some plant extracts and their heat, pH and storage stability. Food Chem 100:1100–1105. Aslam MF, Anwar R, Nadeem U, Rashid TG, Kazi A, Nadeem M (2005) Mineral composition of Moringa oleifera leaves and pods from different regions of Punjab, Pakistan. Asian J Plant Sci 4:417–421. Chen A, Bookstein JJ, Meldrum DR (1991) Diagnosis of a testosterone-secreting adrenal adenoma by selective venous catheterization. Fertil Steril 55:1202–1203. Chen F, Ding M, Castranova V, Shi XL (2001) Carcinogenic metals and NF-kappa B activation. Mol Cell Biochem 222:159–171. Chiang AN, Wu HL, Yen HI (2006) Antioxidant effects of black rice extract through the induction of superoxide dismutase and catalase activities. Lipids 41:797–803. Chiu D, Fredrick H, Tappel AL (1976) Purification and properties of rat lung soluble glutathione peroxidase. Biochim Biophys Acta 445:558–566. Coles EH (1974) Veterinary Clinical Pathology, 1st edn. W. B Saunders Co. Philadelphia, London, Toronto, pp 212–213. Das S, Kanodia L (2012) Effect of ethanolic extract of leaves of moringa olifera lam. On acetic acid induced colitis in albino rats. Asian J Pharm Clin Res 5:110–114. D’cruz M (2005) Effect of piperine on the epididymis of adult male rats. Asian J Androl 7:363–368.
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DeFlora S, Wetterhahn KE (1989) Mechanism of chromium (VI) metabolism and genotoxicity. Life Chem Rep 7:169– 244. Dey SK, Roy S (2009) Effect of chromium on certain aspects of cellular toxicity. Iranian J Toxicol 2:260–267. Doumas BT, Watson WA, Biggs HG (1971) Determination of serum albumin. J Clin Chem Acta 31:87–89. Ezeamuzie IC, Ambakederemo AW, Shode FO, Ekwebelem SC (1996) Anti-inflammatory activity of Moringa oleifera (Moringaceae). Int J Pharmacogn 34:207–212. Fisher CL, Nakamura R (1976) Latex serology test for detection of C-Reactive protein. Am J Clin Pathol 66:840– 847. Habig WH, Pabst MI, Jakoby WB (1974) Glutathione Stransferase: the first enzymatic step in mercapturic acid formation. J Biol Chem 249:1730–1739. Heim KE, Tagliaferro AR, Bobilya DJ (2002) Flavonoid antioxidants: chemistry, metabolism and structure activity relationships. J Nutr Biochem 13:572–584. Hsu R, Midcap S, Lucienne de Witte AL (2006) Moringa oleifera, medicinal and socio-economic uses. Int J Econ Bot 5:1–25. Iqbal S, Bhanger M (2006) Effect of season and production location on antioxidant activity of Moringa oleifera leaves grown in Pakistan. J Food Compost Anal 19:544–551. Kasolo JN, Bimenya GS, Ojok L, Ochieng J, Ogwal-Okeng JW (2010) Phytochemicals and uses of Moringa oleifera leaves in Ugandan rural communities. J Med Plant Res 4:753–757. Koracevic D, Koracevic G (2001) Colorimetric method for determination of total antioxidant capacity. J Clin Pathol 54:356–361. Leonard SS, Harris GK, Shi XL (2004) Metal-induced oxidative stress and signal transduction. Free Radic Biol Med 37:1921–1942. Lobo VC, Phatak A, Chandra N (2010) Antioxidant and free radical scavenging activity of Hygrophila schulli (Buch.Ham.) Almeida and Almeida seeds. Adv Biores 1:72–78. Lukacinova A, Mojzis J, Benacka R, Keller J, Maguth T, Kurila P (2008) Preventive effects of flavonoids on alloxan-induced diabetes mellitus in rats. Acta Vet Brno 77:175–182. Mahajan SG, Banerjee A, Chauhan BF, Padh H, Nivsarkar M, Mehta AA (2009) Inhibitory effect of n-butanol fraction of Moringa oleifera Lam. Seeds on ovalbumin-induced airway inflammation in a guinea pig model of asthma. Int J Toxicol 28:519–527. Misra HP, Fridovich I (1972) The role of superoxide anion in the auto-oxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175. Nilanjan D, Kunal S, Santinath G, Bernard F, Sanjit D (2012) Moringa oleifera leaf extract prevent early liver injury and restores antioxidant status in mice fed with high fat diet. Indian J Exp Biol 50:404–412. Ojala K, Weber TH, Kauhala A (1981) Immunoturbidimetric haptoglobin determination. J Clin Chem Clin Biochem 19:788.
