International Journal of Cosmetic Science, 2008, 30, 443–452

Characterization of cosmetic cream with

Mesembryanthemum crystallinum plant extract:
influence of formulation composition on physical
stability and anti-oxidant activity

I. Bouftira*, , C. Abdelly and S. Sfar*

*Laboratory of Galenic Pharmacy, Faculty of Pharmacy, Monastir and Laboratory of Plant Adaptation to Abiotic
Stresses, Center of Biotechnology, Technoparc of Borj Cédria, Borj Cédria, Tunisia

Received 18 November 2007, Accepted 28 June 2008

Keywords: antioxidant activity, catalase, dermo-cosmetic formulation, Mesembryanthemum crystallinum,

superoxide dismutase

and an anti-radical activity (30% of DPPH

Synopsis
inhibition). The formulation storage (15 days at
Cosmetic or pharmaceutical composition contain- 4C) showed a marked loss of total anti-oxidant
ing superoxide dismutase (SOD) was usually used capacity. The addition of the M. crystallinum
in topical administration, particularly, in fighting extract induced also a reduction in formulation
against skin ageing and in the protection of the viscosity and pH.
skin against radiation exposure. Mesembryanthe-
mum crystallinum is a halophyte plant widely
Résumé
used in the traditional medicine, characterized
by the presence of anti-oxidants enzymes in Une composition cosmétique ou pharmaceutique
responses to abiotic stresses. In the present contenant de la superoxyde dismutase (SOD) est
study, we prepared a formulation with M. crys- habituellement administrée sous forme topique, en
tallinum extract characterized by naturally occur- particulier pour lutter contre le vieillissement cuta-
ring SOD and catalase in association with other née et pour protéger la peau d’une exposition aux
anti-oxidants molecules. The SOD activity was radiations. Mesembryanthemum crystallinum est
measured by 3-(4,5-dimethyldiazol-2-yl)-2,5- une plante halophyte couramment utilisée en
diphenyl-tetrazolium bromide/riboflavin method, médecine traditionnelle et caractérisée par la pré-
catalase by colorimetric method and the total sence d’enzymes anti-oxydantes présentes pour
anti-radical activity was measured by 1,1-diphe- répondre aux stress abiotiques. Dans cette étude,
nyl-2-picrylhydrazyl radical (DPPH) method. For- nous avons préparé une formulation à partir d’un
mulations contain a significant SOD activity extrait de M. crystallinum dans lequel se trouve
(8.33 U mg)1), a catalase activity (0.5 · 107 UC) naturellement de la superoxyde dismutase et de
la catalase en association avec d’autres
molécules anti-oxydantes. L’activité SOD a été
Correspondence: Ibtissem Bouftira, Laboratory of Galenic mesurée par la méthode utilisant le bromure de
Pharmacy, Faculty of Pharmacy of Monastir, Rue Ibn
Sina, 5000 Monastir, Tunisia. Tel.: (0216) 73461000;
3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium/
fax: (0216) 73461830; e-mail: ibtissem.bouftira@ Riboflavine, l’activité catalase par une méthode
laposte.net colorimétrique et l’activité anti-radicalaire totale

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie 443
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

