Ameliorating effect of various fractions of Rumex hastatus roots against hepato- and testicular toxicity caused by CCl4.

Effect of methanolic extract of Rumex hastatus roots (MRR) and its derived fractions, n-hexane (HRR), ethyl acetate (ERR), chloroform (CRR), butanol (BRR), and aqueous extract (ARR), was studied against carbon tetrachloride (CCl4) induced hepato and testicular toxicity in rats. Intraperitoneal dose of 20 percent CCl4 (0.5 ml/kg bw) was administered twice a week for eight weeks to a group of rats. Other groups were given CCl4 and various fractions of R. hastatus roots (200 mg/kg bw). CCl4 treatment depleted glutathione contents and activities of antioxidant enzymes while increased the concentration of lipid peroxides (TBARS) along with corresponding DNA injuries and histopathological damages. Supplementation with various fractions of R. hastatus roots (200 mg/kg body weight) attenuated the toxicity of CCl4 in liver and testis tissues through improvement in the serological, enzymatic, and histological parameters towards the normal. Posttreatment of R. hastatus roots (200 mg/kg body weight) also reversed the alteration in reproductive hormonal secretions and DNA damages in CCl4 treated rats. The results clearly demonstrated that R. hastatus treatment augments the antioxidants defense mechanism and provides the evidence that it may have a therapeutic role in free radical mediated diseases.

1. Introduction

Reactive oxygen species (ROS), like hydroxyl, peroxyl, and superoxide radicals, are very transient and highly reactive causes of the pathogenesis of atherosclerosis, neurodegeneration, inflammation, cardiovascular diseases, diabetes, and cataracts [1, 2]. CCl4 is a toxic chemical, commonly used to induce hepatic cirrhosis [3] and testicular injuries in experimental animals [4]. Metabolic activation of CCl4 by cytochrome P450 resulted in the production of trichloromethyl radical (•CCl3) and peroxy trichloromethyl radical (•OOCCl3) that in turn initiate lipid peroxidation, responsible for injuries in various organs like liver and testis [5]. These free radicals combine with polyunsaturated fatty acids of hepatic and testicular cell membranes, cause elevation of thiobarbituric acid reactive substances (TBARSs) concentration with subsequent necrosis [6], and increase lysosomal enzymes activities [7]. The health promoting effects of antioxidants on oxidative damage are mostly examined through cellular antioxidants enzymes in addition to TBARS and GSH concentration [5, 8]. It is also reported that increase in oxidative damage to sperm membranes, proteins, and DNA is associated with alterations in signal transduction mechanisms that affect fertility [9] and cause degeneration of somniferous tubules showing a relationship between hypogonadism and liver cirrhosis [5]. Rumex hastatus D. Don belongs to the Polygonaceae family and is popularly known as “khatimal.” It is distributed in northern Pakistan, northeast Afghanistan, and southwest China, growing between 700 and 2500 m, and sometimes grows as a pure population. It is reported that the whole plant is used as medicine. It is laxative, alterative, tonic, and is used in rheumatism [10] and sexually transmitted diseases including AIDS [11]. Our previous studies substantiated the R. hastatus leaves as a good antioxidant source with sufficient amount of phenolics [12]. Zhang et al. [13] by referring the use in Chinese herbal system reported seven phenolic compounds from R. hastatus roots. Thus, regarding the cultural/ethnic use present toxicological studies in rat models have been planned to evaluate the protective effect of various fractions of R. hastatus roots against hepato- and testicular toxicity caused by CCl4.

2. Materials and Methods

2.1. Extract Preparation

R. hastatus District roots were collected from Havelian, Abbottabad, Pakistan. Shade dried roots were powdered in a Willy Mill to 60-mesh size and used for solvent extraction. Five kg powder was extracted twice with 10 liters of 95 percent methanol at 25°C for 48 h and filtered. The methanolic solution was dried in a rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) to obtain methanolic crude extract of R. hastatus roots (MRR). In order to resolve the compounds with escalating polarity, a part of the extract was suspended in distilled water and subjected to liquid-liquid partition by using solvents in a sequence of n-hexane (HRR), ethyl acetate (ERR), chloroform (CRR), and butanol (BRR), while the remaining soluble portion was filtered and used as aqueous fraction (ARR). After fractioning, the solvent of the respective fraction was evaporated by rotary evaporator [14].

2.2. In Vivo Evaluation of Fractions

For in vivo studies six-week-old male Sprague-Dawley rats weighting 180 ± 10 g were provided with food and water ad libitum and kept at 20–22°C on a 12 h light-dark cycle. All experimental procedures involving animals were conducted in accordance with the guidelines of National Institutes of Health, Islamabad, Pakistan. The study protocol was approved by Ethical Committee of Quaid-i-Azam University, Islamabad Pakistan. The rats were acclimatized to laboratory condition for 7 days before commencement of experiments.