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Okpuzor J, Ogbunugafor H, Kareem GK (2009) Antioxidative properties of ethyl acetate fraction of Globimetula branuii in normal rats. J Biol Sci 9:470–475. Pakade V, Cukrowska E, Chimuka L (2013) Comparison of antioxidant activity of Moringa oleifera and selected vegetables in South Africa. S Afr J Sci 109:1–5. Peters TJ (1968) Proposals for standardization of total protein assays. Clin Chem 14:1147. Placer ZA, Crushman L, Son BC (1966) Estimation of product of lipid peroxidation (malondialdehyde) in biochemical system. Anal Biochem 16:359–364. Pourmorad F, Hosseinimehr SJ, Shahabimajd N (2006) Antioxidant activity, phenol and flavonoids content of some selected Iranian plants. Afr J Biotechnol 5:1142–1145. Rakesh S, Singh VJ (2010) In vivo antioxidant activity of Moringa oleifera leaf and pod extracts against carbon tetra chloride induced liver damage in albino mice. J Chem Pharm Res 2:275–283. Saalu LC, Adesanya AO, Oyewopo AO, Raji Y (2007) An evaluation of the deleterious effect of unilateral cryptorchidism on the contralateral normally descended testis. Sci Res Essays 2:74–78. Saalu LC, Osinubi AA, Akinbami AA, Yama OE, Oyewopo AO, Enaibe BU (2011) Moringa oleifera Lamarck (drumstick) leaf extract modulates the evidences of
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hydroxyurea–induced testicular derangement. Int J Appl Res Nat Prod 4:32–45. SAS (2002) User’s Guide Statistical Analysis system, Version 6, 4th edn. SAS Institute, Cary, NC, USA. Sedlack J, Lindsay RH (1968) Estimation of total protein bound and non protein sulfhydryl groups in tissues with Ellman reagent. Anal Biochem 86:271–278. Siddhuraju P, Becker K (2003) Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam) leaves. J Agric Food Chem 51:2144– 2155. Sinha KA (1971) Calorimetric assay of catalase. Anal Biochem 47:389–394. Smolin LA, Grosvenor MB (2007) Nutrition Science and Applications, 4th edn. John Wiley & Sons Inc., New York, pp 123–125. Sreelatha S, Padma PR (2010) Antioxidant activity and total phenolic content in Moringa oleifera leaves in two stages of maturity. Plant Foods Hum Nutr 64:303–311. Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18:321–336. Sugiyama M (1992) Role of physiological antioxidant in chromium (VI)-induced cellular injury. Free Radic Biol Med 12:397–407.
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
Chemotherapeutic efficacy of an ethanolic Moringa oleifera leaf extract against chromium-induced testicular toxicity in rats K. M. Sadek Department of Biochemistry, Faculty of Veterinary Medicine, Damanhur University, Damanhur, Egypt
Keywords Antioxidant—chromium—immunostimulant—Moringa oleifera—sperm parameters Correspondence Kadry M. Sadek, PhD, Department of Biochemistry, Faculty of Veterinary Medicine, Damanhur University, Komhamada, Behera, Egypt. Tel.: (+2)045-3693078; Fax: (+2)045-3591018; E-mail: [email protected]
Accepted: September 30, 2013 doi: 10.1111/and.12196
Summary This study was conducted to determine the mechanism underlying the chemotherapeutic efficacy of an ethanolic Moringa oleifera leaf extract (MOLEE) against chromium-induced impairments of rat testes using biochemical methods. Twenty male Wistar rats were divided into four groups of five animals each. Group I (control), group II injected potassium dichromate (8 mg kg 1) i.p., group III gastrogavaged MOLEE (500 mg kg 1) p.o. and group IV received (potassium dichromate plus MOLEE) by the same doses for 60 days. After the blood samples were collected, the animals were sacrificed to determine the testicular antioxidant status and sperm parameters. The chromiumtreated group exhibited a significant decrease in testicular antioxidant enzymatic activities, local immunity and sperm parameters as well as an increase in inflammatory markers when compared with the control and MOLEE-treated group. However, concurrent administration of chromium and MOLEE significantly ameliorated the chromium effects on the sperm parameters, local immunity, inflammatory markers and antioxidant enzymatic activities compared with rats exposed to chromium alone. This study concludes that chronic exposure to chromium produces clear testicular toxicity, which can either be prevented or at least decreased by concomitant administration of MOLEE. Interestingly, the metal ion chelation could attribute partly the antioxidant activities of MOLEE.
