Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 64-72
Published Online November 2013 (
Open Access JCDSA
Dualistic Properties of Cosmetic Formulations Based on
Phenylpropanoids from Ajuga reptans
Silvia Vertuani1, Paola Ziosi2, Roberto Dal Toso3, Chiara Beatrice Vicentini1, Stefano Manfredini1
1Master Course in Cosmetic Science and Technology, Department Life Science and Biotechnology, University of Ferrara, Ferrara,
Italy; 2AmbrosiaLab, Ferrara, Italy; 3Institute for Biotechnological Research (I.R.B.), Altavilla Vicentina, Vicenza, Italy.
Received October 17th, 2013; revised November 13th, 2013; accepted November 20th, 2013
Copyright © 2013 Silvia Vertuani et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Our continued interest in the research and development of cosmetic active ingredients deriving from natural sources led
us to investigate the potential of a purified extract of Ajuga reptans, a plant belonging to the family Labiatae and known
for its traditional use in skin healing. The extracts deriving from a biotechnology platform are composed by meris-
tematic cell culture, developed in the frame of a NTFP (non-timber forest product) project, and characterized by high
content in phenylpropanoid, of which teupolioside represents the majority component. The latter is a phenylpropanoid
glucoside, structurally correlated echinacoside and known in the literature for the antioxidant properties. This study was
conducted with the purpose of evaluating the applicability of the Ajuga reptans extract within different cosmetic for-
mulations. In particular, Photochemiluminescence (PCL) was used to proof the antioxidant capacity of cosmetic formu-
lations containing the product, in relation to the change of the title of teupolioside. Furthermore, UVA and UVB filter-
ing properties were also investigated. The results of the study showed relevantly antioxidant capacity of the finished
formulation against superoxide anion, which is the main reactive oxygen species responsible for skin aging and signifi-
cant synergic capacities to filter UV radiation.
Keywords: Phenylpropanoids; Superoxide Anion; Radical Scavenger; UV Filter; Cosmetic Formulation
1. Introduction
Ajuga reptans, typical of the grassy zones of Europe, West-
ern Asia and Africa, is an annual herbaceous plant be-
longing to the Lamiaceae family (Figure 1).
It is widely present in temperate regions, especially in
mountainous areas, where it is frequently used as forage
for cattle. Ajuga reptans L. is frequently used in the tra-
ditional medicine of many countries especially, the east-
ern part of Europe, for its skin healing properties. The
extracts obtained from “bugle” (Ajuga reptans L.) most
likely own their activity to the content of polyphenols of
the flavonoid and polyphenol-carboxylic acids type (an-
tioxidant, vascular and antimicrobial properties), as well
as of iridoids (anti-inflammatory and wound healing) [1].
Moreover, the research on cosmetic product for sun pro-
tection is evolving towards the development of natural
products. In addition to classic UV filters, active ingre-
dients with antioxidant activity can block the activity of
reactive oxygen species (ROS) when formed during ex-
posure, and these characteristics would be very interest-
Figure 1. Structure of teupolioside.
ing if expressed within the same molecule (dualistic prop-
erties) [2]. The development of an integrated sun protec-
tion system, which would allow taking full advantage of
the beneficial influences of sunlight, but reducing skin
damages, is becoming more important and essential. The
field of solar products (dermoprotective and after-sun)
consists in the use of natural emollient, i.e. lipid vegeta-
ble oils such as coconut, avocado, wheat germ, shea but-
Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans 65
ter, or of different plant extracts, used for antioxidant
activities specific of the plant complex. A few of these
ingredients are also endowed by both antioxidant and UV
protecting (dualistic) activities [3]. Our continued interest
in the research and development of cosmetic ingredients
and nutritional supplements, led us to investigate the po-
tential for use in cosmetics of an extract of Ajuga reptans
obtained with a particular biotechnology platform, em-
ploying in vitro plant cells cultures. Plant cell culture
consents production of the active compounds present in
plants, allows the attainment of concentrated extracts in
phytoconstituents of the plant, which is usually difficult
to obtain with traditional methods (i.e. extraction from
plants) and is highly sustainable. Extracts obtained from
the Ajuga reptans cell culture, are characterized by the
presence of phenylpropanoids (PPs) and responsible of
the activity profile of the plant [4]. As mentioned above,
these compounds perform an important protective func-
tion facing environmental stress, and in virtue of this,
they bear important biological properties. In this study,
because of our interest in dualistic molecules, we invest-
tigated the free radical scavenging and UV filtering po-
tential of formulations based on an extract from plant cell
lines of Ajuga reptans, with high titre (minimum 50%) in
total PPs, of which the majority is the type A (PPA) also
called teupolioside.
