Euphorbia dracunculoides L. abrogates carbon tetrachloride induced liver and DNA damage in rats
© The Author(s). 2017
Received: 15 December 2016
Accepted: 13 April 2017
Published: 20 April 2017
Evaluation of Euphorbia dracunculoides of family Euphorbiaceae during previous studies had established the in vitro antioxidant and in vivo anti-inflammatory activities. The plant is used by the local communities of Pakistan for various disorders including rheumatism and edema. In this investigation we have evaluated the hepatoprotective effects against CCl4 induced toxicity in rat.
Dry powder of the aerial parts of E. dracunculoides was extracted with 95% methanol to get the extract (EDME). To investigate the hepatoprotective effects of EDME the Sprague-Dawley male rats were divided in to 8 groups with 6 rats in each. Group I and II were the normal and vehicle treated while the Groups III-VI were injected intraperitoneally with 1 ml of CCl4 (30% in olive oil). Rats of Group IV were orally administered with silymarin (50 mg/kg) while the Group V and VI with 200 mg/kg and 400 mg/kg of EDME, respectively. Animals of Group VII (200 mg/kg) and VIII (400 mg/kg) were treated with EDME alone. The treatments were given thrice a week for 4 weeks. Effects of EDME were evaluated for the protective effects against oxidative stress and genotoxicity induced with CCl4 in liver of rat.
Analysis of serum indicated significant (p < 0.05) rise in the level of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and globulin whereas decrease was recorded for the total protein and albumin in CCl4 treated rats. In liver tissues the activity level of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione (GSH) was decreased while the level of lipid peroxides; thiobarbituric acid reactant substances (TBARS), nitrite and hydrogen peroxide increased in CCl4 treated rats as compared to the control group. Histopathological injuries and DNA damages were recorded in liver of rat with CCl4 treatment. However, co-administration of EDME, dose dependently, ameliorated the CCl4-induced hepatic toxicity in these parameters.
These results suggested that the phyto-constituents of EDME were able to ameliorate the oxidative stress induced with CCl4 and can be a useful therapeutic agent for oxidative stress related disorders.
KeywordsEuphorbia Dracunculoides Oxidative stress Lipid peroxidation Genotoxicity
The reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced as byproduct of metabolic pathways. The reactive species include hydrogen peroxide (H2O2), hydroxyl radical (OH), nitric oxide (NO) and superoxide anion (O2 −). Production of such species in trivial amounts is required for normal physiological functions but surplus yield can cause oxidative stress. Drugs, chemicals, pollutant and radiations are the common sources for the generation of free radicals which cause damages to cellular organization by affecting membranes, proteins, lipids as well as nucleic acids [1, 2]. Such effects have been observed in pathological conditions like diabetes mellitus, Alzheimer’s disease, liver cirrhosis, rheumatic arthritis, cancer and multiple sclerosis [3, 4].
Carbon tetrachloride (CCl4) can cause hepatotoxicity by producing oxidative stress which leads to steatosis and centrilobular necrosis and its prolonged administration produces chronic liver injury which leads to hepatic fibrosis . In recent years, antioxidants have been isolated from plants to scavenge free radicals. Within the antioxidant compounds, flavonoids and phenolic acids have the considerable antioxidative potential . The phenolic compounds show the antioxidant activity because of the presence of hydroxyl group in conjugated ring in their structure, and hence these can prevent free radical mediated diseases. Antioxidants protect the tissues by scavenging ROS , singlet oxygen  and superoxide anion .
Euphorbiaceae is one of the most varied family, ranges from the herb, shrub and tall tree . The largest among the spurge family is the genus Euphorbia covering over 2000 species. Most of the species of this genus are used conventionally in Chinese medicinal system for curing skin disorders, edemas etc. . Euphorbia dracunculoides Lam. a short-lived perennial herb attaining height up to 10–40 cm is dispersed in valleys, riverbanks, roadsides of filthy areas in Southwest Asia, South Europe and North Africa. It has been used as a traditional medicine in India for its diuretic and laxative effects . A decoction of whole plant is effective in killing lice when applied to the body of cattle . Fruits are known for their potential to exterminate warts from the skin . Leaf paste mixed with powder of the black pepper and ghee is useful in snake bite whereas leaf powder is used in epilepsy . Hepatoprotective effects of E. nematocypha have been investigated on hepatocyte cell lines  whereas in vivo investigation of E. neriifolia against CCl4 induced hepatic damages in rat has been reported by Bigoniya and Rana . Aamir et al.  reported the hepatoprotective potential of E. thymifolia against CCl4 induced hepatic damages in rat.
The medicinal propensity can be assessed by initial qualitative phytochemical screening of plants. Evalaution of E. dracunculoides confirmed the presence of coumarins, terpenoids, tannins, flavonoids, phenols, saponins, alkaloids and betacyanin. GC-MS analysis of n-hexane extract revealed the presence of 30 chemical constituents. Analysis of aqueous extract through HPLC confirmed the presence of four compounds i.e. catechin, rutin, myricetin and caffeic acid. Catechin is a polyphenol and has excellent antioxidant potential against free radicals and provides protection against inflammation, neurological disorders and apoptosis. Rutin is a well reputed secondary metabolite of plants with admirable anti-inflammatory, hepatoprotective and antioxidant activities. Various antioxidant assays exhibited significant correlation with that of total phenolic and total flavonoids contents . Non-toxic attitude with profound anti-inflammatory and antioxidant assertiveness of E. dracunculoides make it a strong candidate for in vivo evaluation against oxidative stress induced by CCl4 in rat. In this concern, activity of hepatic antioxidant enzymes and biochemical investigation of serum was executed. Also to check protective potential of the extract at genetic level, comet assay was conducted.
