European Journal of Integrative Medicine 42 (2021) 101286

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European Journal of Integrative Medicine

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

Research paper

Photoprotective potential of a Tunisian halophyte plant Carpobrotus edulis L

Mohamed Ali Lassoued a,∗, Nour El Houda Ben Fatma a, Mariem Haj Romdhane b, Adel Faidi a,
Hatem Majdoub b, Souad Sfar a
a
University of Monastir, Laboratory of Pharmaceutical, Chemical and Pharmacological Drug Development - LR12ES09, Faculty of Pharmacy of Monastir, Rue Ibn Sina,
5000 Monastir, Tunisia
b
University of Monastir, Laboratory of Interfaces and Advanced Materials - LR11ES55, Faculty of Sciences of Monastir, Avenue de l’environnement, 5019 Monastir,
Tunisia

a r t i c l e i n f o a b s t r a c t

Keywords: Introduction: Different strategies are applicable to protect skin against the harmful effects which may appear
Carpobrotus edulis L. due to an overexposure to UV radiation including the use of sunscreens. Natural polyphenols have shown an
Chemical analysis interesting activity as photoprotectors. This study aimed to assess the potentiality of a Tunisian halophyte plant,
Phenolic compounds
Carpobrotus edulis L. (CE) as a candidate for topical photoprotection.
Antioxidant
Sun Protection Factor Methods: A phytochemical characterization of CE aqueous leaf extract was processed by quantification of total
Photoprotection polyphenols and flavonoids contents as well as by FT-IR, UV and LC-MS analysis. The plant extract was, then, in-
corporated into an oil-in-water (O/W) cream and assessed for its in vitro antioxidant properties and sun protection
factor (SPF).
Results: Phytochemical characterization highlighted the richness of the aqueous extract in polyphenols. LC-MS
analysis permitted the identification of 20 phenolic compounds. Catechin, procyanidin B2, dihydromyricetin
and syringetin-3-O-rutinoside were the most predominant components (19.39%, 16.04%, 12.85% and 14.28%
respectively). UV spectrophotometric analyses showed that CE extract absorbed in the UV region (190–400 nm).
C.E crude extract exhibited, at the concentration of 1000 μg/ml, a high photoprotective effect with a SPF value
of 6.996 ± 0.174 corresponding to a percent of UV blockage ranging from 80 to 90%. Besides, when it was
incorporated into an O/W emulsion at a concentration of 10% (m/m), it presented a good antioxidant activity
(DPPH inhibition (%) ≈ 70%) and an in vitro SPF value of 1.626 ± 0.466 corresponding to a percent of UV
blockage ≈50%.
Conclusion: These results suggest that Carpobrotus edulis L. could be a good topical photoprotective agent.

1. Introduction
Nowadays, sun exposure has increased due to changes in lifestyle.
The electromagnetic radiations provided by the sun have a wide spec-
trum of wavelengths including the ultraviolet (UV) region (100- 400 nm)
[1,2]. An overexposure to UV-B (290 – 320 nm) and UV-A (320 -
400 nm) lights without suitable protection may cause, in human skin,
a variety of undesirable effects such as oxidative stress induction, im-
mune response alteration, erythema, keratosis, sunburn reaction, pre-
mature skin aging and, in extreme cases, carcinogenesis. These acute and
chronic reactions are mediated by the generation of reactive oxygen and
nitrogen species (ROS and RNS, respectively), singlet oxygen and free
radicals, which damage lipids, proteins, nucleic acids (DNA, RNA) and
others cutaneous cell components; and by an impairment or an overflow
of biological antioxidant systems (endogenous enzymatic and nonen-
zymatic antioxidants) [3-6]. Various strategies can protect skin from
UV-induced damage including the use of broad-spectrum sunscreens.
Sunscreen agents such as zinc oxide or titanium dioxide (physical sun-
screens) act by reflecting UV radiation away from the skin. Chemical
sunscreens such as methylene bis-benzotriazolyl tetramethylbutylphe-