2.3. Experimental Plan

For subchronic toxicity, eight-week experiment was designed according to Shyu et al. [3]. Ninety-six rats were randomly divided into sixteen groups (6 rats of each group). Group I animals remained untreated, while Group II animals received olive oil and DMSO twice a week for eight weeks. Animals of Groups III, IV, V, VI, VII, VIII, IX, and X received intraperitoneally 0.5 mL of CCl4, (20 percent in olive oil) twice a week for eight weeks. Group III received only CCl4, while Group IV administered silymarin at a dose of 50 mg/kg bw after 48 h of CCl4 treatment. Groups V, VI, VII, VIII, IX, and X received different fractions at a dose of 200 mg/kg bw, HFC, EFC, CFC, BFC, MFC, and AFC, twice a week for eight weeks orally. However, Groups XI, XII, XIII, XIV, XV, and XVI received fractions (200 mg/kg bw) alone twice a week for eight weeks after 48 hr of CCl4 treatment orally. At the end of eight weeks, after 24 h of the last treatment, animals were given chloroform anesthesia and dissected from ventral side. Blood was drawn and centrifuged at 1500 ×g for 10 min, at 4°C to collect the serum. Liver and testis tissues were perfused with ice cold saline and excised. Subsequently, half of both tissue portions were treated with liquid nitrogen and stored at −80°C for further enzymatic and DNA damage analysis, while other portions were processed for histology.

2.4. Analysis of Serum

For estimation of liver function tests serum samples were assayed for ALT, AST, ALP,-γ-GT, total cholesterol, triglycerides, LDL, and HDL by using standard AMP diagnostic kits (Graz, Austria).

Serum analysis of testicular hormones like FSH, LH, testosterone, prolactin, and estradiol was radioimmunoassayed by using Marseille Cedex 9 France Kits and Czech Republic Kits from Immunotech Company.

2.5. Assessment of Antioxidant Enzymes

Ten percent of homogenates of liver and testis tissues were prepared separately in 100 mM KH2PO4 buffer containing 1 mM EDTA (pH 7.4) and centrifuged at 12,000 ×g for 30 min at 4°C. The supernatant was collected and used for the following experiments as described below. Protein concentration of the supernatant was determined by the method of Lowry et al. [15] using crystalline bovine serum albumin as standard.

2.5.1. Catalase Assay (CAT)

CAT activities were determined by using H2O2 as a substrate [16]. 0.1 mL of the supernatant was mixed with 2.5 mL of 50 mM phosphate buffer (pH 5.0) and 0.4 mL of 5.9 mM H2O2, and change in absorbance was recorded at 240 nm after one min. One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.

2.5.2. Peroxidase Assay (POD)

In this method guaiacol was used as the substrate [16]. For the POD activity determination 0.1 mL of the supernatant was added to the reaction mixture having 2.5 mL of 50 mM phosphate buffer (pH 5.0), 0.1 mL of 20 mM guaiacol, and 0.3 mL of 40 mM H2O2. Changes in absorbance of the reaction solution at 470 nm were determined after one min. One unit of POD activity was defined as an absorbance change of 0.01 units/min.

2.5.3. Superoxide Dismutase Assay (SOD)

In this method NADH was used as the substrate [17]. Reaction mixture of this method contained 0.1 mL of phenazine methosulphate (186 μM), 1.2 mL of sodium pyrophosphate buffer (0.052 mM; pH 7.0), and 0.3 mL of supernatant after centrifugation (1500 ×g for 10 min followed by 10000 ×g for 15 min) of tissue homogenate was added to the reaction mixture. Enzyme reaction was initiated by adding 0.2 mL of NADH (780 μM) 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.

2.5.4. Glutathione-S-Transferase Assay (GST)

Glutathione-S-transferase activity was assayed by the method of Habig et al. [18]. 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 of 1-chloro-2,4-dinitrobenzene (CDNB) (1 mM), and 0.3 mL of supernatant 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 × 103 M−1 cm−1.

2.5.5. Glutathione Reductase Assay (GSR)

Glutathione reductase activity was determined by the method of Carlberg and Mannervik [19]. The reaction mixture consisted of 1.65 mL phosphate buffer (0.1 mol; pH 7.6), 0.1 mL EDTA (0.5 mM), 0.05 mL oxidized glutathione (1 mM), 0.1 mL NADPH (0.1 mmol), and 0.1 mL of supernatant in a total volume of 2 mL. Enzyme activity was quantitated at 25°C by measuring disappearance of NADPH at 340 nm and was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 × 103 M−1 cm−1.

2.5.6. Glutathione Peroxidase Assay (GSH-Px)

Glutathione peroxidase activity was assayed by the method of Mohandas et al. [20]. The reaction mixture consisted of 1.49 mL phosphate buffer (0.1 M; pH 7.4), 0.1 mL EDTA (1 mM), 0.1 mL sodium azide (1 mM), 0.05 mL glutathione reductase (1 IU/mL), 0.05 mL GSH (1 mM), 0.1 mL NADPH (0.2 mM), 0.01 mL H2O2 (0.25 mM), and 0.1 mL of supernatant in a total volume of 2 mL. The disappearance of NADPH at 340 nm was recorded at 25°C. Enzyme activity was calculated as nM NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 × 103 M−1 cm−1.

