Colloids and Surfaces B: Biointerfaces 189 (2020) 110855

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Assessment of antioxidant and dermoprotective activities of gold T

nanoparticles as safe cosmetic ingredient
Maroua Ben Haddadaa,b, Elise Geromettaa,c, Rachid Chawecha, Jonathan Sorresa, Anne Bialeckic,
Sabrina Pesnela, Jolanda Spadavecchiab, Anne-Laure Morela,*
a
TORSKAL nanosciences, 2 rue Maxime Rivière, 97490, Sainte-Clotilde, La Réunion, France
b
CNRS, UMR 7244, CSPBAT, Laboratoire de Chimie, Structures et Propriétés de Biomateriaux et d’Agents Thérapeutiques, Université Paris 13, Sorbonne Paris Cité,
Bobigny, France
c
LCSNSA - Laboratoire de Chimie des Substances Naturelles et des Sciences des Aliments – UR, Faculté des Sciences et Technologies, Université de La Réunion, 15 avenue
René Cassin, France

A R T I C LE I N FO A B S T R A C T

Keywords: Hubertia ambavilla, an endemic plant originating from Reunion Island in the Indian Ocean, is traditionally used
Nanoparticle as an anti-inflammatory and in healing, both for internal and external use. Polyphenolic compounds from
Gold nanoparticle aqueous phase extractions can reduce metal salts into nanoparticles and stabilize them in one step. Although
Plant extract gold nanoparticles are well described in the literature as anti-ageing ingredients, the nanoparticles presented
Medicinal plant
herein are novel and are synthesized using a green process. We demonstrate their efficiency as dermoprotective,
Antioxidant
Dermoprotective
free radical scavenger and antioxidant cosmetic ingredients. Comparison with common nanoparticles obtained
Green chemistry by the Turkevich method clearly emphasizes the necessity to carefully screen the products used for nanoparticle
Cosmetic ingredients coatings, as they play a major role in the biological properties of the product. Hubertia ambavilla mediated gold
nanoparticles are non-toxic to human dermal fibroblasts, possess free radical scavenging potential, and protect
against damage to fibroblast and dermal cells caused by ultraviolet A radiation.

1. Introduction disinfectants, anti-inflammation and anti-ageing creams. Skin is ex-

Nanoparticles are widely used and investigated in several areas such
as medicine, environmental research or cosmetics [1–3]. The European
commission defines a nanomaterial as "a natural, incidental or manu-
factured particulate and where, for 50 % or more of the particles in the
number size distribution, one or more external dimensions is in the size
range 1–100 nm" [4].
Two different protocols were previously described [5] for the
synthesis of gold nanoparticles from either the hydrosoluble crude ex-
tract of the medicinal plants, or from the totum of flavonoids. The use of
hydrosoluble crude extracts led to nanoflower shaped particles,
whereas the totum produced smaller, monodispersed spherical nano-
particles. The nature and the ratio of the phytochemical fractions are of
great significance for the formation of nanoparticles. The major com-
pounds of Hubertia ambavilla are flavonoids, tannins, proanthocyanidins
and carbohydrate complexes that may confer therapeutic anti-in-
flammatory and healing properties, and other therapeutic activities
used to treat renal infections, asthma and diabetes [6–8].
Gold nanoparticles are used in cosmetics, especially as skin wound
posed to many factors that damage it, including pollution, exposure to
solar ultraviolet (UV) rays, cigarette smoke, etc. These factors are re-
sponsible for the production of reactive oxygen species (ROS). An ex-
cess of ROS induces oxidative stress that damages cells, DNA and pro-
tein, thus leading to skin ageing by upregulating the expression of
collagen- and elastin-degrading matrix metalloproteinases (MMP) [9].
It is important to help the skin to protect itself by providing an addi-
tional source of antioxidants [10].
In the present study, we studied the potential application of green
gold nanoparticles as a cosmetic ingredient. We assessed their effects on
normal human fibroblast cells as well as antioxidant activities and
compared them to results obtained with the oldest Turkevich method
[11].
2. Experimental
2.1. Reagents and chemicals
Tetrachloroauric acid (HAuCl4) was purchased from Sigma-Aldrich

⁎
Corresponding author.
E-mail address: annelaure.morel@torskal.fr (A.-L. Morel).

