Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

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Journal of Photochemistry and Photobiology B: Biology

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

Assessment of extracts of Helichrysum arenarium, Crataegus monogyna,

Sambucus nigra in photoprotective UVA and UVB; photostability
in cosmetic emulsions
Anna Jarzycka a, Agnieszka Lewinska b, Roman Gancarz a, Kazimiera A. Wilk a,
a
b

skiego 27, 50-370 Wrocaw, Poland

Organic and Pharmaceutical Technology Group, Faculty of Chemistry, Wrocaw University of Technology, Wybrzez_ e Wyspian
Faculty of Chemistry, University of Wrocaw, Joliot-Curie14 Str., 50-383 Wrocaw, Poland

a r t i c l e

i n f o

Article history:
Received 12 March 2013
Received in revised form 3 July 2013
Accepted 31 July 2013
Available online 20 August 2013
Keywords:
Natural sunscreens
Photoprotective activity
Photostability efcacy
Helichrysum arenarium
Sambucus nigra
Crataegus monogyna

a b s t r a c t
The aim of our study was to investigate the photoprotective activity and photostability efcacy of
sunscreen formulations containing Helichrysum arenarium, Sambucus nigra, Crataegus monogyna extracts
and their combination. UV transmission of the emulsion lms was performed by using diffuse transmittance measurements coupling to an integrating sphere. In vitro photoprotection and photostability
efcacy were evaluated according to the following parameters: sun protection factor (SPF), UVA
protection factor (PF-UVA), UVA/UVB ratio and critical wavelength (kc) before and after UV irradiation.
The results obtained show that the formulations containing polyphenols fulll the ofcial requirements
for sunscreen products due to their broad spectrum of UV protection combined with their high
photostability and remarkable antioxidant properties. Therefore H. arenarium, S. nigra, C. monogyna
extracts represent useful additives for cosmetic formulation.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Chronic exposure to the suns radiation induces in the human
body various skin diseases including premature aging of the skin
(wrinkling, scaling, dryness, dilatation of blood vessel and loss of
collagen) and cancer development [1]. Thus in recent years increased human awareness of the harmful effects of UV radiation
on the human body and health has been observed. In order to
reduce these effects and protect people from the harmful effects
of solar radiation, different photoprotective actions can be taken,
like complete avoidance of sun exposure, keeping in the shade
when disease-inducing wavelengths are relatively intense, wearing
protective clothing and using topical sunscreens [2]. An optimal
sunscreen contains multiple elements which reect, absorb and
scatter UV radiation in order to provide a broad spectrum of
protection throughout the whole UV range (290400 nm) of the
solar radiation reaching the Earths surface [3]. Furthermore the
photostability of the sunscreens has great inuence on their
efcacy and safety as their light-induced degradation leads to a
reduction of their protective capacity during exposure to the sun
and can also generate potentially toxic species, e.g. free radicals
[4,5].
Corresponding author. Tel.: +48 71 320 28 28; fax: +48 71 320 36 78.
E-mail address: kazimiera.wilk@pwr.wroc.pl (K.A. Wilk).
1011-1344/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.07.029

In the last few years the use of natural products has gained
widespread interest [6] mainly due to some limitation of the
organic UV-lters which are characterized by their narrow
spectrum of protection and low photostability. Polyphenols appear
particularly promising as cosmetic sunscreens because they can
absorb a broad spectrum of UV radiation including the UVB and
UVA regions [7]. In addition they reduce the penetration of the
radiations into the skin and decrease inammation, oxidative
stress and DNA damaging effects [6,8]. As a consequence they
have immuno-modulatory and antioxidant properties as they can
react with free radicals produced by UV radiation (singlet oxygen
and hydroxyl free-radicals) and inhibit or delay their harmful
effects [9,10].
The inorescences of Helichrysum arenarium L. (Asteraceae),
Crataegus monogyna Jacq. (Rosaceae) and Sambucus nigra L. (Adoxaceae) have long been known in herbal medicine in Europe, Asia,
North Africa and America. Pharmacological data shows that the
inorescences of elderower (S. nigra L.), hawthorn (C. monogyna
Jacq.) and moench (H. arenarium L.) are rich in phenolic
compounds, including phenolic acids, avonoids, catechins, and
proanthocyanidins. Their benecial effects on the human body
are also related to the presence of other organic compounds
(vitamins, carotenoids, avonoids, phenolic acids, etc.) and
inorganic compounds (such as macro- and micronutrients: Cu,
Fe, Mg, Mn, Zn, etc.), as well [11]. They exhibit antioxidant [12],

