Protective effect of oregano water extract against Carbon tetrachloride induced Hepatotoxicity in female rats

Carbon tetrachloride (CCL4) is one of the most toxic chemicals causes oxidative stress in the living organisms. Our experiment started with twenty female rats that were divided into 4 groups; The 1 group G1 served as control group, 2 group G2 injected twice a week with CCL4 diluted in olive oil (1:1 v/v), 3 group G3 were fed daily for 14 days with 1ml of Oregano water extract and the 4 group G4 were injected with CCL4 plus daily feeding with 1ml of Oregano water extract. The biochemical results indicated a significant elevation in the liver and kidney functions including ALT, AST, creatinine, uric acid and urea for the 2 group due to the toxicity induced by the CCL4. The levels of ALT, AST, creatinine, urea and uric acid were significantly decreased after the administration of oregano water extract in the 4 group. Lipid profile was evaluated in the four groups by measuring the levels of total cholesterol, HDL-C and triglycerides. The variation noticed by the over and down expression of the liver genes due to CCl4 was explored. These up and down regulated genes were tracked using differential display technique.


INTRODUCTION
In the recent days, the rates of human being diseases and organs toxicity have been increased as a result of exposing to multi environmental factors.These factors include xenobiotics, heavy metals, and toxic chemicals in addition to different microbial infections.
The liver is almost involved with all the biochemical pathways related to growth, fight against disease, nutrient supply and energy provision (Palanivel et al., 2008).It is also responsible for the process of detoxification of toxic chemicals.There are some factors that can easily cause liver injury including non-adequate nutrition, alcohol and drug abuse, viral infections and incidental poisoning (Domitrovic´ et al., 2013).
Carbon tetrachloride (CCl4) intoxication is a frequently used model of liver injury (Weber et al., 2003).CCl4 is activated by hepatic microsomal cytochromes to form the trichloromethyl radical, CCl3.This radical induces oxidative stress by binding to cellular molecules, thus impairing numerous cellular processes that lead to necrotic cell damage, inflammation and apoptosis (Sun et al., 2001).Non lethal intoxication triggers liver tissue remodeling and healing through the activation of hepatic stellate cells (HSCs), leading to liver fibrosis (Friedman, 2000).Carbon tetrachloride (CCl4) is extensively used hepatotoxic agent in preclinical animal studies and considered the bestcharacterized chemical to study xenobiotic induced free radical mediated acute liver injury in rats (Recknagel and Glende, 1973).In addition, CCl4 can cause pathological lesions in treated animals that closely resemble the symptoms of cirrhosis in human, and so considered an excellent model to evaluate the efficacy of hepatoprotectants (Desai et al., 2012).
A large variety of methods are used for the treatment of liver diseases include, pharmacotherapy, surgery as well as liver transplantation, all of which have shown limited therapeutic benefits and are associated with serious complications (Mihailovic et al., 2013).Consequently, there is a great demand to figure out an alternative approaches for the treatment of liver diseases (Bishayee et al., 2010).The medicinal plants are considered the most recommended approaches for the treatment of liver disorders (Jaishree and Badami, 2010).
Oregano or Marjoram (Majorana hortensis) is the gray green leaf of the mint family (Lamiaceae or Labiatae).It is widely distributed in Egypt and was known to the ancient Egypt for its healing properties (Badee et al., 2013).It has been used for treatment of arthritis, rheumatism, muscle and nerve pain, headaches, circulatory disorders, respiratory infections (Rashwan, 2011) in addition to its medicinal effect as gastrointestinal tract stimulant, tonic, carminative, diaphoretic, hypoglycemic, diuretic as well as antibacterial (Leeja and Thoppil, 2007) and as antioxidant (Handl et al., 2008).
Our study was targeted to use carbon tetrachloride (CCl4) as hepatotoxic agent with subsequent treatment with marjoram.The efficacy of marjoram was tracked using physiological, biochemical and advanced molecular methods.

Chemicals:
Carbon tetrachloride used in the experiment was purchased from Sigma-Aldrich Co., Germany.All the diagnostic kits assaying liver, kidney function tests and the lipid profile were obtained from Biodiagnostic Company, Egypt.Olive oil used for dissolving CCL4 was purchased from the local market.

Plant material:
Marjoram leaves were obtained as a gift from a farmer in Minia province, Egypt.

Extraction of oregano (marjoram):
15 gm of dry leaves of the plant were grinded and extracted by warming in 300 ml distilled water for 30 min at 80°C and finally cooled.The extract was filtered using small diameter mesh, and the remaining fine particles were filtered using small pores filter paper and stored in 4°C until use.

