WO2021009443A1 - Gold nanoparticles comprising a plant extract and their cosmetic use - Google Patents

Gold nanoparticles comprising a plant extract and their cosmetic use Download PDF

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WO2021009443A1
WO2021009443A1 PCT/FR2020/051223 FR2020051223W WO2021009443A1 WO 2021009443 A1 WO2021009443 A1 WO 2021009443A1 FR 2020051223 W FR2020051223 W FR 2020051223W WO 2021009443 A1 WO2021009443 A1 WO 2021009443A1
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extract
nanoparticles
plant extract
gold
cells
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French (fr)
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Anne-Laure MOREL
Sophie Giraud
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Torskal
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0241—Containing particulates characterized by their shape and/or structure
    • A61K8/0275—Containing agglomerated particulates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/41—Particular ingredients further characterized by their size
    • A61K2800/413—Nanosized, i.e. having sizes below 100 nm
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers

Definitions

  • the present invention relates to the field of gold nanoparticles used in cosmetics. It relates more particularly to the use of nanoparticles obtained from a mixture of gold and plant extracts rich in flavonoids, and their use in cosmetics for preventing skin damage linked to UV rays and free radicals.
  • Hubertia ambavilla is an endemic plant native to Reunion Island, in the Indian Ocean, traditionally used both internally and externally.
  • the main compounds of Hubertia ambavilla are flavonoids, tannins, proanthocyanidins and carbohydrate complex leading to skin therapeutic activities such as anti inflammatory and wound healing properties as well as other therapeutic activities used to treat kidney infections, asthma and diabetes (6, 7, 8).
  • Nanoparticles are widely used and studied in several fields such as medicine, the environment or cosmetics (1, 2, 3). Their size and shape can be adjusted by changing the chemicals and changing their ratio.
  • the European Commission defines nanomaterial as "a natural, accidental or manufactured particle of which, for 50% or more of the particles in the size distribution, one or more external dimensions are between 1 and 100 nm" (4).
  • Gold nanoparticles are also well described in the literature as anti-aging ingredients in the United States. They are used in particular for the disinfection of skin wounds, anti-inflammatory and anti-aging cream. These nanoparticles are obtained by the Turkevich method according to which it is necessary to carefully select the chemicals or the natural product used for the coating of nanoparticles to avoid the risk of toxicity.
  • the skin is the organ most exposed to external factors that damage it. These factors are pollution, exposure to solar ultraviolet (UV) rays, cigarette smoke, etc. These factors are responsible for the production of reactive oxygen species (ROS). A excess ROS induces oxidative stress which damages cells, DNA and proteins, thus leading to skin aging. In the case of sun exposure, sunscreens are generally recommended for skin protection, but their effectiveness is reduced if application is inadequate and spectral protection is incomplete. The body has natural antioxidants to eliminate these free radicals, but in the case of overexposure, these systems can be easily overwhelmed. It is therefore important to help the skin protect itself by providing another source of antioxidants.
  • UV solar ultraviolet
  • nanoparticles comprising a plant extract rich in flavonoids in cosmetics.
  • the invention relates to the use of a composition comprising gold nanoparticles comprising a mixture of gold and a plant extract rich in flavonoids for the prevention of damage due to UV rays and / or the prevention of skin damage due to UV rays. to free radicals.
  • the plant extract used is a crude extract of Hubertia Ambavilla or Hypericum lanceolatum.
  • the inventors have studied and characterized the gold nanoparticles comprising a plant extract rich in flavonoids. They have thus demonstrated that these have very advantageous properties for use in cosmetics.
  • these gold nanoparticles are stable due to the fact that the plant extracts, in particular the polyphenols, allow their stabilization.
  • nanoparticles comprising a raw extract of Hubertia ambavilla is particularly remarkable since it is greater than that of vitamin E.
  • These nanoparticles make it possible in particular to inhibit the oxidation induced by UV-A irradiations by trapping free radicals, offering anti-oxidant protection in the dermis.
  • these nanoparticles are obtained by a “green” process that respects the environment and does not use any toxic product.
  • the invention relates to the use of a composition comprising at least one gold nanoparticle comprising a mixture of gold and a plant extract rich in flavonoids for the prevention of skin damage due to UV rays and / or free radicals.
  • the gold nanoparticles used in this invention are obtained by an ecological method comprising: (a) preparing at least one plant extract rich in flavonoids and (b) mixing at least one of said plant extracts with a aqueous solution of at least one gold salt.
  • the plant extract rich in flavonoids may be a crude total extract of said plant or a total of flavonoids of said plant.
  • the nanoparticles are obtained by mixing a totum of plant flavonoids with an aqueous solution of at least one gold salt, they are spherical.
  • nanoparticles When the nanoparticles are obtained by mixing a total crude plant extract with an aqueous solution of at least one gold salt, they have the shape of a flower and are called “nanoflowers”.
  • Nanoparticles according to the invention and their preparation process are described in application WO2017 / 125695.
  • the plant extract is chosen from an extract of Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Ayapana triplinervis, Camellia sinensis var. assamica, Citrus hystrix, Curcuma longa, Cryptomeria japonica, Dodonaea viscosa, Mussaenda arcuate, Nuxia verticillate, Olea europea Africana, Phyllanthus casticum, Pittosporum senacia, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe or Vepris lanceolata.
  • the extract is chosen from extracts of Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Camellia sinensis var. assamica, Citrus hystrix, Cryptomeria japonica, Mussaenda arcuate, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe or Vepris lanceolata.
  • the plant extract rich in flavonoids is chosen from an extract of Hubertia ambavilla, Hypericum lanceolatum, Citrus hystrix, Curcuma longa, Dodonaea viscosa, Nuxia verticillate, Psidium cattleianum, or Psiloxylon mauritianum,
  • the extract is a raw extract of Hubertia ambavilla; the nanoparticle is then in the shape of a flower and its diameter is between 40 and 80 nm.
  • the particular interest of this plant has been validated by in-depth experiments, presented in the experimental part of this text.
  • the extract is a totum of Hypericum lanceolatum; the nanoparticle is then spherical, its diameter is approximately 15 nm and it is characterized by a reducing / oxidizing ratio of 21.
  • the gold nanoparticles comprising a mixture of gold and a plant extract rich in flavonoids are incorporated into a cosmetic composition.
  • Such a composition can also comprise at least one other cosmetic ingredient.
  • nanoparticles can be combined with other cosmetic ingredients to increase and strengthen its properties.
  • This cosmetic composition can be in the form of a cream, a lotion or any other suitable form.
  • Figure 1 (a) Absorption spectrum of an aqueous solution of dispersed GAuNPs and (b) its characterization by TEM.
  • Figure 2 XPS spectra of (a) Au4f and (b) Ois regions.
  • Figure 3 HPLC chromatogram of a plant extract of Hubertia Ambavilla by detection of the charged aerosol. The extract was separated into 5 fractions noted from A to E.
  • Figure 4 LC-MS / MS and UV chromatograms of the aqueous plant extract.
  • Figure 5 Scheme of conventional synthesis of gold nanoparticles using citrate (Turkevich method) and leading to a spherical AuNP of 15 nm surrounded by citrate.
  • Figure 7 Antioxidant activity by the DPPH method measured on (a) GAuNP and (b) CAuNP.
  • the preparation of nanoparticles from two plant species is illustrated here.
  • the first plant is Hubertia ambavilla which is a shrub endemic to Reunion Island.
  • the second is Hypericum lanceolatum which is a species of arborescent St. John's wort native to Reunion Island.
  • These two plants are particularly rich in flavonoids including rutin and quercetin for Hypericum lanceolatum and isoquercetin and hyperoside for Hubertia ambavilla.
  • Freshly harvested plants are washed with deionized water. 3 grams are mixed with 50 ml of deionized water then the mixture is heated at 60 ° C. for 5 min, which makes it possible to release the biological material by lysis of the plant cells. The supernatant is then cooled to room temperature then on hanging ice for 10 minutes. The cooled supernatant is then filtered through a grade 2 porosity filter.
  • the extract obtained is green.
  • the starting material is Hypericum lanceolatum, the extract obtained is brown.
  • the extraction method used is a cold maceration method.
  • the plants are crushed on a sieve with pores of 10 mm diameter and then left to macerate with stirring at 150 rpm for 20 h at room temperature.
  • a mixture of equal amounts of water and ethanol is added to the mixture in a solid / solvent ratio of 1:20 to obtain the best possible yield of phenolic compounds (Cujic N et al).
  • the maceras are filtered, dried at low pressure (maximum bath temperature: 45 ° C, pressure between 50 and 150 bars) then lyophilized for 48 hours.
  • the nanoparticles obtained have a diameter measured in TEM of approximately 40 nm. b) Preparation of spherical gold nanoparticles with a total of flavonoids
  • the nanoparticles obtained have a diameter measured in TEM of approximately 15 nm.
  • the analyzes were carried out as follows: the extract (10 mg) was taken up in 500 ⁇ l of a water / acetone 4/1 v: v mixture, the addition of water alone leaving undissolved residues. 1 ⁇ L of this extract was injected into our LC-MS / HRMS machine (Dionex Ultimate 3000 HPLC chain with UV-visible diode array detector and Q.- TOF Impact II Bruker mass spectrometer) on a 50 mm x column 4 mm Nucleoshell RP18, 2.7 ⁇ m, with a gradient between the following mobile phases: H2O + 0.1% formic acid / acetonitrile + 0.1% formic acid.
  • the UV-Vis absorption spectrum of the AuNPs solution was recorded by a Perkin Elmer Lambda UV / Vis 950 instrument using standard 1 cm plastic cells at room temperature. The measurements were carried out in the spectral range 200-900 nm.
  • Transmission electron microscope (TEM) images of AuNPs were acquired on a TEM / STEM Technai Osiris (FEI) microscope equipped with a wide angle dark field detector operating at 200 kV. To perform the TEM analysis, a 3 ⁇ l drop of solution AuNP previously treated for 5 min in an ultrasonic bath was deposited on a copper grid covered with carbon. The sample was allowed to dry for 30 minutes in ambient air.
  • the hydrodynamic particle size distribution and the zeta potential were measured on a Zetasizer NanoZSP (Malvern Instruments). The measurements were carried out at 25 ° C.
  • the x-ray spot size was 400 microns for acquisition of surface points and 200 microns for sputtering.
  • a pressure of 10 to 7 Pa was reached in the chamber during the transfer of drops of liquid onto an indium sheet.
  • Full spectra (0-1100 eV) were obtained with constant pass energy of 200 eV and high resolution spectra at constant pass energy of 40 eV. Charge neutralization was applied during analysis.
  • the high resolution C1s, Ois, Au4f spectra were adjusted and quantified using AVANTAGE software supplied by ThermoFisher Scientific (Scofield sensitivity factors used for quantification).
  • the stability of the AuNP suspension stored at 4 ° C for more than a month was evaluated on the basis of changes in the absorption spectrum of the AuNP solution.
  • the hydrodynamic particle size distribution of a fresh suspension of AuNP and an aged suspension (4 months old stored at 4 ° C throughout the period) was compared.
  • NHDF normal human dermal fibroblasts isolated from the foreskin of the newborn were used. NHDF were grown in DMEM (GIBCO ®, Invitrogen TM) supplemented with 10% fetal calf serum (FCS) and antibiotics in a humidified atmosphere at 37 ° C and 5% C02. The cells were cultured routinely in culture flasks of 25 to 75 cm 2 and subcultured regularly before confluence.
  • the toxicity of AuNP was evaluated in NHDF seeded in 96-well microplates at 20 x 10 3 cells per well. Cell viability was assessed by a red neutralization (NRU). AuNP solutions were prepared in complete medium (DMEM + 1% FCS) after ultrasonic homogenization (3 cycles of 5 seconds at 20 kHz). A wide range of concentrations (from 0.005 to 100 pg / ml) expressed in weight / volume of active material was tested. The NRU assay was performed after 24 h and 72 h of incubation.
  • the percentage of antioxidant activity was evaluated by assaying free radicals with DPPH. This method is based on the change in color of the DPPH solution from purple to pale yellow when this stable free radical is converted into a reduced form by the reaction of a substance capable of giving off a hydrogen atom.
  • the test is based on the evaluation of the production of matrix metalloproteinase 1 (MMP-1 or collagenase) by culture of NHDF in the absence or presence of AuNP and exposed to UV-A.
  • MMP-1 or collagenase matrix metalloproteinase 1
  • NHDF from the parent cultures was harvested with trypsin from tissue culture flasks. After counting, the cells were suspended in complete medium (DMEM + FCS) and seeded in 24-well plates at a density of 75 x 10 3 cells per well. 72 hours after plating, the medium was removed and replaced with fresh medium supplemented with 1% FCS and containing various concentrations of AuNP. The NHDF cells were then incubated for 24 hours. Before irradiation, the cell monolayers were washed with HBSS and exposed to UV-A in the presence of HBSS containing the AuNP solution at different concentrations with a parallel row of Philips TL-K 40W ACTINIC BL REFLECTOR tubes.
  • the HBSS solutions were removed and replaced with fresh medium supplemented with 1% FCS and containing the AuNP solution.
  • the NHDF cells were then placed in the incubator at 37 ° C for 24 h.
  • conditioned media from cultures of NHDF were collected and stored at -20 ° C until analysis for MMP-1.
  • the corresponding NHDF monolayers were extracted to quantify the protein content. Protein levels were determined using the BCA Protein Assay Kit.
  • NaOH solution 0.1 N
  • aliquots of lysates were transferred to a 96-well microplate and BCA reagent was added. After a period of 30 minutes, the optical densities were measured at 570 nm.
  • MMP-1 activity was determined using an ELISA assay (Human Pro-MMP-1 Quantikin; R & D Systems ® ) for the detection and quantitative measurement of MMP-1 in supernatants. culture, according to the manufacturer's instructions. Each measurement was performed in triplicate and a positive control (1mM ⁇ -tocopherol) was included in the test.
  • ELISA assay Human Pro-MMP-1 Quantikin; R & D Systems ®
  • the AuNP acute toxicity study is based on OECD Guidance Document No. 129.
  • the toxicity was evaluated on Balb / c 3T3 mouse fibroblasts seeded in 96-well microplates at a rate of 2 ⁇ 10 3 cells per well. Cell viability was assessed by a red neutralization test (NRU). Solutions of AuNP were prepared in DMEM after ultrasonic homogenization. A wide range of concentrations (0.01-125,000 pg / ml) were tested. The NRU assay was performed after 48 hours of incubation. 4.1.2 3T3 NRU phototoxicity test
  • the test compares the cytotoxicity of chemicals applied to mouse fibroblasts (Balb / c 3T3, clone A31) in the presence or absence of exposure to a non-cytotoxic level of UV-A light (5 J / cm2). Cytotoxicity was measured as inhibition of the ability to absorb the essential dye, neutral red (NR), one day after UV-A treatment. This study was based on OECD guideline 432. The phototoxicity was assessed in Balb / c 3T3 mouse fibroblasts seeded in 96-well microplates at 1x10 4 cells per well. The plates were incubated for 24 hours, then the culture medium was removed and the cells were washed with preheated HBSS.
