Cosmetics 06 00008 PDF

cosmetics
Article
Design Methodology for the Development of a New
Cosmetic Active Based on Prunus domestica L.
Leaves Extract
Hortense Plainfossé 1,2,3 , Pauline Burger 3 , Grégory Verger-Dubois 2,3 , Stéphane Azoulay 1 and
Xavier Fernandez 1, *
1 Université Côte d’Azur, CNRS, ICN, Parc Valrose, CEDEX 2, 06108 Nice, France;
hortense.plainfosse@nissactive.com (H.P.); stephane.azoulay@unice.fr (S.A.)
2 Laboratoires JYTA, 5ème rue, 1ère avenue 3283m, 06150 Carros, France; gvd@nissactive.com
3 NissActive, Pépinière Innovagrasse, Espace Jacques-Louis Lions, 4 traverse Dupont, 06130 Grasse, France;
pauline.burger@nissactive.com
* Correspondence: Xavier.Fernandez@unice.fr; Tel.: +33-492076469

Received: 20 December 2018; Accepted: 26 January 2019; Published: 29 January 2019
Abstract: When it comes to the development of new active ingredients for cosmetics, biodiversity
is a rich source for inspiration that must be tapped in a sustainable manner to cause no social nor
ecological damage. Agri-food by-products are therefore more and more considered as available
biomass that can be reused to extract their maximum value to produce new cosmetic ingredients
before returning to the biosphere. The process to transform plant waste materials into powerful
cosmetic actives is thoroughly described in the present paper via the example of the design of
a liquid anti-aging ingredient based on a Prunus domestica L. extract obtained by maceration of
plums’ dried leaves in propylene glycol. The subsequent development of an SPE (solid-phase
extraction) methodology used to remove the propylene glycol to get access to the extracted molecules
is thoroughly described as a means to follow the stability of the ingredient over time once formulated
into a finished product.
Keywords: natural cosmetic ingredient; Prunus domestica L.; agricultural by-products; circular
economy; sustainability; anti-age; cosmetic bioassays; green glycols; SPE
1. Introduction
In the last few decades “green” has become increasingly relevant in various aspects of everyday
life, and personal care (PC) products and cosmetics are no exception. Aware of the impact of increased
consumption and highly concerned with ingredients’ provenance and sustainability, growing practices,
supply chain policies, etc., end consumers are looking more toward ethically conceived PC and
cosmetics products.
With over-reliance on resources and pollution becoming major concerns, the overall waste
management must be profoundly revamped, and a growing number of businesses are taking the
U-turn toward sustainability and integrating the green values of a circular economy into their processes
where residues and by-products are consequently converted into value added products.
Due to the global demographic expansion over the last decades and the subsequent substantial
demand for edible processed goods, agricultural practices and industrial food manufacturing industries
are among the activities that generate enormous quantities of highly environmentally impacting wastes
(seeds, pulp, pomace, peels, etc.) [1]. Urged to decrease its environmental impact, the agri-food sector
is putting significant efforts into the identification and development of new uses for their activities’
by-products [2]. Properly managed, these wastes become main streams of original raw material sources
Cosmetics 2019, 6, 8; doi:10.3390/cosmetics6010008 www.mdpi.com/journal/cosmetics

Cosmetics 2019, 6, 8 2 of 21
for the cosmetics sector [3,4]. These new environmentally-friendly habits offer valuable advantages:
(a) reusing such “waste” material to get as much value as possible from every resource, which offers
a unprecedented degree of traceability on the materials’ origin as sourcing channels already exist
and are secured [5]; (b) developing the potential of waste to be used as raw material enables the
diversification of value chains for one single market segment and may subsequently imply the growth
of farmers/producers’ incomes, hence strengthening those sourcing channels; and (c) they constitute
furthermore fantastic communication opportunities and with the appropriate marketing strategy,
cosmetic brands can really connect with customers through their mutual responsibility toward the
environment preservation and gain new market shares.
This article presents a procedure to design efficient cosmetic ingredients while meeting the
challenging consumer’s demands for sustainability, naturality, transparency and traceability.
In order to integrate those sustainability values into our process of ingredients’ development and
to take advantage of the “green” phenomenon, we accessed a wide range of by-products that were
extracted using several solvents. Given the promising results already shown by our research team
regarding leaves of Prunus domestica L. (Rosaceae) [6], special attention was brought to the potentiality
to create valorisation channels for by-products of the plum industry (food usages mainly: fresh, canned
or dried fruits, more processed products, etc.).
Prunus domestica L. is a species of flowering plant that includes many varieties of plum trees.
P. domestica is the most commonly grown plum in Europe over a wide range of climatic conditions:
the fruits constitute a first-rate dessert, and are appreciated to produce tarts, eau de vie, as well as dried
plums, known as prunes.
The Blue Perdrigon (also known as Violet Perdrigon, Perdrigon Viollette or Brignoles Violette) is
a very old plum variety cultivated in the Provence region and in the Alps, characterized by downy
branches and middle-sized oval fruits ripening from mid-August till late September. The variety name
stems from the colour of the fruit skin: first reddish, it rapidly turns purple. The flesh, rather firm,
sugary and adhering to the stone, presents a greenish to yellowish colour.
In Provence, the dried Brignoles prunes, known as Pistoles, are obtained as follows: the fresh
fruit is peeled (the fruit can be slightly warmed up to ease the skin removal), dried, de-stoned,
hand-flattened and further dried before consumption [7]. A resurgence of interest was observed over
the last few decades for this culinary specialty, the production of which generates quantities of unused
skins that could be valorised somehow rather than simply discarded as they are nowadays.
Young plum trees are immature and do not produce fruits; according to varieties, it sometimes
takes four to six years to bear fruits. As many other stone fruits, plum trees are alternate bearing
ones, meaning the plant will produce more fruits every other year. Even if this behaviour is internally
regulated by the plant (under the control of plant hormones, e.g., gibberellins, among others), it can be
controlled by appropriate crop management including regular tree thinning and pruning [8]. The huge
quantities of leaves and thin branches, usually burned, might, if properly valued, constitute a new
source of revenue for the plum producers, generating incomes even during the immaturity period
before the exploitation of fruits themselves.
If P. domestica fruits and seeds have already been valorised in PC products, no cosmetic ingredients
based on leaves or skin exist to our knowledge. Hence, both leaves and skins of Brignoles prunes
have therefore been included in our screening of the cosmetic potential of agricultural by-products:
organic solvent extracts obtained from some 30 plants and plants by-products were investigated for
their anti-aging properties. From this survey, the ethanolic crude extract of P. domestica leaves displays
the best unprecedented anti-aging activities and was then selected for further investigations leading to
the development of an innovative anti-aging active.
2. Materials and Methods

All chemicals were obtained from Sigma-Aldrich (Saint-Louis, Missouri, MI, USA) unless
otherwise stated.
Cosmetics 2019, 6, 8 3 of 21
2.1. Plant Material

Leaves and branches of P. domestica were collected in Brignoles, in Provence-Alpes-Côte d’Azur,
France. Skins were recovered from Brignoles plums warmed up to produce pistoles. A voucher
specimen of P. domestica leafy branches has been deposited in the botanical collection at the Natural
History Museum, Nice, under the reference number NICE-D-4432.
2.2. Plant Extraction

Both leaves on one side and skins of P. domestica on the other side were air dried and stored
in a ventilated area. The plant material was crushed into a fine powder directly before extraction.
For the initial activities’ screening, extracts of plant by-products were obtained via accelerated
solvent extraction (ASE), using the Dionex ASE 350 system from Thermo Scientific (Waltham,
Massachusetts, MA, USA). These solid–liquid extractions were performed at 70 ◦ C, at a pressure
of 100 bar, using ethanol. Powdered plant samples mixed with diatomaceous earth were placed in
stainless steel extraction cells with volumes of 66 mL. After a 5 min heating step of the extraction cell
at 70 ◦ C, three successive static extraction stages of 5 min were performed by pumping fresh solvent
through the system to rinse the sample and the tubing. All the solvent present in the system was then
purged with compressed nitrogen, and the resulting extracts (total solvent volume, e.g., extractant and
rinsing) were collected in septum-topped vials.
In the second phase of the study, extraction of plant material was performed via direct maceration
in ethanol (extraction ratio 1/5) at room temperature (RT) using a magnetic stirrer (500 rpm) for 2 h.
The resulting extracts were then filtered over 8–12 µm filter paper.
In the final development phase, extraction of the plant material was performed via direct
maceration in propylene glycol (in fact, transposition of the initial ASE extraction is not possible
using propylene glycol (PG), as this solvent might be far too viscous to be used for such assays).
About 1 g of plant material was extracted with approximately 10 g of solvent (extraction ratio 1/10) at
RT or at 70 ◦ C using a magnetic stirrer (500 rpm) for 4 h, 7 h or 48 h. The resulting extracts were then
filtered over 8–12 µm filter paper.
PG extraction yields are calculated as follow: the amount of PG added to the dried raw material
was weighted and compared to the weight of the PG extract collected after maceration and removal of
the plant material by filtration. A further squeezing step of the filter paper was initially performed to
gain access to the residual PG soaking the plant material; as no significant increase of the resulting
extraction yield was found, this step was dismissed in further experiments at the lab scale.
2.3. Fractionation of P. domestica Leaves Extract