Experimental design:
A total of twenty female rats were used for the study; divided into four groups with total of five rats for each.The first group served as control group which fed orally on normal diet with distilled water, the second group served as treated group which injected interperitoneally with CCl4 dissolved in olive oil (1:1 V/V), the third group served as positive control group which only fed orally on oregano extract and the last group served as treated group injected interperotineally with CCl4 and orally fed on oregano extract.
The study was run for two weeks with injection of 0.25 ml of CCl4 twice a week and daily feeding of 1 ml oregano/ rat.
At the end of the study, the rats were sacrificed under ether anesthesia and serum samples were collected in test tubes and preserved at 4 o C, while, liver samples were kept in phosphate buffer and preserved in -80 o C for the molecular analysis.Total proteins (TP) were determined by means of the Biuret reaction as described by Gornall et al. (1949).In the presence of an alkaline cupric sulfate, the protein produces a violet color, the intensity of which is proportional to their concentration.Alanine aminotanseferase (ALT) and Aspartate aminotransefrase (AST) were determined following the method of Reitman and Frankel (1957).The catalytic activity was measured by spectrophotometery at 505nm.

Measurement of kidney function markers:
Creatinine level was determined by colorimetric kinetic method as described by Larsen (1972), where Creatinine in alkaline solution reacts with picric acid to form a colored complex.Urea levels in the serum were measured by means of the coupled reactions described by Fawcett and Soctt (1960).The blue dye indophenol product reaction absorbs light between 530 nm and 560 nm proportional to initial urea concentration.Uric acid level was determined as described by Barham and Trinder (1972), where the uric acid was hydrolyzed enzymatically to release hydrogen peroxide which reacts with 4aminoantipyrine in the presence of 3,5 Dichloro-2-hydroxybenzen sulphonate to form a quinoneimine which has color measured at 510 nm.

Lipid profile:
The total cholesterol was determined after enzymatic hydrolysis and oxidation according to Allain et al. (1974).The quinoneimine is formed from hydrogen peroxide and 4aminoantipyrine in the presence of phenol and peroxidase.The HDLcholesterol was determined by enzymatic colorimetric method as described by Lopez-Virella et al. (1977) where phosphotungstic acid and magnesium ions selectively precipitating all lipoproteins except the HDL fraction, cholesterol present in the supernatant can be determined by the same method used for total cholesterol.

Measurement of glutathione peroxidase (GPx):
The assay is an indirect measure of the activity of c-GPx according to Paglia and Valentine (1967).Oxidized glutathione (GSSG), produced upon reduction of organic peroxide by c-GPx, and is recycled to its reduced state by the enzyme reductase (GR).

Statistical analysis:
The data were given as individual values and as mean  standard error.Comparisons between the means of various treatment groups were analyzed using one way ANOVA.Differences were considered significant at P < 0.05.All statistical analyses were performed using the statistical software SPSS, version 10.

Molecular methods: RNA extraction:
The frozen liver tissues were thawed, grinded and submitted to total RNA extraction according to the manual instructions of the used RNA extraction kit (Qiagen, Germany).The RNA was separately extracted from all the rats groups whether the controls or the treated ones.
The successful extraction of RNA was ensured through agarose gel electrophoresis.Ten micro liters of the extracted RNA was injected into the wells of 1% agarose gel, submitted to electrical migration for 30 min at 120V and finally photographed using gel documentation system (Bio Rad, GelDoc, 2000).

Reverse transcriptase PCR (cDNA Synthesis)
At first, five micro liters of total RNA was treated with DNase1 enzyme to emphasize complete absence of the genomic DNA.The RNA was eluted with 100 µl of an elution buffer followed by incubation with 70 units of DNase1 at 37 o C for 1 h.The enzyme was then inactivated at 75 o C for 10 min and the RNA was precipitated using 100 µl of isopropanol followed by centrifugation at maximum speed (13.000 rpm) for 30 min at 4 o C. Two 70% ethanol washes were followed, using the same centrifuge settings.The ethanol was removed and the RNA pellets were allowed to air dry for 15 min, at which point 32 µL RNase-Free water were added.The mRNA was then transcribed to cDNA using a TaqMan Reverse Transcriptase kit (Applied Biosystems) at the presence of oligo(dT) 16-mers primers according to the manufacturer's instructions.The mixture was incubated at 37 o C for 1 h followed by enzyme inactivation step at 75 o C for 10 min.
The up and down expressed genes were excised from the gel and submitted to PCR-gel purification kit (Qiagene) and the purified bands were submitted to sequencing (Macrogen Inc., Korea).

