Phytotherapy in reducing glycemic index and testicular oxidative stress resulting from induced diabetes : a review

Oxidative stress plays a main role in the development of diabetes complications. The impairment of gonadal antioxidant potential and endocrine disturbance in diabetic males causes testicular damage and failure in sperm production. Plants have been widely used to control diabetes due their hypoglycemic and antioxidant potential, contributing towards the recovery of testicular function. Current study comprises a review of the literature on the main medicinal plants used in the recovery of testicular oxidative damage in animals with experimental diabetes. Eighteen plant species in the nineteen studies selected from the search strategy were evaluated. Plant extracts were evaluated according to their effects on blood glucose and insulin levels, antioxidant enzymes and oxidant levels, lipid peroxidation, total protein, testosterone levels, gonadosomatic index, diameter of seminiferous tubules, seminiferous epithelium height and integrity, number of germ cells at stage VII and apoptosis in the seminiferous epithelium, sperm production, motility, viability and morphology. After the analysis of the studies, it was observed that plant species, used alone or in combination, may control testicular oxidative damage triggered by diabetes. The antioxidant potential varies among species, with some plants proving to have a better performance in the recovery of reproduction parameters than others.


Introduction
Diabetes is a metabolic disease characterized by increased blood glucose levels due to failure in insulin production or function.The persistence of high levels of blood glucose compromises homeostasis and promotes body damage, including retinopathy, nephropathy, cardiovascular complications and sexual dysfunction (Van Belle et al., 2011).
Oxidative stress is the main cause of diabetes complications (Maritim et al., 2003).It mainly originates from high blood glucose levels, where the reactive oxygen species (ROS) and the free radicals (FR) production exceeds the defense capacity of the organism and disrupt the cellular reduction-oxidation balance (Dowling and Simmons, 2009;Cansian et al., 2015;Takao et al., 2015).
The impairment of gonadal antioxidant potential of the male reproduction system causes testicular disturbance and failure in sperm production (Mallick et al., 2007;Hamden et al., 2008).Furthermore, disorders in the endocrine control of spermatogenesis have also been observed in diabetic patients, with reduced levels of essential hormones, such as FSH, LH and testosterone (Ballester et al., 2004;Agbaje et al., 2007).
The body's natural antioxidants act by neutralizing ROS or by preventing the release of ions responsible for initiating lipid peroxidation (Shah and Khan, 2014).Superoxide dismutase (SOD) and catalase (CAT) are key enzymes that act against toxicity by controlling ROS levels (Singh et al., 2013).It has been reported that SOD and CAT levels are reduced in diabetic patients, which, along with increased Bcl-2 and Bax protein, lead to apoptosis of the testicular germ cells (Chatterjee et al., 2012).
Thus, glycemic control and increased antioxidant protection are necessary to reduce the body damage in diabetic conditions (Singh et al., 2013).The most common way to achieve nearly-normal glucose levels is the daily administration of insulin or insulin pumps (Ratner et al., 2000).Although other methods such as the use of sulfonylureas and biguanides may also be applied, they are expensive and may cause complications involving toxicity, gastrointestinal disorders and weight gain (Modak et al., 2007).
Therefore, plants have been widely used as a method for the control of diabetes due to their products in secondary metabolism and their hypoglycemic and antioxidant potential (Singh et al., 2013;Ghosh and Roy, 2013).These products minimize the damage caused by the accumulation of free radicals and contribute towards the recovery of testicular functions (Modak et al., 2007;Shah and Khan, 2014).Thus, the aim of current literature review is to report on the main medicinal plants with antioxidant and hypoglycemic potential used in the recovery of testicular oxidative damage in animals with experimental diabetes.

Material and Methods
Articles were identified by searching the MedLine, PubMed and Scopus databases in October 2014.There was no article restriction related to the year of publication.
In the search strategy, the following combinations of words were used: 1. plants AND oxidative stress AND diabetes AND testes AND rat, 2. plants AND oxidative stress AND diabetes AND sperm AND rat, 3. plants AND oxidative stress AND diabetes AND spermatogenesis AND rat, 4. plants AND antioxidant AND diabetes AND testes AND rat, 5. plants AND antioxidant AND diabetes AND spermatogenesis AND rat, 6. plants AND antioxidant AND diabetes AND sperm AND rat.