a été mesurée par la méthode utilisant le radical mation of sun-burn cells in skin [8]. Mizushima
1,1-diphenyl-2-picrylhydrazyl (DPPH). La formu- et al. [9] and Niwa [10] showed that a topically
lation possédait une activité SOD significative applied SOD cream was effective against several
(8.33 U/mg), une activité catalase (0.5 · 107 UC) skin diseases associated with increased reac-
et une activité anti-radicalaire (inhibition de 30% tive oxygen species (ROS) levels in humans.
du DPPH). La conservation de la formulation (15 Nevertheless, the use of SOD as an active phar-
jours à 4C) a montré une perte marquée de la maceutical ingredient has been a problem because
capacité anti-oxydante totale. L’addition de of the fragility of this enzyme. Most enzymes lose
l’extrait M. crystallinum a également induit une biological activity at room temperature within
diminution de la viscosité de la formulation et de a few months. Topical formulations consist of
son pH. various oils and surfactants that may disturb
enzyme structure and consequently deactivate the
enzyme [11].
Introduction
In fact, one of most challenging tasks in the
Skin is our biological border to environment. development of formulations with proteins is to
Because of its barrier function, it is a potential tar- deal with the physical and chemical instabilities of
get organ for oxidative stress from external insults, proteins [12]. Proteins perform molecular tasks
such as ultraviolet (UV) irradiation, ionizing radia- with unparalleled speed and specificity, which
tion and various toxic chemicals. This oxidative make them useful as pharmaceutical drugs, but
stress, which can be an initiator in the pathogene- their applications are often hampered by their
sis of skin cancer and photoageing [1], may be low stability [12]. Proteins in formulations are
described as disequilibrium between pro-oxidant highly susceptible to both chemical and physi-
and anti-oxidant, with predominant pro-oxidant cal instabilities when compared with traditional
status of skin. It is known that prolonged exposure drugs. Chemical instability results in the genera-
to UV light results in a severe decrease in the tion of a new chemical entity by bond formation
anti-oxidant capacity of skin. Moreover, an addi- or cleavage. Physical instability involves changes
tional increase in active oxygen intermediates in the secondary, tertiary or quaternary structure
such as dangerous free radicals have been demon- of the molecule, which can be manifested as dena-
strated [2]. turation, adsorption, aggregation and precipita-
Superoxide dismutase (SOD) is an essential tion. Both chemical and physical changes in
enzyme which protects cells from oxidative dam- proteins can result in a loss of biological activity
age by catalysing the reduction of the O2) to
H2O2 and molecular oxygen (O2) [3]. The H2O2
 [13].
In this study, we investigate the potential
produced is then scavenged by catalase and sev- anti-oxidant activity of the halophyte plant
eral classes of peroxidases. Catalase, which is extract Mesembryanthemum crystallinum alone
found in peroxisomes, cytosol and mitochondria, and after addition in formulation. Salt tolerant
dismutate H2O2 into H2O and O2. The combined plants (Halophyte) are highly evolved with spe-
action of SOD and catalase reduces the formation cialized organisms with well-adapted morphologi-
of the hydroxyl radical (OH·) which is the most cal and physiological characteristics allowing
toxic of oxygen radicals. them to proliferate in soils possessing high salt
These anti-oxidant enzymes already have been concentrations [14]. During normal metabolism,
shown to influence ageing, cancer and some very plants generate ROS, including superoxide radical
important diseases, such as arteriosclerosis and (O2)), hydrogen peroxide (H2O2), hydroxyl radi-
age-dependent immune deficiency [4]. Inal et al. 
cal (HO ) and singlet oxygen (O) [15]. ROS is
[5] and Andersen et al. [6] reported that there overproduced in plants under stress, including
were age-related decreases in SOD activity. In drought and desiccation, salt stress, chilling, heat
addition, the enzyme activity was found to be shock, heavy metals, UV radiation and air pollu-
decreased immediately after solar-simulated tants such as ozone and SO2, mechanical stress,
UV-irradiation, and UVB can be considered to be nutrient deprivation, pathogen attack and high
mainly responsible [7]. light stress [16, 17].
Treatment with exogenous SOD can reduce There is increasing evidence that salinity is one
the activity loss and prevent the UV-induced for- factor leading to oxidative stress in plants cells

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
444 International Journal of Cosmetic Science, 30, 443–452
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

