Involvement of Bitter Leaf Extracts in Testicular Metabolic Stress Induced by Oil Contaminated Diets in Rats The environment we live in is increasingly inundated with several types of environmental toxic substances that tend to cause injury and metabolic stress to plants, animals and humans. Several industrial pollutants including unrefined petroleum and its related products such as kerosene, gas flares, premium motor gasoline, diesel, 3,1-dinitrobenzene or nonylphenol, methanoxyethanol, ether glycol and brake oils, are known to exert oxidative metabolic stress and testicular atrophy. A significant danger to people exposed to environmental toxicants is the increased risk of being infertile, defined as the inability of a sexually active, non-contraceptive-using individual or couple to achieve a spontaneous pregnancy. within one year (WHO, 2010; Zegers et al., 2009). Many studies reveal that most male infertility problems are the result of testicular oxidative stress which would affect seminal plasma antioxidants and increased lipid peroxidation (altered sperm morphology, impaired sperm motility. Say no to plagiarism Get a tailor-made essay on “Why violent video games should not be banned”?Get the original essayMechanistic defense against oxidative stress depends on the ability of the body and cells to enhance the buffering capacities of antioxidants. , which will help eliminate oxidative radicals generated by various metabolic processes Today, Vernonia amygdalina, a well-known vegetable common to many tribes in Nigeria, has been elucidated for its antioxidant buffering capabilities. as an alternative diet against malaria It has been used individually as a protective agent and ameliorate of the deleterious effects of many toxic products such as cyanide, carbon tetrachloride, unrefined petroleum and cycasin. From the above, there is no doubt that while there is ample evidence for the ability of unrefined oil to induce various forms of metabolic oxidative stress, there is little evidence for the possible role of oil poisoning. unrefined oil in the induction of testicular damage caused by adulterated unrefined oil foods, as well as the ability of Vernonia amygdalina to induce the possible restoration or control of activated metabolic stress parameters. . This study was therefore carried out to clearly show the research evidence to fill these existing gaps. Materials and methods Ripe bitter leaves (Vernoniaamygdalina Del) were collected from a farm in Abraka, Nigeria and preliminary identification was carried out at the Department of Botany, Delta State University, Abraka, Nigeria by Dr. Erhenhi AH The leaf was then authenticated at the Forestry Research Institute of Nigeria, Jericho Hill, Ibadan, Nigeria, where a specimen bearing the reference number F101963 has been deposited in the herbarium. Thirty-six male albino rats (Rattusnorvegicus), with an average weight of 150–182 g, were acquired from the Delta State University Animal Facility, Abraka, Nigeria. Rats were housed in a wooden cage and allowed to acclimatize for a week on producer mash (a product of Rainbow Feed Limited). The compositionof the food declared by the manufacturer was previously published by Achuba (2018). All other reagents used for biochemical analysis were of analytical grade. Preparation of Bitter Leaf Extract Bitter leaf was washed, chopped and air-dried at room temperature for one week in an open space within the laboratory of Department of Biochemistry, Delta State University, in Abraka. After drying, the bitter leaf was chopped and macerated using a Warren blender to obtain a dry, smooth powder. Bitter leaf extract was prepared using methanol as described by Yin et al. (2013). One hundred (100 g) powdered leaves were dissolved in 400 ml of methanol by sonication for 10 minutes and then filtered with Whatman No.1 using a vacuum pump. The obtained extract is concentrated via a rotary evaporator at 40-50℃ under reduced pressure to obtain the bitter leaf methanolic extract (BLME). The extract was stored at -8 ℃ until needed. Experimental design and treatment The distribution of six rats per group was carried out according to the following description: Group A = Food Group B = Food + 100 mg kg-1 body weight of BLME Group C = Food + 200 mg kg -1 body weight from BLMEGroup D = Food (100 g food + 4 ml unrefined oil) Group E = Food (100 g food + 4 ml unrefined oil) + 100 mg kg -1 body weight from BLME Group F = Food (100 g food + 4 ml unrefined oil) + 200 mg kg -1 body weight of BLME. The bitter leaf extract used was freshly prepared at the time of administration. To obtain 200 mg ml-1, 20 grams of the extract was dissolved in 100 ml of distilled water, from which aliquots of the freshly dissolved extract were administered by gavage according to the body weight of the rat once per day. Rats in groups A and D did not receive the extracts while all rats had free access to water. All treatments lasted a period of 30 days. Sample collection After an exposure period of 30 days, the rats were sacrificed by cervical decapitation on the 31st day after an overnight fast. Testes were collected in pre-refrigerated labeled containers. Wet