The latter is produced as a secondary metabolite by
Ajuga reptans, but in our cell cultures, it appears to be
the primary component. Smaller amounts of the phenyl-
propanoid B (PPB, methoxy-teupolioside) and the phenyl-
propanoid C (PPC, verbascoside) are also present (Fig-
ures 2 and 3).
From the structural point of view, the phenylpropanoid
glycosides (PPGs) are characterized by a cinnamic acid
and a phenylethanolmoyeties bound to a same molecule
of glucopyranose (generally glucose), with an ester bond
and glycosidic bond respectively [5]. The agliconic por-
tion is responsible for the antioxidant activity [6], antim-
icrobial, anti-inflammatory and anti-mutagenic whereas
the glycosidic part is responsible for hydrosolubility.
Moreover, cinnamic acid derivative, due to its structure,
possesses interesting protective properties against ery-
thema induced by UVB [7], and on the other hand,
phenylethanol is a potent antioxidant. Synthetic deriva-
tives of cinnamic acid are widely used as filters in com-
mercial sun formulations. The reference compound for
this category is the octylmethoxycinnamate (Parsol MCX),
which represents one of the filters most used in cosmetics,
by virtue of moderate cost and low toxicity.
Taking this into account, we focused on the investiga-
tion of phenylpropoanoids obtained from Ajuga reptans,
because of many existing studies on two other related
phenylpropanoid glycosides: echinacoside (from Echina-
cea angustifolia), and verbascoside (from Verbascum sinu-
atum). From the “cosmeceutical” point of view, these
Figure 2. Structure of verbascoside.
Figure 3. Structure of methoxy-teupolioside.
molecules are known for the protection of collagen type
III, from the degradation caused by oxygen free radicals
[8]. The echinacoside possesses in vivo healing and anti-
inflammatory activity by virtue of anti-hyaluronidase
properties, involved in the maturation and organization
of the fibrous tissue [9]. Based on these premises, we
have undertaken the present study, evaluating 1) the an-
tioxidant capacity of the finished cosmetic formulations,
following the approach previously developed by us [10-
15], and 2) determining the Sun Protection Factor (SPF)
following the directive of Cosmetic Europe [16]. Un-
expectedly, antioxidant capacity against superoxideanion,
and the main reactive oxygen species, responsible for
skinaging, increased during accelerated stability studies.
Moreover, the extract obtained was endowed by interest-
ing UV filtering capabilities.
2. Stability and Antioxidant Capacity of
Cosmetic Formulations
The antioxidant potential of a finished product is usually
attributed by the concentration of contained antioxidants,
using traditional analytical methods. This evaluation does
not take into account a number of variables, i.e. possible
interactions between the functional ingredients, the pres-
ence of so-called “minor ingredients” (compared to the
active substances) that compones the formulation. Be-
cause all the ingredients in a finished cosmetic product
determine the antioxidant effectiveness, we recently de-
veloped a new protocol for the instrumental assessment
of the antioxidant capacity of the cosmetic products, ac-
tive ingredients and excipients comprised. Using our ap-
Open Access JCDSA
Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans
proach and in order to assess the contribution of the in-
gredients, two different emulsions have been developed.
These differ in the kind of ingredients, the first charac-
terized by ingredients chosen for their antioxidant poten-
tial, the other with ingredients devoided by this ability.
Furthermore, the Ajuga reptans extract was inserted at
two different percentages (0.3% and 0.6%), to evaluate
the variation in the activity at the variation of the con-
centration of active.
