Oxidative Medicine and Cellular Longevity

Volume 2013 (2013), Article ID 325406, 11 pages

http://dx.doi.org/10.1155/2013/325406

Research Article

Ameliorating Effect of Various Fractions of Rumex hastatus Roots against Hepato- and Testicular Toxicity Caused by CCl₄

Sumaira Sahreen,¹ Muhammad Rashid Khan,² and Rahmat Ali Khan³

¹Botanical Sciences Division, Pakistan Museum of Natural History, Garden Avenue, Shakarparian, Islamabad, Pakistan

²Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan

³Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology, Bannu, Khyber Pakhtunkhwa 28100, Pakistan

Received 14 February 2013; Revised 4 April 2013; Accepted 4 April 2013

Academic Editor: Kota V. Ramana

Copyright © 2013 Sumaira Sahreen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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 -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  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 (), 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 .

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 () augmented the concentrations of serum marker enzymes of liver which was attenuated significantly () 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 () 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 ( number).

**indicate significance from the control group at probability level.

++indicate significance from the CCl₄ group at 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.

(means with different letters) indicate significance at .

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 () decreased the testosterone, FSH, and LH concentration of serum while significantly () raised the prolactin and estradiol concentration. The serum concentrations of LH, testosterone, prolactin, and estradiol were restored () 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 stimulating

hormone (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.

(means with different letters) indicate significance at .

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 () 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 (M/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.

(means with different letters) indicate significance at .

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.

(means with different letters) indicate significance at .

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.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

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)).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

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.

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