Plant material was collected in April 2015 from District Lakki Marwat, Pakistan. Then recognized by its native name and confirmed by Dr. Rizwana Aleem Qureshi, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. Voucher specimen, with Accession No. 127962 was deposited at the Herbarium of Pakistan, Quaid-i-Azam, University, Islamabad, Pakistan.
Preparation of extract
The aerial part of the plant was washed to get free of dust constituents and shade dried for 3 weeks at room temperature (20–25 °C). The fully dried plant was ground with an electric grinder. The powder (1.2 kg) was macerated twice at room temperature with 3 l of 95% methanol for 48 h. Filtrate was dried in rotary vacuum evaporator at 40 °C to obtain crude methanol extract of E. dracunculoides (EDME).
Acute toxicity study
Acute toxicity of EDME was investigated on Sprague-Dawley rats including females and males. The rats in each group were three (females 2; male 1) and were orally administered during morning (fasting conditions) with EDME at 50, 250, 500, 1000, 2000, 3000, 4000 mg/kg. Saline (10 ml/kg) was administered to the control rats. The animals were examined once daily for 14 days for mortality, behavioral pattern (lethargy, sleep, salivation), changes in physical appearance, injury, pain, and signs of illness.
The protocol of Shyu et al.  was trailed to do this experiment. Forty eight (48) male Sprague Dawley rats (180–200 g) were used for experiment. Guidelines of National Institute of Health, Islamabad were strictly followed in order to conduct experiment effectively. The designed protocol was ratified (Bch#0265) by the Ethical Committee of Quaid-i-Azam University, Islamabad, Pakistan. Animals were kept at room temperature with a dark/light 12 h phase in ordinary cages. Animals were properly fed on usual laboratory feed and water. The arbitrarily distribution of rats was done in eight different groups and each group was treated with its respective dose or combination of doses in calculated amount. Group I was remained untreated and only standard food supply was offered. Group II labeled as vehicle control was orally administered with 10% DMSO in olive oil (1 ml/kg). Rats of Group III were intraperitoneally injected with 1 ml/kg of 30% CCl4 (in olive oil). Animals of Group IV were administered with 1 ml/kg of 30% CCl4 (in olive oil) + silymarin dissolved in DMSO (50 mg/kg). Rats of group V and group VI were treated with 1 ml/kg of 30% CCl4 (in olive oil) + EDME dissolved in DMSO (200 mg/kg and 400 mg/kg, respectively) whereas Group VII and VIII were administered with EDME alone dissolved in DMSO (200 mg/kg and 400 mg/kg, respectively). The treatments of CCl4 and EDME were given in the morning on alternate days thrice a week for 4 weeks. After last treatment rats were unfed for 24 h and were euthanized after sedation with chloroform. Blood was collected for serum analysis. The liver was excised and placed in saline solution. A part of the liver was stored in liquid nitrogen for enzymatic and biochemical studies while the other portion was stored in 10% formalin solution histopathological studies.
For assessment of liver function tests, serum mockups were analyzed for AST, ALT, ALP, albumin, globulin and total protein by means of AMP diagnostic kits (Graz, Austria) according to the instructions of the manufacturer.
Assessment of antioxidative profile
Tissues of liver from various groups were homogenized in 10X prepared by the addition of 100 mM potassium phosphate buffer having 1 mM EDTA (pH 7.4). Centrifugation of homogenate was done at 12000×g at 4 °C for 30 min. In the supernatant antioxidant enzyme assays which are listed below were performed.
Catalase assay (CAT)
Catalase activity was measured by the procedure of Chance and Maehly . The reaction mixture for the catalase assay contained 2500 μl of phosphate buffer (pH 5.0; 50 mM), 100 μl of the supernatant and 400 μl of H2O2 (5.9 mM). Change in absorbance was determined at 240 nm after a minute. One unit catalase activity was demarcated as an absorbance variation of 0.01 units per minute.
Peroxidase assay (POD)
Peroxidase commotion was measured by the Chance and Maehly  method. Reaction mixture of peroxidase assay contained 2500 μl of phosphate buffer, (pH 5.0; 50 mM), 150 μl of guaiacol (20 mM), 300 μl of H2O2 (50 mM) and 1000 μl of the supernatant. Change in absorbance after a minute was recorded at 470 nm. One unit peroxidase activity was described as an absorbance change of 0.01 units per minute.
Superoxide dismutase, assay (SOD)
SOD assay was performed by following the Kakkar et al.  method. The reaction mixture contained 100 μl of phenazine methosulphate (186 μM), 1200 μl of sodium pyrophosphate buffer (pH 7.0; 0.052 mM) and 300 μl of supernatant obtained from liver homogenate which was incorporated to reaction amalgam after centrifugation at 1500 rpm for 10 min and then at 10000 rpm for 15 min. The reaction was started by adding 0.2 ml of NADH (780 μM) and then stopped on adding 1000 μl of glacial acetic acid after 1 min. Absorbance was recorded at 560 nm and results were articulated in units/mg protein.
