Automated analyzer

Blood samples were collected after an overnight fast to avoid potential confounding influences due to hormonal rhythmicity. Biochemical evaluation included glucose, urea, creatinine, uric acid, cholesterol and triglyceride levels. Serum glucose was analyzed by an automated analyzer (Roche/Hitachi, Switzerland). Urea, creatinine, uric acid, total cholesterol and triglyceride concentrations were determined by enzymatic spectrophotometric methods (Roche, Basel, Switzerland). Cholesterol/Triglyceride ratio was calculated for predicting the presence of small, dense LDL [12 (link), 19 (link)] and Triglycerides/glucose index was calculated to estimate insulin resistance [20 (link), 21 (link)].

The animals were separated into four groups consisting of six animals each: the first group served as control, the second group was treated with a known hypolipidemic drug- losartan (10 mg/kg, p.o) [14 (link)], and the third and fourth groups received H. pubescens extract orally at two different doses of 250 mg/kg and 500 mg/kg [10 (link)]. The treatment was given for 28 days, and the serum levels of cholesterol, HDL-cholesterol, and triglycerides were determined using an automated analyzer (Roche, St. Louis, MO, USA).

We collected data on age, gender, blood pressure (BP), body mass index (BMI), and waist circumference (WC) of participants. We collected blood samples from participants under fasting conditions. Venipuncture was performed using vacuum blood collection tubes, and blood was stored in blood collection tubes, serum separator tubes, and EDTA Vacutainer (Becton Dickinson, Franklin Lakes, NJ, USA). Blood samples were centrifuged at 1,500 × g for 15 min at 4°C. Standard laboratory methods and certified biochemical and hematological tests were performed using an automated analyzer (Roche Diagnostics, Mannheim, Germany).
Participants’ fasting blood glucose (FBG), Glycated albumin (GA), triglycerides (TG), total cholesterol (TC), High-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), Small dense low-density lipoprotein-cholesterol (sdLDL-C), Apolipoprotein A1 (ApoA1), Apolipoprotein B (ApoB), Apolipoprotein E (ApoE) and lipoprotein (a) [Lp (a)].

From the blood obtained on fourth and sixth week, serum was separated by centrifuging at 4000 rpm and 4°C for 10 min. The concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), glucose (Gluc), gamma-glutamyl transferase (GGT), aspartate amino-transferase (AST), alanine amino-transferase (ALT), uric acid (UA), phosphorus (phosp.) and alkaline phosphatase (ALP) were estimated on Automated Analyzer (Roche Cobas c-111) using commercially available kits. Atherogenic index (AI) was calculated as the ratio of Non-HDL and HDL [21 (link)]. TC/HDL, LDL/HDL and AST/ALT ratios were also calculated. Serum Nitric oxide concentration was estimated by Griess method [88 (link)]. At the end of experiment (sixth week), thoracic aortae were isolated and endothelial nitric oxide synthase (eNOS) enzyme activity was assayed using the Nitric Oxide Synthase Assay Kit, Colorimetric (Calbiochem, Cat. No. 482702) following the manufacturer’s instructions.

After fasting for 15 hours and undergoing glucose (2 g/kg body weight) gavage, blood glucose levels of rats were measured at 0, 30, 60, and 120 min via automated analyzer (Roche Diagnostics, Mannheim, Germany) to calculate the following:
AUCg = (G₀+G₁₂₀)/4+(G₃₀+G₆₀)/4+(G₆₀+G₁₂₀)/2
HOMA-β = 20×FINS/(FBG-3.5)
HOMA-IR = FBG× FINS/22.5.
AUCg: area under the curve for glucose; G: blood glucose level (0 min [G₀], 30 min [G₃₀], 60 min [G₆₀], and 120 min [G₁₂₀]); FINS: fasting plasma insulin; FBG: fasting blood glucose level