Abbreviations: UV, Ultraviolet; ROS, Reactive oxygen species; RNS, Reactive nitrogen species; C.E, Carpobrotus edulis; FT-IR, Fourier-transform infrared; LC-MS,
Liquid chromatography–mass spectrometry; O/W, Oil-in-water; SFP, Sun protection factor; TPC, Total phenols content; GAE, Gallic acid equivalent; TFC, Total
flavonoid content; QE, Quercetin equivalent; PAD, photodiode array detector; DPPH, 2,2-diphenyl-1-picrylhydrazyl.
∗
Corresponding author: Faculty of Pharmacy of Monastir, Laboratory of Pharmaceutical, Chemical and Pharmacological Drug Development LR12ES09, Rue
Avicenne, 5000 Monastir, Tunisia.
E-mail addresses: lassoued98@yahoo.fr (M.A. Lassoued), nour.benfatma@gmail.com (N.E.H. Ben Fatma), maryem.hajromdhane92@gmail.com (M. Haj Romd-
hane), adelfaidi_mima@yahoo.fr (A. Faidi), hatemmajdoub.fsm@gmail.com (H. Majdoub), souad.sfar@laposte.net (S. Sfar).

https://doi.org/10.1016/j.eujim.2021.101286
Received 1 September 2020; Received in revised form 6 January 2021; Accepted 8 January 2021
1876-3820/© 2021 Elsevier GmbH. All rights reserved.
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al.
nol or isoamyl 4-methoxycinnamate act by absorbing UV rays. However,
these synthetic agents may interact with cutaneous cells causing various
skin effects such as contact dermatitis, photo-irritation or photosensitiv-
ity reactions [4]. For these reasons, in recent years, there has been an
increasing interest in finding natural compounds from plants such as
herbal oils, plant extracts, etc. as photoprotective agents [4,7,8]. Carpo-
brotus edulis L. specie (Order: Caryophyllales, Family: Aizoaceae), also
known as “sea fig”, is a medicinal and an edible ground creeping halo-
phyte plant growing, mainly, in sandy coastal areas [9-11]. In Tunisia,
its leaves were used, in traditional medicine, for their wound healing
properties and for treating warts [9]. Moreover, several published stud-
ies reported that this specie has several in vitro biological activities such
as antioxidant, antibacterial, anti-tumoral, anticholinestrease and anti-
inflammatory properties [12-16]. This halophyte plant is known to be
rich in phenolic compounds. Natural polyphenols, applied topically,
have shown promising photoprotective effects against photo-damage
and inflammatory response produced by UV irradiation [4,17-19]. In
the present study, we investigated the potential photoprotective effect
of Carpobrotus edulis L. (C.E) aqueous leaf extract, collected from a
Tunisian biotope. The phytochemical characterization of the plant mate-
rial was carried out by Fourier-transform infrared spectroscopy (FT-IR),
by spectrophotometric methods (total contents of polyphenols (TPC)
and flavonoids (TFC)) and by Liquid chromatography–mass spectrom-
etry (LC-MS). The plant extract was, then, incorporated into an oil-in-
water (O/W) emulsion and its effects on the physicochemical proper-
ties of the formulation (pH, viscosity and spreadability) were studied.
Finally, its in vitro antioxidant properties and photoprotective activity
(UV spectrum analysis, sun protection factor (SPF)) were evaluated.
2. Material and methods
2.1. Chemical reagents
Extra virgin olive oil was purchased from the local market. POE
(20) sorbitan monostearate (HLB=14.9) was purchased from PROLABO
(France). Sorbitan monostearate (HLB=4.7) and Sepicide® HB were ob-
tained from SEPPIC (France). Glycerin purified was purchased from CDH
(India). Cetyl alcohol was purchased from AppliChem (Germany). Par-
sol® MCX (Octyl methoxycinnamate) was purchased from ROCHE Inc
(Switzerland). Ethanol absolute was purchased from LOBA Chemie (In-
dia).
2.2. Plant material
Carpobrotus edulis L. (C.E) was collected from its natural biotope
(Falaise, Monastir; Tunisia) in January 2018. The plant material was
identified by Dr. Amer Elaissi, Ph.D. (Department of the botany, Fac-
ulty of Pharmacy of Monastir, Tunisia). A voucher specimen (No. 0241)
was deposited at the herbarium of this Department.
2.3. Preparation of the extract
C.E aqueous leaf extract was prepared according to the protocol de-
scribed by Bouftira et al. [12]: Fresh leaves were well rinsed with dis-
tilled water, cut into small pieces, boiled in distilled water for 15 min
followed by filtration using a filter paper (Munktell® 3 m/N, Germany).
The obtained filtrate was, then, lyophilized (FLEXI-DRY® Freeze Dryer,
USA) and stored at −10 °C, in a hermetically sealed glass bottle, until
further use. The ratio of C.E leaf to water, for decoction, was 2:1 (w/v).