A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

anticancer, immune-stimulating, anticoagulant, antiplatelet

[13,14], antibacterial, antiallergic, antitussive, bronchodilatory,
and antiviral activity [15]. Due to such biological properties medical natural products are widely used in the management and prevention of age-related diseases, heart, cardiovascular ailments,
gastro-intestinal disorders, rheumatism and respiratory infection,
as food additives and nutraceuticals. [16,17]. Polyphenols present
in diet are associated with a reduced risk of certain degenerative
diseases like cancer and artherosclerosis [18].
In the light of the plant polyphenol properties presented above,
the aim of this study also as a continuation of our interest in novel drug carriers and encapsulation processes [1921] was to
investigate whether topical cosmetic formulation containing polyphenolic extracts from moench, elderower and hawthorn can exert a photoprotective effect against UVA and UVB. Moreover, we
have investigated their efcacy as broad spectrum UVB and UVA
photoprotective sunscreens by measuring: (1) the sun protection
factor (SPF) the universal indicator related to UVB radiation and
the UVA protection factor (PF-UVA); (2) the UVA/UVB ratio and
the critical wavelength (kc). In addition, the photostability of the
sunscreens was evaluated by the values of its SPF, PF-UVA before
and after UV irradiation in different solar simulation systems
[22,23].
2. Materials and methods

51

Fig. 1. A schematic diagram of the extraction procedure.

further extracted several times with ethyl acetate (5  20 mL) in

order to obtain an ethyl acetate fraction and an aqueous fraction
III, respectively and the solvents were evaporated to dryness under reduced pressure.

2.1. Materials
2.3. Determination of total avonoids content (TF)
The medicinal plant raw materials (inorescences of H. arenarium L. (Asteraceae), C. monogyna Jacq. (Rosaceae) and S. nigra L.
(Adoxaceae) were purchased at a pharmacy and originated from
the Flos (Mokrsko, Poland) herbal enterprise. The cetyl alcohol
and parafnum liquidum FP IV were purchased from StearinerieDubois Fils (Boulogne, France) and Aopa (Pabianice, Poland),
respectively. The carnauba wax was supplied by C.E. Roeper Gmbh
(Hamburg, Germany). The sweet almond and coconut oils were
obtained from Provital Group (Lubon, Poland). The caprylic/capric
triglyceride (Crodamol GTCC) and sorbitan monostearate (Span
60) were purchased from Croda Poland (Warszawa, Poland). The
cyclopentasiloxane was from (Dow Corning 245 Fluid) Dow Corning Poland (Warszawa, Poland) and Phenonip from Clariant International Ltd. (Muttenz, Switzerland). The sucrose palmitate
(PS750-C) was obtained from Sisterna B.V. (Roosendaal, The Netherlands). The allantoin was supplied by Merck (Warszawa, Poland)
and glycerin by Synthezis (Katowice, Poland).
2.2. Polyphenolic fraction extraction procedure
The extraction procedure was carried out using the method of
Wolski et al. [24] with some modications. The dried plant material was extracted with chloroform at elevated temperature followed by purication in several steps (Fig. 1). Dried plant
material (50.0 g) was put into a thimble lter and placed into the
main chamber of a Soxhlet apparatus. Then the plant material
was extracted 10-fold with chloroform (500 mL) at 60 C during
12 h to remove the hydrophobic compounds and then the fraction
marked as residual plant material I was ltered off. The separated solid part, residual plant material I, was subjected to
exhaustive extraction with methanol in the boiling solvent for
5 h and nally the methanol was evaporated under reduced pressure. The dry methanolic fraction was diluted with hot distilled
water (100 mL), and ascorbic acid (0.5 mg/g) was added. Then
the whole was left in a refrigerator for 24 h. The aqueous fraction
I obtained in this way was extracted with diethyl ether (5-fold,
20 mL) yielding the aqueous fraction II. The last residues were

