Detoxifying and antioxidant effect of ellagic acid nano particles in rats intoxicated with sodium nitrites

Sodium nitrite is used as a preservative in food products to stabilize color and reduce rancidity. Its absorption into the body causes many diseases. Ellagic acid is a natural polyphenol that contains powerful antioxidants, but it is taken as a poorly absorbed food. Therefore, chitosan-coated nanoparticles (EANP@CS) were loaded to enhance their bioactivity and bioavailability after oral administration. EANP@CS was administrated in rats given water containing sodium nitrite to reduce toxins. Ellagic acid was extracted from pomegranate and manufactured EANP@CS. EANP@CS was identified by FT-IR, UV, X-ray diffraction and TEM. Average EANP@CS size ranges from 20–62 nm. Rats were divided into five groups: normal, treated using EANP@CS; rats were receiving nitrite for 8 weeks, and the last two groups were treated with EANP@CS. Serum and liver NO, MDA, and DNA fragmentation were reduced. Liver thiol and GSH levels, and Gpx, catalase and GST activities increased in rats treated with EANP@CS rats compared to rats drinking nitrites. Liver NOS activity was reduced 7 and 4.9 times in rats treated with EANP@CS during or after discontinuation of nitrite administration, respectively. Liver arginase activity was raised in rats that drink nitrite or were treated using EANP@CS. Inflammatory infiltrations of the liver, kidney, and spleen were observed in the tissues of rats that received nitrites and improved when the rats were given EANP@CS. EANP@CS improved oral bioavailability and reduce the risk of sodium nitrite in rats. EANP@CS can be used as a therapeutic goal to detoxify any unwanted toxic substance in food.


Introduction
Sodium nitrite is used to stabilize meat color and reduce rancidity by inhibiting lipid peroxidation and growth of microorganisms on food. Nitrites are used in the medical field such as vasodilator, bronchodilator, and detoxifying of cyanide poisoning. But the use of nitrite leads to human exposure to many diseases [1].
Sodium nitrite is one of the causes of colorectal cancer. Sodium nitrite is added to preserved meat or any other foods to prevent the growth of microorganisms such as Clostridium botulinum [2]. Sodium nitrite consumption cancer cells [7]. Ellagic acid is taken with many drugs that cause oxidative stress [7,8]. Ellagic acid is insoluble in water, so it has low bioavailability, because of its metabolites, and is quickly eliminated. So its therapeutic advantage is restricted because most clinical drugs are administered orally [9].
Chitosan is a polymer compound derived from chitin by N-deacetylation. It is commonly used in food, bioengineering, enzyme loading, drug conduction carrier, plant growth factor, and antimicrobial. It has the property of biodegradation, and biological compatibility and activity, [10]. Chitosan nanoparticles are more precisely applied to cells, and positively charged chitosan nanoparticles give greater convergence to negative-charge biological membrane and site-specific site targeting, allowing their use as drug-coated materials, to accelerate drug release in a target cells. Chitosan nanoparticles are used in the food industry to control food stability [11].
Ellagic acid nanoparticles that are coated with chitosan polymer lead to increased absorption and delivery of the ellagic acid (EA) to the target cells. Chitosan-coated ellagic nanoparticles (EANP@CS) can increase EA stability and improve drug absorption, have been used as a system to release the drug and provide an appropriate dose of the drug to the target tissues. EANP@CS increases the affinity of these nanoparticles with protein receptors in the target cells and may prevent the accumulation of EA molecules [12].
This study aimed to extract EA from pomegranate and convert it into nanoparticles with the help of chitosan polymer (EANP@CS) to enhance biological availability. Antioxidants, nitric oxide, NOS, arginase, and DNA fragmented were then evaluated in the liver and kidney to study the efficacy of EANP@CS in rats intoxicated with sodium nitrite.