[15, 18, 19]. To mitigate the oxidative damage

Materials and methods
initiated by ROS, plants have developed a complex
anti-oxidative system [18] involving both enzy-
Plant material
matic and non-enzymatic mechanisms [15].
Non-enzymatic anti-oxidants such as ascorbic acid, Aerial parts from M. crystallinum were collected
a-tocopherol, flavonoids, tannins and phenolics from their native biotope in Monastir (Tunisia)
acids are present in the halophytes plants. For coasts during March 2004.
example, the halophyte plant Crithmum maritimum
(Apiacea) contains vitamin C, carotenoids [20],
Extraction with phosphate buffer pH 7.8
flavonoids and tannins [21]. Limonium axillare
(Limoniacea) contain flavonoids [22]. In the halo- Twenty-five grams of fresh leaves were homoge-
phyte plant M. crystallinum, high photosynthetic nized at 4C with 500 mL of medium: 50 mmol L)1
active radiation and UV irradiance induce the K phosphate buffer (pH 7.8), 10 mmol L)1 MgCl2,
accumulation of flavonoids [23]. Many methods 10% (V/V) glycerol, 1 mmol L)1 EDTA and 1%
were used to evaluate the anti-radical/anti-oxidant (W/W) and polyvinylpyrrolidone (PVP). The
activity in plant tissues. For example, the 2,2- homogenate was then filtered at 4C. The filtrate
diphenyl-2-picrylhydrazyl radical (DPPH) test is was lyophilized and used for anti-oxidant activity
already reported to evaluate in a relatively short determination. Protein concentration was deter-
time the anti-radical activity [24]. The absorbance mined according to Bradford [35] method, using
decreases as a result of a colour change from pur- bovine serum albumin as a standard.
ple to yellow as the radical is scavenged by anti-
oxidants through donation of hydrogen to form
Superoxide dismutase activity
the stable DPPH-H molecule [25]. The DPPH
method can be used for solid or liquid samples and Total SOD activity was assayed according to
is not specific to any particular anti-oxidant com- Scebba et al. [36]. The reaction mixture contained
ponent, but applies to the overall anti-oxidant 50 mmol L)1 potassium phosphate buffer (pH
capacity of the sample [26]. The species M. crys- 7.8), 0.1 mmol L)1 EDTA, 13 mmol L)1 l-methio-
tallinum (Family: Aizoaceae, Order: Caryophyllales) nine, 2 lmol L)1 riboflavin and 75 lmol L)1
also termed the ‘common ice plant’ has emerged 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium
as a model organism in plant molecular physiol- bromide (MTT). The reaction was started by expos-
ogy [27]. The plant is characterised by the stress ing the mixture to cool white fluorescent light at a
inducible switch from C3 photosynthesis to Cras- photosynthetic photon flux of 50 lmol m)2 s)1 for
sulacean acid metabolism [28, 29]. A second 15 min. The blue colour developed in the reaction
major area of interest centres on the plant’s was measured spectrophotometrically at 560 nm
extreme stress tolerance, particularly tolerance to (Cam spec M230/330 UV visible spectrophotome-
high salinity. The plant thrives in coastal area ter, Cambridge, United Kingdom). The volume of
under climatic conditions characterized by short, sample causing 50% inhibition in colour develop-
cool, moist winters and long dry summers ment was taken as one unit of SOD activity [37]
[30–32]. M. crystallinum shows five distinct and the reaction was expressed as units per mL.
growth phases during its life cycle (that is, germi-
nating seedlings, juvenile, adult, flowering and
Catalase activity
seed forming) [33]. The plant is a demulcent and
a diuretic. In ancient times, physicians used Catalase activity was measured by hydroperoxide
leaf juice to soothe inflammation of the mucous reduction method [38]. Extract of 0.5 mL was
membranes of the respiratory or urinary system. mixed with 7.5 mL of distilled water containing
In Europe, the fresh juice has been used to treat 1 mL of 1% H2O2. After incubation for 30 min at
water retention, painful urination and to soothe 25C, the reaction was stopped by adding 5 mL of
lung inflammation [34]. The purpose of this 10% H2SO4. The content of H2O2 reduced by cata-
study is to investigate the potential anti-oxidant lase was determined by a solution of 1N KMnO4
activity of the plant extract containing SOD and by titration.
catalase and the effect of the formulation on this The enzymatic activity can be established by the
anti-oxidant activity. following formula:

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
International Journal of Cosmetic Science, 30, 443–452 445
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

Activity (mg H2 O2 Þ : 1:7  V=30  0:5 were made at room temperature in triplicates for
each analysed sample.
where V, volume (mL) of 1N KMNO4, incubation
time = 30 min and extract volume = 0.5 mL.
Phytochemical screening