The total avonoids content was determined using the method

of Ordonez et al. [25]. Shortly 0.5 mL of 2% aluminum trichloride
(AlCl3) methanol solution was added to 0.5 mL of the plant
extract diluted in methanol (1.0 mg/mL). The absorbance value
has been measured at the wavelength of 420 nm using Metertech
Inc. UVVis spectrophotometer SP-8001 against a blank, after
60 min of incubation at room temperature. The nal absorbance
of each sample was compared with a standard calibration
curve made for quercetin (R2 = 0.998). The data were expressed
as mg of quercetin equivalents (QRE) per 1.0 g of a dry extract
weight.
2.4. Determination of total polyphenolic content (TP)
The total polyphenols content was determined according to the
method described by the International Organization for Standardization (ISO) 14502-1 [26]. Shortly described, 1.0 mL of the plant
extract dissolved in methanol (1.0 mg/mL) was mixed with
5.0 ml (diluted 10 times in water) of the FolinCiocalteus reagent.
After 8 min 4.0 mL of the sodium carbonate solution (7.5% w/v)
was added and let to stand in room temperature. After 60 min
the absorbance value at 765 nm using Metertech Inc. UVVis spectrophotometer SP-8001 against a blank was measured. The concentration of the polyphenols in samples was derived from a
standard calibration curve of gallic acid in the range of 1050 lg/
mL (R2 = 0.996). The data were expressed as mg of the gallic acid
equivalents (GAE) per 1.0 g of the dry extract weight.
2.5. HPLC-DAD analysis of polyphenols and avonoids
The identication of polyphenolic-rich ethyl acetate extracts
obtained by the extraction route C (5 mg in 25% methanol solution
with ascorbic acid (100 mg/L)) were carried out using Merck-Hitachi L-7455 HPLC instrument with the diode array detector
(DAD) and quaternary pumps L-7100, equipped with D-7000
HSM multisolvent delivery system (Merck-Hitachi, Tokyo, Japan)

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A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

and autosampler L-7200. The separation was performed on

Cadenza CDC18 (75 mm  4.6 mm, 5 lm) column Imtakt, Japan.
Oven temperature was set to 20 C. The mobile phase was
composed of eluent A (4.5% formic acid, v/v) and eluent B (acetonitrile). The gradient program began with a linearly, staring from 0%
of B to 25% of B in 30 min, followed by washing and reconditioning
of the column resin. The ow rate was 1.0 mL/min, and the runs
were monitored at the following wavelength: for avan-3-ols at
280 nm, for hydroxycinnamates at 320 nm, and for avonol
glycosides at 360 nm. DAD spectra were recived in the wavelength
range of 200600 nm in the steps of 2 nm. The retention time
parameters and spectra received were compared with those of
pure standards within 200600 nm range. The calibration curves
were for: ()-epicatechin, (+)-catechin, chlorogenic acid, quercetin-3-O-glucoside, isorhamentin, p-cumaric acid, kaempferol-3-Oglucoside, rosmarinic acid as standards were prepared.

For each formulation three plates were prepared and the measurements were repeated ve times for each sample. The product lm
was left to equilibrate in a dark place under ambient temperature
(20 2 C) for 1530 min. UV transmission measurements between 290 and 400 nm were performed using a spectrophotometer
equipped with an integrating sphere (UV Transmittance Analyzer
UV-2000S, Labsphere, North Sutton, US). As a standard emulsion
the P2 reference sunscreen formulation was used mandated by
the COLIPA Monograph [27].
In accordance with the recommendations of the European Union (2006/647/EC) [28] and the guidelines of the European Cosmetics Association (COLIPA) concerning all sunscreen products
marketed in the European Union the value of the UVA protection
factor (PF-UVA) must be at least 1/3 of the labeled SPF and a critical
wavelength (kc) higher than 370 nm.
Values of the in vitro SPF and PF-UVA factors were calculated
according to the following Eqs. (1) and (2) [29,30]:

R 400

2.6. Preparation of sunscreen emulsion

The basic emulsion system was prepared in the laboratory by
adding xed concentrations of the individual polyphenolic fractions
(ethyl acetate fractions) (where x = 2.0; 5.0 and 10.0 wt.%) and their
various combinations into the formulation components. The combinations were made using the same percentage of each extract
(10.0 wt.%). The emulsions were made by the authors and a detailed
description of the emulsion studied is presented in Table 1. At the
beginning, both phases, oil and aqueous, were heated separately
to about 7080 C. Then the aqueous phase was placed into a
high-speed homogenizer (UltraTurrax T25 Basic, IKA, Staufen,
Germany) and the oil phase was slowly added under intensive stirring (20,000 rpm for 2 min). The whole mixture was homogenized
(3400 rpm) until the temperature was cooled to 3040 C and nally the phase C was incorporated. Afterwards the formulation was
mixed to cool the emulsion to room temperature (2025 C). As a
blank, sample formulation without any sunscreen was used.

Ek  Ikdk
SPFin v itro R 400290
Ek  Ik  Tkdk
290
where
I(k) is
T(k) is
length

E(k) is the erythema action spectrum at wavelength k [31];

the spectral irradiance of sunlight at wavelength k [32];
the spectral transmittance of the sunscreen layer at wavek.