To identify the active fractions, the ethanolic extract of P. domestica leaves was then fractionated
over silica gel (normal phase). The fractionation of the bulk extract led to the recovery of six distinct
fractions: F1 (300 mL cyclohexane/diethyl ether 50/50 v/v), F2 (300 mL diethyl ether/methanol 80/20
v/v), F3 (300 mL diethyl ether/methanol 50/50 v/v), F4 (300 mL diethyl ether/methanol 20/80 v/v),
F5 (300 mL methanol) and F6 (300 mL methanol/water 50/50 v/v). The resulting fractions were further
evaluated for their bioactivities, and their respective compositions were addressed by HPLC (high
performance liquid chromatography).
2.4. Bioassays
The bioassays were performed as presented previously [9].
2.4.1. Materials
Untreated 96-well plates were purchased from Thermo Nunc (Villebon-sur-Yvette, Ile-de-France,
France), whereas the UV-transparent ones were obtained from Costar, Sigma-Aldrich (Saint-Quentin
Fallavier, Auvergne-Rhône-Alpes, France). Adhesive films (Greiner Bio-One, Courtaboeuf, Île-de-France,
Cosmetics 2019, 6, 8 4 of 21
France) were used to seal the 96-well plates during incubation. Samples (extracts, fractions, standards
and controls) were prepared at a concentration of 3.433 mg/mL (to reach a final sample’s concentration
of 100 µg/mL in the well once all the reagents had been added) in dimethyl sulfoxide (DMSO) in
1.5 mL Eppendorf tubes, appropriate for the use of the automated pipetting system epMotion®5075
(Eppendorf, Montesson, Île-de-France, France).
A hydroglycerinated active containing, among others, some Prunus persica leaves extract,
commercialized for its claimed lightening and anti-aging activities, was tested alongside our samples to
perform direct comparison between Prunus species-based ingredients. DMSO tested alone constitutes
the negative control (ODcontrol , with OD stating for optical density) in each plate; it appeared to have
no activity on its own. Two positive controls were added in each plate (Table 1).
Table 1. Positive controls used in each bioassay performed.
Assay Positive Control 1 Positive Control 2

Rosmarinus officinalis L. commercial extract
DPPH radical scavenging assay Trolox (3607.8 µM in DMSO)
(3.433 mg/mL in DMSO)
Arctostaphylos uva-ursi (L.) Spreng. extract
Tyrosinase assay Kojic acid (3.433 mM in DMSO)
Centella asiatica (L.) Urb. commercial extract
Lipoxygenase assay Quercetin hydrate (1000 µM in DMSO)
Rubus idaeus L. commercial extract
Elastase assay Quercetin hydrate (8583.33 µM in DMSO)
Rubus idaeus L. commercial extract
Hyaluronidase assay Tannic acid (0.435 mg/mL in DMSO)
Collagenase assay Tannic acid (1.72 µM in DMSO) Betulinic acid (3.433 mg/mL in DMSO)
2.4.2. Instrumentation
An automated pipetting system Eppendorf epMotion®5075 was used to carry out the bioassays.
A microplate reader (Spectramax Plus 384, Molecular Device, Wokingham, Berkshire, UK) was used to
measure absorbance values. Data were acquired with the SoftMaxPro software (Molecular Devices,
Wokingham, Berkshire, UK) and the Prism software (GraphPad Software, La Jolla, California, CA,
USA) was used to calculate inhibition percentages.
The results are presented as inhibition percentages (I%) calculated as follows (for the DPPH
radical scavenging assay, and tyrosinase, lipoxygenase and elastase assays):
ODcontrol − ODsample

I% = × 100
ODcontrol
or as follows (for the hyaluronidase and collagenase assays):
ODsample

I% = × 100
ODblank − ODcontrol
Similarly, all OD (apart the hyaluronidase ones) were corrected with the blank measurement
corresponding to the absorbance of the sample before addition of the substrate.
2.4.3. DPPH Radical Scavenging Assay

The antioxidant activity of extracts was evaluated based on the scavenging activity of the stable
1,1-diphenyl-2-picrylhydrazyl radical (DPPH) [10,11]: 150 µL of a solution of ethanol/acetate buffer
0.1 M (50/50) was distributed in each well, together with 7.5 µL of the samples evaluated. A first OD
reading was performed at 517 nm (ODblank ). Then, 100 µL of a DPPH solution (386.25 µM in ethanol)
were distributed in each well. The sealed plate was incubated in the dark at RT for 30 min before the
final OD reading.
Cosmetics 2019, 6, 8 5 of 21
2.4.4. Tyrosinase Assays

Tyrosinase is a copper-containing enzyme that plays a key role in melanogenesis; it is mainly
involved in the hydroxylation of L-tyrosine into L-DOPA (l-3,4-dihydroxyphenylalanine) and its
further oxidation to dopaquinone [12]. The assays were performed via distribution of 150 µL of a
solution of mushroom tyrosinase (171.66 U/mL in phosphate buffer) in each well, together with 7.5 µL
of the extracts evaluated. The filmed plate was incubated at RT for 20 min. Then, 100 µL of a solution
of substrate (either L-tyrosine or L-DOPA, 1 mM in phosphate buffer) were distributed in each well,
and the final OD reading was performed at 480 nm after 20 min of incubation.
2.4.5. Lipoxygenase Assay

Lipoxygenase is an iron-containing enzyme known to play a key role in inflammation [13].
The assays were performed as follows: 150 µL of a solution of soybean lipoxygenase (686.66 U/mL
in phosphate buffer) was distributed in each well, together with 7.5 µL of the extracts evaluated.
The filmed plate was incubated in the dark for 10 min. Then, 100 µL of a solution of linoleic acid in
phosphate buffer were distributed in each well. After incubation for 2 min in the dark, a first OD
reading was performed at 235 nm; the final OD reading was performed after a further incubation of
50 min.
2.4.6. Elastase Assay

Elastase is an endopeptidase that preferentially digests elastin and is the highly elastic protein
responsible for cutaneous firmness, together with collagen [14]. The assays were performed as follows:
150 µL of a solution of porcine pancreatic elastase (0.171 U/mL in Tris buffer) was distributed in
each well, together with 7.5 µL of the extracts. The filmed plate was incubated at RT for 20 min.
A first OD reading was performed at 410 nm before the addition of 100 µL of a solution of
N-succinyl-Ala-Ala-Ala-p-nitroanilide (2.06 mM in Tris buffer). The final OD reading was performed
after a 40-min incubation.
2.4.7. Hyaluronidase Assay

Hyaluronidases are enzymes that degrade hyaluronic acids, which are widely distributed in the
body and notably at the periphery of collagen and elastin fibres, that play therefore a major role in skin
aging [15,16]. The assays were performed as follows: 150 µL of a solution of hyaluronidase (13.3 U/mL
in hyaluronidase buffer) was distributed in each well, together with 7.5 µL of the extracts evaluated.
The filmed plate was incubated at 37 ◦ C for 20 min, and a first OD reading was performed at 405 nm.
Then, 100 µL of a solution of hyaluronic acid (150 µg/mL in pH 5.35 buffer) was distributed in each
well. After 30 min incubation at 37 ◦ C, 50 µL of cetyltrimethylammonium bromide (40 mM in a 2 %
NaOH solution) was added and a final OD reading was performed.
2.4.8. Collagenase Assay

Collagenases constitute a family of enzymes that cleave collagen and that are more generally
involved in the degradation of the extracellular matrix components, thus leading to sagging skin [17].
The assays were performed as follows: 150 µL of a solution of collagenase (53 U/mL in tricine buffer)
was distributed in each well, together with 7.5 µL of the extracts. The filmed plate was incubated at
RT for 15 min before the first OD reading was performed at 345 nm. Then, 100 µL of a solution of
2-furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine (5.15 mM in tricine buffer) was distributed in each
well, and a final OD reading was performed after a 30 min-incubation.
2.5. Solid Phase Extraction