  • the NRU assay was used to assess changes in cell viability after incubation of NHDF cells with AuNP at various concentrations (from 0.01 to 200 pg / ml) for 24 or 72 hours of incubation. .
  • Control cells were incubated with complete medium (DMEM + 1% FCS) and the experiments were repeated six times.
  • KeratinoSens cells were first plated on 96-well plates and cultured for 24 hours at 37 ° C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of AuNP and positive controls. The treated plates were then incubated for 48 hours at 37 ° C. At the end of the treatment, the cells were washed and the production of luciferase was measured by flash luminescence. At the same time, cytotoxicity was measured by an MTT reduction test and taken into account in the interpretation of the sensitization results. Four independent analyzes were performed as part of this study.
  • the AuNP solution is deposited on the surface of the epidermis and is gently spread over the entire surface. After 15 minutes of exposure, EpiskinTM was rinsed with PBS, then incubated at 37 ° C for 6 hours. After incubation, the reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred to a tube containing 1 ml of Qjazol reagent and 2 steel balls. The epidermis was homogenized using the TissueLyser II. After centrifugation, the supernatant was collected and stored at -20 ° C until RNA extraction and analysis of 61 genes related to the irritation process, SENS-IS and ARE.
  • the study design is based on OECD guideline 439.
  • the skin irritation potential of AuNP was assessed using the Episkin TM reconstructed human epidermis model. AuNP and negative and positive controls were applied topically to tissues in triplicate and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at + 37 ° C, 5% CO in a humidified incubator. Cell viability was then assessed using the MTT reduction colorimetric test. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).
  • the acute ocular irritation potential of AuNP was evaluated by measuring their cytotoxic effect on the corneal epithelial model EpiOcular TM.
  • AuNP and negative and positive controls were applied topically to duplicate tissues and incubated at + 37 ° C for 30 minutes.
  • each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test material from the tissue, applied to absorbent material, and then incubated for an additional 2 hours at 37 ° C, 5% C0 in a humidified incubator.
  • Cell viability was then assessed using the MTT reduction colorimetric test. The mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).
  • the study design is based on OECD Guideline 490.
  • AuNPs diluted in water for injection, were tested in a single experiment, with and without a metabolic activation system (S9 mix) prepared from a fraction of microsomes hepatic (fraction S9) from rats induced with Aroclor 1254.
  • S9 mix metabolic activation system
  • Cytotoxicity was measured by evaluating the adjusted relative total growth, the relative growth of the adjusted relative suspension and the efficiency of cloning after expression time. The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.
  • the objective of this study was to assess the potential of AuNPs to induce an increase in the frequency of micronuclear cells in the L5178Y TK +/- mouse cell line.
  • the study design is based on OECD Guideline No.487.
  • the AuNPs diluted in water for injections were tested in a single cytogenetic experiment, with and without a metabolic activation system, the S9 mixture, prepared from a fraction Hepatic microsomal (fraction S9) of rats induced by Aroclor 1254. Each treatment was associated with an evaluation of cytotoxicity at the same doses. Cytotoxicity was assessed by determining the PD (population doubling) of cells. After the final cell count, the cells were washed and fixed.
  • the GAuNPs were synthesized as described previously by Morel et al (5).
  • the addition of plant extracts to the heated gold precursor solutions caused a color change from pale yellow to blue.
  • This color change attributed to the excitation of the surface plasmon (SP) band, constitutes the first evidence of the formation of AuNPs.
  • SPD surface plasmon band
  • We observed in UV-Vis spectra the disappearance of the surface plasmon band (SP) of Au (III) at 290 nm in favor of SP at 524 nm, which is due to the reduction of gold salts and the formation of Au (0), the presence of bioreducing agents which are plant extracts (14).
  • FIG. 1 shows a typical spectrum obtained with crude extracts; the surface plasmon band (SP) is wide and between 550 and 590 nm with a maximum at 550 nm; which is characteristic of anisotropic nanoparticles where we find different contributions of SP.
  • SP surface plasmon band
  • Table 1 below indicates that the mean hydrodynamic diameter of the GAuNPs is 97.7 ⁇ 7.1 nm. This diameter is Brownian motion and takes into account the layer of plant extract formed around the nanoparticle. The hydrodynamic diameter is larger than the "dry" diameter due to the effects of solvation, hydrogen bonding and van der Waals. The zeta potential of GAuNPs is -33.8 ⁇ 7.4 mV due to the negative charge of the hydroxyl group of the flavonoids covering the NPs and responsible for their stability.
  • AuNPs appeared in TEM as a polydisperse population consisting of 40 nm flower-shaped particles associated with organic components from plant extracts.
  • the flavonoids present in the plant extract are able to reduce gold ions and act as effective styling agents, stabilizing GAuNPs.
  • TEM images show nanoparticles surrounded by a thin layer with a poor electron density attributed to an organic layer, which stabilizes the gold nanoparticles.
  • the Au4f region has well-separated spin-orbit components: Au4f7 / 2 and Au4f5 / 2 at 84.2ev and 87.89ev, respectively.
  • the Au4f7 / 2 peak may be a useful binding energy reference at 84.00eV. Changes in binding energy can be observed with Au nanoparticles, but two gold states, Au (0) and Au (I) have been reported. The Au (I) state has been shifted by 2eV from the Au (0) state. From Figure 2 and Table 2 below, no Au4f peak was observed at 86.2ev. Table 2 also indicates the presence of major peaks Au4f 7/2 at 84.2ev and 86.5ev, which corresponds to Au (0).
  • Tanaka and Negishi (15) attributed the high Au4f binding energy to the binding of the surface gold atoms of the nanoparticles to the molecules surrounding the gold nanoparticles that stabilize them. This shift is attributed to an electrodonation of the NPs to the stabilizing agents.
  • Polyphenols are bidentate ligands which chelate to Au (III) forming a stable ring. Orthophenolic hydroxyls can chelate to Au (III), leading to the formation of a stable Au (III) complex.
  • the change in binding energy is attributed to the chelation effect between Au ions and ligands.
  • Region 01 shows a single peak at 532.94 eV, demonstrating a carbonyl coordination bond to the surface gold atom of the nanoparticle.
  • the crude medicinal plant extract exhibits a higher solubility in water than that of the isolated flavonoids when introduced into the reaction mixture. This suggests a synergistic interaction between the flavonoids which therefore leads to an efficient reduction reaction, nucleation and growth of nanoparticles.
  • the aqueous extract obtained with the dried leaves was analyzed by LCR spectroscopy, LC-MS / MS and 1 H NMR in reverse phase.
  • the complex nature of this mixture is attributed to the wide variety of chemical compounds all belonging to the flavonoid family. This could be considered as a complete mixture comprising a reducing agent and a styling agent.
  • CAuNPs were prepared according to the Turkevich method. A red solution was obtained at the end of the synthesis. This method is the oldest and the most widely used (11), which makes it possible to simplify the development of 15 nm gold nanoparticles functionalized by ligand exchange (FIG. 5). The nanoparticles obtained are stabilized with citrate in aqueous solution.
  • the NRU assay was used to assess changes in cell viability. After 24 h of incubation, GAuNP and CAuNP had no significant cytotoxic effect on the viability of NDHF at concentrations between 0.005 and 50 pg / ml. After 72h of incubation, as shown in Figure 7a, GAuNP did not have a significant cytotoxic effect on the viability of NHDF cells up to 25 pg / ml, whereas a dose-dependent cytotoxic effect was observed from 50 pg / ml. The concentration of 30 pg / ml was retained as the maximum dose tested to study the biological activity of GAuNP. As regards CAuNP, after 72 h of incubation, no significant cytotoxic effect on viability was observed up to 40 pg / ml, while a cytotoxic effect was observed at 80 pg / ml.
  • MMP-1 matrix metalloproteinase I
  • collagenase collagenase
  • the concentrations of MMP-1 in the various samples were corrected by the protein contents (pg / well) in the corresponding culture wells.
  • UV-A irradiation markedly increased production of MMP-1 in control NHDF cells.
  • An 8-fold increase in baseline MMP-1 was observed after UV-A exposure.
  • Treatment of cells with GAuNP and CAuNP showed a significant decrease in MMP-1 production in a dose dependent manner.
  • the IC 50 value of the antioxidant activity of GAuNP and CAuNP was found to be 9.25 pg / ml and 30.1 pg / ml, respectively.
  • Ga-tocopherol reduced by 52% the collagenase induced by UV-A irradiation.
  • GAuNPs Topical application of GAuNPs did not alter cell viability compared to control explants (not exposed or exposed to UV-A without GAuNPs), but the formation of oxidized proteins was reduced. In the absence of UV-A irradiation, GAuNPs reduced the level of endogenous oxidized proteins in the dermis. In addition, they completely inhibited the formation of oxidized proteins induced by UV-A irradiation, thus providing antioxidant protection in the dermis. GAuNPs therefore have good antioxidant activity.
  • the second set of tests was to assess the potential for skin sensitization to AuNP.
  • the KeratinoSens test the potential of AuNPs to activate the transcription factor Nrf2 was evaluated. Four analyzes were performed using different concentrations from 0.2 to 400 pg / ml. The end result is negative, in accordance with the OECD guideline, so that AuNPs do not have the potential to activate the transcription factor Nrf2.
  • a SENS-IS test was carried out to evaluate the capacity of AuNPs to induce the expression of specific biomarkers of irritation and sensitization in a model of the epidermis reconstructed in 3D.
  • the expression profile of 61 genes divided into three sets was analyzed: a set of 23 genes linked to the irritation process and the other two sets of genes, named “SENS-IS” and “ARE”, with 21 and 17 biomarkers, respectively, involved in skin sensitization (19-20).
  • SENS-IS skin-IS
  • ARE a set of 23 genes linked to the irritation process
  • 21 and 17 biomarkers respectively, involved in skin sensitization (19-20).
  • AuNPs can be classified as non-sensitizing.
  • the third set of tests was to assess skin and eye irritation. For skin irritations, the principle of the test is that the irritant chemicals are cytotoxic to the model of the reconstructed epidermis Episkin TM after short-term exposure.
  • Irritating chemicals can enter the stratum corneum and are cytotoxic enough to cause cell death in the underlying cell layers.
  • the mean relative viability of the tissues treated with AuNPs was 95% with a standard deviation of 3%.
  • AuNPs are considered non-irritant to the skin.
  • the mean relative viability of tissues treated with AuNP is 96%, with a difference of 4% between duplicate tissues.
  • the mean viability is greater than 60% after reduction of MTT, the results meet the criteria for a "non-irritant" response.
  • AuNPs are considered to be non-irritant to the reconstructed human corneal-like epithelium.
  • the fourth series of tests aimed to assess the genotoxicity of AuNPs.
  • dose levels chosen were 156.3, 312.5, 625. 1250, 2500 and 5000 pg / mL, with and without S9 admixture.
  • No precipitate was observed in the culture medium, at any dose, neither at the start nor at the end of the 3 hour treatment period.
  • No significant increase in mutation frequency was observed compared to the corresponding control vehicle, at any dose, with or without S9 admixture (IMF ⁇ FEM of 126 x 10-6).
  • the Hubertia ambavilla extract contains bioactive constituents used for the synthesis of gold nanoparticles with interesting properties, in particular for cosmetic applications.
  • Green AuNPs are not toxic, neither for fibroblast cells nor for dermal cells, and are able to scavenge free radicals efficiently. They exhibit a protective effect against damage caused by UV-A to fibroblast cells and dermal cells. Regulatory tests to ensure the safety of gold nanoparticles as ingredients in cosmetics have been performed and have shown that AuNPs are not toxic, genotoxic, irritant or sensitizing, in accordance with OECD guidelines . These results suggest that green AuNPs are a promising ingredient in cosmetics.
  • Adsersen A Adsersen H. Plants from Réunion Island with alleged antihypertensive and diuretic effects-an experimental and ethnobotanical evaluation. J Ethnopharmacol, 2017, 53 (3): 189-206.
  • Kumar B Smita K, Cumbal L, et al. Synthesis of silver nanoparticles using Sacha inchi (Plukenetia volubilis L.) leaf extracts. Saudi J Biol Sci. 2014, 21: 605-609.

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Abstract

The present invention relates to the field of gold nanoparticles used in cosmetics. The invention more particularly relates to the use of nanoparticles obtained from a mixture of gold and plant extract rich in flavonoids and to the use thereof in cosmetics.

Description

NANOPARTICULES D'OR COMPRENANT UN EXTRAIT DE PLANTES ET LEUR UTILISATION COSMETIQUE GOLD NANOPARTICLES INCLUDING A PLANT EXTRACT AND THEIR COSMETIC USE
La présente invention a trait au domaine des nanoparticules d'or utilisées en cosmétique. Elle concerne plus particulièrement l'utilisation de nanoparticules obtenues à partir d'un mélange d'or et d'extrait de plantes riches en flavonoïdes, et leur utilisation en cosmétique pour prévenir les dommages cutanés liés aux UV et aux radicaux libres. The present invention relates to the field of gold nanoparticles used in cosmetics. It relates more particularly to the use of nanoparticles obtained from a mixture of gold and plant extracts rich in flavonoids, and their use in cosmetics for preventing skin damage linked to UV rays and free radicals.
Domaine technique Technical area
Hubertia ambavilla est une plante endémique originaire de La Réunion, dans l'océan Indien, traditionnellement utilisée à la fois en usage interne et externe. Les principaux composés de Hubertia ambavilla sont les flavonoïdes, les tanins, les proanthocyanidines et le complexe glucidique conduisant à des activités thérapeutiques de la peau telles que des propriétés anti inflammatoires et cicatrisantes, ainsi que d'autres activités thérapeutiques utilisées pour traiter les infections rénales, l'asthme et le diabète (6, 7, 8). Hubertia ambavilla is an endemic plant native to Reunion Island, in the Indian Ocean, traditionally used both internally and externally. The main compounds of Hubertia ambavilla are flavonoids, tannins, proanthocyanidins and carbohydrate complex leading to skin therapeutic activities such as anti inflammatory and wound healing properties as well as other therapeutic activities used to treat kidney infections, asthma and diabetes (6, 7, 8).
Les nanoparticules sont largement utilisées et étudiées dans plusieurs domaines tels que la médecine, l'environnement ou les cosmétiques (1, 2, 3). Leur taille et leur forme peuvent être ajustées en modifiant les produits chimiques et en modifiant leur rapport. La Commission européenne définit le nanomatériau comme "une particule naturelle, accidentelle ou manufacturée et dont, pour 50% ou plus des particules dans la répartition de la taille, une ou plusieurs dimensions extérieures sont comprises entre 1 et 100 nm" (4). Nanoparticles are widely used and studied in several fields such as medicine, the environment or cosmetics (1, 2, 3). Their size and shape can be adjusted by changing the chemicals and changing their ratio. The European Commission defines nanomaterial as "a natural, accidental or manufactured particle of which, for 50% or more of the particles in the size distribution, one or more external dimensions are between 1 and 100 nm" (4).
