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Antioxidant potential of Carica papaya Linn ( Caricaceae ) leaf extract in mice with cyclophosphamide induced oxidative stress

METHODS: The male Swiss mice received 15 days of treatment with the extract (500 mg kg-1, via gavage) and intraperitoneal injection of cyclophosphamide (75 mg kg-1) or saline (0.9%) on the 15th day. After 24 h the last treatment, the animals were anesthetized for blood withdrawal, sacrificed and removal of the organs for analyses (liver, kidney and heart). In the biochemical tests were determined: hematological parameters in blood, aminotransferases, alkaline phosphatase, glucose and total cholesterol dosages in plasma, enzymatic and non-enzymatic antioxidants and lipid damage marker were evaluated in different tissues, besides genotoxic and histopathological analyzes. RESULTS: In the extract of Carica papaya leaves, the flavonoids quercetin-3β-D-glucoside and rutin were identified, besides present positive results for alkaloids, saponins and tannins. This extract increased the activity of glutathione-S-transferase and catalase enzymes in the liver and reduced the levels of reduced glutathione in the kidneys and hematocrit levels, red cell count, and hemoglobin. It promoted the decrease of the reactive species of thiobarbituric acid (TBARS) in the kidneys and the activity of enzyme aspartate aminotransferase in the plasma and was antimutagenic in the micronucleus test.


INTRODUCTION
Free radicals are molecules or molecular fragments containing one or more unpaired electrons, a condition that confers high reactivity and may present considerable interference in cellular integrity [1]. The adverse effects of free radicals occur when there is an overproduction of reactive species and a deficiency of antioxidant enzymes and non-enzymatic antioxidants (reduced glutathione, ascorbic acid, tocopherols, carotenoids, vitamins A and E) [2].
The organism has an antioxidant defense system, enabling the evaluation of oxidative stress by means of analysis of some antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione-S-transferase (GST) and others such as reduced such as reduced gluthatione (GSH) levels as even though it is a non-enzymatic antioxidant agent, GSH can act as a substrate in the reactions catalyzed by enzymes, for example GST, or can act directly in free radical scanning [3].An additional parameter useful in evaluating oxidative stress is the lipid peroxidation generated in the cellular membranes. This triggers several actions harmful to the cell, which can result in its death. As the free radical has a very short halflife, it is only possible to be measured by markers such as malondialdehyde through TBARS analysis (thiobarbituric acid reactive substances) [4].
Cyclophosphamide (CP) is a widely used drug for the treatment of chronic diseases, autoimmune diseases and cancer [5]. The antineoplastic activity of CP is due to phosphoramide mustard, which promotes the alkylation of DNA, in addition to the other metabolite, acrolein, which interferes with the antioxidant system producing reactive species, superoxide radical and hydrogen peroxide, leading to toxicity of various organs [6].
The Carica papaya Linn, known as mamoeiro (in Brazil) is a tree present in tropical and subtropical regions of the world, with its fruit known as papaya.
The fruit stands out as having a pleasant taste and aroma and high nutritional value, being rich in sugars, calcium, carotenoids and vitamin C [7].
The fruits, leaves, flowers, roots, seeds and even latex are all widely used in traditional medicine to treat a variety of diseases. In particular, the leaves are used in healing, in the treatment of dengue, jaundice and malaria [8]. Some studies have investigated these medicinal properties of the leaves, for example the methanolic extract promoted inhibition of sickle hemoglobin formation and hemolysis in vitro tests [9] and antioxidant and cytoprotective action of the hydrometanolic extract in human liver cell lines oxidatively stressed with tert-butyl peroxide [10]. The ethanolic extract presented analgesic action compared to the aspirin action in an experimental model with mice [11]; antibacterial and antithrombocytopenic activity in Wistar rats using aqueous extract and lastly antiproliferative and antimetastatic activity of dry leaf extract on prostate cancer cell lines was observed [12].
The objective of the present study was to investigate the effects of raw aqueous extract of

Preparation of Carica papaya Linn extract
For the preparation of the extract, the leaves of C. papaya were collected and cleaned with distilled water and exposed to the fan-forced oven drying process at an average temperature of 40 °C for seven days. After drying, the leaves were crushed, yielding a weight of 220 g. The crushed leaves were infused with distilled water (4.5 L) under a stable temperature of 70 °C for 1 hour in a water bath. After this time, the material was filtered and rotated with vacuum pump under reduced pressure and water bath at 60 °C. Subsequently, the samples were frozen and lyophilized to obtain the final crude extract (CE) of 68.47 g.
The aqueous extract was produced to resemble that which is used by the general population, in which it is commonly used to make tea or as an infusion. The selected dose was 500 mg kg -1 ,in accordance with [13].

Determination of flavonoids and DPPH • test
The determination of the amount of total flavonoids was performed using quercetin as a standard curve in a reaction with aluminum chloride, in accordance with [14]. The result was expressed in milligrams of quercetin equivalent per gram of extract (mg EQ g -1 ).
The antioxidant potential of the vegetable extract was evaluated based on the methodology of [15]. From the consumption of the DPPH • free radical (2,2-diphenyl-1-picrylhydrazine) in the samples, the absorbance of the solutions read at wavelength 515 nm using rutin and ascorbic acid as standards was measured.