SPE cartridges (Waters, Guyancourt, Ile-de-France, France; Oasis HLB6cc, 500 mg) were selected
as they are filled with polymeric reversed-phase sorbent adequate for the extraction of a wide range of
Cosmetics 2019, 6, 8 6 of 21
acidic, basic, and neutral compounds from various matrices. The cartridges were fitted into stopcocks
and connected
Cosmetics 2018, 5, xto a vacuum
FOR manifold. The sorbent was conditioned with 10 mL of methanol (MeOH)
PEER REVIEW 6 of 20
and equilibrated with 10 mL water as recommended by the supplier. The propylene glycol extract
extract
of of P. domestica
P. domestica leaves toleaves
analysetowas
analyse wasindiluted
diluted in water
water (PG (PG extract/water
extract/water 1/3) its
1/3) to lower to viscosity,
lower its
viscosity,
hence hence facilitating
facilitating its furtherits furtheronto
loading loading onto the with
the cartridge cartridge with the stopcocks
the stopcocks opened and opened and the
the vacuum
turned on. The presence of extracted molecules in the effluent was monitored by regular regular
vacuum turned on. The presence of extracted molecules in the effluent was monitored by HPLC
HPLC analyses.
analyses. Once the Once the cartridge
cartridge was saturated
was saturated and theandPGthe
wasPGeliminated,
was eliminated,
elutionelution
of theofsample
the sample
was
was achieved with 4 mL MeOH / dichloromethane (DCM) 1/1 (v/v) to elute all the
achieved with 4 mL MeOH/dichloromethane (DCM) 1/1 (v/v) to elute all the molecules retained on molecules retained
on the
the cartridge.
cartridge.
2.6.
2.6. HPLC
HPLC Analysis
Analysis
P.
P.domestica
domesticaextracts
extractsand
andSPESPE
effluent samples
effluent werewere
samples analysed using using
analysed an HPLC AgilentAgilent
an HPLC 1200 system
1200
(Courtaboeuf, Ile-de-France,
system (Courtaboeuf, France) France)
Ile-de-France, equipped with a DAD
equipped with a(diode array detector)
DAD (diode and anand
array detector) ELSDan
(evaporative light scattering detector) operating under the following conditions: injection
ELSD (evaporative light scattering detector) operating under the following conditions: injection volume of
20 µL andofflow
volume 20 µLrate wasflow
and set at 1.0was
rate mL/min.
set at Separations
1.0 mL/min.were performed
Separations wereonperformed
a C18 column on (Phenomenex,
a C18 column
Torrance, CA, USA,
(Phenomenex, Luna®5
Torrance, California, mm ×Luna®
µm, 150 USA, 4.6 mm5 i.d.). The mm
µm, 150 mobile phase
× 4.6 mmconsisted
i.d.). Theof a multistep
mobile phase
gradient
consistedofofwater (A), acetonitrile
a multistep gradient(B)
of and
water2-propanol (C), all (B)
(A), acetonitrile acidified with 0.1% formic
and 2-propanol (C), allacid (Figure
acidified 1):
with
0–5
0.1%min, 5%acid
formic B; 5–40 min,1):5–45%
(Figure B; 40–50
0–5 min, min, min,
5% B; 5–40 100%5–45%
B; 50–55 min, min,
B; 40–50 100%100%
B; 55–68 min,min,
B; 50–55 100% C,
100%
68–70 min,
B; 55–68 100%
min, 100%C. The DAD min,
C, 68–70 was set
100%at 366 nm,DAD
C. The and ELSD
was setconditions
at 366 nm, were setELSD
and as follows: nebulizer
conditions were
gas pressure
set as 3.7nebulizer
follows: bars, evaporative tube3.7
gas pressure temperature 40 ◦ C and
bars, evaporative tubegain 4.
temperature 40 °C and gain 4.
100
90
80
70
60
%A (Water)
50
%B (Acetonitrile)
40
%C (2-propanol)
30
20
10
0
0 5 40 50 55 68 Time (min)
Figure 1. Multistep
Figure 1. Multistep gradient
gradient of
of water
water (A),
(A), acetonitrile
acetonitrile (B),
(B), and
and 2-propanol
2-propanol (C),
(C), all
all acidified
acidified with
with 0.1%
0.1%
formic acid used for the HPLC analyses.
formic acid used for the HPLC analyses.
2.7. Gas Chromatography-Mass Spectrometry (GC-MS)
2.7. Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS analyses were performed using an Agilent 6890 gas chromatograph (Palo Alto, CA,
GC-MS analyses were performed using an Agilent 6890 gas chromatograph (Palo Alto,
USA) equipped with an Agilent MSD5973N mass selective detector, a multifunction automatic
California, USA) equipped with an Agilent MSD5973N mass selective detector, a multifunction
sampler (Combi-Pal, CTC Analytics, Zwingen, Switzerland), both on a DB-5HT capillary column
automatic sampler (Combi-Pal, CTC Analytics, Zwingen, Switzerland), both on a DB-5HT capillary
((5%-phenyl)-methylpolysiloxane; 0.25 mm × 30 m; film thickness, 0.10 µm) and on an HP-1 MS
column ((5%-phenyl)-methylpolysiloxane; 0.25 mm × 30 m; film thickness, 0.10 µm) and on an HP-1
capillary column (100% polydimethylpolysiloxane; 0.2 mm × 50 m; film thickness, 0.33 µm). Samples
MS capillary column (100% polydimethylpolysiloxane; 0.2 mm × 50 m; film thickness, 0.33 µm).
(1 µL) were injected in splitless mode (split vent: 50 mL/min, 30 s) and the injector was set at a
Samples (1 µL) were injected in splitless mode (split vent: 50 mL/min, 30 s) and the injector was set
temperature of 250 ◦ C. The carrier gas was helium in constant flow mode at 1 mL/min. The oven
at a temperature of 250 °C. The carrier gas was helium in constant flow mode at 1 mL/min. The oven
temperature was programmed to rise from 60 ◦ C to 180 ◦ C at 2 ◦ C/min, then from 180 ◦ C to 300 ◦ C
temperature was programmed to rise from 60 °C to 180 °C at 2 °C/min, then from 180 °C to 300 °C at
at 6 ◦ C/min and kept isothermally at 300 ◦ C for 5 min. Acquisition was performed in scan mode
6 °C/min and kept isothermally at 300 °C for 5 min. Acquisition was performed in scan mode (35–500
(35–500 a.m.u. (atomic mass unit)/s; scan rate: 3.15 scans/s) and mass spectra were generated at 70 eV.
a.m.u. (atomic mass unit)/s; scan rate: 3.15 scans/s) and mass spectra were generated at 70 eV.
Compound identifications were based on the comparison of mass spectra with literature,
Compound identifications were based on the comparison of mass spectra with literature,
commercial libraries (NIST, Wiley, Indianapolis, USA) and laboratory MS libraries built up from
commercial libraries (NIST, Wiley, Indianapolis, USA) and laboratory MS libraries built up from pure
substances, combined with comparison of the GC linear retention index (LRI) [18,19]. Retention
indices were determined with a series of linear alkanes C8–C24 used as a reference.
2.8. Stability Assays

Cosmetics 2019, 6, 8 7 of 21
pure substances, combined with comparison of the GC linear retention index (LRI) [18,19]. Retention
indices were determined with a series of linear alkanes C8–C24 used as a reference.
2.8. Stability Assays

Stability assays were undertaken to ensure that the ingredient developed meets aesthetic and
quality standards when stored under specific conditions. Some accelerated stability assays were carried
out as follows: the liquid ingredient directly developed in propylene glycol was stored in glass vials
at 42 ◦ C, and weekly monitoring was undertaken to assess eventual aesthetic changes in colour and
odour, or modification of its chemical composition using HPLC-DAD-ELSD after separation from the
cosmetic support via SPE using the methodology presented in Section 2.5.
3. Results
3.1. Bioactivities of Crude Extracts

As already stated, a series of by-products of Mediterranean origin were initially selected based
on their accessibility (easy recovery after the initial industrial processing or during agricultural
activity, considerable volumes recovered that allow to picture promising revalorization, etc.) and their
originality regarding the cosmetic activity, and more particularly the anti-aging one. The initial plant
screening was performed using ASE, an appropriate technique to extract metabolites covering a large
range of structures and polarities, while enabling considerable reduction of extraction time and solvent
consumption, which is ideal for the large-scale screening of numerous samples [20]. Some thirty
extracts were recovered, and their anti-aging properties were evaluated in vitro.
Skin aging is a complex mechanism that is driven by both intrinsic (natural age-dependent/
chronological aging, genetic, etc.) and extrinsic factors (premature environmentally-driven
aging induced by exposure to ultraviolet light, weather changes and environmental pollution,
which aggravates the natural aging process) [21]. Skin exposure to UV-light, stress, etc., leads to
an increased generation of ROS (reactive oxygen species) and subsequent induction of oxidative stress,
which constitutes one of the greatest cutaneous threats. High levels of ROS might, among others,
activate hyaluronidase, collagenase and elastase, enzymes that specifically degrade structural building
blocks of the skin, namely the hyaluronic acid in charge of retaining the moisture of the skin, as well as
collagen and elastin, responsible for its elasticity and strength, hence contributing to skin aging [21,22].
The free radical scavenging and anti-inflammatory activities, as well as specific enzymes’
inhibitory activities of natural extracts were investigated to assess their potentiality to slow down the
skin aging process. From this survey, a total of six plants were identified as potentially interesting for
the development of anti-aging ingredients. Furthermore, it appears that the crude extract of P. domestica
leaves displays the best unprecedented anti-aging activities, while no remarkable bioactivity was
observed for P. domestica dried skins (Figure 2).
The easiness of the raw material supply (plant growing in the region, processing activities
generating both by-products), and the attractive marketing potential of the resulting cosmetic active
conveying the circular economy concepts, induced the selection of P. domestica for further investigations.
A larger P. domestica leaves extract was obtained via maceration in ethanol (plant/solvent ratio 1/5
w/w) at RT (extraction yield 12.3 ± 0.9%). Its HPLC profile (Figure 3) revealed the presence of some
highly polar compounds (e.g., sugars) eluted at the beginning of the analysis, as well as a large group
of polyphenols and flavonoids eluted between 12 and 35 min and a series of terpenic compounds.
The free radical scavenging and anti-inflammatory activities, as well as specific enzymes’
inhibitory activities of natural extracts were investigated to assess their potentiality to slow down the
skin aging process. From this survey, a total of six plants were identified as potentially interesting for
the development of anti-aging ingredients. Furthermore, it appears that the crude extract of P.
domestica
Cosmetics 2019,leaves
6, 8 displays the best unprecedented anti-aging activities, while no remarkable 8 of 21
bioactivity was observed for P. domestica dried skins (Figure 2).
100
80
Inhibition (%)
Cosmetics 60
2018, 5, x FOR PEER REVIEW 8 of 20
Figure 2. Bioactivities of P. domestica extracts obtained via ASE using EtOH compared with the
40
bioactivities of commercial cosmetic ingredients used for the respective activities tested (positive
controls) and to the bioactivities of a commercial ingredient based on a Prunus persica extract.
20
The easiness of the raw material supply (plant growing in the region, processing activities
generating both by-products), and the attractive marketing potential of the resulting cosmetic active
0
conveying theAnticircular economy concepts,
-hy aluronidase activity induced
Anti -i nflammator y act ivity theAnti-oxi
selection
dant act ivity of P. domestica fory further
Anti -elastase activit
investigations.
Aerial parts (ASE extract) Skins (ASE extract) Positive controls Commercial ingredient
A larger P. domestica leaves extract was obtained via maceration in ethanol (plant / solvent ratio
1/5 w/w) at RT (extraction yield 12.3 ± 0.9%). Its HPLC profile (Figure 3) revealed the presence of some
Figure 2. Bioactivities of P. domestica extracts obtained via ASE using EtOH compared with the
highly polar compounds (e.g., sugars) eluted at the beginning of the analysis, as well as a large group
bioactivities of commercial cosmetic ingredients used for the respective activities tested (positive
of polyphenols and
controls) and to theflavonoids
bioactivitieseluted
of a between
commercial 12ingredient
and 35 min andona aseries
based Prunusofpersica
terpenic
extract.compounds.
Intensity of
absorbance
(mAU) 140
Terpenic compounds
Sugars Polyphenols and flavonoids
120
100
80
60
40
20 366 nm
0
0 10 20 30 40 50 60 min
Time (min)
mV
14
12
10
2 ELSD
0
0 10 20 30 40 50 60
Time (min)
HPLC chromatograms
chromatograms obtained ® column (150 × 4.6 mm; 5 µm) at 366 nm and
Figure 3.
Figure 3. HPLC obtained on
on aa Luna
Luna® C
C18
18 column (150 × 4.6 mm; 5 µm) at 366 nm and
with ELSD, presenting the major families of compounds identified in

