Dualistic Properties of Cosmetic Formulations Based on Phenylpropanoids from <i>Ajuga reptans</i>

doi:10.4236/jcdsa.2013.33A2014

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Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 64-72

Published Online November 2013 (http://www.scirp.org/journal/jcdsa)

http://dx.doi.org/10.4236/jcdsa.2013.33A2014

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.

Email: Stefano.manfredini@unife.it

Received October 17th, 2013; revised November 13th, 2013; accepted November 20th, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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-

Teupolioside

HO OH

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

HO OH

Verbascoside

Figure 2. Structure of verbascoside.

HO OH

MeO OH

Methoxy-Teupolioside

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-

tive;

 ALAB-2 and ALAB-AOX-2 containing 0.6% of ac-

tive.

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

properties.

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

Formulations

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-

mined.

For the determination of SPF factor the equation Dif-

fey and Robson is applied.

  

 

400

400 290

400

290

SPF EB











where:







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.

Formulations:

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-

AOX-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

days.

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

zero.

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

Open Access JCDSA

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

formulation.

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

irradiation.

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 **** *** ****

Description MAXIMUMSUPERIOR MAXIMUM

Cosmetic

Europe

Standard

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

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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-

tion.

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

properties.

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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