Pineapple (Ananas comosus L.) cv. Pattavia which is grown in Thailand was used in this study. The pineapples were weighed, sliced, and dried at 60 °C for 96 h using a hot air oven. The dried pineapple slices were weighed and ground into fine powder, then stored at -20 °C until further study.
Evaluation of active compounds, antioxidant capacity and approximate analysis
Total phenolic content was determined by using Folin–Ciocalteu’s colorimetric method . 10 mg of dried pineapple was incubated in 1 mL ethanol for 48 h and then sonicated for 20 min. The samples were filtered, then 20 µL of the solution was transferred into a 96-well microplate and mixed with 100 µL of Folin–Ciocalteu’s working reagent. After 5 min 80 µL of 7.5% (w/v) Na2CO3 was added and incubated in a dark room for 30 min. The absorbance was measured at 765 nm and the total phenolic content was calculated by using the linearity of gallic acid.
Total flavonoid content was determined using the method described by Chang et al. . 10 mg of dried pineapple was incubated with 1 mL ethanol for 48 h followed by sonication for 20 min. The samples were filtered, then 500 µL of the solution was mixed with 1.5 mL of ethanol. After that, 100 μL of 10% (w/v) Aluminum chloride (AlCl3) and 100 μL of 1 M Potassium acetate (CH3COOK) were added. The volume was made up to 5 mL using distilled water and placed in the dark at room temperature for 30 min. Quercetin was used as a standard curve at a wavelength 415 nm.
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay described by Brand-Williams et al.  was performed to evaluate antioxidant capacity of the pineapple in this study. Dried pineapple was mixed with ethanol at the concentrations of 375, 750, 1,500, 3,000, 6,000, and 12,000 µg/mL, then 100 μL of the samples were transferred into a 96-well microplate and mixed with 100 μL of 200 μM DPPH followed by incubation in the dark for 30 min. The absorbance was measures at 517 nm and the antioxidant capacity was determined by using the linearity of the standard Trolox curve.
The 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) method was described by Arnao et al.  to determine the pineapples antioxidant capacity. The dried pineapple was prepared with similar concentrations as above by using 50 μL of the samples mixed with 100 μL of working ABTS solution and then incubated for 15 min, followed by the Trolox being used as a standard curve at a wavelength of 734 nm.
The proximate components of the dried pineapple, which included ash, carbohydrates, fat, moisture, protein, and dietary fibre were analyzed at the Central Lab Thai, Bangkok, Thailand, according to standard protocols.
All animal experiments were approved by the Animal Ethics Committee of Naresuan University, Thailand (approval number: NU-AE610409), and performed according to the regulations of the institutional guidelines for the care and use of laboratory animals. Four week old Four-week-old male Sprague–Dawley rats that weighed 150–200 g were obtained from Nomura Siam International Co., Ltd. Bangkok, Thailand, and housed at the Centre for Animal Research, Naresuan University in Phitsanulok, Thailand. The rats were maintained in a controlled temperature environment at 25 ± 2 °C, a 12 h light‑dark cycle with a standard diet and filtered water ad libitum for 1 week. After the rats were acclimatized, they were randomly divided into 5 groups (n = 5–6) and treated for 8 weeks: control with standard diet and normal drinking water (control), high-cholesterol diet (standard diet + 1.5% (w/w) of cholesterol, 0.37% (w/w) of cholic acid) (HCD), high-cholesterol diet with a low-dose of pineapple; LPA (100 mg/kg/day) (HCD+LPA), high-cholesterol diet with a high-dose of pineapple; HPA (200 mg/kg/day) (HCD+HPA), and high-cholesterol diet with simvastatin (40 mg/kg/day) (HCD+S). Their body weights were determined weekly, and their total food consumptions were measured daily. The powdered pineapple and the simvastatin were administered by oral gavage.