The selected studies were analyzed and organized according to the year of publication, drugs used to induce diabetes, plant species used for treatment, treatment duration, testicular and reproductive parameters evaluated and treatment efficiency.

Results and Discussion
The literature comprised 429 articles.After the removal of duplicates and after analysis in accordance with the study purposes, 19 articles were selected.Search strategy and the selection of items are summarized in Figure 1.
Table 1 shows the features of the selected studies according to authors, year of publication, drug used to induce experimental type 1 diabetes, plant species used in the extract, type and duration of treatment and testicular parameters used for assessing the treatments' efficiency.
Plant extract had its antioxidant potential assessed in only one diabetic experimental group in 38% of the studies, whereas in 14.2% of the studies, the treatment had been provided to a group of healthy animals, which were used as a control group.The above methodology is important due to the possibility of identifying toxic effects in the treatment by the plant species.Extracts were also used in different doses (14.2%) and with different chemical compositions (14.2%), to determine the most effective treatment.Moreover, the protective role of the extract was evaluated in 9.4% of studies; in other words, the treatment started before the induction of experimental diabetes.
Blood glucose level during the experiment or on euthanasia day was assessed in 52.6% of the studies.The authors with the greatest success on hypoglycemic potential of plant species were Mallick et al. (2007), Chatterjee et al. (2012) and Shah and Khan (2014), who restored glucose levels to those in the control group (see Table 2).
It has been generally observed that increase in the treatment period results in a better response and, consequently, a greater reduction in blood glucose levels.However, Shah and Khan (2014) had better results with higher concentrations in the plant extract.Whereas Chlorophytum borivilianum, Morus alba and Urtica dioica had low efficiency in the reduction of blood glucose levels, Dracaena arborea had no effect and the treated animals revealed similar rates as those for untreated diabetics, regardless of extract concentration.
In 31.6% of studies that evaluated the blood insulin concentration, Mallick et al. (2010) obtained the most efficient results, restoring the low levels observed in diabetic animals to those of control group.However, Mallick et al. (2007), Chatterjee et al. (2012, 2013), Hajizadeh et al. (2014) and Shah and Khan (2014) reported a recovery compared to untreated diabetic animals.According to Chatterjee et al. (2013), the extract used for treatment of diabetic animals increased the production of the transforming growth factor β1 (TGF-β1) which prevents the infiltration of lymphocytes in pancreatic islet and protects β-cells while providing a safe environment for cellular regeneration.
Gonadosomatic index (GSI), the ratio between testicular weights and body weight, was measured in only 21% of the studies.All studies revealed that rates for untreated diabetic animals were significantly lower than those of the other experimental groups and the treatment with plant species resulted in recovery.In spite of the absence of testicular weight in such studies, the reduction of GSI, or rather, the percentage of body weight allocated in the gonads, indicated a decrease in testis size.Specifically in Chatterjee et al. (2013), in which body weight of experimental groups had no significant difference, the reduction of such index features drastic testicular damage.Wankeu-Nya et al. ( 2013) also observed a testicular atrophy in diabetes-induced animals, partially recovered by treatment.
According to França and Russell (1998), testicular weight is an important assessor of androgenic activity and sperm production in mammals.Chatterjee et al. ( 2013) and Ghosh et al. (2014) reported that decrease in GSI and, consequently, in testis weight, is the result of diabetic low testosterone and insulin levels.
Levels of antioxidant enzymes, including catalase, glutathione reductase, glutathione s-transferase, glutathione peroxidase, peroxidase and superoxide dismutase, were investigated in 63% of studies.All papers reported reduced levels of antioxidant enzymes in untreated diabetic animals.However, only 10.4% of the studies measured oxidant levels, such as nitric oxide, nitrite/nitrate and hydrogen peroxide.Giribabu et al. (2014) and Shah and Khan (2014) observed increased levels of these oxidants respectively   2014) noted a reduction in the levels of these oxidative stress indicators in the sperm of diabetic rats and underscored the extract's capacity to eliminate free radicals.More efficient results were obtained when higher extract concentration for ROS levels were administered, also corroborated by Shah and Khan (2014).Thereby, the plants' active principles play an important role in reducing blood glucose levels and in recovering testicular antioxidant defense (Hamden et al., 2008).Oxidative stress is a main feature in diabetic conditions and results from persistent high blood glucose levels.There is a reduction in the levels of critical enzymes for antioxidant protection under these conditions, leading to increased formation of reactive oxygen species and, consequently, to tissue damage, such as lipid peroxidation and increased rate of apoptosis (Maritim et al., 2003;Ghosh, et al., 2014).Superoxide dismutase (SOD) and peroxidase are the principal enzymes that protect the male reproduction organs (Chatterjee et al., 2013).All studies that assessed the concentration of antioxidant enzymes, such as catalase, peroxidase, superoxide dismutase and glutathione in diabetic animals, reported decrease in their levels.
