Hura crepitans L. (HC) belonging the Euphorbiaceae family, is commonly known as “Monkey-no-climb”. It is toxic, but usually used in ethnomedicinal applications for rheumatism, treatment of skin diseases, intestinal worms in leprosy. The aim of the present work was the evaluation of nanoformulation for improving the biological activity in cells. The leaves of H. crepitans were collected in Venezuela in 2018, dried, powdered, and subjected to different extraction methods. Preliminary data obtained by different in vitro antioxidant in tube test and cell assays, as well as the chemical profile, allowed the selection of the macerated extract (HC-M) as the most performing to be encapsulated into liposomes in order to promote cell interaction and enhance the biological activity. The ability of liposomes to maintain and accommodate the stability of encapsulated compounds is the major advantage of this formulation. For this reason, the liposome technology is employed in manufacturing of functional food, nutraceutical, cosmetic, and pharmaceutical products. Liposomes were prepared by a simple method involving the sonication of the phospholipid (soy lecithin) and HC-M extract in water. Empty liposomes were also prepared and characterized to evaluate the effect of the incorporation of the extract on the vesicle arrangement. The characterization of empty liposomes displayed small size (73 nm), good homogeneity (P.I. 0.25), and highly negative zeta potential (∼ −50 mV). When the HC-M extract was incorporated, there was a slight increase in size (84 nm) with an improvement in the homogeneity (P.I. 0.20). Then, HC-M extract and extract-loaded liposomes (LHC-M) were subjected to analysis of bioactivity on human hepatoma cells (HepG2) cells. In particular, the dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA) were used to evaluate the cell viability for 24 h and the intracellular reactive oxygen species (ROS) level, respectively. The cytotoxicity of liposomal formulation is dose-dependent with an IC50 value approximately of 30 µg/mL. Therefore, concentrations lower than the IC50 value were used assessing the antioxidant activity of the liposomal formulation of LHC-M. Interestingly, the liposomes were able to maintain ROS levels close to endogenous ones, preventing the oxidative stress caused by ROS already at the lower concentration (3.125 µg/mL), without statistical difference between the tested concentrations. Moreover, in order to identify the compounds involved in these effects, the macerated extract was subjected to liquid chromatography–mass spectrometry analyses. Fourteen compounds were identified and the most abundant were the phenols caffeic acid, gallic acid, and quercetin. These results suggest the use of H. crepitans as antioxidant source, especially when incorporated into liposomes. In fact, the extract incorporates in liposomes is an advantage to facilitate the interaction with cells and allow the release of the payload in the cytoplasm, where the antioxidant activity is exerted.

Hura crepitans L. extract: chemical characterization, nanoformulation and bioactivity in cell model

Faraone I.;Russo D.;Vassallo A.;Armentano M. F.;Ostuni A.;Bisaccia F.;Milella L
2021

Abstract

Hura crepitans L. (HC) belonging the Euphorbiaceae family, is commonly known as “Monkey-no-climb”. It is toxic, but usually used in ethnomedicinal applications for rheumatism, treatment of skin diseases, intestinal worms in leprosy. The aim of the present work was the evaluation of nanoformulation for improving the biological activity in cells. The leaves of H. crepitans were collected in Venezuela in 2018, dried, powdered, and subjected to different extraction methods. Preliminary data obtained by different in vitro antioxidant in tube test and cell assays, as well as the chemical profile, allowed the selection of the macerated extract (HC-M) as the most performing to be encapsulated into liposomes in order to promote cell interaction and enhance the biological activity. The ability of liposomes to maintain and accommodate the stability of encapsulated compounds is the major advantage of this formulation. For this reason, the liposome technology is employed in manufacturing of functional food, nutraceutical, cosmetic, and pharmaceutical products. Liposomes were prepared by a simple method involving the sonication of the phospholipid (soy lecithin) and HC-M extract in water. Empty liposomes were also prepared and characterized to evaluate the effect of the incorporation of the extract on the vesicle arrangement. The characterization of empty liposomes displayed small size (73 nm), good homogeneity (P.I. 0.25), and highly negative zeta potential (∼ −50 mV). When the HC-M extract was incorporated, there was a slight increase in size (84 nm) with an improvement in the homogeneity (P.I. 0.20). Then, HC-M extract and extract-loaded liposomes (LHC-M) were subjected to analysis of bioactivity on human hepatoma cells (HepG2) cells. In particular, the dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA) were used to evaluate the cell viability for 24 h and the intracellular reactive oxygen species (ROS) level, respectively. The cytotoxicity of liposomal formulation is dose-dependent with an IC50 value approximately of 30 µg/mL. Therefore, concentrations lower than the IC50 value were used assessing the antioxidant activity of the liposomal formulation of LHC-M. Interestingly, the liposomes were able to maintain ROS levels close to endogenous ones, preventing the oxidative stress caused by ROS already at the lower concentration (3.125 µg/mL), without statistical difference between the tested concentrations. Moreover, in order to identify the compounds involved in these effects, the macerated extract was subjected to liquid chromatography–mass spectrometry analyses. Fourteen compounds were identified and the most abundant were the phenols caffeic acid, gallic acid, and quercetin. These results suggest the use of H. crepitans as antioxidant source, especially when incorporated into liposomes. In fact, the extract incorporates in liposomes is an advantage to facilitate the interaction with cells and allow the release of the payload in the cytoplasm, where the antioxidant activity is exerted.
978-88-85915-26-8
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11563/155345
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