RESULTS AND DISCUSSION Biochemical analysis:
The serum biochemical parameters are considered the internal mirror that reflects the actual effect of both of the CCl4 toxin and marjoram as its counter.The measured biochemical parameters were as follows: ALT, AST, TP, creatinine, uric acid, urea, total cholesterol, HDL-C, TG and GPx.The results indicated that the injection of rats with CCl4 was able to induce hepatotoxicity as shown in Figs.1-7.
The activity levels of ALT and AST liver enzymes, in addition to the levels of total protein, creatinine, urea, total cholesterol, TG were significantly increased in CCl4 group when compared with the control group (p< 0.05).On the other hand, the levels of uric acid and HDL-C were non significant (p> 0.05) in CCl4 group when compared with the control group.
These results are in agreement with Leelaprakash, et al. (2011), who reported statistically significant increase in plasma activities of AST and ALT.They reported general statistically significant losses in activity of GPx which agree with our results that indicate a significant decrease in the activity levels of GPx in the CCl4 group in comparison with that of control group.It was observed in our study that the feeding of rats with marjoram was able to reduce and sometimes completely remove the toxic effect of CCl4.Some parameters including ALT, AST, creatinine, urea, total cholesterol and TG were significantly decreased (p< 0.05) in the group injected and fed with CCl4 and marjoram compared with CCl4 group.In contrast, total protein, uric acid and HDL-C showed no significance (p> 0.05) under the same conditions.Leelaprakash, et al. (2011), had also reported counteract of all the damage caused by CCl4 using Vernonia cinerea.The slight difference between our results and Leelaprakash results may back to the type of the used medicinal plant or the sex of animal model.
Protective effect of oregano water extract against Carbon tetrachloride Hepatotoxicity 71       The increased activities of liver marker enzymes such as AST and ALT in the serum of CCl4 induced animals indicate damage to hepatic cells (Wolf, 1999).CCl4-mediated acute toxicity increased permeability of the hepatocyte membrane and cellular leakage (Paduraru et al., 1996;Huang et al., 2011).The obtained results indicate the ability of marjoram to help liver cells to overcome the bad effect caused by CCl4 toxicity.This may achieved via stabilization of plasma membrane as well as repair of hepatic tissue damages caused by CCl4 (Ravikumar and Gnanadesigan, 2011).It had been reported that CCl4 is metabolized by cytochrome P450 system and converted to trichloromethyl and trichloromethyl peroxy radicals (Zhou et al., 2010).
These radicals initiates peroxidation of polyunsaturated fatty acid constituents of various membranes with secondary damage and severe enzymatic disturbances (Stehbens, 2003).The body has a set of endogenous antioxidant enzymes which are able to detoxify free radicals by converting them back to more stable molecules within the cell, to be used or disposed accordingly (Saeed et al., 2005).Marjoram is a medicinal plant that has antioxidant activity (Yasin and Abou-Taleb, 2007;Badee et al., 2013).
We attribute the ability of marjoram to help the liver to restore its activity to this antioxidant activity of marjoram.

Molecular part:
Differential display is considered one of the most recently valuable molecular techniques.It is able to provide researchers with general overview of the down and up expressed genes under specific treatments without previous knowledge about the extent of their expression or identity.In our study; we used differential display to compare between the genetic status of liver tissues in normal and treated conditions.The treated condition include exposure to specific dose of CCl4 alone, exposure to specific dose of CCl4 plus marjoram and finally exposure to specific dose of marjoram alone.After two weeks of treatments, total RNA was extracted from all the mentioned tissues and mRNA was submitted to reverse transcription to cDNA using oligo dT primers.At the end, the obtained cDNA was randomly amplified through traditional PCR using OP1 and OP2 arbitrary primers.
As shown in figure 8, many genes with different sizes were amplified.Most of these genes were normally amplified in control samples (C) while same genes were down regulated in samples treated with toxin alone (T).On the other hand, the oral feeding of rats with marjoram was able to return the re-expression of these genes again wheather toxin is injected (T+M) or not (M).One of the down regulated genes in only toxin group (d) was excised from the gel and submitted to sequencing.The sequence result of this gene cleared that the gene is Mus musculus TBC1 domain family, member 30 (Tbc1d30), with somewhat similarity of (96%).As shown in Fig. 9, Tbc1d30gene is highly expressed in some but not all the body tissues.Liver tissue is considered one of them (http://www.genecards.org/cgi-bin/ carddisp.pl? gene = TBC1D30).This Tbc1d30 gene has the function of Rab GTPase activator activity.Rab GTPases are master regulators of membrane trafficking (Stenmark, 2009).They exist in an active, GTP-bound state and an inactive, GDP-bound state (Ishibashi et al., 2009).When active, they bind a number of specific effector proteins that mediate their diverse roles in transport vesicle formation, motility, docking, and fusion (Goueli et al., 2012).

CONCLUSION
The use of natural products extracted from the medicinal plants found in Egypt is considered an alternative solution for the treatment of many diseases especially the liver diseases instead of the use of synthetic drugs.However, this needs further studies to know the active ingredients of these natural products like those found in marjoram which affect the physiological and genetic status of the body.

Fig. 1 :
Fig. 1: Serum activity levels of AST and ALT liver enzymes in the different experimental rat groups.

Fig. 2 :
Fig. 2: Serum levels of Total Protein (TP) in the different experimental rat groups.

Fig. 3 :
Fig. 3: Serum levels of Creatinine and uric acid in the different experimental rat groups.

Fig. 4 :
Fig. 4: Serum levels of urea in the different experimental rat groups.

Fig. 5 :
Fig. 5: Serum levels of total cholesterol and HDL-cholesterol in the different experimental rat groups.

Fig. 6 :
Fig. 6: Serum levels of triglycerides in the different experimental rat groups.

Fig. 7 :
Fig. 7: Serum activity levels of glutathione peroxidase enzyme (GPx) in the different experimental rat groups.

Fig. 8 :
Fig. 8: DD-PCR of rat groups submitted to CCl4 toxicity and treated with marjoram as medicinal plant.C: control group, T: CCl4 injected group, M: marjoram feeding group, T+M: CCl4 injected and marjoram feeding group, L: 1 kb DNA ladder, d: the down regulated gene.