https://doi.org/10.1016/j.colsurfb.2020.110855
Received 20 July 2019; Received in revised form 13 January 2020; Accepted 6 February 2020
Available online 11 February 2020
0927-7765/ © 2020 Elsevier B.V. All rights reserved.
M. Ben Haddada, et al.
(Saint-Quentin Fallavier, France) and potassium citrate from ACROS
Organics (Geel, Belgium). Plants originated from natural habitats in
Reunion Island and were collected with the necessary permissions.
2.2. Synthesis of gold nanoparticles
2.2.1. Green synthesis of gold nanoparticles using plant extracts [5]
Green gold nanoparticles (GAuNP) were prepared according to
previously described procedure (5). Briefly, 50 mL of an aqueous so-
lution of HAuCl4, 1 mM, was refluxed with vigorous stirring, in a flask
equipped with a reflux condenser and protected from light. Then, once
the fine droplets appeared on the walls, 20 mL of acidic aqueous solu-
tion of total brut plant extract was added very quickly. The solution
turned blue rapidly in 1 min.
2.2.2. Synthesis of gold nanoparticles using Turkevich method
100 mL of an aqueous solution of 1 mM HAuCl4 was refluxed with
vigorous stirring in a flask equipped with a reflux condenser and pro-
tected from light. Then, once fine droplets appeared on the walls, 10 mL
of potassium citrate (38.8 mM) were added.
2.3. Characterization of AuNP
All the measurements were performed at least in triplicate.
The UV–vis absorption spectra (wavelength range: 200−900 nm)
were recorded by a Perkin Elmer Lambda UV/Vis 950 using standard
1 cm plastic cells at room temperature.
3 μl drop of AuNP solution deposited on a carbon-coated copper
grid, were analysed by Transmission electron microscopy (TEM) ac-
quired on a TEM/STEM Technai Osiris microscope (FEI) equipped with
a high angle dark field detector operating at 200 kV.
Hydrodynamic particle size distributions and zeta potential were
measured on a Zetasizer NanoZSP (Malvern Instruments) at 25 °C.
2.4. Stability of GAuNP suspensions
Stability of green gold nanoparticles (GAuNP) suspensions stored at
4 °C for over one month was evaluated based on changes in the ab-
sorption spectra of the GAuNP solutions. We additionally compared the
hydrodynamic particle size distributions of fresh and aged (4 months
stored at 4 °C) suspensions.
2.5. Characterization of medicinal plant extracts for the synthesis of green
gold nanoparticles
2.5.1. HPLC-UV-ESI-HR-MS/MS analysis
The extract (10 mg) was taken up in 500 μl of a 4/1 v:v water/
acetone mixture, since the addition of water alone left undissolved re-
sidues. 1 μL of this extract was injected into our LC–MS / HRMS ma-
chine (Dionex Ultimate 3000 HPLC chain with UV–vis diode array de-
tector and Q-TOF Impact II Bruker mass spectrometer) on a Nucleoshell
RP18 column 50 mm × 4 mm i.d., 2.7 μm, with a gradient between the
following mobile phases: H2O+0.1 % formic acid/acetonitrile + 0.1 %
formic acid. The analysis was carried out in negative mode with an
electrospray source. Two injections were made: a simple MS mode
analysis, and a data-dependent mode analysis, called autoMS/MS (i.e.
alternating between single MS and MS/MS on the majority ions of the
previous MS spectrum).
2.5.2. 1H NMR analysis
1
H-NMR spectra were obtained using a Bruker Ascend 600 NMR
Spectrometer operating at 600 MHz in CD3OD. In a typical experiment,
5 mg of fraction C was dissolved in 1 mL of CD3OD in an NMR tube and
readings were taken between 0–14 ppm. The residual methanol re-
sonance was used as the internal reference. Coupling constants are
given in Hertz. The chemical shifts are expressed in δ (ppm).
2
Colloids and Surfaces B: Biointerfaces 189 (2020) 110855
2.6. Biological activities
2.6.1. In vitro cell culture
Normal human dermal fibroblasts (NHDF) isolated from newborn
foreskin were used. NHDF were cultured in DMEM (GIBCO®,
Invitrogen™) supplemented with 10 % fetal calf serum (FCS) and anti-
biotics in a humidified atmosphere at 37 °C and 5 % CO2. Cells were
routinely grown into 25- 75-cm² culture flasks and subcultured reg-
ularly before confluence.
2.6.2. In vitro cytotoxicity
The AuNP toxicity was evaluated in NHDF seeded in 96-well mi-
croplates at 20 × 103 cells per well. Cell viability was assessed by
Neutral Red Uptake (NRU) assay. Solutions of AuNP were prepared in
complete medium (DMEM + 1 % FCS) after ultrasound homogeniza-
tion (3 cycles of 5 s at 20 kHz). A broad range of concentrations (from
0.005 to 100 μg/mL) expressed in w/v of active material was tested.
NRU assays were performed after 24 h and 72 h of incubation.
2.6.3. In vitro DPPH assay
The percentage of antioxidant activity was assessed using the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free radical assay. Solutions of AuNP
were prepared in ethanol after ultrasound homogenization (3 cycles of
5 s at 20 kHz). Concentrations ranging from 0.05 % to 10 % were tested.
The reduction of DPPH was evaluated by the change of absorbance at
540 nm of a DPPH solution (0.126 Mm in ethanol) after 30 min in-
cubation at 37 °C with the AuNP. Each measure was performed in tri-
plicate and a positive control (α-tocopherol at 50 μM) was included in
the assay. DPPH activity (%) was calculated according to the formula:
[DPPH] = [Abs Treated / Abs Control] x 100.
2.6.4. In vitro anti-collagenase activity
The assay was based on the evaluation of matrix metalloproteinase
1 (MMP-1 or collagenase) production by NHDF culture in the absence
or presence of the GAuNP and exposed to ultraviolet A rays (UV-A).
Ultraviolet A (UVA) has a longer wavelength and is associated with skin
aging.
After counting, cells were suspended in complete medium
(DMEM + FCS) and seeded in 24-well plates at a density of 75 × 103
cells per well. 72 h after plating, the medium was removed and replaced
by fresh medium supplemented with 1% FCS and containing various
concentrations of GAuNP. NHDF cells were then incubated for 24 h.
Before irradiation, cell monolayers were washed with HBSS and ex-
