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Lipid Lowering Effects of Hydroalcoholic Extract of Anethum graveolens L. and Dill Tablet in High Cholesterol Fed Hamsters
Cholesterol, Volume 2015, pp 1-7; doi:10.1155/2015/958560
Abstract: Objective. This study was aimed to determine the effect of Anethum graveolens extract and Anethum graveolens (dill) tablet on lipid profile, liver enzymes, and gene expression and enzymatic activity of HMG-CoA reductase in high cholesterol fed hamsters. Materials and Methods. Golden Syrian male hamsters (130 ± 10 g) were randomly divided into 6 groups () and received daily the following: group 1 received chow + 2% cholesterol + 0.5% cholic acid (HCD), groups 2 and 3 received HCD diet plus 100 and 200 mg/kg hydroalcoholic extract of dill, respectively, and groups 4 and 5 received HCD diet plus 100 and 200 mg/kg dill tablet, respectively. Group 6 received only chow. After 1 month feeding serum biochemical factors were determined. HMG-CoA reductase mRNA level was measured (real-time PCR) and its activity was determined spectrophotometrically. Results. Compared with hypercholesterolemic group 1, lipid profile, blood glucose, and liver enzymes significantly decreased in all dill tablet or dill extract treated groups (). The changes in HMG-CoA reductase gene expression level and enzyme activity significantly reduced in animals that received 200 mg/kg of extract or tablet. Conclusion. Dill extract and dill tablet showed potential hypocholesterolemic properties in hamsters by inhibition of HMG-CoA reductase activity.1. IntroductionHypercholesterolemia is a common disorder which is known as main cause of coronary heart disease (CHD) . This disease is recognized as cause of the most of the deaths in developed countries. High levels of total cholesterol (TC), triglycerides (TG), and low density lipoprotein-cholesterol (LDL-C) have been implicated as contributing risk factors in progress of CHD and atherosclerosis . Accordingly, different lipid lowering drugs have been applied for the treatment of hypercholesterolemia . Statins are the effective hypocholesterolemic drugs which competitively inhibit the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis . Since chemical drugs, for example, statins, have adverse effects such as rhabdomyolysis, myopathy, myalgia, and myotoxicity , applicability of herbal medicine for treatment of many diseases has attracted much more attention. Some herbal medicines have been known to exhibit hypocholesterolemic properties in clinical studies . One of the useful and well-known plants is Anethum graveolens L., commonly known as a dill, an annual plant growing in Europe, Mediterranean region, and Asia . In traditional medicine, this plant has been used for the treatment of gastrointestinal disorder including indigestion, flatulence, and stomacha colic and also for its antibacterial, antifungal, antispasmodic, antisecretory, mucosal protective, and hypoglycemic effects. Dill tablet (DT) which is administrated as lipid lowering agent contains Citrus aurantifolia sp. (4%), Cichorium intybus (5%), Fumaria parviflora (5%), and Anethum graveolens (68%) . Previous studies have reported the presence of phenolic and flavonoids, flavonol, alkaloids, anthocyanin, tannin, and saponin contents in dill . Studies have established that phenolic and flavonoids have potential antioxidant and hypocholesterolemic effects . The hypocholesterolemic activity of dill has been shown in different studies, but the lipid lowering mechanism has remained unknown so far. Therefore, the aim of this study was to investigate the hypocholesterolemic effects of the dill in high cholesterol fed hamsters and to determine gene expression level and enzymatic activity of HMG-CoA reductase in liver.2. Materials and Methods2.1. Preparation of Dill ExtractDill was purchased from Hamadan (west of Iran) daily market and identified by our colleagues in the Department of Biology, Borujerd Azad University (Borujerd, Iran). Hydroalcoholic extract was prepared according to the previously described method . Dill tablet (DT) was purchased from Iran Darouk Company (Tehran, Iran).2.2. Determination of Total Phenolic ContentThe amount of total phenolic content of dill extract and/or dill tablet was determined according to previously reported method with small modification using Folin-Ciocalteu reaction . Briefly, 1 mg of dill was dissolved in 3.8 mL of deionized water, 2 mL of Na2CO3 (2%), and 0.1 mL of Folin-Ciocalteu reagent (50%). The samples were then incubated for 30 min at room temperature and the absorbance of the reaction mixture was measured at 750 nm against deionized water. Total phenolic content was calculated per mg equivalents of gallic acid (GAE) per gram of each extract.2.3. Determination of Total FlavonoidsTotal flavonoids were measured using aluminum chloride colorimetric assay, according to the previously described method . Briefly 0.5 mL of the sample (1.0 mg/mL in methanol) was mixed with 1.5 mL of alcohol (95%), 0.1 mL of AlCl3 (10%), 0.1 mL of potassium acetate (1 M), and 2.8 mL of deionized water. After that, the