Corresponding author: Denitsa Aluani ( denitsa.aluani@gmail.com ) Academic editor: Plamen Peikov
© 2022 Denitsa Aluani, Magdalena Kondeva-Burdina, Alexandra Tosheva, Krassimira Yoncheva, Virginia Tzankova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Aluani D, Kondeva-Burdina M, Tosheva A, Yoncheva K, Tzankova V (2022) Improvement of in vitro antioxidant activity of kaempferol by encapsulation in copolymer micelles. Pharmacia 69(1): 25-29. https://doi.org/10.3897/pharmacia.69.e77678
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Antioxidant capacity of poorly soluble natural antioxidant kaempferol, in particular free or loaded in two types of cationic micelles, was studied on non-enzyme induced lipid peroxidation (LPO) in vitro. The micelles were based on triblock copolymers - poly(2-(dimethylamino)ethyl methacrylate-b-poly(propylene oxide)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA-PPO-PDMAEMA) and poly(2-(dimethylamino)ethyl methacrylate-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA-PCL-PDMAEMA). The lipid peroxidation was induced by incubating of rat liver microsomes with iron sulphate and ascorbic acid (Fe2+/AA). The effect of free and micellar kaempferol (at concentrations 25, 50 and 75 μg/ml) was assessed after 20 min incubation time. In the non-enzyme lipid peroxidation model, the kaempferol-loaded micelles significantly decreased the formation of malondialdehyde (MDA). The effect of kaempferol loaded in PDMAEMA-PCL-PDMAEMA micelles was more pronounced, showing an improved antioxidant activity in the conditions of oxidative stress and lipid peroxidation in vitro.
kaempferol, in vitro, antioxidant, copolymer micelles
Natural antioxidants are very attractive active substances because of their high potential to prevent or treat various diseases associated with oxidative stress. Oxidative stress occurs due to the lost balance between the prooxidant and antioxidant reactions in living organisms (
The flavonoids (quercetin, rutin, kaempferol etc.) and hydroxycinnamic acids (caffeic, ferulic etc.) are intensively studied natural antioxidants. Kaempferol is a flavonol showing a variety of activities, including antioxidant, cytoprotective, anticancer etc. (
The aim of the present study was to evaluate the potential of two polymeric micellar systems to improve antioxidant activity of kaempferol in the model of iron/ascorbic acid (Fe2+/AA) induced lipid peroxidation in rat liver microsomes.
Kaempferol (KF) was purchased from Sigma Aldrich (Germany). The triblock copolymers poly(2-(dimethylamino)ethyl methacrylate-b-poly(propylene oxide)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA13-PPO69-PDMAEMA13) and poly(2-(dimethylamino)ethyl methacrylate-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA9-PCL70-PDMAEMA9) were synthesized in the Institute of Polymers (Bulgarian Academy of Science) according to previously established procedure (
Kaempferol loading in PDMAEMA-PPO-PDMAEMA and PDMAEMA-PCL-PDMAEMA micelles was performed by solvent evaporation method. In a brief, the copolymer and kaempferol were dissolved in 5 ml dioxane. After their incubation for 30 min, 2 ml purified water was slowly added to the organic phase. Dioxane was evaporated under reduced pressure (Buchi-144, Switzerland) and the aqueous micellar dispersions were filtered (0.22 µm). The filter was rinsed with ethanol and these fractions were determined for non-encapsulated kaempferol.
Iron/ascorbic acid (Fe2+/AA) induced lipid peroxidation in rat liver microsomes
The microsomes were isolated from the livers of male Wistar rats (200.0–220.0 g) purchased from the National Breeding Center, Sofia, Bulgaria. At least 7 days of acclimatization were allowed before the commencement of the study. The vivarium (certificate of registration of farm No. 0072/01.08.2007) was inspected by the Bulgarian Drug Agency to check the husbandry conditions (No. A-11-1081/03.11.2011). All performed procedures were approved by the Institutional Animal Care Committee and made according Ordinance No. 15/2006 for humaneness behaviour to experimental animals.