testicular tissue (0.5 g) was homogenized with 9.0 ml of normal saline using a pre-chilled mortar and pestle and the resulting supernatant was stored at -8° C in cold room and used for biochemical analysis within 48 hours. the determination of the level of lipid peroxidation (MDA) (Gutteridge and Wilkins, 1982) and enzymatic markers of oxidative stress as follows; aldehyde oxidase (AO) (Omarov et al. 1998), sulfite oxidase (Macleod et al. 1961); monoamine oxidase (MO) and xanthine oxidase (XO) (McEwen, 1971). The non-enzymatic antioxidant profile assay in the testes was determined using the methods of Ellman (1959) for the assay of reduced glutathione, while the vitamin C assay used the methods reported by Achuba (2008) . Analyzes of specific enzymatic antioxidant activities were carried out using the methods of Misra and Fredorich (1972) for superoxide dismutase (SOD), Cohen et al., (1972) for Catalase, Habig et al. (1974) for glutathione-s-transferases (GST) and Khan et al. (2009) for glutathione peroxidase (GPx). Histological examination A known portion of the testes from each rat was harvested and fixed in 10% formalin saline for 48 hours and processed for paraffin wax embedding with an automatic tissue processor by 70% dehydration, 90 %, 95% and two changes of absolute ethanol for 90 minutes each. Clarification was achieved through two changes of xylene for 2 hours each; and infiltration with two changes of paraffin for 2 hours. THESections were cut at 5 μm with a rotary microtome. Sections were stained with hematoxylin and eosin (H and E) using the method of Odoulaet al. (2009), examined and photographed using an optical microscope. Statistical analysis Data analysis was performed using one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences version 17 (SPSS 17). Post-hoc analysis (comparisons between groups) was performed using Bonferroni at a significance level of P < 0.05. ResultThe presented result revealed a significant increase in lipid peroxidation (MDA) levels in rats that received both doses of BLME without a contaminated diet (B and C) compared to positive control (A) that was fed a contaminated diet. normal food. This did not differ significantly between rats fed contaminated diets without treatment (groups D) and rats fed contaminated diets and given both doses of BLME (groups E and F). contaminated with unrefined oil. Furthermore, increased activities of AO, SO, MO and a significant reduction compared to rats fed unrefined petroleum. spoiled food (group D). Administration of both doses of BLME to rats fed unrefined oil-contaminated food (groups E and F) significantly increased the activities of oxidases (AO, SO, MO and XO) compared to the rat fed with untreated food (group A) and to rats. fed food contaminated with unrefined petroleum (group D). However, there was no significant difference between groups E and F when taken together. As shown, CuZnSOD activities were not significantly increased in rats fed 100 mg Kg-1 body weight of BLME (group B) compared to normal control group A fed only normal diet. However, it increased significantly in rats receiving 200 mgKg-1 body weight compared to the control group. Furthermore, CuZnSOD activities in rats fed both doses were significantly higher than in rats fed an oil-contaminated diet without treatment and in those fed a contaminated diet and treated with both doses of BLME. . It was observed that rats fed only contaminated diets showed reduced CuZnSOD activities compared to normal control, but were significantly increased compared to those fed contaminated diets and treated with 200 mg Kg-1 wt. bodily. MnSOD activities did not change in rats in groups A and B but increased significantly when rats in group C and group A were relative. MnSOD activities were significantly reduced in rats fed a contaminated diet in group D compared to normal control and in rats fed a normal diet and treated with both doses of BLME in groups B and C . Treatment of rats fed a diet contaminated with 100 mgKg-1 and 200 mgKg-1. 1 of BLME showed no significant difference compared to untreated rats in group D. Total SOD activities showed no significant difference between groups AE but were significantly reduced in group F which was exposed to contaminated diets and treated with 200 mgKg-1 of BLME compared to AD groups. The results presented reveal that there was no significant change in vitamin C levelsin all groups. It was observed that GSH levels showed no significant change in rats fed 100 mg Kg-1 and 200 mg Kg-1 body weight of BLME (B and C) compared to normal control group A, but increased significantly compared to rats fed an oil-polluted diet. Administration of 100 MgKg-1 body weight of BLME significantly increased GSH levels compared to those fed only spoiled diets, but reduced them compared to normal control (group A). Those fed polluted diets and given 200 mg Kg-1 body weight of BLME (group E) remained unchanged compared to group D but reduced compared to all other groups. The activity of the antioxidant enzyme catalase was significantly elevated in rats treated with 100 mgKg-1 body weight, but not with 200 mgKg-1 compared to