with ELSD, presenting the major families of compounds identified in P.
P.domestica
domesticaleaves
leavesextract.
extract.
This extract was then fractionated over silica gel. The fractionation led to the recovery of six
This extract was then fractionated over silica gel. The fractionation led to the recovery of six
distinct fractions: F1 (cyclohexane/diethyl ether 50/50 v/v), F2 (diethyl ether/methanol 80/20 v/v),
distinct fractions: F1 (cyclohexane / diethyl ether 50/50 v/v), F2 (diethyl ether / methanol 80/20 v/v),
F3 (diethyl ether/methanol 50/50 v/v), F4 (diethyl ether/methanol 20/80 v/v), F5 (methanol) and F6
F3 (diethyl ether / methanol 50/50 v/v), F4 (diethyl ether / methanol 20/80 v/v), F5 (methanol) and F6
(methanol / water 50/50 v/v). The resulting fractions F1 to F6 were analysed using HPLC (Figure 4)
and further evaluated for their bioactivities. To do so, fraction solutions were concentrated at 3.433
mg/mL in DMSO and their activities were assessed the same way as the one of the crude extract.
Results of these bioassays are presented in Table 2. The bulk extract obtained via ethanolic maceration
presents slightly less interesting activities compared to the one obtained using ASE; these differences
Cosmetics 2018, 5, x FOR PEER REVIEW 9 of 20
Table 2. Bioactivities of the bulk P. domestica extract and of the corresponding fractions F1–F6.
Cosmetics 2019, 6, 8 9 of 21
P. domestica
P. domestica
Bioactivities Maceration F1 F2 F3 F4 F5 F6
ASE Extract
Extract
(methanol/water 50/50 v/v). The resulting fractions F1 to F6 were analysed using HPLC (Figure 4) and
Anti-inflammatory
further evaluated for their
++ bioactivities. +To do so, fraction
+ solutions
+++ were concentrated
++ - at 3.433 mg/mL
- -
activity
in DMSO and their activities
Anti-elastase activity ++
were assessed
+++
the same -
way as++++
the one of+++
the crude extract.
-
Results
-
of -
these bioassays
Anti-oxidant activity are presented
++ in Table 2.
+++ The bulk extract
- obtained
- via ethanolic
++++ maceration
+ presents
++++ ++++
slightly less interesting activities compared to the one obtained using ASE; these differences might
Anti-hyaluronidase
++++ + - + ++++ + - ++
be due to the short maceration time (only 2 h), or due to the lower plant/solvent ratio used for the
activity
maceration. However,
(−): inhibition < 30%; one can<note
(+): 30% that F3
inhibition displayed
< 50%; some
(++): 50% very interesting
< inhibition anti-oxidant
< 70%; (+++): potency,
70% < inhibition
as well as strong
< 90%; (++++): anti-hyaluronidase,
inhibition > 90%. anti-inflammatory and anti-elastase activities.
mV
175
Polyphenols and flavonoids

150
125
100 F6
F5
75
F4
50
F3
25
F2
F1
0
0 10 20 30 40 50 60 min
Luna®® C18
Figure 4. ELSD profiles obtained on aa Luna 18 column
column (150
(150 ××4.6
4.6mm;
mm;55 µm)
µm) for
for the
the six fractions
obtained via fractionation of the P.
P. domestica
domestica leaves
leaves extract.
extract.
Table 2. Bioactivities of the bulk P. domestica extract and of the corresponding fractions F1–F6.
A positive relationship between the anti-oxidant activity of fractions F3, F5 and F6 and the
occurrence of compounds eluting in the phenolic
P. domestica / flavonoid region was found (Figure 4). Even
P. domestica
Bioactivities F1 F2 F3 F4 F5 F6
though some further phytochemical ASE Extractanalysis is needed
Maceration Extractto properly characterise the compounds
constituting these fractions,
Anti-inflammatory activity one can++ mention the identification
+ +of kaempferol,
+++ ++ quercetin
- -and rutin,
- as
Anti-elastase
well as some of activity
their respective++glycosylated derivatives
+++ in- this++++
eluting+++region- from - several
- P.
Anti-oxidant activity ++ +++ - - ++++ + ++++ ++++
domestica extracts previously studied
Anti-hyaluronidase activity ++++
by our research +
team [6].-Such +observations
++++ +
are also
-
consistent
++
with correlations between chemical composition and bioactivities reported in the literature [9,23–27].
(−): inhibition < 30%; (+): 30% < inhibition < 50%; (++): 50% < inhibition < 70%; (+++): 70% < inhibition < 90%;
The
(++++):bulk extract,
inhibition > 90%.as well as fractions F1–F6, were further analysed using GC-MS. The
compounds identified are listed in Table 3; no compound was identified with certainty in F2. One
mustAkeep in mind
positive that this between
relationship volatile fraction only represents
the anti-oxidant activitya ofsmall proportion
fractions F3, F5ofand
theF6molecular
and the
content of the respective samples analysed.
occurrence of compounds eluting in the phenolic/flavonoid region was found (Figure 4). Even though
some further phytochemical analysis is needed to properly characterise the compounds constituting
theseTable 3. Major compounds identified by GC-MS in the bulk P. domestica leaves extract, as well as in
fractions, one can mention the identification of kaempferol, quercetin and rutin, as well as some
fractions F1 to F6.
of their respective glycosylated derivatives in this eluting region from several P. domestica extracts
previously studied by our research team [6]. Such observations are also consistent with correlations
RI Exp/RI
Extract/Fraction Constituent References
between chemical composition and bioactivities Litareported in the literature [9,23–27].
bulk extract, aspyrocatechol
Theextract
Bulk
1198/1197
well as fractions F1–F6, were further analysed using -GC-MS. The compounds
para vinyl guaiacol 1300/1311
identified are listed in Table 3; no compound was identifiedidentified in P.
with armeniacaindried
certainty fruitsmust
F2. One extract [28] in
keep
Cosmetics 2019, 6, 8 10 of 21
mind that this volatile fraction only represents a small proportion of the molecular content of the
respective samples analysed.
Table 3. Major compounds identified by GC-MS in the bulk P. domestica leaves extract, as well as in
fractions F1 to F6.
Extract/Fraction Constituent RI Exp/RI Lit a References

pyrocatechol 1198/1197 -
para vinyl guaiacol 1300/1311 identified in P. armeniaca dried fruits extract [28]
Bulk extract
eugenol 1348/1348 identified in P. domestica dried fruits extract [29]
identified in P. armeniaca dried fruits extract [28], in P.
palmitic acid 1964/1972
laurocerasus fruits extract [30] and P. mahaleb seed oil [31]
eugenol 1327/1330 identified in P. domestica dried fruits extract [29]
F1 6,10,14-trimethyl-2-pentadecanone 1827/1830 identified in P. domestica oil [32]
3,7,11,15-tetramethyl-2-hexadecene * 1863/1854 -
coumarane 1192/1188 identified in P. mahaleb honey [33]
F3 eugenol 1325/1330 identified in P. domestica dried fruits extract [29]
neophytadiene * 1833/1829 -
ethyl linoleate 2140/2153 identified in P. domestica shoots extract [32]
2-hydroxy-2-cyclopenten-1-one * 897/905 -
2,3-dihydro-3,5-dihydroxy-6-methyl-
F4 1113/1120 identified in P. domestica dried fruits [34]
4H-pyran-4-one
F5
hexadecenoic acid 1939/1943
2-hydroxy-2-cyclopenten-1-one * 897/905 -
F6 coumarane 1196/1188 identified in P. mahaleb honey [33]
hexadecenoic acid 1939/1943
aRI = retention indices determined on HP-1 column (grey cells) or on DB5 column (white cells) using the homologous
series of n-alkanes (C8–C24); and compared to the RI available in the literature. * tentative identification.
3.2. Liquid Cosmetic Ingredient