posed to UV-A in the presence of HBSS containing the AuNP solution at
different concentrations with a parallel bank of Philips TL-K 40 W
ACTINIC BL REFLECTOR tubes. Immediately after irradiation, the HBSS
solutions were withdrawn and replaced by fresh medium supplemented
with 1% FCS and containing the AuNP solution. NHDF cells were then
replaced in the incubator at 37 °C for 24 h. At the end of the assay, the
conditioned media from NHDF cultures were collected and stored at
−20 °C until MMP-1 analysis. Concurrently, corresponding NHDF
monolayers were extracted to quantify protein content.
The protein levels were determined using the BCA protein Assay Kit.
After rinsing cells with HBSS, NaOH solution (0.1 N) was added into
each well. After 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 min, optical densities were measured at
570 nm.
MMP-1 activity was determined using an ELISA assay (Human Pro-
MMP-1 Quantikine; R&D Systems®) for the detection and quantitative
measurement of MMP-1 in cell culture supernatants, according to the
manufacturer’s instructions. Each measure was performed in triplicate
and a positive control (α-tocopherol at 1 mM) was included on the
assay.
M. Ben Haddada, et al.
2.6.5. Ex vivo evaluation of the antioxidant activity on human skin explants
15 skin explants were prepared from an abdominoplasty of a
Caucasian woman, phototype II, 55 years-old. They were cultured in
BEM in a humidified atmosphere at 37 °C and 5 % CO2. Topical ap-
plication of GAuNP were realized with 2 μL of solution per explant
(2 mg/cm²). After 4 days, some explants were placed in HBSS and ex-
posed to UV-A (18 J/cm²). Immediately after irradiation, the HBSS so-
lutions were withdrawn and replaced by fresh medium and then re-
placed in the incubator at 37 °C for 24 h. The explants were then cut in
half. One part was fixed in a buffered formalin solution and the second
part was frozen and stored at −80 °C. After 24 h, 5 μm slices were
prepared to observed cell viability (Masson’s trichrome stain) and oxi-
dized proteins (immunolabelling).
2.7. Safety studies
2.7.1. Toxicity studies
2.7.1.1. Acute toxicity. The AuNP acute toxicity study was based on the
OECD Guidance document n°129. The toxicity was evaluated in Balb/c
3T3 mouse fibroblasts seeded in 96-well microplates at 2 × 103 cells
per well. Cell viability was assessed by Neutral Red Uptake (NRU)
assay. Solutions of AuNP were prepared in DMEM after ultrasound
homogenization. A broad range of concentrations (from
0.01–125000 μg/mL) was tested. NRU assay was performed after 48 h
incubation.
2.7.1.2. 3T3 NRU phototoxicity tests. The assay 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 the
inhibition of the capacity to take up the vital dye, Neutral Red (NR),
one day after UV-A treatment. This study was based on the OECD
Guideline n° 432. The phototoxicity was evaluated in Balb/c 3T3 mouse
fibroblasts seeded in 96-well microplates at 1 × 104 cells per well.
Plates were incubated for 24 h, then culture medium was removed, and
cells were washed with pre-warmed 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 one hour. At the end of the treatment period, one plate
per test item or positive control was exposed to UV light while the other
plate remained in the dark. For the irradiated plates, the cells were
irradiated with 5 J/cm2 at room temperature in an UVACUBE 400 (Sol-
500) equipped with an H1 filter through the lid of the 96-well plate.
50 min after the start of light treatments, the solutions from each well of
all plates were removed and the cells were washed twice with pre-
warmed HBSS. The NRU assay was used to assess changes in cell
viability after NHDF cells were incubated with AuNP at various
concentrations (from 0.01–200 μg/mL) for 24 or 72 h incubation.
Control cells were incubated with complete medium (DMEM + 1 %
FCS) and experiments were repeated six times.
2.7.2. Sensitization assays
2.7.2.1. Keratinosens test. The KeratinoSens cells were first plated on
96-well plates and grown for 24 h at 37 °C. Then the medium was
removed and the cells were exposed to the vehicle control or to
different concentrations of AuNP and of positive controls. The treated
plates were then incubated for 48 h at 37 °C. At the end of the
treatment, cells were washed, and luciferase production was
measured by flash luminescence. In parallel, the cytotoxicity was
measured by the MTT reduction test and was taken into consideration
in the interpretation of the sensitization results.
2.7.2.2. SENS-IS test. The AuNP solution was deposited on the
epidermis surface and gently spread on the entire surface. After
15 min of exposure, the Episkin™ was rinsed with PBS and then
incubated at 37 °C for 6 h. After incubation, reconstructed epidermis
3
Colloids and Surfaces B: Biointerfaces 189 (2020) 110855
was removed from the inserts with forceps and placed in a cryotube for
freezing in liquid nitrogen. The epidermis was transferred into a tube
containing 1 mL of Qiazol reagent and 2 steel beads, then 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.
2.7.3. Irritation tests
2.7.3.1. In vitro skin irritation test. The study design was based on the
OECD Guideline No. 439. The skin irritation potential of AuNP was
assessed using the Episkin™ reconstructed human epidermis model. The
AuNP and both negative and positive controls were applied topically
onto triplicate tissues and incubated at room temperature for 15 min. At
the end of the treatment period, each tissue was rinsed with D-PBS and
incubated for 42 h at +37 °C, 5% CO2 in a humidified incubator. Cell
viability was then assessed by means of the colorimetric MTT reduction
assay.
2.7.3.2. In vitro eye irritation test. The acute eye irritation potential of
AuNP was assessed by measurement of their cytotoxic effect on the
EpiOcular™ cornea epithelial model. The AuNP and both negative and
positive controls were applied topically onto duplicate tissues and