Free radical scavenging activity Chemical screening was carried out on the pow-
dered samples using standard procedures to iden-
Free radical scavenging activity of plant extract tify the constituents as described by Denoël and
was determined by using a stable free radical, Paris [40–43]. By using PVP in the extraction pro-
(1,1-diphenyl-2-picrylhydrazyl) (DPPH), according cedure, we do not include polyphenol compounds
to slightly modified method of Blois [39]. DPPH in the identified molecules. PVP is known for
solution was prepared at the concentration of removal of polyphenols through the filtration step.
0.024 mg mL)1 DPPH in ethanol. During assay, Saponins: About 2 g of the powdered plant sam-
1 mL of the crude extract (2 mg mL)1) was ple was boiled in 10 mL of distilled water. Six mil-
mixed with 1 mL DPPH solution. The mixture lilitre of the filtrate was mixed vigorously and
was incubated at the room temperature for observed for the formation of emulsion.
30 min; absorbance was recorded at 517 nm Steroids: A portion of powdered plant sample
(Cam spec M230/330 UV visible spectrophoto- was heated with chloroform and filtered. To one-
meter). Butylated hydroxytoluene (BHT) was fifth of the filtrate, acetic anhydride and H2SO4
used as a standard for the investigation of the was added. The colour changed from violet to blue
anti-radical activity. or green indicating the presence of steroid.
The percentage of the remaining DPPH (%
DPPHREM) at steady state was determined as fol-
 Coumarines: A portion of powdered plant sample
was mixed with 95% alcohol, heated over a steam
lows: bath and filtered. NaOH was added and the
%DPPHREM ¼ 100 CDPPH =CDPPHðt¼0Þ changes in colour observed under a UV lamp indi-
cated the presence of coumarines.
Where CDPPH (t = 0) is the initial DPPH concen- Irridoı̈des: About 2 g of the powdered plant sam-
tration and CDPPH is the DPPH concentration at ple was boiled with distilled water for 10 min.
the steady state. After filtration, 1 mL HCl was added to the filtrate
and heated over a steam bath. The detection of
black precipitate indicates the presence of irri-
Cream formulation
doı̈des.
Polyacrylamide (3%), C13–14 isoparaffine (3%), Cardiotonic heterosides: A portion of the powdered
Laureth 7 (3%), Huile de paraffine (6%), Cétéoryl sample was mixed with 95% alcohol for 24 h and
ethyl hexanoate (5%), Glycérine (5%), Sepicide HB then the acetate of plumbum was added. After
(0.5%), plant extract (%, 2 mg mL)1) in cream,
Water QSP100.
100
% DPPH inhibition

Physical stability evaluation of the formulation 80

60
Physical stability was evaluated by subjecting the
formulation to storage at 4C. Samples were col- 40
lected for the evaluation of rheological behaviour
20
and pH measurements at the initial time and then
at 15-day intervals. The rheological measurements 0
were made using a rotational rheometer (Contraves A B C D
Rheometer 6806 STV, Contraves AG, Zurich,
Figure 1 Anti-radical activity measured by the percent-
Switzerland). Measurements were made at progres- age of 2,2-diphenyl-2-picrylhydrazyl radical (DPPH) inhi-
sively higher rotation speeds. The pH of formula- bition. (A) Formulation after addition of plant extract, (B)
tions diluted to 1 : 10 in distilled water was formulation without plant extract, (C) plant extract
measured using Thermo Orion model 410 (2 mg mL)1) and (D) butylated hydroxytoluene
(Thermo Orion, Beverly, MA, USA). Measurements (2 mg mL)1).

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
446 International Journal of Cosmetic Science, 30, 443–452
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

activity (30.66 ± 3.05% of DPPH inhibition for

30
1 g of formulation). There was no anti-oxidant

SOD activity U mg–1

25 activity in the cream formulated without plant
20 extract. The different results of the anti-radical
15 activity were compared with the synthetic anti-
10 oxidant BHT (99.7 ± 1.14 % of DPPH inhibition
5 at concentration of 2 mg mL)1).
0
A B C D E
Superoxide dismutase activity
Figure 2 Superoxide dismutase (SOD) activity. (A) For-
The plant extract showed an SOD activity of
mulation after addition of plant extract, (B) formulation
20 U mg)1 in the lyophilized extract which corre-
without plant extract, (C) plant extract (2 mg mL)1), (D)
spond to 40 U mg)1 of proteins. The SOD activity
SOD activity in a medicament (Zymo Radical; 0.1 U SOD
per mg) and (E) SOD activity in a cosmetic product in the plant extract was more than that of the
(Extramel C: 6 U SOD per mg). medicaments commercialized (Fig. 2). This activity
was reduced in the cream that contains the plant
extract. The SOD activity in the cream with plant
filtration, NaOH and 0.5 mL (Reactive of Buljet) extract (8.33 U mg)1) was more than that of a
was added to the filtrate. The red colouration indi- commercialized dermocosmetic cream, Extramel C
cates the presence of cardiotonic heterosides. (6 U mg)1).
Anthracenic heteroside: A portion of the powdered
sample was mixed in distilled water and heated for
15 min and filtered. The filtrate was mixed with Catalase activity
ammoniac 1/2. Red colouration indicates the pres- Catalase activity was detected in the same plant
ence of anthracenic heteroside. extract (Fig. 3). This activity was compared with a
commercialized medicament (Pulvo Neomycine)
Results containing a purified catalase from blood horse
(2 · 107 UC). The catalase activity in the plant
Anti-radical activity extract was 1.35 · 107 UC corresponding to
2.7 · 107 UC mg)1 of proteins. A reduction of cat-
The results of the anti-radical activity (Fig. 1) alase activity was noted in the cream formulation
showed that the activity was more than 50% of (0.5 · 107 UC).
DPPH inhibition in the lyophilized plant extract
(67 ± 2% of DPPH inhibition at concentration of
2 mg mL)1). An anti-oxidant activity was detected Anti-oxidant activity in formulation
in the cream, but we noted a reduction of this The results of the anti-oxidant activity in the for-
mulation after addition of the plant extract are
3 shown in the Table I. The decrease in the anti-oxi-
Catalase activity (107 UC)