Botanical extracts that display interesting bioactivities are usually not directly incorporable into
cosmetic formulations as they may display undesirable qualities (colour, odour, viscosity, etc.) [35,36].
The addition of an appropriate cosmetic support, either liquid or solid, can somehow ease their
incorporation into a cosmetic formulation. By being willing to develop liquid ingredients as they
are often preferred when formulating cosmetics, propylene glycol was hence used to directly extract
the active metabolites of P. domestica by-products to free the researchers from the time-consuming
steps of solvent evaporation and resolubilisation of the dry extract in PG. P. domestica skins were
also incorporated in this experimentation as PG, owing to its lower polarity compared to ethanol,
might extract other components that might present some cosmetic activities.
To prepare such extracts, maceration parameters defined earlier for the development of liquid
ingredients were used in the first instance [9]: dried P. domestica leaves and skin macerate in PG (ratio
dried by-product/PG 1/10 w/w) under stirring for 7 h at RT. The resulting extracts were then filtered
over filter paper, and their activities, as well as the ones of propylene glycol alone (no activity reported;
data not shown) were assessed using in vitro bioassays (Figure 5).
As shown in Figure 5, the P. domestica skins PG extract again displayed no significant activities;
this bio-inactivity could be explained either by the absence of active molecules in plum skins or
by the heating applied to ease the fruit peeling during the preparation of Pistoles [7], which may
lead to degradation of thermolabile active compounds (for example, loss of anti-oxidant activity
linked to thermal treatment and to the subsequent decrease of phenolic content was already reported
in the literature [37,38]). Some exhaustive phytochemical characterisation could be considered to
understand its bio-inactivity (and to potentially rule out one of these hypothesis), but given the
absence of anti-aging properties, P. domestica skins were subsequently dropped from this study without
further experimentation.
Cosmetics 2019, 6, 8 11 of 21
100
80
Inhibition (%)
60
40
20
0
Anti -hy aluronidase activity Anti -i nflammator y act ivity Anti-oxi dant act ivity Anti -elastase activit y
Aerial parts (PG) Skins (PG) Positive controls Commercial ingredient
Figure5.5. Bioactivities
Figure Bioactivities of
of P. domestica
domestica extracts obtained
obtained using
using propylene
propylene glycol
glycol compared
comparedtotothethe
bioactivities of commercial cosmetic ingredients used for the respective activities tested
bioactivities of commercial cosmetic ingredients used for the respective activities tested (positive(positive
controls)and
controls) andtotothe
thebioactivities
bioactivitiesof
of aa commercial
commercial ingredient
ingredient based
based on
on aa Prunus
Prunus persica
persica extract.
extract.
As shown
Focus was in Figureon
placed 5, the
the P.development
domestica skins of PG extractingredient
a liquid again displayed
basednoonsignificant activities;
P. domestica leaves
this
as thebio-inactivity
relevant PG could be explained
extract displays either
even by the absence
better of (particularly
activities active molecules in plum
better skins or and
antioxidant by
the heating appliedactivities
anti-inflammatory to ease the fruitevidenced)
were peeling during
than the
thepreparation
corresponding of Pistoles
EtOH [7], which
extract may lead
obtained to
using
degradation of thermolabile active compounds (for example, loss of anti-oxidant
ASE (see Section 3.1). Several assumptions can be made to explain these differences: PG may be a more activity linked to
thermal treatment
appropriate solventandthantoEtOH
the subsequent
to extract decrease of phenoliccontained
active compounds content was
in P.already reported
domestica leaves,inorthe
on
literature
the contrary,[37,38]).
it couldSome
also beexhaustive
less efficientphytochemical
as EtOH to extractcharacterisation could be considered
antagonist compounds and thwart theto
understand
active ones. its bio-inactivity (and to potentially rule out one of these hypothesis), but given the
absence of anti-aging properties, P. domestica skins were subsequently dropped from this study
3.2.1. Optimization
without of Extraction Parameters
further experimentation.
Focus was placed
Attempts were then on undertaken
the development of a liquid
to optimize the ingredient
PG extractionbasedprocedure
on P. domestica leaves as the
to potentialize the
relevant PG extract displays even better activities (particularly better antioxidant and anti-
bioactivity of the resulting P. domestica leaves extract (subsequently named PDL) while keeping
inflammatory activities were evidenced) than the corresponding EtOH extract obtained using ASE
in mind further industrial scale-up and formulation: the effects of variations of several extraction
(see Section 3.1). Several assumptions can be made to explain these differences: PG may be a more
parameters on the bioactivities of the resultant extract were monitored.
appropriate solvent than EtOH to extract active compounds contained in P. domestica leaves, or on
•the contrary,
First, theiteffect
couldofalso
thebeplant/PG
less efficient as on
ratio EtOH thetoresultant
extract antagonist compounds and
extracts’ bioactivities wasthwart the
evaluated:
active ones.
the plant/PG ratio of 1/10 (w/w) was set as the minimal convenient ratio to properly extract
the plant material and to further manipulate the resulting extract (using a lower plant/PG ratio
3.2.1. Optimization
means that not of
allExtraction Parameters
the plant material was soaked in PG, so the extraction would be incomplete).
The bioactivities
Attempts of the
were then resultant extract
undertaken obtained
to optimize the after a 7h-maceration
PG extraction procedure performed at RT were
to potentialize the
compared
bioactivity to those
of the obtained
resulting respectively
P. domestica leaves with plant/PG
extract ratios named
(subsequently of 1/15PDL)
(w/w)while
and keeping
1/20 (w/w)
in
(Figure 6).
mind further industrial scale-up and formulation: the effects of variations of several extraction
parameters on the bioactivities of the resultant extract were monitored.
Independent of the plant/PG ratio, the corresponding extraction yield was roughly similar
• First, the effect of the plant / PG ratio on the resultant extracts’ bioactivities was evaluated: the
(≈80%);plant
however, the extract
/ PG ratio obtained
of 1/10 (w/w) wasusing
set as the
the plant/PG ratio of 1/10
minimal convenient ratio(w/w) displays
to properly the the
extract best
bioactivities. Therefore,
plant material andthe
to plant/PG ratio of 1/10
further manipulate (w/w) was
the resulting used(using
extract for further optimisation
a lower plant / PG of the
ratio
extraction parameters.
means that not all the plant material was soaked in PG, so the extraction would be incomplete).
The bioactivities of the resultant extract obtained after a 7h-maceration performed at RT were
• Then, the effect of the duration of the contact between the dried plant material and the PG on the
compared to those obtained respectively with plant / PG ratios of 1/15 (w/w) and 1/20 (w/w)
resultant extracts’ bioactivities was evaluated (Figure 7). No matter this duration, the extraction
(Figure 6).
yields were similar (≈73–74%) and all three extracts displayed very interesting antioxidant
and anti-hyaluronidase activities. Some promising anti-inflammatory activity was also found.
100
Cosmetics 2019, 6, 8 12 of 21
80
However, given the fact that a long extraction process would be less interesting from an economical
Inhibition (%)
point of view, one can conclude that maceration times of between 4 h and 7 h are appropriate.
60
• Finally, the effect of the temperature at which the maceration was performed was evaluated: to do
so, both 4 h- and 7 h-macerations were performed either at RT or at 70 ◦ C, respectively, using a
40
plant/PG ratio of 1/10 (w/w) and the resultant extracts’ bioactivities were evaluated using in vitro
assays (Figure 8).
Cosmetics
202018, 5, x FOR PEER REVIEW 12 of 20
0
100 Anti-hy aluronidase Anti -i nflammatory Anti -oxidant Anti -elastase Whitening activi ty Whitening activi ty
act ivity act ivity act ivity act ivity (L-Tyr) (L-DO PA)
PDAE 1/10 RT 7h PDAE 1/15 RT 7h PDAE 1/20 RT 7h

80
Figure 6. Effect of the plant / PG ratio on the resulting extracts’ bioactivities (plant / PG ratios of 1/10,
Inhibition (%)
60 and 1/20 (w/w) respectively, with maceration performed at RT for 7 h).

1/15
Independent
40 of the plant / PG ratio, the corresponding extraction yield was roughly similar
(≈80%); however, the extract obtained using the plant / PG ratio of 1/10 (w/w) displays the best
bioactivities. Therefore, the plant / PG ratio of 1/10 (w/w) was used for further optimisation of the
20
• Then, the effect of the duration of the contact between the dried plant material and the PG on
0 resultant extracts’ bioactivities was evaluated (Figure 7). No matter this duration, the
the Anti-hy aluronidase Anti -i nflammatory Anti -oxidant Anti -elastase Whitening activi ty Whitening activi ty
extraction yields were actsimilar
act ivity ivity (≈73–74%)
act ivity and all three
act ivity extracts displayed
(L-Tyr) very(L-DO
interesting
PA)
antioxidant and anti-hyaluronidase

PDAE 1/10 RT 7h activities. PDAE 1/15Some RT 7h promising PDAE 1/20anti-inflammatory
RT 7h activity was
also found. However, given the fact that a long extraction process would be less interesting
from6.6.an
Figure
Figure economical
Effect
Effect of the
of point
the plant/PG
plant / PG ofratio
view,
ratio onone
on can conclude
the resulting
the resulting that
extracts’ maceration
bioactivities
bioactivities times
(plant
(plant/PG/ PG of ratios
between
ratios of
of1/10,4 h and
1/10,
7 hand
1/15
1/15 are1/20
and appropriate.
1/20 (w/w)
(w/w)respectively,
respectively,with withmaceration
macerationperformed
performedatatRT RTfor
for7 7h).h).
Independent of the plant / PG ratio, the corresponding extraction yield was roughly similar
(≈80%); however, the extract obtained using the plant / PG ratio of 1/10 (w/w) displays the best
100
bioactivities. Therefore, the plant / PG ratio of 1/10 (w/w) was used for further optimisation of the
• 80 Then, the effect of the duration of the contact between the dried plant material and the PG on
the resultant extracts’ bioactivities was evaluated (Figure 7). No matter this duration, the
Inhibition (%)
extraction yields were similar (≈73–74%) and all three extracts displayed very interesting
60
antioxidant and anti-hyaluronidase activities. Some promising anti-inflammatory activity was
also found. However, given the fact that a long extraction process would be less interesting
40
from an economical point of view, one can conclude that maceration times of between 4 h and
7 h are appropriate.
20
100
0
Anti -hy aluronidase Anti-inflammatory Anti-oxi dant Anti -elastase Whitening activi ty Whitening activi ty
80 PDAE 1/10 RT 4h PDAE 1/10 RT 7h PDAE 1/10 RT 48h