Glutathione-S-transferase assay (GST)
GST was assessed by following Habig et al.  method. The reaction assortment for this assay contained 1475 μl of phosphate buffer (pH 6.5; 0.1 M), 200 μl of reduced glutathione (1 mM), 25 μl of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 300 μl of supernatant in 2000 μl of total volume. Change in absorbance was recorded at 340 nm. The enzyme activity was calculated as nM CDNB conjugate formed per min/mg protein and the molar extinction coefficient used here was 9.6 × 10- 3 M−1 cm−1.
Reduced glutathione assay (GSH)
Reduced glutathione assay was performed by the method of Jollow et al. . An aliquot of 1000 μl of supernatant of the homogenate was initially precipitated with 1000 μl (4%) of sulfosalicylic acid. After an hour it was centrifuged at 1200×g at 4 °C for 20 min. From the filtrate 100 μl was mixed with 2700 μl of phosphate buffer (pH 7.4; 0.1 M), and 200 μl of 100 mM 1,2-dithio-bis nitro-benzoic acid (DTNB). The absorbance of the reaction mixture was measured immediately at 412 nm. Reduced glutathione activity was determined as μM GSH/g tissue.
Lipid peroxidation (TBARS) estimation
Lipid peroxidation assay was executed by following the protocol of Iqbal et al. . The reaction mixture for this assay comprised of 580 μl of phosphate buffer (pH 7.4; 0.1 M), 200 μl of supernatant of the homogenate, 20 μl of ferric chloride (100 mM) and 200 μl of ascorbic acid (100 mM) in a total 1000 μl volume. The incubation of reaction mixture was executed at 37 °C in water bath for an hour. The reaction was stopped by adding 1000 μl of 10% trichloroacetic acid. After the addition of 1000 μl of 0.66% thiobarbituric acid, the conduits were retained for 20 min in boiling water and then placed on ice bath and centrifuged for 10 min at 2500×g. The quantity of TBARS (lipid peroxidation) was calculated by taking the absorbance of supernatant contrary to a reagent blank at 535 nm. The results were articulated as nM TBARS/min/mg tissue at 37 °C using molar extinction coefficient of 1.56 × 10- 5 M−1 cm−1.
The method of Lowry et al.  was followed to estimate total soluble protein of liver tissues by using bovine serum albumin (BSA) standard curve.
Hydrogen peroxide assay (H2O2)
Hydrogen peroxide (H2O2) assay was performed by following the protocol of Pick and Keisari  using the principle of H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red. The reaction mixture contained 3 ml of tissue homogenate recessed in 1000 μl of 0.28 nM of phenol red, 5.6 nM dextrose, 0.06 M phosphate buffer (pH 7.0) and 8.5 units of horseradish peroxidase and incubated at 37 °C for 60 min. A volume of 10 μl of 10 N of NaOH was added to stop the reaction. After centrifugation for 5 min at 800×g the absorbance of the supernatant was measured at 610 nm against reagent blank. Quantity of H2O2 produced was given as nM H2O2/min/mg tissue by using H2O2 oxidized phenol red standard curve.
The procedure of Grisham et al.  was used to quantify nitrite contents in the samples. After homogenization of the tissue equal volume (100 μl) of 0.3 M NaOH and 5% ZnSO4 was mixed for deproteinization. After centrifugation for 15–20 min at 6400×g an aliquot of 30 μl of the supernatant was mixed with 2 ml of Griess reagent in cuvette. The absorbance was recorded at 540 nm. Sodium nitrite curve was used for determining the nitrite amount in tissue samples.
DNA mutilation was evaluated by following the protocol of Dhawan et al. . The slides were dipped in methanol then burned over blue flame to get rid of dust and machine oil. About three quarters of sanitized slides were immersed in 1% solution of normal melting point agarose (NMPA) and was endorsed to set at room temperature. A tiny piece of liver tissue was minced into small pieces in 1 ml cold lysing solution. Before coating on already coated slides, the minced tissue was assorted with 85 μl of low melting point agarose solution. The slide was covered gently by placing a cover slip over it and placed for 10–12 min on ice packs. For a second time low melting agarose was added on slide by removing cover slip. Then it was permitted to solidify on ice packs. After coating the slide thrice with low melting agarose, it was retained in lysing solution for 10 min and then in freezer for about 2 h. After performing electrophoresis, staining was done with ethidium bromide (1%) and examined under the fluorescent microscope. The level of DNA damage was appraised by CASP 1.2.3.b software for image analysis. In every sample almost 50–100 cells were examined for head length, comet length, tail moment, tail length and amount of DNA in head of hepatic cell’s nuclei.
Histopathological study of tissues
Alterations in histopathology were evaluated via paraffin-embedded staining procedure. The multistep process entails fixation of liver samples in a fixative and were further rinsed and processed in the course of an ascending sequence of alcohol (50, 70, 90 and 100%). The tissues were then secured on hard solid blocks via paraffin-embedding. In the final step, slides were prepared by sectioning 3–4 μm thin layers of the embedded-tissue samples followed by staining with hematoxylin and eosin. Afterwards, these slides were examined under the light microscope (DIALUX 20 EB) at 40X and photographed via HDCE-50B camera.
The values for all the assays were articulated as mean ± standard deviation. Tukey’s multiple comparison tests centered on parametric scrutiny of variance was used to appraise the enormities of altered treatments to rats in vivo by the use of computer software Statistix 8.1. Statistical significance for demeanors was done at P-value ≤0.05.
