الفهرس 
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Abstract
N
Summary
onalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. The major characteristic of NAFLD is the metabolic syndrome dyslipidemia. It is generally related to a high calorie intake and sedentary life style.
While liver biopsy remains the gold standard for diagnosis, staging and prognosis for the NAFLD spectrum disorders, this invasive procedure is not recommended due to high risks and cost. Advanced diagnostic approach are demanded for novel management strategies for NAFLD.
Lipidomics, a novel technique, refers to the quantitative determination of all lipids in a given cellular system at a certain time point. Due to its sensitivity and specificity, mass spectrometry (MS) is the underlying technology of choice for simultaneous determination of large sets of lipids. Lipidomics of plasma and urine became a potential candidate for metabolic biomarkers in several metabolic diseases as NAFLD.
Various pharmacologic and nutritional agents have been suggested for the treatment of NAFLD but no successful consensus has been reached.
Herbs have recently attracted attention as a source material for drug development. Herbal medicines derived from plant extracts are being utilized increasingly to treat NAFLD such as Eclipta prostrata (E.prostrata) and Bauhinia retusa (B.retusa)
E.prostrata is an annual herbaceous plant belonging to Asteraceae family. The plant has a wide spread medicinal use. It contains a range of health-promoting phytochemicals including poly phenols, flavonoids and coumarins which have hypolipidemic effect.
B.retusa is a genus of trees or shrubs belonging to Liguminosea (Caesalpiniaceae) family. It contains a wide range of antioxidants, polyphenols, flavonoids and tanins. Various Bauhinia species act as hypocholesterolemic agents.
This study aimed to evaluate the protective and treated effects of methanolic extract of E. prostrata aerial parts (stem and leaves) and seeds of B.retusa on NAFLD induced by high fat diet in rats in comparison to the hypocholesteremic drug (lipanthyl® 300) using lipidomic approach and to determine metabolites involved for early diagnosis of NAFLD in serum and urine.
Toxicity assays of methanolic extract of Eclipta prostrata and Bauhinia retusa were determined by observing mortility rate in rats and using biochemical assays (liver, kidney function tests and lipid profile). No mortality was recorded in rats following oral administration of methanolic extracts of both Eclipta prostrata and Bauhinia retusa in doses starting from 20 mg/Kg BW and ending with 600 mg/Kg BW. However, biochemical assays of rats΄ sera improved that methanolic extract of Bauhinia retusa showed a significant toxicity by increasing liver enzymes and lipid profile in contrast to E.prostrata which showed safe administration. So, this study was continued using methanolic extract of E. prostrata only.
Animals in this study were orally administered the methanolic extract of E.prostrata plant in doses equal to 50, 100, 200, 300 mg/Kg BW to determine the most effective dose.
Phyto-chemical screening of methanolic extract of the aerial part (stem and leaves) of E.prostrata using HPLC showed that the major flavonoids were rutin and quercetin which represented 4.78 and 1.93 mg/g dry weight, respectively and the major phenolic compound was gallic acid which represented 2.12 mg/g dry weight of the total extract. Wedelolactone is the characteristic chemical of coumarin constituent of E. prostrata with average content 5.00 mg/g dry weight.
The present study included 84 adult male Wistar albino rats divided into 3 main groups as follows. Control groups (GI) included 24 rats divided equally into 2 subgroups: Normal healthy control (GIa): 12 normal rats received standard chow (SC) and tap water ad libitum, half of them were sacrificed after 8 weeks as a control group of protected groups and the other half was sacrificed after 12 weeks as a control group of treated groups. Control with induced fatty liver (GIb): 12 normal rats fed a high-fat diet containing SC supplemented with cholesterol and cholic acid, half of them were sacrificed after 8 weeks as a control group of protected groups and the another half was sacrificed after 12 weeks as a control group of treated groups.
Protected groups (GII) included 30 rats divided into two subgroups: Eclipta prostrata protected groups (GIIa): 24 normal rats fed high-fat diet containing SC supplemented with cholesterol and cholic acid followed by oral administration of methanolic extract of Eclipta prostrata at doses 50, 100, 200, 300 mg/kg body weight; through stomach tube for 8 weeks in the same time (6 rats for each dose).
Lipanthyl protected group (GIIb): 6 normal rats received a high-fat diet containing SC supplemented with cholesterol and cholic acid followed by the lipid-lowering agent Lipanthyl® drug (300mg) at a dose 5.35 mg/200g rat.
Treated groups (GIII) included 30 rats divided into 2 subgroups: Eclipta prostrata treated groups (GIIIa): 24 normal rats fed a high-fat diet containing SC supplemented with cholesterol and cholic acid for 8 weeks, followed by oral administration of methanolic extract of E. prostrata at doses of 50, 100, 200, 300 mg/kg body weight respectively for extra 4 weeks (6 rats for each dose).