Introduction The pervasiveness of heavy metals in the environment has led to an increase in the incidence of the human organ toxicity. Arsenic, cadmium, lead, mercury, chromium and their inorganic compounds are possibly the most potentially toxic metals in the environment. These metals have many industrial uses, which increase the possibility of human exposure (Leonard et al., 2004). The chromium compounds have been reported to exert toxic effects on body tissues (Sugiyama, 1992). There were at least three forms of chromium compounds have been identified, chromium III, IV and VI with the two latter being more toxic than the former because of their easy permeation at physiological pH through the permease system (DeFlora & Wetterhahn, 1989). The same authors reported that inside the cells, chromium (VI) is reduced to reactive intermediates such as chromium (V), (IV) and finally to the more stable chromium (III) by cellular reductants including © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
glutathione (GSH), cysteine, ascorbic acid and riboflavin as well as NADPH-dependent flavoenzymes such as microsomal cytochrome P450. Chromium contaminates the testes by generating reactive radicals, which results in cellular damage, such as a reduction of enzyme activity and damage to the lipid bilayer and DNA (Stohs & Bagchi, 1995), resulting in amplified oxidative stress damage in the membranes, proteins and DNA in the sperm. The reactive oxygen species (ROS) contribute to cellular ageing, mutagenesis, carcinogenesis and coronary heart disease (Heim et al., 2002). The antioxidants provide protection against degenerative diseases including cancer, coronary heart and Alzheimer’s disease (Iqbal & Bhanger, 2006). Moringa oleifera (Moringaceae) is one of the 14 species from the Moringaceae family, which is native to India, Africa, Arabia, Southeast Asia, South America and the Pacific and Caribbean Islands (Iqbal & Bhanger, 2006). The Moringa oleifera tree (also known as drumstick tree) is a rapidly growing deciduous shrub or small desert tree 1047
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approximately 13 m tall and 35 cm in diameter with an umbrella-shaped open cap (Anjorin et al., 2010). This plant was utilised by the ancient Romans, Greeks and Egyptians. Almost all parts of the plant are used culturally for its nutritional value and supposed medicinal properties and for flavouring as a vegetable and seed (Kasolo et al., 2010). The leaves of Moringa oleifera can be eaten fresh or cooked, and reports have shown that it can be stored as a dried powder for many months without any major loss of its nutritional value (Arabshahi et al., 2007). Moringa oleifera leaves contains specific plant compounds that have been demonstrated to possess the same powerful antioxidant ability as vitamins C, E and A from oranges, pomegranates and carrots, respectively, as well as to contain caffeoylquinic acids, carotenoids-lutein, alpha- and beta carotene, kaempferol, quercetin and rutin (Siddhuraju & Becker, 2003; Aslam et al., 2005; Smolin & Grosvenor, 2007). Moringa oleifera oil and its micronutrients contain antitumor, antioxidant, antiepileptic, antidiuretic, antiinflammatory, hepatoprotective and antidiabetic properties (Hsu et al., 2006; Sreelatha & Padma, 2010). Because of aforementioned medicinal properties of Moringa oleifera, the objective of this study was to investigate the chemotherapeutic efficacy of an ethanolic Moringa oleifera leaf extract (MOLEE) against chromium-induced testicular toxicity and the mechanism responsible for this action, which had not been previously determined.