2.1. Material and Methods
The extract from cell culture of Ajuga reptans, as a yel-
low powder, water soluble, was kindly supplied by the
IRB Srl (Institute for Biotechnological Research, Alta-
villa Vicentina, Vicenza). The study was carried on two
cosmetic bases, termed ALAB and ALAB-AOX, both at
different concentrations of extract:
ALAB-1 and ALAB-AOX-1 containing 0.3% of ac-
ALAB-2 and ALAB-AOX-2 containing 0.6% of ac-
The bases tested were the following, ingredient and
preservative system have been chosen as simple as pos-
sible to avoid interference:
ALAB emulsion (O/W): (formulated with emollients,
emulsifiers and other ingredients that do not have anti-
oxidant properties).
INCI: Aqua, paraffinum liquidum, Cetyl Alcohol,
Glyceryl stearate, PEG-75 Stearate, Ceteth-20, Steareth-
20, Octildodecanol, Cetearyl alcohol, Ciclotetrasiloxane,
Dimethicone, Phenethyl Alcohol, Caprylyl Glycol, Xan-
than Gum, Disodium EDTA. From the cosmetic tech-
nology point of view, is an O/W formulation character-
ized by the presence of a nonionic emulsifier, able to
stabilize the system by the formation of liquid crystals
structures. The emollients part is composed by a mixture
of mineral oils and silicone. As a stabilizer of the aque-
ous phase was employed Xanthan gum.
ALAB-AOX emulsion (O/W): (formulated with emol-
lients, emulsifiers and other ingredients that have anti-
oxidant properties)
INCI: Aqua, Olive Oil, Cetearyl Olivate, Sorbitan Oli-
vate, Triticum Vulgare, Limnanthes Alba, Rice Wax,
Fitic Acid, Phenethyl Alcohol, Caprylyl Glycol, Xanthan
Gum, BHT.
From the cosmetic technology point of view is an
emulsion O/W, formulated with a nonionic emulsifier of
vegetable origin (contains the oleic fraction of olive oil
esterified with cetearyl alcohol and sorbitan). It differs
from the formulation ALAB for the presence of compo-
nents such as rice wax, wheat germ oil, olive oil and
Phytic acid was added to the chelating and antioxidants
2.2. Analysis
2.2.1. HPLC
The different cosmetic formulations were subjected to
accelerated aging at 40˚C, and monitored by HPLC in
order to assess phenylpropanoids concentration over the
time. Quantitative determination of the total phenylpro-
panoids in Ajuga reptans extracts was performed with a
Agilent 1100 Series HPLC System equipped with a
G1315A DAD and with an Hydro RP18 Sinergi 80A
column (4.6 × 150 mm, 4 μm) from Phenomenex. The
method involves a run of 20 minutes in isocratic condi-
tions with 0.01 M H3PO 4 in H2O:CH3CN = 82:18 at
room temperature, with a flow 0.8 mL/min. The teupo-
lioside PPA was identified and quantified by the external
standard method. All solvents, analyticals and reagents
were from Sigma-Aldrich srl, Milan, Italy.
2.2.2. Pr e paration o f th e S amples
The samples of each emulsion were accurately weighed
(500 mg) and diluted in 10 ml mixture of H2O:THF =
50:50. Each sample is shaken in a vortex mixer for about
60 seconds until complete dissolution of the cosmetics
matrix. The solutions are then diluted 1:2 with H2O and
each sample is filtered with 0.45 μ cellulose acetate fil-
ters. The volume injected into the column for each run
was of 20 μl and three injections for each sample were
carried out.
