Protective effects of Carissa opaca fruits against CCl4-induced oxidative kidney lipid peroxidation and trauma in rat

Background Carbon tetrachloride (CCl4) is a potent nephrotoxin, as it causes acute as well as chronic toxicity in kidneys. Therefore, this study was carried out to assess the pharmacological potential of different fractions of Carissa opaca fruits on CCl4-induced oxidative trauma in the kidney. Methods The parameters studied in this respect were the kidney function tests viz, serum profile, urine profile, genotoxicity, characteristic morphological findings, and antioxidant enzymatic level of kidneys. Result The protective effects of various fractions of C. opaca fruits against CCl4 administration were reviewed by rat renal function alterations. Chronic toxicity caused by 8-week treatment of CCl4 to the rats significantly decreased the pH level, activities of antioxidant enzymes, and glutathione contents, whereas a significant increase was found in the case of specific gravity, red blood cells, white blood cells, level of urea, and lipid peroxidation in comparison to control group. Administration of various fractions of C. opaca fruit with CCl4 showed protective ability against CCl4 intoxication by restoring the urine profile, activities of antioxidant enzymes, and lipid peroxidation in rat. CCl4 induction in rats also caused DNA fragmentation and glomerular atrophy by means of dilation, disappearance of Bowmen's space, congestion in the capillary loops, dilation in renal tubules, and foamy look of epithelial cells of tubular region, which were restored by co-admiration of various fractions of C. opaca. Conclusion Results revealed that the methanolic fractions of C. opaca are the most potent and helpful in kidney trauma.

C arbon tetrachloride (CCl 4 ) is a toxic chemical, widely used in the dry cleaning industry, in filling fire extinguishers, in the fumigation of grains, and as an insecticide (1). Recent studies have shown that CCl 4 is associated with advanced production of free radicals leading to dysfunction of several organs (2). Chronic CCl 4 treatment is a common practice to induce hepatic fibrosis (3,4), renal (5,6), pulmonary (7) and testicular injuries (8), and cardiac tissue damage (9,10) in rats as an experimental model. Tissue damage by CCl 4 depends on the amount of dosage and duration of exposure of the experimental animals to this toxicant. Its action is based on membrane lipid peroxidation and induction of trichloromethyl radical ( CCl 3 ), resulting in severe cell damage (11). It is evidenced that metabolic activation of CCl 4 by cytochrome P 450 resulted in the production of trichloromethyl radical ( CCl 3 ) and peroxy trichloromethyl radical ( OOCCl 3 ) that, in turn, initiate subsequent lipid peroxidation, responsible for injuries in various organs such as liver and kidney (12Á14). Therefore, it can be stated that CCl 4 is the best-characterized tool for the study of oxidative stress trials as it consistently generates free radicals with the implication of pathological environment. These free radicals damage the integrity of liver cell membranes by releasing the cytosolic enzymes such as alanine transaminase, aspartate transaminase, alkaline phosphatase, and lactate dehydrogenase into the blood stream and elevating thiobarbituric acid reactive substances (TBARS) level with subsequent necrosis and inflammatory cell infiltration; affect physical parameters of kidney such as urinary and serum profile; increase lysosomal enzymes activities of testis and kidney;  and decrease the activity of a diagnostic marker enzyme creatinine phosphokinase (an enzyme responsible for ATP regeneration) in cardiac tissue (15). The healthpromoting effect of antioxidants on oxidative damage is mostly examined through the cellular antioxidants including the enzymatic behavior of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR), in addition to TBARS (a product of lipid peroxidation) and reduced glutathione (GSH) level (16) among various tissues. Previous studies have shown that antioxidants, including naringenin, N-acetyl cysteine, vitamin E, silymarin, quercetin, and rhein decrease lipid peroxidation, and partially ameliorate tissue injuries (16). Over the years, many researchers have reported that plants containing phenolics and flavonoids exhibit a large array of biological activities such as hepatoprotection, cardio-protection, and reversal of fibrosis. These phytochemicals are widely found in fruits and vegetables as an important part of the human diet. Consequently, plants like dietary substances may be reliable guards against tissue damage, as well as a mechanism in curtailing the progression to lethal diseases like cancer (17). Carissa opaca Stapf ex Hanes is a 2Á3 m tall evergreen shrub containing glabrous or puberulous branches with opposite and ovate glabrous leaves, hard and sharp spines arising between the petiole widely found in Pakistan and in some areas of India, Burma, and Sri Lanka (17). Traditionally, this plant is used for the treatment of asthma and pulmonary diseases (18), anticancer (19), diarrhea (20), hepatoprotection (21), and reproductive dysfunction (22). Therefore, the present study aimed to assess the nephro-protective potential of different fractions of C. opaca fruits on CCl 4 -induced oxidative trauma in the kidney.