Extraction of ellagic acid
Ellagic acid was extracted from the pomegranate peel powder (Punica granatum), the pomegranate peel was purchased from the Egyptian market and was recognized by the Faculty of Science, University of Tanta (Herbarium-TANE, Index Herbariorum New York Botanical Garden), according to Lu and Yuan [13] with some modification. In brief, ground crushed pomegranate peel was immersed in 60% aqueous ethanol and stirred over a hot plate with a stirring The tannin that results from this extract was filter and hydrolyzed by 5% H 2 SO 4 for 5 h and left overnight. The dark brown crude precipitate EA was collected and dissolved in ethanol, filtered and left to dry in the oven at 50°C.

Synthesis of EA nanoparticles coated with chitosan (EANP@ CS)
EANP@CS was synthesized using sodium triphosphate as a gelatin agent with some modification. Chitosan polymer (MW = 60-90 kDa, ≥ 85% decyl, sigma) was dissolved in 2% glacial acetic acid. The mixture of chitosan and glacial acetic acid was stirred overnight at ambient temperature and the pH was adjusted to 5.0 using sodium hydroxide 1 M. Ellagic acid was dissolved in 0.5% dimethyl sulfoxide (DMSO), added to the fresh chitosan dispersion and stirred for about an hour. An equal amount of sodium tripolyphosphate (Sigma Aldrich, USA) 1 mg/mL was added drop wise to the CS-EA solution under moderate stirring. The obtained mixture was stirred for 1 h to form EA coated chitosan nanoparticles. EANP@CS was separated by centrifugation. The pellets were collected and stored at −20°C for further use [14].

Identification of ellagic acid and EANP Fourier transforms Infrared spectroscopy (FT-IR)
To identify the functional groups of the active compound, an infrared spectroscopy (Model-JASCO FT-IR 4100 LE, made in Japan, range: 4000-400 cm -1) was performed in the infrared field in the (Microanalytical Unit, Faculty of Science, Tanta University, Egypt). The extract of Ellagic Acid and EANP @ CS (1-2 mg) was ground to a powder with mortar and mixed with KBr (3-4 mg).

Ultraviolet spectroscopy (UV)
Absorption of ellagic acid and EANP @ CS extract was determined by a double visible UV spectrophotometer in the ultraviolet region (200 nm-800 nm) at a 5 nm interval and readings versus blank. The graph is drawn between absorption and wavelength to determine the maximum wavelength for ellagic acid and EANP@CS (Microanalytical Unit, Faculty of Science, Tanta University, Egypt).

X-ray diffraction
X-ray diffraction patterns for EA and EANP@CS (Philips, Netherlands) were recorded with Cu Kα (1.5418 Å) as the source and the spectrum was collected with an average scanning rate of 10 per min.

Transmission electron microscopy (TEM)
Transmission electron mcroscopy (TEM) was taken to capture different image snapshots of EANP@ CS. EANP@CS image size determined using JEM 2010F electron microscope with 200 kV operating voltage (JEOL, Ltd., Tokyo, Japan). EANP@CS was ultrasonicated for 30 min, one drop of ellagic acid nanoparticle sample was placed on the smooth film of carbon-coated TEM copper grid and dried. A drop of 0.5% of phosphotungstic acid was diffused on the TEM grid.

Experimental animals
Fifty adult white male albino Wister rats, 3-4 months old with an average body weight of 180-200 g, were used in the experimental study. The mice were purchased from the Animal House at Tanta University, Egypt. The mice lived in a specially controlled room. The rats were treated according to the instructions of the Animal Ethics Committee (Tanta University, Egypt). The animals were adapted 2 weeks before the experiment.
Rats were divided into two main groups: the normal group and the sodium nitrite group, the normal group was subdivided into control rats that did not receive any drugs (GI), the normal rats received EANP@CS (G. II), the rats were received orally EANP@CS (20 mg/kg b.Wt.) 4 day/week [15]. The sodium nitrite group was divided into three subgroups, the untreated nitrite group (G. III) (rats received 2 g/L nitrite in drinking water for 8 weeks) [16], Sodium nitrite and EANP @ CS treated during drinking (G. IV) (rats received nitrite in drinking water at 2 g/L concentrations for 8 weeks, and treated with EANP@CS orally 4 days/weeks in the last 4 weeks). The Last one sodium nitrite and EANP@ CS treated after stopping sodium nitrite (G. V) (rats treated with EANP@CS orally 4 days/weeks in the last 4weeks; (rats received 2 g/L nitrites in the drinking-water for 4 weeks and then treated with EANP@ CS 4 days/weeks in the last 4 weeks). The nitrite was replaced by drinking water in the last 4 weeks in G.V.
By the end of 8 weeks, the rats fasted overnight, then anesthetized and sacrificed. Blood was collected plasma and serum were separated. The serum was used for evaluated various biochemical parameters. The liver, kidney and spleen were separated and washed with saline. Part of the liver and kidney samples were homogenized at 4° C. The second part was fixed in formalin for 24 h and then stained with hematoxylin and eosin [17].