2.2.3. Photoch em iluminescen ce (PCL)
PCL assay, based on the methodology of Popov and
Lewin [14,15], was used to measure the antioxidant ac-
tivity of extracts with a Photochem® apparatus (Analytik
Jena, Leipzig, Germany) against superoxide anion radi-
cals generated from luminol, a photo-sensitizer, when
exposed to UV light (Double Bore® phosphor lamp, out-
put 351 nm, 3 m Watt/cm2). The antioxidant activity was
measured using both ACW (Antioxidant Capacity of
Water soluble substance) (data not shown) and ACL
(Antioxidant Capacity of Liposoluble substance) kits
provided by the manufacturer designed to measure the
antioxidant activity of hydrophilic and lipophilic com-
pounds, respectively [13,14]. For ACW studies, the lu-
minol reagent and Trolox work solution were freshly
prepared according to the ACW protocol. The presence
of Trolox (or any other antioxidants from the extracts)
retarded luminescence for a period: hence, a lag time was
noted before a signal was measured. The duration of the
lag, which is calculated by the computer software from
the first derivative of the detector signal at its turning
point and intersection with the X-axis, was plotted
against the concentration of Trolox added to the assay
medium. The concentration of the added extract solution
was such that the generated luminescence fell within the
limits of the standard curve. Therefore, the lag time
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Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans 67
(seconds) for the ACW assay was used as the radical
scavenging activity and the antioxidant capacity calcu-
lated by comparison with a Trolox standard curve and
then expressed as micromoles of Trolox per gram of
matter. In ACL studies, the kinetic light emission curve,
which exhibits no lag phase, was monitored for 180 s and
expressed as micromoles of Trolox per gram of dry
matter. The areas under the curves were calculated using
the PCL soft control and analysis software. As greater
concentrations of Trolox working solutions were added
to the assay medium, a marked reduction in the magni-
tude of the PCL signal and hence the area calculated
from the integral was observed. This inhibition was used
as a parameter for quantification and related to the de-
crease in the integral of PCL intensities caused by vary-
ing concentrations of Trolox. The observed inhibition of
the signal was plotted against the concentration of
Trolox added to the assay medium. The concentration
of the added extract solution was such that the generated
luminescence during the 180 s sampling interval fell
within the limits of the standard curve. The extracts for
ACW and ACL measurements were centrifuged (5 min
at 16000 g) prior to analysis. The antioxidant assay was
carried out in triplicate for each sample, and 20 μL of the
diluted extract (1:40, v/v) in HPLC-grade water (ACW)
or HPLC-grade methanol (ACL) was sufficient to corre-
spond to the standard curve.
2.2.4. Preparation of Samples
An accurately weighed quantity of each formulation pre-
viously prepared was suspended in 10 ml of mixture
MeOH/esano/Et2O (1:1:1) sonicated for 20 min at 20˚C
and subsequently centrifuged for 5 - 7 min at 3000 rpm.
The antioxidant capacity was evaluated by the means of a
calibration curve obtained by using as a standard the
Trolox® and the results obtained were expressed in
Trolox® nmol/mg of cream.
2.2.5. Accelerated Stability Study
The cosmetic formulations were subjected to accelerated
aging at 40˚C, and the samples for the analysis were
drawn at the following times:
T0 = at the moment of preparation;
T1 = one week after preparation (7 days);
T2 = two weeks after preparation (14 days);
T3 = one month after preparation (30 days);
T4 = three months after preparation (90 days).
3. UV Filtering Capacity of Cosmetic
As stated above, the conjugated aromatic portion of te-
upolioside, presents a structural similarity with that of
Parsol MCX. It is thus reasonable that teupolioside can
also possess filtering properties against UVB rays. To
support this hypothesis we have carried out some pre-
liminary analysis to verify its photoprotective activity
and its potential use in solar formulations. Two kind of
study have been conducted, 1) stability of the teupo-
lioside against solar radiation by using a solar simulator
and 2) determination the SPF factor. At first photostabil-
ity was assessed, the extract was introduced win different
cosmetic formulations, and subjected to a series of radia-
tion intensity and energy equal to that of the sun, to ver-
ify the possible alterations. After irradiation the formula-
tions were analyzed in relation to the content of the ac-
tive principle (teupolioside) and the variation of the an-
tioxidant capacity. The two parameters were monitored
before and after irradiation, through HPLC and PCL re-
spectively. After checking the stability of the extract, in
order to quantify the possible photoprotective capacity,
the value of SPF was determined spectrophotometrically
according to the international method of Diffey and
Robson [17].
3.1. Material and Methods
3.1.1. Simula tion of Solar Radiation
The test was performed on cosmetic formulations con-
taining 0.1% of extract with titre >50%, irradiation was
continued for two hours in a solar simulator Suntest CPS
+ (Atlas, Germany), consisting of a Xenon lamp (inten-
sity of the radiant energy emitted: 250 W/m2), with opti-
cal filters with IR reflective coating and a UV filter/Su-
prax, to simulate the conditions of exposure to the sun (λ
from 300 to 900 nm). Formulations: we designed stan-
dard formulations, in order to be able to assess if the ve-
hicle or the different components can in some way influ-
ence the degradation of teupolioside. The preservative
system has been selected in view of its performance and
safety of use. Each product contained 0.1% of extract.