Extraction yield (%) was 0.965%.
2.4. Fourier transform infrared spectroscopy analysis
Fourier transform infrared spectrum of C.E leaf extract was obtained
on a Shimadzu 8400 FT-IR Spectrophotometer. Data were collected over
a spectral region from 4000 to 400 cm−1 with a resolution of 4 cm−1 .
2
European Journal of Integrative Medicine 42 (2021) 101286
Data analysis was performed using Hyper® 1.57 software (Shimadzu
Corporation, Japan).
2.5. Polyphenolic compounds analysis
2.5.1. Determination of total phenolic content
Total phenols content (TPC) was determined spectrophotometri-
cally (Evolution® 60 UV-Visible spectrophotometer, USA), according
to Folin-Ciocalteu method, using gallic acid as standard [20]. 0.5 ml
of extract stock solution (1 mg/ml) were mixed with 2.5 ml of 10%
Folin-Ciocalteu reagent and 2.5 ml of 7.5% NaHCO3 solution. Next, the
mixture was kept for 45 min at 45 °C before measuring absorbance at
765 nm. The experiment was performed in triplicate (n = 3). Results, ex-
pressed as μg gallic acid equivalent per ml of sample (μg GAE/ml), were
determined from a calibration curve of gallic acid (calibration curve
equation: y = 8.6231x – 0.0197, r2 = 0.9988).
2.5.2. Determination of total flavonoid content
Total flavonoid content (TFC) was assessed according to Lamaison
and Carnat method (flavonoids-aluminium’s complex formation), us-
ing quercetin as standard [21]. 2 ml aliquots of extract stock solution
(1 mg/ml) were mixed with 2 ml of 2% AlCl3 solution. Next, absorbance
was measured at 430 nm (Evolution® 60 UV-Visible spectrophotome-
ter, USA), after 10 min of incubation at room temperature. Experiment
was repeated 3 times (n = 3). Results, expressed as μg quercetin equiv-
alent per ml of sample (μg QE/ml), were determined from a calibration
curve of quercetin (calibration curve equation: y = 0.045x – 0.0328;
r2 = 0.9994).
2.5.3. Characterization of phenolic compounds by LC-MS
The aqueous extract (5 mg/ml in distilled water) was analyzed us-
ing a Shimadzu Europe - LCMS-2010EV system (Germany) equipped
with an auto-sampler (kept at 5 °C), a degasser, a quaternary pump,
a photodiode array detector (PAD) coupled to an electrospray ioniza-
tion mass detector (LC-DAD-ESI/MS) and an automated thermostatted
column compartment. Chromatographic separation was achieved with
a Waters Spherisorb S3 ODS-2C18 3 𝜇m (4.6 mm × 150 mm) column
thermostatted at 35 °C. The solvents used were: (A) Methanol, (B) 0.1%
formic acid in water. The elution gradient established was isocratic 15%
A for 5 min, 15–30% A over 5 min, 30–50% A over 5 min, 50–60% B
over 5 min, 60–70% A over 5 min, 70–80% A over 5 min, 80–90% A
over 5 min, 90–50% A over 5 min and re-equilibration of the column,
using a flow rate of 0.5 ml/min. Detection was carried out in the PAD
using 280 nm as preferred wavelength and in mass spectrometer (MS).
The injection volume was 50 μl. MS detection was performed in negative
mode, using a Shimadzu Europe - LCMS-2010EV system equipped with
an electrospray ionization (ESI) source. Nitrogen served as the sheath
gas (50 psi); the system was operated with a spray voltage of 5 kV, a
source temperature of 325 °C and a capillary voltage of −20 V. The tube
lens offset was kept at a voltage of - 66 V. The full scan covered the mass
range from m/z 100–1500. The collision energy used was 35 (arbitrary
units). Data acquisition was carried out with Xcalibur® data system.
Phenolic compounds were identified according to their retention times,
mass spectra and UV–Vis spectra compared with standards when avail-
able, or, were tentatively identified by comparing the obtained results
with available data reported in the literature.
2.6. Preparation of O/W emulsions
2.6.1. Preparation of Carpobrotus edulis L. leaf extract formulations
Qualitative and quantitative compositions of the cream base (0% of
C.E leaf extract (F1 )) and the formulations containing various plant ex-
tract concentrations (2% and 10% of plant extract for F2 and F3 , re-
spectively) are reported in Table 1. O/W emulsions were prepared as
follow: Both phases A (oil phase) and B (aqueous phase) were heated
separately to 75 °C under gentle stirring (250 rpm). Next, the aqueous
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al. European Journal of Integrative Medicine 42 (2021) 101286

Table 1
Qualitative and quantitative compositions of cream formulations containing various
concentrations of Carpobrotus Edulis L. extract.