Determination of Hemoglobin; urea level, ALT and AST activities
Hemoglobin level was measured. Serum urea level as a biomarker for kidney disease, ALT and AST activities for liver disease were determined by using kits purchased from Randox Labs, UK. Liver and kidney protein were measured [18].

Determination of L-MDA; GSH, total protein thiol (PTH) and non-protein thiol (NPTH) levels; Gpx; GST and catalase activities
The concentration of liver and kidney L-MDA was evaluated by the reaction MDA with thiobarbituric acid (TBA) which gives a pink color. The pink color is extracted by n-butanol and measured at 532 nm. The MDA level was calculated using molar absorptivity according to Draper and Hadley [19].
Glutathione levels were measured in the liver and kidneys by the interaction of 5, 5´-dithiobis-2-nitrobenzoic acid (DTNP) with GSH at pH 8.2. The obtained yellow color was read at 412 nm. The level of GSH in the liver or kidney was calculated using the standard curve for different known GSH concentrations ranging from 0.25 to 5 mM [20]. Total protein thiol (PTH) and non-protein thiol (NPTH) were performed in the liver and kidney. Thiol reacts with DTNB (5, 5-Dithiobis (2-nitrobenzoic acid) in the presence of pH Tris-buffer 8.2 and methanol, and absorption was measured at 412 nm [21].
Glutathione peroxidase (Gpx) activity was determined in liver and kidney after the consumption of GSH and H 2 O 2 to form GSSG. In summary, a mixture of GSH, H 2 O 2 , EDTA salt, sodium azide, 10% liver and kidney homogenate, and distilled water were incubated for ten minutes. The GSH decrease was determined by the Ellman method and Gpx activity was expressed as nmole/ min/mg [22]. Liver and kidney glutathione S-transferase (GST) activity was determined by kinetic method. In brief, 1-Chloro-2, 4-dinitrobenzene (CDNB, Sigma-Aldrich) was coupled with reduced glutathione (GSH, Sigma-Aldrich) in a quartz cuvette, this coupling leads to a marked increase in measured absorption, and the reaction was done in the presence of 0.05 M buffer potassium phosphate pH 7.4 [23]. Liver catalase was determined by the H 2 O 2 degradation rate at 240 nm. The unit of the catalase enzyme was expressed by mmol of the consumed H 2 O 2 /min/mg protein [24].