Approximately 1 kg of fresh tender leaves of Moringa oleifera collected during January–February was used for the study. The plant was identified and authenticated by Dr. Ahmed M Ismail (Faculty of Agriculture, Damanhour University, Egypt). The plant material was air-dried at room temperature. The dried leaves were grounded to a fine powder and stored in an air tight container. Three hundred grams of the dry powder obtained was soaked in 95% ethanol for 24 h in a percolator. After 24 h, it was allowed to percolate slowly and the extract was collected in Petri dishes. The extract was concentrated in vacuum using a rotary flash evaporator. There was a net yield of 30.5 g of the concentrated extract (10.16% w/w). The crude extract was suspended in distilled water before administration to rats on the subsequent day.
Materials and methods
Animal grouping and treatment
Animals Twenty male Wistar rats (Rattus norvegicus) weighing approximately 140–170 g and aged 8 weeks were used for this study and were obtained from the College of Science at Tanta University in Egypt. The animals were housed in stainless steel cages at standard atmospheric temperature (25 5 °C) with a 12-h light/dark cycle and were fed a standard diet and allowed access to water ad libitum. The animals were acclimatised for 2 weeks in their new environment prior to experimentation. All of the animals received humane care in compliance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the study protocol was approved by the local authorities. Chemicals Potassium dichromate (K2Cr2O7), nitric acid, 5, 5-dithiobis(2-nitrobenzoic acid; DTNB, Ellman’s reagent) and 1chloro-2, 4-dinitrobenzene (CDNB) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fresh leaves of the drumstick tree (Moringa oleifera) were obtained 1048
from a local garden at the Badr Centre in the Behera governorate in Egypt. Atomic spectrophotometry standard solutions for iron (Fe in 3% nitric acid) and copper (Cu in 3% nitric acid) were purchased from Ricca Chemical Company (Fenton, MO, USA). Reduced glutathione (GSH) and thiobarbituric acid (TBA) were purchased from Fluka (Buchs, Switzerland). All the other reagents were of analytical, high-performance liquid chromatography (HPLC), or the best available pharmaceutical grade. Preparation of the extract
The rats were randomly divided into the following experimental groups: I (control), II (chromium-treated), III (MOLEE-treated) and IV (chromium + MOLEE combination treated), with each group containing five animals. The chromium-treated, MOLEE-treated and chromium + MOLEE combination group received potassium dichromate (8 mg kg 1, i.p.), MOLEE (500 mg kg 1) p.o. or both daily, respectively, whereas the control group was not given any supplementation. The dose of Potassium dichromate and MOLEE were selected on the basis of previous reports of (Dey & Roy, 2009 and Das & Kanodia, 2012) respectively. The experiment lasted for 60 days. Blood sampling, animal sacrifice and organ harvesting At the end of the experiment and after a night of fasting, blood samples (2 ml) were collected using the retro-orbital plexus method into heparinised tubes. The plasma was separated by centrifugation at 704 g for 15 min, and the samples were frozen at 20 °C until further analysis. The plasma was used to determine the total amount of protein (Peters, 1968), albumin (Doumas et al., 1971), globulin (Coles (1974), C-reactive protein (CRP; Fisher © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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& Nakamura, 1976), Total Antioxidant Capacity (TAC; Koracevic & Koracevic, 2001), IgA (Ojala et al., 1981) and testosterone (TT) (Chen et al., 1991). After the rats were sacrificed by cervical dislocation, the abdominal cavity was opened up through a midline abdominal incision to expose the reproductive organs. The testes were detached, cleared free of the surrounding tissue and weighed with an electronic analytical balance. The testes were used to determine the malonaldehyde (MDA; Placer et al., 1966), superoxide dismutase (SOD; Misra & Fridovich, 1972), catalase (CAT; Sinha, 1971), (GSH; Sedlack & Lindsay, 1968), glutathione S-transferase (GST; Habig et al. (1974) and glutathione peroxidase (GPx; Chiu et al., 1976). A VARIAN spectroAA-20 plus GTA-96 flameless graphite furnace AAS was used to quantify Cu and Fe levels in the testes.
Results Testicular oxidative stress The activities of the testicular antioxidant enzymes SOD, CAT and TAC and GSH in the chromium-treated group decreased significantly (P < 0.05) when compared with the control and MOLEE-treated group. However, administration of chromium induced no significant changes in the activities of GPx and GST when compared with the control group. Following co-administration with MOLEE, there was an improvement in the testicular antioxidant status when compared with the chromium-treated group (Tables 1 and 2). The testicular content of MDA in the chromium-treated group was significantly elevated when compared with the control and MOLEE-treated group (P < 0.05). There was a significant decrease in the MDA levels of the chromium + MOLEE combination group when compared with the chromium-treated group (P < 0.05; Table 2).