Lipanthyl treated group (GIIIb): 6 normal rats fed a high-fat diet containing SC supplemented with cholesterol and cholic acid for 8 weeks, followed by oral administration of the lipid-lowering agent Lipanthyl® drug (300mg) at a dose 5.35 mg/200g rat for extra 4 weeks.
At the end of the experimental periods, urine samples were collected from all groups using experimental animal collectors over 24 hr. and stored at −80 C for lipidomic analysis. Blood samples were taken from retro-orbital vein of animals after fasting 14 hr. during light anesthesia and placed in sterilized tubes for serum separation by centrifugation at 5000 r.p.m for 15 min. The obtained serum samples were then stored at −80 C for biochemical assays and lipidomic analysis. Rats were decapitated and livers were removed for histopathological examination.
Biochemical analysis:
The following parameters are determined in serum
A. Liver function test:
• Alanine amino transferase activity
• Aspartate amino transferase activity.
B. Kidney function tests:
• Levels of urea and creatinine
C. Lipid profile:
• Levels of total cholesterol, triacylglycerols, HDL-cholesterol, LDL-cholesterol.
• HDL-C/LDL-C ratio.
Lipidomic analysis:
The lipidomic analysis performed in serum and urine included three steps:
Lipid extraction: by using methyl tert- butyl ether (MTBE).
Lipid derivatization: for transformation of free fatty acids into methyl or silyl esters in order to enhance their volatility by using N-methyl N- (trimethylsilyl) trifluoroacetamide (MSTFA) with trimethylchlorosilane (TMCS).
Lipid detection: Gas chromatography / Mass spectrometer (GC/MS) will be used for detection of different lipid metabolites.
The results obtained can be summarized as follows:
Biochemical analysis
• Administration of E. prostrata extract had no effect on the all studied parameters compared to normal control group indicating safe administration.
• Results showed a significant elevation in the levels of ALT, AST, TC, TAGs, LDL-C, Urea, Creatinine and also a significant depletion in the levels of HDL-C and HDL-C/LDL-C ratio in the serum of rats bearing NAFLD compared to healthy control. It is also noticed that liver enzymes, lipid profile and kidney function test were markedly ameliorated in protected and treated animals with high and moderate doses (200 & 300 mg/kg BW) of E. prostrata compared to NAFLD group.
Lipidomic analysis based on GC-MS
• Concerning protected group, lipidomic analysis of sera΄s results showed significant elevation in β-hydroxybutyric acid, phosphoric acid, urea, cholest-5-en-3-ol (3β) acetate, α-glycerophosphate, glucose, xylose, oleic acid, myristic acid, palmitoleic acid, galactose, stearic acid, glycerol and significant depletion in linoleic acid and arachidonic acid levels in NAFLD group compared to healthy control.
• Concerning treated group, serum cholest-5-en-3-ol (3β) - acetate, cholesta-4, 6-dien-3-ol, (3β), urea, 1, 3 dipalmitin, glycerol were significantly elevated while linoleic acid and arachidonic acid levels were depleted in NAFLD compared to healthy group.
• These results indicated that cholest-5-en-3-ol (3β) - acetate, glycerol, linoleic acid and arachidonic acid, were the most significant lipidomic metabolites in both protective and treated groups and can discriminate between healthy and NAFLD ones for early detection of disease in serum .
• Concerning protected group, lipidomic analysis of urine΄s results showed significant elevation in 1, 3 dipalmitin, cholesterol, myristic acid, oleic acid, glycerol in NAFLD group compared to control group. While lipidomic analysis results in treated groups showed significant increase in the levels of palmitic, oleic, stearic, myristic, and palmitoleic acids, in addition to glycerol, 1-monostearin and 1, 3-dipalmitin in the urine of rats bearing NAFLD compared to healthy control. These results indicated that oleic acid, myristic acid, glycerol and 1, 3-dipalmitin were the most significant lipidomic metabolites in both protective and treated groups and can discriminate between healthy and NAFLD ones for early detection of disease in urine.
• Administration of E. prostrata extract as protective and treating agents ameliorated the abnormalities in the levels of these metabolites. The results concluded that E. prostrata in high dose had the potential efficacy to alleviate NAFLD in both serum and urine.
• Histopathological examination showed damaging effect, lobular hepatic architecture, hepatocyte ballooning, severe micro and macrovesicular steatosis and moderate lobular inflammation of liver cells with nonalcoholic fatty liver score activity (NAS) equals 2.8 and 3.5 after administration of high fat diet in protective and treated groups respectively and a marked improvement was seen in protected and treated groups especially rats administered dose of 300 mg/kg BW.
• In conclusion, the current study represented the main altered lipid metabolites related to NAFLD in serum and urine samples using a lipidomic approach to act as key points to control this disease in the early stage. E.prostrata extract possessed prophylactic and therapeutic effects against experimentally induced liver injury in rats. However, the prophylactic role was more efficient than the therapeutic one.