2.5.7. Quinone Reductase Assay (QR)

The activity of quinone reductase was determined by the method of Benson et al. [21]. The 3.0 mL reaction mixture consisted of 2.13 mL Tris-HCl buffer (25 mM; pH 7.4), 0.7 mL BSA, 0.1 mL FAD, 0.02 mL NADPH (0.1 mM), and 0.l mL of supernatant. 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 × 104 M−1 cm−1.

2.6. Reduced Glutathione Assay (GSH)

Reduced glutathione was estimated by the method of Jollow et al. [22]. 1.0 mL sample of supernatant was precipitated with 1.0 mL of (4 percent) sulfosalicylic acid. The samples were kept at 4°C for 1 h and then centrifuged at 1200 ×g for 20 min at 4°C. 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 of 1,2-dithio-bis-nitrobenzoic acid DTNB (100 mM). The yellow color developed was read immediately at 412 nm on a SmartSpec Plus Spectrophotometer. It was expressed as μM GSH/g tissue.

2.7. Estimation of Lipid Peroxidation (TBARS)

The assay for lipid peroxidation was carried out following the modified method of Iqbal et al. [23]. One milliliter of 20 percent TCA aqueous solution and 1.0 mL of 0.67 percent TBA aqueous solution was added to 0.6 mL of phosphate buffer (0.1 M; pH 7.4) and 0.4 mL of homogenate sample. The reaction mixture was heated in a boiling water bath for 20 min and then shifted to crushed ice-bath before centrifuging at 2500 ×g for 10 min. The amount of TBARS formed in each of the samples was assessed by measuring 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 37°C using molar extinction coefficient of 1.56 × 105 M−1 cm−1.

2.8. Hydrogen Peroxide Assay (H2O2)

Hydrogen peroxide (H2O2) was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari [24]. 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 was incubated at 37°C 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 H2O2 produced was expressed as nM H2O2/min/mg tissue based on the standard curve of H2O2 oxidized phenol red.

2.9. DNA Fragmentation Assay

DNA fragmentation assay was conducted using the procedure of Wu et al. [25]. Tissue samples (50 mg) were homogenized in 10 volumes of a TE solution pH 8.0 (5 mM Tris-HCl, 20 mmol EDTA) and 0.2 percent triton X-100. 1.0 mL aliquot of each sample was centrifuged at 27,000 ×g for 20 min to separate the intact chromatin (pellet, B) from the fragmented DNA (supernatant, T). The pellet and supernatant fractions were assayed for DNA content using a freshly prepared DPA (Diphenylamine) solution for reaction. Optical density was read at 620 nm at (SmartSpec Plus Spectrophotometer catalog no. 170-2525) spectrophotometer. The results were expressed as an amount of percent fragmented DNA by the following formula:

2.10. DNA Ladder Assay

DNA was isolated from tissue samples by using the method of Wu et al. [25] to estimate DNA damages. 5 μg of DNA of rats separately was loaded in 1.5 percent agarose gel containing 1.0 μg/mL ethidium bromide including DNA standards (0.5 μg per well). After electrophoresis gel was studied under gel doc system and was photographed through digital camera.

2.11. Histopathological Studies

For microscopic evaluation tissues were fixed in a fixative (absolute alcohol 60 percent, formaldehyde 30 percent, glacial acetic acid 10 percent) and embedded in paraffin, sectioned at 4 μm, and subsequently stained with hematoxylin and eosin. Sections were studied under light microscope (DIALUX 20 EB) at 10x magnifications. Slides of all the treated groups were studied and photographed.

2.12. Statistical Analysis

Data are expressed as means ± SD (n = 6), and significant differences between the groups were statistically analyzed by Duncan's multiple range test (Statistica Software, 1990). Concentration of significance among the various treatments was determined at P < 0.05.

3. Result

3.1. Effects of R. hastatus Roots on Liver Function Test and Biochemical Markers

The serological concentrations of AST, ALT, ALP, and γ-GT are highly susceptible to oxidative stress in liver tissue as shown in Table 1. Chronic CCl4 treatment considerably (P < 0.05) augmented the concentrations of serum marker enzymes of liver which was attenuated significantly (P < 0.05) by oral administration of various fraction of R. hastatus roots. However, various fractions of R. hastatus roots alone showed the same serum enzyme concentration like that of control group. Hepatotoxin also reacts with polyunsaturated fatty acids to cause lipid peroxidation by disturbing lipid profile as summarized in Table 2. These parameters were significantly restored (P < 0.05) by various fractions of R. hastatus roots near to control. For serological investigations fractions can be ordered as BRR > MRR > ARR > CRR > ERR > HRR.

Table 1

Effects of various fractions of R. hastatus roots on liver function tests.