Phytochemical screening
The presence of other secondary metabolites in the extract was evaluated through qualitative tests; the colorimetric tests were used to verify the presence of alkaloids, coumarins, steroids, saponins, polysaccharides, purines and tannins following the methodology of [16]. ionizationaccording to [17], source temperature 300 °C, and desolvation temperature 250 °C.  Biochemical analyzes were performed on superoxide dismutase (SOD) based on [19], the result being expressed as UI SOD mg protein -1 .

Animals and experimental design
Catalase activity (CAT) was measured according to [20] and the results expressed in μmol min -1 mg protein -1 . GST analysis followed the methodology [21], with the GST activity expressed in μmol GS-DNB min -1 mg protein -1 . Reduced glutathione (GSH) was quantified according to [22], with thiolate anion formation evaluated and compared to a standard GSH curve. The result was expressed in μmol GSH mg protein -1 . The thiobarbituric acid reactive substances (TBARS) followed the method

Micronucleus test
The micronucleus test was performed in accordance with the methodology of [25] where A formula was used to verify the percent of harm reduction as the mean frequency decrease of micronucleated cells according to [26]and [27] using the formula:

STATISTICAL ANALYSIS
In order to compare the differences in the biochemical variables between pretreatment (water with vehicle or extract) and treatment (CP or saline), statistical analyses were performed using one-way or two-way analysis of variance (ANOVA) followed by the Tukey's test. Bartlett's test was performed to compare the homogeneity of variances among the groups. Data were expressed as mean ± standard deviation. For the micronucleus frequency test, the chi-square test was used according to [28].

Flavonoid content and antioxidant activity in vitro
The CE extract presented low antioxidant activity in vitro when compared to ascorbic acid and rutin standards, not being able to reach 50% elimination of the DPPH • radical. On the total flavonoids, the value of 21.7 mg EQ g -1 was obtained in the CE (data not shown).

Presence of compounds by phytochemical screening
In the phytochemical screening of CE, the tests showed positive results only for the presence of alkaloids, saponins and tannins (data not shown).  Table 1.    Table 2 shows CAT activity in the hepatic, renal and cardiac tissues. In the hepatic tissue a significant reduction of the CAT (p < 0.05) in the CP group was observed, a reduction of 24% in comparison to the control. However, CE significantly restarted CAT activity (p < 0.05), the action of CP. The other tissues did not present significant differences. The GSH levels (

Blood parameters
Biochemical parameters of the plasma of the treated animals were evaluated (   Table 6.

Histological analysis of the liver
No significant histological differences were observed between the treated groups, as observed in Figure 4 (A, B, C and D).