Inhibition (%)
Figure7.7.Effect
Figure Effect of
of the
the maceration
maceration duration
duration on
on the
the resulting
resulting extracts’
extracts’ bioactivities
bioactivities(plant / PG ratio
(plant/PG ratio 1/10
1/10
60
(w/w), with maceration performed at RT for either 4 h, 7 h or 48
(w/w), with maceration performed at RT for either 4 h, 7 h or 48 h). h).
40
20
0
PDAE 1/10 RT 4h PDAE 1/10 RT 7h PDAE 1/10 RT 48h

• Finally, the effect of the temperature at which the maceration was performed was evaluated:
to do so, both 4 h- and 7 h-macerations were performed either at RT or at 70 °C, respectively,
Cosmeticsusing
2019, 6,a8 plant / PG ratio of 1/10 (w/w) and the resultant extracts’ bioactivities were evaluated
13 of 21
using in vitro assays (Figure 8).
100
80
Inhibition (%)
60
40
20
0
PDAE 1/10 RT 4h PDAE 1/10 RT 7h PDAE 1/10 70 4h PDAE 1/10 70 7h
Figure8.8.Effect
Figure Effectof
of the
the maceration
maceration temperature on
on the
the resulting
resultingextracts’
extracts’bioactivities
bioactivities(plant / PG ratio
(plant/PG ratio
1/10 (w/w),
1/10 (w/w), with
with maceration
macerationperformed
performedfor
foreither
either44hhoror77h,h,atatRT ◦ C,respectively).
RToror7070°C, respectively).
No
Nosignificant
significantchanges
changeswere
wereobserved
observedininterms
termsofofbioactivities between
bioactivities between extracts obtained
extracts obtainedafter 4h-
after
and
4h- 7h-macerations (the 7 h-ones
and 7h-macerations (the were only slightly
7 h-ones moreslightly
were only active, particularly
more active, against lipoxygenase,
particularly e.g.,
against
lipoxygenase, e.g.,activity),
anti-inflammatory anti-inflammatory
which wereactivity), which were
either performed at RTeither ◦ C. However,
performed
or at 70 at RTthe orapplication
at 70 °C.
ofHowever, the application
heating induced of heating
a significant induced
energetic a significant
consumption that energetic
brought no consumption
added valuethat brought
to the no
resultant
added value to the resultant extract in terms of bioactivity; further experimentation will therefore
extract in terms of bioactivity; further experimentation will therefore be performed at RT, which is also be
performed
more at RT, relevant
ecologically which is for
alsoa more
futureecologically relevant for a future process scale-up.
process scale-up.
3.2.2.
3.2.2.Impact
Impactof
ofthe
theIngredient’s
Ingredient’s Colour
Colour
Solvents
Solvents used
used to to extract
extract metabolites
metabolites from a plant or a by-product by-product alsoalso extract
extract the
the molecules
molecules
responsible
responsiblefor forthe
thelatter’s
latter’s colour,
colour, while
while odourlessness and colourlessness
colourlessnessare arepreferable
preferablefor forcosmetic
cosmetic
purposes[35,36,39]
purposes [35,36,39] andand need
need therefore
therefore to to be
be submitted
submitted to further
further processing,
processing, including
includingnotably
notablyaa
discolorationprocedure.
discoloration procedure. SuchSuch a discoloration
a discoloration can becan be achieved
achieved via adsorption
via adsorption of compoundsof compounds
responsible
responsible
for the colour forontheactivated
colour oncarbon:
activated carbon:
the powderedthe powdered
activatedactivated
carbon is carbon
easilyisadded
easily added
to the to the
liquid
liquid extract and removed by settling and filtration. However, such adsorption
extract and removed by settling and filtration. However, such adsorption is not selective and active is not selective and
active molecules
molecules can be can be retained
retained on theon the activated
activated carbon, carbon, implying
implying a consequent
a consequent lossloss of activity
of activity of
of the
the resulting
resulting ingredient
ingredient [9]. Some
[9]. Some loss ofloss of ingredient
ingredient mass was massalsowas also reported
reported in some in some
cases cases
after after
activated
activated
carbon’s carbon’s elimination
elimination [40]. Many[40]. Many time-consuming
time-consuming trials
trials are often are oftentonecessary
necessary determine to the
determine the
appropriate
appropriate discoloration parameters (varying activated carbon / ingredient
discoloration parameters (varying activated carbon/ingredient (w/w) ratio, duration of the contact (w/w) ratio, duration of
the contact
between the between
activatedthe activated
carbon and carbon and the etc.).
the ingredient, ingredient, etc.). Discoloration
Discoloration attempts usingattempts using
molecular
molecular distillation could also be performed but imply the cost
distillation could also be performed but imply the cost afferent to this equipment. afferent to this equipment.
Toavoid
To avoidsuch
suchlosses
lossesof of ingredient’s
ingredient’s mass
mass andand activity,
activity, some
some preliminary
preliminaryformulation
formulationtrialstrialshave
have
been performed to establish whether this discoloration procedure is mandatory
been performed to establish whether this discoloration procedure is mandatory in the precise case in the precise case of
the PDL ingredient; this ingredient was incorporated at 1% and 4.5% in
of the PDL ingredient; this ingredient was incorporated at 1% and 4.5% in a typical oil-in-watera typical oil-in-water (O/W)
emulsion
(O/W) to observe
emulsion its impact
to observe its on the finished
impact products’products’
on the finished aspect (Figure
aspect9).(Figure
The use 9).ofThe
theuse
non-of
discoloured
the ingredient
non-discoloured only resulted
ingredient in a slight
only resulted in beige coloration,
a slight which is convenient
beige coloration, for cosmetic
which is convenient for
formulations.
cosmetic formulations.
Cosmetics 2019, 6, 8 14 of 21
Figure 9. O/W
Figure emulsions
9. O/W formulated
emulsions formulatedwith
withno
no(left),
(left), 11 (centre) or4.5%
(centre) or 4.5%(right)
(right)
of of
thethe
PDLPDL ingredient.
ingredient.
3.3. Green
3.3. Green Liquid
Liquid Cosmetic
Cosmetic Ingredient
Ingredient
To develop an even greener ingredient, consistent with the notions of naturality and circular
To develop an even greener ingredient, consistent with the notions of naturality and circular
economy mentioned earlier, these optimized maceration conditions (7 h, RT, plant/PG ratio 1/10
economy mentioned earlier, these optimized maceration conditions (7 h, RT, plant / PG ratio 1/10
w/w) were then transposed to maceration assays using several green glycols issued from the biomass
w/w)obtained
were then fromtransposed to maceration
different suppliers. assays using
The bioactivities of theseveral green
resultant glycols
extracts issued from
(subsequently namedthegPDL
biomass
obtained from different suppliers. The bioactivities of the resultant extracts (subsequently
standing for green PDL extract) were assessed using in vitro assays and compared to the ones with the named
gPDL standing forPDL
corresponding green PDLobtained
extract extract)using
wereconventional
assessed using in vitro
PG (Table 4).assays and compared
Apart from green glycolto2 the
thatones
withpresented
the corresponding PDL extract activity,
some anti-hyaluronidase obtained using
none conventional
of the green glycolsPG (Table
tested shared4).responsibility
Apart from ingreen
glycolthe2bioactivities found.some anti-hyaluronidase activity, none of the green glycols tested shared
that presented
Extraction
responsibility in thetrials with green
bioactivities glycols were obtained with extraction yields similar to the ones
found.
calculated
Extractionfor PDLwith
trials obtained
greenwith conventional
glycols PG (≈73–74
were obtained with %). All the yields
extraction seven gPDL displayed
similar to the ones
anti-hyaluronidase and anti-oxidant activities like the ones of conventional PDL; gPDL6 displayed
calculated for PDL obtained with conventional PG (≈73–74 %). All the seven gPDL displayed anti-
some additional interesting anti-inflammatory activity.
hyaluronidase and anti-oxidant activities like the ones of conventional PDL; gPDL6 displayed some
All the green glycols tested here are therefore convenient solvents to obtain a greener version of
additional interesting anti-inflammatory activity.
this ingredient based on P. domestica leaves extract. The final decision about the green PG that will be
All the
used green
at the glycolsscale
industrial tested
willhere are therefore
correspond to the convenient solvents
best compromise to obtain
between a greener version
the bioactivities of the of
this ingredient based on P. domestica leaves extract. The final decision
resultant ingredient and more trivially, the green glycols’ prices per kilogram. about the green PG that will be
used at the industrial scale will correspond to the best compromise between the bioactivities of the
resultant ingredient and more trivially, the green glycols’ prices per kilogram.
Cosmetics 2019, 6, 8 15 of 21
Table 4. Bioactivities of PDL extracts obtained using several green glycols.
PDL 1/10 RT
Extract gPDL1 gPDL2 gPDL3 gPDL4 gPDL5 gPDL6 gPDL7
7H
green glycol 5 green glycol 6
conventional green glycol 1 green glycol 2 green glycol 4 green glycol 7
Solvent green glycol 3 (pentylene (pentylene glycol
propylene (propan-1.2-diol (dipropylene (propan-1.2-diol (propan-1.3-diol
(composition) (propan-1.2-diol) glycol and and glyceryl
glycol 99.6 %) glycol) >99.0 %) >99.5 %)
phenylpropanol) caprylate/caprate)
Anti-inflammatory activity +* (-)** + (-) ++ (-) + (-) + (-) ++ (-) +++ (-) + (-)
Anti-oxidant activity ++++ (-) ++++ (-) +++ (-) +++ (-) ++++ (-) ++++ (-) ++++ (-) ++++ (-)
Anti-hyaluronidase activity ++++ (-) +++ (-) ++++ (+) ++++ (-) +++ (-) +++ (-) +++ (-) +++ (-)
* (-): inhibition <30%; (+): 30 % <inhibition <50%; (++): 50 % <inhibition <70%; (+++): 70 % <inhibition <90%; (++++): inhibition >90%. ** Activities reported in brackets correspond to the
bioactivities of the corresponding glycol vehicle alone.
Cosmetics 2019, 6, 8 16 of 21
3.4. HPLC Analysis of Liquid Ingredients