R 400

Pk  Ikdk
PF-UVAin v itro R 400320
Pk
 Ik  Tkdk
320

Sunscreen product was precisely applied (0.75 mg/cm ) to

roughened polymethylmethacrylate PMMA plates (Sa = 2 lm,
50  50 mm, Helioscreen Helioplate HD, Creil, France) and was distributed evenly over the whole surface using a cot-coated nger.

kc

Akdk 0:9

wt.%

Phase A (oil phase)

Cetyl alcohol
Carnauba wax
Parafnum liquidum FP IV
Cyclopentasiloxane (Dow Corning 245 Fluid)
Sweet almond oil
Caprylic/Capric triglyceride (Crodamol GTCC)
Coconut oil
Sorbitan monostearate (Span 60)

6.0
3.0
2.0
2.0
4.0
3.0
1.5
2.0

Phase B (aqueous phase)

Glycerin
Allantoin
Sucrose palmitate (PS750-C)
Deionized water
Phase C
Phenoxyethanol, methylparaben, ethylparaben, buthylparaben,
isobuthylparaben, propylparaben (Phenonip)
Polyphenolic (acetate) fraction (sunscreen)

3.0
0.5
2.0
qsp
100.0
0.5
x

400

Akdk

where A is the absorption, kc is the critical wavelength (nm) and k is

the wavelength (nm).
The UVA/UVB ratio is a criterion that reects the ratio of the
integral UVA to UVB attenuation. The closer the ratio is to 1, the
more similar are the integral UVA- and UVB-attenuation values.
For its determination the Eq. (4) (UVA/UVB) was used [30]:

UVA=UVB

Ingredients

290

R 400
Table 1
Composition of sunscreen emulsion.

where P(k) is the persistent pigment darkening action spectrum at

wavelength k [33]; I(k) is the spectral irradiance of sunlight at
wavelength k [32]; T(k) is the spectral transmittance of the sunscreen layer at wavelength k.
The critical wavelength was calculated with the following Eq.
(3) [29,30]:

290

2.7. In vitro determination of photoprotective efcacy of sunscreens

320
R 320
290

Akdk=
Akdk=

R 400
320

dk

290

dk

R 320

where A is the absorption and k is the wavelength [nm].

2.8. In vitro study of photostability of sunscreens
The photostability study was determined using the method of
Choquenet et al. [22]. Plates prepared according to previous Section 2.6 were irradiated for 2 h with a solar simulator apparatus
(Suntest CPS+; Atlas, Warszawa, Poland) equipped with a xenon
arc lamp (1500 W) and special UV glass lters cutting off radiation
below 290 nm. The light source emission was maintained at
650 W/m2 in accordance with global solar spectral irradiance
[19,20]. Before and after irradiation, all the characteristic parameters such as the SPF, the PF-UVA, the critical wavelength (kc) and
the UVA/UVB ratio of the formulations were measured in vitro.
The degree of photostability was expressed as the percentage
effectiveness after exposure of both protection factors: the SPF
in vitro (% SPFeff) and the UVA-PF (%UVA-PFeff) and was calculated
according to Eqs. (5) and (6), respectively [29]. The results were
calculated as the average of at least three experiments.

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A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057
Table 2
The total content of avonoids in the extracts of H. arenarium, C. monogyna and S.
nigra expressed as mg of quercetin per 1.0 g of the dry extract weight and as mg of
gallic acid per 1.0 g of the dry extract weight. All analyzes are the mean of triplicate
measurements standard deviation.
Fractions of:

H. arenarium

C. monogyna

S. nigra

As mg quercetin
Ethyl acetate
Aqueous 3
Chloroform
Ether 2
Ether 3
Bicarbonate

84.00 3.20
43.36 0.32
2.23 0.03
32.43 1.92
13.80 0.34
1.23 0.03

95.30 4.14
66.11 3.42
27.47 0.45
32.71 0.54
38.14 0.04
2.26 0.01

103.01 5.73
43.90 2.02
19.72 0.21
4.62 0.04
9.48 0.11
0.42 0.04

As mg gallic acid
Ethyl acetate
Aqueous 3
Chloroform
Ether 2
Ether 3
Bicarbonate

265.29 7.51
143.13 4.87
16.40 1.03
110.91 2.65
69.81 16.84
4.21 0.84

283.67 8.93
184.72 6.82
67.98 3.04
103.65 13.45
96.20 4.09
6.87 1.03

311.30 15.87
139.17 9.56
77.78 0.62
42.43 0.90
51.62 0.07
4.22 0.06

%SPFeff

SPFin v itro after irradiation

 100
SPFin v itro before irradation

%PF-UVAeff

PF-UVAin v itro after irradation

 100
PF-UVAin v itro before irradation

2.9. Statistical analysis

Experiments were repeated at least three times, and the data
were analyzed statistically. All results are given as mean
value standard deviation (SD). The statistical analysis of the data
was performed using Students t-test, the analysis of variance
(ANOVA) and Tukeys posttest. P-values < 0.05 were signicantly
considered. All calculations using the statistical software Statgrath
were carried out.