Determination of serum, liver and kidney NO, liver NOS, liver and kidney arginase activities and DNA fragmentation
Serum, liver, and NO were determined after converting nitrate to nitrite using cadmium. Total nitrite was determined by Griess's reagents. The level of nitric oxide (NO) (nitrite and nitrate) was determined in the blood serum and liver homogeneity as described previously [25]. Briefly, 30% of zinc sulfate was added to the serum or 10% of the liver homogenate to precipitate the protein.
After centrifugation, 100 µL of supernatant was added to 0.5 g granulated cadmium and 0.4 mL of deionized water. After overnight rotating and centrifugation, 0.25 mL of the final supernatant was completed with distilled water, after which Griess's reagent contained 1 g/L sulphanilamide, 25 g/L phosphoric acid and 0.1 g/L N-1-naphthylethylen diamine reacted with nitrites measured at 540 nm. The sum of total nitrites (NO) was studied by the aiding of the nitrites standard curve (5 to 100 μM).
The activity of liver nitric oxide synthase (NOS) was determined by releasing nitric oxide from l-arginine A reaction mixture contained 3.4 mM of arginine, 2 mM of tetrahydrobiopterin, 0.5 mM of FAD, 12 mM of NADPH + , 50 µL of 20 mM CaCl 2 , 50 µL of 40 mM of dithiothreitol, the reaction was completed to 1 mL with 0.1 M Tris-HCl buffer, pH 7.5 after adding 50 µL of 10% liver homogenates and incubated at 37 o C for 3 h [26]. Total nitrites were measured in the reaction and subtracted from reaction control (not containing enzymes) as described above [25]. The unit of NOS was expressed as 1 nmol NO formed per hour/mg protein.
The activity of the liver and kidneys arginase was evaluated by the formation of urea from arginine. Briefly, 100 µL of 200 mM arginine and 100 µL of 0.5 mM MnCl 2 were added to 100 µL 10% liver homogenate. The reaction was completed to 1 mL with 100 mM of sodium bicarbonate buffer, pH 9.5 and incubated at 37 °C for 1 h and 1 mL of 0.5 M perchloric acid was added [27]. Urea was determined to using the diacetyl monoxime method [28].
DNA fragmentation was performed according to the method described by Gerçel-Taylor [29].

Preparation of samples for histopathology
Part of the liver, kidneys and spleen were placed directly into formalin for approximately 24 h. The tissue samples were then dried, embedded in paraffin and divided at 5 mm. The wax was removed from various parts, rehydrated and treated to stain hematoxylin and eosin [30].

Statistical analysis
Excel 2013, Origin 6 and SPSS 19 were used to perform statistical analysis and plot numbers of obtained results. Values are expressed as mean ± SD. One-way ANOVA with post hoc Tukey's tests were carried out. Also Ryan-Einot-Gabriel-Welsch F-test (R-E-G-WF) was performed to separate means by using SPSS program. The one way Anova F-test is to compare significant values between groups. p values less than 0.05 are considered significant.

Characterization of extracted ellagic and ellagic acid nanoparticles
The ellagic acid was extracted from pomegranate and encapsulated with chitosan as the nanoparticles of ellagic acid EANP@CS. EANP@CS. The characterization has been demonstrated by FT-IR, UV, XRD diffraction, and TEM. The FT-IR procedure was to recognize the interaction between CS and EA confirming the embodiment of EA in the nanoparticle (Fig. 1a, b). The FT-IR spectrum of ellagic acid exhibits a broadband at 3400 cm −1 which is attributed to the -OH expansion. While EANP@CS was shifted to 3425 cm −1 , the broadband C = O was changed at 1705 cm −1 and in EANP@CS to 1653 cm −1 . The aromatic ring range observed at 1613 cm −1 and 1509 cm −1 was observed due to peak vibrations, the range at 1190 and 1052 cm −1 is due to the ester linkage of the expansion of C-O, and the band is assigned to 758 cm −1 to CH aromatic. The bending vibration and 2927 cm −1 and 2848 cm −1 can be attributed to the expansion of the aromatic ring CH. The shifted bands that noted in these results confirmed that the phenolic OH group had participated during the EA reaction with CS-NP. Thus, this shift at peak sites is due to the interaction between EA and CS-NP that is consistent with Arulmozhi [15].
UV spectroscopy was used to verify whether EA interference in chitosan led to changes in its optical properties. UV spectra for both ellagic and EANP@CS molecules indicated that ellagic acid has a maximum λ of 264, 295 and 393.8 (Fig. 2a, b).
XRD data for both EA and EANP@CS extracted are shown in (Fig. 3a, b) The observed diffraction peaks were at the values of theta 9.7, 12.27, 13.51, 17.48, 24.99, 25.4 and 28.13. On the other hand, EANP@CS displayed integration of peaks as a result of nano size (Fig. 3b). XRD data for the extracted EA reveal that the compound is crystalline, with a sharp and dense peak with a diffraction angle (2θ) of 28.13° and 25.4 small and dull peaks at different diffraction angles. Furthermore, the XRD of Chitosan shows two distinct crystal peaks close at 10° and 20° due to reflections 020 and 110, this may be the regularity of the polymer chain structure due to the strong hydrogen bonds between the molecules formed between the hydroxyl and amino groups present in the chitosan [31].
Electron microscopy transmittance graphs of EANP@ CS coated are shown in (Fig. 4). It was observed that the size of the nanoparticles was of average size varying from 20 to 62 nm and appeared as spherical clusters in the shape. Most of the characteristic peaks of ellagic acid and chitosan appeared in the refractive patterns of EANP@ CS but at low intensity. This also demonstrated that crystal penetration and Chitosan decreased but still occur as most of the drug was converted to an amorphous form during installation, EANP@CS showed the integrity of the peaks as a result of the nano-compound. Nanoparticles covered with ellagic acid appear as groups of average molecular size ranging from 20 to 62 nm, which may be due to the tendency of chitosan molecules of ellagic acid to merge with each one through a broad hydrogen bond. The slight increase in the size of some nanoparticles can be due to the drug's incorporation between the polymeric networks [32].
Ellagic acid coated nanoparticles in target tissues were reached by passive targeting, and nanoparticles reached the target organ due to leaking connections [33].