Les nanoparticules d'or sont également bien décrites dans la littérature en tant qu'ingrédients anti-âge aux États-Unis. Elles sont utilisées notamment pour la désinfection des plaies cutanées, crème anti-inflammatoire et anti-âge. Ces nanoparticules sont obtenues par la méthode de Turkevich selon laquelle il est nécessaire de sélectionner avec soin les produits chimiques ou le produit naturel utilisés pour le revêtement de nanoparticules pour éviter les risques de toxicité. Gold nanoparticles are also well described in the literature as anti-aging ingredients in the United States. They are used in particular for the disinfection of skin wounds, anti-inflammatory and anti-aging cream. These nanoparticles are obtained by the Turkevich method according to which it is necessary to carefully select the chemicals or the natural product used for the coating of nanoparticles to avoid the risk of toxicity.
Deux protocoles différents ont été décrits dans des études antérieures (5) pour la synthèse de nanoparticules d'or extraites de l'extrait brut hydrosoluble de plantes médicinales ou du totum de flavonoïdes. Le premier protocole a conduit à des particules de forme nanofleurs à partir d'extrait brut, le deuxième à la préparation de nanoparticules sphériques plus petites et monodispersées obtenues avec le totum. La nature et le rapport des fractions phytochimiques sont d'une grande importance pour la formation de nanoparticules. Two different protocols have been described in previous studies (5) for the synthesis of gold nanoparticles extracted from the water-soluble raw extract of medicinal plants or from the flavonoid totum. The first protocol led to nanoflower-shaped particles from crude extract, the second to the preparation of smaller, monodisperse spherical nanoparticles obtained with the totum. The nature and the ratio of phytochemicals are of great importance for the formation of nanoparticles.
La peau est l'organe le plus exposé aux facteurs extérieurs qui l'endommagent. Ces facteurs sont la pollution, l'exposition aux rayons ultraviolets solaires (UV), la fumée de cigarette, etc. Ces facteurs sont responsables de la production d'espèces réactives de l'oxygène (ROS). Un excès de ROS induit un stress oxydatif qui endommage les cellules, l'ADN et les protéines, conduisant ainsi au vieillissement cutané. Dans le cas d'une exposition au soleil, les écrans solaires sont généralement recommandés pour la protection de la peau, mais leur efficacité est réduite si l'application est inadéquate et si la protection spectrale est incomplète. L'organisme dispose d'antioxydants naturels pour éliminer ces radicaux libres, mais dans le cas d'une surexposition, ces systèmes peuvent être facilement dépassés. Il est donc important d'aider la peau à se protéger en apportant une autre source d'antioxydants. The skin is the organ most exposed to external factors that damage it. These factors are pollution, exposure to solar ultraviolet (UV) rays, cigarette smoke, etc. These factors are responsible for the production of reactive oxygen species (ROS). A excess ROS induces oxidative stress which damages cells, DNA and proteins, thus leading to skin aging. In the case of sun exposure, sunscreens are generally recommended for skin protection, but their effectiveness is reduced if application is inadequate and spectral protection is incomplete. The body has natural antioxidants to eliminate these free radicals, but in the case of overexposure, these systems can be easily overwhelmed. It is therefore important to help the skin protect itself by providing another source of antioxidants.
Exposé de l'invention Disclosure of the invention
Dans le cadre de la présente invention, l'application potentielle de nanoparticules d'or vert en tant qu'ingrédient cosmétique a été étudiée. Ainsi, des tests pour évaluer les effets sur les cellules de fibroblastes humains normaux, ainsi que les activités antioxydantes et anti collagénase de ces nanoparticules ont été réalisés et comparés aux résultats obtenus avec des nanoparticules d'or synthétisées par la méthode la plus utilisée : la synthèse de Turkevich (11). In the context of the present invention, the potential application of green gold nanoparticles as a cosmetic ingredient has been investigated. Thus, tests to evaluate the effects on cells of normal human fibroblasts, as well as the antioxidant and anti-collagenase activities of these nanoparticles were carried out and compared with the results obtained with gold nanoparticles synthesized by the most widely used method: Turkevich synthesis (11).
De manière très intéressante, les inventeurs ont validé l'intérêt d'utiliser des nanoparticules comprenant un extrait de plante riche en flavonoïdes en cosmétique. Very interestingly, the inventors have validated the benefit of using nanoparticles comprising a plant extract rich in flavonoids in cosmetics.
Ainsi, l'invention concerne l'utilisation d'une composition comprenant des nanoparticules d'or comprenant un mélange d'or et un extrait de plante riche en flavonoïdes pour la prévention des dommages dus aux UV et/ou la prévention des dommages cutanés dus aux radicaux libres. Thus, the invention relates to the use of a composition comprising gold nanoparticles comprising a mixture of gold and a plant extract rich in flavonoids for the prevention of damage due to UV rays and / or the prevention of skin damage due to UV rays. to free radicals.
Dans un mode de réalisation préféré, l'extrait de plante utilisé est un extrait brut de Hubertia Ambavilla ou d'Hypericum lanceolatum. In a preferred embodiment, the plant extract used is a crude extract of Hubertia Ambavilla or Hypericum lanceolatum.
Avantages de l'invention Advantages of the invention
Les inventeurs ont étudié et caractérisé les nanoparticules d'or comprenant un extrait de plante riche en flavonoïdes. Ils ont ainsi démontré que celles-ci présentent des propriétés très intéressantes pour une utilisation en cosmétique. The inventors have studied and characterized the gold nanoparticles comprising a plant extract rich in flavonoids. They have thus demonstrated that these have very advantageous properties for use in cosmetics.
En effet, ces nanoparticules d'or sont stables du fait que les extraits de plantes, notamment les polyphénols, permettent leur stabilisation. Indeed, these gold nanoparticles are stable due to the fact that the plant extracts, in particular the polyphenols, allow their stabilization.
De plus, elles présentent des propriétés anti-oxydantes qui peuvent être attribuées à l'enrobage d'extrait de plante riche en flavonoïdes. Le pouvoir antioxydant des nanoparticules comprenant un extrait brut d'Hubertia ambavilla est particulièrement remarquable puisqu'il est supérieur à celui de la vitamine E. Ces nanoparticules permettent en particulier d'inhiber l'oxydation induite par les irradiations UV-A par piégeage des radicaux libres, offrant une protection anti-oxydante dans le derme. In addition, they exhibit antioxidant properties which can be attributed to the coating of plant extract rich in flavonoids. The antioxidant power of nanoparticles comprising a raw extract of Hubertia ambavilla is particularly remarkable since it is greater than that of vitamin E. These nanoparticles make it possible in particular to inhibit the oxidation induced by UV-A irradiations by trapping free radicals, offering anti-oxidant protection in the dermis.
L'innocuité de ces nanoparticules a été validée en conformité avec les règlementations en vigueur, ce qui est un point important. Il a été démontré qu'elles ne sont pas phototoxiques, ne sont pas sensibilisantes pour la peau, ni irritantes pour la peau et les yeux. Elles ne sont pas non plus génotoxiques. The harmlessness of these nanoparticles has been validated in accordance with the regulations in force, which is an important point. They have been shown to be not phototoxic, not sensitizing to the skin, nor irritating to the skin and eyes. They are also not genotoxic.
De plus, ces nanoparticules sont obtenues par un procédé « vert », respectueux de l'environnement et ne mettant pas en œuvre de produit toxique. In addition, these nanoparticles are obtained by a “green” process that respects the environment and does not use any toxic product.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
L'invention concerne l'utilisation d'une composition comprenant au moins une nanoparticule d'or comprenant un mélange d'or et un extrait de plantes riche en flavonoïdes pour la prévention des dommages cutanés dus aux UV et/ou aux radicaux libres. The invention relates to the use of a composition comprising at least one gold nanoparticle comprising a mixture of gold and a plant extract rich in flavonoids for the prevention of skin damage due to UV rays and / or free radicals.
Les nanoparticules d'or utilisées dans cette invention sont obtenues par un procédé écologique comprenant : (a) la préparation d'au moins un extrait de plante riche en flavonoïdes et (b) le mélange d'au moins un desdits extraits de plante avec une solution aqueuse d'au moins un sel d'or. The gold nanoparticles used in this invention are obtained by an ecological method comprising: (a) preparing at least one plant extract rich in flavonoids and (b) mixing at least one of said plant extracts with a aqueous solution of at least one gold salt.
L'extrait de plante riche en flavonoïdes peut-être un extrait total brut de ladite plante ou un totum de flavonoïdes de ladite plante. The plant extract rich in flavonoids may be a crude total extract of said plant or a total of flavonoids of said plant.
Lorsque les nanoparticules sont obtenues par mélange d'un totum de flavonoïdes de plante avec une solution aqueuse d'au moins un sel d'or, elles sont sphériques. When the nanoparticles are obtained by mixing a totum of plant flavonoids with an aqueous solution of at least one gold salt, they are spherical.
Lorsque les nanoparticules sont obtenues par mélange d'un extrait total brut de plante avec une solution aqueuse d'au moins un sel d'or, elles ont une forme de fleur et sont appelées « nanofleurs ». When the nanoparticles are obtained by mixing a total crude plant extract with an aqueous solution of at least one gold salt, they have the shape of a flower and are called “nanoflowers”.
Des nanoparticules selon l'invention et leur procédé de préparation sont décrites dans la demande WO2017/125695. Nanoparticles according to the invention and their preparation process are described in application WO2017 / 125695.
L'extrait de plante est choisi parmi un extrait de Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Ayapana triplinervis, Camellia sinensis var. assamica, Citrus hystrix, Curcuma longa, Cryptomeria japonica, Dodonaea viscosa, Mussaenda arcuate, Nuxia verticillate, Olea europea Africana, Phyllanthus casticum, Pittosporum senacia, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe ou Vepris lanceolata. The plant extract is chosen from an extract of Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Ayapana triplinervis, Camellia sinensis var. assamica, Citrus hystrix, Curcuma longa, Cryptomeria japonica, Dodonaea viscosa, Mussaenda arcuate, Nuxia verticillate, Olea europea Africana, Phyllanthus casticum, Pittosporum senacia, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe or Vepris lanceolata.
De préférence, l'extrait est choisi parmi les extraits de Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Camellia sinensis var. assamica, Citrus hystrix, Cryptomeria japonica, Mussaenda arcuate, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe ou Vepris lanceolata. Preferably, the extract is chosen from extracts of Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Camellia sinensis var. assamica, Citrus hystrix, Cryptomeria japonica, Mussaenda arcuate, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe or Vepris lanceolata.
Dans un autre mode de réalisation préféré, l'extrait de plante riche en flavonoïdes est choisi parmi un extrait de Hubertia ambavilla, Hypericum lanceolatum, Citrus hystrix, Curcuma longa, Dodonaea viscosa, Nuxia verticillate, Psidium cattleianum, ou Psiloxylon mauritianum, In another preferred embodiment, the plant extract rich in flavonoids is chosen from an extract of Hubertia ambavilla, Hypericum lanceolatum, Citrus hystrix, Curcuma longa, Dodonaea viscosa, Nuxia verticillate, Psidium cattleianum, or Psiloxylon mauritianum,
Dans un mode de réalisation tout à fait préféré, l'extrait est un d'extrait brut de Hubertia ambavilla ; la nanoparticule est alors en forme de fleur et son diamètre est compris entre 40 et 80 nm. L'intérêt particulier de cette plante a été validé par des expériences approfondies, présentées dans la partie expérimentale de ce texte. In a very preferred embodiment, the extract is a raw extract of Hubertia ambavilla; the nanoparticle is then in the shape of a flower and its diameter is between 40 and 80 nm. The particular interest of this plant has been validated by in-depth experiments, presented in the experimental part of this text.
Dans un autre mode réalisation préféré, l'extrait est un totum de Hypericum lanceolatum ; la nanoparticule est alors sphérique, son diamètre est d'environ 15 nm et elle est caractérisée par un ratio réducteur/oxydant de 21. In another preferred embodiment, the extract is a totum of Hypericum lanceolatum; the nanoparticle is then spherical, its diameter is approximately 15 nm and it is characterized by a reducing / oxidizing ratio of 21.
Les nanoparticules d'or comprenant un mélange d'or et un extrait de plantes riche en flavonoïdes sont incorporées dans une composition cosmétique. The gold nanoparticles comprising a mixture of gold and a plant extract rich in flavonoids are incorporated into a cosmetic composition.
Une telle composition peut comprendre en outre au moins un autre ingrédient cosmétique. En effet, les nanoparticules peuvent être combinées à d'autres ingrédients cosmétiques pour augmenter, renforcer ses propriétés. Such a composition can also comprise at least one other cosmetic ingredient. Indeed, nanoparticles can be combined with other cosmetic ingredients to increase and strengthen its properties.
Cette composition cosmétique peut se présenter sous forme de crème, de lotion ou toute autre forme appropriée. This cosmetic composition can be in the form of a cream, a lotion or any other suitable form.
BREVE DESCRIPTION DES FIGURES BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : (a) Spectre d'absorption d'une solution aqueuse de GAuNPs dispersés et (b) sa caractérisation par TEM. Figure 1: (a) Absorption spectrum of an aqueous solution of dispersed GAuNPs and (b) its characterization by TEM.
Figure 2 : Spectres XPS des régions (a) Au4f et (b) Ois. Figure 2: XPS spectra of (a) Au4f and (b) Ois regions.
Figure 3 : Chromatogramme HPLC d'un extrait de plante d'Hubertia Ambavilla par détection de l'aérosol chargé. L'extrait a été séparé en 5 fractions notées de A à E. Figure 4 : Chromatogrammes LC-MS / MS et UV de l'extrait aqueux de plante. Figure 3: HPLC chromatogram of a plant extract of Hubertia Ambavilla by detection of the charged aerosol. The extract was separated into 5 fractions noted from A to E. Figure 4: LC-MS / MS and UV chromatograms of the aqueous plant extract.
Figure 5 : Schéma de synthèse conventionnelle de nanoparticules d'or à l'aide de citrate (méthode de Turkevich) et conduisant à une AuNP sphérique de 15 nm entourée de citrate. Figure 5: Scheme of conventional synthesis of gold nanoparticles using citrate (Turkevich method) and leading to a spherical AuNP of 15 nm surrounded by citrate.
Figure 6 : (a) Cytotoxicité de l'AuNP sur les cellules NHDF après 72h d'incubation, (b) Effets de l'AuNP sur la production de MMP-1 induite par les UV-A. Les résultats sont exprimés en moyenne ± ET de trois expériences distinctes. Une solution de vitamine E (VE 1 mM) a été utilisée comme étalon de référence. * p <0,001 (test de Student). Figure 6: (a) Cytotoxicity of AuNP on NHDF cells after 72 h of incubation, (b) Effects of AuNP on the production of MMP-1 induced by UV-A. The results are expressed as the mean ± SD of three separate experiments. A solution of vitamin E (1 mM VE) was used as a reference standard. * p <0.001 (Student's test).
Figure 7 : Activité antioxydante par la méthode DPPH mesurée sur (a) GAuNP et (b) CAuNP. Figure 7: Antioxidant activity by the DPPH method measured on (a) GAuNP and (b) CAuNP.