2.5 dant activity was enhanced by the time of formu-

lation storage. After 7 days of formulation storage,
2
the anti-radical activity was reduced by 12%, SOD
1.5
activity by 25% and catalase activity by 40%.
1 After 15 days of formulation storage, the anti-radi-
0.5 cal activity was reduced by 95%, the SOD activity
0 decreased by 62% and catalase activity by 80%.
A B C D

Figure 3 Catalase activity. (A) Formulation after addi- Physical stability of the formulation
tion of plant extract, (B) formulation without plant
extract, (C) plant extract (2 mg mL)1), (D) catalase activ- The results of the formulations viscosity (Fig. 4)
ity used in a medicament (Pulvo neomycine: indicates a variation by addition of the plant
2 · 107 UC). UC: Quantity of enzyme needed to reduce extract. We can note a decrease in initial formula-
1 lmol H2O2 per minute in the condition of experience. tion viscosity (1174.5 cP) after addition of the

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
International Journal of Cosmetic Science, 30, 443–452 447
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

Table I Anti-oxidant activity in 1 g

SOD activity Catalase activity of formulation (after the addition of
Anti-radical activity (Unit found in (UC found in the plant extract)
Time (% DPPH inhibition) 1 g of cream) 1 g of cream)

0h 33.83 ± 0.76 8.33 0.5 · 107

24 h 30.66 ± 3.05 8.33 0.5 · 107
7 days 29.98 ± 0.97 6.25 0.3 · 107
15 days 1.39 ± 0.53 3.125 0.1 · 107

DPPH, 2,2-diphenyl-2-picrylhydrazyl radical; SOD, superoxide dismutase.

1200 Table II Chemical screening of the lyophilized plant

extract
1000
Viscosity (cP)

800 Plant extract

600 Chemicals compounds (lyophilized)

400
Saponins )
200 Steroids )
0 Alkaloids )
A B C Coumarines )
Irridoı̈des )
Figure 4 Variation in the viscosity of the formulation. Cardiotonic heterosides )
(A) Formulation without plant extract, (B) formulation Anthracenic heterosides )
after addition of the plant extract (48 h) and (C)
formulation after addition of the plant extract (15 days).
()), absence of constituents.

7.4
plant extract. We obtained a decrease in pH from
7.2 7.2 to 6.8 just after plant extract addition (2 h).
When the period of formulation storage increased,
7 the pH values increased in the formulation from
pH

6.8 to 7.14 (after 15 days).

6.8

6.6 Phytochemical screening

The results of the qualitative screening (Table II)

6.4
of the plant extract showed the absence of the dif-
A B C D E
ferent molecules like coumarines, saponins, ste-
Figure 5 Variation of the pH values in the formulation roids, alkaloids, irridoı̈des, cardiotonic heterosides
before and after the addition of the plant extract. (A) For- and anthracenics heterosides.
mulation without plant extract, (B) formulation after
addition of plant extract (2 h), (C) formulation after addi-
tion of plant extract (48 h), (D) formulation after addi- Discussion
tion of plant extract (7 days) and (E) formulation after Many noxious substances and ROS from UV light
addition of plant extract (15 days).
irradiation penetrate into the skin and are
involved in various skin diseases including skin
plant extract (808.1 cP). This decrease in viscosity tumours, skin wrinkling and skin ageing. Multiple
was intensified by the increase in the period of for- lines of anti-oxidant defense have evolved and
mulation storage (733.5 cP after 15 days of stor- served to protect human skin from oxidative stress,
age). We also can note a variation in pH values including prevention and repair. Primary defense
(Fig. 5) of the formulation after addition of the mechanisms prevent oxidative damage by scaveng-