Group	AST (U/L)	ALT (U/L)	ALP (U/L)	γ-GT (U/L)
Control	78.25 ± 2.09++	65.23 ± 1.78++	121.65 ± 2.19++	1.94 ± 0.07++
Oil + DMSO	76.46 ± 2.94++	64.74 ± 2.02++	123.29 ± 2.55++	1.95 ± 0.16++
CCl4	240.15 ± 4.19**	198.93 ± 4.27**	350.79 ± 5.31**	5.11 ± 0.53**
Silymarin + CCl4	104.23 ± 3.24++	89.28 ± 2.19++	171.54 ± 2.63++	2.27 ± 0.13++
HRR + CCl4	213.23 ± 4.21+	130.94 ± 4.18++	235.67 ± 4.86++	3.15 ± 0.48++
ERR + CCl4	202.18 ± 5.23+	128.32 ± 3.15++	229.92 ± 5.95++	3.01 ± 0.36++
CRR + CCl4	176.33 ± 3.09++	106.23 ± 2.56++	207.11 ± 4.23++	2.79 ± 0.11++
BRR + CCl4	142.01 ± 3.29++	94.28 ± 2.66++	189.61 ± 2.16++	2.72 ± 0.13++
MRR + CCl4	139.28 ± 4.13++	99.91 ± 2.73++	193.26 ± 3.42++	2.57 ± 0.32++
ARR + CCl4	170.45 ± 4.27++	90.34 ± 3.11++	175.24 ± 3.18++	2.33 ± 0.12++
HRR alone	78.45 ± 1.29++	63.26 ± 1.45++	124.65 ± 1.27++	1.93 ± 0.10++
ERR alone	75.37 ± 1.55++	65.34 ± 1.34++	122.15 ± 2.67++	1.90 ± 0.09++
CRR alone	79.43 ± 0.95++	68.86 ± 0.93++	123.15 ± 1.23++	1.92 ± 0.17++
BRR alone	76.76 ± 1.56++	66.56 ± 2.20++	120.55 ± 2.32++	1.99 ± 0.04++
MRR alone	74.51 ± 1.34++	62.02 ± 0.74++	125.34 ± 1.22++	1.97 ± 0.07++
ARR alone	77.45 ± 1.75++	63.36 ± 1.34++	122.20 ± 2.85++	2.01 ± 0.07++

Mean ± SE (n = 6 number).

**indicate significance from the control group at P < 0.01 probability level.

++indicate significance from the CCl4 group at P < 0.01 probability level.

Table 2

Effects of various fractions of R. hastatus roots on lipid profile.

Group	Triglycerides (mg/dL)	Total cholesterol (mg/dL)	HDL (mg/dL)	LDL (mg/dL)
Control	140.00 ± 3.57e	29.14 ± 1.59d	41.23 ± 1.44d	23.25 ± 1.01d
Oil + DMSO	139.89 ± 4.73e	30.00 ± 1.34d	40.92 ± 1.71d	23.98 ± 1.26d
CCl4	263.67 ± 2.62a	72.09 ± 1.99a	60.80 ± 2.89a	37.21 ± 1.98a
Sily + CCl4	182.14 ± 2.36d	40.80 ± 2.18c	47.29 ± 1.70c	27.83 ± 1.71c
HRR + CCl4	205.23 ± 3.17c	64.30 ± 1.79b	56.36 ± 0.77b	34.38 ± 0.16b
ERR + CCl4	213.85 ± 3.25b	60.96 ± 2.64b	54.50 ± 1.24b	33.65 ± 0.84b
CRR + CCl4	199.13 ± 4.44c	56.36 ± 3.60b	50.97 ± 1.12c	30.35 ± 1.14c
BRR + CCl4	189.16 ± 3.82c	42.51 ± 2.84c	46.54 ± 1.56c	27.93 ± 1.28c
MRR + CCl4	194.83 ± 2.92c	44.89 ± 2.06c	45.27 ± 1.46c	29.51 ± 2.08c
ARR + CCl4	190.65 ± 3.54c	54.57 ± 2.35b	49.35 ± 0.43c	31.23 ± 0.66c
HRR alone	138.45 ± 1.35e	31.55 ± 1.44d	40.56 ± 1.33d	24.78 ± 0.15d
ERR alone	137.96 ± 2.50e	32.14 ± 1.32d	41.66 ± 1.45d	23.25 ± 1.35d
CRR alone	140.26 ± 1.64e	30.23 ± 1.74d	42.55 ± 1.56d	22.98 ± 1.01d
BRR alone	138.76 ± 1.41e	28.15 ± 1.46d	40.12 ± 1.68d	24.98 ± 0.23d
MRR alone	141.01 ± 1.62e	33.10 ± 2.81d	42.20 ± 0.72d	22.25 ± 0.95d
ARR alone	139.78 ± 3.84e	29.14 ± 1.36d	39.23 ± 1.45d	23.25 ± 0.16d

Values are mean ± SD (06 number). Sily: Silymarin.

a–d(means with different letters) indicate significance at P < 0.05.