Defensive effect of EDME on liver serum enzymes
Effect of EDME on biochemical markers of liver in serum
Serum proteins (mg/dl)
44.6 ± 2.22e
64.3 ± 3.15d
43.0 ± 2.08d
8.5 ± 0.37a
5.6 ± 0.2a
2.9 ± 0.24c
44.97 ± 2.16e
63.8 ± 2.12d
43.3 ± 2.25d
8.9 ± 0.40a
5.5 ± 0.23a
3.0 ± 0.26bc
CCl4 1 ml/kg
173.2 ± 4.19a
136.3 ± 4.11a
89.3 ± 3.26a
5.6 ± 0.15d
2.1 ± 0.18d
3.5 ± 0.16a
CCl4+ Sily 50 mg/kg
61.8 ± 3.29d
71.6 ± 2.41c
48.0 ± 1.15e
7.5 ± 0.19b
4.3 ± 0.19b
3.2 ± 0.13b
CCl4 + EDME 200 mg/kg
104.2 ± 3.21b
93.1 ± 3.22b
67.1 ± 2.09b
6.3 ± 0.22c
3.5 ± 0.15c
2.8 ± 0.17c
CCl4 + EDME 400 mg/kg
67.4 ± 3.23c
71.7 ± 3.39c
53.8 ± 2.33c
7.7 ± 0.24b
4.7 ± 0.42b
3.0 ± 0.18bc
EDME 200 mg/kg
46.2 ± 2.16e
65.8 ± 3.13d
45.3 ± 2.15d
8.7 ± 0.16a
5.8 ± 0.20a
2.9 ± 0.12c
EDME 400 mg/kg
45.7 ± 2.31e
62.32 ± 3.17d
43.3 ± 1.28d
8.8 ± 0.21a
5.7 ± 0.29a
3.1 ± 0.14bc
Defensive effect of EDME on serum protein profile
For the evaluation of protective effects of EDME against the CCl4 induced toxicity on liver metabolism the level of albumin, globulin and total serum protein were examined (Table 1). In serum the level of albumin and total protein decreased (p < 0.05) while the level of globulin increased with CCl4 toxicity in rats as compared to the control group (Table 1). The results showed that EDME have significant protective effects and the altered level of albumin, total protein and globulin induced with CCl4 were improved after co-administration of EDME in a dose dependent manner. The protective effects of silymarin for albumin, globulin and total protein were comparable (p > 0.05) with the higher dose of EDME 400 mg/kg to rats. Treatment of rats with EDME alone did not (p > 0.05) change the level of protein profile as compared to the control group.
Defensive effect of EDME on liver antioxidant enzymes
Effect of EDME antioxidant enzymes of liver
5.47 ± 0.21a
8.66 ± 1.21a
3.28 ± 0.17a
21.33 ± 2.13a
5.43 ± 0.18a
8.61 ± 1.16a
3.25 ± 0.28a
21.04 ± 2.38a
CCl4 1 ml/kg
1.86 ± 0.12e
3.93 ± 0.23e
1.12 ± 0.16e
8.94 ± 0.92d
CCl4 + Sily 50 mg/kg
4.41 ± 0.26b
7.89 ± 1.29b
2.73 ± 0.28b
21.3 ± 1.33a
CCl4 + EDME 200 mg/kg
2.48 ± 0.14d
4.75 ± 0.35d
1.78 ± 0.21d
16.26 ± 1.07c
CCl4 + EDME 400 mg/kg
4.05 ± 0.29c
6.56 ± 0.72c
2.46 ± 0.16c
19.87 ± 2.17b
EDME 200 mg/kg
5.46 ± 0.25a
8.63 ± 0.92a
3.26 ± 0.22a
21.32 ± 2.19a
EDME 400 mg/kg
5.38 ± 0.32a
8.37 ± 0.87a
3.31 ± 0.31a
21.26 ± 2.45a
Defensive effect of EDME on liver biochemicals
Effect of EDME biochemical parameters in liver
Protein (μg/mg tissue)
TBARS (nM/min/mg protein)
2.55 ± 0.14a
19.35 ± 1.18a
22.33 ± 2.12d
0.23 ± 0.06e
50.61 ± 2.13e
2.41 ± 0.05ab
19.03 ± 1.11a
22.07 ± 2.1d
0.24 ± 0.05e
50.93 ± 2.15e
CCl4 1 ml/kg
0.98 ± 0.08d
6.75 ± 0.57e
44.52 ± 3.26a
0.77 ± 0.19a
92.96 ± 3.17a
CCl4+ Sily 50 mg/kg
2.43 ± 0.23ab
18.33 ± 1.08b
29.28 ± 2.08c
0.27 ± 0.08d
55.03 ± 2.07c
CCl4 + EDME 200 mg/kg
1.66 ± 0.29c
9.33 ± 1.18d
36.87 ± 2.38b
0.58 ± 0.07b
73.47 ± 3.2b
CCl4 + EDME 400 mg/kg
2.11 ± 0.06b
14.82 ± 1.33c
28.13 ± 2.07c
0.44 ± 0.07c
60.23 ± 3.24d
EDME 200 mg/kg
2.47 ± 0.11a
20.24 ± 2.13a
23.09 ± 2.2d
0.27 ± 0.05e
50.62 ± 2.21e
EDME 400 mg/kg
2.52 ± 0.16a
20.33 ± 1.37a
22.67 ± 2.43d
0.24 ± 0.03e
51.69 ± 2.18e
After the completion of treatment, in the liver samples of CCl4 intoxicated rats an escalation in the content of TBARS, H2O2 and nitrite was recorded i.e. 44.52 ± 3.26 nM/min/mg protein, 0.77 ± 0.19 μM/ml and 92.96 ± 3.17 μM/ml, respectively (Table 3). The toxicity of CCl4 was ameliorated by the co-administration of EDME in a dose dependent fashion. The level of TBARS, H2O2 and nitrite in liver samples of rat with co-treatment of the lower dose of EDME 200 mg/kg were; 36.87 ± 2.38 nM/min/mg protein, 0.58 ± 0.07 μM/ml and 73.47 ± 3.2 μM/ml, respectively. The high dose of EDME 400 mg/kg remarkably (p < 0.05) lowered the level of TBARS, H2O2 and nitrite content; 28.13 ± 2.07 nM/min/mg protein, 0.44 ± 0.07 μM/ml and 60.23 ± 3.24 μM/ml, respectively. Moreover, the rats treated with EDME alone did not exhibit alteration in the level of TBARS, H2O2 and nitrite in liver samples as compared to the control group.