Sperm characteristics As described by Saalu et al. (2007), sperm motility, concentration and progressive motility were determined by removing the caudal part of epididymis and placing it in a beaker containing 1 ml of physiological saline solution, after which, each section was quickly incised with a pair of sharp scissors and left for a few minutes to release its spermatozoa into the saline solution. Semen drops were placed on a slide, and two drops of warm 2.9% sodium citrate were added. The slide was covered with a cover slip and examined with a light microscope using a 409 objective to determine sperm motility. The sperm concentration was performed using an enhanced Neubauer hemocytometer. The sperm morphology was carried out by staining technique.
Trace mineral levels Compared with the control animals, the testicular Fe and Cu contents were significantly higher (P < 0.05; Table 2) in chromium-treated rats. The MOLEE + chromium-treated rats had significantly decreased levels of Fe and Cu when compared with the group treated with chromium alone. Body weight changes Table 3 reported that rats in the chromium-treated group lost body weight when compared with rats in both the control and MOLEE-treated group. The rats in the chromium + MOLEE combination group had a significant increase in body weight when compared with the chromium-treated group (P < 0.05), it was just portrayed in the Table 3.
Statistical analysis The results are expressed as the mean SE. The analysis of variance for the obtained data was performed using SAS (2002). Values of P < 0.05 were considered to be statistically significant.
Table 1 Effects of chromium and/or MOLEE on TAC, SOD, CAT, GPx and GST in rats
Groups Group I (control) Group II (chromium) Group III (MOLEE) Group IV (chromium/ MOLEE)
1
TAC (mM l ) 0.98 0.38 2.79 1.85
c
0.13 0.08d 0.24a 0.16b
SOD (U mg protein) 89.65 67.65 119.91 90.37
1
CAT (K s protein) b
5.43 6.56c 6.77a 4.49b
96.47 52.34 95.63 89.46
1
mg
a
3.35 3.87cd 4.42a 4.54ab
1
GPX Activity IU g Wet tissue 69.42 63.38 69.74 67.86
a
1.72 3.19ab 2.42a 2.23a
1
GST Activity mol CDNB per min 1 g 396.1 397.5 479.7 433.7
1
Wet tissue
c
7.42 6.61c 9.87a 6.37b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract; TAC, total antioxidant capacity; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferase.
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Table 2 Effects of chromium and/or MOLEE on MDA, GSH, testicular Fe and Cu levels in rats 1
Groups
MDA (nmol g
Group Group Group Group
119.5 5.26 158.44 4.67a 87.3 5.75c 121.5 6.71b
I (control) II (chromium) III (MOLEE) IV (chromium/MOLEE)
wet tissue)
GSH (lmol g
b
68.34 43.43 89.28 73.36
1
wet tissue)
Fe (lg g
bc
2.68 3.78de 3.69a 4.64b
106.68 215.77 83.68 177.78
1
wet tissue) c
1.39 2.33a 1.39d 1.81b
Cu (lg g 33.16 67.55 31.96 52.89
1
wet tissue)
1.21c 0.96a 1.15c 1.37b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract; MDA, malonaldehyde; GSH, reduced glutathione; Fe, iron; Cu, copper.