Plant collection
The ripened fruits of C. opaca were collected in MarchÁ April 2011 from the Quaid-i-Azam University Islamabad and Abbottabad of Northern Pakistan, respectively. The plants were recognized by their local names and then validated by Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, and Dr. Saleem Ahmad, Curator, Pakistan Museum of Natural History, Islamabad. A voucher specimen with Accession No. 24561 (C. opaca) was deposited at the Herbarium of Pakistan, Museum of Natural History, Islamabad.

Extract preparation
The collected plant samples were cleaned to get rid of dust particles and then dried under shade for 1Á2 weeks. Willy Mill of 60-mesh size was used to prepare powder of dried samples, and then each powdered plant sample was used for further solvent extraction. First of all, 5 kg of powdered sample was extracted twice with 10 L of 95% methanol at 258C for 48 h. For filtration, Whatman No. 1 filter paper was used, and then the filtrate was concentrated on rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) under reduced pressure at 408C. To resolve the compounds with escalating polarity, a part of the extract was suspended in distilled water and subjected to liquidÁliquid partition by using solvents in the sequence of n-hexane, ethyl acetate, and methanol. After fractioning, the solvent of respective fractions was also evaporated by rotary evaporator. Fractions of n-hexane (HFC), ethyl acetate (EFC), and methanol (MFC) were obtained and were stored at 48C for further in vivo investigation.

Experimental plan
A total of 42, 6-week-old, male SpragueÁDawley rats weighing 180910 g were purchased from the National Institute of Health (NIH), Islamabad. Rats were given food and water ad libitum and kept at 20Á228C on a 12-h lightÁdark cycle. The study protocol was approved by ethical committee of Quaid-i-Azam University, Islamabad. The rats were acclimatized to laboratory condition for 7 days before commencement of experiments and then randomly divided into seven groups (six rats per group). Administration of CCl 4 (0.5 ml/kg b.w., 20% CCl 4 /olive oil) was intraperitoneally (i.p.) administered twice a week for 8 weeks. At the same time, the rats were individually administered silymarin (50 mg/kg b.w.) in DMSO and various fractions including HFC, EFC, and MFC (200 mg/kg b.w.) orally twice a week for 8 weeks. At the end of 8 weeks, 24 h after the last treatment, urine was collected and stored at (708C for further analysis, and then animals were given chloroform anesthesia and then dissected. All animals were sacrificed; blood was drawn prior to the excision of organ tissues from the ventral side through cardiac puncture. The serum was stored at (808C after separation until it was assayed, as described below. Kidneys were removed and washed in ice-cold saline. Subsequently, half of the organs were treated with liquid nitrogen and stored at (808C for further enzymatic and DNA damage analysis while the other portion was processed for histology (5).

Biochemical investigations
Evaluation of the pharmacological effects of different fractions of C. opaca against the toxicity induced with CCl 4 in rats has been done following assays as described by Khan et al. (5).
Physical analysis of urine Urine samples were assayed for red blood cells (RBCs) count, white blood cells (WBCs) count, urobilinogene, pH, and specific gravity by using standard diagnostic kits (MediScreen Urine Strips, Orgenics, France).

Biochemical analysis of urine and serum
Estimation of total protein, albumin, urea, creatinine, and creatinine clearance was done by using standard diagnostic kits (MediScreen kit France).

Assessment of antioxidant enzymes
In total, 10% homogenate of tissue was prepared in 100 mM KH 2 PO 4 buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA; pH 7.4) and centrifuged at 12,000)g for 30 min at 48C. The supernatant was collected and used for the following parameters as described below. Superoxide dismutase assay SOD activity was estimated by the method of Kakkar et al. (24). A mixture containing 0.1 ml of phenazine methosulfate (186 mM), 1.2 ml of sodium pyrophosphate buffer (0.052 mM; pH 7.0), and 0.3 ml of supernatant after centrifugation (1,500 )g for 10 min followed by 10,000 )g for 15 min) of kidney homogenatewasadded to this reaction mixture. Enzyme reaction was initiated by adding 0.2 ml of NADH (780 mM) and stopped after 1 min by adding 1 ml of glacial acetic acid. Amount of chromogen formed was measured by recording color intensity at 560 nm. Results are expressed in units/mg protein.