EXEMPLES EXAMPLES
A - Matériels et méthodes A - Materials and methods
1. Synthèse de nanoparticules d'or (AuNPs) 1. Synthesis of gold nanoparticles (AuNPs)
1.1 Synthèse de nanoparticules d'or à l'aide d'extraits de plantes (5) 1.1 Synthesis of gold nanoparticles using plant extracts (5)
1.1.1 Préparation des extraits de plantes de Hubertia ambavilla et de Hypericum lanceolatum 1.1.1 Preparation of plant extracts of Hubertia ambavilla and Hypericum lanceolatum
La préparation de nanoparticules à partir de deux espèces de plantes est illustrée ici. La première plante est Hubertia ambavilla qui est un arbuste endémique de l'île de la Réunion. La deuxième est Hypericum lanceolatum qui est une espèce de millepertuis arborescent originaire de l'île de la Réunion. Ces deux plantes sont particulièrement riches en flavonoïdes dont la rutine et la quercétine pour Hypericum lanceolatum et l'isoquercétine et l'hyperoside pour Hubertia ambavilla. The preparation of nanoparticles from two plant species is illustrated here. The first plant is Hubertia ambavilla which is a shrub endemic to Reunion Island. The second is Hypericum lanceolatum which is a species of arborescent St. John's wort native to Reunion Island. These two plants are particularly rich in flavonoids including rutin and quercetin for Hypericum lanceolatum and isoquercetin and hyperoside for Hubertia ambavilla.
1.1.2 Préparation d'un extrait total brut de plantes 1.1.2 Preparation of a crude total plant extract
Des plantes fraîchement récoltées sont lavées à l'eau déionisée. 3 grammes sont mélangés à 50 mL d'eau déionisée puis le mélange est chauffé à 60°C pendant 5 min, ce qui permet de libérer la matière biologique par lyse des cellules végétales. Le surnageant est ensuite refroidi à température ambiante puis sur la glace pendante 10 minutes. Le surnageant refroidi est ensuite filtré sur un filtre de porosité de grade 2. Freshly harvested plants are washed with deionized water. 3 grams are mixed with 50 ml of deionized water then the mixture is heated at 60 ° C. for 5 min, which makes it possible to release the biological material by lysis of the plant cells. The supernatant is then cooled to room temperature then on hanging ice for 10 minutes. The cooled supernatant is then filtered through a grade 2 porosity filter.
Lorsque le matériel de départ est Hubertia ambavilla, l'extrait obtenu est vert. Lorsque le matériel de départ est Hypericum lanceolatum, l'extrait obtenu est marron. When the starting material is Hubertia ambavilla, the extract obtained is green. When the starting material is Hypericum lanceolatum, the extract obtained is brown.
Aucun solvant organique n'est utilisé dans cette préparation. No organic solvent is used in this preparation.
1.1.3 Isolement du totum de flavonoïdes de plantes 1.1.3 Isolation of totum flavonoids from plants
La méthode d'extraction utilisée est une méthode de macération à froid. Les plantes sont écrasées sur un tamis à pores de diamètre de 10 mm puis laissées à macérer sous agitation à 150 rpm pendant 20h à température ambiante. Un mélange à quantité égale d'eau et d'éthanol est ajouté au mélange dans un ratio solide/solvant de 1:20 pour obtenir le meilleur rendement possible en composés phénoliques (Cujic N et al). Après extraction, les macéras sont filtrés, séchés à basse pression (température maximale du bain : 45°C, pression entre 50 et 150 bars) puis lyophilisés pendant 48h. The extraction method used is a cold maceration method. The plants are crushed on a sieve with pores of 10 mm diameter and then left to macerate with stirring at 150 rpm for 20 h at room temperature. A mixture of equal amounts of water and ethanol is added to the mixture in a solid / solvent ratio of 1:20 to obtain the best possible yield of phenolic compounds (Cujic N et al). After extraction, the maceras are filtered, dried at low pressure (maximum bath temperature: 45 ° C, pressure between 50 and 150 bars) then lyophilized for 48 hours.
Le rendement d'extraction pour Hubertia ambavilla dans ces conditions est proche de 50%. The extraction yield for Hubertia ambavilla under these conditions is close to 50%.
1.1.4 Préparation des nanoparticules d'or par mélange avec des extraits de plantes Hubertia ambavilla et de Hypericum lanceolatum a) Préparation des nanoparticules d'or en forme de fleur avec les extraits totaux bruts 50 mL d'une solution aqueuse d'acide chloraurique (HAuCI4) à 1 mM sont refluées sous agitation vigoureuse dans un ballon bicol surmonté d'un réfrigérant à reflux à l'abri de la lumière. Lorsque de fines gouttelettes apparaissent sur les parois, 20 mL d'une solution aqueuse d'extraits totaux brut de plantes sont ajoutés très rapidement. La solution devient alors rapidement bleu-nuit en 1 minute. Le ballon est ensuite retiré du bain d'huile et la solution est maintenue sous agitation vigoureuse pendant 15 minutes supplémentaires. La solution est finalement maintenue à 4°C à l'abri de la lumière. 1.1.4 Preparation of gold nanoparticles by mixing with extracts of Hubertia ambavilla and Hypericum lanceolatum plants a) Preparation of flower-shaped gold nanoparticles with total crude extracts 50 mL of an aqueous solution of chlorauric acid (HAuCl4) at 1 mM are refluxed with vigorous stirring in a two-necked flask surmounted by a reflux condenser protected from light. When fine droplets appear on the walls, 20 mL of an aqueous solution of total crude plant extracts are added very quickly. The solution then quickly turns dark blue in 1 minute. The flask is then removed from the oil bath and the solution is kept under vigorous stirring for a further 15 minutes. The solution is finally maintained at 4 ° C. protected from light.
Les nanoparticules obtenues ont un diamètre mesuré en MET d'environ 40 nm. b) Préparation des nanoparticules d'or sphériques avec un totum de flavonoïdes The nanoparticles obtained have a diameter measured in TEM of approximately 40 nm. b) Preparation of spherical gold nanoparticles with a total of flavonoids
4 mL d'une solution aqueuse de totum de flavonoïdes sont refluées sous agitation vigoureuse dans un ballon bicol surmonté d'un réfrigérant à reflux à l'abri de la lumière. Lorsque de fines gouttelettes apparaissent sur les parois, 4 mL d'une solution aqueuse de HAuCI4 sont ajoutés très rapidement. La solution devient alors rapidement rouge brun en 1 minute. Le ballon est ensuite retiré du bain d'huile et la solution est maintenue sous agitation vigoureuse pendant 15 minutes supplémentaires. La solution est finalement maintenue à 4°C à l'abri de la lumière. 4 ml of an aqueous solution of flavonoids totum are refluxed with vigorous stirring in a two-necked flask topped with a reflux condenser protected from light. When fine droplets appear on the walls, 4 mL of an aqueous solution of HAuCl4 are added very quickly. The solution then quickly turns reddish brown within 1 minute. The flask is then removed from the oil bath and the solution is kept under vigorous stirring for a further 15 minutes. The solution is finally maintained at 4 ° C. protected from light.
Les nanoparticules obtenues ont un diamètre mesuré en MET d'environ 15 nm. Un ratio molaire spécifique entre les réactifs permet d'obtenir des nanoparticules d'or sphériques. Ce ratio est le suivant : n(flavonoïdes)/n(HAuCI4) = 21 The nanoparticles obtained have a diameter measured in TEM of approximately 15 nm. A specific molar ratio between the reagents makes it possible to obtain spherical gold nanoparticles. This ratio is as follows: n (flavonoids) / n (HAuCI4) = 21
1.2 Synthèse de nanoparticules d'or par la méthode de Turkevich (CAuNP) 1.2 Synthesis of gold nanoparticles by the Turkevich method (CAuNP)
On a chauffé au reflux sous agitation vigoureuse 100 ml d'une solution aqueuse de HAuCI4 1 mM dans un ballon équipé d'un réfrigérant à reflux et protégé de la lumière. Ensuite, une fois que les fines gouttelettes sont apparues sur les parois, 10 ml de citrate de potassium (38,8 mM) ont été ajoutés. 30 minutes plus tard, le ballon a été retiré du bain d'huile et la solution est restée sous agitation vigoureuse pendant 15 minutes supplémentaires. La solution rouge a été centrifugée et redispersée dans de l'eau désionisée. La solution a finalement été maintenue à 4° C, à l'abri de la lumière. 100 ml of a 1 mM aqueous HAuCl4 solution were heated to reflux with vigorous stirring in a flask fitted with a reflux condenser and protected from light. Then, after the fine droplets appeared on the walls, 10 ml of potassium citrate (38.8 mM) was added. 30 minutes later, the flask was removed from the oil bath and the solution remained under vigorous stirring for a further 15 minutes. The red solution was centrifuged and redispersed in deionized water. The solution was finally kept at 4 ° C, protected from light.
2. Caractérisation des AuNPs 2. Characterization of AuNPs
Les analyses ont été réalisées de la manière suivante : l'extrait (10 mg) a été repris dans 500 pl d'un mélange eau / acétone 4/1 v: v, l'addition d'eau seule laissant des résidus non dissous. 1 pL de cet extrait a été injecté dans notre machine LC-MS / HRMS (chaîne HPLC Dionex Ultimate 3000 avec détecteur de réseaux de diodes UV-visible et spectromètre de masse Q.- TOF Impact II Bruker) sur une colonne de 50 mm x 4 mm Nucleoshell RP18, 2,7 pm, avec un gradient entre les phases mobiles suivantes: H20 + 0,1% acide formique / acétonitrile + 0,1% acide formique. The analyzes were carried out as follows: the extract (10 mg) was taken up in 500 μl of a water / acetone 4/1 v: v mixture, the addition of water alone leaving undissolved residues. 1 μL of this extract was injected into our LC-MS / HRMS machine (Dionex Ultimate 3000 HPLC chain with UV-visible diode array detector and Q.- TOF Impact II Bruker mass spectrometer) on a 50 mm x column 4 mm Nucleoshell RP18, 2.7 µm, with a gradient between the following mobile phases: H2O + 0.1% formic acid / acetonitrile + 0.1% formic acid.
L'analyse a été effectuée en mode négatif avec une source d'électrospray. Deux injections ont été effectuées une analyse en mode MS simple et une analyse en mode dépendant des données, appelée « autoMS / MS » (c'est-à-dire en alternance entre MS unique et MS / MS sur les ions majoritaires du spectre MS précédent). Analysis was performed in negative mode with an electrospray source. Two injections were performed a single MS mode analysis and a data dependent mode analysis, called "autoMS / MS" (that is, alternating between single MS and MS / MS on the majority ions of the MS spectrum. previous).
2.1 Méthode 2.1 Method
Toutes les mesures ont été effectuées au moins en triple afin de valider la reproductibilité des procédures synthétiques et analytiques. All measurements were performed at least in triplicate in order to validate the reproducibility of the synthetic and analytical procedures.
Le spectre d'absorption UV-Vis de la solution AuNPs a été enregistré par un appareil Perkin Elmer Lambda UV / Vis 950 en utilisant des cellules en plastique standard de 1 cm à la température ambiante. Les mesures ont été effectuées dans la gamme spectrale 200-900 nm. Les images au microscope électronique à transmission (TEM) des AuNPs ont été acquises sur un microscope TEM / STEM Technai Osiris (FEI) équipé d'un détecteur de champ noir à grand angle fonctionnant à 200 kV. Pour effectuer l'analyse TEM, une goutte de 3 pl de solution AuNP préalablement traitée pendant 5 min dans un bain à ultrasons a été déposée sur une grille de cuivre recouverte de carbone. L'échantillon a été laissé sécher pendant 30 minutes à l'air ambiant. The UV-Vis absorption spectrum of the AuNPs solution was recorded by a Perkin Elmer Lambda UV / Vis 950 instrument using standard 1 cm plastic cells at room temperature. The measurements were carried out in the spectral range 200-900 nm. Transmission electron microscope (TEM) images of AuNPs were acquired on a TEM / STEM Technai Osiris (FEI) microscope equipped with a wide angle dark field detector operating at 200 kV. To perform the TEM analysis, a 3 µl drop of solution AuNP previously treated for 5 min in an ultrasonic bath was deposited on a copper grid covered with carbon. The sample was allowed to dry for 30 minutes in ambient air.
La distribution granulométrique hydrodynamique et le potentiel zêta ont été mesurés sur un Zetasizer NanoZSP (Malvern Instruments). Les mesures ont été réalisées à 25° C. The hydrodynamic particle size distribution and the zeta potential were measured on a Zetasizer NanoZSP (Malvern Instruments). The measurements were carried out at 25 ° C.
Un spectromètre ThermoFisher Scientific K-ALPHA a été utilisé pour l'analyse XPS avec une source d'AIKa monochromatisée (hv = 1486,6 eV). La taille du spot de rayons X était de 400 microns pour l'acquisition de points de surface et de 200 microns lors de la pulvérisation. Une pression de 10 à 7 Pa a été atteinte dans la chambre lors du transfert de gouttes de liquide sur une feuille d'indium. Les spectres complets (0-1100 eV) ont été obtenus avec une énergie de passe constante de 200 eV et des spectres haute résolution à une énergie de passe constante de 40 eV. La neutralisation de la charge a été appliquée pendant l'analyse. Les spectres haute résolution Cls, Ois, Au4f ont été ajustés et quantifiés à l'aide du logiciel AVANTAGE fourni par ThermoFisher Scientific (facteurs de sensibilité Scofield utilisés pour la quantification). A ThermoFisher Scientific K-ALPHA spectrometer was used for XPS analysis with a monochromatized AIKa source (hv = 1486.6 eV). The x-ray spot size was 400 microns for acquisition of surface points and 200 microns for sputtering. A pressure of 10 to 7 Pa was reached in the chamber during the transfer of drops of liquid onto an indium sheet. Full spectra (0-1100 eV) were obtained with constant pass energy of 200 eV and high resolution spectra at constant pass energy of 40 eV. Charge neutralization was applied during analysis. The high resolution C1s, Ois, Au4f spectra were adjusted and quantified using AVANTAGE software supplied by ThermoFisher Scientific (Scofield sensitivity factors used for quantification).
2.2 Stabilité de la suspension AuNP 2.2 Stability of the AuNP suspension
La stabilité de la suspension AuNP stockée à 4°C pendant plus d'un mois a été évaluée sur la base des modifications du spectre d'absorption de la solution AuNP. Ainsi, la distribution granulométrique hydrodynamique d'une suspension fraîche d'AuNP et d'une suspension âgée (âgée de 4 mois stockée à 4°C pendant toute la période) a été comparée. The stability of the AuNP suspension stored at 4 ° C for more than a month was evaluated on the basis of changes in the absorption spectrum of the AuNP solution. Thus, the hydrodynamic particle size distribution of a fresh suspension of AuNP and an aged suspension (4 months old stored at 4 ° C throughout the period) was compared.