control group A. It was, however, significantly increased for both doses compared to group D rats fed a spoiled diet without treatment. . treatment with both doses resulted in further reduction in catalase activities compared to groups A and D. It was observed that GPX and GST activities had no significant changes for rats treated with 100 mg Kg-1 wt. body without contamination compared to the control (group A) and reduced. significantly for GPX while increasing for GST compared to rats fed only polluted diets (group D). Treatment with both doses of BLME significantly reduced GSTS activities compared to control group A, while GPx activities were only significantly reduced at the dose of 200 mg Kg-1 body weight. However, compared to group D, it was observed that GST activities remained significantly unchanged at both doses (100 mgKg-1 and 200 mgKg-1) of body weight. GPx, on the other hand, was significantly reduced for both doses. DiscussionUnrefined oil contamination remained a significant contributor to several endocrine-induced metabolic stress and dysfunction. On the other hand, testicular oxidative stress is believed to be responsible for the majority of the many cases of infertility worldwide. The result presented in this study revealed increased MDA levels and oxidative enzyme activities (AO, SO, MO and XO) in the testes of rats fed contaminated diets compared to normal control. Increasing MDA levels are increasingly reported as a potent marker of the negative effects of unrefined oil consumption and other unrefined oil-related exposures. The increase in oil-induced tissue peroxidation is said to go hand in hand with a possible increase in oxidative enzymes that are necessary to initiate the eventual elimination of peroxides and superoxides generated by oil contamination. It is important to indicate that, depending on the physiological position and nature of the testicles, they are said to be very vulnerable to toxins, therefore there is an enhanced antioxidant buffer built in due to the presence of non-toxic antioxidants. enzymatic antioxidants (vitamins such as vitamin C, vitamin E and GSH) and enzymatic antioxidants (SOD, GST, GPX and catalase). Therefore, for oxidative damage to occur, there must be evidence based on the antioxidant defense capacity of the tissues and biological membranes involved. The observed induction of lipid peroxidation and oxidative enzymes in the testes of rats fed petroleum-polluted diets without BLME treatment is consistent with the observed reduced levels of defensive markers..

Essay / Involvement of Bitter Leaf Extracts in Testicular Metabolic Stress Induced by Oil Contaminated Diets in Rats The environment we live in is increasingly inundated with several types of environmental toxic substances that tend to cause injury and metabolic stress to plants, animals and humans. Several industrial pollutants including unrefined petroleum and its related products such as kerosene, gas flares, premium motor gasoline, diesel, 3,1-dinitrobenzene or nonylphenol, methanoxyethanol, ether glycol and brake oils, are known to exert oxidative metabolic stress and testicular atrophy. A significant danger to people exposed to environmental toxicants is the increased risk of being infertile, defined as the inability of a sexually active, non-contraceptive-using individual or couple to achieve a spontaneous pregnancy. within one year (WHO, 2010; Zegers et al., 2009). Many studies reveal that most male infertility problems are the result of testicular oxidative stress which would affect seminal plasma antioxidants and increased lipid peroxidation (altered sperm morphology, impaired sperm motility. Say no to plagiarism Get a tailor-made essay on “Why violent video games should not be banned”?Get the original essayMechanistic defense against oxidative stress depends on the ability of the body and cells to enhance the buffering capacities of antioxidants. , which will help eliminate oxidative radicals generated by various metabolic processes Today, Vernonia amygdalina, a well-known vegetable common to many tribes in Nigeria, has been elucidated for its antioxidant buffering capabilities. as an alternative diet against malaria It has been used individually as a protective agent and ameliorate of the deleterious effects of many toxic products such as cyanide, carbon tetrachloride, unrefined petroleum and cycasin. From the above, there is no doubt that while there is ample evidence for the ability of unrefined oil to induce various forms of metabolic oxidative stress, there is little evidence for the possible role of oil poisoning. unrefined oil in the induction of testicular damage caused by adulterated unrefined oil foods, as well as the ability of Vernonia amygdalina to induce the possible restoration or control of activated metabolic stress parameters. . This study was therefore carried out to clearly show the research evidence to fill these existing gaps. Materials and methods Ripe bitter leaves (Vernoniaamygdalina Del) were collected from a farm in Abraka, Nigeria and preliminary identification was carried out at the Department of Botany, Delta State University, Abraka, Nigeria