Effect of EDME on DNA damages of liver cells by comet assay
Comet length (μm)
Head length (μm)
Tail length (μm)
%DNA in head
%DNA in tail
42.3 ± 1.3d
37.6 ± 1.3a
4.2 ± 1.2d
95.3 ± 1.1a
4.13 ± 0.31d
0.17 ± 0.08e
41.6 ± 1.94d
37.1 ± 0.8a
4.87 ± 0.7d
94.18 ± 0.9a
5.32 ± 0.26d
0.25 ± 0.09e
CCl4 1 ml/kg
64.5 ± 1.4a
23.4 ± 1.2d
40.81 ± 0.8a
73.9 ± 1.13d
25.7 ± 1a
10.48 ± 0.6a
CCl4+ Sily 50 mg/kg
41.9 ± 1d
35.2 ± 1.78b
6.0 ± 1.19d
94.9 ± 0.7a
5.03 ± 0.5d
0.30 ± 0.07d
CCl4 + EDME 200 mg/kg
53.7 ± 1.8b
32.8 ± 1.4c
19.7 ± 1.7b
86.5 ± 1.2c
13.43 ± 0.7b
2.64 ± 0.14b
CCl4 + EDME 400 mg/kg
48.3 ± 1.21c
35.9 ± 1.1b
11.63 ± 1c
89.6 ± 0.8b
10.16 ± 0.9c
1.18 ± 0.16c
EDME 200 mg/kg
42.5 ± 1.4d
37.4 ± 0.9a
5.1 ± 0.5d
94.92 ± 1.2a
5.06 ± 0.8d
0.25 ± 0.09e
EDME 400 mg/kg
41.1 ± 1.53d
37.9 ± 1.87a
3.3 ± 0.4d
95.71 ± 0.6a
4.27 ± 0.5d
0.14 ± 0.08e
Defensive effect of EDME on histoarchitecture of liver
Traditional consumption of plants as medicines is considered non-damaging since centuries. To check the new promising natural antioxidants in plants or to exploit new drug development for tissue related human and animal health, CCl4 was used as chemical toxicant for different organs to produce ROS which cause disturbance in the antioxidant system. The current research was conducted to check the defensive prospective of E. dracunculoides methanol extract on liver against oxidative stress induced by CCl4.
Determination of enzymes such as AST, ALT and AST in the serum provides an important indicator of the hepatic membrane functional integrity. In the present study, elevated levels of liver serum markers ALT, ALP and AST might reflect the hepatic damages due to CCl4 intoxication in rats. Enhanced generation of ROS in hepatocytes lead to cellular death due to DNA damage, protein oxidation, lipid peroxidation and also added to the hepatic damaging action . Co-administration of silymarin along with CCl4 displayed the protective ability and the values of ALT, ALP and AST were found near to the level of control group. The methanol extract of plant showed its protective aptitude against the CCl4 induced hepatotoxicity in rat in a dose dependent fashion. The lower dose (200 mg/kg) of EDME exerted protective aptitude and the lower level of ALT, ALP and AST was recorded whereas the higher dose of EDME (400 mg/kg) exhibited potent toxico-suppressive effects and the level of above parameters was found near to the level of control animals. The elevated level of ALP, AST and ALT in serum with CCl4 treatment might be associated with membrane damage of liver cells. Restoration of the hepatic enzymes towards the control level with EDME indicated the hepatoprotective potential of the extract [30, 31].
On the other hand a marked decline in the level of total proteins and albumin while increase of globulin in serum of CCl4 treated group was determined in this study. Presence of albumin and globulin in normal range is essential for the proper physiology of a person. Globulin helps to regulate the function of the circulatory system. Globulins consist of four proteins; alpha-1, alpha-2, beta and gamma globulin and among them liver produces predominantly the alpha and beta globulins. Increase in the level of globulin in serum might be due to the prolong treatment of CCl4 to rat. Elevated level of globulin in serum can be considered a reliable non-invasive marker towards the degree of liver fibrosis . Co-treatment with low dose of the EDME (200 mg/kg) was protective and revert the levels of serum proteins towards the control samples while the higher dose (400 mg/kg) showed more promising results and restored serum proteins to levels that are analogous to the control group. The protective abilities of EDME might be attributed by the presence of antioxidant phyto-constituents .