Amount (%)
Compound F1 F2 F3 Reference

Phase Olive oil 15 15 15 12.3

A Cetyl alcohol 5.5 5.5 5.5 5.5
Octyl methoxycinnamate 0 0 0 2.7
Sorbitan monostearate 2.75 2.75 2.75 2.75
Phase POE (20) sorbitan monostearate 2.25 2.25 2.25 2.25
B Glycerin 5 5 5 5
Carpobrotus Edulis extract 0 2 10 0
Water up to 100
Phase C Sepicide HB 0.5

phase was added to the oil phase at a stirring rate of 500 rpm and mixing
was maintained for 10 min. During this step, the temperature was kept
at 75 °C. Next, the formed emulsion was passed through a homogenizer
(ERWEKA® HO, Germany) and kept under stirring until cooling to room
temperature. Sepicide® HB (preservative) (phase C) was added at the
end when temperature was within 40 °C. Stirring was, then, maintained
for 5 more minutes.
2.6.2. Preparation of the reference Octyl methoxycinnamate (2.7%) as the
standard sunscreen
A reference formulation (Table 1) containing 2.7% of Octyl
methoxycinnamate (UV-B filter) was realized according to the same pro-
cedure described in Section 2.6.1. It was prepared to ensure the repro-
ducibility of results in SPF determination. Expected SPF value of the
reference formulation is 4 [22].
2.7. Characterization of formulated creams
Cream formulations with and without 2% of CE extract (F1 and F2 ,
respectively) were evaluated for their pH, viscosity and spreadability
in the aim to assess the plant extract effect on these physicochemical
properties.
2.7.1. pH
1 g of sample was dispersed in 9 ml of distilled water and pH mea-
surements were carried out at room temperature. Results were expressed
as mean ± standard deviation (s.d.) of 6 repetitions (n = 6).
2.7.2. Apparent viscosity
Apparent viscosity was determined, at room temperature, using
Brookfield® DV-III Ultra Rheometer (USA) equipped with an RV5 and
RV2 spindle for F1 and F2 , respectively. Measurements were obtained
at a spindle speed rate of 1 rpm. Results (n = 6) were expressed, in cP,
as mean ± s.d.
2.8. Antioxidant activity of cream formulations by DPPH radical
scavenging
1 g of each cream formulation was weighted, transferred to 100 ml
volumetric flask and diluted to volume with ethanol absolute for F1 or
ethanol-water mixture (40:60 v/v) for F2 and F3 . Next, samples were,
first, vortexed for 1 min, then kept for ultrasonication for 10 min (Pro-
labo® Transsonic 275, T460 model, 35 khz, Germany) and finally mixed
using a magnetic stirring at 500 rpm for 10 min. Afterward, they were
filtered through a pleated filter paper (Munktell® 3 m/N, Germany).
Free radical scavenging activity was measured from the bleaching
of purple-coloured ethanol solution of 2,2-diphenyl-1-picrylhydrazyl
(DPPH), a stable free radical, according to Bouftira et al. [24]. 1 ml
of each sample was mixed with 1 ml of DPPH ethanolic solution and in-
cubated in darkness at room temperature for 30 min. Absorbance values
of the different samples were determined at 517 nm (Evolution® 60 UV-
Visible spectrophotometer, USA) against a blank solution containing a
mixture of 1 ml of DPPH solution and 1 ml of ethanol. DPPH quenching
ability was determined according to Falleh et al. [25] as follow:
Ablank − Aassay
%DPPH inhibition = × 100
Ablank
Where: A blank and A assay are the absorbance values, at 517 nm, of
the blank and the samples solutions, respectively.
All measurements were repeated in triplicate (n = 3) and results
were expressed as percentage inhibition of DPPH ± s.d. ANOVA test
and Fisher’s least significant difference (LSD) multiple range test at 95%
were applied to determine significant differences between groups. Stat-
graphics Centurion XV® software (StatPoint, version 15.1.02) was used
for statistical analysis.
2.9. In vitro photoprotective assessment
2.9.1. UV spectrophotometric analyses
Absorbances of C.E crude extract in water were measured with Evo-
lution® 201 UV-Visible spectrophotometer (USA). Scans were realized
in the UV region (190–400 nm) after appropriate dilution of the samples.