Catalase assay
Glutathione-S-transferase assay GST activity was assayed by the method of Habig et al. (25). The reaction mixture consisted of 1.475 ml phosphate buffer (0.1 mol, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025 ml (CDNB) (1 mM), and 0.3 ml of homogenate in a total volume of 2.0 ml. The changes in the absorbance were recorded at 340 nm, and enzymes activity was calculated as nM CDNB conjugate formed/ min/mg protein using a molar extinction coefficient of 9.6 )10 3 M (1 cm (1 .
Glutathione reductase assay GR activity was determined by the method of Carlberg and Mannervik (26). The reaction mixture consisted of 1.65 ml phosphate buffer: (0.1 mol; pH 7.6), 0.1 ml (0.5 mM EDTA), 0.05 ml oxidized glutathione (1 mM), 0.1 ml nicotinamide adenine dinucleotide phosphate (NADPH), and 0.1 ml of homogenate in a total volume of 2 ml. Enzyme activity was quantitated at 258C by measuring the disappearance of NADPH at 340 nm and was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 )10 3 M (1 cm (1 .

Quinone reductase assay
The activity of quinone reductase (QR) was determined by the method of Benson et al. (28). The 3.0 ml reaction mixture consisted of 2.13 ml Tris-HCl buffer (25 mM; pH 7.4), 0.7 ml bovine serum albumin, 0.1 ml FAD, 0.02 ml NADPH (0.1 mM), and 0.l ml of homogenate. The reduction of dichlorophenolindophenol (DCPIP) was recorded at 600 nm, and enzyme activity was calculated as nM of DCPIP reduced/min/mg protein using molar extinction coefficient of 2.1 )10 4 M (1 cm (1 .

Reduced glutathione assay
Reduced GSH was estimated by the method of Jollow et al. (29). A volume of 1.0 ml of homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 48C for 1 h and then centrifuged at 1,200 )g for 20 min at 48C. The total volume of 3.0 ml assay mixture contained 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M; pH 7.4), and 0.2 ml DTNB (100 mM). The yellow color developed was read immediately at 412 nm on a SmartSpecTM plus Spectrophotometer. It was expressed as mM GSH/g tissue.

