Ozonated sunflower oil (OSO) is an established therapeutic agent and nutraceutical harboring various therapeutic values, including antiallergic, derma-protective, and broad-spectrum antimicrobial activity. Conversely, the medicinal aspects of OSO for wound healing, tissue regeneration, and treatment of inflammation in dyslipidemia have yet to be fully elucidated. Herein, a comparative effect of OSO and sunflower oil (SO) was investigated to heal cutaneous wound and tissue regeneration of zebrafish impediment by carboxymethyllysine (CML) toxicity, following impact on hepatic inflammation and blood lipid profile. After OSO (final 2%, 1 μL) and SO's (final 2%, 1 μL) treatment, substantial healing was elicited by OSO in the cutaneous wound of zebrafish impaired by CML (final 25 mg). As an important event of wound healing, OSO scavenges the reactive oxygen species (ROS), rescues the wound from oxidative injury, and triggers the essential molecular events for the wound closer. Furthermore, the intraperitoneal injection of OSO was noted to counter the CML-induced adversity and prompt tissue regeneration in the amputated tail fin of zebrafish. Additionally, OSO counters the CML-induced neurotoxicity and rescues the zebrafish from acute mortality and paralysis, along with meticulous recovery of hepatic inflammation, fatty liver changes, and diminished ROS and proinflammatory interleukin (IL)-6 production. Besides, OSO efficiently ameliorated CML-induced dyslipidemia by alleviating the total blood cholesterol (TC), triglyceride (TG) and increasing high-density lipoproteins cholesterol (HDL-C). The outcome of multivariate assessment employing principal component analysis and hierarchical cluster analysis supports a superior therapeutic potential of OSO over SO against the clinical manifestation of CML. Conclusively, OSO owing to its antioxidant and anti-inflammatory potential, counters CML-induced toxicity and promotes wound healing, tissue regeneration, hepatoprotection, improved blood lipid profile, and survivability of zebrafish.

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.

Ozonated sunflower oil (OSO) is well known functional oil with its antioxidant, antimi-crobial, anti-allergic, and skin-moisturizing properties. However, studies on the effects of OSO on high-cholesterol diet (HCD)-induced metabolic disorders have been scarce. In the current study, we aimed to determine the anti-inflammatory effects of OSO on lipid me-tabolism in adult hypercholesterolemic zebrafish and its embryo. Microinjection of OSO (final 2%, 10 nL) into zebrafish embryos under presence of carboxymethyllysine (CML, 500 ng) protected acute embryo death upto 61% survival, while sunflower oil (final 2%) showed much less protection around 42% survival. The microinjection of OSO was more effective than SO to inhibit reactive oxygen species (ROS) production and apoptosis in the CML induced embryo toxicity. Intraperitoneal injection of OSO under presence of CML protected acute death from CML-induced neurotoxicity with improved hepatic inflamma-tion, less detection of ROS and interleukin (IL)-6, and lowering blood total cholesterol (TC) and triglyceride (TG), while SO-injected group did not protect the CML-toxicity. Long-term supplementation of OSO (final 20%, wt/wt) with HCD for 6 months resulted higher sur-vivability than HCD alone group or HCD+SO group (final 20%, wt/wt) with significant lowering of plasma TC and TG levels. The HCD+OSO group showed the least hepatic in-flammation, fatty liver change, ROS, and IL-6 production. In conclusion, short-term treat-ment of OSO by injection exhibited potent anti-inflammatory activity against acute neuro-toxicity of CML in zebrafish and its embryo. Long-term supplementation of OSO in diet also revealed the highest survivability and blood lipid-lowering effect through potent an-ti-oxidant and anti-inflammatory activity.