DISCUSSION
Plants are sources of remarkable active molecules that can modulate oxidative stress [29].
Currently, this mechanism has been extensively studied. Particularly, the leaves of C. papaya have aroused interest for these studies. In the present work, we investigated the effect of the crude extract of [30] and [31] where the fragmentary ion of the rutin corresponds to the loss of two glycans [32], and the quercetin-3β-D-glycoside fragment comprises the cleavage of the glycoside group.
As regards phytochemical screening, the tests were positive for the presence of alkaloids, saponins and tannins as previously described in the literature by [33] also with aqueous extract.
In biological tests, CP caused a significant reduction (p < 0.05) in both SOD and CAT activity of liver tissue.In addition, we observed a decrease in the GSH for all tissues, as described by [34] and [35]. This occurs because during the metabolism of CP ROS is generated that lead to the depletion of antioxidant enzymes in different tissues [36][37][38][39]. In his studies [37] found that acrolein induces the irreversible inactivation of SOD activity by attacking its amino acid residues, histidine for example, which is pointed out as more susceptible since it is an essential amino acid for SOD activity and for increasing protein carbonylation. Other amino acids are susceptible to attack by acrolein, lysine, cysteine, serine, arginine, and threonine.
Acrolein may have its production triggered by various conditions; some are metabolized by cyclophosphamide or oxidation of metal-catalyzed polyunsaturated fatty acids. Point to the inactivation process of SOD, the superoxide radical induces the inactivation of the CAT enzyme [38]. The reduction of GSH after CP exposure occurs because GSH conjugates with acrolein to form mercapturic acid making it less likely to exert its toxic effects on the body and facilitates its elimination through the urine [39].
In contrast, CE restored liver CAT activity, helped decrease lipid peroxidation in liver and kidney, increased liver GST and kidney GSH. In a similar study, using papaya epicarp in human cells that had oxidative stress induced by hydrogen peroxide, the extract increased CAT activity and GSH levels, in addition to minimizing lipid peroxidation [40]. In other work [41], rats that had the oxidative stress induced by Fe 2+ ions and received aqueous extract of the green fruit, saw a decrease in TBARS.
Another study showed that fruit extract was also used against acrylamide-induced oxidative stress, resulting in a decrease in TBARS in the liver and kidney, as well as an increase in GSH and CAT in the tissues mentioned [42]. GST showed a significant increase in its activity, showing that the enzyme was not depleted by CP, similarly to [43]. This may be a response of the body of animals treated to combat the effects of CP. The role of GST is to protect against oxidants by catalyzing the conjugation of the sulfur atom of glutathione to an electrolytic center of toxic xenobiotics in order to produce compounds that facilitate its metabolism and excretion [44]. CE also induced an increase in GST activity, such as has been observed by [45] which identified a similar effect of fruit investigations, found that ethyl acetate extract from the fruits of C. papaya on GST increase rat liver cell lines at the 25 mg mL -1 concentration.
Considering that the extract of the present study contains rutin and quercetin-3-β-d-glucoside, we suggest that these flavonoids may be interfering with these findings regarding the various oxidative stress parameters, since there are studies that confirm their antioxidant effects [48][49][50].
Although CP is widely used in clinical practice, its use produces several side effects in the organism, among them the elevation of liver enzymes [51].
Increased serum levels of AST and ALT are clinical markers used to assess hepatocellular toxicity [52]. CP treatment induced a significant increase of AST and ALP enzymes, as well as increased TBARS in liver and kidney, indicating hepatic and renal damage by CP administration, results already obtained in the literature [53][54][55]. This increase in TBARS levels is due to the fact that the production of free radicals mediated by CP metabolites stimulates the lipid peroxidation process [56].
CE resulted in a significant increase in enzymes ALT and ALP besides TBARS in the livers of the group receiving only the extract, suggesting toxicity. The increase in ALP has already been observed by [57] in Wistar rats using aqueous extract of C. papaya leaves for 7 days of administration. This liver toxicity caused by CE may be due to the presence of other substances that may exhibit toxic effects to the body or its prolonged use is not advised. In the phytochemical screening of CE, the tests were positive for the presence of tannins and alkaloids; in low doses these substances have a positive effect, but their excess can lead to hepatotoxicity as already reported by [58]. In addition [59], verified an abundance of calcium oxalate in leaves of C.
papaya by micromorphology and chemical tests.
The presence of these compounds may have led to the toxic event.
Although hepatotoxic action of the extract on hepatic enzymes was observed, no lesions were observed in the hepatocytes or any structure of the liver in the histological analysis of the treated animals. The same was observed by Ismail et al. [60] where rats treated with aqueous extract of leaves C. papaya at a concentration of 140 mg kg -1 for 13 weeks showed no histopathological differences in hepatic tissue. In that study the increase of the ALP enzyme was also observed.
In the histological analysis no damage was observed in the hepatic tissue by CP, although it is common to find works that show that this drug

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hepatocellular necrosis is observed at high doses or in conjunction with busulfan or BCNU [61]. In addition, the dose administrated of CP in our work was 75 mg kg -1 for 24 hours, so it is probably that was a short time exposure to cause histological alterations.
A significant increase in the levels of HCT, Hb, and RBC was observed with decrease of the counts of WBC in the groups CP and CE + CP. The number of WBC from peripheral blood can directly reflect the degree of myelosuppression of chemotherapeutic agents because myelosuppression often first manifests as a decline in white blood cells, followed by a series of hematopoietic impairments [62].
The number of WBC changes most obviously because of its short life cycle [63]. CE also caused a significant increase (p < 0.05) in HCT, Hb and RBC levels. Data similar has been observed in the work of Song et al. [64], where treatment with aqueous extract of leaves C. papaya during a period of 5 days (twice a day) was administered to a dengue patient, increasing, among other parameters, the HCT and RBC indexes. In this context, the work of Ahmad et al. [65] demonstrated that the leaf extract of papaya saw healthy increased levels of RBC in mice, indicating strong eritropoietic activation. In the studies of Dharmarathna et al. [66] it has also been observed that the ethanolic extract of the leaves of C. papaya promotes an eritropietic stimulation when analyzing cells of the bone marrow of mice. Increases in blood components may be related to the presence of rutin in CE, since it has already been associated with the ability to attenuate myelosuppression and increase eritropoietic production [67]. In addition, the rutin is attributed to the ability to ameliorate ROS action [55].
In the micronucleus test the CP induced an increase of PCEMN in comparison with control, results already observed in the works done with mouse bone marrow cells using a dose of 50 mg kg -1 [68][69][70]. The results show that CP induced chromosomal damage, because the drug does not specifically act on tumor cells, binds covalently to DNA and interferes with the cell cycle [71].
The CE showed antimugenic activity, decreasing significantly the PCEMN frequency by 28%, proving this effect. There are no papers in the literature using leaves of C. papaya with antimugenic action; on the other hand [72], verified that the aqueous extract of roots of C. papaya was antimutagenic in the micronucleus test with bone marrow of Wistar rats. Another study points to antiproliferative and anti-metastatic activity of papaya leaf extract on prostate cancer cell lines [12].The rutin has already been attributed to the ability to repair DNA damage, Wistar rats that were supplemented for two weeks with rutin (10 mg/100g) prior to induction of carcinogenic damage had reduced damage [73] and in our study rutin is one of the flavonoids present in CE used in the treatment of animals.

CONCLUSION
The present study showed that the crude

Funding
This study did not receive financial support from external sources