3.2. Effects of R. hastatus Roots on Male Reproductive Hormones of Rats

To estimate the testicular toxicity, reproductive hormones act as effective biomarkers. CCl4 intoxication alters the secretion of pituitary and reproductive hormonal concentration. The effects of various fractions of R. hastatus roots against CCl4 toxicity on hormonal concentration of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, and estradiol are summarized in Table 3. CCl4 intoxicated rats considerably (P < 0.05) decreased the testosterone, FSH, and LH concentration of serum while significantly (P < 0.05) raised the prolactin and estradiol concentration. The serum concentrations of LH, testosterone, prolactin, and estradiol were restored (P < 0.05) by oral administration of various fractions of R. hastatus roots near to control group except HRR and ERR that showed no significance for luteinizing hormone.

Table 3

Effects of various fractions of R. hastatus roots on male reproductive hormonal concentration.

Group	Testosterone (ng/mL)	Luteinizing hormone (ng/mL)	Follicle stimulatinghormone (ng/mL)	Prolactin (ng/mL)	Estradiol (ng/mL)
Control	2.87 ± 0.09h	3.08 ± 0.06d	45.63 ± 0.27h	10.23 ± 1.41f	15.23 ± 0.78g
Oil + DMSO	2.82 ± 0.05h	2.98 ± 0.09d	45.28 ± 0.37h	12.26 ± 1.45f	16.74 ± 0.45g
CCl4	1.12 ± 0.06a	1.21 ± 0.10a	20.69 ± 0.39a	25.61 ± 0.32a	32.45 ± 0.19a
Sily + CCl4	2.55 ± 0.08g	2.67 ± 0.14c	38.96 ± 0.60g	15.45 ± 0.74e	19.48 ± 0.90f
HRR + CCl4	1.40 ± 0.04b	1.34 ± 0.06a	22.34 ± 0.42b	22.05 ± 0.80b	29.04 ± 0.45b
ERR + CCl4	1.45 ± 0.10b	1.33 ± 0.09a	22.31 ± 0.38b	23.14 ± 0.62b	28.92 ± 0.36b
CRR + CCl4	1.75 ± 0.11c	1.64 ± 0.10b	25.62 ± 0.71c	20.55 ± 0.70c	25.24 ± 0.74c
BRR + CCl4	2.30 ± 0.07f	2.26 ± 0.11c	33.61 ± 0.32e	17.90 ± 0.63d	23.51 ± 0.17d
MRR + CCl4	2.25 ± 0.06e	2.44 ± 0.12c	34.37 ± 0.25f	18.26 ± 0.56d	21.35 ± 0.78e
ARR + CCl4	2.02 ± 0.05d	2.04 ± 0.13c	28.55 ± 0.65d	20.31 ± 0.38c	25.37 ± 0.28c
HRR alone	2.80 ± 0.08h	2.93 ± 0.11d	45.22 ± 0.47h	12.11 ± 0.65f	16.73 ± 0.47g
ERR alone	2.78 ± 0.16h	2.88 ± 0.18d	45.53 ± 0.60h	10.53 ± 0.71f	17.03 ± 0.80g
CRR alone	2.99 ± 0.09h	3.00 ± 0.10d	45.48 ± 0.49h	13.06 ± 1.06f	16.61 ± 0.35g
BRR alone	2.75 ± 0.05h	3.18 ± 0.09d	45.78 ± 0.46h	9.76 ± 1.93f	16.74 ± 0.65g
MRR alone	2.98 ± 0.09h	3.11 ± 0.06d	46.05 ± 0.26h	10.45 ± 1.48f	14.63 ± 0.97g
ARR alone	2.93 ± 0.10h	3.08 ± 0.07d	46.13 ± 0.58h	12.17 ± 1.42f	15.47 ± 0.87g

Values are mean ± SD (06 number). Sily: Silymarin.

a–h(means with different letters) indicate significance at P < 0.05.

3.3. Effects of R. hastatus Roots on Testis Enzymatic Antioxidant Concentrations

In the present study scavenging effects of various antioxidant enzymes were assessed. The effects of various fractions of R. hastatus roots against CCl4 intoxication on tissue soluble protein and antioxidant enzyme system such as CAT, POD, SOD, TBARS, and H2O2 testis are reported in Table 4. Free radicals generated by CCl4 injection, disturbing the cell membrane by reacting with phospholipids, leading to lipid peroxidation, caused significant elevation of TBARS and H2O2 content. Our results point to the ability of CCl4 on tissue to cause significant damage by decreasing the tissue protein as well as CAT, POD, and SOD activities in addition to increasing the lipid peroxidation and hydrogen peroxide contents versus control group. Posttreatment of various fractions of R. hastatus roots with CCl4 improved the activity of reduced enzymes and the soluble protein whereas reduced the concentration of TBARS and H2O2. The effects of various fractions of R. hastatus roots on phase II antioxidant enzymes like GST, GPx, GR, GSH, QR, and DNA fragmentation percent of testicular tissue are presented in Table 5. Administration of CCl4 significantly (P < 0.05) decreased the glutathione status of GST, GPx, GR, GSH, and QR while amplifying the percent fragmentation of DNA. Posttreatment of various fractions of R. hastatus roots along with CCl4 treatment markedly improved the activities of GST, GPx, GR, GSH, QR, and percent DNA fragmentation.