3.4. HPLC Analysis of Liquid Ingredients
HPLC analysis of such liquid ingredients for compositional or stability purposes revealed
HPLCtoanalysis
themselves of such
be useless givenliquid ingredients
the prevalence for propylene
of the compositionalglycolor signal
stability purposes
masking revealed
the extracted
themselvessignals.
molecules’ to be useless given the prevalence
No experimental methodology of the propylene
developed glycol signal
to remove masking
the glycol and the extracted
to gain access
molecules’ signals. No experimental methodology developed to remove the glycol
to the extracted molecules could be found in the literature, so we had to develop such a protocol. and to gain access
to the
SPE wasextracted
selectedmolecules
to achievecould be found
this PG removalin the
andliterature, so we had to
several parameters develop
had such a protocol.
to be settled SPEa
on to obtain
was selected
convenient PGtoelimination
achieve this PG removal
(sorbent and several
type, saturation parameters
volume, etc.). had
The to be settled
closest accesson
wetohad
obtain a
to the
convenient PG elimination (sorbent type, saturation volume, etc.). The closest access
chemistry of the plum leaves’ extract was the HPLC profile obtained for the ethanolic P. domestica we had to the
chemistry
leaves’ of the
extract, plumisleaves’
which extract was
not completely the HPLC profile
representative of theobtained
chemicalforprofile
the ethanolic
of the PGP. extract
domesticaas
leaves’ extract, which is not completely representative of the chemical profile of the PG extract as the
the different polarities of both solvents enable the extraction of various families of molecules, a fact
different polarities of both solvents enable the extraction of various families of molecules, a fact
evidenced by the bioactivity discrepancies observed between both extracts. By extrapolation, given
evidenced by the bioactivity discrepancies observed between both extracts. By extrapolation, given
the nature of the families of compounds identified in this extract, two cartridges were tested (Oasis
the nature of the families of compounds identified in this extract, two cartridges were tested (Oasis
HLB6cc, Waters, Guyancourt, Ile-de-France, France and Strata XL, Phenomenex, Torrance, California,
HLB6cc, Waters, Guyancourt, Ile-de-France, France and Strata XL, Phenomenex, Torrance, California,
USA). Both were composed of a reversed phase polymeric sorbent that strongly retained neutral,
USA). Both were composed of a reversed phase polymeric sorbent that strongly retained neutral,
acidic, and basic compounds and gave similar results. For convenience, further optimisation of our
acidic, and basic compounds and gave similar results. For convenience, further optimisation of our
SPE methodology was then achieved using Oasis HLB6cc cartridges available at the laboratory.
SPE methodology was then achieved using Oasis HLB6cc cartridges available at the laboratory.
After proper conditioning and equilibration steps, saturation of the Oasis HLB6cc cartridge was
After proper conditioning and equilibration steps, saturation of the Oasis HLB6cc cartridge was
assessed via sequential addition on the cartridge of the liquid ingredient obtained via maceration of
assessed via sequential addition on the cartridge of the liquid ingredient obtained via maceration of
P. domestica leaves in PG, properly diluted in water to lower its viscosity, 0.5 mL at a time. Regular
P. domestica leaves in PG, properly diluted in water to lower its viscosity, 0.5 mL at a time. Regular
HPLC analysis was performed to monitor the saturation of the cartridge that was supposed to be
HPLC analysis was performed to monitor the saturation of the cartridge that was supposed to be
reached when P. domestica extracted molecules appeared in the subsequent HPLC chromatogram.
reached when P. domestica extracted molecules appeared in the subsequent HPLC chromatogram.
HPLC
HPLCanalysis
analysis ofof the
the effluent’s
effluent’s composition
composition enabled
enabled thethe identification
identification of P. domestica
of P. domestica extracted
extracted
molecules
molecules(the
(the major
major one being identified
one being identifiedasaschlorogenic
chlorogenicacid)acid)
in in
thethe effluent
effluent as soon
as soon as 3asmL3 of
mLtheof
the ingredient were loaded on the cartridge, indicating its saturation
ingredient were loaded on the cartridge, indicating its saturation (Figure 10). (Figure 10).
mAU mAU
366 nm 366 nm
140 ELSD 140 ELSD
120 120
Chlorogenic acid
100 100
80 80
60 60
40 40
20 20
0 0
0 5 10 15 20 25 30 35 40 min 0 5 10 15 20 25 30 35 40 min
Figure 10.SPE
Figure10. SPEeffluent’s
effluent’scomposition
composition after
after load
load of the cartridge using respectively
respectively 1.5
1.5 (left)
(left)and
and33mL
mL
(right)
(right)ofofthe
the ingredient, monitoredusing
ingredient, monitored usingHPLC
HPLCanalysis
analysis at 366
at 366 nmnm (blue
(blue signal)
signal) and with
and with ELSDELSD
(red
(red signal).
signal).
Once
Oncethe
thevolume
volumeofofthe
theingredient
ingredientnecessary
necessarytotosaturate
saturatethethecartridge
cartridgewaswasdetermined,
determined,we wehad
hadto
establish thethe
to establish volume
volume necessary
necessary to toelute
eluteallall
thethemolecules
moleculesretained
retainedon onthe
the cartridge.
cartridge. ToTo do
do so,
so,elution
elution
ofofthe sample was achieved by adding MeOH/DCM (1/1 v/v), 1 mL
the sample was achieved by adding MeOH/DCM (1/1 v/v), 1 mL at a time; we found that the at a time; we found that the
addition of 4 mL of MeOH/DCM (1/1 v/v) ensured the complete elution
addition of 4 mL of MeOH/DCM (1/1 v/v) ensured the complete elution of the compounds retained of the compounds retained
on
onthethecartridge.
cartridge.
Hence
Hencethetheappropriate
appropriate SPESPE conditions
conditions to to gain
gain access
access to
to the
the extracted molecules using
extracted molecules using Oasis
Oasis
HLB6cc
HLB6cccartridges
cartridgesconsisted
consistedofofa aload
loadofof2.5
2.5mL mLofofthe
theingredient
ingredientononthe thecartridge,
cartridge,and
andafter
afterremoval
removal of
the
of glycol, in the
the glycol, in successive use use
the successive of 4 of
mL4 of
mLMeOH/DCM
of MeOH/DCM v/v)
(1/1(1/1 to to
v/v) collect thethe
collect molecules
molecules retained
retainedon
the
oncartridge.
the cartridge.
To
Tovalidate
validatethis
thismethodology,
methodology,aasolution
solutionof ofchlorogenic
chlorogenicacid acid(5(5 mg/mL
mg/mL in in MeOH)
MeOH)was wasanalysed
analysed
using
usingHPLCHPLCandandthethearea
areaof
ofthe
thechlorogenic
chlorogenic acid acid peak
peak was determined. In In parallel,
parallel,the
thePGPGingredient
ingredient
diluted
diluted inin water
water (1/3)
(1/3) was
was supplemented
supplemented with with 12.5
12.5 mg mg ofof chlorogenic
chlorogenic acidacid toto reach
reach thethe final
final
concentration
concentrationofof55mg/mL
mg/mL andand loaded on a SPE cartridge. Elution Elution waswas performed
performedas asdetermined
determinedand and
the resulting extract was concentrated and analysed using HPLC in the same analytical conditions.
Cosmetics 2019, 6, 8 17 of 21

the resulting extract was concentrated and analysed using HPLC in the same analytical conditions.
areaofofthethe
The area chlorogenic
chlorogenic acidacid
peakpeak was determined
was determined and compared
and compared to the onetodetermined
the one determined
previously
previously
(Figure 11);(Figure
one can11); one canthat
conclude conclude that this methodology
this methodology enabled
enabled a signal a signal amplification
amplification by a factor byof 6.a
factor
Such aof 6. Such a enabling
procedure procedure enabling
the removaltheof removal
glycol andof the
glycol andto
access the
the access to themolecules
extracted extractedismolecules
essential
is
to essential to a) geton
(a) get a glimpse a glimpse on thecomposition
the molecular molecular composition
of the ingredientof theobtained
ingredientandobtained andmolecule
identify the identify
the molecule
responsible forresponsible for the
the bioactivities bioactivities
evidenced; andevidenced;
to (b) ensure andthetostability
b) ensure the stability
monitoring monitoring
of the ingredient’s of
the ingredient’s
composition composition
over time, which is aover time, pre-requisite
mandatory which is abefore mandatory pre-requisite In
its commercialisation. before
fact, itits
is
commercialisation. In fact, it is absolutely necessary to certify the post-manufacture
absolutely necessary to certify the post-manufacture stability of a cosmetic ingredient and to establish stability of a
cosmetic ingredient
its appropriate and toofestablish
conditions storage its
andappropriate
use (type ofconditions
container of storageitand
in which mayuse
be (type
stored,ofmaximum
container
in which of
duration it may be stored,
storage, maximum
ideal storage duration of
temperature, storage,
etc.) to shareideal
withstorage temperature,
customers, namelyetc.) to share
the finished
with customers,
cosmetic products’ namely the finished cosmetic products’ formulators.
formulators.
Chlorogenic acid peak area : 13621

mAU
80 Chlorogenic acid peak area : 83965
60
40
20
0 10 20 30 40 50 60 min
Figure 11.
Figure Determination of
11. Determination of the
the chlorogenic
chlorogenic acid
acid peak
peak area
area monitored
monitored by by HPLC
HPLC analysis
analysis at
at 366
366 nm:
nm:
solution of
solution of chlorogenic
chlorogenic acid
acid in
in MeOH
MeOH (red
(red signal)
signal) and
and PG
PG ingredient
ingredient diluted
diluted in
in water
water (1/3)
(1/3) and
and
supplemented with
supplemented with chlorogenic
chlorogenic acid
acid (blue
(blue signal). The artificial
signal). The artificial shift
shift between
between both
both chlorogenic
chlorogenic acid
acid
peaks
peaks eases
eases the
the reading
reading of
of the
the figure.
figure.
3.5. Stability Testing