3 Activités biologiques in vitro et ex vivo 3 Biological activities in vitro and ex vivo
3.1 Culture de cellules 3.1 Cell culture
Des fibroblastes du derme humain normal (NHDF) isolés à partir du prépuce du nouveau-né ont été utilisés. Les NHDF ont été cultivés dans du DMEM (GIBCO®, Invitrogen™) additionné de 10% de sérum de veau fœtal (SVF) et d'antibiotiques dans une atmosphère humidifiée à 37°C et à 5% de C02. Les cellules ont été cultivées en routine dans des flacons de culture de 25 à 75 cm2 et sous-cultivées régulièrement avant la confluence. Normal human dermal fibroblasts (NHDF) isolated from the foreskin of the newborn were used. NHDF were grown in DMEM (GIBCO ®, Invitrogen ™) supplemented with 10% fetal calf serum (FCS) and antibiotics in a humidified atmosphere at 37 ° C and 5% C02. The cells were cultured routinely in culture flasks of 25 to 75 cm 2 and subcultured regularly before confluence.
3.2 Cytotoxicité 3.2 Cytotoxicity
La toxicité de AuNP a été évaluée dans du NHDF ensemencé dans des microplaques à 96 puits à raison de 20 x 103 cellules par puits. La viabilité cellulaire a été évaluée par un test de neutralisation du rouge (NRU). Des solutions de AuNP ont été préparées dans un milieu complet (DMEM + 1% de FCS) après homogénéisation par ultrasons (3 cycles de 5 secondes à 20 kHz). Une large gamme de concentrations (de 0,005 à 100 pg / ml) exprimées en poids / volume de matière active a été testée. Le dosage du NRU a été effectué après 24h et 72h d'incubation. The toxicity of AuNP was evaluated in NHDF seeded in 96-well microplates at 20 x 10 3 cells per well. Cell viability was assessed by a red neutralization (NRU). AuNP solutions were prepared in complete medium (DMEM + 1% FCS) after ultrasonic homogenization (3 cycles of 5 seconds at 20 kHz). A wide range of concentrations (from 0.005 to 100 pg / ml) expressed in weight / volume of active material was tested. The NRU assay was performed after 24 h and 72 h of incubation.
3.3 Dosage DPPH 3.3 DPPH assay
Le pourcentage d'activité antioxydante a été évalué par dosage des radicaux libres au DPPH. Cette méthode est basée sur le changement de couleur de la solution de DPPH du violet au jaune pâle lorsque ce radical libre stable se convertit en une forme réduite par la réaction d'une substance capable de donner un atome d'hydrogène. The percentage of antioxidant activity was evaluated by assaying free radicals with DPPH. This method is based on the change in color of the DPPH solution from purple to pale yellow when this stable free radical is converted into a reduced form by the reaction of a substance capable of giving off a hydrogen atom.
Des solutions de AuNP ont été préparées dans de l'éthanol après homogénéisation par ultrasons (3 cycles de 5 secondes à 20 kHz). Plusieurs concentrations allant de 0,05 à 10% ont été testées. La réduction de DPPH est évaluée par le changement d'absorbance à 540 nm d'une solution de DPPH (0,126 Mm dans de l'éthanol) après 30 minutes d'incubation à 37°C avec l'AuNP. Chaque mesure a été réalisée en triple et un contrôle positif (a-tocophérol à 50 mM) a été inclus dans le test. L'activité DPPH (%) a été calculée selon la formule suivante : Solutions of AuNP were prepared in ethanol after ultrasonic homogenization (3 cycles of 5 seconds at 20 kHz). Several concentrations ranging from 0.05 to 10% were tested. The reduction of DPPH is evaluated by the change in absorbance at 540 nm of a solution of DPPH (0.126 Mm in ethanol) after 30 minutes of incubation at 37 ° C. with AuNP. Each measurement was performed in triplicate and a positive control (50 mM α-tocopherol) was included in the test. DPPH activity (%) was calculated according to the following formula:
[DPPH] = [Abs traité / contrôle absolu] x 100. [DPPH] = [Abs treated / absolute control] x 100.
3.4 Activité anti-collagénase 3.4 Anti-collagenase activity
Le test est basé sur l'évaluation de la production de métalloprotéinase 1 de matrice (MMP-1 ou collagénase) par culture de NHDF en l'absence ou en présence de AuNP et exposée aux UV- A. The test is based on the evaluation of the production of matrix metalloproteinase 1 (MMP-1 or collagenase) by culture of NHDF in the absence or presence of AuNP and exposed to UV-A.
La NHDF des cultures-mère a été récoltée avec de la trypsine provenant de flacons de culture tissulaire. Après comptage, les cellules ont été mises en suspension dans du milieu complet (DMEM + FCS) et ensemencées dans des plaques à 24 puits à une densité de 75 x I03 cellules par puits. 72 heures après le placage, le milieu a été retiré et remplacé par un milieu frais additionné de 1% de SVF et contenant diverses concentrations de AuNP. Les cellules NHDF ont ensuite été incubées pendant 24h. Avant l'irradiation, les monocouches de cellules ont été lavées avec du HBSS et exposées aux UV-A en présence de HBSS contenant la solution AuNP à différentes concentrations avec une rangée parallèle de tubes Philips TL-K 40W ACTINIC BL REFLECTOR. Immédiatement après l'irradiation, les solutions de HBSS ont été retirées et remplacées par du milieu frais additionné de 1% de FCS et contenant la solution de AuNP. Les cellules NHDF ont ensuite été replacées dans l'incubateur à 37°C pendant 24h. À la fin de l'analyse, les milieux conditionnés provenant de cultures de NHDF ont été recueillis et stockés à -20°C jusqu'à l'analyse de la MMP-1. En même temps, les monocouches de NHDF correspondantes ont été extraites pour quantifier la teneur en protéines. Les niveaux de protéines ont été déterminés en utilisant le kit de dosage de protéines BCA. Après rinçage des cellules avec HBSS, une solution de NaOH (0,1 N) a été ajoutée dans chaque puits. Après une incubation de 10 min à la température ambiante, des aliquotes de lysats ont été transférées dans une microplaque à 96 puits et du réactif BCA a été ajouté. Après une période de temps de 30 minutes, les densités optiques ont été mesurées à 570 nm. NHDF from the parent cultures was harvested with trypsin from tissue culture flasks. After counting, the cells were suspended in complete medium (DMEM + FCS) and seeded in 24-well plates at a density of 75 x 10 3 cells per well. 72 hours after plating, the medium was removed and replaced with fresh medium supplemented with 1% FCS and containing various concentrations of AuNP. The NHDF cells were then incubated for 24 hours. Before irradiation, the cell monolayers were washed with HBSS and exposed to UV-A in the presence of HBSS containing the AuNP solution at different concentrations with a parallel row of Philips TL-K 40W ACTINIC BL REFLECTOR tubes. Immediately after irradiation, the HBSS solutions were removed and replaced with fresh medium supplemented with 1% FCS and containing the AuNP solution. The NHDF cells were then placed in the incubator at 37 ° C for 24 h. At the end of the assay, conditioned media from cultures of NHDF were collected and stored at -20 ° C until analysis for MMP-1. At the same time, the corresponding NHDF monolayers were extracted to quantify the protein content. Protein levels were determined using the BCA Protein Assay Kit. After rinsing the cells with HBSS, NaOH solution (0.1 N) was added to each well. After a 10 min incubation at room temperature, aliquots of lysates were transferred to a 96-well microplate and BCA reagent was added. After a period of 30 minutes, the optical densities were measured at 570 nm.
L'activité de la MMP-1 a été déterminée à l'aide d'un dosage ELISA (Human Pro-MMP-1 Quantikine; R & D Systems®) pour la détection et la mesure quantitative de la MMP-1 dans des surnageants de culture cellulaire, conformément aux instructions du fabricant. Chaque mesure a été effectuée en triple et un contrôle positif (a-tocophérol à 1 mM) a été inclus dans le test. MMP-1 activity was determined using an ELISA assay (Human Pro-MMP-1 Quantikin; R & D Systems ® ) for the detection and quantitative measurement of MMP-1 in supernatants. culture, according to the manufacturer's instructions. Each measurement was performed in triplicate and a positive control (1mM α-tocopherol) was included in the test.
3.5 Evaluation ex vivo de l'activité antioxydante des GAuNPs sur des expiants de peau humaine 3.5 Ex vivo evaluation of the antioxidant activity of GAuNPs on explants of human skin
15 expiants de peau ont été préparés à partir d'une abdominoplatie d'une femme de type européenne blanche, phototype II, âgée de 55 ans. Ils ont été cultivés dans du BEM dans une atmosphère humidifiée à 37°C et 5% de CO2. L'application topique de GAuNPs a été réalisée avec 2 pL de solution par expiant (2 mg / cm2). Au bout de 4 jours, certains expiants ont été placés dans du HBSS et exposés à des UV-A (18 J / cm2). Immédiatement après l'irradiation, les solutions de HBSS ont été retirées et remplacées par du milieu frais, puis replacées dans l'incubateur à 37°C pendant 24 heures. Les expiants ont ensuite été coupés en deux. Une partie a été fixée dans une solution de formol tamponnée et la seconde partie a été congelée et stockée à -80°C. Après 24h, des tranches de 5 pm ont été préparées afin d'observer la viabilité cellulaire (coloration au trichrome de Masson) et les protéines oxydées (immunomarquage). 15 skin explants were prepared from the abdominoplasty of a 55-year-old white European-type woman, phototype II. They were grown in BEM in a humidified atmosphere at 37 ° C and 5% CO2. The topical application of GAuNPs was carried out with 2 μL of solution per explant (2 mg / cm 2 ). After 4 days, some explants were placed in HBSS and exposed to UV-A (18 J / cm 2 ). Immediately after irradiation, the HBSS solutions were removed and replaced with fresh medium, then returned to the incubator at 37 ° C for 24 hours. The explants were then cut in half. One part was fixed in buffered formalin solution and the second part was frozen and stored at -80 ° C. After 24 h, 5 μm slices were prepared in order to observe the cell viability (Masson trichrome staining) and the oxidized proteins (immunolabelling).
4. Etudes de sécurité 4. Safety studies
4.1 Études de toxicité 4.1.1Toxicité aiguë 4.1 Toxicity studies 4.1.1 Acute toxicity
L'étude de toxicité aiguë AuNP est basée sur le document d'orientation n°129 de l'OCDE. La toxicité a été évaluée sur des fibroblastes de souris Balb/c 3T3 ensemencés dans des microplaques à 96 puits à raison de 2xl03 cellules par puits. La viabilité cellulaire a été évaluée par un test de neutralisation du rouge (NRU). Des solutions de AuNP ont été préparées dans du DMEM après homogénéisation aux ultrasons. Une large gamme de concentrations (de 0,01 à 125 000 pg / ml) a été testée. Le dosage du NRU a été effectué après 48h d'incubation. 4.1.2 Test de phototoxicité 3T3 NRU The AuNP acute toxicity study is based on OECD Guidance Document No. 129. The toxicity was evaluated on Balb / c 3T3 mouse fibroblasts seeded in 96-well microplates at a rate of 2 × 10 3 cells per well. Cell viability was assessed by a red neutralization test (NRU). Solutions of AuNP were prepared in DMEM after ultrasonic homogenization. A wide range of concentrations (0.01-125,000 pg / ml) were tested. The NRU assay was performed after 48 hours of incubation. 4.1.2 3T3 NRU phototoxicity test
Le test compare la cytotoxicité de produits chimiques appliqués à des fibroblastes de souris (Balb / c 3T3, clone A31) en présence ou en l'absence d'exposition à un niveau non cytotoxique de lumière UV-A (5 J / cm2). La cytotoxicité a été mesurée en tant qu'inhibition de la capacité à absorber le colorant essentiel, le rouge neutre (NR), un jour après le traitement UV-A. Cette étude était basée sur les lignes directrices de l'OCDE n°432. La phototoxicité a été évaluée dans des fibroblastes de souris Balb/c 3T3 ensemencés dans des microplaques à 96 puits à lxlO4 cellules par puits. Les plaques ont été incubées pendant 24 heures, puis le milieu de culture a été retiré et les cellules ont été lavées avec du HBSS préchauffé. Ensuite, pour chaque plaque, huit concentrations de AuNP et de CPZ (témoin positif) ont été appliquées aux cellules (six réplicats / concentration). Les cellules ont été exposées au produit pendant 1 heure. À la fin de la période de traitement, une plaque par condition de test ou contrôle positif a été exposée aux rayons ultraviolets, tandis que l'autre plaque est restée dans l'obscurité. Pour les plaques irradiées, les cellules ont été irradiées avec 5 J / cm2 à température ambiante dans un UVACUBE 400 (Sol-500) équipé d'un filtre H1 à travers le couvercle de la plaque à 96 puits. 50 minutes après le début des traitements légers, les solutions de chaque puits de toutes les plaques ont été retirées et les cellules ont été lavées deux fois avec du HBSS préchauffé. Le dosage du NRU a été utilisé pour évaluer les changements de la viabilité cellulaire après l'incubation de cellules de NHDF avec de l'AuNP à diverses concentrations (de 0,01 à 200 pg / ml) pendant 24 ou 72 heures d'incubation. Les cellules témoins ont été incubées avec du milieu complet (DMEM + 1% de SVF) et les expériences ont été répétées six fois. The test compares the cytotoxicity of chemicals applied to mouse fibroblasts (Balb / c 3T3, clone A31) in the presence or absence of exposure to a non-cytotoxic level of UV-A light (5 J / cm2). Cytotoxicity was measured as inhibition of the ability to absorb the essential dye, neutral red (NR), one day after UV-A treatment. This study was based on OECD guideline 432. The phototoxicity was assessed in Balb / c 3T3 mouse fibroblasts seeded in 96-well microplates at 1x10 4 cells per well. The plates were incubated for 24 hours, then the culture medium was removed and the cells were washed with preheated HBSS. Then, for each plate, eight concentrations of AuNP and CPZ (positive control) were applied to the cells (six replicates / concentration). The cells were exposed to the product for 1 hour. At the end of the treatment period, one plate per test condition or positive control was exposed to UV light, while the other plate was left in the dark. For the irradiated plates, the cells were irradiated with 5 J / cm 2 at room temperature in a UVACUBE 400 (Sol-500) equipped with an H1 filter through the cover of the 96-well plate. 50 minutes after the start of the light treatments, solutions from each well of all plates were removed and cells were washed twice with pre-warmed HBSS. The NRU assay was used to assess changes in cell viability after incubation of NHDF cells with AuNP at various concentrations (from 0.01 to 200 pg / ml) for 24 or 72 hours of incubation. . Control cells were incubated with complete medium (DMEM + 1% FCS) and the experiments were repeated six times.