Table 4

Effects of various fractions of R. hastatus roots on tissue proteins and antioxidant enzyme concentrations.

Group	Protein (µg/mg tissue)	CAT (U/min)	POD (U/min)	SOD (U/mg protein)	TBARS (nM/min/mg protein)	H2O2 (μM/mL)
Control	2.27 ± 0.020f	4.80 ± 0.10d	14.31 ± 0.25d	4.33 ± 0.46d	2.30 ± 0.41c	1.89 ± 0.10c
Oil + DMSO	2.30 ± 0.010f	4.75 ± 0.15d	13.90 ± 0.20d	4.10 ± 0.28d	2.08 ± 0.33c	1.78 ± 0.12c
CCl4	1.05 ± 0.021a	2.10 ± 0.07a	6.67 ± 0.17a	1.23 ± 0.54a	6.52 ± 0.58a	3.54 ± 0.26a
Sily + CCl4	1.91 ± 0.023e	3.97 ± 0.06c	11.67 ± 0.44c	3.14 ± 0.43c	3.11 ± 0.80b	2.18 ± 0.31b
HRR + CCl4	1.42 ± 0.009b	2.92 ± 0.32b	9.09 ± 0.34b	1.96 ± 0.16b	6.00 ± 0.54a	3.40 ± 0.17a
ERR + CCl4	1.43 ± 0.010b	3.07 ± 0.20b	9.57 ± 0.21b	2.05 ± 0.21b	6.18 ± 0.33a	3.29 ± 0.11a
CRR + CCl4	1.50 ± 0.013c	3.63 ± 0.17c	10.13 ± 0.64c	2.38 ± 0.31b	5.03 ± 0.26b	2.89 ± 0.20b
BRR + CCl4	1.82 ± 0.085e	3.99 ± 0.15c	11.01 ± 0.24c	2.90 ± 0.15c	4.18 ± 0.92b	2.69 ± 0.31b
MRR + CCl4	1.78 ± 0.060e	4.00 ± 0.26c	11.61 ± 0.75c	3.32 ± 0.43c	3.65 ± 0.75b	2.27 ± 0.42b
ARR + CCl4	1.65 ± 0.014d	3.80 ± 0.10c	10.60 ± 0.55c	2.96 ± 0.18c	4.62 ± 0.71b	2.77 ± 0.25b
HRR alone	2.30 ± 0.011f	4.71 ± 0.12d	14.68 ± 0.62d	4.21 ± 0.50d	2.19 ± 0.74c	1.82 ± 0.34c
ERR alone	2.33 ± 0.019f	4.65 ± 0.24d	14.71 ± 0.71d	4.78 ± 0.36d	2.34 ± 0.82c	1.84 ± 0.57c
CRR alone	2.44 ± 0.010f	4.91 ± 0.27d	14.99 ± 0.53d	4.64 ± 0.45d	2.52 ± 0.67c	1.96 ± 0.77c
BRR alone	2.26 ± 0.009f	4.96 ± 0.41d	14.66 ± 0.45d	4.71 ± 0.33d	2.24 ± 0.63c	1.93 ± 0.61c
MRR alone	2.38 ± 0.028f	4.86 ± 0.21d	15.07 ± 0.23d	4.73 ± 0.21d	2.18 ± 0.64c	2.01 ± 0.49c
ARR alone	2.35 ± 0.012f	4.84 ± 0.15d	15.01 ± 0.10d	4.58 ± 0.26d	2.39 ± 0.45c	1.92 ± 0.70c

Values are mean ± SD (06 number). Sily: Silymarin.

a–f(means with different letters) indicate significance at P < 0.05.

Table 5

Effects of various fractions of R. hastatus roots on phase II antioxidant enzymes and DNA fragmentation.