The formulations tested are the following:
ALAB1: Hydroxyethylcellulose gel.
INCI: Aqua, Glycerin, Hydroxyethylcellulose, Pro-
pylen glycol, Phenethyl Alcohol, Caprylyl Glycol,
Ajugareptans cell culture extract.
ALAB2: O/W emulsion designed and formulated to
avoid possible interactions between the cosmetics matrix
and the extract.
The emulsifier used in the formulation gives a good
stability and allows easy dispersion of the active princi-
ples. The lipophilic component is composed of a mixture
of mineral oils and silicone.
INCI: Aqua, Glyceryl Stearate, Ceteareth-20, Ceteareth-
12, Cetearyl Alcohol, CetylPalmitate, Dicaprylylcabon-
ate, CetearylIsononyl, Glycerin, Oleylerucate, Phenethyl
Alcohol, Caprylyl Glycol, Dimethicone, Disodium EDTA,
Ajugareptans cell culture extract.
As a reference, an aqueous solution containing 0.1%
of cell extract of Ajuga reptans was used.
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Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans
500 mg, accurately weighed, of each cosmetic formu-
lation was sampled, and then distributed in a homogene-
ous and uniform layer on the bottom of a beaker, whereas
for the aqueous solution were accurately weighed 2.5 g
of solution.
The samples are then placed inside the analysis cham-
ber of the simulator and irradiated for 2 hours at a tem-
perature of 37˚C.
3.1.2. HPLC
The quantitative determination of the teupolioside in the
samples has been carried out as described above, before
and after irradiation.
3.1.3. Sa m ple Preparation
The 0.1% solution is diluted 1:50 in H2O; Accurately
weighed 500 mg of gel ALAB1 are diluted with 10 ml of
H2O; 500 mg accurately weighed O/W ALAB2 emulsion
is diluted with 10 ml mixture of H2O:THF = 50:50 and
placed under mixing with the aid of a vortex mixer for
about 60 seconds until the complete dissolution of the
cosmetics matrix. The solutions are next diluted 1:2 with
H2O and filtered with 0.45 μ regenerated cellulose filter
before each injection.
3.1.4. Photoch em iluminescen ce (PCL)
Determination of antioxidant capacity of cosmetic for-
mulations was conducted before and after irradiation as
described above.
3.1.5. Determination of the SPF in Vitro
The test is carried out according to guide line COLIPA
2009 (now Cosmetic Europe), to the recommendation EC
647/2006 of the 22/09/2006 about the efficacy of sun
protecting products and to the evaluation of Boots star
rating system. Employ a UV-VIS spectrophotometer
Shimadzu (Shimadzu Italy, Milan) mod. UV-2600 pro-
vided with 60 mm integration sphere ISR-2600Plus and
dedicate software package for the determination of
SPF/UVA (Sunny Detection) it features dual-beam opti-
cal system, single monochromator with spectral range up
to 1400 nm with a Czenry-Turner optycalbanch endowed
with proprietary correction of chromatic aberration
(low-raylight) and UV-Probe software control. Support
for the determination is PMMA WW2-2 μm-Plates
(SCHÖN-BERG GmbH, Hamburg). Quantity of sample
(cosmetic preparation to be analyzed) at a dose of 2 ±
0.04 mg/cm2 follows Cosmetic Europe Guidelines.
The measurement is repeated several times (at least 6)
and at the end of the program through acquisitions and
analytical processing spectral data by the Sunny Detec-
tion package a mean absorbance spectrum was deter-
For the determination of SPF factor the equation Dif-
fey and Robson is applied.
  
 
400 290
is the spectral irradiance of sunlight, measured
at noon in the summer in southern Europe, at latitude 40˚
North, 20˚ zenith angle, thickness of the ozone layer
0.305 cm.
is the erythema action spectrum of solar radia-
tion, obtained by McKinlay and Diffey comparing over
12 erythema inducing spectra measured between 1929
and 1985.
is the monochromatic spectral transmittance.
ALAB8: was an hydroxyethyl cellulose gel, function-
alized with 4% of the extract of Ajuga reptans. This for-
mulation is “neutral” in the respect of SPF because the
gel matrix does not have photoprotective activity. The
preservative system (Akema, Italy) has been selected in
view of its performance and safety of use. [18].