3. Results and discussion

It is well-known that H. arenarium, C. monogyna and S. nigra extracts show very good antioxidant properties and potential ability
to protect against both UVA and UVB rays [16,17]. The highest
measured TF values for ethyl acetate fractions were observed.
The measured quercetin equivalents (QRE) were 103.01 5.73 mg
per 1.0 g of the dry extract weight (dew) forS. nigra extract,
95.30 4.14 mg QRE/g of dew for C. monogyna extract, and
84.00 3.20 mg QRE/g of dew for H. arenarium extract. The lowest
values of the total avonoids content observed for bicarbonate
fractions of H. arenarium, C. monogyna and S. nigra were detected.
The corresponding values were between 0.31 0.07 to
1.46 0.12 mg QRE/g of dew. The highest value assessed with the
TP method for the ethyl acetate fraction was observed. The corre-

Table 3
The content of the individual componends, like phenolic acids, avonoids present in the extracts of H. arenarium C. monogyna and S. nigra detected using HPLC method and DAD
detection system.
Peak No.

Individual substances

Retention time (min)

Concentration (%)

The extract of H. arenarium

1
2
3
4
5
6
7
8
9
10
11
12

Neochlorogenic acid
Cryptochlorogenic acid
Chlorogenic acid
Naringenin
Naringenin-4-O- glucoside
Naringenin-5-O- glucoside
Dicaffeoylquinic acid derivatives
Dicaffeoylquinic acid derivatives
Kaempferol-3-O-glucoside
Quercetin-3-O-glucoside
Apigenin-7-O-glucoside
Isosalipurposide

8.55
14.16
14.91
22.00
22.81
24.02
27.10
28.21
28.71
26.00
29.50
30.52

0.01
0.03
1.74
0.08
2.50
6.69
3.37
0.78
5.59
0.98
0.69
12.14

Neochlorogenic acid
Chlorogenic acid
p-Cumaric acid glucoside
()epicatechin
p-Coumaroylquinic acid
Rutin
Hyperoside
Quercetin-3-O-glucoside
Rosmarinic acid
Kaempferol-3-O-glucoside
Vitexin-200 -O-rhamnoside

8.01
12.17
13.55
15.23
15.85
21.99
23.23
23.79
25.68
26.89
29.05

0.30
6.65
0.19
6.86
0.66
0.55
12.62
3.13
6.50
0.57
1.88

Neochlorogenic acid
3-O-p-coumaroylquinic acid
Chlorogenic acid
Cryptochlorogenic acid
5-O-p-coumaroylquinic acid
Rutin
Quercetin-3-O-glucoside
3,4-di-O-caffeoylquinic acid
3,5-di-O-caffeoylquinic acid
Quercetin-3-O-(600 -acetyl-glucoside)
Kaempferol-3-O-rutinoside
Isorhamentin-3-O- rutinoside
4,5-di-O-caffeoylquinic acid

7.75
9.43
10.54
10.85
13.13
18.98
19.61
20.61
21.45
21.93
22.67
23.31
23.75

0.32
0.06
4.94
0.66
0.55
5.13
5.99
1.59
10.81
0.46
1.26
1.09
9.91

The extract of C. monogyna

1
2
3
4
5
6
7
8
9
10
11
The extract of S. nigra
1
2
3
4
5
6
7
8
9
10
11
12
13

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A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

Fig. 2. UVVis spectra of polyphenolic fractions isolated fromHelichrysum arenarium, Crataegus monogyna and Sambucus nigra (30 lg/mL).

Fig. 3. Formulations containing various concentrations of polyphenolic fraction vs.

the photoprotective effectiveness in the UVB range.