2.7.3. Spreadability 2.9.2. In vitro SPF determination

Spreadability was evaluated at room temperature using the scanned
image analysis method developed by Rigo et al. [23]. A total of 20 wt
plates were used. Scanned images were obtained by a HP® Deskjet 1050
scanner (China) and pictures were treated using ImageJ® software (ver-
sion 1.50i, National Institutes of Health, USA). Spreadability factor (Sf ),
expressed in mm2 .g−1 , was calculated as follow:
𝐴
𝑆𝑓 =
𝑊
where: A (mm2 ) corresponds to the maximum spread area after addition
of all the plate weights and W (g) corresponds to the total weight added
in the experiment. Results were expressed as mean ± s.d. of 3 repetitions
(n = 3).
2.9.2.1. Carpobrotus edulis L. crude extract. Lyophilized crude extract
was dissolved in water to a final concentration of 200 and 1000 μg/ml.
Afterward, the samples were assayed spectrophotometrically (Evolu-
tion® 201 UV-Visible spectrophotometer, USA) by measuring the ab-
sorbance in UV-B wavelength range (290–320 nm) with 5-nm incre-
ments.
In vitro SPF values were calculated by applying Mansur et al. equa-
tion [26] and results were expressed as mean ± s.d of 6 repetitions
(n = 6).
∑320
SPF = CF x EE(𝜆) x I(𝜆) x Abs(𝜆)
290
Where: EE (𝜆) corresponds to the erythemogenic effect of radiation at
𝜆; I (𝜆) is the intensity of solar light at 𝜆; Abs (𝜆) is the absorbance of the