The methods employed were effective in recovering the levels of most antioxidant enzymes to control group Different letters in the same study represent a significant difference (p<0.05),their absence indicates lack of statistical information.
levels.Nevertheless, Singh et al. (2013) found no significant increase in testicular glutathione S-transferase levels (GST) although Raza et al. (2004), Mallick et al. (2010), Shah and Khan (2014), Chatterjee et al. (2012, 2013) reported increase of GST levels in testis and/or sperm to the levels of control group.In studies by Mallick et al. (2007Mallick et al. ( , 2010)), Hamden et al. (2008), Giribabu et al. (2014) and Shah and Khan (2014), the treatment with plant extracts resulted in the recovery of catalase, SOD and glutathione peroxidase (GPx) levels.Mallick et al. (2007) achieved the recovery of catalase and glutathione peroxidase levels and thus antioxidant protection to the testicular tissue.Hamden et al. (2008) noted that the levels of antioxidant enzymes were more efficiently recovered in animals treated with the extract prior to diabetes induction rather than those treated after induction.Further, there was an increase in enzyme cofactors levels, such as zinc, copper and iron, providing a protective effect against damage and death induced by the accumulation of free radicals.Reduction in enzyme cofactors levels is directly related to SOD decrease and to high blood glucose levels (Shah and Khan, 2014).
Chatterjee et al. ( 2012) noted that levels of catalase, peroxidase and superoxide dismutase increased after treatment, whereas Shah and Khan (2014) observed that extract administration enhanced the recovery of enzymes levels.Moreover, better results were achieved with higher extract doses.Ghosh et al. (2014) and Chatterjee et al.
(2013) obtained the recovery of catalase, peroxidase and superoxide dismutase levels similar to the control group.In the study by Hajizadeh et al. (2014), the treatment recovered glutathione levels to higher rates than those of the control group.Moreover, Ukwenya et al. (2013) observed a recovery in SOD, with an increase in levels similar to control group and higher than insulin group.Mallick et al. (2007), Chatterjee et al. (2012, 2013) and Ghosh et al. (2014) reported a decrease in oxidative markers that indicate the absence of metabolic toxicity of the used compounds.Thereby, medicinal plants comprise components, such as flavonoids and alkaloids, with hypoglycemic properties and antioxidant potential.They also play a fundamental role in the stimulation of endogenous production of protective enzymes and in the monitoring of the blood glucose levels in diabetic subjects (Mallick et al. 2010;Chatterjee et al., 2013;Ukwenya et al., 2013).
Further, 52.4% of studies measured the levels of testicular lipid peroxidation.In lipid peroxidation, the oxidizing substances, mostly ROS, attack the lipids with carbon-carbon double bond, especially polyunsaturated fatty acids in membranes, and cause apoptosis or necrosis (Yin et al., 2011;Ayala et al., 2014).Sperm plasma membrane has great amounts of the polyunsaturated fatty acid called docosahexaenoic acid, which makes it highly susceptible to oxidation (Suresh et al., 2013;Chatterjee et al., 2013;Giribabu et al., 2014).Increase in ROS levels, as observed in diabetic patients, changes membrane permeability, while the attack on mitochondrial membrane leads to reduced ATP production and alteration of sperm motility and morphology (Amaral et al., 2006;Chatterjee et al., 2013).