Effect of EANP@CS on hemoglobin, liver and kidney functions, oxidant, antioxidants and nitric oxide, DNA fragmentation
Some rats under study drinking water contaminated with sodium nitrite (2 g/L) without taking EA nanoparticles for 8 weeks resulted in some of these rats died, one of them was paralyzed on one side of the body, some deformities occurred to them and two of them were tumors in their abdomen. Hemoglobin level showed a significant decrease in rats drinking the sodium nitrite group compared to the normal group. Whereas the hemoglobin level was improved to the normal level in EANP@CS-treated mice during or off drinking nitrites (Table 1). Prolonged use of sodium nitrite may result in death from methemoglobin in the blood. Moreover, low doses have had a harmful health effect, which includes birth faults, respiratory disease, nervous system injury and paralysis [34]. Prolonged ingestion of nitrite may lead to carcinogenicity and mutation [35]. Prolonged drinking of sodium nitrite may lead to toxic heme or iron, nitrite is believed to create free radicals, and oxidation of ferrous in oxyhemoglobin may form of methemoglobin. [36].
The urea level as a biomarker of kidney disease as well as serum AST activity as a biomarker for the liver, heart, and muscle showed a significant increase in mice that drink nitrite compared to normal rats. It was observed that blood urea level and AST activity showed almost normal levels and significantly decreased in rats that drink nitrite and treated it with EANP@CS during or stopping nitrite (Table 1).
Serum AST activity was high in rats that drank nitrites, possibly due to increased methemoglobin in the blood from hypoxia in liver or heart cells causing the enzymes to leak into the blood [37,38].
Also, serum urea was higher in rats given nitrite. It indicated that the glomerular filtration rate and tubular reabsorption may decrease due to the formation of peroxynitrites [37,38].
Nitric oxide consists of nitrates and nitrites. Food nitrate is converted to nitrite by bacteria in the large intestine and by protein enzymes containing heme and changed to nitrite. Nitrites are a reservoir of NO and bind with thiol hem-protein to form nitrosylation of hems [39].
F-test of serum NO, liver and kidney NO levels were 144.73, 16.71 and 12.93 respectively. Serum NO and liver NO levels (nitrite/nitrate) increased 5.2 folds and 2 folds respectively compared to normal rats (Fig. 5a, b). Whereas, serum NO level showed a significant decrease in EANP@CS-treated rats after nitrite was discontinued and no change was observed compared to the normal control group (Fig. 5a). On the other hand, the NO level of the liver and kidneys showed a significant decrease in EANP@CS-treated rats after nitrites were discontinued (G. V) compared to the sodium nitrite group (Fig. 5b).
It was reported that the plasma nitric oxide level increased about twice as much in rats that drink sodium nitrite by gavage (45 mg/kg/day) for 7 days [40]. Ellagic acid inhibitor the level of nitric oxide in the liver and kidneys in rats that cause alcohol and endrin substances and treated with EA [41].
On the contrary, liver activity of NOS was reduced 7 and 4.9 times in rats drinking NaNO 2 and rats treated with EANP@CS during drinking NaNO 2 respectively compared to normal group. Serum or liver NO level was raised which may prevent NOS expression. F-test of liver NOS activity was 123.89. Liver NOS activity was raised in rats that drank nitrites and treated with EANP@CS after discontinuation of nitrite compared to intoxicating rats with nitrite (Fig. 5b).
Arginase prevents l-arginine from producing NO. Arginase produces ornithine forms the polyamine responsible for tissue repair and cellular growth and inhibits NOS. EA inhibits gene expression of NOS in liver cells and increase the activity of arginase in cancer cells [42]. F-test of liver and kidney arginase activity were 8.43 and 3.63 respectively. Liver arginase activity showed a significant increase in the sodium nitrite group, EANP@ CS with and after stopping nitrites compared to the normal control. On the other hand, the renal arginase activity showed a significant increase in the sodium nitrite group compared to the normal group, but the liver arginase activity showed a significant decrease in both EANP@ CS with and after stopping nitrites (G. IV and GV) compared to the sodium nitrite group (G. III) (Fig. 5c).
The spleen can produce NO when it is driven by any poison [43]. The data obtained in this study showed that the sodium nitrite group resulted in a significant increase in both the spleen NO and L-MDA compared to the normal group whereas EANP@CS significantly improved spleen disorder for each of the treated group compared to the sodium nitrite group (Data have not shown).