INCI: Aqua, Ajuga reptans, Hydroxyethylcellulose,
Propylene Glycol, Phenethyl Alcohol, Caprylyl Glycol.
ALAB9 emulsion (O/W): in this formulation have
been inserted photoprotective ingredients of natural ori-
gin, to assess the value of SPF obtainable using only
plant products. Contains ferulic acid complexed in cyclo-
dextrins, Aloe extract, rice bran oil, γ-oryzanol.
INCI: Aqua, Cetyl Alcohol, Glyceryl stearate, PEG-75
Stearate and Ceteth-20, Steareth-20, Ferulic Acid, Cyclo-
dextrin, Cetearyl alcohol, ButyrospermumParkii, Elaeis-
guineensis, Rice Bran Oil, Tocopheryl Acetate, Buxus
Chinensis, Olive Oil, Rice Wax, Aloe Barbadensis, γ-
orizanol, Phenethyl Alcohol, Caprylyl Glycol, Xanthan
Gum, Fitic Acid.
ALAB10 emulsion (O/W) the same formula described
above that is functionalized with 4% of extract of Ajuga
(Titred > 50% in PP) to assess the contribution to the
final value of the SPF.
INCI: Aqua, Cetyl Alcohol, Glyceryl stearate, PEG-75
Stearate, Ceteth-20, Steareth-20, Ajuga reptans, Ferulic
Acid, Cyclodextrin, Cetearyl alcohol, Butyrospermum-
Parkii, Elaeisguineensis, Rice Bran Oil, Tocopheryl Ace-
tate, BuxusChinensis, Olive oil, Rice Wax, Aloe Bar-
badensis, Phenethyl Alcohol, Caprylyl Glycol, Xanthan
Gum, Fitic Acid.
4. Results and Discussion
4.1. Antioxidant Activity
Among ACW and ACL values, only this last one was
selected, after preliminary evaluations, for the study. As
a general consideration we can say that all formulations
containing the extract of Ajuga reptans, compared with
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Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans 69
the respective base, have a very significant antioxidant
capacity. As predictable the emulsions containing the
highest concentration of extract (0.6%) have the higher
antioxidant capacity (Figure 4). At a closer analysis is
possible to observe that the antioxidant capacity of the
formulations under examination, varies in function of the
choice of excipients. In fact, the value of the antioxi-
dant capacity (ACL) of the formulation ALAB-1 at T0
(12.6 nmol of Trolox/mg cream), is approximately 32%
lower compared to the value of ALABAOX-1 (18.5
nmoles Trolox®). This trend is also found in the formu-
lations at higher concentration (ALAB and ALAB-2-
The values of higher ACL for formulations ALAB-
AOX are attributable to the presence of ingredients en-
dowed with significant antioxidants capacity such as
phytic acid, rice wax and oils of vegetable origin. How-
ever, as can be seen from the Figure 4, increasing con-
centration of extract of Ajuga reptans, does not corre-
spond to a proportional increase in antioxidant capacity.
In fact, as regards the formulations ALAB, the ALAB-2
despite containing twice extract with respect to ALAB-1
has an ACL value only 13% higher as compared to
ALAB-1. It is possible to observe a similar trend also
regarding formulations ALAB-AOX. The variation of the
antioxidant capacity of the formulations under acceler-
ated ageing and compared with the respective bases, are
shown in Figure 5. As might be expected the antioxidant
capacity at first decreases in function of time.
However, unattendently, the antioxidant capacity of
the formulations ALAB-AOX undergoes, in the first
week, a reduction greater than that of ALAB but, in-
creases after two weeks. In fact, for example, from T0 to
T1, the value of ACL for ALAB-1 goes from 12.6 nmol
of Trolox®/mg of cream to 10.62 (which corresponds to a
reduction of 10%), whereas for the ALAB-AOX-1 shows
a decline of 43%. During the next time interval (T1 to
T2), there is a slight increase of the antioxidant activity
of ALAB, whereas the ACL value of formulations
ALAB-AOX (different from the previous ones for the
presence of components with potential antioxidant activ-
ity) decreases in both emulsions although to a greater
extent in ALAB-AOX-2.