Antioxidant defense system includes the enzymatic antioxidants (CAT, POD, SOD, GST and GSH) which have vital role in defense system. Catalase (CAT) is a very important enzyme which can neutralize H2O2 through catalytic conversion to H2O and O2 at higher concentrations or through peroxidation activity at lower concentration. SOD enzyme dismutates the superoxide anion radical to less toxic O2 and H2O2 by oxidation reduction mechanism of its active metal ion. Glutathione peroxidase (POD) is involved in the catalytic reduction of H2O2 and lipid peroxides at the expenditure of glutathione. GSH is a free radical scavenger and shielded the toxic effects of various peroxides. During oxidative stress the level of these antioxidants is compromised. In the present study, CCl4 injection to rats showed the reduced levels of CAT, POD, SOD, GST and GSH. This condition might reflect the excessive generation of ROS which inhibit the synthesis of antioxidant enzymes. Decreased level of GSH might be due to its more consumption by the hepatocytes in scavenging toxic radicals generated by CCl4. Younis et al.  also reported the reduced levels of all above enzymes and GSH content in liver tissue by CCl4 administration. The antioxidant effects of EDME might improves the levels of antioxidant enzymes including CAT, POD, SOD and GST, and GSH that was declined due to CCl4 action. Presence of flavonoids, tannins, sterols, terpenoids might be suspected for the attribution of antioxidant activities . The same study indicated the presence of strong antioxidant phyto-constituents i.e. rutin, myricetin, catechin and caffeic acid in E. dracunculoides . Similar protective effects comments were also reported by using Alnus nitida against CCl4 induced oxidative stress in liver . Presence of various polyphenols in the extract might be responsible for the scavenging of free radicals and in the inhibition of lipid peroxidation . Different studies have also reported protective effects of various plant extracts against the CCl4 induced hepatic damages in rat [17, 18, 31].
CCl4 also exerts its toxic effects by changing the level of TBARS, H2O2, tissue protein and nitrite content. In CCl4 intoxication the tissue protein level decreases but there is noteworthy increase in the lipid peroxidation (TBARS), H2O2 and nitrite content. Concentration and duration of CCl4 treatments to experimental animals exhibit varied pattern of oxidative injuries. Administration of CCl4 for short duration to rat regulates the endogenous expression of interleukin (IL)-6 and IL-10 which in turn suppresses the synthesis of tumor necrosis factor-β1 (TGF-β1) and consequently inhibits the synthesis of collagen. However, longer duration of the treatment of mice with CCl4 causes the infiltration of neutrophils and fibrosis of liver [36, 37]. Parola and Robino  elaborated that peroxidation of lipids induce overexpression of fibrogenic cytokines by stimulating the synthesis of collagen and activating hepatic stellate cells. It might be assumed that that long term administration of CCl4 to rats might increase the oxidative stress and consequently the hepatic fibrosis. In this study hepatic histopathology of rats treated with CCl4 exhibited macro steatosis in hepatocytes of rat. Co-administration of silymarin to rats diminished the CCl4 toxic effects and showed the level of TBARS, H2O2 and nitrite content of liver near to the level of control rats. Administration of EDME to CCl4 intoxicated rats decrease the level of TBARS, H2O2 and nitrite content of liver homogenates and the level of above parameters lies near the level of control group. Our findings are supported by the work of Sajid et al.  who reported the in vivo antioxidant effects of Alnus nitida in rat liver intoxicated with CCl4.
Oxidative stress induced with CCl4 in liver causes the single stranded or the double stranded break in the DNA and damages the DNA integrity . In the present study, comet assay was performed to check the DNA damage in terms of comet length, head length, %DNA in head, tail length, %DNA in tail and tail moment. A noteworthy elevation in comet length, tail length, tail moment while sharp decline in the level of DNA in head of comet was observed in the hepatocyte of CCl4 intoxicated rats in comparison to control group. DNA damages induced with CCl4 toxicity in liver has tremendously contributed towards the migration of DNA (22.63%) from head to the tail that consequently resulted in the increase of tail length, %DNA in tail and in tail moment of hepatocytes . Oxidative damage caused by CCl4 to DNA in the hepatic cells of rat has also been reported in previous study . Co-administration of EDME, dose dependently, lessened the toxic effects of CCl4 on DNA damages. Co-administration of EDME causes an increase in head length and %DNA in head of comet along with a decrease in the mean values of comet length, tail length, tail moment and %DNA in tail suggesting the protective abilities of EDME against the CCl4 induced DNA damages. Treatment of EDME to CCl4 intoxicated rats substantially inhibited the migration of DNA from head to tail in comet of hepatocytes. At lower dose of EDME (200 mg/kg) there was 9.75% DNA migration from head to tail while 5.07% DNA migration from head to tail of comet was recorded at higher dose of EDME (400 mg/kg) suggesting the toxico-suppressive effect of EDME.
Number of DNA strand breaks (tail moment −1) provides a useful estimate for the protective abilities of the extract. Generation of ROS is considered to be involved in the oxidative DNA damages. In this investigation DNA strand breaks were not determined in the control group as well as the groups treated with EDME alone at both 200 mg/kg and 400 mg/kg treatment to rats. However, treatment of CCl4 to rats caused 9.48 number of DNA strand breaks in the comet of hepatocytes. The co-administration of EDME to CCl4 treated rats decreased the number of DNA stand breaks; 1.64 (200 mg/kg) and 0.181 (400 mg/kg) of EDME, respectively. DNA strand breaks were not recorded with the co-administration of silymarin to CCl4 intoxicated rats. These results suggested that EDME is effective in decreasing the ROS level, DNA fragmentation and in increasing the cell viability. DNA damaging effects of CCl4 might be ameliorated by the various phytochemical classes reported in E. dracunculoides . Protection of DNA damages induced with hydrogen peroxide in human lymphocytes has been demonstrated with various herbal extracts .