incubated at 37 °C for 30 min. At the end of the treatment period, each
tissue was rinsed with D-PBS, incubated for 12 min at room temperature
to remove any remaining test item from the tissue, blotted on absorbent
material, and then incubated for another 2 h at 37 °C, 5% CO2 in a
humidified incubator. Cell viability was then assessed by means of the
colorimetric MTT reduction assay.
2.7.4. Mutation and micronucleus tests
2.7.4.1. In vitro mammalian cell gene mutation test. The study design was
based on the OECD Guideline No. 490. AuNP, diluted in water for
injections, were tested in a single experiment, with and without a
metabolic activation system (S9 mix) prepared from the liver
microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
Cultures of 20 mL at 5 × 105 L5178Y TK+/− mouse lymphoma cells/
mL were exposed to the AuNP or controls in the presence or absence of
S9 mix (final concentration of S9 fraction 2%). During the 3 -h
treatment period, the cells were maintained as a suspension culture in
RPMI 1640 culture medium supplemented by heat inactivated horse
serum at 5% in a 37 °C, 5% CO2 humidified incubator. Cytotoxicity was
measured by assessment of the adjusted relative total growth, adjusted
relative suspension growth and cloning efficiency following the
expression time. The number of mutant clones (differentiated by
small and large colonies) was evaluated after expression of the
mutant phenotype.
2.7.4.2. In vitro micronucleus test. The objective of this study was to
evaluate the potential of AuNP to induce an increase in the frequency of
micronucleated cells in the mouse cell line L5178Y TK+/−. The study
design was based on the OECD Guideline No. 487. After a preliminary
cytotoxicity test, AuNP diluted in water for injections were tested in a
single cytogenetic experiment, with and without a metabolic activation
system, the S9 mix, prepared from a liver microsomal fraction (S9
fraction) of rats induced with Aroclor 1254. Each treatment was
coupled to an assessment of cytotoxicity at the same dose levels.
Cytotoxicity was evaluated by determining the PD (Population
Doubling) of cells. After the final cell counting, the cells were washed
and fixed. Then, cells from three dose levels of AuNP treated cultures
were dropped onto clean glass slides. The slides were air-dried before
being stained in 5% Giemsa. Slides from vehicle and positive controls
cultures were also prepared as described above. For each main
experiment (with or without S9 mix), micronuclei were analyzed for
three dose levels of AuNP, for the vehicle and the positive controls, in
1000 mononucleated cells per culture (total of 2000 mononucleated
cells per dose).
M. Ben Haddada, et al.
2.8. Statistical analysis
Results are expressed as a mean ± SD of three samples. Student's t-
test was used to evaluate the statistical significance of the results.
Differences between groups were considered statistically significant
when p value < 0.001.
3. Results and discussion
3.1. Synthesis and characterization of green gold nanoparticles
GAuNP were synthetized as previously described by Morel et al. [5].
The addition of plant extracts to the heated gold precursor solutions
induced a color change from pale yellow to blue. This color shift at-
tributed to the excitation of Surface Plasmon (SP) is the first evidence of
formation of AuNP. In UV Vis spectra, we observed the disappearance
of the SP band of Au (III) at 290 nm in favor of SP between
524−550 nm, which is due to the reduction of gold salts and formation
of Au(0), in the presence of bioreducing agents in the plant extracts
[12].
UV-Vis spectroscopy allows the assessment of the formation and the
stability of nanoparticles in aqueous solution. Fig. 1a shows a typical
spectrum; the maximal absorbance was obtained for λmax between
524−550 nm which corresponds to 40−80 nm size nanoparticles ac-
cording to the literature [13].
The average hydrodynamic diameter of GAuNP is 97.7 ± 7.1 nm.
The polydispersity index (PDI) is 0.25 ± 0.04. This diameter is based
on Brownian movement and takes into account the plant extract layer
formed around the nanoparticle. The hydrodynamic diameter is larger
than the “dry” diameter due to solvation, hydrogen bonding and van
der Waals effects. The zeta potential is -33.8 ± 7.4 mV due to the ne-
gative charge of hydroxyl group of flavonoids coating the nanoparticles
which is responsible for their stability.
In Fig. 1b and d, GAuNP appear in TEM as polydisperse flower-
shaped particles with average size of 50 nm. These particles are asso-
ciated with organic components coming from plant extracts. Flavonoids
present in the plant extract are able to reduce gold ions and act as ef-
ficient capping agents, stabilizing the GAuNP. TEM images show na-
noparticles surrounded by a thin layer with a poor electronic density
attributed to an organic layer, which stabilizes AuNP.
Stability studies were performed on the nanoparticle suspension
(Fig. 1c). No redshift was observed for one month, indicating an ab-
sence of aggregation.
3.2. Characterization of medicinal plant extracts for the synthesis of green
gold nanoparticles
The medicinal plant crude extract showed higher water solubility
Fig. 1. (a) The absorption spectrum of an aqueous solution of dispersed GAuNPs, (b) its characterization by transmission electronic microscopy, (c) the size
distribution histogram and (d) a stability study for 25 days.
4
Colloids and Surfaces B: Biointerfaces 189 (2020) 110855
than the isolated flavonoids when introduced into the reaction mixture.
This suggests a synergistic interaction between flavonoids which con-
sequently leads to an efficient reducing reaction.
First of all, the aqueous extract was analyzed by HPLC-HR-MS-ESI-
MS/MS. This crude extracts have then been fractionated by MPLC
(Medium Pressure Liquid Chromatography) in 5 fractions A–E (Fig. 2a).
The strategy in phytochemistry consisted in identifying the funda-