3
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al.
Table 2
Normalized EE (𝜆) × I (𝜆) values used
in SPF calculation [27].
𝜆 (nm) EE (𝜆) × I (𝜆) (normalized)
290 0.015
295 0.0817
300 0.2874
305 0.3278
310 0.1864
315 0.0837
320 0.018
Total 1 3.3. Physicochemical parameters of cream formulations
preparation at 𝜆 and CF represents the correction factor determined so
that the reference formulation containing 2.7% octyl methoxycinnamate
presented a SPF value of 4. EE(𝜆) × I(𝜆) values are reported in Table 2
[26].
2.9.2.2. O/W cream formulations. 100 mg of cream samples were
weighted in test tubes, added with 10 ml of absolute ethanol for F1
and the reference formulation (containing 2.7% of Octyl methoxycin-
namate) or ethanol-water mixture (40:60 v/v) for F2 and F3 and vor-
texed for 1 min. Next, samples were ultrasonicated for 10 min, vor-
texed for 1 min and filtered through a pleated filter paper (Munk-
tell® 3 m/N, Germany). Afterward, they were appropriately di-
luted using the same solvent for their preparation and assayed spec-
trophotometrically (Evolution® 201 UV-Visible spectrophotometer,
USA).
Absorbance measurements as well as SPF values of each cream sam-
ple were determined as described above (Section 2.9.2.1.). SPF results
corresponded to mean ± s.d. of 6 determinations.
2.10. Statistical analysis
Statistical analysis was computed with Microsoft Office® Excel 2007
software. Student’s t-test was used to compare average values for F1
versus F2 formulations. Differences observed were considered significant
at p ≤ 0.05.
3. Results
3.1. Infrared spectroscopy analysis
C.E leaf extract functional groups were determined by FT-IR spec-
troscopy. The main vibrations detected in the FT-IR spectrum of the
plant extract were as follow (Fig. 1): a broad and intense absorp-
tion band due to O–H stretching vibrations at 3324 cm−1, an ab-
sorption band due to C = O stretching vibrations at 1606 cm−1 , an
absorption band due to O–H bending vibrations at 1434 cm−1 and
two absorption bands due to C–O stretching vibrations at 1249 and
1067 cm−1 .
3.2. Phytochemical analysis of Carpobrotus edulis L. aqueous leaf extract
Phytochemical studies of C.E extract included the determination of
total phenolic content (TPC) and total flavonoid content (TFC), as well
as an LC-MS analysis.
According to the linear regression line equations of the calibration
curves of gallic acid and quercetin, total phenol and total flavonoid
concentrations were found to be 81.84 ± 27.92 μg of GAE / ml and
3.07 ± 0.51 μg of QE / ml of extract, respectively.
The investigation of the chromatographic profile of C.E aqueous ex-
tract (Fig. 2) and the mass spectrum data revealed that this plant is rich
in phenolic compounds. These later were identified by comparing their
retention time, mass spectra, and UV spectra with those obtained from
4
European Journal of Integrative Medicine 42 (2021) 101286
standard compounds. Moreover, compounds were tentatively charac-
terized comparing the obtained information with data reported in lit-
erature. In the current study, 21 phenolic compounds were recorded
but only 20 compounds were successfully identified (Table 3). The de-
tected phenolic compounds in C.E aqueous extract belong mostly to the
group of flavonoids with catechin, procyanidin B2 and dihydromyricetin
being the most predominant flavonoids (19.39, 16.04 and 12.85%, re-
spectively), followed by the group of flavonoid-O-glycosides, essentially
syringetin-3-O-rutinoside (14.28%).
3.3.1. pH measurements
pH measurements were done after appropriate dilution of samples
with distilled water. pH values of F1 and F2 formulations were found
6.50 ± 0.20 and 5.61 ± 0.17, respectively. The presence of C.E leaf
extract in the formulation led to a significant decrease in the pH value
in comparison with the cream base (p < 0.05).
3.3.2. Apparent viscosity
Apparent viscosity, expressed in cP, was assessed using a Brook-
field® DV-III Ultra Rheometer equipped with an RV5 (for F1 ) and RV2
(for F2 ) spindles at a speed rate of 1 rpm. Apparent viscosity values were
found 43.792 × 103 ± 5.751 × 103 cP and 14.770 × 103 ± 0.515 × 103 cP
for F1 and F2 respectively. The addition of C.E extract (F2 ) led to a sta-
tistically significant decrease in apparent viscosity value in comparison
with the cream base (F1 ) (p < 0.05).
3.3.3. Spreadability
The scanned image analysis method developed by Rigo et al.
[23] was used to evaluate the spreadability of the different formulations.
Experiments were performed at room temperature. The formulation F2
showed a higher spreadability, and thus an easier skin application, than
the formulation F1 with a calculated spreadability factor (Sf ) equal to
3.557 ± 0.464 mm2 .g − 1 and 2.169 ± 0.098 mm2 .g − 1 , respectively.
3.4. Anti-radical activity of formulated creams
The results of DPPH free-radical scavenging activity of the cream
base (F1 ) and of those containing 2% and 10% of C.E leaf extract (F2
and F3 , respectively) are reported in Fig. 3. DPPH inhibition percentage
was found 3.05 ± 0.82%, 24.11 ± 3.71% and 69.02 ± 2.81% for F1 ,
F2 and F3 , respectively. According to these results, there is a statisti-
cally significant difference in DPPH inhibition percentage between the
three formulations (p < 0.05). The cream base (F1 ) showed a very low
anti-radical activity, whereas F3 (10% of Carpobrotus edulis L. leaf ex-
tract) exhibited the highest percent of inhibition in comparison to other
formulations.
3.5. In vitro photoprotective assessment: SPF determination
To be effective in preventing UV-induced skin damage, C.E extract
should have a wide range of absorbance in the UV region, viz, between
290 nm and 400 nm. For this purpose, both UV spectrum analysis and
sun protection factor (SPF) estimation were conducted.
UV spectrophotometric analyses in UV region (Fig. 4) indicated that
C.E aqueous leaf extract absorbs in the UV-C (190–290 nm), UV-B (290–
320 nm) and UV-A (320–400 nm) ranges. This result suggests that C.E
may provide protection against both UV-B and UV-A radiations with a
good score.
In vitro SPF values were determined spectrophotometrically. SPF
value of the reference formulation (2.7% OMC) was 3.664 ± 0.320
which corresponds to 91.6% when compared to the expected SPF (=
4). This result indicates the good reliability of this in vitro method to
measure SPF. The correction factor (C.F), calculated, on the basis of the
standard sunscreen result, by applying Mansur et al. equation [26] is
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al.
Fig. 2. Chromatographic profile of Carpobrotus Edulis L. aqueous leaves extract, at 280 nm, analyzed by HPLC-MS in negative ionization mode. The numbers
correspond to the compounds listed in Table 3.
10.98. This latter was used to determine the SPF values of C.E. crude
extract samples (200 and 1000 μg/ml) as well as those of F1 , F2 and F3
formulations.
SPF values of C.E crude extract were 1.426 ± 0.031 and
6.996 ± 0.174, for a concentration of 200 and 1000 μg/ml, re-
spectively. Cream base formulation (F1 ) exhibited the lowest SFP
value (0.206 ± 0.026) when compared to the others formulations
(0.646 ± 0.151 and 1.626 ± 0.466 for F2 and F3 , respectively). The
highest value was obtained when 10% (m/m) of plant extract was in-
corporated.
5
European Journal of Integrative Medicine 42 (2021) 101286
Fig. 1. FT-IR spectrum of aqueous extract of Carpobrotus
Edulis L. leaves.
4. Discussion
This study aimed to assess the potential photoprotective activ-
ity of Carpobrotus edulis L. (CE) aqueous leaf extract which could
justify its use as ingredient for pharmaceutical formulations. This
work was divided, mainly, into two parts, viz, 1) a phytochemical
characterization of CE aqueous leaf extract by quantification of to-
tal polyphenols and flavonoids contents (TPC and TPC, respectively)
as well as by FT-IR and LC-MS analysis; and 2) the assessment of
its in vitro antioxidant properties (DPPH inhibition percent) and pho-
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al. European Journal of Integrative Medicine 42 (2021) 101286