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
448 International Journal of Cosmetic Science, 30, 443–452
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

ing reactive species directly. Secondary defense a-tocopherol showed 70% of DPPH inhibition, for-
mechanisms complete processes elicited by ROS mulation with Ginkgo biloba extract showed 60%
such as lipid peroxidation. One approach is the of DPPH inhibition and a formulation with
supplementation of the skin with anti-oxidants Glycyrrhiza glabra extract showed 20% of DPPH
and thereby strengthening its anti-oxidative inhibition [12].
capacity [2]. Enzymes are one of the potential active ingredi-
Several non-animal SOD sources for cosmetic use ents in pharmaceutical and cosmetic products.
are available today. Most of them include SOD alone SOD has a high capacity of removing free radicals
or in association with other anti-oxidants com- that are one of the main causes for skin ageing.
pounds like the enzyme catalase, which act in cor- Topical administration of anti-oxidants is capable
relation with SOD. In the present study, we used a of diminishing oxidative injury, but it is necessary
total plant extract containing SOD, catalase and to develop a stable formulation that maintains the
other anti-oxidants compounds. The extract is char- enzymatic activity.
acterized by a mixture of anti-oxidants enzymes and In our study, besides the total anti-oxidant
molecules isolated from M. crystallinum leaves. activity, SOD and catalase activities were demon-
The DPPH· assay originally developed by Blois strated in the plant extract. These enzymes were
[39] is widely used for the measurement of free widely used in the treatment of the skin fibrosis
radical scavenging capacity in phytotechnology, induced by radiation therapy. Reactive oxygen
food technology and pharmacology/toxicology. metabolites including superoxide anion (O)2),
DPPH· is a free radical that easily accepts an elec- hydrogen peroxide (H2O2) and hydroxyl radical
tron or hydrogen radical to become a stable dia- (OH·) are involved in radiofibrosis. Therefore, pro-
magnetic molecule. It can accommodate large tective enzymes like catalase and SOD, which
number of samples within a short period and is decreases post-irradiation, might be useful in pre-
sensitive enough to detect low concentrations of venting and/or treating these effects [46].
the active principles [44]. The extract samples In vitro and in vivo studies have demonstrated
tested in the present study for their H-donor ability the radioprotective effect of the percutaneous
measured by the stable DPPH· showed an anti- administration of SOD (Cu/Zn) [19]. Our results
radical activity in the plant extract that exceed demonstrate a significant SOD activity in the plant
60% of DPPH inhibition for a concentration of extract (20 U mg)1), this activity was reduced
2 mg mL)1. This can be because of the presence (8.33 U mg)1) in the cream but was higher than
of anti-oxidant molecules like chlorophylls, carote- in Extramel C and Zymo Radical (0.1 U mg)1).
noids, vitamins or polysaccharides [45]. In Extramel C is a commercialized cream containing
response to the stress of the environment, it was SOD from Cucumis Melo (Melon) fruit extract, with
indicated that M. crystallinum tissues contain neg- activity of 6 U mg)1. In this commercialized
ligible amounts of phenolics compounds [33]. It cream, SOD was stabilized by micro-encapsulation.
was demonstrated in another study that this plant In our study, catalase activity was reduced in the
contained polysaccharides belonging to the fairly cosmetic formulation (0.5 · 107 UC) in compari-
methylated pectin class [45]. In the formulation, son with plant extract (1.35 · 107 UC).
results showed the presence of anti-radical activity Di Mambro and Fonseca [12] demonstrate that
which was approximately 30% of DPPH inhibition. the SOD alone and in formulations maintained the
This anti-radical activity was because of probably activity when stored at 4C; the addition of SOD
one anti-oxidant molecule or an association of dif- even in higher concentration did not change the
ferent anti-oxidants that can be present in the physical parameters of the formulations. Also, the
same extracts. addition of this macromolecule does not affect
In another study, investigations of topical for- the formulation viscosity, once it was constant
mulations were evaluated following inclusion of concerning time, temperature and SOD concentra-
different plant extracts containing flavonoids [12]. tion. Proteins can influence physical stability of a
The anti-oxidant effect of these plant extracts formulation because they adsorb to oil-water inter-
alone and after addition in the formulation was faces. Adsorption then results in the reduction of
evaluated using the stable DPPH·. A formulation the interfacial tension and a formation of a large
added with a-tocopherol was used to compare interface area in emulsion. Upon adsorption, glob-
anti-oxidant activity. The formulation with ular proteins unfold to an extension depending on