mental fractions required for the gold reduction, the synthesis of na-
noparticles and their stabilization. The objective is to perform a pre-
liminary elucidation of the reaction mechanism by analyzing the major
compounds (18 molecules).
We carried out the nanoparticle synthesis with the different frac-
tions A–E. The absorption spectrum of GAuNP obtained with poly-
phenolic fractions (B–E) are shown in Fig. 3. Fractions A and B led to
the formation of very large and unstable particles (gray color) which
precipitate rapidly. The redshift of the plasmon band towards 580 nm
with high absorbance around 780 nm indicated the formation of ag-
gregates. Fraction C interestingly demonstrated a plasmon band at
530 nm and similar nanoparticles obtained with the whole crude ex-
tracts. Finally, GAuNP obtained with fractions D and E gave respec-
tively a plasmon band at 538 nm (more intense than C) and at 540 nm
(same intensity as D). Another difference with D is that we do not have
the same formation kinetics of NPs, E takes more time than D (60 s
versus 30 s).
HPLC-UV of the aqueous extract of the leaves of Hubertia ambavilla
was presented in Fig. 2b. At the wavelengths of 190, 220, 325 and
354 nm, which were used for detection of chlorogenic acids derivatives
and flavonoids, the aqueous extract displayed a higher diversity of
polyphenols compound. Phytochemical analysis of this extract by
HPLC-ESI-HR-MS/MS in negative mode showed the presence of two
families of phenolic compound. Compounds were identified and
quantified by comparing retention times, spectral data (LC-ESI-MS/MS)
and according to literature [14–17]. A total of eighteen phenolic
compounds of which sixteen belonging to the family of chlorogenic
acids (1-8, 11-18) and two flavonoids (9, 10) were identified in this
study (Table 1). The structures of all compounds were presented in
Fig. 4.
The relevant assignments of the chemical shifts in the 1H NMR
spectrum of the fraction C are presented in Fig. 5. The main signals
were observed in aromatic portion (δ > 6 ppm) as doublets at
δH = 9.17 (d, J =15.9 Hz, D1); 9.10 (d, J =15.8 Hz, D2); 7.90 (d, J
=15.9 Hz, D3) and 7.77 ppm (d, J =15.8 Hz, D4). These protons are
coupled and give trans-olefins confirming the presence of caffeic acids.
The other signals of the aromatic part (between δH = 6.70 and
8.62 ppm) reveal the presence of aromatic cycles confirming the pre-
sence of flavonoids. Moreover, the aliphatic part between δH = 2.90
and 5.00 ppm was attributed to the protons of quinic acid. The signal to
9.83 ppm attributed to hydroxyl groups of phenolic compounds.
M. Ben Haddada, et al.
Fig. 2. (a) HPLC chromatogram of a plant extract from Hubertia ambavilla using Charged Aerosol Detection. The extract was separated in 5 fractions A–E, (b) LC–MS/
MS (red) and UV (green) chromatograms of the aqueous plant extract. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
3.3. Synthesis and characterization of citrate gold nanoparticles
Citrate was used as reducer (CAuNP) in the Turkevich method (Fig. I
in Supplementary file). A red solution was obtained at the end of the
synthesis [11] leading to 15 nm gold nanoparticles (Fig. II in Supple-
mentary file). The obtained nanoparticles are stabilized by citrate in
aqueous solution.
3.4. Biological activities
The NRU assay was used to assess changes in cell viability. After
24 h incubation, GAuNP and CAuNP did not have a significant cytotoxic
effect on the viability of NDHF for concentrations between 0.005 and
50 μg/mL. After 72 h of incubation, as shown in Fig. 6a, GAuNP did not
have a significant cytotoxic effect on the viability of NHDF cells up to
25 μg/mL while a dose dependent cytotoxic effect was observed from
50 μg/mL. The 30 μg/mL concentration was retained as the highest
tested dose to study biological activity of GAuNP. After 72 h of in-
cubation, CAuNP had no significant cytotoxic effect on the viability of
NHDF cells up to 40 μg/mL while a cytotoxic effect was observed at
80 μg/mL.
The anti-radical properties of AuNP were assessed. As shown in
Fig. 7a, GAuNP had a significant DPPH radical scavenging activity. A
dose-dependent inhibition of DPPH was observed in the range of tested
5
Colloids and Surfaces B: Biointerfaces 189 (2020) 110855
concentrations allowing calculation of the IC50 from the regression
[DPPH (%) = f(concentration)]. The IC50 value for antioxidant activity
of GAuNP was found to be 16.5 μg/mL. Under the same experimental
conditions, DPPH signal was inhibited by 96 % with 50 μM α-toco-
pherol (commonly named Vitamin E) and no inhibition was observed
with CAuNP (Fig. 7b). This strongly suggests that active nanoparticle
coatings that are not produced by Turkevich method are required for
antioxidant activity. In addition, the electrostatic interactions between
negatively charged phytochemicals and GAuNP seem to contribute sy-
nergistically in improving the inherent bioactivity of medicinal plants
[18,19]. We can conclude that GAuNP from these plant extracts had
much higher antioxidant activity than vitamin E.
The dermoprotective potential of NPs to human skin cells was as-
sessed. As shown in Fig. 6b, UV-A irradiation markedly increased MMP-
1 production in control NHDF cells. An 8-fold increase in basal MMP-1
was observed after UV-A exposure. The treatment of cells with GAuNP
and CAuNP showed a significant decrease of MMP-1 production in a
dose-dependent manner. IC50 values for antioxidant activity of GAuNP
and CAuNP were found to be 9.25 μg/mL and 30.1 μg/mL, respectively.
Under the same experimental conditions, α-tocopherol reduced UV-A
irradiation-induced collagenase activity by 52 %.
The topical application of GAuNP did not change the cell viability
compared to the control explants (non-exposed or exposed to UV-A
without GAuNP) but the formation of oxidized proteins lower. In the
M. Ben Haddada, et al. Colloids and Surfaces B: Biointerfaces 189 (2020) 110855