Estimation of lipid peroxidation assay
The assay for lipid peroxidation was carried out following the modified method of Iqbal et al. (30). The reaction mixture in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 M; pH 7.4), 0.2 ml homogenate sample, 0.2 ml ascorbic acid (100 mM), and 0.02 ml ferric chloride (100 mM). The reaction mixture was incubated at 378C in a shaking water bath for 1 h. The reaction was stopped by the addition of 1.0 ml of 10% trichloroacetic acid (TCA). Following the addition of 1.0 ml of 0.67% thiobarbituric acid, all the tubes were placed in a boiling water bath for 20 min and then shifted to a crushed icebath before centrifuging at 2,500 )g for 10 min. The amount of TBARS formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm using spectrophotometer against a reagent blank. The results were expressed as nM TBARS/ min/mg tissue at 378C using molar extinction coefficient of 1.56 )10 5 M (1 cm (1 . Hydrogen peroxide assay Hydrogen peroxide (H 2 O 2 ) was assayed by H 2 O 2mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari (31). A volume of 2.0 ml of homogenate sample was suspended in 1.0 ml of solution containing phenol red (0.28 nM), horse radish peroxidase (8.5 units), dextrose (5.5 nM), and phosphate buffer (0.05 M; pH 7.0) and incubated at 378C for 60 min. The reaction was stopped by the addition of 0.01 ml of NaOH (10 N) and then centrifuged at 800)g for 5 min. The absorbance of the supernatant was recorded at 610 nm against a reagent blank. The quantity of H 2 O 2 produced was expressed as nM H 2 O 2 / min/mg tissue based on the standard curve of H 2 O 2 oxidized phenol red.
Molecular studies DNA has been isolated and its fragmentation percent was quantified in molecular studies of in vivo toxicity.
DNA fragmentation assay with diphenylamine reaction DNA fragmentation from tissue extract was determined using the procedure of Wu et al. (32). A quantity of 100 mg tissue was homogenized in Tris triton EDTA (TTE) solution. A volume of 0.1 ml of homogenate was labeled B, centrifuged at 200)g at 48C for 10 min, and the supernatant was collected and labeled S. S tubes were centrifuged at 20,000 )g for 10 min at 48C to separate intact chromatin and was labeled T. A volume of 1.0 ml of 25% TCA was added to all tubes T, B, S and incubated overnight at 48C. After incubation, precipitated DNA was recovered by pelleting for 10 min at 18,000 )g at 48C. A volume of 160 ml of 5% TCA was added to each pellet and heated for 15 min at 908C, then 320 ml of freshly prepared DPA solution was added, vortexed, and incubated for 4 h 378C. Optical density was read at 600 nm with a spectrophotometer (Smart spec TM Plus, catalogue # 170-2525).
DNA isolations and ladder assay DNA was isolated by using the methods of Wu et al. (32). A quantity of 100 mg of tissue in a Petri dish was washed with DNA buffer and homogenized in 1 ml of lysis buffer. A volume of 100 ml of proteinase K (10 mg/ml) and 240 ml of 10% SDS were added shaken gently, and incubated overnight at 458C in a water bath, then 0.4 ml of phenol was added and shaken for 5Á10 min and centrifuged at 3,000 rpm for 5 min at 108C. Supernatant was mixed with 1.2 ml of phenol, 1.2 ml of chloroform/isoamyl alcohol (24:1); shaken for 5Á10 min and centrifuged at 3,000 rpm for 5 min at 108C. A volume of 25 ml of 3 M sodium acetate (pH 5.2) and 5 ml ethanol was added with supernatant, shaken until DNA was precipitated. DNA was washed with 70% ethanol, dried, dissolved in TE buffer and its concentration checked at 260 and 280 nm. A quantity of 5 mg of total DNA and 0.5 mg of DNA standard per well were loaded on 1.5% agarose gel containing ethidium bromide. Electrophoresis was performed for 45 min with 100 V batteries, and DNA was observed under digital gel doc system and photographed.
Histopathological study of tissue After weighting the portion specified for histology, small pieces of each tissue were fixed for 3Á4 h in fixative sera followed by dehydration with ascending grades of alcohol (80, 90, and 100%) and transferred in cedar wood oil. When tissue became clear, then all tissues were embedded in paraplast and prepared blocks for further microtomy. Thin slides of 3Á4 mm were prepared with microtome; wax was removed, stained with hematoxylinÁeosin and photographed under a light microscope at 10 ) and 40 ).

Statistical analysis
To find the different treatment effects of in vivo studies, one-way analysis of variance was carried out by using the computer software SPSS 13.0. The level of significance among the various treatments was determined by LSD at 0.05% level of probability.

Effects of C. opaca fruit against CCl 4 -induced nephrotoxicity in rat
The present achievements were used to assess the medicinal potential of various fractions of C. opaca fruit against CCl 4induced oxidative damages in rat kidney. The biochemical alterations in different groups of rats were collaborated by the renal histological architectures of respective groups. The results obtained in this context are given below.

Effects of C. opaca fruits on urine profile of rat
The protective effects of various fractions of C. opaca fruits against CCl 4 administration were reviewed by rat renal functions alterations. Table 1 presents urine profile including urinary pH, specific gravity, RBCs, WBCs, and level of urea. Chronic toxicity caused by 8-week treatment of CCl 4 to the rats significantly decreased the pH level, whereas a significant increase was found in the cases of specific gravity, RBCs, WBCs, and level of urea in comparison to control group. Table 2 demonstrates a marked increase in urinary creatinine and urobilinogen whereas creatinine clearance, urinary albumin, and level of urinary proteins were significantly decreased by CCl 4 administration (p B0.05) in contrast to control group. Administration of various fractions of C. opaca fruit with CCl 4 showed protective ability against CCl 4 intoxication by restoring the urine levels of rat. Results of urine analysis for the groups treated with fractions alone did not show any toxic effects as their values were near to control. Data show that almost all fractions had significant pharmacological effects for urine versus diseased group.
Effects of C. opaca fruits on serum profile of rats Effects of different fractions of C. opaca fruits on serum profile of kidneys are summarized in Tables 3 and 4.  Table 3 presents serum protein, albumin, globulin, total bilirubin, and direct bilirubin of serum. There was a significant reduction in protein, albumin, and globulin level of serum in CCl 4 -toxicated group, while total bilirubin and direct bilirubin of serum were improved in CCl 4 -toxicated group. Table 4 summarizes the blood urea nitrogen (BUN), urobilinogen, creatinine, creatinine clearance, and nitrite level of serum. Administration of CCl 4 significantly enhanced the BUN, urobilinogen, creatinine, and nitrite level of serum, whereas reduced the creatinine clearance of serum. Silymarin was used as a reference drug to reduce nephrotoxicity.