sponding values were 311.30 15.87 mg GAE per g of dry extract

weight (dew) for elderower extract 283.67 8.93 mg GAE/g of
dew for hawthorn extract, and 265.29 7.51 mg GAE/g of dew
for moench extract. Values between 1.52 0.02 and
5.64 0.26 mg GAE/g of dew obtained for all bicarbonate fractions,
to be the lowest values of total polyphenolic content in the studied
extracts were found (Table 2).
Only the ethyl acetate fractions were characterized as containing the highest total avonoids content, and polyphenolic content
as well. Thus, they were further analyzed using HPLC method. The
content of the individual phenolic acids, avonoids and their derivatives in the studied extracts are presented in Table 3. The results
have indicated that the H. arenarium, C. monogyna andS. nigra extracts are rich in various polyphenolic compounds including phenols and hydroxy-phenols, and their acids, alcohol or
carbohydrate derivatives. Each extract is a combination of many
individual, specic compounds; interactions between themselves
might have a great impact on their antioxidant and photoprotective activity.
Spectra proles for moench, hawthorn and elderower extracts
indicated that they absorb UV radiation in both, UVA and UVB
regions (Fig. 2). For C. monogyna ethyl acetate fraction the wavelength of maximum absorption was kmax = 320, 360 nm and for
H. arenarium and S. nigra kmax = 341 and 319 nm, respectively.
Spectrophotometric measurements were performed in order to
determine the photoprotective effectiveness and photostability of
cosmetic emulsions containing of the studied plant extracts.

Furthermore, the effectiveness and photostability were evaluated

using the spectrophotometric in vitro method in accordance with
the research [5,29] and the characteristic photoprotective parameters such as, the sun protection factor (SPFin vitro), the protection
factor against the UVA (PF-UVAin vitro), the critical wavelength (kc)
and the ratio of UVA/UVB were determined before and after
irradiation.
3.1. In vitro determination of photoprotective efcacy of sunscreens
The results obtained of photoprotective efcacy of the formulations studied before and after exposure to ultraviolet radiation, are
collected in Tables 2 and 3. The SPFin vitro values ranged from
6.00 0.42 to 9.88 1.66 for the individual plant extracts
(10 wt.% ) solubilized in the emulsions. According to the recommendations of the European Union (2006/647/EC) concerning the
effectiveness of sunscreen products [28] the preparations studied
meet the current standards for protection against UVA. The values
of PF-UVAin vitro for the compositions studied (from 3.64 0.07 to
6.96 0.21) were much greater than the minimal recommended
value, which should be at least 2.00. Ranging from 376.5 to 387.0
for all the sunscreen emulsions studied, they were in compliance
with the minimum requirements of at least 370 nm of the critical
wavelength [28]. In comparison with the P2 reference standard
formulation the emulsions studied provided higher protection
against UVA radiation, in particular, they had much higher values
of critical wavelength (kc) and PF-UVA factor. As has been proved

Table 4
Photoprotective activity and photostability of formulations containing individual plant extracts before and after 120 min of irradiation (mean standard deviation).

a
b
c
d
e
f

Helichrysum arenarium (10 wt.%)

Crataegus monogyna (10 wt.%)

Sambucus nigra (10 wt.%)

P2 reference sunscreen formulations

Pre-irradiation

SPFin vitroa = 6.80 (0.26)

PF-UVAin vitrob=6.96 (0.21)
UVA/UVBc = 1.06 ()
kcd (nm) = 387.0

SPFin vitro = 6.00 (0.42)

PF-UVAin vitro = 3.64 (0.07)
UVA/UVB = 0.81 ()
kc (nm) = 377.3

SPFin vitro = 9.88 (1.66)

PF-UVAin vitro = 5.18 (0.15)
UVA/UVB = 0.73 ()
kc (nm) = 376.5

SPFin vitro = 15.41 (2.00)

PF-UVAin vitro = 2.00 (0.06)
UVA/UVB = 0.83 ()
kc (nm) = 357.6

Post-irradiation

SPFin vitro = 5.60 (0.17)

PF-UVAin vitro = 6.35 (0.06)
UVA/UVB = 0.82 ()
kc (nm) = 382.0
%SPFeff.e = 82.35%
%PF-UVAeff.f = 91.24%

SPFin vitro = 4.88 (0.34)

PF-UVAin vitro = 3.06 (0.27)
UVA/UVB = 0.77 ()
kc (nm) = 379.0
%SPFeff. = 81.33%
%PF-UVAeff. = 84.07%

SPFin vitro = 8.70 (1.31)

PF-UVAin vitro = 4.73 (0.97)
UVA/UVB = 0.76 ()
kc (nm) = 378.6
%SPFeff. = 88.05%
%PF-UVAeff. = 91.31%

SPFin vitro = 11.81 (0.80)

PF-UVAin vitro = 1.85 (0.04)
UVA/UVB = 0.73 ()
kc (nm) = 353.6
%SPFeff. = 76.63%
%PF-UVAeff. = 92.50%

In vitro sun protection factor, calculated according to Eq. (1).