Table 3 Effects of chromium and/or MOLEE on body weight, body weight difference, testes weight, testes weight/body weight ratio and plasma testosterone level in rats
Groups
Initial body weight (g)
Group Group Group Group
160.8 172.5 165.3 170.5
I (control) II (chromium) III (MOLEE) IV (chromium/MOLEE)
4.7cd 5.4a 4.9bc 5.2ab
Final body weight (g) 169.8 143.3 162.7 161.9
4.3a 2.0cd 3.9b 5.6b
Body weight difference (g)
Testis weight (g)
9.0b 29.2a 2.6c 8.6b
1.61 0.53 1.78 1.27
0.5ab 0.3de 0.5a 0.2c
Testis weight per Body weight Ratio
Testosterone (ng ml 1)
0.0095ab 0.0037de 0.010a 0.0078c
5.63 1.39 5.59 3.45
1.1a 0.2c 1.7a 0.6b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract. Table 4 Effects of chromium and/or MOLEE on total protein, albumin and globulin, IgA and CRP in rats Groups
Total protein (g dl 1)
Group Group Group Group
7.92 7.69 8.27 8.38
I (control) II (chromium) III (MOLEE) IV (chromium/MOLEE)
0.33ab 0.21bc 0.35a 0.29a
Albumin (g dl 1) 4.60 3.97 4.57 4.63
0.33a 0.31b 0.36a 0.28a
Globulin (g dl 1) 3.32 3.72 3.70 3.75
0.08ab 0.06a 0.13a 0.05a
IgA (mg dl 1) 98.66 87.45 104.83 102.58
5.43ab 4.56c 4.52a 5.18a
CRP (mg l 1) 5.82 41.91 6.23 31.46
2.51cd 3.78a 1.21cd 2.69b
Means within the same column carrying different letters are significantly different (P < 0.05). MOLEE, Moringa oleifera leaf ether extract; IgA, immunoglobulin A; CRP, C-reactive protein.
Changes in testis weight Table 3 revealed that the testicle weight and testicle weight to body weight ratios of the rats in the chromium-treated group were significantly lower than those of the control, MOLEE-treated and chromium + MOLEE combination group (P < 0.05). Testosterone level Table 3 showed a significant decrease in the TT levels of the chromium-treated group when compared with the control and MOLEE-treated group (P < 0.05). Following co-treatment with MOLEE, the TT levels significantly increased when compared with the chromium-treated group (P < 0.05).
MOLEE-treated group (P < 0.05). However, the chromium + MOLEE combination group showed a significant increase in the total protein and albumin levels when compared with the chromium-treated group (P < 0.05). Immunoglobulin A and C-reactive protein concentration Table 4 showed a significant decrease in the IgA levels and increase in the CRP levels of the chromium-treated group when compared with the control and MOLEE-treated group (P < 0.05). When co-treated with MOLEE, the level of IgA increased and the level of CRP decreased significantly (P < 0.05) when compared with the group treated with chromium alone.
Sperm parameters Changes in total protein, albumin and globulin Table 4 revealed a significant decrease in the total protein and albumin of the chromium-treated group with no effect on globulin when compared with the control and 1050
Morphology As shown in Table 5, the sperm morphology of the control, MOLEE-treated and chromium + MOLEE combination group was normal when compared with the © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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Okpuzor J, Ogbunugafor H, Kareem GK (2009) Antioxidative properties of ethyl acetate fraction of Globimetula branuii in normal rats. J Biol Sci 9:470–475. Pakade V, Cukrowska E, Chimuka L (2013) Comparison of antioxidant activity of Moringa oleifera and selected vegetables in South Africa. S Afr J Sci 109:1–5. Peters TJ (1968) Proposals for standardization of total protein assays. Clin Chem 14:1147. Placer ZA, Crushman L, Son BC (1966) Estimation of product of lipid peroxidation (malondialdehyde) in biochemical system. Anal Biochem 16:359–364. Pourmorad F, Hosseinimehr SJ, Shahabimajd N (2006) Antioxidant activity, phenol and flavonoids content of some selected Iranian plants. Afr J Biotechnol 5:1142–1145. Rakesh S, Singh VJ (2010) In vivo antioxidant activity of Moringa oleifera leaf and pod extracts against carbon tetra chloride induced liver damage in albino mice. J Chem Pharm Res 2:275–283. Saalu LC, Adesanya AO, Oyewopo AO, Raji Y (2007) An evaluation of the deleterious effect of unilateral cryptorchidism on the contralateral normally descended testis. Sci Res Essays 2:74–78. Saalu LC, Osinubi AA, Akinbami AA, Yama OE, Oyewopo AO, Enaibe BU (2011) Moringa oleifera Lamarck (drumstick) leaf extract modulates the evidences of
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hydroxyurea–induced testicular derangement. Int J Appl Res Nat Prod 4:32–45. SAS (2002) User’s Guide Statistical Analysis system, Version 6, 4th edn. SAS Institute, Cary, NC, USA. Sedlack J, Lindsay RH (1968) Estimation of total protein bound and non protein sulfhydryl groups in tissues with Ellman reagent. Anal Biochem 86:271–278. Siddhuraju P, Becker K (2003) Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam) leaves. J Agric Food Chem 51:2144– 2155. Sinha KA (1971) Calorimetric assay of catalase. Anal Biochem 47:389–394. Smolin LA, Grosvenor MB (2007) Nutrition Science and Applications, 4th edn. John Wiley & Sons Inc., New York, pp 123–125. Sreelatha S, Padma PR (2010) Antioxidant activity and total phenolic content in Moringa oleifera leaves in two stages of maturity. Plant Foods Hum Nutr 64:303–311. Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18:321–336. Sugiyama M (1992) Role of physiological antioxidant in chromium (VI)-induced cellular injury. Free Radic Biol Med 12:397–407.