Group	GST (nM/mg protein)	GPx (nM/mg protein)	GR (nM/mg protein)	GSH (µM/g tissue)	QR (nM/mg protein)	Percent DNA injuries
Control	150.21 ± 4.11g	110.71 ± 3.23e	198.47 ± 4.72h	17.69 ± 1.11e	105.33 ± 1.34g	9.21 ± 1.30e
Oil + DMSO	146.34 ± 4.10g	104.56 ± 3.10e	190.69 ± 4.39h	15.40 ± 1.30e	107.12 ± 1.46g	8.38 ± 1.63e
CCl4	87.48 ± 3.45a	57.86 ± 2.67a	120.76 ± 3.02a	7.63 ± 0.75a	63.34 ± 1.01a	51.11 ± 2.02a
Sily + CCl4	122.22 ± 3.18e	94.62 ± 2.47d	158.61 ± 2.35g	13.73 ± 0.73d	91.54 ± 2.11f	22.43 ± 1.57d
HRR + CCl4	94.05 ± 2.56b	68.44 ± 2.37b	127.11 ± 2.34b	10.08 ± 0.39b	69.69 ± 1.72b	42.22 ± 1.27b
ERR + CCl4	95.67 ± 2.48b	70.37 ± 2.42b	127.51 ± 2.55b	10.25 ± 0.36b	71.47 ± 1.95b	35.33 ± 2.15c
CRR + CCl4	100.28 ± 2.27c	76.22 ± 2.71c	132.17 ± 2.33c	11.17 ± 0.28c	75.56 ± 1.38c	26.44 ± 1.73d
BRR + CCl4	119.16 ± 3.15e	80.51 ± 2.33c	144.43 ± 2.26e	12.19 ± 0.43d	83.24 ± 2.82e	25.53 ± 2.15d
MRR + CCl4	130.53 ± 2.34f	88.61 ± 2.20c	150.39 ± 2.41f	12.36 ± 0.15d	90.97 ± 2.61f	23.78 ± 1.23d
ARR + CCl4	108.26 ± 3.68d	85.47 ± 2.62c	139.40 ± 2.16d	11.53 ± 0.32c	79.87 ± 2.28d	25.73 ± 1.56d
HRR alone	149.67 ± 3.63g	107.34 ± 3.57e	192.22 ± 2.23h	16.22 ± 0.50e	108.34 ± 1.41g	7.66 ± 1.27e
ERR alone	155.87 ± 4.58g	106.48 ± 4.53e	201.47 ± 3.50h	18.37 ± 0.38e	100.44 ± 1.70g	7.22 ± 1.43e
CRR alone	148.51 ± 3.45g	109.38 ± 3.22e	191.71 ± 3.00h	15.30 ± 0.38e	109.34 ± 1.24g	7.80 ± 1.28e
BRR alone	156.63 ± 4.43g	107.68 ± 4.34e	195.45 ± 3.45h	17.04 ± 0.38e	108.45 ± 1.89g	8.34 ± 0.28e
MRR alone	156.52 ± 3.23g	111.51 ± 3.38e	200.10 ± 2.69h	18.10 ± 0.23e	110.71 ± 1.57g	8.23 ± 0.93e
ARR alone	157.77 ± 3.76g	102.57 ± 3.43e	197.53 ± 4.02h	15.37 ± 0.20e	109.34 ± 1.45g	6.78 ± 1.73e

Values are mean ± SD (06 number). Sily: Silymarin.

a–h(means with different letters) indicate significance at P < 0.05.

3.4. Effects of R. hastatus Roots on DNA Damages (Ladder Assay)

CCl4 induces DNA damages in the testicular tissues of rats. DNA ladder assay showed that intact genomic DNA was found in control as well as DMSO treated group. Conversely, CCl4 group showed severe DNA damages. Postadministration of silymarin and different fractions of R. hastatus roots showed reduction in DNA damages as DNA band patterns of these groups were more similar to control group (Figure 1).

Figure 1

Agarose gel showing DNA damage by CCl4 and protective effects of various fractions of R. hastatus leaves in testicular tissue. Lanes from left (M) low molecular weight marker, (1) control, (2) DMSO + olive oil group, (3) CCl4 group, (4) silymarin + CCl4 group, (5) MRR + CCl4 group, (6) BRR + CCl4 group, (7) ARR + CCl4 group, (8) CRR + CCl4 group, (9) ERR + CCl4 group, and (10) HRR + CCl4 group.

3.5. Effects of R. hastatus Roots on Testis Histoarchitecture

Figure 2 illustrates the histological examination of testicular tissues of different treatment groups. Microscopic assessment of male reproductive system revealed the normal seminiferous tubules, sperms with normal morphology, and concentration in control as shown in Figure 2(a). Histological structure of germ cells was found to be normal in appearance. Figure 2(b) demonstrates that CCl4 intoxication caused degenerative changes such as loss of germ cells, abnormality of germinative epithelium, interruption in meiosis, sperm with abnormal shape and concentration, and delocalization of seminiferous tubules. These changes were markedly reduced with oral administration of various fractions of R. hastatus roots or silymarin revealing a marked repairing of testicular abnormalities. Among all the tested samples of R. hastatus roots, MRR and BRR as shown in Figures 2(c) and 2(d) demonstrated maximum antioxidant and healing effects against CCl4 induced damage showing sperm with normal morphology and concentration near to control group. Histopathological findings are in accord with the results of above studied parameters for testicular toxicity.

Figure 2

Microphotograph of rat testis (H&E stain). (a) Representative section of testis from the control group showing normal histology, (b) CCl4 group, (c) MRR + CCl4 group, (d) BRR + CCl4 group, (e) ARR + CCl4 group, (f) CRR + CCl4 group, (g) ERR + CCl4 group, and (h) HRR + CCl4 group.

3.6. Effects of R. hastatus Roots on Histopathology of Liver

Slides of liver tissues were prepared for histopathological study and stained with hematoxylin and eosins as shown in Figure 3. Figure 3(c) depicts that administration of CCl4 causes fatty changes like ballooning of cells, inflammatory cells infiltrations, dilation of central vein, cellular hypertrophy, necrosis, and degeneration of the lobular architecture. CCl4 administration for eight weeks resulted in chronic injury in the form of hepatic cirrhosis. Postadministration of various fractions of R. hastatus roots attenuated the hepatic injuries and percent them such as with very less or no fatty changes, no dilation of blood vessel, and uniform morphology of hepatocytes near to control group as shown in Figures 3(c)–3(h). Abnormal changes were not found in the morphology of control group (Figure 3(a)).