4.2 Test de sensibilisation 4.2 Sensitization test
4.2.1 Test Keratinosens 4.2.1 Keratinosens test
Les cellules KeratinoSens ont d'abord été étalées sur des plaques à 96 puits et cultivées pendant 24 heures à 37°C. Ensuite, le milieu a été retiré et les cellules ont été exposées au véhicule témoin ou à différentes concentrations de AuNP et de témoins positifs. Les plaques traitées ont ensuite été incubées pendant 48 heures à 37°C. A la fin du traitement, les cellules ont été lavées et la production de luciférase a été mesurée par luminescence éclair. Parallèlement, la cytotoxicité a été mesurée par un test de réduction au MTT et prise en compte dans l'interprétation des résultats de sensibilisation. Quatre analyses indépendantes ont été réalisées dans le cadre de cette étude. KeratinoSens cells were first plated on 96-well plates and cultured for 24 hours at 37 ° C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of AuNP and positive controls. The treated plates were then incubated for 48 hours at 37 ° C. At the end of the treatment, the cells were washed and the production of luciferase was measured by flash luminescence. At the same time, cytotoxicity was measured by an MTT reduction test and taken into account in the interpretation of the sensitization results. Four independent analyzes were performed as part of this study.
4.2.2 SENS-IS test 4.2.2 SENS-IS test
La solution AuNP est déposée à la surface de l'épiderme et est doucement étendue sur toute la surface. Après 15 minutes d'exposition, EpiskinTM a été rincé avec du PBS, puis incubé à 37° C pendant 6 heures. Après incubation, l'épiderme reconstruit a été retiré des inserts avec une pince et placé dans un cryotube pour la congélation dans de l'azote liquide. L'épiderme a ensuite été transféré dans un tube contenant 1 ml de réactif Qjazol et 2 billes d'acier. L'épiderme a été homogénéisé au moyen du TissueLyser II. Après centrifugation, le surnageant a été recueilli et stocké à -20°C jusqu'à l'extraction de l'ARN et l'analyse de 61 gènes liés au processus d'irritation, SENS-IS et ARE. The AuNP solution is deposited on the surface of the epidermis and is gently spread over the entire surface. After 15 minutes of exposure, EpiskinTM was rinsed with PBS, then incubated at 37 ° C for 6 hours. After incubation, the reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred to a tube containing 1 ml of Qjazol reagent and 2 steel balls. The epidermis was homogenized using the TissueLyser II. After centrifugation, the supernatant was collected and stored at -20 ° C until RNA extraction and analysis of 61 genes related to the irritation process, SENS-IS and ARE.
4.3 Tests d'irritation 4.3 Irritation tests
4.3.1 Test In vitro d'irritation de la peau 4.3.1 In vitro skin irritation test
Le plan de l'étude repose sur les lignes directrices de l'OCDE n ° 439. Le potentiel d'irritation cutanée de l'AuNP a été évalué à l'aide du modèle de l'épiderme humain reconstruit Episkin™. Les AuNP et les contrôles négatifs et positifs ont été appliqués localement sur des tissus en triple exemplaire et incubés à température ambiante pendant 15 minutes. À la fin de la période de traitement, chaque tissu a été rincé avec du D-PBS et incubé pendant 42 heures à + 37°C, 5% de CO dans un incubateur humidifié. La viabilité cellulaire a ensuite été évaluée au moyen du test colorimétrique de réduction au MTT. Les valeurs de viabilité relative ont été calculées pour chaque tissu et exprimées sous forme de pourcentage de la viabilité moyenne des tissus de contrôle négatifs qui a été fixée à 100% (en tant que viabilité de référence). The study design is based on OECD guideline 439. The skin irritation potential of AuNP was assessed using the Episkin ™ reconstructed human epidermis model. AuNP and negative and positive controls were applied topically to tissues in triplicate and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at + 37 ° C, 5% CO in a humidified incubator. Cell viability was then assessed using the MTT reduction colorimetric test. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).
4.3.2 Test In vitro d'irritation des yeux 4.3.2 In vitro eye irritation test
Le potentiel d'irritation oculaire aiguë de l'AuNP a été évalué par la mesure de leur effet cytotoxique sur le modèle épithélial cornéen EpiOcular™. Les AuNP et les contrôles négatifs et positifs ont été appliqués localement sur des tissus en double et incubés à + 37°C pendant 30 minutes. À la fin de la période de traitement, chaque tissu a été rincé avec du D-PBS, incubé pendant 12 minutes à température ambiante pour éliminer tout élément de test restant du tissu, appliqué sur un matériau absorbant, puis incubé pendant 2 heures supplémentaires à 37°C, 5% de CÜ dans un incubateur humidifié. La viabilité cellulaire a ensuite été évaluée au moyen du test colorimétrique de réduction au MTT. Les valeurs de viabilité moyenne ont été calculées pour chaque tissu et exprimées sous forme de pourcentage de la viabilité moyenne des tissus de contrôle négatifs qui a été fixée à 100% (comme viabilité de référence). The acute ocular irritation potential of AuNP was evaluated by measuring their cytotoxic effect on the corneal epithelial model EpiOcular ™. AuNP and negative and positive controls were applied topically to duplicate tissues and incubated at + 37 ° C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test material from the tissue, applied to absorbent material, and then incubated for an additional 2 hours at 37 ° C, 5% C0 in a humidified incubator. Cell viability was then assessed using the MTT reduction colorimetric test. The mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).
4.4 Tests micronucléaire et mutations 4.4 Micronuclear tests and mutations
4.4.1 Test in vitro de mutation génique sur cellules de mammifères 4.4.1 In vitro mammalian cell gene mutation test
Le plan de l'étude repose sur les lignes directrices de l'OCDE n°490. Les AuNP, diluées dans de l'eau pour préparation injectable, ont été testées au cours d'une expérience unique, avec et sans système d'activation métabolique (S9 mix) préparé à partir d'une fraction de microsomes hépatiques (fraction S9) de rats induits avec Aroclor 1254. Des cultures de 20 mL à 5 x 105 cellules de lymphome de souris L5178Y TK +/- / mL ont été exposées à l'AuNP ou aux éléments de contrôle, en présence ou en l'absence du mélange S9 (concentration finale de la fraction S9 de 2%). Au cours de la période de traitement de 3 heures, les cellules ont été maintenues sous forme de culture en suspension dans un milieu de culture RPMI 1640 complété par du sérum de cheval inactivé par la chaleur à 5% dans un incubateur humidifié à 37°C et 5% de CO2. La cytotoxicité a été mesurée par évaluation de la croissance totale relative ajustée, de la croissance relative de la suspension relative ajustée et de l'efficacité du clonage après le temps d'expression. Le nombre de clones mutants (différenciant petites et grandes colonies) a été évalué après expression du phénotype mutant. The study design is based on OECD Guideline 490. AuNPs, diluted in water for injection, were tested in a single experiment, with and without a metabolic activation system (S9 mix) prepared from a fraction of microsomes hepatic (fraction S9) from rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 10 5 mouse lymphoma cells L5178Y TK +/- / mL were exposed to AuNP or controls, in the presence or in the absence of the S9 mixture (final concentration of the S9 fraction of 2%). During the 3 hour treatment period, cells were maintained as a suspension culture in RPMI 1640 culture medium supplemented with 5% heat inactivated horse serum in a humidified 37 ° C incubator. and 5% CO2. Cytotoxicity was measured by evaluating the adjusted relative total growth, the relative growth of the adjusted relative suspension and the efficiency of cloning after expression time. The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.
4.4.2 Test In vitro micronucléaire 4.4.2 Micronuclear In Vitro Test
L'objectif de cette étude était d'évaluer le potentiel des AuNP à induire une augmentation de la fréquence des cellules micronucléées dans la lignée cellulaire de souris L5178Y TK +/-. Le plan de l'étude repose sur les directives de l'OCDE n°487. Après un test préliminaire de cytotoxicité, les AuNP diluées dans de l'eau pour préparations injectables ont été testées au cours d'une seule expérience cytogénétique, avec et sans système d'activation métabolique, le mélange S9, préparé à partir d'une fraction microsomique hépatique (fraction S9) de rats induite par Aroclor 1254. Chaque traitement a été associé à une évaluation de la cytotoxicité aux mêmes doses. La cytotoxicité a été évaluée en déterminant le PD (doublage de population) des cellules. Après le comptage final des cellules, les cellules ont été lavées et fixées. Ensuite, des cellules provenant de trois niveaux de dose de cultures traitées avec AuNP ont été déposées sur des lames de verre propres. Les lames ont été séchées à l'air avant d'être colorées dans du Giemsa à 5%. Les lames de cultures témoins et positives ont également été préparées comme décrit ci-dessus. Pour chaque expérience principale (avec ou sans mélange S9), les micronoyaux ont été analysés pour trois doses d'AuNP, pour le véhicule et les témoins positifs, dans 1000 cellules mononucléées par culture (total de 2000 cellules mononucléées par dose). Le nombre de cellules avec des micronoyaux et le nombre de micronoyaux par cellule ont été enregistrés séparément pour chaque culture traitée et témoin. The objective of this study was to assess the potential of AuNPs to induce an increase in the frequency of micronuclear cells in the L5178Y TK +/- mouse cell line. The study design is based on OECD Guideline No.487. After a preliminary cytotoxicity test, the AuNPs diluted in water for injections were tested in a single cytogenetic experiment, with and without a metabolic activation system, the S9 mixture, prepared from a fraction Hepatic microsomal (fraction S9) of rats induced by Aroclor 1254. Each treatment was associated with an evaluation of cytotoxicity at the same doses. Cytotoxicity was assessed by determining the PD (population doubling) of cells. After the final cell count, the cells were washed and fixed. Next, cells from three dose levels of cultures treated with AuNP were spotted onto clean glass slides. Slides were air dried before staining in 5% Giemsa. Slides of control and positive cultures were also prepared as described above. For each main experiment (with or without S9 mixture), micronuclei were analyzed for three doses of AuNP, for vehicle and positive controls, in 1000 mononuclear cells per culture (total of 2000 mononuclear cells per dose). The number of cells with micronuclei and the number of micronuclei per cell were recorded separately for each treated and control culture.
B-RESULTATS B-RESULTS
1. Synthèse et caractérisation des GAuNPs 1. Synthesis and characterization of GAuNPs
Les GAuNP ont été synthétisées comme décrit précédemment par Morel et al (5). L'ajout d'extraits de plantes aux solutions de précurseurs d'or chauffées a provoqué un changement de couleur du jaune pâle au bleu. Ce changement de couleur, attribué à l'excitation de la bande de plasmons de surface (SP), constitue la première preuve de la formation d'AuNPs. Nous avons observé dans les spectres UV-Vis la disparition de la bande de plasmons de surface (SP) de Au (III) à 290 nm au profit de SP à 524 nm, ce qui est dû à la réduction des sels d'or et à la formation de Au (0), présence d'agents de bioréduction qui sont des extraits de plantes (14). The GAuNPs were synthesized as described previously by Morel et al (5). The addition of plant extracts to the heated gold precursor solutions caused a color change from pale yellow to blue. This color change, attributed to the excitation of the surface plasmon (SP) band, constitutes the first evidence of the formation of AuNPs. We observed in UV-Vis spectra the disappearance of the surface plasmon band (SP) of Au (III) at 290 nm in favor of SP at 524 nm, which is due to the reduction of gold salts and the formation of Au (0), the presence of bioreducing agents which are plant extracts (14).
La spectroscopie UV-Vis permet d'évaluer la formation et la stabilité de nanoparticules en solution aqueuse. La figure 1 montre un spectre typique obtenu avec des extraits bruts ; la bande de plasmon de surface (SP) est large et comprise entre 550 et 590 nm avec un maximum à 550 nm ; ce qui est caractéristique des nanoparticules anisotropiques où l'on retrouve différentes contributions de SP. UV-Vis spectroscopy makes it possible to evaluate the formation and the stability of nanoparticles in aqueous solution. Figure 1 shows a typical spectrum obtained with crude extracts; the surface plasmon band (SP) is wide and between 550 and 590 nm with a maximum at 550 nm; which is characteristic of anisotropic nanoparticles where we find different contributions of SP.
Le tableau 1 ci-dessous indique que le diamètre hydrodynamique moyen des GAuNPs est de 97,7 ± 7,1 nm. Ce diamètre est le mouvement brownien et prend en compte la couche d'extrait de plante formée autour de la nanoparticule. Le diamètre hydrodynamique est plus grand que le diamètre « sec » en raison des effets de solvatation, de liaison hydrogène et de van der Waals. Le potentiel zêta des GAuNPs est de -33,8 ± 7,4 mV en raison de la charge négative du groupe hydroxyle des flavonoïdes recouvrant les NP et responsable de leur stabilité. Table 1 below indicates that the mean hydrodynamic diameter of the GAuNPs is 97.7 ± 7.1 nm. This diameter is Brownian motion and takes into account the layer of plant extract formed around the nanoparticle. The hydrodynamic diameter is larger than the "dry" diameter due to the effects of solvation, hydrogen bonding and van der Waals. The zeta potential of GAuNPs is -33.8 ± 7.4 mV due to the negative charge of the hydroxyl group of the flavonoids covering the NPs and responsible for their stability.
Figure imgf000015_0001
Figure imgf000015_0001
Tableau 1 : Diamètre hydrodynamique (RH), indice de polydispersité (PDI) et potentiel zêta (ZP) mesurés sur une solution de GAuNP. Les résultats sont exprimés en moyenne ± SD de cinq expériences distinctes. Table 1: Hydrodynamic diameter (RH), polydispersity index (PDI) and zeta potential (ZP) measured on a solution of GAuNP. Results are expressed as the mean ± SD of five separate experiments.
Ces résultats corroborent les données TEM et XPS des Figures 1 et 2. These results corroborate the TEM and XPS data in Figures 1 and 2.
En effet, les AuNPs apparaissaient dans le TEM sous forme d'une population polydispersée constituée de particules en forme de fleur de 40 nm associées à des composants organiques provenant d'extraits de plantes. Les flavonoïdes présents dans l'extrait de plante sont capables de réduire les ions d'or et d'agir comme agents de coiffage efficaces, stabilisant les GAuNPs. Les images TEM montrent des nanoparticules entourées d'une couche mince avec une densité électronique médiocre attribuée à une couche organique, qui stabilise les nanoparticules d'or. Indeed, AuNPs appeared in TEM as a polydisperse population consisting of 40 nm flower-shaped particles associated with organic components from plant extracts. The flavonoids present in the plant extract are able to reduce gold ions and act as effective styling agents, stabilizing GAuNPs. TEM images show nanoparticles surrounded by a thin layer with a poor electron density attributed to an organic layer, which stabilizes the gold nanoparticles.