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induced disturbances in Cu/Fe homeostasis reflected in increased their levels in the testes. The mechanism related to these disturbances has not been clearly defined. Simultaneous treatment with MOLEE, which has been shown to contain bioflavonoids and other effective antioxidants (Saalu et al., 2011), resulted in a notable amelioration of the unbalanced sperm parameters of the testes in the chromium + MOLEE combination group. Pre-treatment with the MOLEE has been shown to shield the testes from a variety of toxic substances (Stohs & Bagchi, 1995). Siddhuraju & Becker (2003) and Saalu et al. (2011) reported that Moringa oleifera contains fundamental antioxidants and phenolic compounds that help to protect against oxidative changes brought about by toxic materials and certain antineoplastic agents. Saalu et al. (2011) showed the antioxidative properties of Moringa oleifera and its ability to increase the antioxidant capability. Similarly, the phenolic compounds in Moringa oleifera are a class of antioxidant agents that act as free radical terminators and are involved in the impedance of oxidative degradation of lipids (Pourmorad et al., 2006). Pre-treatment with Moringa oleifera hydro-alcoholic leaf and aqueous pod extracts improved the SOD, CAT, glutathione and peroxidase levels significantly and reduced lipid peroxidation in CCl4-induced hepatocellular damage and high-fat diet-induced nonalcoholic fatty liver disease respectively (Rakesh & Singh, 2010; Nilanjan et al., 2012). The total phenolic content and the total flavonoid content of the Moringa leaves were almost two and three times that of the vegetables, respectively (Pakade et al., 2013). It is known that phenolic and flavonoid contents are directly linked to antioxidant properties (Siddhuraju & Becker, 2003). Flavonoids can exert their antioxidant activities by various mechanisms, such as scavenging or quenching free radicals and inhibiting enzymatic systems responsible for free radical generation (Lukacinova et al., 2008). Unexpectedly, the decreased testicular Fe++ and Cu+ by MOLEE in this study added another mechanism of chelating metal ions. Thereby, chemotherapeutic action of MOLEE against chromium-induced testicular toxicity could be attained partly by mechanism related to mineral homeostasis or binding the redox-active metals such as Fe and Cu. Antioxidant properties also can be due to the presence of carotenoids, alkaloids, and proanthocyanidins in this plant. The increased antioxidant enzymatic activity after administration of MOLEE could partially be due to the plant’s ability to prevent activity impairment. MOLEE effectively scavenges the free radicals generated and subsequently reduces the destructive effects of oxidative stress as well as decreases the need of antioxidant enzymes to counteract the increased free radicals (Arabshahi et al., 2007). The elevated activity of SOD and CAT may suggest an induction of the enzymes by MOLEE in the rats. SOD 1052
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and CAT are both inducible enzymes whose production can be stimulated. Previous reports support this finding; including applications with black rice extract (Chiang et al., 2006) and the ethyl acetate fraction of the Globimetula branuii leaf extract (Okpuzor et al., 2009). Increased activity of the enzymes could mean enhanced antioxidative capacity of the animals (Lobo et al., 2010). However, high vitamin C content in the leaves of Moringa oleifera has been reported (Siddhuraju & Becker, 2003; Aslam et al., 2005). Vitamin C can act as a pro-oxidant as well as an antioxidant. It could be suggested that vitamin C may play a pro-oxidant role in the observed induction of antioxidant enzymes. The decreased lipid peroxidation observed in this study correlates with the induction of antioxidant enzymes above basal levels by MOLEE. Regarding the anti-inflammatory effect of Moringa oleifera, Ezeamuzie et al. (1996) demonstrated that the roots of Moringa oleifera contain anti-inflammatory properties that may be useful in the treatment of the acute inflammatory conditions such as carrageenaninduced oedema. Amelioration of airway inflammation associated with ovalbumin treatment in guinea pigs