الفهرس 
Liver AnatomyOnly 14 pages are availabe for public view
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Abstract
Carbon tetrachloride (CCl4) is regarded as the most environmental contaminant. CCl4 is one of the most extensively studied hepato and nephro- toxicants. The most frequent mechanism of hepatocellular and nephrocellular injury involves the production of toxic metabolites by the cytochrome P450 system.
Carbone tetrachloride (CCl4) is metabolized by the cytochrome P450 enzyme system resulting in the production of trichloromethyl (CCl3•) and trichloromethyl peroxyl (CCl3OO•) radicals. These free radicals have the ability to bind to proteins and/or lipids of the plasma membrane leading to lipid peroxidation and damage to the plasma membrane and DNA that is responsible for liver and kidney damage.
Currently, natural products play an important role in the prevention, limitation and treatment of liver and kidney diseases due to their ability to scavenge the free radicals. Ginger and silymarin are among the natural products that have hepatoprotective and nephroprotective effects against CCl4 toxicity due to their antioxidant and anti-inflammatory properties.
Ginger is the rhizome of the plant Zingiber officinale Roscoe, belongs to the family Zingiberaceae, it is used as a common condiment for various foods and beverages. The most abundant compounds in ginger are [6]-gingerol and [6]-shogaol. So ginger possesses hepatoprotective and nephroprotective effects. It exerts its protective actions through its antioxidant and free radical scavenging mechanisms.
Regardless of their attractive properties, ginger still has very limited application in supplements and clinical use due to their poor solubility in water and consequently inadequate oral bioavailability. To overcome these drawbacks, ginger was ground by ball milling technique into nano - size to produce ginger nanoparticles. Also, in an attempt to increase
the solubility of ginger, ginger was mixed with sodium bicarbonate and ground together to produce ginger nanobase in nanosize.
Silymarin (as herbal drug) is an antioxidant flavonoid complex derived from the herb milk thistle (Silybum marianum), has the ability to scavenge free radicals, chelate metal ions and inhibit lipid peroxidation. It has hepatoprotective and nephroprotective effects due to its antioxidant properties.
The aim of the present work is to evaluate the total phenols and flavonoids and identification of the bioactive components of ginger, also to investigate the effect of ginger (G), ginger nanoparticles (GNPs) and ginger nanobase (GNB) on hepatotoxicity and nephrotoxicity induced by carbon tetrachloride (CCl4) in rats in comparison with silymarin (SM).
Ginger powder was chemically analyzed for their total phenolic and flavonoid contents and the main phenolic constituents of ginger powder were determined using HPLC technique. Also, ginger nanoparticles (GNPs) was prepared by milling ginger powder at (350 round/sec) for 16 hours. While ginger nanobase (GNB) was prepared by milling ginger powder with sodium bicarbonate at (350 round/sec) for 15 hours. The particle size of GNPs and GNB was measured by using DLS technique. Ginger powder (G), GNPs, GNB and silymarin (SM) were separately suspended in distilled water immediately before administration to rats and administered to each rat according to its body weight.
Hepatotoxicity and nephrotoxicity were induced by intraperitoneal (i.p) injection with a single dose of carbon tetrachloride (1ml / kg B.W) which was diluted with corn oil (1:1) immediately before administration to rats at the end of the fourth week of the experiment.
Fifty-four adult male albino rats Sprague -Dawely strain weighing 150-180 g received the commercial diet and water ad libitum for 7 days (acclimatization period) and continued to have the same diet throughout the 8 weeks of the experiment (experimental period). All rats were divided into six groups each of nine rats as the following; group 1: a healthy untreated group which served as normal control and received water by gastric tube daily and injected i.p with corn oil (1ml / kg B.W). group 2: CCl4 treated group; rats received water by gastric tube daily and injected i.p with CCl4. group 3: CCl4 + G group; rats administered ginger powder (50 mg/ kg B.W/day) and injected i.p with CCl4. group 4: CCl4 + GNPs group; rats administered GNPs (50 mg / kg B.W/ day) and injected i.p with CCl4. group 5: CCl4 + GNB group; rats administered GNB (50 mg / kg B.W/ day) and injected i.p with CCl4. group 6: CCl4 + SM group; rats administered silymarin (100 mg /kg B.W/day) and injected i.p with CCl4.
During the experimental period, food intake was recorded and animals were weighed weekly to calculate body weight change and feed efficiency ratio. At the end of the experimental period (8 weeks) and after overnight fasting, all rats were weighed, sacrificed under ether anesthesia and blood samples were collected from the hepatic portal vein. Serum was obtained by centrifugation at 3000 rpm for 15 min. the liver and kidneys of rats were removed for biochemical analysis and microscopic examination, respectively.
The following biological and biochemical parameters were measured:
Body weight change, food intake and feed efficiency ratio (FER)
Relative liver and kidney weights
Liver function tests [serum alanine aminotransferase (ALT), aspartate aminotransferase enzyme (AST) and alkaline phosphatase (ALP) activities].