by Dr. Erhenhi AH The leaf was then authenticated at the Forestry Research Institute of Nigeria, Jericho Hill, Ibadan, Nigeria, where a specimen bearing the reference number F101963 has been deposited in the herbarium. Thirty-six male albino rats (Rattusnorvegicus), with an average weight of 150–182 g, were acquired from the Delta State University Animal Facility, Abraka, Nigeria. Rats were housed in a wooden cage and allowed to acclimatize for a week on producer mash (a product of Rainbow Feed Limited). The compositionof the food declared by the manufacturer was previously published by Achuba (2018). All other reagents used for biochemical analysis were of analytical grade. Preparation of Bitter Leaf Extract Bitter leaf was washed, chopped and air-dried at room temperature for one week in an open space within the laboratory of Department of Biochemistry, Delta State University, in Abraka. After drying, the bitter leaf was chopped and macerated using a Warren blender to obtain a dry, smooth powder. Bitter leaf extract was prepared using methanol as described by Yin et al. (2013). One hundred (100 g) powdered leaves were dissolved in 400 ml of methanol by sonication for 10 minutes and then filtered with Whatman No.1 using a vacuum pump. The obtained extract is concentrated via a rotary evaporator at 40-50℃ under reduced pressure to obtain the bitter leaf methanolic extract (BLME). The extract was stored at -8 ℃ until needed. Experimental design and treatment The distribution of six rats per group was carried out according to the following description: Group A = Food Group B = Food + 100 mg kg-1 body weight of BLME Group C = Food + 200 mg kg -1 body weight from BLMEGroup D = Food (100 g food + 4 ml unrefined oil) Group E = Food (100 g food + 4 ml unrefined oil) + 100 mg kg -1 body weight from BLME Group F = Food (100 g food + 4 ml unrefined oil) + 200 mg kg -1 body weight of BLME. The bitter leaf extract used was freshly prepared at the time of administration. To obtain 200 mg ml-1, 20 grams of the extract was dissolved in 100 ml of distilled water, from which aliquots of the freshly dissolved extract were administered by gavage according to the body weight of the rat once per day. Rats in groups A and D did not receive the extracts while all rats had free access to water. All treatments lasted a period of 30 days. Sample collection After an exposure period of 30 days, the rats were sacrificed by cervical decapitation on the 31st day after an overnight fast. Testes were collected in pre-refrigerated labeled containers. Wet testicular tissue (0.5 g) was homogenized with 9.0 ml of normal saline using a pre-chilled mortar and pestle and the resulting supernatant was stored at -8° C in cold room and used for biochemical analysis within 48 hours. the determination of the level of lipid peroxidation (MDA) (Gutteridge and Wilkins, 1982) and enzymatic markers of oxidative stress as follows; aldehyde oxidase (AO) (Omarov et al. 1998), sulfite oxidase (Macleod et al. 1961); monoamine oxidase (MO) and xanthine oxidase (XO) (McEwen, 1971). The non-enzymatic antioxidant profile assay in the testes was determined using the methods of Ellman (1959) for the assay of reduced glutathione, while the vitamin C assay used the methods reported by Achuba (2008) . Analyzes of specific enzymatic antioxidant activities were carried out using the methods of Misra and Fredorich (1972) for superoxide dismutase (SOD), Cohen et al., (1972) for Catalase, Habig et al. (1974) for glutathione-s-transferases (GST) and Khan et al. (2009) for glutathione peroxidase (GPx). Histological examination A known portion of the testes from each rat was harvested and fixed in 10% formalin saline for 48 hours and processed for paraffin wax embedding with an automatic tissue processor by 70% dehydration, 90 %, 95% and two changes of absolute ethanol for 90 minutes each. Clarification was achieved through two changes of xylene for 2 hours each; and infiltration with two changes of paraffin for 2 hours. THESections were cut at 5 μm with a rotary microtome. Sections were stained with hematoxylin and eosin (H and E) using the method of Odoulaet al. (2009), examined and photographed using an optical microscope. Statistical analysis Data analysis was performed using one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences version 17 (SPSS 17). Post-hoc analysis (comparisons between groups) was performed using Bonferroni at a significance level of P < 0.05. ResultThe presented result revealed a significant increase in lipid peroxidation (MDA) levels in rats that received both doses of BLME without a contaminated diet (B and C) compared to positive control (A) that was fed a contaminated diet. normal food. This did not differ significantly between rats fed contaminated diets without treatment (groups D) and rats fed contaminated diets and given both doses of BLME (groups E and F). contaminated with unrefined oil. Furthermore, increased activities of AO, SO, MO and a significant reduction compared to rats fed unrefined petroleum. spoiled food (group D). Administration of both doses of BLME to rats fed unrefined oil-contaminated food (groups E and F) significantly increased the activities of oxidases (AO, SO, MO and XO) compared to the rat fed with untreated food (group A) and to rats. fed food contaminated with unrefined petroleum (group D). However, there was no significant difference between groups E and F when taken together. As shown, CuZnSOD activities were not significantly increased in rats fed 100 mg Kg-1 body weight of BLME (group B) compared to normal control group A fed only normal diet. However, it increased significantly in rats receiving 200 mgKg-1 body weight compared to the control group. Furthermore, CuZnSOD activities in rats fed both doses were significantly higher than in rats fed an oil-contaminated diet without treatment and in those fed a contaminated diet and treated with both doses of BLME. . It was observed that rats fed only contaminated diets showed reduced CuZnSOD activities compared to normal control, but were significantly increased compared to those fed contaminated diets and treated with 200 mg Kg-1 wt. bodily. MnSOD activities did not change in rats in groups A and B but increased significantly when rats in group C and group A were relative. MnSOD activities were significantly reduced in rats fed a contaminated diet in group D compared to normal control and in rats fed a normal diet and treated with both doses of BLME in groups B and C . Treatment of rats fed a diet contaminated with 100 mgKg-1 and 200 mgKg-1. 1 of BLME showed no significant difference compared to untreated rats in group D. Total SOD activities showed no significant difference between groups AE but were significantly reduced in group F which was exposed to contaminated diets and treated with 200 mgKg-1 of BLME compared to AD groups. The results presented reveal that there was no significant change in vitamin C levelsin all groups. It was observed that GSH levels showed no significant change in rats fed 100 mg Kg-1 and 200 mg Kg-1 body weight of BLME (B and C) compared to normal control group A, but increased significantly compared to rats fed an oil-polluted diet. Administration of 100 MgKg-1 body weight of BLME significantly increased GSH levels compared to those fed only spoiled diets, but reduced them compared to normal control (group A). Those fed polluted diets and given 200 mg Kg-1 body weight of BLME (group E) remained unchanged compared to group D but reduced compared to all other groups. The activity of the antioxidant enzyme catalase was significantly elevated in rats treated with 100 mgKg-1 body weight, but not with 200 mgKg-1 compared to control group A. It was, however, significantly increased for both doses compared to group D rats fed a spoiled diet without treatment. . treatment with both doses resulted in further reduction in catalase activities compared to groups A and D. It was observed that GPX and GST activities had no significant changes for rats treated with 100 mg Kg-1 wt. body without contamination compared to the control (group A) and reduced. significantly for GPX while increasing for GST compared to rats fed only polluted diets (group D). Treatment with both doses of BLME significantly reduced GSTS activities compared to control group A, while GPx activities were only significantly reduced at the dose of 200 mg Kg-1 body weight. However, compared to group D, it was observed that GST activities remained significantly unchanged at both doses (100 mgKg-1 and 200 mgKg-1) of body weight. GPx, on the other hand, was significantly reduced for both doses. DiscussionUnrefined oil contamination remained a significant contributor to several endocrine-induced metabolic stress and dysfunction. On the other hand, testicular oxidative stress is believed to be responsible for the majority of the many cases of infertility worldwide. The result presented in this study revealed increased MDA levels and oxidative enzyme activities (AO, SO, MO and XO) in the testes of rats fed contaminated diets compared to normal control. Increasing MDA levels are increasingly reported as a potent marker of the negative effects of unrefined oil consumption and other unrefined oil-related exposures. The increase in oil-induced tissue peroxidation is said to go hand in hand with a possible increase in oxidative enzymes that are necessary to initiate the eventual elimination of peroxides and superoxides generated by oil contamination. It is important to indicate that, depending on the physiological position and nature of the testicles, they are said to be very vulnerable to toxins, therefore there is an enhanced antioxidant buffer built in due to the presence of non-toxic antioxidants. enzymatic antioxidants (vitamins such as vitamin C, vitamin E and GSH) and enzymatic antioxidants (SOD, GST, GPX and catalase). Therefore, for oxidative damage to occur, there must be evidence based on the antioxidant defense capacity of the tissues and biological membranes involved. The observed induction of lipid peroxidation and oxidative enzymes in the testes of rats fed petroleum-polluted diets without BLME treatment is consistent with the observed reduced levels of defensive markers..

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