Fig. 3. Absorption spectra of GAuNP obtained with polyphenolic fractions A-E.

absence of UV-A irradiation, GAuNP decreased the rate of endogenous exposure, no change in cell morphology was observed and there was no
oxidized proteins within the dermis. In addition, they prevented the decrease in NR uptake at any tested concentrations in the irradiated and
formation of oxidized proteins induced by UV-A irradiation, thereby non-irradiated plates. Under the experimental conditions of this study,
providing antioxidant protection within the dermis. the AuNP tested up to 1000 μg/mL were determined not to be photo-
toxic, according to the classifications presented in the OECD guideline
432.
3.5. Safety studies
The second series of tests was to evaluate the skin sensitization
potential of AuNP. With the KeratinoSens test, the potential of AuNP to
The objectives of these tests consisted in evaluating the safety of
activate the Nrf2 transcription factor was assessed. Four runs were
AuNP as raw ingredients for cosmetics. Since the worldwide ban on
performed using different concentrations from 0.2–400 μg/mL. The
animal testing for cosmetics came into effect, several in vitro tests have
final outcome is negative in agreement with the OECD guideline, so
been established in order to assess: toxicity, in vitro skin sensitization, in
AuNP were considered to have no potential to activate the Nrf2 tran-
vitro irritation and genetic toxicology.
scription factor. In complement to this test, a SENS-IS assay was per-
For the acute toxicity test, after 48 h of incubation with AuNP, no
formed to evaluate the capacity of the AuNP to induce the expression of
decrease in cell viability was noted at any concentrations, therefore no
specific irritation and sensitization biomarkers in a 3D-reconstructed
IC50 and as a result no LD50 was estimated. So according to this study,
epidermis model. The expression profile of 61 genes divided in three
AuNP are not considered cytotoxic. Several concentrations in a range
sets were analyzed: one set of 23 genes related to the irritation process
between 0.32 and 1000 μg/mL were then used to assess phototoxicity.
and the two other sets of genes, named “SENS-IS” and “ARE”, with 21
Phototoxicity is defined as a toxic response from a substance which is
and 17 biomarkers respectively, involved in skin sensitization [20,21].
either elicited or increased after exposure to light. 24 h after light