The histology of the liver is the direct mean of assessing the protective effects of therapeutic agents against CCl4 induced hepatic damages. CCl4 induced the high degree of damage in liver cells, by inducing fibrosis, necrosis, cellular hypertrophy and central lobule disruption. Co-administration of silymarin as well as low and high dose of EDME ameliorated the toxic effects of CCl4 and decreased the damages induced with CCl4 intoxication. The results of this study are in accordance with previous reports . The protective effects exhibited by the EDME might be attributed by the presence of flavonoids, terpenoids, sterols and tannins. E. dracunculoides was found to be constituted by rutin, catechin, myricetin and caffeic acid .
Our study recommends that E. dracunculoides have the aptitude to ameliorate the hepatic damage provoked by CCl4 and has potential to restore the levels of enzyme activity, serum markers, DNA damages and histopathological amendments. The defensive properties of EDME might probably be concomitant with its phytochemical profile and antioxidant properties.
Euphorbia dracunculoides methanol extract
Thiobarbituric acid reactive substances
MRK is intensely acknowledged for his kind supervision, expert guidance and substantial facilitations of all necessary materials and equipment.
The project was funded by the Department of Biochemistry Quaid-i-Azam University Islamabad Pakistan.
Availability of data and materials
All the data is contained in the manuscript.
RB made significant contribution to experimentation, acquisition and drafting of the manuscript. MRK has made substantial contribution to designing, analyzing and drafting of the manuscript. MM made a contribution in the experimentation and acquisition of the data. All authors read and approved the final manuscript.
MRK did his Diploma in Unani Medicine and Surgery (DUMS) and is a registered practitioner of the National Council for Tibb of Pakistan. He is working as Associate Professor at the Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study makes use of rats and the experimental protocol for the use of animal was approved (Bch#0267) by the ethical board of Quaid-i-Azam University, Islamabad Pakistan.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Gutteridge J. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem. 1995;41(12):1819–28.PubMedGoogle Scholar
- Tirkey N, Pilkhwal S, Kuhad A, Chopra K. Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney. BMC Pharmacol. 2005;5(1):2.View ArticlePubMedPubMed CentralGoogle Scholar
- Kasprzak KS. Oxidative DNA and protein damage in metal-induced toxicity and carcinogenesis 1, 3. Free Radic Biol Med. 2002;32(10):958–67.View ArticlePubMedGoogle Scholar
- Jacob RA, Sotoudeh G. Vitamin C function and status in chronic disease. Nutr Clin Care. 2002;5(2):66–74.View ArticlePubMedGoogle Scholar
- Hernández-muñoz R, Díaz-muñoz M, Suárez J, de Sánchez VC. Adenosine partially prevents cirrhosis induced by carbon tetrachloride in rats. Hepatology. 1990;12(2):242–8.View ArticlePubMedGoogle Scholar
- Dimitrios B. Sources of natural phenolic antioxidants. Trends Food Sci Technol. 2006;17(9):505–12.View ArticleGoogle Scholar
- Torel J, Cillard J, Cillard P. Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry. 1986;25(2):383–5.View ArticleGoogle Scholar
- Husain SR, Cillard J, Cillard P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry. 1987;26(9):2489–91.View ArticleGoogle Scholar
- Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochem Pharmacol. 1988;37(5):837–41.View ArticlePubMedGoogle Scholar
- Perveen A, Qaiser M. Pollen flora of Pakistan-XLVII. Euphorbiaceae. Pak J Bot. 2005;37(4):785.Google Scholar
- Jassbi AR. Chemistry and biological activity of secondary metabolites in Euphorbia from Iran. Phytochemistry. 2006;67(18):1977–84.View ArticlePubMedGoogle Scholar
- Wang L, Cao P, Zang Z, Ma Y-T, Liu F-Y, Li F, Wu X-H, Huang S-X, Zhao Y. Two new triterpene rhamnosides from Euphorbia dracunculoides lam. Phytochem Lett. 2015;12:168–72.View ArticleGoogle Scholar
- Sikarwar R, Bajpai A, Painuli R. Plants used as veterinary medicines by aboriginals of Madhya Pradesh, India. Pharm Biol. 1994;32(3):251–5.View ArticleGoogle Scholar
- Mossa JS, Al-Yahya MA, Al-Meshal IA: Medicinal plants of Saudi Arabia. 1987.Google Scholar
- Sharma J, Painuli R, Gaur R. Plants used by the rural communities of district Shahjahanpur, Uttar Pradesh. Indian J Tradit Knowl. 2010;9(4):798–803.Google Scholar
- Ito M, Shimura H, Watanabe N, Tamai M, Hanada K, Takahashi A, Tanaka Y, Arai K, Zhang PL, Chang R. Hepatoprotective compounds from Canarium album and Euphorbia nematocypha. Chem Pharm Bull (Tokyo). 1990;38(8):2201–3.View ArticleGoogle Scholar