La région Au4f présente les composantes spin-orbite bien séparées: Au4f7 / 2 et Au4f5 / 2 à respectivement 84,2ev et 87,89ev. Selon Casaletto et al., le pic Au4f7 / 2 peut être une référence d'énergie de liaison utile à 84,00eV. Des changements d'énergie de liaison peuvent être observés avec des nanoparticules d'Au, mais deux états d'or, Au (0) et Au (I) ont été rapportés. L'état Au (I) a été décalé de 2eV de l'état Au (0). D'après la figure 2 et le tableau 2 ci-dessous, aucun pic de Au4f n'a été observé à 86,2ev. Le tableau 2 indique également la présence de pics majeurs Au4f 7/2 à 84,2ev et 86,5 ev, ce qui correspond à Au (0). The Au4f region has well-separated spin-orbit components: Au4f7 / 2 and Au4f5 / 2 at 84.2ev and 87.89ev, respectively. According to Casaletto et al., The Au4f7 / 2 peak may be a useful binding energy reference at 84.00eV. Changes in binding energy can be observed with Au nanoparticles, but two gold states, Au (0) and Au (I) have been reported. The Au (I) state has been shifted by 2eV from the Au (0) state. From Figure 2 and Table 2 below, no Au4f peak was observed at 86.2ev. Table 2 also indicates the presence of major peaks Au4f 7/2 at 84.2ev and 86.5ev, which corresponds to Au (0).
Figure imgf000016_0001
Figure imgf000016_0001
Tableau 2 : Pics d'énergie de liaison obtenus en XPS pour GAuNP Table 2: Binding energy peaks obtained in XPS for GAuNP
Tanaka et Negishi (15) ont attribué la haute énergie de liaison Au4f à la liaison des atomes d'or de surface des nanoparticules aux molécules entourant les nanoparticules d'or qui les stabilisent. Ce décalage est attribué à une électrodonation des NP vers les agents stabilisants. Les polyphénols sont des ligands bidentés qui se chélatent en Au (III) en formant un cycle stable. Les hydroxyles orthophénoliques peuvent chélater en Au (III), conduisant à la formation d'un complexe stable Au (III). Tanaka and Negishi (15) attributed the high Au4f binding energy to the binding of the surface gold atoms of the nanoparticles to the molecules surrounding the gold nanoparticles that stabilize them. This shift is attributed to an electrodonation of the NPs to the stabilizing agents. Polyphenols are bidentate ligands which chelate to Au (III) forming a stable ring. Orthophenolic hydroxyls can chelate to Au (III), leading to the formation of a stable Au (III) complex.
Ensuite, le complexe est réduit à Au (0) in situ. Les autres parties des groupes hydroxyle orthophénoliques sont oxydées en quinone correspondante, à savoir l'orthoquinone. La stabilisation peut être effectuée via OH libre ou carbonyl quinones. Then the complex is reduced to Au (0) in situ. The other parts of the orthophenolic hydroxyl groups are oxidized to the corresponding quinone, namely orthoquinone. Stabilization can be carried out via free OH or carbonyl quinones.
Le changement d'énergie de liaison est attribué à l'effet chélateur entre les ions Au et les ligands. The change in binding energy is attributed to the chelation effect between Au ions and ligands.
On peut en conclure que l'or Au (III) a été réduit par des extraits de plantes, ce qui a donné des nanoparticules d'or Au (0) stabilisées par des polyphénols. It can be concluded that Au (III) gold was reduced by plant extracts, resulting in Au (0) gold nanoparticles stabilized by polyphenols.
La région 01 présente un pic unique à 532,94 eV, démontrant une liaison de coordination carbonyle à l'atome d'or de surface de la nanoparticule. Region 01 shows a single peak at 532.94 eV, demonstrating a carbonyl coordination bond to the surface gold atom of the nanoparticle.
Des études de stabilité ont été effectuées sur la suspension de nanoparticules. Pendant un mois, des spectres d'absorption sur une suspension d'AuNP stockées à 4°C ont été régulièrement mesurés pour analyser la présence d'agrégats. Le pic d'absorption était similaire pendant toute cette période, indiquant une absence d'agrégation. De plus, le diamètre hydrodynamique a été mesuré sur une solution fraîche et 4 mois plus tard. Les valeurs obtenues pour la suspension fraîche et la suspension vieillie étaient comparables et presque inchangées. Stability studies were carried out on the suspension of nanoparticles. For one month, absorption spectra on a suspension of AuNP stored at 4 ° C were regularly measured to analyze the presence of aggregates. The absorption peak was similar throughout this period, indicating an absence of aggregation. In addition, the hydrodynamic diameter was measured on a fresh solution and 4 months later. The values obtained for the fresh suspension and the aged suspension were comparable and almost unchanged.
2. Composition chimique de l'extrait de plante médicinale utilisé comme agent de bioréduction 2. Chemical composition of medicinal plant extract used as a bioreduction agent
L'extrait brut de plante médicinale présente une solubilité dans l'eau supérieure à celle des flavonoïdes isolés lorsqu'il est introduit dans le mélange réactionnel. Ceci suggère une interaction synergique entre les flavonoïdes qui conduit par conséquent à une réaction de réduction efficace, à la nucléation et à la croissance des nanoparticules. The crude medicinal plant extract exhibits a higher solubility in water than that of the isolated flavonoids when introduced into the reaction mixture. This suggests a synergistic interaction between the flavonoids which therefore leads to an efficient reduction reaction, nucleation and growth of nanoparticles.
L'extrait aqueux obtenu avec des feuilles séchées a été analysé par spectroscopie LCR, LC-MS / MS et RMN 1H en phase inverse. La nature complexe de ce mélange est attribuée à la grande variété de composés chimiques appartenant tous à la famille des flavonoïdes. Cela pourrait être considéré comme un mélange complet comprenant un agent réducteur et un agent coiffant. The aqueous extract obtained with the dried leaves was analyzed by LCR spectroscopy, LC-MS / MS and 1 H NMR in reverse phase. The complex nature of this mixture is attributed to the wide variety of chemical compounds all belonging to the flavonoid family. This could be considered as a complete mixture comprising a reducing agent and a styling agent.
Le chromatogramme HPLC, utilisant la détection des aérosols chargés (CAD) sur la figure 3, a montré 21 pics majeurs. L'extrait a été séparé en 5 fractions A-E. The HPLC chromatogram, using charged aerosol detection (CAD) in Figure 3, showed 21 major peaks. The extract was separated into 5 fractions A-E.
L'analyse LC-MS / MS de l'extrait aqueux de la figure 4 permet l'identification et la quantification relative de 18 composés. Cette quantification a été établie en utilisant l'apigénine comme référence. Nous en avons déduit que les 18 composés identifiés représentaient 7,7% de l'extrait sec. À partir de cette première information, des hypothèses structurelles pourraient être formulées, en référence à la bibliographie (4, 16). LC-MS / MS analysis of the aqueous extract of Figure 4 allows the identification and relative quantification of 18 compounds. This quantification was established using apigenin as a reference. We deduced that the 18 compounds identified represented 7.7% of the dry extract. From this first information, structural hypotheses could be formulated, with reference to the bibliography (4, 16).
3. Synthèse et caractérisation de CAuNP 3. Synthesis and characterization of CAuNP
Les CAuNPs ont été préparées selon la méthode de Turkevich. Une solution rouge a été obtenue à la fin de la synthèse. Cette méthode est la plus ancienne et la plus utilisée (11), ce qui permet de simplifier la mise au point de nanoparticules d'or de 15 nm fonctionnalisées par échange de ligand (figure 5). Les nanoparticules obtenues sont stabilisées par du citrate en solution aqueuse. CAuNPs were prepared according to the Turkevich method. A red solution was obtained at the end of the synthesis. This method is the oldest and the most widely used (11), which makes it possible to simplify the development of 15 nm gold nanoparticles functionalized by ligand exchange (FIG. 5). The nanoparticles obtained are stabilized with citrate in aqueous solution.
4. Activités biologiques 4. Biological activities
Le dosage du NRU a été utilisé pour évaluer les changements de la viabilité cellulaire. Après 24h d'incubation, GAuNP et CAuNP n'avaient pas d'effet cytotoxique significatif sur la viabilité du NDHF à des concentrations comprises entre 0,005 et 50 pg / ml. Après 72h d'incubation, comme le montre la figure 7a, GAuNP n'avait pas d'effet cytotoxique significatif sur la viabilité des cellules NHDF jusqu'à 25 pg / ml, alors qu'un effet cytotoxique dépendant de la dose était observé à partir de 50 pg / ml. La concentration de 30 pg / ml a été retenue comme dose maximale testée pour étudier l'activité biologique du GAuNP. En ce qui concerne CAuNP, après 72h d'incubation, aucun effet cytotoxique significatif sur la viabilité n'a été observé jusqu'à 40 pg / ml, tandis qu'un effet cytotoxique a été observé à 80 pg / ml. The NRU assay was used to assess changes in cell viability. After 24 h of incubation, GAuNP and CAuNP had no significant cytotoxic effect on the viability of NDHF at concentrations between 0.005 and 50 pg / ml. After 72h of incubation, as shown in Figure 7a, GAuNP did not have a significant cytotoxic effect on the viability of NHDF cells up to 25 pg / ml, whereas a dose-dependent cytotoxic effect was observed from 50 pg / ml. The concentration of 30 pg / ml was retained as the maximum dose tested to study the biological activity of GAuNP. As regards CAuNP, after 72 h of incubation, no significant cytotoxic effect on viability was observed up to 40 pg / ml, while a cytotoxic effect was observed at 80 pg / ml.
Les propriétés anti-radicalaires de l'AuNP ont été évaluées à l'aide d'une méthode basée sur le balayage du radical DPPH. Différentes concentrations de AuNP ont été testées de 0,05 à 10% et chaque mesure a été réalisée en triple. Un contrôle positif (a-tocophérol à 50 pM) a été inclus dans le test. Comme le montre la figure 7a, GAuNP possède une activité de piégeage radical des DPPH. Une inhibition dépendante de la dose de DPPH a été observée dans la plage des concentrations testées, ce qui a permis de déterminer la CI50 à partir de la courbe de régression [DPPh (%) = f (concentration)]. La valeur IC50 pour l'activité antioxydante de GAuNP s'est avérée être de 3,29%, soit 16,5 pg / ml. Dans les mêmes conditions expérimentales, le signal DPPH était inhibé à 96% avec 50 pM d'a-tocophérol (VE) et aucune inhibition n'était observée avec CAuNP (Figure 7b). Les CAuNPs synthétisées avec le protocole standard n'ont pas d'activité antioxydante. Donc, obtenir de « simples » nanoparticules d'or ne suffit pas pour obtenir des propriétés antioxydantes. L'enrobage d'extrait de plante améliore la propriété d'élimination des radicaux libres des nanoparticules d'or. De plus, les interactions électrostatiques entre les composés phytochimiques chargés négativement et les GAuNP semblent contribuer de manière synergique à l'amélioration de la bioactivité inhérente des plantes médicinales (17, 18). Nous pouvons en conclure que l'extrait de plante a permis la formation de nanoparticules hautement antioxydantes, beaucoup plus élevées que la vitamine E. The anti-radical properties of AuNP were evaluated using a method based on scanning for the DPPH radical. Different concentrations of AuNP were tested from 0.05 to 10% and each measurement was performed in triplicate. A positive control (50 µM α-tocopherol) was included in the test. As shown in Figure 7a, GAuNP possesses radical scavenging activity for DPPH. Dose dependent inhibition of DPPH was observed over the range of concentrations tested, allowing the IC50 to be determined from the regression curve [DPPh (%) = f (concentration)]. The IC50 value for the antioxidant activity of GAuNP was found to be 3.29%, or 16.5 pg / ml. Under the same experimental conditions, the DPPH signal was 96% inhibited with 50 pM α-tocopherol (VE) and no inhibition was observed with CAuNP (Figure 7b). CAuNPs synthesized with the standard protocol have no antioxidant activity. So just getting "simple" gold nanoparticles is not enough to get antioxidant properties. The plant extract coating improves the free radical scavenging property of gold nanoparticles. Additionally, electrostatic interactions between negatively charged phytochemicals and GAuNPs appear to synergistically contribute to the enhancement of the inherent bioactivity of medicinal plants (17, 18). We can conclude that the plant extract allowed the formation of highly antioxidant nanoparticles, much higher than vitamin E.
Afin d'évaluer le potentiel dermoprotecteur des NP dans les cellules de la peau humaine, une approche expérimentale in vitro basée sur la mesure de l'activité de la métalloprotéinase I (MMP-1 ou collagénase) de la matrice dans des cellules NHDF exposées au rayonnement UV- A a été réalisée. Les taux de MMP-1 ont été quantifiés dans le milieu de culture après traitement et irradiation UV-A. Les cellules NDHF ont été traitées avec AuNP avant (24h) et après (24h) irradiation UV. Chaque condition expérimentale a été analysée trois fois et un contrôle positif (a-tocophérol à 1 mM) a été inclus dans l'essai. Les concentrations de MMP- 1 dans les différents échantillons (ng / puits) ont été corrigées par les teneurs en protéines (pg / puits) dans les puits de culture correspondants. Comme le montre la figure 6b, l'irradiation UV-A a nettement augmenté la production de MMP-1 dans les cellules NHDF témoins. Une augmentation de 8 fois de la MMP-1 de base a été observée après une exposition aux UV-A. Le traitement des cellules avec GAuNP et CAuNP a montré une diminution significative de la production de MMP-1 de manière dépendante de la dose. La valeur IC50 de l'activité antioxydante de GAuNP et CAuNP s'est avérée être de 9,25 pg / ml et de 30,1 pg / ml, respectivement. Dans les mêmes conditions expérimentales, Ga-tocophérol réduit de 52% la collagénase induite par l'irradiation UV-A. L'application topique de GAuNPs n'a pas modifié la viabilité cellulaire par rapport aux expiants témoins (non exposés ou exposés aux UV-A sans GAuNPs), mais la formation de protéines oxydées a été réduite. En l'absence d'irradiation UV-A, les GAuNPs ont diminué le taux de protéines oxydées endogènes dans le derme. En outre, ils ont totalement inhibé la formation de protéines oxydées induites par les irradiations UV-A, offrant ainsi une protection antioxydante dans le derme. Les GAuNPs ont donc une bonne activité antioxydante. In order to assess the dermoprotective potential of NPs in human skin cells, an in vitro experimental approach based on the measurement of the activity of matrix metalloproteinase I (MMP-1 or collagenase) in NHDF cells exposed to UV- A radiation was carried out. The levels of MMP-1 were quantified in the culture medium after treatment and UV-A irradiation. The NDHF cells were treated with AuNP before (24 h) and after (24 h) UV irradiation. Each experimental condition was analyzed three times and a positive control (1 mM α-tocopherol) was included in the test. The concentrations of MMP-1 in the various samples (ng / well) were corrected by the protein contents (pg / well) in the corresponding culture wells. As shown in Figure 6b, UV-A irradiation markedly increased production of MMP-1 in control NHDF cells. An 8-fold increase in baseline MMP-1 was observed after UV-A exposure. Treatment of cells with GAuNP and CAuNP showed a significant decrease in MMP-1 production in a dose dependent manner. The IC 50 value of the antioxidant activity of GAuNP and CAuNP was found to be 9.25 pg / ml and 30.1 pg / ml, respectively. Under the same experimental conditions, Ga-tocopherol reduced by 52% the collagenase induced by UV-A irradiation. Topical application of GAuNPs did not alter cell viability compared to control explants (not exposed or exposed to UV-A without GAuNPs), but the formation of oxidized proteins was reduced. In the absence of UV-A irradiation, GAuNPs reduced the level of endogenous oxidized proteins in the dermis. In addition, they completely inhibited the formation of oxidized proteins induced by UV-A irradiation, thus providing antioxidant protection in the dermis. GAuNPs therefore have good antioxidant activity.