F-test of liver and kidney MDA level were 16.28 and 6.49 respectively. In the present study, liver and kidney MDA level was higher in rats that were drinking nitrites consistent with Adewale et al. [44]. Whereas, Liver and kidney L-MDA level showed a significant decrease in the two groups (G. IV and G. V) compared to (G. III) and showed insignificant differences between rats that treated with EANP during or discontinuation drink NaNO 2 Kidney L-MDA level was reduced in rats treated with EANP@CS after discontinuation of NaNO2 compared with rats that drank sodium nitrate (Fig. 6a).
Nitrite is known as a highly reactive compound that has the ability to oxidize, reduce or reduce nitrosylating compounds. It can also be converted into a trillion of the same compounds, nitrous acid, nitric oxide and nitrite [45].
Lipid peroxidation is one of the main causes of cell toxicity with sodium nitrite. Sodium nitrite binds with amines in the stomach that produce nitrosamine and free radicals. Nitrosamine can increase the lipid peroxide cell membrane, thereby dissolving the liver membrane and infecting the cell [46].
It was reported that the EA reduces the liver MDA level in skin necrosis in rats due to two groups of lactone in the EA structure that act as scavenging of free radicals and lipid peroxide [41].
Ellagic acid contains powerful antioxidants. The main antioxidant technique is direct scavenging of free radicals, nitrogen reactive species and ROS, including hydroxyl radicals, peroxyl radicals, NO 2 radicals, and peroxynitrite. Another technique involves protecting the DNA from any attack of mutation by direct combination with this large molecule, the suppression of produced ROS, and chelation of metals as copper [47].
F-test of liver and kidney GSH level (15.57 and 6.43), and liver PSH and NPSH levels (8.74 and 4.63) were observed. F-test of liver and kidney Gpx activities (20.52 and 1.491), liver and kidney GST activites (12.68 and 3.34), and liver catalase activity (16.70) were detected. The current study, liver and kidney GSH and both liver PSH and NPSH levels, and kidney and liver Gpx, GST and catalase activities showed a significant decrease in rats that drank nitrite, while these antioxidant parameters did not show changes after stopping the nitrite and treated with EANP@CS compared to control (Fig. 6b-d) and (Fig. 7). GSH may combine with nitrites or its metabolites and act as a non-enzymatic detoxification action or be catalyzed by GST. GSH was depleted as a substrate and cofactors for both GST and Gpx activities, and thus these enzymatic activities were reduced. It was reported that serum L-MDA, NO levels and GST activity were raised. On the other hand, the activities of GSH, catalase, and arginase have been depleted in rats given to nitrite [48]. Ellagic acid inhibits the oxidation of glutathione and lipid peroxidation and increases genetic expressions and Gpx, GST and catalase activities in liver cells [7].
Liver, kidney, GSH, and PSH and NSPH levels of both groups (G. IV and G. V) showed a significant increase compared to the sodium nitrite group (G. III) but did not reach the normal value ( Fig. 6b-d). Liver GPx, GST and catalase activities showed a significant increase in EANP@CS-treated rats and a significant increase in kidney GST activity in (G. V) compared to rats taking sodium nitrite numbers (Fig. 7a-c).
The chemical composition of EA has conjugated double bonds and accepts electrons from free radical compounds to form stable compounds. Ellagic acids can improve antioxidant status in both liver and kidneys by scavenging superoxide, hydroxyl anion, and peroxynitrites. EA is a potent antioxidant compared to α-tocopherol and can enhance GSH-based protection. EA inhibits the cytokines produced by reactive oxygen species and inflammation [49].
F-test of hepatic DNA fragmentation was 29.54. Hepatic DNA fragmentation was raised in the sodium nitrite group (43.32%) compared to the normal group. On the other hand, the proportion of DNA fragmentation showed a significant decrease in the two groups treated with EANP@CS (G.IV and G. V) (27.30 and 26.4%), respectively (Fig. 8).The obtained data were an agreement with Sherif and Al-Gayyer [50].
Sodium nitrite causes hepatic failure through various mechanisms, for example, oxidative stress and DNA fragmentation [51]. Also, the lipid peroxide induced by the free radicals of sodium nitrite binds with DNA and thus accelerates the fragmentation of DNA [52].
The nitric oxide created as a result of daily consumption of sodium nitrite can deaminate the bases of purine and pyrimidine in the DNA, and thus can be the cause of the increase in mutations and the breaking of the DNA strand [53].

