The LHP content was determined using a molar absorption coefficient of 4.3 × 104 M−1 cm−1. Results were expressed as μmol LHP/g LDL protein. The haemoglobin oxidation assay was modified from the method of Marouf, Zalzala, Al-Khalifa, Aziz, and Hussain (2011). Erythrocytes from healthy volunteers

were washed 3 times with 10 mM PBS and lysed in a hypotonic solution (5 mM PBS) for 1 h at 4 °C. Then, the solution was centrifuged at 1000g for 10 min and the supernatant was collected. The haemolysate (0.75 ml) was mixed with 0.5 ml of B. racemosa leaf extract, stem extract or gallic acid (0–1000 μg/ml). Subsequently, 50 μl of freshly prepared sodium nitrite (0.65 mM) were added to induce oxidation of haemoglobin (Hb) to methaemoglobin (MetHb). The formation of MetHb was monitored at 631 nm every 5 min up to 30 min. The amount of MetHb was determined using a molar Sirolimus mw extinction coefficient of 3.7 mM−1 cm−1. Data were expressed as means ± standard deviation of triplicate analyses. Data were statistically analysed using the SPSS statistical

software, version 15 (SPSS Inc, Chicago, IL). Independent t-test was used for comparison of means between groups. One-way analysis of variance (ANOVA) and Tukey’s Honestly Significant Different test was used to compare means among Proteasome inhibitor groups. The level of significance was set at p < 0.05. Graph Pad Prism Version 5.1 software (GraphPad Software Inc., San Diego, CA) was used to predict the time needed to convert 50% of the Hb to MetHb, using a non-linear regression model. Fig. 1 shows the UHPLC chromatograms of the leaf and stem extracts of the shoots of B. racemosa, prepared through the freeze drying method. Six polyphenolic compounds were identified, consisting of three phenolic acids and three flavonoids. The phenolic acids were gallic acid, protocatechuic acid and ellagic acid, while the flavonoids were rutin, quercetin

and kaempferol. Hussin et al. (2009) detected six polyphenols in the leaves of B. Benzatropine racemosa, consisting of gallic acid, rutin, kaempferol, ferulic acid, naringin and luteolin. We did not detect the presence of ferulic acid, naringin and luteolin and this could be due to variation in the extraction method ( Ignat, Volf, & Popa, 2011). Due to variation in the absorption spectra of the polyphenolic compounds and to ensure maximum detection, two wavelengths, 280 and 325 nm, were utilised to observe the separated polyphenols. The λmax of all the polyphenols in this study corresponded to the λmax reported from the literature ( Table 1). Identification of the polyphenolic compounds was done by comparing the retention times (tR) of the sample peaks ( Fig. 1(a–d)) with those of authentic standards ( Fig. 1(e)). For further validation, the UV–Vis spectrum and the λmax of the eluted peaks generated from the diode array detector were compared with the spectrum of the authentic standards ( Fig. 2). Fig.