Heart and serum collection
At the end of the 8-weeks dietary schedule, the rats were fasted overnight then anesthetized with pentobarbital sodium (Nembutal® Sodium Solution CII; 100 mg/kg; Akorn, Inc., Lake Forest, IL, USA) and lithium heparin (150 U; Government Pharmaceutical Organization, Bangkok, Thailand) via an intraperitoneal injection. Then an operation on the rats was carried out to expose their thoracic cavities, after the incision area had been cleaned with70% (v/v) ethanol. Their hearts were weighed then snap-frozen in liquid nitrogen and stored at -80 °C. The blood samples were collected rapidly from the thoracic cavity and centrifuged at 3000×g for 10 min at 4 °C. The Serum was then collected and immediately snap-frozen in liquid nitrogen and kept at − 20 °C, followed by its biochemical parameters being measured within 24 h.
Determination of blood biochemistry
Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), Low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), aspartate amino-transferase (AST) activity, alanine amino-transferase (ALT) activity, blood urea nitrogen (BUN) as well as creatinine (Cr) were analyzed using an automate biochemistry analyzer (cobas c 111 analyzer). Atherogenic Coefficient (AC) was calculated by (TC-HDL-C)/HDL-C and Cardiac risk ratio (CRR) was evaluated following this formula; CRR = TC/HDL-C .
Hearts homogenization and protein extraction
The frozen heart tissues were thawed and homogenized in cold phosphate buffer saline (PBS) containing a protease inhibitor cocktail (CAT. 0589295301, ROCHE, Germany; 100 µL/100 mg) by using a pestle and mortar. The homogenized tissues were then centrifuged at 14,000×g for 10 min at 4 °C and the supernatants were collected for determining the protein concentration by using the Bradford assay (BIO-RAD, USA) as previously described .
Protein carbonyl assay
To determine the protein carbonyls content in oxidized protein, the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay was performed as previously describe . Pre-analytical quality controls that included linearity test, within-run precision assay, and between day precision were determined using the control serum (HUMATROL P) with the DNPH spectrophotometric assay.
Determination of inflammatory cytokine levels
The determination of inflammatory cytokine levels that included tumor necrosis factor-alpha (TNF-α; CAT. 900-M73; Prepotech®, USA), interleukin 1-beta (IL1-β; CAT. 900-M91; Prepotech®, USA), and interleukin 6 (IL-6; CAT. 900-M86; Prepotech®, USA), were performed using the enzyme-linked immunosorbent assay (ELISA) Buffer Kit (CAT. 900-K00; Prepotech®, USA) according to the manufacturer’s instructions.
Lipid peroxidation assay
Malondialdehyde (MDA) levels were measured by using the Lipid Peroxidation Assay Kit (ab118970; Abcam, Cambridge, UK). The hearts were homogenized on ice in 303 µL MDA lysis buffer and centrifuged at 14,000×g for 10 min at 4 °C. This was followed by 200 µL of the supernatant being mixed with 600 µL of the thiobabituric acid (TBA) solution, incubated at 95 °C for 60 min and then cooled on ice for a further 10 min, after which 200 µL of the mixed solution was added to a 96-well microplate. The MDA level was calculated with standard curve at a wavelength 532 nm and calculated following the manufacturer's protocols.
Measurements of total antioxidant capacity of the heart tissue
The Total Antioxidant Capacity Assay Kit (ab65923; Abcam, Cambridge, UK) was used to evaluate the total antioxidant capacity (TAC) of the heart tissues. Then they were washed with cold phosphate buffer saline (PBS) and subsequently homogenized. The heart homogenate was incubated on ice for 10 min and centrifuged at 14,000×g for 10 min at 4 °C. The supernatant was transferred to a 96-well microplate, then a 100 µL CU2+ of the working solution was added. After being incubated on a shaker for 90 min at room temperature in the dark, the absorbance was measures at 570 nm and the antioxidant capacities were determined by using the linearity of the standard Trolox curve according to the manufacturer's protocols.
All the data was expressed as the mean ± SEM. One-way analysis of variance (ANOVA) followed by Turkey’s post hoc analysis being performed using GraphPad Prism 5.0. p value < 0.05 was considered as statistically significance.