الفهرس 
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Abstract
Liver diseases are a worldwide health problem with acute liver injury associated with high mortality rates. The acute liver injury is known to involve a complex role of oxidative stress, apoptosis, inflammation and necrosis.
Carbon tetrachloride (CCL4) is a strong hepatotoxic agent causing hepatic necrosis, and is widely used in animal models for induction of acute and chronic liver injury. The hepatotoxic effect induced by CCL4 could be reduced by treatment with dietary phytochemicals and antioxidants such as silymarin and marjoram.
Origanum majorana (Marjoram) is an important source of flavonoids and antioxidants. In addition to its nutritional value, marjoram is very important for its medicinal value and it is employed for the treatment of different ailments in medicine.
Silybum marianum (Milk thistle) is a well-known herb for treatment of hepatic diseases. Also it is used for treatment for other diseases which may be due to its antioxidant, anti-inflammatory and anticancer effect.
The present study was designed to investigate the hepatoprotective effects of Origanum majorana in dose (250 mg/ kg bwt.) and Silybum marianum (100 mg/ kg bwt.) against CCl4 induced liver injury in rats.
To achieve the objectives, the present work included the following main points:
Preparation of Origanum majorana and Silybum marianum ethanolic extracts:
Two hundred grams of Silybum marianum powdered seeds were refluxed in a Soxhlet apparatus with petroleum ether for 24 h. the defatted seeds were extracted with ethanol. Two hundred grams of the Origanum majorana powdered leaves were extracted with ethanol. The ethanolic extracts of Origanum majorana and Silybum marianum were filtered using filter paper, followed by percolation till complete exhaustion and then concentrated in vacuum at 40 oC using a rotary evaporator yielding 32.5g of Origanum majorana and 39.2 g of Silybum marianum extract. The extracts were kept in dark vials at - 4 oC until used. The extracts were freshly suspended in sterile distilled water and given to the rats orally.
The biological trial: Sixty four male wistar rats weighing 180-200 g were received the commercial diet and water ad-libitum for 7 days (acclimatization period). Rats were divided into 8 groups, eight rats in each group. group 1 (normal control): Rats were received distilled water daily by oral for 8 weeks and administered olive oil single dose (1ml/kg b.wt. i.p.) at the end of the fourth week of the experiment. group 2: Rats were received distilled water daily by oral for 8 weeks and single dose of CCl4 (1ml/kg b.wt. i.p.) at the end of the fourth week of the experiment. group 3, 4 and 5: Rats were received marjoram extract (250mg/kg b.wt), silymarin extract (100mg/kg b.wt) and mixture of them, respectively daily by oral for 8 weeks and were received olive oil single dose (1ml/kg b.wt. i.p.) at the end of the fourth week of the experiment. group 6, 7 and 8: Rats were received marjoram extract (250mg/kg b.wt.), Silymarin extract (100mg/kg b.wt) and mixture of them, respectively daily by oral for 8 weeks and were received single dose of CCl4 (1ml / kg b.wt. i.p.) at the end of the fourth week of the experiment.
At the end of the experimental period (8 weeks) and after overnight fasting, all rats were weighed and sacrificed under ether anesthesia and blood samples were collected from hepatic portal vein and serum was separated. Liver was separated immediately, washed and weighed to record the absolute liver weight and calculate the relative liver weight. Part of liver was stored for biochemical analysis and the other part of liver was preserved in 10% formalin for microscopical examination.
A- Evaluation of Biochemical Parameters:
1- Serum transaminases (ALT, AST) enzymes activity
2- Alkaline phosphatase (ALP) enzyme activity
3- Gamma Glutamyl transferase (γGT) enzyme activity
4- Serum total antioxidant capacity (TAC) levels
5- Liver antioxidant enzymes activity (CAT and SOD)
6- Liver inflammatory biomarkers (TNF-α and CYP)
B- Microscopic examination of liver tissues.
The results of this study showed that:
1-Final Body Weight, Absolute and Relative Liver Weight:
Final body weight was markedly decreased in CCl4 intoxicated group compared to normal control group and it significantly increased in intoxicated groups which treated with marjoram and/ or silymarin as compared to intoxicated group. Also, there was a significant increase in absolute and relative liver weights of (G2) CCl4 intoxicated group compared to normal control. On the other hand, there is no significant change in absolute and relative liver weight of other groups if compared to normal group.
2-Serum ALT, AST, ALP and γGT Enzymes Activity:
There was a significant increase in serum ALT, AST, ALP and γGT enzymes activity in CCl4 intoxicated group (G2) comparing to normal control group (G1). On the other hand, our results showed a significant decrease in the enzymes activity of CCl4 intoxicated groups which treated with marjoram (G6), silymarin (G7) or combination of them (G8) as compared to +ve control group. This indicated that treatment with marjoram and silymarin caused an improvement of toxicity in CCl4 intoxicated groups and these results were confirmed by the histological examination. The best result was for the group treated with combination of marjoram and silymarin when compared to toxicity group which prove the synergic effect of marjoram and silymarin in treatment of liver injury. Also, the results of healthy groups treated with marjoram and/or silymarin showed a non-significant change of ALT and ALP enzymes activity. While, it showed a significant decrease of AST and γGT enzymes activity as compared to normal control group. This indicated that treatment with marjoram and silymarin caused a protection against liver injury in healthy groups.
3-Serum TAC Level, Liver CAT and SOD Activity:
Serum TAC, liver CAT and SOD activity showed a significant decrease in toxicity group if compared to normal group. On the other hand, the CCl4 intoxicated groups (G6, G7 and G8) which treated with marjoram, silymarin or combination of them, respectively showed a significant increase in serum TAC level, liver CAT and SOD activity as compared to intoxicated group. The statistical analysis clarified that administration of marjoram, silymarin or combination of them, caused a significant increase of the serum total antioxidant capacity and liver CAT and SOD enzymes activity in groups (G3, G4 and G5) as compared to normal group. This elevation clarifies the antioxidant content of marjoram and silymarin.
4-Liver TNF-α Level and CYP Activity:
The results clarified the significant increase of liver TNF-α levels and CYP activity in intoxicated group if compared to normal group. This elevation clarifies the toxic effect of CCl4 on liver tissue. In contrast, liver TNF-α and CYP were significantly decreased in intoxicated groups which treated with marjoram (250mg/kg b.wt.), silymarin (100mg/kg b.wt) or combination of them, respectively if compared to non-treated intoxicated group which clarify the strong effect of each of them or combination in protection of liver tissues. On the other side, the results of liver TNF-α levels and CYP activity showed the non-significant change between the groups that administrated marjoram or silymarin as compared to normal group which proves the non-toxic effect of the administrated doses.
5-Microscopic Examination of Liver:
Microscopic examination of liver showed that CCl4 dose affect the hepatic tissues in the form of inflammation around the portal tract, cytoplasmic vaculation and inflammation as well as areas of apoptosis. On the other hand, the ethanolic extracts of Origanum majorana leaves and/or Silybum marianum seeds improved the architecture of hepatic tissues. Liver sections of CCl4 intoxicated rats which administrated marjoram and silymarin showed preserved hepatocytes arrangement with minimal affection in the form of venous congestion and scattered inflammation in cells. It could be concluded that using marjoram and silymarin extracts were more effective than using each of them alone in preserving hepatocytes and decreasing the inflammation which may be due to marjoram and silymarin have antioxidants and anti-inflammatory properties.