The trend of the variation of the antioxidant capacity
continues to be non-linear even in later times. At the final
time (T4), however, despite the three months of acceler-
ated aging, all the tested formulations maintain a signifi-
cant antioxidant capacity. In particular, for the formula-
tions ALAB-AOX, there was a reduction of the values of
ACL equal to 65% compared to T0, while the antioxi-
dant capacity of ALAB decreases to a lesser extent (50%
compared to the initial value). To complete the study, in
parallel HPLC analysis was conducted, to assess the de-
crease over the time of teupolioside present in the extract.
In Figure 6, it is shown that the concentration of PPA
Figure 4. Antioxidant capacity of formulations based on
phenylpropanoids obtained from Ajuga reptans (titre > 50%
in total phenylpropanoids). The basic formulation was used
as a positive control.
Figure 5. Variation of the antioxidant capacity cosmetic
formulations based extract of Ajuga reptans (titre > 50% in
total phenylpropanoids) during accelerated aging (40˚C).
Legend: T1 = 7 days; T2 = 14 days; T3 = 30 days; T4 = 120
Figure 6. Change of the concentration of teupolioside in
emulsions during accelerated aging (40˚C).
decreases in function of time. The percentage values of
residual PPA, at various times, show, however, that the
disappearance of teupolioside is slower and the % recov-
ery greater in ALAB than in the formulations ALAB-
AOX. Indeed, at the time of the final observation, in
ALAB and ALAB-1 and -2, the amount of active ingre-
dient present is 40% and 45%, respectively, while in
ALAB-AOX-2 recovery % of teupolioside is practically
The increase of antioxidant activity at T3 paralleled by
disappearance of teupolioside can be explained by the
formation of the by-product iso-teupolioside, due to the
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Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans
migration of caffeic acid moiety from position 4 to posi-
tion 6 that may occur and is largely dependent from the
kind of formulation used [19].
4.2. UV Filtering Activity and Stability
Stability Study
Analyzing the concentration of teupolioside before and
after irradiation (Figure 7), it is possible to observe that
the molecule slowly disappear proportionally in all for-
mulations tested.
In fact, a similar decrease in aqueous solution (6%), in
hydroxyethyl cellulose gel (7%) and in the emulsion
O/W emulsion (4%) was recorded. Also, the variation of
the antioxidant capacity (Figure 8) is in line with the
amount of decrease of the teupolioside. This finding dif-
fers from the trend above observed where the decrease in
teupolioside was not accompanied by a concomitant de-
crease in antioxidant capacity. This behaviour can be
explained by the different kind of formulation used.
From HPLC, no by-products appeared after irradiation.
The final determination with the solar simulator pro-
vides an additional parameter to evaluate the stability of
phenylpropanoids upon solar irradiation, which may be
different from the behavior observed in solutions.
4.3. Determination of in Vitro SPF
The Gel ALAB8 (Ajuga 4%) showed a 1.8 SPF, whereas
the emulsion ALAB9 6.5 and ALAB10 (Ajuga 4%) 7.2.
The values are significant for a natural ingredient based
The ALAB8 (gel) provides a picture of the ability to
absorb UV radiation by the phenylpropanoids. In fact,
the lattice of the gel does not influence the absorption of
UV, so the radiation is shielded solely by the chemical
structure of the phenylpropanoids. The formulation
ALAB9, due to the content of natural ingredients with
filtering properties, has an interesting value of SPF (6.5).
With the addition of 4% extract from cell cultures of
Ajuga reptans, the value increases to 7.2.
Comparing the value of the gel, with that of the emul-
sion functionalized with the extract of Ajuga reptans, it is
clear that the vehicle is critical for the SPF value given
by the ingredient. The spectrophotometric determination
also allows to check other interesting parameters such as
the UVA/UVB ratio (UVA Ratio), between the total ab-
sorption in the UVA region (400 - 320 nm) and UVB
(320 - 280 nm). This is important to understand whether
the formulation provides a good overall protection over
the whole range of the UV rays that are involved in sun
exposure. Based on the value of the ratio five categories
have been defined (Table 1) [17].
As for this classification the following classification
can be drawn for our formulations (Table 2). The extract
Figure 7. Concentration of teupolioside before and after
Figure 8. Antioxidant capacity of the formulations before
and after irradiation.
Table 1. Classification categories UVA-UVB ratio.