Effects of C. opaca fruits on antioxidant levels
Nature has developed metabolic systems in various organisms to reduce the oxidative damage and avoid different lethal damage to cells and tissues. During this natural process, various oxidative metabolites are formed as a by-product in the cells. Antioxidant enzymes reduce these metabolites in a scavenging process. In this study, activities and scavenging effects of various antioxidative enzymes were studied. Effects of various fractions of C. opaca fruits and CCl 4 on tissue soluble protein and antioxidant defense enzyme systems such as CAT, POD, SOD, TBARS, and H 2 O 2 are shown in Table 5. CCl 4 administration to rats significantly decreased tissue protein and disturbs the antioxidative status by lessening the renal catalase, peroxidase, and superoxidase levels, and increased the lipid peroxidation and hydrogen peroxide levels in comparison to control group. Co-administration of various fractions of C. opaca fruits with CCl 4 enhanced the activity of suppressed enzymes and the soluble protein, whereas reduced the activity of elevated enzymes. However, non-significant change was recorded with the treatment of fractions alone against the control group. Table 6 shows the levels of phase II metabolizing antioxidant enzymes including GST, GPx, GR, GSH, QR, and DNA fragmentation in renal tissues of different  Effects of C. opaca fruits on DNA damages (ladder assay) DNAwas extracted from renal tissues of the treated rats by a stepwise method and then was loaded on 1.5% agarose gel. Figure 1 details the banding pattern of DNA. In case of CCl 4 -intoxicated rats, a peculiar fragmentation of DNA can be observed, which was absent from the renal tissues of control rats. DNA extracted from the group treated with various fractions of C. opaca fruits along with CCl 4 showed a markedly repaired DNA. The groups treated with only various fractions did not show any kind of DNA damage.

Effects of C. opaca fruits on kidney histoarchitecture
Histoarchitecture of kidneys of different groups is summarized in Fig. 2. The histopathologically observed changes showed that the intraperitoneal injection of CCl 4 for 8 weeks resulted in severe impairment to corticular region of kidneys. The damage induced was in different forms of degenerations showing glomerular atrophy by means of dilation and disappearance of Bowmen's space, congestion in the capillary loops, dilation in renal tubules, and foamy look of epithelial cells of tubular region (Fig. 2c). In addition, subchronic administration of CCl 4 exhibited interstitial inflammatory cells infiltration in both corticular and medullary regions. The evaluation of kidney section of control and DMSO group had normal histological architecture as shown in Fig. 2a and b. Renal sections of rats treated with various fractions of C. opaca fruits (HFC, EFC, and MFC) reversed the CCl 4 intoxication and showed mild injury in contrast to control group (Fig. 2dÁf). The histological appearance of glomeruli and Bowman's capsule was almost normal, and only some of the glomeruli were degenerated. Mild necrosis in the glomeruli and tubules was observed. As a measure of protective effects, silymarin was also induced to a group of rats (Fig. 2d). Silymarin treatment erased the CCl 4 pathogenesis, relative to control group. These findings are in agreement with the rest of the parameters studied.