3.5. Stability Testing
Accelerated stability testing was then undertaken to ensure that the liquid ingredient developed
Accelerated stability testing was then undertaken to ensure that the liquid ingredient developed
meets the intended quality standards when stored under specific conditions. To do so, the P. domestica
meets the intended quality standards when◦ stored under specific conditions. To do so, the P. domestica
ingredient was stored in a glass vial at 42 C, and weekly monitoring will be undertaken to evaluate
ingredient was stored in a glass vial at 42 °C, and weekly monitoring will be undertaken to evaluate
critical aesthetic properties (changes in colour and odour), and to track any change in its chemical
critical aesthetic properties (changes in colour and odour), and to track any change in its chemical
composition by HPLC-DAD-ELSD. Those stability assays are not over, but so far, i.e., three weeks after
composition by HPLC-DAD-ELSD. Those stability assays are not over, but so far, i.e., three weeks
their beginning, no alteration of the ingredient was noticed.
after their beginning, no alteration of the ingredient was noticed.
Once the definite formulation of the ingredient has been determined, further stability assays will
Once the definite formulation of the ingredient has been determined, further stability assays will
be performed by putting the finished product in an aging chamber to monitor the potential changes
be performed by putting the finished product in an aging chamber to monitor the potential changes
that could take place from the initial formulation to the final consumer’s everyday use of the product.
that could take place from the initial formulation to the final consumer’s everyday use of the product.
4. Discussion and Conclusions
4. Discussion and Conclusion
This article presents an integrated strategy to develop innovative natural cosmetic actives to fully
This
satisfy article presents
green-minded an integrated
consumers by usingstrategy to develop
agricultural innovative
by-products naturalraw
as starting cosmetic actives
material, henceto
fully satisfy green-minded consumers by using agricultural by-products as starting
placing notions of naturality and circular economy at the very centre of this kind of design. Such araw material,
hence placing
conversion notions of naturality
of by-products, originallyand circular
heading for economy at the
destruction, intovery
highcentre of this
value raw kind ofpresents
materials design.
Such a conversion of by-products, originally heading for destruction, into high value
benefits for various actors of the integrated supply chain generated. In fact, it represents a wasteraw materials
presents
management benefits for various
solution actors of sector
for the agri-food the integrated
and brings supply chain generated.
more sourcing In fact,
transparency anditsustainability
represents a
waste management solution for the agri-food sector and brings more sourcing transparency
to the cosmetic sector, while constituting a fantastic opportunity for both actors to communicate about and
sustainability to the cosmetic sector,
their environmentally-friendly actions. while constituting a fantastic opportunity for both actors to
communicate about their environmentally-friendly actions.
However, before a formulator will consider using such a natural cosmetic active in an actual
finished-product formulation, the ingredient must still pass several crucial steps: its efficacy, quality,
shelf life and safety/tolerability must be controlled prior to being launched on the market. In fact, the
Cosmetics 2019, 6, 8 18 of 21
However, before a formulator will consider using such a natural cosmetic active in an actual
finished-product formulation, the ingredient must still pass several crucial steps: its efficacy, quality,
shelf life and safety/tolerability must be controlled prior to being launched on the market. In fact,
the safety of finished cosmetic products is based on the safety of the individual ingredients it contains.
Even if not mandatory, it is highly recommended to assess the stability of ingredients over time to
establish that no chemical changes generating undesired molecules (harmful molecules, compounds
responsible for an unwanted colour or odour, etc.) takes place. The objective is to ensure that the
challenged ingredient preserves its physical and chemical integrities, its microbiological qualities and
functionalities under appropriate storage conditions [41]. No method enabling such an assessment
exists for liquid cosmetic ingredients directly developed in glycolic solvents. To determine that the
liquid ingredients developed during this project meets the intended quality standards, a simple SPE
method was developed to eliminate their glycol content to gain access to their molecular composition
using HPLC analysis. This method can easily be transferred with some eventual adjustments (type of
sorbent, washing solvent, etc.) to any type of natural cosmetic ingredients developed using glycolic
solvent, whether the raw material consists of plant or an agri-food by-product.
The toxicological assessment of the cosmetic ingredient is mandatory before its commercial launch.
Regularly revised guidelines regarding all aspects of safety evaluation of cosmetic substances are
published by the Scientific Committee on Consumer Safety (SCCS) at the European level, while the
U.S. Food and Drug Administration (FDA) guarantees the vigilance about worldwide regulation
compliance [42]. A thorough examination of the literature available about the species studied usually
already brings some clues and might lead to its abandonment at the very beginning of the development
if too many toxicological risks are identified. If nothing is reported in the literature, the ingredient
development is undertaken, and the proper toxicology assessment is usually performed at the very end
of the process for cost reasons. However, this assessment is more and more integrated directly into the
cosmetic ingredient R&D process to avoid the unpleasant discovery of some toxicity issue at the end of
the process, which could lead to the restriction or even the abandonment of the ingredient’s use due to
safety concerns [43]. It is generally recommended to at least assess the potency of the ingredient
to induce some skin or ocular irritability [44,45], skin sensitization [46] and phototoxicity [47];
some additional mutagenicity, carcinogenicity, reprotoxicity assays, etc., might complete the safety
assessment of a cosmetic active.
Even if no pesticides were used on the crop from where the starting material comes from,
the presence of pesticides, and more widely, of any contaminant (herbicides, pollutants, etc.) in
the final cosmetic ingredient cannot be ruled out. Analysis of these residues must be undertaken using
GC-MS/MS and LC-MS/MS screening via multi-residue analyses before commercialisation of the
finished cosmetic ingredient.
Therefore, the development of new natural ingredients for cosmetics is a long-term and not
always rewarding procedure but using agri-food by-products as a starting material opens new
innovation pathways.
Author Contributions: Conceptualization, H.P., G.V.D. and X.F.; methodology, H.P., S.A. and X.F.; validation,
H.P., P.B., S.A. and X.F.; formal analysis, H.P.; investigation, H.P.; resources, G.V.D. and X.F.; data curation, H.P.
and P.B.; writing—original draft preparation, H.P. and P.B.; writing—review and editing, H.P., P.B., S.A. and
X.F.; visualization, H.P. and P.B.; supervision, G.V.D. and X.F.; project administration, G.V.D. and X.F.; funding
acquisition, G.V.D. and X.F..
Funding: This research was funded by the Association Nationale de la Recherche et de la Technologie,
grant number 0036/2016.
Acknowledgments: H.P. is grateful to the CIFRE—Conventions Industrielles de Formation par la REcherche—
(ANRT JYTA) for her Ph.D. financing. The authors wish to thank Marc Richard for kindly giving us access to the
Prunus plant material and Thibault Allanic for the formulation assays.
Conflicts of Interest: The authors declare no conflict of interest.
Cosmetics 2019, 6, 8 19 of 21
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Cosmetics 2019, 6, 8; doi:10.3390/cosmetics6010008 www.mdpi.com/journal/cosmetics

2. Materials and Methods

2.1. Plant Material

2.2. Plant Extraction

2.3. Fractionation of P. domestica Leaves Extract

Table 1. Positive controls used in each bioassay performed.

Assay Positive Control 1 Positive Control 2

or as follows (for the hyaluronidase and collagenase assays):

2.4.3. DPPH Radical Scavenging Assay

2.4.4. Tyrosinase Assays

2.4.5. Lipoxygenase Assay

2.4.6. Elastase Assay

2.4.7. Hyaluronidase Assay

2.4.8. Collagenase Assay

2.5. Solid Phase Extraction

2.8. Stability Assays

2.8. Stability Assays

3.1. Bioactivities of Crude Extracts

with ELSD, presenting the major families of compounds identified in

Polyphenols and flavonoids

Extract/Fraction Constituent RI Exp/RI Lit a References

3.2. Liquid Cosmetic Ingredient

Aerial parts (PG) Skins (PG) Positive controls Commercial ingredient

PDAE 1/10 RT 7h PDAE 1/15 RT 7h PDAE 1/20 RT 7h

60 and 1/20 (w/w) respectively, with maceration performed at RT for 7 h).

antioxidant and anti-hyaluronidase

80 PDAE 1/10 RT 4h PDAE 1/10 RT 7h PDAE 1/10 RT 48h

PDAE 1/10 RT 4h PDAE 1/10 RT 7h PDAE 1/10 RT 48h

PDAE 1/10 RT 4h PDAE 1/10 RT 7h PDAE 1/10 70 4h PDAE 1/10 70 7h

Table 4. Bioactivities of PDL extracts obtained using several green glycols.

3.4. HPLC Analysis of Liquid Ingredients

Cosmetics 2018, 5, x FOR PEER REVIEW 17 of 20

Chlorogenic acid peak area : 13621

80 Chlorogenic acid peak area : 83965

3.5. Stability Testing

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