Figure 3

Microphotograph of rat liver (H&E stain). (a) Representative section of liver from the control group showing normal histology, (b) CCl4 group, (c) MRR + CCl4 group, (d) BRR + CCl4 group, (e) ARR + CCl4 group, (f) CRR + CCl4 group, (g) ERR + CCl4 group, and (h) HRR + CCl4 group.

4. Discussion

Medicinal plants extracts and their bioactive metabolites play important role in the prevention of oxidative damages especially CCl4 induced hepatic and testicular injuries in experimental animals [7, 26]. In the present investigations administration of various fractions of R. hastatus revealed reduction in elevated concentrations of AST, ALT, ALP, and γGT to maintain the structural consistency of the hepatocellular structure. Our findings are in agreement with Singh et al. [6] who reported that rise in serum markers has association with immense centrilobular necrosis, cellular infiltration, and ballooning of liver. CCl4 treatment caused alteration in cholesterol profile which was significantly reversed with postadministration of various fractions of R. hastatus. Similar investigations were reported by Wang et al. [27] while working on female rats to assess hepatic protection of Noni fruit juice against CCl4 induced chronic liver damage. Like testicular histology, serum gonadotropin releasing hormone (GnRH) including LH and FSH concentrations may facilitate in discovering conclusion about toxicosis. The reduction in serum testosterone concentrations indicates either a direct effect of chemical (CCl4) at Leydig cell concentration or an indirect effect by disturbing the hormonal environment at hypothalamopituitary axis [28] due to oxidative trauma in CCl4 treated rats. Tohda et al. [29] also reported that abnormal concentration of intratesticular testosterones inhibits spermatogenesis. The production of testosterone in Leydig cells is stimulated by LH, which activates FSH to bind with Sertoli cells to stimulate spermatogenesis [30]. CCl4 insults revealed the suppression in FSH concentration of serum that was in consistency with Khan and Ahmed [5] who reported significant reduction in serum FSH concentration. CCl4 intoxicated rats show the malfunctioning of pituitary to secrete FSH and LH indicating that testicular dysfunction leads to infertility. Estradiol directly stimulates the pituitary by determining prolactinemia, with hypothalamic dysfunction in case of hypogonadism. Thus, increased concentration of estradiol and prolactin may also be liable for the origin of hypogonadism in the present study. GSH concentrations are dependent upon the activities of glutathione reductase (GR) and NADH [31]. Glutathione system including GPx, GR, GST, as well as SOD and CAT represents a mutually loyal team of defense against ROS [32]. Enhanced lipid peroxidations expressed in terms of TBARS determine structural and functional alterations of cellular membranes [33]. In the present study, administration of various fractions of R. hastatus improved the activities of antioxidant enzymatic (SOD, CAT, POD, GPx, GST, GR, and QR) as well as nonenzymatic (GSH, TBARS, and H2O2). Hence, the present results regarding chronic toxicity of CCl4 are in accordance with previous reports of Khan and Ahmed [5] while studying the protective effects of Digera muricata (L.) Mart on testis against CCl4 oxidative stress. It was reported that CCl4 resulted in the oxidative damage to testicular proteins and DNA in rats [4, 34]. From the present study, it can be assumed that various fractions of R. hastatus ameliorated the toxic effects on DNA as revealed by percent DNA fragmentation and ladder assay. The present study clearly augments the defensive mechanism of various samples against oxidative stress induced by CCl4 and provides confirmation about its therapeutic use in reproductive abnormalities. Hepatohistology of CCl4 intoxicated rats revealed necrosis, fatty changes, cellular hypertrophy, infiltrated kupffer cells and lymphocyte, cirrhosis, and nuclear degeneration in some areas, which was markedly diminished by induction of various fractions of R. hastatus. Our study revealed similar investigation which is in agreement with earlier findings [3], while evaluating the medicinal activity of plants against CCl4 stimulated hepatotoxicity in rats. The CCl4 challenge revealed testicular destruction [35] and degeneration in histological architecture like that of profenofos that was recorded by Moustafa et al. [36]. Data of the present study revealed that CCl4 may cause proliferative behavior of testicular cells and obstruct reproduction. However, groups administered various fractions of R. hastatus demonstrated a quality active spermatogenesis, thin basement membranes, and normal seminiferous tubules in most of the part of testis. Same histopathology was noticed by Manjrekar et al. [37] while evaluating the protective effects of Phyllanthus niruri Linn. on testis against CCl4 intoxication.

5. Conclusions

It can be concluded from the current study that various fractions of R. hastatus roots have the ability to recover the metabolic enzymatic activities and repair cellular injuries, thus providing scientific evidence in favour of its pharmacological use in hepatic and testicular dysfunctioning.