Materials
Arctium lappa L. Root was purchased from Anqiu vegetable farm center (Weifang, Shandong, China) and identified by Dr. Jingying Sun (Shandong Academy of Medicine Pharmacy Institute). Arctium lappa L. is a well established traditional Chinese medicine herb, since the material used in this study met the quality standards set by the Shandong Food and Drug Administration (SDFDA), no further specimen deposit was performed. Simvastatin was purchased from Hainan Pharmaceutical Factory Co., Ltd. (Hainan, China) (20 mg/Tablet, Batch number: 130406). 1,4-dioxane was purchased from Tianjin Ruijin Chemicals Co. Ltd. (Tianjin, China). Isopropyl alcohol (ISO) was obtained from Tianjin Fuyu Fine Chemical Co., LTD. (Tianjin, China). Nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione peroxidase (GSH-Px) assay kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China).
Preparation of AREs
Preparation of the ethanol extract (AEE)
The dried roots of A. Lappa (0.6 kg) were soaked in 4 L of 80 % ethanol for 24 h, and then reflux extracted twice with 80 and 60 % ethanol for 5 h respectively. The resulting residue was dried and used for next extraction. The organic solutions were combined and concentrated under reduced pressure to give 301.8 g of the ethanol extract. 231.6 g starch was added to facilitate the formation of powder, resulting in a total of 533.4 g AEE.
Preparation of the aqueous extract (AAE)
The dried pretreated sample was dipped in distilled water (9 L) and boiled twice for 2 h. The combined filtrate was then concentrated under reduced pressure and evaporated to dryness and the yield of the aqueous extract is 59.1 g. 21.5 g starch was added, resulting in a total of 80.6 g AAE.
Preparation of the chloroform fraction (ACE)
The dried roots of A. Lappa (10 kg) were reflux extracted twice with methanol for 5 h. The methanol solution was contration under reduced pressure to obtain a residue. The extract was suspended in water (6 L) and extracted with chloroform (6 L × 3 times). The resulting fraction was collected and then concentrated in vacuo to afford 48.3 g the chloroform fraction. 68.6 g starch was added to form powder. 116.9 g ACE was the final total yield.
Preparation of the flavones extract (AFE)
The remaining solution from chloroform extract was heated to evaporate the organic solvent. Cooled solution was added into column loading-treated Macroporous resin DM-130 for absorption, and then washed with water and 10 % ethanol, respectively, to get rid of impurities. The total flavonoids in column was eluted with 80 % ethanol and dried in a vacuum condition until powder was formed (157.7 g). 17.1 g starch was added to form a total of 174.8 g AFE.
Identification of the AREs
Extracted AREs were analyzed in a previous study carried out by our group. Refer to this work for information about detailed compositions and identified specific chemicals in different AREs [20].
Animal treatment and sample collection
Male quails (3 weeks old, body weight about 100 g) were purchased from Lanke Poultry Breeding Center (Jimo, Qingdao, China). All experimental protocols were approved by the Institutional Animal Use and Care Committee of Qingdao University (Qingdao, China). Quails were kept on a 12 h day/night lighting schedule and had access to standard quail chow and water ad libitum. After one week environment adaption, the quails were randomly divided into treatment groups. Due to limited handling capacity, two separated batches were used. First batch included: control group, model group, positive control group and AAE groups (AAE 0.75, 1.5 or 3 g/kg/day); second batch included: control group, model group, positive control group, AEE groups (AEE 1 or 2 g/kg/day), ACE groups (ACE 100 or 200 mg/kg/day) and AFE groups (150 or 300 mg/kg/day). Quails in the control group were fed standard chow (0 % cholesterol). Quails in all other groups were fed with high fat diet (1 % cholesterol and 14 % pork oil, w/w), along with specified treatments via gavage. After 4.5 weeks treatment, 2 mL blood were collected from quail right jugular vein. After 10 weeks treatment, terminal body weights were recorded, then venous blood sample (3 mL) was taken from the right jugular vein of each quail. Collected blood samples were incubated at 37 °C for 10 min and then centrifuged at 3000 rpm for 10 min, resulting serum samples were collected and archived at -80 °C until further analysis. The animals were then sacrificed and aorta were dissected for further analysis.
Serum lipid profile assessment
Serum collected at the beginning of the experiment, after 4.5 weeks treatment and after ten weeks treatment were subjected to automatic biochemistry analyzer Beckman AU5400 (Brea, CA, US) for serum lipid profile assessment. The levels of serum total cholesterol (TC), low density lipoprotein (LDL), and high density lipoprotein (HDL) were assessed.
Anti-oxidant and pro-oxidative status assessment in serum
All the biochemical parameters (NO, MDA, SOD, GSH, NADPH and GSH-Px) were measured with commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) following manufacturer’s protocols. All the kits were based on colorimetric methods, and were carried out on a spectrophotometer 722E (Shanghai Spectrum Instruments, Shanhai, China).
Histology of quail aorta
Dissected aortas (aorta arch) were fixed in 4 % formaldehyde for 24 h, embedded in paraffin, and then sectioned at 6 μm on a rotary microtome (Leica RM2016, Wetzlar, Germany). Hematoxylin and eosin (Beyotime, Jiangsu, China) staining was performed following manufacturer’s protocol. Pictures were taken with a microscope (Olympus BX51, Tokyo, Japan) and analyzed with ImageJ (NIH, US). The ratio of atherosclerotic area to total aorta area was calculated as an assessment of atherosclerosis.
Transmission electronic microscopy on quail aorta
Dissected aortas were fixed in 5 % glutaraldehyde for 24 h, dehydrated with graded ethanol, embedded with epoxy resin 618, sectioned and observed under a transmission electronic microscope JEM-1200EX (JEOL, Tokyo, Japan).
Aorta lipid profile assessment
Aortas were homogenized in 1 % 1,4-dioxane. Samples were then incubated in a 37 oC incubator shaker for 72 h. For each sample, 1 mL of supernatant was collected and dried, and then dissolved in 50 μL of isopropanol. Lipid profile was then assessed by subjecting dissolved samples to automatic biochemical analyzer Beckman AU5400 (Brea, CA, US).
Statistical analysis
Drawing lots method was used to ensure that quails were randomly assigned into each treatment groups. SPSS 17.0 was used to perform statistical analysis. All data were expressed as mean ± standard derivation. Normal distribution was confirmed with Levene’s test, then one-way analysis of variance (ANOVA) was performed. When P-values were less than 0.05, the statistical significance was determined and post-hoc least significant difference (LSD) tests were performed for the differences among groups.