UVA protection factor, calculated according to Eq. (2).
The ratio of the integral UVA to UVB attenuation, calculated according to Eq. (4). Approximation by UVA Star Rating System [29].
Critical wavelength, calculated according to Eq. (3).
The percentage effectiveness after exposure of the SPF in vitro, calculated according to Eq. (5).
The percentage effectiveness after exposure of the SPF in vitro, calculated according to Eq. (6)

A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

55

Fig. 4. Absorbance proles of formulations studied containing the individual polyphenolic fractions (10.0% w/w) and their various combinations (10.0% w/w of each one): (a)
Sambucus nigra extract; (b) Crataegus monogyna extract; (c) Helichrysum arenarium extract; (d) Helichrysum arenarium and Crataegus monogyna extracts; (e) Crataegus
monogyna and Sambucus nigra extracts and (f) Helichrysum arenarium and Sambucus nigra extracts. Full line: before sunlight exposure, dashed line: after sunlight exposure.
The curves presented are the mean values resulting from three PMMA plates; the reproducibility of the values was within 10%.

before [29,30], the critical wavelength may well describe the width
of the spectrum of photoprotection, but is not suitable for measuring the intensity of protection. In addition, the ability to protect
against UVA radiation was also evaluated according to the Boots
Star System in which product photoprotection is graded by the
stars denoting the level of protection against UVA [34]: No stars
(UVA/UVB < 0.60 before and < 0.56 irradiation) small, Three stars
() medium (>0.60 before, >0.57 after); Four stars () (>0.80
before, >0.76 after) high and Five stars () (>0.90 before,

>0.86 after) very high. According to the guidelines of the Boots

Star System the formulations studied containing moench, hawthorn, and elderower fractions could be appropriately classied
as preparations with a very high (), high () and medium
() degree of protection against UVA, respectively (see Table 4).
Moreover, for each fraction, we analyzed the inuence of concentration on their effectiveness in the UVB range (Fig. 3). As has been
previously demonstrated by other scientists [22,35], the concentration of UV lters has great inuence on their efcacy. The results

56

A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

Table 5
Photoprotective activity and photostability of formulation containing a mixture of plant extracts before and after 120 min of irradiation (mean standard deviation).

a
b
c
d
e
f

Helichrysum arenarium (10 wt.%)

andCrataegus monogyna (10 wt.%)

Crataegus monogyna (10 wt.%)

and Sambucus nigra (10 wt.%)

Sambucus nigra (10 wt.%)

and Helichrysum arenarium (10 wt.%)

Pre-irradiation

SPFin vitroa = 19.51 (4.19)

PF-UVAin vitrob = 16.58 (1.67)
UVA/UVBc = 0.95 ()
kcd (nm) = 385.6

SPFin vitro = 18.21 (5.24)

PF-UVAin vitro = 7.54 (0.68)
UVA/UVB = 0.76 ()
kc (nm) = 377.6

SPFin vitro = 16.94 (3.76)

PF-UVAin vitro = 11.57 (1.47)
UVA/UVB = 0.89 ()
kc (nm) = 383.9

Post-irradiation

SPFin vitro = 17.51 (4.09)

PF-UVAin vitro = 16.00 ( 0.56)
UVA/UVB = 0.97 ()
kc (nm) = 386.0
%SPFeff.e = 89.74%
%PF-UVAeff.f = 96.50%

SPFin vitro = 15.33 (4.26)

PF-UVAin vitro = 6.83 (1.02)
UVA/UVB = 0.78 ()
kc (nm) = 378.7
%SPFeff. = 84.18%
SPF-UVAeff. = 90.58%

SPFin vitro = 14.75 (3.04)

PF-UVAin vitro = 10.65 (2.02)
UVA/UVB = 0.91 ()
kc (nm) = 385.0
%SPFeff. = 87.07%
%PF-UVAeff. = 92.05%

In vitro sun protection factor, calculated according to Eq. (1).

UVA protection factor, calculated according to Eq. (2).
The ratio of the integral UVA to UVB attenuation, calculated according to Eq. (4). Approximation by UVA Star Rating System [29].
Critical wavelength, calculated according to Eq. (3).
The percentage effectiveness after exposure of the SPF in vitro, calculated according to Eq. (5).
The percentage effectiveness after exposure of the SPF in vitro, calculated according to Eq. (6).