The livers were thoroughly perfused with 1.15% KCl and homogenized with ice-cold 0.1 mol/L potassium phosphate buffer (pH = 7.4). The liver homogenate was centrifuged at 9,000×g for 30 min at 4 °C and the resulting postmitochondrial fraction was centrifuged at 105,000×g for 60 min at 4 °C. The microsomal pellets were dispersed in 0.1 mol/L potassium phosphate buffer, containing 20% glycerol. Microsomal protein content was determined according to the method of Lowry et al. (
The microsomes were preincubated with the solutions of the pure kaempferol or with micellar dispersions of kaempferol at 37 °C for 15 min. The lipid peroxidation was started with a 20 μmol/L solution of iron sulphate and 0.5 mmol/L of ascorbic acid (
Statistical analysis was performed by one-way analysis of variance analysis of variance with Dunnett’s post hoc test. Differences were accepted to be significant when P < 0.05. All statistical analysis was carried out on Graph Pad 6 software (GraphPad Software, Inc., La Jolla, CA, USA).
Physicochemical properties of nanosized drug delivery systems are very important for their in vivo behaviour. In particular, the size is a key factor for cellular uptake ability and distribution. Here, the micelles having PPO-core were larger in size than those prepared with a copolymer possessing PCL-core (Fig.
The objective of this study was to investigate the potential of free kaempferol and kaempferol loaded into two types of polymeric micelles to protect rat liver microsomes against iron/ascorbic acid (Fe2+/AA) induced lipid peroxidation. Rat liver microsomes were selected as a relevant model for evaluation of antioxidant activity of natural antioxidants on microsomal lipid peroxidation (
Initially, we evaluated the effects of the empty and kaempferol loaded micelles (PDMAEMA-PCL-PDMAEMA and PDMAEMA-PPO-PDMAEMA) on non-treated rat liver microsomes. The microsomes were treated with three different concentrations (80, 160 and 240 µg/ml) of the empty polymeric micelles. The results showed no apparent induction of lipid peroxidation by empty micelles in non-treated rat liver microsomes (Fig.
The results are expressed as means ± SD of triplicate assays (n = 3). All groups were compared statistically vs untreated controls by one-way Anova with Dunnet’s post-test. *p < 0.05; ***p < 0.001 vs control.
The results are expressed as means ± SD of triplicate assays (n = 3). All groups were compared statistically vs untreated controls by one-way Anova with Dunnet’s post-test. *p < 0.05 vs control.
In the next experiments, we evaluated the protective activity of free and micellar kaempferol (25, 50 and 75 µg/ml) in a lipid peroxidation model оn iron/ascorbic acid treated microsomes. The results related to iron/ascorbic acid treated microsomes are presented on Fig.
Protective effects of free kaempferol (KF) (25, 50, 75 μg/ml) and kaempferol loaded PDMAEMA-PPO-PDMAEMA (PPO-KF) micelles on the level of malondialdehyde (MDA) in iron/ascorbic acid (Fe2+/AA) treated microsomes. Mean values ± SN (n = 6). *** p < 0.001 compared to untreated control group; +++ < 0.001 vs Fe2+/AA is considered to be statistically significant.
Mean values ± SN (n = 6). *** p < 0.001 compared to untreated control group; +++ < 0.001 vs Fe2+/AA is considered to be statistically significant.
The comparison between the protective activity of micellar kaempferol revealed a significant differences depending on the different polymeric micelles. When kaempferol was loaded into PDMAEMA-PCL-PDMAEMA micelles, the protective effects on Fe2+/AA induced lipid peroxidation were more evident compared to free kaempferol and PDMAEMA-PPO-PDMAEMA micelles. For example, at 75 µg/ml kaempferol loaded PDMAEMA-PCL-PDMAEMA micelles (KF-PCL) decreased the level of MDA by 94% (Fig.
The data in the present study suggest that the loading of kaempferol in micelles, especially based on PDMAEMA-PCL-PDMAEMA copolymer, might improve its antioxidant activity in the conditions of oxidative stress and lipid peroxidation.
The authors are grateful to Prof. Petar Petrov for the synthesis of copolymers.
D. Aluani is grateful for the support by the National Program “Young Scientists and 342 Postdoctoral Candidates” of the Ministry of Education and Science, Bulgaria.