Enhanced Antioxidant and Anti-Glycation Abilities of Beeswax Alcohol in Reconstituted High-Density Lipoprotein with Potent Wound-Healing Activity and Protection of Embryo from Acute Toxicity of Carboxymethyllysine in Zebrafish
Authors: Cho Kyung-Hyun , Baek Seung-Hee , Nam Hyo-Seon , Bahuguna Ashutosh 
Published: September 2023

The antioxidant and anti-inflammatory abilities of beeswax alcohol (BWA) are well reported in animal and human clinical studies, with a significant decrease in malondialdehyde (MDA) in blood, reduction of liver steatosis, and decrease in insulin. On the other hand, there is insufficient information to explain the in vitro antioxidant activity of BWA because it is extremely insoluble in aqueous buffer system. The BWA mixture was incorporated into reconstituted HDL (rHDL) with apoA-I, and the physiological functions of BWA in a water system were evaluated. After synthesis of rHDL with palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apolipoprotein A-I (apoA-I), and the BWA at molar ratios of 95:5:1:0 (rHDL-0), 95:5:1:0.5 (rHDL-0.5), and 95:5:1:1 (rHDL-1) for POPC:FC:apoA-I:BWA, the particle size of rHDL-1 was increased 15% compared to rHDL-0. rHDL-1 exhibited the strongest anti-glycation activity, up to 18% less glycation than rHDL-0 treated HDL3, with the highest protection of apoA-I from proteolytic degradation: a 28% larger band intensity than that of rHDL-0 treated HDL3 in the presence of fructose (final 250 mM). The antioxidant ability to inhibit cupric ion-mediated LDL oxidation increased as the BWA content in rHDL increased. The rHDL-1-treated LDL exhibited the smallest oxidation extent in electromobility with the least quantification of oxidized species (MDA). The antioxidant activities of HDL, ferric ion reduction ability (FRA), and paraoxonase (PON) were enhanced by the BWA in rHDL treatment with a concomitant increase in the molar ratio. rHDL-1-treated HDL showed 20–22% higher FRA and PON activities than rHDL-0-treated HDL. A microinjection of each rHDL into zebrafish embryos was performed in the presence of carboxymethyllysine (CML). The rHDL-1 injected embryo exhibited the highest survivability (~63%), whereas the CML alone group showed 28% survivability. A higher BWA content in rHDL helped neutralize the CML toxicity, resulting in higher survivability and normal developmental morphology. In contrast, the CML alone injected embryo showed severe retardation of the developmental speed and morphological defect. The CML-alone group showed the highest extent of reactive oxygen species (ROS) production and cellular apoptosis in embryos, but a co-injection of rHDL-1 resulted in a remarkable decrease in ROS and apoptosis. The dermal application of rHDL containing BWA resulted in higher potent wound-healing activity in a dose-dependent manner with decreased reactive oxygen species and cellular apoptosis in the cutaneous wound area in the presence of CML. Conclusively, incorporating BWA in rHDL significantly enhanced the anti-glycation and antioxidant activities in rHDL via more stabilization of apoA-I with a larger particle size. The rHDL containing BWA facilitated enforced inherent antioxidant ability of HDL and anti-inflammatory activity to suppress the CML toxicities in zebrafish embryos and to ameliorate CML-aggravated chronic wounds in adult zebrafish.