obtained show also a positive linear correlation between the concentration of the polyphenolic extracts and their effectiveness in
the UVB range, for the H. arenarium extract (R2 = 0.9877), C. monogyna (R2 = 0.9760) and S. nigra (R2 = 0.9859) (see Fig. 4).
Additionally, we investigated the photoprotective properties of
formulations containing the various combinations of the polyphenolic fraction in the concentration equals 10 wt.% of each one
(Table 5). This allowed us to observe the interaction between the
extracts studied, whether they showed an additive, synergistic or
antagonistic effects. The SPFin vitro and PF-UVAin vitro values for the
emulsions containing a mixture of H. arenarium (10 wt.%) and C.
monogyna (10 wt.%) 19.51 (4.19) and 16.58 (1.67), C. monogyna
and S. nigra 18.21 (5.24) and 7.54 (0.68) and S. nigra and H. arenarium 16.94 (3.76) and 11.57 (1.47) were found, respectively.
The results obtained suggest that the photoprotective activity
against UVA and UVB of the formulations studied containing combinations of the extracts has been signicantly reinforced (synergistic effect). These results are in accordance with the previous
publications [4,22,30,35,36], in which the combination of several
types of UV lters in the preparation, either natural, mineral or
chemical, ensured the maximum protection, broad spectrum of
effectiveness, and improved the photostability of such formulation,
as well. These products, like the formulations containing individual
polyphenolic fractions, also met current standards of the European
Union (2006/647/EC) for protection against UV radiation, classifying them as the products of medium photoprotection
SPFlabeled = 15. However, according to the Boots Star System[34],
emulsions containing S. nigra and H. arenarium, C. monogyna and
H. arenarium could be classied as the products of very high protection against UVA (), and C. monogyna and S. nigra for cosmetics with a medium degree of protection (), respectively.

3.2. In vitro study of photostability of sunscreens

Photostability is another essential parameter characterizing
the effectiveness and safety of sunscreen products, since ultraviolet radiation can lead to reduction of the photoprotective ability
of cosmetics and can also generate free radicals. [5,28]. To determine the photostability of the formulations the cosmetic emulsions were irradiated with ultraviolet radiation of 650 W/m2
intensity within 2 h according to the intensity of global solar radiation (290400 nm) at sea level measured in accordance with the
guidelines of the International Commission on Illumination (CIE)
[31,32]. The results are shown in Fig. 2 as the absorption spectra
of the products before and after irradiation. Preparations for

which the values of%SPFin vitro and%PF-UVAin vitro were at least

80% were considered as photostable products [29]. Analysis of
the results leads to the conclusion that the emulsions studied,
regardless of the intensive UV irradiation, exhibit excellent photostability, especially in the UVA region. For the majority of them
the values of%PF-UVAin vitro exceeded 90%. Similar results were
obtained by Choquenet et al. [22] who measured the photostability of emulsion systems containing individual polyphenolic compounds: quercetin and rutin. They found high photostability
(more than 90%) and compatibility with other commercially
available sunscreen lters. Moreover, as was described in previous publications, avonoids such as quercetin signicantly reduce
the photodegradation of the chemical UV lters without any
changes in the effectiveness of the sunscreen formulations
[4,28,36]. The multiple effects of polyphenolic compounds, as
antioxidants, photostabilizers and UV lters, incorporated into
sunscreen formulations represent a promising strategy for the
development of broad-spectrum sunscreen products with enhanced efcacy and safety.

4. Conclusions
Most classical sunscreens are a combination of UVA and UVB
chemical lters and other physical components in order to provide
broad spectrum protection. However they are not based on renewable resources and cause many side effects i.e. under certain conditions can generate dangerous free radicals [37]. Thus natural
products characterized by high SPF and PF-UVA values must be
taken into consideration. All results of this study indicate that
the individual polyphenolic fractions isolated from H. arenarium,
S. nigra and C. monogyna and their various combinations provide
good protection against ultraviolet radiation (UVR), certainly
higher than the UVR lters widely used in the skin care market.
Furthermore,H. arenarium, S. nigra and C. monogyna extracts and
many of their components show strong antioxidant activity. The
combination of these two features moves up the extracts studied
to the class of cosmeceuticals, and makes them potential active
ingredients of commercial sunscreen formulations mostly due to
their cosmetic, protective and preventive properties. Moreover,
their natural origin meets consumer expectations for green sun
care products.
Use of polyphenolic complexes isolated from plants of C.
monogyna, H. arenarium and S. nigra, and mixtures thereof allow
to obtain an emulsion with a broad spectrum against UV irradiation, both UVB and UVA. The use of non-ionic sugar surfactants

A. Jarzycka et al. / Journal of Photochemistry and Photobiology B: Biology 128 (2013) 5057

as a cosmetic emulsier system, allows to obtain favorable rheological parameters, a desired appearance and texture.
Acknowledgment
The work was nanced by a statutory activity subsidy from the
Polish Ministry of Science and Higher Education for the Faculty of
Chemistry of Wrocaw University of Technology.
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