UVA Ratio Star Category Description
from 0.0 to <0.2 - LOW
from 0.2 to <0.4 * MODERATE
from 0.4 to <0.6 ** GOOD
from 0.6 to <0.8 *** SUPERIOR
0.8 **** MAXIMUM
Table 2. UVA-UVB of formulations analyzed.
Formulation Gel ALAB8
Ajuga 4% Emulsion
ALAB9 Emulsion ALAB10
Ajuga 4%
UVA Ratio 1.2 0.77 0.98
Category **** *** ****
from cell cultures of Ajuga reptans offers not only a cov-
erage over the whole range of UV, as evident from the
value obtained for the gel, but can also improve the
UVA-UVB ratio. The growing importance of having
Open Access JCDSA
Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from Ajuga reptans 71
preparations that can offer a protection also against UVA
prompted us to evaluate a protection factor (data in Ta-
bles 1-3) that was calculated concomitantly with SPF.
The values are specific for these particular formula-
tions and need to be determined case by case as varia-
tions occur in the vehicle, excipients, and emulsifiers.
Future studies will be addressed to verify how the vehicle
can affect the photostability and the SPF value, and
eventual synergies with common filters used in cosmetics
consenting lowering of the concentrations of these latter.
5. Conclusions
The dermo-cosmetic industry is strongly oriented to the
concept of nature as a source of raw materials, with par-
ticular attention paid in recent time to renewable and
sustainable sources. In this work, we described a study
related to a particular type of extract, which is obtained
from in vitro cultures of plant cells of Ajuga reptans and
characterized by a high titre in teupolioside, a phenyl-
propanoid with high antioxidant activity. The innovative
nature of this technology platform has attracted our re-
search interest, since the in vitro culture enables rapid
propagation of selected plants based on the characteris-
tics of the active ingredients of interest present in them.
This innovative approach can account for the pharma-
ceutical, cosmetic and herbal fields; it is a strategic al-
ternative for the supply of active ingredients, which is
highly standardized and not directly obtainable by ex-
traction from traditional plant due to the low content (i.e.
secondary metabolites). A number of studies have been
performed in order to evaluate the potential use of this
extract in the cosmetic field. The antioxidant capacity of
the extract was evaluated, using the technique PCL
within prototype formulations at 0.3% - 0.6%. Parallel
evaluation of the concentration of phenylpropanoid by
HPLC was also conducted leading to the finding that the
antioxidant activity is maintained or even increased due
to the partial conversion of teupolioside to isoteupo-
lioside over the time.
Due to the presence of a cynnamic acid moiety, UV
filtering properties were also investigated disclosing in-
teresting UVB and UVA protecting activity, good UVB/
UVA ratio and good photostability. These findings ac-
compained by the potent antioxidat activity which is dis-
played by finished products indicate a promising role of
these extracts in solar product claiming natural composi-
In relation to the possible application in dermatologi-
cal field and close relationships between echinacoside
and teupolioside, the extract from cell cultures of Ajuga
reptans deserves further investigations as possible anti-
inflammatory agent.
In conclusion, the preliminary assessment described
here has highlighted new applications of Ajuga reptans
Table 3. Factors of protection against the UVA of the for-
mulations analyzed.
Formulation UVA Protection Factor
Gel RB8 Ajuga 4% 1.5
Emulsion ALAB9 3.7
Emulsion ALAB10 Ajuga 4% 4.2
extracts for the first time. In particular, the quite interest-
ing features confirm that the plant cell cultures are a
good method to obtain extracts with high content valu-
able active molecules, which are found only in trace
amounts as plant secondary metabolites. The same tech-
nology can also be applied to plants for ethical reasons
(rare plants) or difficulty in their extensive growth which
can not be collected in large quantities. Such technology
finally allows obtaining extracts with a high degree of
standardization which are hardly obtained by direct ex-
traction from plants due to the variability of the life cy-
cles and different soils compositions.
This first phase of work has helped highlight just some
of the potential applications of the extract of Ajuga rep-
tans, and further studies are currently ongoing with the
aim to investigate the possible lenitive and healing
6. Acknowledgements
We thank the Ministry of Education and Research (PRIN,
Grant 20082L3NFT_003) and Ambrosialab for financial
support. Elisa Durini and Alberto Casolari are gratefully
acknowledged for technical assistance.
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