could be possible with the potent anti-inflammatory activity of Moringa oleifera bioactive compounds (Mahajan et al., 2009). The ethanolic extract of the Moringa oleifera leaves showed significant reduction of experimentally induced colitis, which may be attributed to its anti-inflammatory property (Das & Kanodia, 2012). This result could provide a rationalisation for the observations in this study as to why the group treated with chromium and MOLEE combination showed enhanced sperm parameters and increases in antioxidant enzymes activity and IgA as well as decreased testicular MDA and CRP inflammatory marker levels. In this regard, the results of this study are consistent with another report (D’cruz, 2005) which showed that the sperm cytoplasm contained low concentrations of free radical scavenging enzymes and, as a result, that an increase in the antioxidant enzyme system levels after Moringa oleifera treatment can favour reproductive potential. Regarding the effects of Moringa oleifera on total protein and albumin levels, this study showed that MOLEE increased the total amount of protein and albumin levels in chromium-treated rats, which further indicated the nutritional properties of Moringa oleifera and its hepatoprotection efficacy. Herein, the globulin level in Chromium- and/or MOLEE-treated group remained unchanged compared with control group. There was shortage in literature which discussed the effects of chromium and moringa oleifera on serum globulin level. The increased IgA in MOLEE-treated group and decreased IgA in chromium-treated group might be associated with decreased or increased in other subclasses of globulin respectively. © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 1047–1054
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Conclusion The increased oxidative stress and inflammation and decreased local immunity resulting from chromium intoxication in testicular tissue might be a cause of abnormal sperm parameters. However, ethanolic MOLEE exerted a therapeutic effect against the toxicity, which was shown by the improvement in the sperm parameters. The antioxidant, anti-inflammatory and immunostimulatory potential of Moringa oleifera was reflected by decreases in the oxidative stress and CRP levels and increased IgA levels in the chromium + MOLEE-treated combination group. The metal ion chelation could partly attribute the antioxidant activities of MOLEE. Thus, Moringa oleifera could provide multiple health benefits to humans by having various biological activities. Acknowledgements The author gratefully acknowledges to Mr. Abdel Rahman Saleh for providing the plant leaves and to Dr. Ahmed M. Ismail for plant identification and authentication. References Anjorin ST, Ikokoh PS, Okolo A (2010) Mineral composition of Moringa oleifera leaves pods and seeds from two regions in Abuja. Nigeria. Int J Agric Biol 12:1560–1569. Arabshahi DS, Devi V, Urooj A (2007) Evaluation of antioxidant activity of some plant extracts and their heat, pH and storage stability. Food Chem 100:1100–1105. Aslam MF, Anwar R, Nadeem U, Rashid TG, Kazi A, Nadeem M (2005) Mineral composition of Moringa oleifera leaves and pods from different regions of Punjab, Pakistan. Asian J Plant Sci 4:417–421. Chen A, Bookstein JJ, Meldrum DR (1991) Diagnosis of a testosterone-secreting adrenal adenoma by selective venous catheterization. Fertil Steril 55:1202–1203. Chen F, Ding M, Castranova V, Shi XL (2001) Carcinogenic metals and NF-kappa B activation. Mol Cell Biochem 222:159–171. Chiang AN, Wu HL, Yen HI (2006) Antioxidant effects of black rice extract through the induction of superoxide dismutase and catalase activities. Lipids 41:797–803. Chiu D, Fredrick H, Tappel AL (1976) Purification and properties of rat lung soluble glutathione peroxidase. Biochim Biophys Acta 445:558–566. Coles EH (1974) Veterinary Clinical Pathology, 1st edn. W. B Saunders Co. Philadelphia, London, Toronto, pp 212–213. Das S, Kanodia L (2012) Effect of ethanolic extract of leaves of moringa olifera lam. On acetic acid induced colitis in albino rats. Asian J Pharm Clin Res 5:110–114. D’cruz M (2005) Effect of piperine on the epididymis of adult male rats. Asian J Androl 7:363–368.
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