Histopathology of liver, kidney, and spleen
Control of normal hepatic mice showed average pyloric ducts, and average central veins surrounded by intermediate hepatic cells arranged in single cell cords with average overlapping blood sinusoids. On the other hand, the sodium nitrite group (G. III) showed a marked expansion of the dilated central and portal veins and hepatocytes showed more marked micro-and macro-vesicular steatosis in periportal areas than peri-venular areas with an expanded portal tract by inflammatory infiltrate. Moreover, in both groups treated with EANP @ CS (G. IV and G. V) they showed central and dilated portal veins and hepatocytes showed more steatosis in the areas surrounding peri-venular than the areas surrounding the portal (Fig. 9). Furthermore, control in normal rats of the kidney showed average renal capsule average and average glomeruli with average bowman area and average proximal tubules with preserved brush borders, average distal tubules, and average interstitial. However, in the sodium nitrite group, the kidney showed atrophied glomeruli with dilated Bowman's spaces and some proximal tubules showed loss of brush borders. The sodium nitrite group treated with EANP@CS average glomeruli with average Bowman's spaces and average proximal tubules with preserved brush borders (Fig. 10).
The spleen from the normal control group showed average lymphoid follicles (white bulb) with central arterioles, average sinusoids in the blood (red bulb), and average fibrous septa. Whether the spleen from the NaNO 3 group showed average lymph follicles with central arterioles and sinus dilatation in the blood with bleeding areas. The group treated with EANP@CS showed average lymphoid follicles, mildly dilated blood sinusoids and scattered giant cells (Fig. 11).
Kidney and hepatocyte infiltration and vascular degeneration were observed in rats consuming 20 mg/kg/day of nitrite for 4 weeks, and in rats that drink nitrite 200 mg/ kg for 6 months. The cytoplasmic vacuolization of centrilobular hepatocytes and tubular degeneration and congestion of glomerular capillaries and swelling in glomerular cells in the kidney of mice and rats were given nitrites [37,54]. Reactive oxygen species and nitric oxide can be the main cause of degenerative and histopathological alterations of tissues [54]. Ellagic acid can reduce fibrosis and inhibit the formation of NF-κB as biomarkers of inflammation in rats with diabetic nephropathy [55]. EANP@CS can be beneficial to protect against the negative effects of nitrite.