Discussion
As we previously investigated, the methanol fraction of C. opaca is rich in active compounds such as orientin, isoquercetin, myricetin, and apigenin (20). Hence, it is proposed that these fractions might have protection against degenerative diseases caused by oxidative stress. CCl 4 intoxication generates free radicals that trigger a cascade of events resulting in nephrotoxicity. Activation of cytochrome P 450 oxygenase system yields highly reactive trichloromethyl peroxy radical that initiate lipid peroxidation (33). The lipid peroxidation may cause peroxidative tissue damage (34). Relevant to the latter findings, plant extract and its fractions comprehensively ameliorate the injuries induced through CCl 4 intoxication and affect the activity of biochemical enzymes, DNA strand breakage, and increase the activity of telomerase cancer marker enzyme of kidney tissue (35). The protective effects might seem to be related to their molecular structure, more precisely to the presence and number of hydroxyl groups, and to double bond conjugation and resonance effects substituents in the phenyl ring and on the position of its nitrogen atom relative to the NÁH bond as was previously reported (36,37). Previous studies reported that the urine analysis provides information about the functional status of kidneys. It can be said that malfunctioning kidneys result in distressed urine profiles. Urobilinogen, RBCs, and acidic pH are not the components of urine in normal conditions; however, the presence of significantly higher amount of urobilinogen, RBCs, and acidic pH was observed in urine that may be the result of oxidative damage in kidney of CCl 4intoxicated rats. Besides this, the elevated levels of WBCs, urinary creatinine, and urea, and decline in creatinine clearance, albumin, and proteins of urine were an indication of severe kidney damage. Similarly, the specific gravity of urine correlates with osmolality (36), and increased specific gravity is one of the symptoms of kidney damages such as dehydration, renal artery steatosis, necrosis, decreased blood flow to kidneys, and proteinuria (37). The present study exposed that various fractions of tested plant samples showed different levels of ameliorating effects by normalization of urinary profile.
Sedlak and Snyder (38) reported that the bilirubin is an important physiological cytoprotectant due to its antioxidant ability. At present, administration of CCl 4 indicated the risk of impaired-renal function by elevation of serum  (44) who reported that CCl 4 -induced oxidation caused an increase in TBARS content and lessened antioxidant enzymatic and non-enzymatic indicators of serum and renal tissue. Present observations are in agreement with previous demonstrations that hesperidin is a bioflavonoid, which reduces the production of CCl 4 -induced free radical by improving GSH levels of kidneys and decreases the concentration of 8-hydroxydeoxyguanosine (8-OH-dG), a marker of DNA fragmentation (45). Consequently, improvement in antioxidant enzyme levels such as CAT, POD, SOD, and phase II metabolizing enzymes (GST, GR, GPx, and QR) in contrast to CCl 4 -injected rats revealed the antioxidative trend of different fractions of tested plant samples. Increased DNA damage ultimately turns into cell proliferation and apoptosis (46Á48). CCl 4 administration is the major cause of oxidative stress and reactive oxygen species in the cell. It enhances the formation of the lipid peroxide radicals, which accelerate the breakdown of polyunsaturated fatty acids (PUFAs). Persistent oxidative stress induces DNA damage by modifying base products and results in strand breaks, which may lead to further mutation and chromosomal aberrations (49). Endonucleases degrade the cellular DNA to induce tubular epithelial cell death of kidney tissue. Overproduction of free radicals triggers the activity of Deoxyribonuclease I (DNase I) (50). DNase I, a highly reactive renal endonuclease I, attributes to about 80% of the total endonuclease action in renal tissues (51). Under these circumstances, it is possible that prevention of CCl 4induced renal DNA damage might be due to modulation of DNase I activity. Our findings are consistent with Nagwani and Tripathi (52), who reported similar results in amelioration of cisplatin-induced nephrotoxicity by PTY, a herbal preparation. The histopathological findings in kidney correlate with biochemical estimations of studied experimental groups. CCl 4 challenges produced an ischemic environment leading to glomerular and tubular lesions with vasocongestion in kidney. Similar histopathological architecture was observed by Dogukan et al. (53) in renal tissue of rats in response to chronic administration of CCl 4 for 7 weeks. It is also thought that histopathological alterations may be associated with the absorption power of renal tubules that introduce functional overloading of nephrons with subsequent renal dysfunction (12). Regarding other kidney function tests, it can be revealed that the mechanism of CCl 4 to intend nephrotoxicity and hepatotoxicity is probably the same. It can be concluded that the natural antioxidants such as flavonoids or plant fractions are direct means to protect the cell against potent toxins and chemicals; hence, the present study verifies the enhanced repairing effects of different fractions of tested plant materials. Our findings are in harmony with other studies who reported that different fractions of plants have pharmacological effects by erasing the CCl 4 insults and reverting toward normalcy (41).

Conclusion
The present study provides additional scientific evidences verifying that methanol extracts of C. opaca are strong antioxidants capable of protecting the kidney from CCl 4 -induced trauma. The protective effect observed in this study provides some mechanistic evidence for why indigenous people of Southeast Asia found it useful for treating kidney ailments as well as being used as a food additive.
Authors' contributions SS made significant contribution to acquisition and interpretation of data, conception, and drafting of the manuscript. MRK, HMA, and RAK (ORCID ID: 0000-0003-0453-2090) have made substantial contribution to conception and design, interpretation of data, and drafting for intellectual content.

Conflict of interest and funding
The authors have not received any funding or benefits from industry or elsewhere to conduct this study.