mixture was incubated at room temperature for 40 min and absorbance of the mixture was measured at 415 nm against deionized water. The content of flavonoids was calculated per mg equivalents of quercetin per gram of each extract.2.4. Determination of Total FlavonolsThe flavonols content was measured according to the previously published method . The dill extract (1 mg/mL) was added to 2 mL of AlCl3 (20 mg/mL) and 6 mL sodium acetate solution (50 mg/mL). The mixture was then incubated for 150 min at room temperature and the absorbance was determined at 440 nm. The content of total flavonols was calculated per mg equivalents of quercetin per gram of each extract.2.5. Experimental DesignA total of 36 male golden Syrian hamsters weighing g were used in this experiment. Animals were kept for one week before experimentation and adaptation. Regular conditions with light/dark cycle (12 hour for each), relative humidity of %, and temperature at °C were applied during the experiments. After adaptation, hamsters were randomly divided into six experimental groups () and fed as follows: group 1: chow + 2% cholesterol + 0.5% cholic acid; group 2: chow + 100 mg/kg hydroalcoholic extract of dill + 2% cholesterol + 0.5% cholic acid; group 3: chow + 200 mg/kg hydroalcoholic extract of dill + 2% cholesterol + 0.5% cholic acid; group 4: chow + 100 mg/kg dill tablet + 2% cholesterol + 0.5% cholic acid; group 5: chow + 200 mg/kg dill tablet + 2% cholesterol + 0.5% cholic acid; and group 6: chow. At the end of 30 days’ experiment, animals were anesthetized and sacrificed after 12 h of fasting. Blood samples were collected from the heart and serum was separated by centrifugation of samples at 3000 rpm for 10 min.2.6. Determination of Serum FactorsLiver enzymes, total cholesterol, fasting blood sugar, triglyceride, and HDL-C were measured by colorimetric methods using the commercial available kits (Pars Azmun, Tehran, Iran), whereas LDL-C and VLDL-C were calculated by Friedwald’s formula [8, 9].2.7. RNA Extraction and cDNA SynthesisTotal RNA was isolated from liver tissue samples (100 mg) using TRIZOL reagent according to the manufacturer’s protocol (Fermentas Life Sciences, Vilnius, Lithuania). The integrity of extracted RNA was determined by 1% agarose gel while its concentration and purity were assessed by Nano-Drop spectrophotometer (Bio-TeK, USA). After that, 1 microgram of RNA was applied for reverse transcription process using QuantiTect Reverse Transcription Kit (Fermentas Life Sciences, Vilnius, Lithuania). Integrity, purity, and concentration of cDNA were also measured as described for RNA .2.8. Real-Time qRT-PCR AssayDetermination of HMG-CoA reductase mRNA level was carried out by real-time PCR using QuantiTect SYBR Green PCR Kit (Qiagen Ltd., Hilden, Germany) in a PCR detection machine (Bio Rad Ltd., USA).Primers of reaction were designed by AlleleID7 software (Premier Biosoft Corporation, USA) for HMG-CoA (forward: 5′-AAGGAGCGTGCAAAGACAATC-3′ and reverse: 5′-TGAACCATGTGACTTCTAACAAG-3′) and GAPDH (forward: 5′-TGGCCTTCCTTCCTACG-3′ and reverse: 5′-TAGCCCAGGATGCCCTTCAG-3′) as internal control. The RT-PCR reaction mixture was prepared by the addition of 1 μL cDNA template (<100 ng), 10 μL SYBR Premix (1X), and 1 μL of each primer (0.4 μM) and adjusting of volume to 20 μL by adding deionized water. HMG-CoA reductase mRNA level was measured in triplicate in 38 cycles using thermal cycler.2.9. HMG-CoA Reductase ActivityLiver tissue samples were homogenized in 500 μL lysis buffer containing 1% protein inhibitor (Sigma-Aldrich Co., St. Louis, MO, USA) and centrifuged for 15 minutes at 14000 rpm and 4°C. Supernatant was used for the measurement of enzyme activity using HMG-CoA reductase assay kit (Sigma-Aldrich Co., St. Louis, MO, USA) based on spectrophotometrical determination of oxidized NADPH at 340 nm.2.10. Statistical AnalysisData are expressed as means ± SEM of three replicate measurements and then analyzed by SPSS package (version 16, SPSS, Inc.). One-way analysis of variance (ANOVA) followed by Dunnett’s test were used. The values less than 0.05 were regarded statistically significant.3. ResultsTotal phenol, flavonoid, and flavonol contents of dill tablet and dill extract are shown in Table 1.Table 1: Total phenols, flavonoids, and flavonols content of dill tablet and dill extract.Figure 1 shows mean of FBS (fasting blood sugar), total cholesterol, triglyceride, LDL-C, HDL-C, and VLDL-C levels in the experimental groups. Serum levels of total cholesterol, triglyceride, LDL-C, and VLDL-C significantly reduced () in dill tablet and dill extract treated hamsters. These reductions were higher in hamsters treated with 200 mg/kg of dill extract or dill tablet. Significant increase was observed in HDL-C level by treatment with 100 and 200 mg/kg of dill extract or dill tablet. Treatment of hamsters with 100 mg/kg of dill extract or dill tablet did not affect body weight compared with untreated high cholesterol diet fed h
Keywords: antioxidant activity / cholesterol / Statins / anthocyanin / dill / RNA Extraction / flavonoids / coronary heart disease / lipoprotein