Table 3
Tentative identification of bioactive compounds detected in Carpobrotus Edulis L. leaves aqueous extract obtained using LC-MS in negative
ionization mode, according to their retention times (Rt ), wavelengths of maximum absorption (𝜆max ) and mass spectral data.

Peak Rt (min) 𝜆max (nm) [M-H]− m/z Tentative identification Classification Identified compound (%)

1 3.20 264 329 Vanillic acid-glucoside Hydrolyzable tannins 0.64

2 3.35 264 533 Kaempferol 3-O-(6″-malonyl-glucoside) Flavonoid-O-glycosides 4.75
3 4.75 – 391 not identified ——————————- 0.47
4 5.08 257 443 Rothindin Flavonoid glucosides 0.95
5 5.50 274 685 Niazinin A Thiocarbamate glycosides 0.19
6 6.28 257 475 Diosmetin-7-O-glucuronide Flavonoid-O- glycosides 0.95
7 6.52 248 613 Catechin-di-O-glucoside Flavonoid O-glycosides 1.66
8 6.73 276 455 Epicatechin 3-(3-methylgallate) Flavonoids 0.24
9 10.25 277 315 Quercetin 3-O-methyl-ether Methylated flavonoids 0.47
10 12.50 278 577 Procyanidin B2 Flavonoids 4.04
11 13.03 279 491 Isorhamnetin 4′-glucuronide Flavonoid glycosides 6.42
12 14.08 279 289 (+) – Catechin Flavonoids 9.16
13 14.59 279 577 Procyanidin B2 Flavonoids 12.00
14 15.23 279 319 (±) - Dihydromyricetin Flavonoids 12.85
15 16.42 279 289 (+) – Catechin Flavonoids 10.23
16 17.37 279 413 Asperuloside Monoterpenoid glycosides 6.66
17 19.63 350 623 Isorhamnetin-O-rutinoside Flavonoid O-glycosides 3.80
18 20.05 350 623 Isorhamnetin-O-rutinoside Flavonoid O-glycosides 4.52
19 20.62 349 639 Laricitrin-3-O-rutinose Flavonoid O-glycosides 3.34
20 21.99 351 653 Syringetin-3-O-rutinoside Flavonoid O-glycosides 14.28
21 22.53 349 507 Syringetin-O-glucoside Flavonoid O-glycosides 2.38
Σ=100%