ª 2008 The Authors. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
International Journal of Cosmetic Science, 30, 443–452 449
Role of Mesembryanthemum crystallinum on physicochemical properties in cosmetic cream I. Bouftira et al.

their intrinsic structural stability and progressively ester microspheres [50]. Catalase was encapsulated
constitute an interfacial film exhibiting viscoelastic by spray drying [51] and this enzyme was more
properties [12]. stable in the presence of a stabilisers. However,
The rheograms obtained showed a decrease in spray-dried catalase without stabiliser was found
viscosity after the addition of the plant extract. to undergo rapid inactivation.
This could be related to the composition of the To maintain the protein stability in formula-
plant extract which may cause a possible internal tions, the native structure is required to be intact.
disarrangement in the cream. It is thought that sugars stabilise proteins during
In another study, the addition of plant extract rapid dehydration through either vitrification or
decreased the viscosity of a formulation [12]. water substitution mechanisms. Water substitution
When plant extracts with lower viscosity (Ginkgo involves the formation of hydrogen bonds between
biloba extract and soybean proteins extract) are the sugar and protein, which is believed to be
added to the aqueous phase, they dilute the system responsible for the inhibition of the unfolding of
and probably destabilize the cream structure. the proteins, whereas vitrification depends upon
Anchisi et al. [47] found a decrease in viscosity to the immobilization of protein molecules that
about 20% in formulations containing 2% Salvia accompanies glass formation [52, 53].
officinalis, Centella asiatica or Calendula. This result
may be due to either the higher viscosity of the
Conclusion
extract or the quantity of protein present. The
structure of proteins and their physical and chemi- In conclusion, we confirmed by this study the pos-
cal properties (as net electric charge) may contrib- sible use of a crude plant extract from M. crystalli-
ute for the viscoelastic properties of oil-water num for a cosmetic formulation. This extract is
interface thus, reducing the destabilization of characterized by the presence of anti-oxidant
cream structure caused by the extract addition. enzymes SOD and catalase and a total anti-radical
Thus, when emulsions for topical use are prepared, activity because of the presence in the same
the chemical composition of the plant extracts extract of anti-oxidants molecules. The anti-oxi-
should be considered to obtain a formulation with dant activity was reduced in the formulation. This
the intended viscosity. was confirmed by the decrease after the addition
The measurement of pH of the formulations is of plant extract to the formulation of the DPPH
necessary to detect pH alterations during time scavenging activity, the activity of SOD and the
storage. We obtained a decrease in pH from 7.2 to activity of catalase. The addition of the plant
6.8 by addition of the plant extract. After formula- extract induced also a decrease in the viscosity
tion storage, the pH values increased to stabilize and the pH of the formulation. The changes
around 7.1. Marquele-Oliveira et al. [48] demon- observed in the physical stability of the formula-
strates that the addition of the propolis extracts in tion could be related to a possible internal struc-
the formulations caused the decrease in their pH tural disarrangement because of the composition
values within the range of 5.5–5.11 and 4.55– of the plant extract.
4.4.
In our study, after 15 days of storage, formula-
Acknowledgements
tions showed a marked loss of SOD and catalase
activity. Storage time can affect enzymatic stability The author wishes to acknowledge Prof. Chekir-
by changes in enzymatic structure leading to pro- Ghedira Leila (Laboratory of Biotechnology and
tein unfolding and/or aggregation. It was reported Molecular Toxicology) for her help in the applica-
that SOD subjected to covalent modifications with tion of the antiradical activity by DPPH method
polyethylene glycol showed remarkable preserva- and Dr Ben Hamed Karim (Laboratory of Plant
tion of activity and exhibited much longer plasma Adaptation to Abiotic Stresses) for his help in the
lifetimes in mice. It appears that attachment of 2/ determination of the superoxide dismutase activity.
4 PEG chains per glycoprotein causes minimal loss
of enzymatic activity [49]. In another study, it
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452 International Journal of Cosmetic Science, 30, 443–452