Kidney function tests (serum urea, creatinine, uric acid and cystatin C levels)
Inflammatory markers [serum tumor necrosis factor- alpha (TNF-α) and interleukin 1 beta (IL-1β levels)]
Oxidative markers [serum malondialdehyde (MDA) and nitric oxide (NO) levels]
Antioxidant status [serum total antioxidant capacity (TAC) level, hepatic superoxide dismutase (SOD) and catalase (CAT) activities]
Also, the microscopic examination of liver and kidney tissues were performed.
The results of the current study are summarized as follows:
1- Total phenols and flavonoids content of ginger
The result illustrated that each 100 g of the tested ginger extract contains 2171.2 mg as gallic acid equivalent (GAE) for total phenols and 343.75 mg as quercetin equivalent (QE) for total flavonoids.
2- The Main Phenolic Compounds of Ginger
Our results showed that [6]-gingerol represents the highest percentage of ginger phenolic components when compared to all other compounds, while [8]-shogaol and [10]-shogaol were found in the lowest percentage.
3- Characterization of GNPs and GNB
Mechanical ball milling of ginger for 16 hours produces GNPs with size 38.29 nm while milling of ginger with sodium bicarbonate for 15 hours produces GNB with size 22.05 nm.
4- Body weight change, food intake, FER, relative liver and kidney weights
Administration of CCl4 to rats caused a significant decrease in body weight, food intake, FER, relative kidney weight while relative liver weight significantly increases. CCl4 intoxicated rats administrated with G, GNPs, GNB and SM showed a marked elevation in body weight, food intake, FER and relative kidney weight with a significant decrease in relative liver weight.
5- Liver functions
Intraperitoneal injection with CCl4 led to a significant elevation in serum ALT, AST and ALP activities which indicate liver toxicity. Treatment with CCl4 along with G, GNPs, GNB and SM caused an amelioration of liver functions parameters, The most significant enhancement was seen in (CCl4 + GNB) group in serum ALT and ALP enzymes activities followed by (CCl4 + SM) group, while the CCl4 group treated with SM showed the most significant enhancement on serum AST enzyme activity followed by (CCl4 + GNPs) group.
6- Kidney functions
Administration of CCl4 to rats significantly increased serum urea, creatinine, uric acid and cystatin C levels as indicative of nephrotoxicity. CCl4 treated groups and groups administered with G, GNPs, GNB and SM exhibited a significant improvement in kidney function parameters. The best improvement in serum creatinine and urea were found in the SM group followed by the GNPs group, while serum uric acid and cystatin C showed the best improvement in CCl4 intoxicated groups treated with GNPs followed by GNB. The treated groups shown a nephroprotective effect due to it is antioxidant activity.
7- Inflammatory biomarkers
CCl4 toxicity induces a massive increase in the levels of serum TNF-α and IL-1β. Administration of CCl4 and G, GNPs, GNB and SM attenuated the inflammatory effect of CCl4. Treatment with GNB caused the most significant improvement in serum TNF-α level, while treatment with SM has a remarkable enhancement in serum IL-1β followed by GNPs. These improvements are due to the anti-inflammatory effect of ginger and silymarin.
8- Oxidative biomarkers and antioxidant status in serum and liver
Injection with CCl4 remarkably elevated serum MDA and NO levels and significantly declined serum TAC level, hepatic CAT and SOD activities as a result of oxidative stress caused by CCl4. Treatment of CCl4 intoxicated rats with G, GNPs, GNB and SM decreased the oxidative stress induced by CCl4 and caused a significant enhancement in the serum TAC level, hepatic CAT and SOD activities, the best improvement in MDA level was seen in the GNB group, while administration of GNPs caused the best enhancement of NO level and CAT activity as well as hepatic SOD activity and serum TAC level showed the best enhancement in the group treated with SM followed by GNB group.
9- Microscopic examination of liver and kidneys tissues
Microscopic examination for liver tissues from the CCl4 group showed focal hepatocellular necrosis and apoptosis associated with mononuclear inflammatory cells infiltration. While, the administration of CCl4 and G showed liver section with congestion of central vein and slight vacuolization of some hepatocytes. Meanwhile, administration of CCl4 with GNPs and GNB showed slight cytoplasmic vacuolization of centrilobular hepatocytes. Moreover, rats treated with CCl4 and SM revealed congestion of central vein and hepatic sinusoids.
Microscopic examination for kidney tissues from the CCl4 group showed vacuolization of epithelial lining renal tubules and endothelial lining glomerular tuft as well as periglomerular inflammatory cells infiltration. While, kidney sections from groups treated with CCl4 and G, GNPs and GNB showed slight vacuolization of epithelial lining renal tubules and endothelial lining glomerular tuft. Whereas, the group treated with CCl4 and SM showed vacuolization of epithelial lining renal tubules and endothelial lining glomerular tuft.