Table 1
Compounds identified from Hubertia ambavilla aqueous extract.
No Chemical Formula Name of Compounds

1 C16H18O9 3-O-caffeoylquinic acid

2 C16H18O10 3-O-caffeoyl-2-Hydroxy-quinic acid
3 C16H18O9 4-O-caffeoylquinic acid
4 C24H24O13 4-O-caffeoyl-2-hydroxy-3-O-[(1-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl]quinic acid
5 C24H24O13 3-O-caffeoyl-4-O-[(1-hydroxy-2-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl]quinic acid
6 C24H24O12 3-O-caffeoyl-5-O-[(1-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl] quinic acid
7 C30H30O15 5-O-caffeoyl-3-O-succinyl-4-O-[(1-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl]methyquinate
8 C24H24O12 3-O-caffeoyl-4-O-[(1-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl] quinic acid
9 C21H20O12 Hyperin
10 C21H20O12 Isoquercetin
11 C38H36O18 3,5-O-diferuloyl-4-O-[(1-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl]-1-methoxyoxaloyquinic acid
12 C25H24O12 3,4-di-O-caffeoyl quinic acid
13 C38H36O18 3,4-O-diferuloyl-5-O-[(1-hydroxy-4-oxocyclohexa-2,5-dienyl)acetyl]-1-methoxyoxaloyquinic acid
14 C33H30O16 3,5-di-O-caffeoyl-2-hydroxy-4-O-[(1-hydroxy-4-oxocyclohexa-2,5-denyl)acetyl]quinic acid
15 C25H24O12 4,5-di-O-caffeoyl quinic acid
16 C33H30O15 3,4-di-O-caffeoyl-5-O-[(1-hydroxy-5-oxocyclohexa-2,5-denyl) acetyl]quinic acid
17 C33H30O15 4,5-di-O-caffeoyl-2-hydroxy-3-O-[(4-hydroxyphenyl]quinic acid
18 C33H30O14 3,4-di-O-caffeoyl-5-O-[(4-hydroxyphenyl)acetyl]quinic acid

6
M. Ben Haddada, et al. Colloids and Surfaces B: Biointerfaces 189 (2020) 110855

Fig. 4. Chemical structures of identified compounds 1–18.

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M. Ben Haddada, et al. Colloids and Surfaces B: Biointerfaces 189 (2020) 110855

Fig. 5. 1H NMR spectrum of fraction C obtained from aqueous extract of H. ambavilla at 600 MHz in CD3OD.