The methods used by Mallick et al. (2010), Chatterjee et al. (2012, 2013), Suresh et al. (2013), Ukwenya et al. (2013) and Hajizadeh et al. (2014) were highly effective in reducing the high levels of lipid peroxidation in diabetic animals to the levels of control group.Mallick et al. (2007) and Singh et al. (2013) also achieved good results and Giribabu et al. (2014) and Shah and Khan (2014) observed that dose increase caused better recovery.Hamden et al. (2008) reported that preventive treatment before the onset of diabetes was more efficient for controlling lipid peroxidation levels than the treatment with diabetic animals.
Reactive oxygen species may also cause cell damage by the activation of apoptosis-inducing proteins, such as Bax (Chatterjee et al., 2012).Furthermore, induction of pro-apoptotic factors in germ cells is often related to testosterone concentration (Mallick et al., 2010).Thus, diabetic patients have an increase in apoptosis levels within the seminiferous tubules which may be linked to the reduction of steroidogenesis and increase in ROS production.
In 21% of the studies analyzed, apoptosis was evaluated within the seminiferous tubules through the identification of cell death in the germ cells and the expression of anti-apoptotic factors, such as Bcl-2, and pro-apoptotic factors, such as Bax.
In diabetic animals without treatment, Chatterjee et al. (2012,2013) and Ghosh et al. (2014) observed increased rates of Bax gene, and Ghosh et al. (2014) noted the reduction of Bcl-2 expression.However, in the three studies, treatment with plant extracts were efficient in the recovery of Bax levels and Bcl-2 expression to the levels of the control group.Mallick et al. (2010) also observed a reduction in the number of apoptotic germ cells in diabetic animals throughout the treatment.According to Mallick et al. (2010) and Chatterjee et al. (2012, 2013), reductions in cell death correspond to decrease in ROS peroxidation of the lipid membrane.
The control of apoptotic rates within the seminiferous tubules is important in regulating the population of germ cells and directly reflects sperm production (Clermont, 1972;Russell et al., 1990).The death of a large number of cells during the spermatogenic process may cause infertility due to cell loss (Ghosh et al., 2014).Besides the number of germ cells turning into spermatozoa, male fertility also depends on sperm motility and morphology (Venkatesh et al., 2009). In fact, 47.4, 31.6, 21 and 15.8% of the studies respectively evaluated sperm production, sperm motility, sperm viability and sperm morphology.
All studies demonstrated that untreated diabetic rats had significant reductions in sperm number, motility and viability when compared to control group.Furthermore, Azeez et al. (2010) and Suresh et al. (2013) also observed alteration in sperm morphology.Treatments performed by Mallick et al. (2007, 2010), Chatterjee et al. (2012, 2013), Suresh et al. (2013) and Ghosh et al. (2014) resulted in some recovery of sperm parameters, whereas Giribabu et al. (2014) reported significant improvement with an increase in extract concentration.Hamden et al. (2008) had better results with preventive actions.
On the other hand, Azeez et al. (2010) did not report any improvement in sperm motility and morphology.Only the experimental group that received the highest extract dose (1000 mg/kg) showed recovery signs in sperm viability.The authors attributed the changes in sperm parameters of diabetic animals to oxidative damage.Since satisfactory results were not obtained, it seems that Cnidoscolus aconitifolius extract and the treatment used are not effective in controlling the production of oxidants.
In the study by Suresh et al. (2013), the Mucuna pruriens extract was effective in restoring the high number of spermatozoa with cytoplasmic droplets and protected the sperm DNA against damages in diabetic animals.These authors revealed that the plant species played an important role in suppressing ROS production and in stimulating antioxidant activity.Giribabu et al. (2014) observed a partial recovery in plasma membrane integrity and sperm morphology, directly related to the control of lipid peroxidation, hydrogen peroxide and nitric oxide production.
According to Chatterjee et al. ( 2013), the amount of sperm in diabetic animals decreases due to an increase in fructose concentration in the seminal plasma, as observed in 10.5% of studies.However, the methods used by Chatterjee et al. ( 2013) and Ghosh et al. (2014) reduced the semen fructose levels to levels of control animals.