5. Etudes de sécurité 5. Safety studies
Les objectifs de ces tests étaient d'évaluer l'innocuité des AuNP en tant que matière première pour les cosmétiques. Depuis l'entrée en vigueur de l'interdiction mondiale des tests sur les produits cosmétiques pour animaux, plusieurs tests in vitro ont été réalisés pour évaluer : la toxicité, la sensibilisation cutanée in vitro, l'irritation in vitro et la toxicologie génétique. The objectives of these tests were to assess the safety of AuNP as a raw material for cosmetics. Since the entry into force of the worldwide ban on testing of cosmetic products for animals, several in vitro tests have been carried out to assess: toxicity, in vitro skin sensitization, in vitro irritation and genetic toxicology.
Pour l'essai de toxicité aiguë, après 48 incubations avec AuNP, aucune diminution de la viabilité cellulaire n'a été constatée, quelle que soit la concentration ; par conséquent, aucune CI50 et donc aucune DL50 n'a été estimée. Donc, selon cette étude, les AuNP ne sont pas considérés comme cytotoxiques. Plusieurs concentrations comprises entre 0,32 et 1 000 pg / ml ont ensuite été utilisées pour évaluer la phototoxicité. La phototoxicité est définie comme une réponse toxique d'une substance qui est soit déclenchée, soit augmentée après une exposition à la lumière. 24 heures après l'exposition à la lumière, aucune modification de la morphologie des cellules n'a été observée et il n'y avait aucune diminution de l'absorption de NR à aucune des concentrations testées dans les plaques irradiées et non irradiées. Dans les conditions expérimentales de cette étude, il a été déterminé que les AuNP testées jusqu'à 1 000 pg / ml n'étaient pas phototoxiques, conformément aux classifications présentées dans la directive 432 de l'OCDE. For the acute toxicity test, after 48 incubations with AuNP, no decrease in cell viability was observed, whatever the concentration; therefore, no IC50 and therefore no LD50 was estimated. Therefore, according to this study, AuNPs are not considered cytotoxic. Several concentrations between 0.32 and 1000 pg / ml were then used to assess phototoxicity. Phototoxicity is defined as a toxic response of a substance that is either triggered or increased after exposure to light. 24 hours after light exposure, no change in cell morphology was observed and there was no decrease in NR uptake at any of the concentrations tested in irradiated and non-irradiated plates. Under the experimental conditions of this study, it was determined that AuNPs tested up to 1000 pg / ml were not phototoxic, in accordance with the classifications presented in OECD guideline 432.
La deuxième série de tests visait à évaluer le potentiel de sensibilisation de la peau aux AuNP. Avec le test KeratinoSens, le potentiel des AuNP pour activer le facteur de transcription Nrf2 a été évalué. Quatre analyses ont été effectuées en utilisant différentes concentrations de 0,2 à 400 pg / ml. Le résultat final est négatif, en accord avec la directive de l'OCDE, de sorte que les AuNP n'ont pas le potentiel d'activer le facteur de transcription Nrf2. En complément de ce test, un test SENS-IS a été réalisé pour évaluer la capacité des AuNP à induire l'expression de biomarqueurs spécifiques d'irritation et de sensibilisation dans un modèle d'épiderme reconstruit en 3D. Le profil d'expression de 61 gènes divisés en trois ensembles a été analysé : un ensemble de 23 gènes liés au processus d'irritation et les deux autres ensembles de gènes, nommés «SENS-IS» et «ARE», avec 21 et 17 biomarqueurs respectivement, impliqués dans la sensibilisation de la peau (19-20). En présence d'AuNP, moins de 7 gènes des groupes de gènes «SENS-IS» et «ARE» ont été exprimés, conduisant à un test négatif. En conclusion, les AuNP peuvent être classées comme non sensibilisantes. La troisième série d'essais consistait à évaluer l'irritation de la peau et des yeux. Pour les irritations cutanées, le principe de l'essai repose sur le fait que les produits chimiques irritants sont cytotoxiques pour le modèle de l'épiderme reconstruit Episkin™ après une exposition de courte durée. Les produits chimiques irritants peuvent pénétrer dans la couche cornée et sont suffisamment cytotoxiques pour entraîner la mort cellulaire dans les couches cellulaires sous- jacentes. Après une exposition de 15 minutes et une période de récupération de 42 heures, la viabilité moyenne relative des tissus traités avec les AuNP était de 95% avec un écart type de 3%. Dans les conditions expérimentales de cette étude, les AuNP sont considérées comme non irritantes pour la peau. En ce qui concerne l'irritation oculaire, la viabilité moyenne relative des tissus traités avec les AuNP est de 96%, avec une différence de 4% entre les tissus dupliqués. Comme la viabilité moyenne est supérieure à 60% après la réduction du MTT, les résultats répondent aux critères de réponse « non irritante ». Dans les conditions expérimentales de cette étude, les AuNP sont considérées comme non irritantes pour l'épithélium humain reconstruit ressemblant à la cornée. The second set of tests was to assess the potential for skin sensitization to AuNP. With the KeratinoSens test, the potential of AuNPs to activate the transcription factor Nrf2 was evaluated. Four analyzes were performed using different concentrations from 0.2 to 400 pg / ml. The end result is negative, in accordance with the OECD guideline, so that AuNPs do not have the potential to activate the transcription factor Nrf2. In addition to this test, a SENS-IS test was carried out to evaluate the capacity of AuNPs to induce the expression of specific biomarkers of irritation and sensitization in a model of the epidermis reconstructed in 3D. The expression profile of 61 genes divided into three sets was analyzed: a set of 23 genes linked to the irritation process and the other two sets of genes, named “SENS-IS” and “ARE”, with 21 and 17 biomarkers, respectively, involved in skin sensitization (19-20). In the presence of AuNP, less than 7 genes from the “SENS-IS” and “ARE” gene groups were expressed, leading to a negative test. In conclusion, AuNPs can be classified as non-sensitizing. The third set of tests was to assess skin and eye irritation. For skin irritations, the principle of the test is that the irritant chemicals are cytotoxic to the model of the reconstructed epidermis Episkin ™ after short-term exposure. Irritating chemicals can enter the stratum corneum and are cytotoxic enough to cause cell death in the underlying cell layers. After an exposure of 15 minutes and a recovery period of 42 hours, the mean relative viability of the tissues treated with AuNPs was 95% with a standard deviation of 3%. Under the experimental conditions of this study, AuNPs are considered non-irritant to the skin. Regarding eye irritation, the mean relative viability of tissues treated with AuNP is 96%, with a difference of 4% between duplicate tissues. As the mean viability is greater than 60% after reduction of MTT, the results meet the criteria for a "non-irritant" response. Under the experimental conditions of this study, AuNPs are considered to be non-irritant to the reconstructed human corneal-like epithelium.
La quatrième série d'essais visait à évaluer la génotoxicité des AuNP. Pour la recherche de mutations de gènes cellulaires, en utilisant une solution d'AuNP à la concentration de 500 mg / mL dans le véhicule et un volume de traitement de 1% (v / v) dans un milieu de culture, les niveaux de dose choisis étaient 156,3, 312,5, 625. 1250, 2500 et 5000 pg / mL, avec et sans mélange S9. Aucun précipité n'a été observé dans le milieu de culture, à quelque dose que ce soit, ni au début ni à la fin de la période de traitement de 3 heures. Aucune augmentation notable de la fréquence de mutation n'a été constatée par rapport au véhicule témoin correspondant, à quelque dose que ce soit, avec ou sans mélange S9 (IMF <FEM de 126 x 10- 6). De plus, aucune relation dose-réponse n'a été démontrée par la régression linéaire. Ainsi, ces résultats répondaient aux critères de réponse négative. Dans les conditions expérimentales de cette étude, les AuNP n'ont montré aucune activité mutagène dans le test du lymphome de souris, que ce soit en présence ou en l'absence d'un système de métabolisation du foie chez le rat. Pour l'analyse micronucléaires, les doses choisies pour l'analyse du micronoyau étaient les suivantes : 1250, 2500 et 5000 pg / mL, ce dernier étant le niveau de dose recommandé le plus élevé. Après les traitements de 3 heures avec et sans mélange S9 ou le traitement de 24 heures sans mélange S9, aucune augmentation statistiquement significative ni liée à la dose de la fréquence des cellules micronucléées n'a été notée à aucune des doses analysées par rapport au témoin correspondant. De plus, aucune des doses analysées n'a montré une fréquence de cellules micronuclées des deux cultures répliquées au-dessus de la plage historique du véhicule correspondante. Ainsi, ces résultats répondaient aux critères d'une réponse négative. Dans les conditions expérimentales de l'étude, l'AuNP n'a provoqué aucun dommage chromosomique ni aucun dommage à l'appareil de division cellulaire, dans des cellules somatiques de mammifères en culture, en utilisant des cellules de lymphome de souris L5178Y TK +/-, en présence ou en l'absence de foie système métabolisant. C- CONCLUSION The fourth series of tests aimed to assess the genotoxicity of AuNPs. For testing for cellular gene mutations, using a solution of AuNP at a concentration of 500 mg / mL in vehicle and a treatment volume of 1% (v / v) in culture medium, dose levels chosen were 156.3, 312.5, 625. 1250, 2500 and 5000 pg / mL, with and without S9 admixture. No precipitate was observed in the culture medium, at any dose, neither at the start nor at the end of the 3 hour treatment period. No significant increase in mutation frequency was observed compared to the corresponding control vehicle, at any dose, with or without S9 admixture (IMF <FEM of 126 x 10-6). In addition, no dose-response relationship was demonstrated by linear regression. Thus, these results met the criteria for a negative response. Under the experimental conditions of this study, AuNPs did not show any mutagenic activity in the mouse lymphoma test, either in the presence or absence of a liver metabolism system in rats. For micronuclear analysis, the doses chosen for the micronucleus analysis were: 1250, 2500 and 5000 pg / mL, the latter being the highest recommended dose level. After the 3 hour treatments with and without S9 admixture or the 24 hour treatment without S9 admixture, no statistically significant or dose-related increase in micronuclear cell frequency was noted at any of the doses analyzed compared to the control. corresponding. In addition, none of the doses analyzed showed a micronuclear cell frequency of the two replicated cultures above the corresponding historical vehicle range. Thus, these results met the criteria for a negative response. Under the experimental conditions of the study, AuNP did not cause any chromosome damage or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK + mouse lymphoma cells. / -, in the presence or absence of liver metabolizing system. C- CONCLUSION
Cette étude montre l'intérêt des nanoparticules en tant que matériau pour les applications cosmétiques. L'extrait d 'Hubertia ambavilla contient des constituants bioactifs utilisés pour la synthèse de nanoparticules d'or aux propriétés intéressantes, notamment pour les applications cosmétiques. Les AuNPs vertes ne sont pas toxiques, ni pour les cellules de fibroblastes, ni pour les cellules dermiques, et sont capables de piéger efficacement les radicaux libres. Ils présentent un effet protecteur contre les dommages causés par les UV-A aux cellules de fibroblastes et aux cellules dermiques. Des tests réglementaires visant à garantir la sécurité des nanoparticules d'or en tant qu'ingrédients dans les cosmétiques ont été réalisés et ont démontré que les AuNP ne sont pas toxiques, ni génotoxiques, ni irritants ni sensibilisants, conformément aux directives de l'OCDE. Ces résultats suggèrent que les AuNP vertes sont un ingrédient prometteur dans les cosmétiques. This study shows the interest of nanoparticles as a material for cosmetic applications. The Hubertia ambavilla extract contains bioactive constituents used for the synthesis of gold nanoparticles with interesting properties, in particular for cosmetic applications. Green AuNPs are not toxic, neither for fibroblast cells nor for dermal cells, and are able to scavenge free radicals efficiently. They exhibit a protective effect against damage caused by UV-A to fibroblast cells and dermal cells. Regulatory tests to ensure the safety of gold nanoparticles as ingredients in cosmetics have been performed and have shown that AuNPs are not toxic, genotoxic, irritant or sensitizing, in accordance with OECD guidelines . These results suggest that green AuNPs are a promising ingredient in cosmetics.
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Claims

REVENDICATIONS
1. Utilisation d'une composition comprenant au moins une nanoparticule d'or comprenant un mélange d'or et un extrait de plantes riche en flavonoïdes pour la prévention des dommages cutanés dus aux UV et/ou aux radicaux libres. 1. Use of a composition comprising at least one gold nanoparticle comprising a mixture of gold and a plant extract rich in flavonoids for the prevention of skin damage due to UV rays and / or free radicals.
2. Utilisation selon la revendication 1 dans laquelle ledit extrait est un extrait brut. 2. Use according to claim 1 wherein said extract is a crude extract.
3. Utilisation selon la revendication 1 dans laquelle ledit extrait est un totum. 3. Use according to claim 1 wherein said extract is a totum.
4. Utilisation selon l'une revendications 1 à 3 dans laquelle l'extrait de plante est choisi parmi un extrait de Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Ayapana triplinervis, Camellia sinensis var. assamica, Citrus hystrix, Curcuma longa, Cryptomeria japonica, Dodonaea viscosa, Mussaenda arcuate, Nuxia verticillate, Olea europea Africana, Phyllanthus casticum, Pittosporum senacia, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe ou Vepris lanceolata. 4. Use according to one of claims 1 to 3, in which the plant extract is chosen from an extract of Hubertia ambavilla, Hypericum lanceolatum, Aphloia theiformis, Ayapana triplinervis, Camellia sinensis var. assamica, Citrus hystrix, Curcuma longa, Cryptomeria japonica, Dodonaea viscosa, Mussaenda arcuate, Nuxia verticillate, Olea europea Africana, Phyllanthus casticum, Pittosporum senacia, Psidium cattleianum, Psiloxylon mauritianum, Terminalia bentzoe or Vepris lanceolata.
5. Utilisation selon l'une revendications 1 à 4 dans laquelle ledit extrait de plante est choisi parmi un extrait de Hubertia ambavilla, Hypericum lanceolatum, Citrus hystrix, Curcuma longa, Dodonaea viscosa, Nuxia verticillate, Psidium cattleianum, ou Psiloxylon mauritianum, 5. Use according to one of claims 1 to 4 wherein said plant extract is chosen from an extract of Hubertia ambavilla, Hypericum lanceolatum, Citrus hystrix, Curcuma longa, Dodonaea viscosa, Nuxia verticillate, Psidium cattleianum, or Psiloxylon mauritianum,
6. Utilisation selon l'une des revendications précédentes dans laquelle ledit extrait de plante est un extrait brut de Hubertia ambavilla. 6. Use according to one of the preceding claims wherein said plant extract is a crude extract of Hubertia ambavilla.
7. Utilisation selon l'une des revendications précédentes dans laquelle ledit extrait de plante est un totum de Hypericum lanceolatum. 7. Use according to one of the preceding claims wherein said plant extract is a totum of Hypericum lanceolatum.
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