In the presence of AuNP, fewer than 7 genes in the “SENS-IS” and
“ARE” gene groups were expressed, leading to a negative test. In con-
clusion, AuNP can be classified as a non-sensitizer.
The third series of tests was to assess the irritation of the skin and of
the eyes. For skin irritation, the principle of the assay is based on the
fact that irritant chemicals are cytotoxic to the Episkin™ reconstructed
human epidermis model after a short-term exposure. Irritant chemicals
can penetrate the stratum corneum and are sufficiently cytotoxic to
cause cell death in the underlying cell layers. Following a 15 min ex-
posure and 42 h recovery period, the relative mean viability of the
tissues treated with the AuNP was 95 % with a standard deviation of
3%. Under the experimental conditions of this study, AuNP are con-
sidered to be non-irritant to skin. For corneal cells, the relative mean
viability of the tissues treated with AuNP was 96 % with a difference of
4% between duplicate tissues. As the mean viability was > 60 % after
the MTT reduction, the results met the criteria for a non-irritant re-
sponse. Under the experimental conditions of this study, AuNP are non-
irritant to reconstructed human cornea-like epithelia.
The fourth series of tests was to assess the genotoxicity of AuNP. For
the research of cell gene mutations, using a AuNP solution at the con-
centration of 500 mg/mL in the vehicle and a treatment volume of 1%
(v/v) in culture medium, the selected dose levels were 156.3, 312.5,
625, 1250, 2500 and 5000 μg/mL, both with and without S9 mix. No
precipitate was observed in the culture medium, at any dose levels,
either at the beginning or the end of the 3 -h treatment period. No
noteworthy increase in the mutation frequency was noted relative to
the corresponding vehicle control, at any dose levels with or without S9
mix (IMF < GEF of 126 × 10−6). Moreover, no dose-response

Fig. 6. (a) Cytotoxicity of AuNP on NHDF cells after 72 h incubation. Results are expressed as mean ± SD (n = 6) (b) Effects of AuNP on UV-A induced MMP-1
production. Results are expressed as mean ± SD (n = 3). Vitamin E solution (VE 1 mM) was used as reference standard. *p < 0.001 (Student’s test).

8
M. Ben Haddada, et al.
Fig. 7. Antioxidant activity by DPPH method measured on GAuNP (a) and (b) CAuNP. Results are expressed as mean ± SD (n = 3).
relationship was demonstrated by the linear regression. Thus, these
results met the criteria for a negative response. Under the experimental
conditions of this study, AuNP did not show any mutagenic activity in
the mouse lymphoma assay, either in the presence or absence of a rat
liver metabolizing system. For micronucleus analysis, the dose levels
selected for the micronucleus analysis were: 1250, 2500 and 5000 μg/
mL, the latter being the highest recommended dose level. Following the
3 -h treatments with and without S9 mix or the 24 -h treatment without
S9 mix, neither statistically significant nor dose-related increases in the
frequency of micronucleated cells were noted at any of the analyzed
dose levels relative to the corresponding vehicle control. Moreover,
none of the analyzed dose levels showed a frequency of micronucleated
cells above the corresponding vehicle historical range. Thus, these re-
sults met the criteria of a negative response. Under the experimental
conditions of the study, AuNP did not induce any chromosome damage,
or damage to the cell division apparatus, in cultured mammalian so-
matic cells, using L5178Y TK+/- mouse lymphoma cells, either in the
presence or absence of a rat liver metabolizing system.
4. Conclusion
This study demonstrates the potential of nanoparticles as a material
for cosmetic applications. Hubertia ambavilla extract contains bioactive
constituents used for the synthesis of gold nanoparticles with inter-
esting properties, particularly for cosmetic applications. GAuNP are
non-toxic to fibroblasts cells and dermal cells, are able to efficiently
scavenge free radicals, they also protect against damage to fibroblasts
cells and dermal cells by UV-A. Regulatory tests to ensure the safety of
gold nanoparticles as ingredients in cosmetics demonstrated that AuNP
are not toxic, not phototoxic, not genotoxic, non-irritant and non-sen-
sitizing according to OECD guidelines. These results suggest that green
AuNP are a promising ingredient for cosmetic applications.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Authors contribution
The conceptualization of this study has been done by ALM.
MBH and ALM synthesized and characterized the gold nano-
particles. MBH provided Figs. 1a, c, 3 . ALM provided Fig. 1b, d & Figs. I
and II from Supplementary file.
JSp supervised the physical characterization of the nanoparticles in
CSPBAT.
ALM wrote the physico chemical (nanoparticle) part of the manu-
script.
9
Colloids and Surfaces B: Biointerfaces 189 (2020) 110855
SP wrote the biological part of the manuscript. She provided the
statistical analyses and the Figs. 6, 7.
EG and RC wrote the phytochemical part of the manuscript.
RC provided the Table 1, Fig. 4, Fig. 5.
EG and JSo provided the Fig. 2, and data on Fig. 4.
AB supervised the phytochemical analyses in LCSNSA.
All authors reviewed the final manuscript.
Acknowledgments
This work has been performed at Torskal, CSPBAT/University Paris
XIII, LCSNSA/University La Réunion, GIP CYROI, BioHC, BioEC and
IJPB.
We thank Dr M. Cesari from CYROI platform, Dr F. Perreau and Dr
G. Mouille from IJPB. Support was provided by Sandra Casale from
University of Paris VI. Plants are originated from CAHEB. This work has
been partly funded by BPI France.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2020.110855.
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