Few studies investigated the characteristics of the seminiferous tubules: 21% observed tubular diameter and seminiferous epithelium integrity; 15.8% evaluated the number of germ cells at stage VII of the seminiferous epithelium cycle; 5.3% measured the seminiferous epithelium height.All authors observed injury in these parameters caused by diabetes.Mallick et al. (2007Mallick et al. ( , 2010)) Plasma testosterone level, performed in 42% of the assessed papers, was another parameter used for the evaluation of the testicular function.Untreated diabetic animals had a decrease in this hormone level in all studies.Such reductions have been attributed to the functionality of the Leydig cells.However, only Mallick et al. (2007) performed an analysis of the Leydig cells nuclear diameter and correlated it with decrease in testosterone levels.
Diabetes is associated with alterations in the hypothalamic-pituitary-testicular axis, resulting in significant reductions in the levels of hormones involved in spermatogenesis (Ballester et al., 2004;Agbaje et al., 2007).In addition, Leydig cells possess insulin receptors, or rather, they are dependent on optimal insulin concentrations for the efficient testosterone production (Brüning et al., 2000).Mallick et al. (2007) reported a reduction in Δ5.3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase enzymatic activities, both involved in steroidogenesis, and a reduction in the nuclear area of Leydig cells.Decrease indicates the cells' functionality failure due to testosterone reduction in diabetic animals.Additionally, Chatterjee et al. ( 2012) observed an increase in cholesterol levels of diabetic animals, which impaired androgen production due to a lower usage of the cholesterol molecule.In Hamden et al. (2008), testicular total cholesterol levels were reduced in untreated diabetic animals.This may be due to the cholesterol glycation reactions or cholesterol-carriers with glucose, inhibiting the transportation to the testicle.
Despite the differences between the experimental results, the administration of plant extracts was effective in the rise of circulating testosterone levels, the restoration of the normal levels of cholesterol and the recovering of Leydig cell morphology.
All studies reported the increase of testosterone levels in treated groups.However, not all experiments succeeded in raising testosterone levels to the rates of the control group.The methods used by Mallick et al. (2010), Shahreari et al. (2010), Michael et al. (2013) and Ghosh et al. (2014) with plant extracts of Coccinia indica, Musa paradisiaca, Danae racemosa, Eugenia jambolana, and Phoenix dactylifera, respectively, proved to be highly efficient in recovering testosterone levels, with rates very close to and even higher than control group.Hamden et al. (2008) observed that the improvement of testosterone levels was more efficient in pre-diabetes induction group.Consequently, the extract had a protective effect, minimizing the reduction in testosterone levels.Nevertheless, the hormone levels were not as similar to the levels of the control group.
The amount of total protein was measured in 15.79% of the studies.All papers revealed reductions in protein levels in untreated diabetic animals.In a hyperglycemic state, glucose molecules may interact with proteins in non-enzymatic reactions to form advanced glycation end-products.High glycation leads to pathological complications that modify the functionality of various biological structures and results in the production of ROS (Barbosa et al., 2008).After treatment with plant extracts, the parameter was recovered.Shah and Khan (2014) observed an improvement directly related to the extract concentration, and the animals with the highest dose achieved results close to control group levels.

Conclusion
Results from all the studies under analysis showed that plant species have active principles in their composition that effectively help to control blood glucose levels and testicular oxidative damage triggered by diabetes.Although the antioxidant potential and the recoverability of reproductive parameters vary among species, these plants may be used as new alternatives to mitigate the effects of diabetes and improve the life quality of diabetic patients.Nevertheless, further analyses in testicular cells are required for a more complete conclusion on the benefits that the treatment provides to the organ´s morphology and function.Additionally, toxicological analyses in organs, such as liver, kidneys and spleen, may be required to have a more precise picture of the extracts' applicability.

Figure 1 .
Figure 1.Search strategy in MedLine, Scopus and PubMed databases and selection of articles on the use of plants for the recovery of testicular oxidative damage resulting from experimental type I diabetes.

Table 1 .
Methodological characteristics of selected studies on the use of plants as therapy against testicular oxidative effect of experimental Type 1 diabetes.
(Chatterjee et al., 2013)o auto-oxidation of glucose and non-enzymatic glycation during hyperglycemia, with the subsequent increase in the production of hydrogen peroxide and superoxide radical(Chatterjee et al., 2013).Giribabu et al. (

Table 2 .
Glucose levels after induction of experimental Type 1 diabetes and under treatment with the plant species in the selected studies.