- Bigoniya P, Rana AC. Protective effect of Euphorbia neriifolia saponin fraction on CCl4-induced acute hepatotoxicity. Afr J Biotechnol. 2010;8:7148–56.Google Scholar
- Aamir S, Tippeswamy BS, Kulkarni VH, Karunakar H. Hepatoprotective effects of Euphorbia thymifolia whole plant extract on CCl4 induced hepatic damages in rats. Int J Res Ayurveda Pharm. 2011;2:681–6.Google Scholar
- Majid M, Khan MR, Shah NA, Haq IU, Farooq MA, Ullah S, Sharif A, Zahra Z, Younis T, Sajid M. Studies on phytochemical, antioxidant, anti-inflammatory and analgesic activities of Euphorbia dracunculoides. BMC Complement Altern Med. 2015;15(1):1.View ArticleGoogle Scholar
- Shyu M-H, Kao T-C, Yen G-C. Hsian-tsao (Mesona procumbens Heml.) prevents against rat liver fibrosis induced by CCl4 via inhibition of hepatic stellate cells activation. Food Chem Toxicol. 2008;46(12):3707–13.View ArticlePubMedGoogle Scholar
- Chance B, Maehly A.  assay of catalases and peroxidases. Methods Enzymol. 1955;2:764–75.View ArticleGoogle Scholar
- Kakkar P, Das B, Viswanathan P. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 1984;21(2):130–2.PubMedGoogle Scholar
- Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130–9.PubMedGoogle Scholar
- Jollow D, Mitchell J. Zampaglione Na, Gillette J: Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3, 4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology. 1974;11(3):151–69.View ArticlePubMedGoogle Scholar
- Iqbal M, Sharma S, Rezazadeh H, Hasan N, Abdulla M, Athar M. Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate mediated hepatic injury. Redox Rep. 1996;2(6):385–91.View ArticlePubMedGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–75.PubMedGoogle Scholar
- Pick E, Keisari Y. Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages-induction by multiple nonphagocytic stimuli. Cell Immunol. 1981;59(2):301–18.View ArticlePubMedGoogle Scholar
- Grisham MB, Johnson GG, Lancaster JR. Quantitation of nitrate and nitrite in extracellular fluids. Methods Enzymol. 1996;268:237–46.View ArticlePubMedGoogle Scholar
- Dhawan A, Bajpayee M, Parmar D. Comet assay: a reliable tool for the assessment of DNA damage in different models. Cell Biol Toxicol. 2009;25(1):5–32.View ArticlePubMedGoogle Scholar
- Rashid U, Khan MR, Sajid M. Hepatoprotective potential of Fagonia Olivieri DC. Against acetaminophen induced toxicity in rat. BMC Complement Altern Med. 2016;16:449.View ArticlePubMedPubMed CentralGoogle Scholar
- Rashid U, Khan MR. Fagonia olivieri prevented hepatorenal injuries induced with gentamicin in rat. Biomed Pharmacother. 2017;2017(88):469–79.View ArticleGoogle Scholar
- Schmilovitz-Weiss H, Tovar A, Halpern M, Sulkes J, Braun M, Rotman Y, Tur-Kaspa R, Ben-Ari Z. Predictive value of serum globulin levels for the extent of hepatic fibrosis in patients with chronic hepatitis B infection. J Viral Hepat. 2006;13(10):671–7.View ArticlePubMedGoogle Scholar
- Younis T, Khan MR, Sajid M. Protective effects of Fraxinus xanthoxyloides (wall.) leaves against CCl4 induced hepatic toxicity in rat. BMC Complement Altern Med. 2016;16:407.View ArticlePubMedPubMed CentralGoogle Scholar
- Sajid M, Khan MR, Shah NA, Shah SA, Ismail H, Younis T, Zahra Z. Phytochemical, antioxidant and hepatoprotective effects of Alnus nitida bark in carbon tetrachloride challenged Sprague Dawley rats. BMC Complement Altern Med. 2016;16:268.View ArticlePubMedPubMed CentralGoogle Scholar
- Khan RA, Khan MR, Khan A. Comparative antioxidant scavenging and lipid peroxidation activity of rutin and gallic acid. Bangladesh J Pharmacol. 2015;10:637–8.View ArticleGoogle Scholar
- Louis H, Laethem J-LV WW, Quertinmont E, Degraef C, Berg KVD, Demols A, Goldman M, Moine OL, Deviere J. Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice. Hepatology. 1998;28(6):1607–15.View ArticlePubMedGoogle Scholar
- Natsume M, Tsuji H, Harada A, Akiyama M, Yano T, Ishikura H, Nakanishi I, Matsushima K, Kaneko S, Mukaida N. Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice. J Leukoc Biol. 1999;66(4):601–8.PubMedGoogle Scholar
- Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis. J Hepatol. 2001;35(2):297–306.View ArticlePubMedGoogle Scholar
- Sajid M, Khan MR, Shah NA, Ullah S, Younis T, Majid M, Ahmad B, Nigussie D. Proficiencies of Artemisia scoparia aagainst CCl4 induced DNA damages and renal toxicity in rat. BMC Complement Altern Med. 2016;16:149.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin K-H, Yang Y-Y, Yang C-M, Huang M-Y, Lo H-F, Liu K-C, Lin H-S, Chao P-Y. Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes. BMC Res Notes. 2013;6:490.View ArticlePubMedPubMed CentralGoogle Scholar
- Alkreathy HM, Khan RA, Khan MR, Sahreen S. CCl4 induced genotoxicity and DNA oxidative damages in rats; hepatoprotective effect of Sonchus arvensis. BMC Complement Altern Med. 2014;14:452.View ArticlePubMedPubMed CentralGoogle Scholar