[14,25,31,32] conducted several studies to assess C.E phytochemical

Fig. 3. Graphic representation of the antiradical activity, measured by the per-
centage of DPPH inhibition, for F1 (formulation without Carpobrotus Edulis L.
extract), F2 (formulation containing 2% of plant leaf extract) and F3 (formula-
tion containing 10% of plant leaf extract). Experiments were repeated in trip-
licate (n = 3) and results are expressed as mean ± S.D. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article.)
toprotective effect (UV spectrum analysis and sun protection factor
estimation).
Phytochemical analysis results (TPC, TFC and LC-MS) revealed the
richness of C.E leaf extract in phenolic compounds. Catechin, procyani-
din B2, dihydromyricetin and syringetin-3-O-rutinoside were the most
predominant components among the 20 phenolic compounds identi-
fied by LC-MS analysis. Catechin (peaks 12 and 15), which belongs to
the family of flavonoids (group of flavan-3-ol), represents 19.39% from
the total phenolic composition. This compound is known for its anti-
aging potential, as well as preventing and repairing of damaged skin
[27]. Besides, catechin is widely used as a positive standard in differ-
ent antioxidant bioassays [28]. Peaks 10 and 13, which were assigned
to procyanidin B2, represents 16.04% of the total polyphenol composi-
tion. This latter compound possesses strong antioxidant properties to-
wards Lipoperoxidation and Anti-Elastase Activity [29]. Syringetin-3-
O-rutinoside (14.28% of the total phenolic composition of C.E aqueous
extract), which belongs to flavonol group, has shown a protective effect
of the skin cells against oxidative stress and cellular aging [30].
When comparing our results to those reported in literature,
qualitative and quantitative differences were noticed. Falleh et al.
composition from the region of Jerba (south of Tunisia, arid bioclimatic
stage). Methanolic extracts analysis showed qualitative and quantita-
tive variation as function of used plant organs (shoots, roots, leaves and
stems) and harvest period (spring, summer, autumn and winter). TPC
ranged from 68.75 ± 1.07 mg GAE/g dry weight (DW) to 122.3 ± 6.4 mg
GAE/g DW and TFC varied from 22.21 ± 0.45 mg Catechin equivalent
(CE)/g DW to 66.0 ± 2.1 mg CE/g DW. Phloretin, Quercitrin, Avicularin,
(+)-Catechin, Procyanidin B2, (-)-Epicatechin, reyotrin and hydroxycin-
namic acid (in shoots methanolic extract) and Procyanidin B2 and (-)-
Epicatechin (in roots methanolic extract) were the major phenolic com-
pounds identified. The same authors [31,32] studied the phytochemical
composition of C.E shoots from Monastir (center of Tunisia, superior
semi-arid bioclimatic stage) and from Bizerte (North of Tunisia, humid
bioclimatic stage). Harvest periods were summer and spring, for Monas-
tir and Bizerte, respectively. For material plant harvested from Monastir,
TPC and TFC were 48.5 ± 0.4 mg GAE/g DW and 35.7 ± 0.7 mg CE/g
DW, respectively. Quercitrin, avicularin, phloretin were the main phe-
nolic compounds found in the methanolic extract for the plant collected
from the region of Monastir, meanwhile, avicularin, isorhamnoside ruti-
nol, Reyotrin, Catechin and epicatechin were the major ones identified
from shoots collected from the region of Bizerte. Hafsa et al. [33] de-
termined the phytochemical content of C.E aqueous (AQe) and ethanol-
water (EWe) leaf extracts harvested from the region of Sousse (center of
Tunisia, superior semi-arid bioclimatic stage). TPC was much higher in
hydroalcoholic extract compared to the aqueous extract (66.3 ± 6.2 mg
GAE/g DW and 151.9 ± 4.1 mg GAE/g DW for AQe and EWe, respec-
tively). Same finding was noticed for TFC (21.9 ± 1.7 mg Quercetin
equivalent (QE)/g DW and 38.84 ± 0.8 mg QE/g DW for AQe and EWe,
respectively). HPLC analysis showed the presence of seven major com-
pounds, including, isoquercitin (6.442 and 3.963 mg/g residue for EWe
and AQe, respectively), luteolin7-O-glucoside (4.013 and 3.741 mg/g
residue for EWe and AQe, respectively) and ferulic acid (1.556 and
1.580 mg/g residue for EWe and AQe, respectively). Finally, a HPLC
analysis, conducted by Meddeb et al. [34], on water-acetone extracts
of C.E leaves harvested from Monastir (Collection period: spring) high-
lighted the presence of 4 major phenolic compounds, viz, Chlorogenic
acid, Catechin, 1,3-Dicaffeoylquinic acid and Gallic acid. TPC was found
184 ± 5 mg GAE/100 g of fresh matter.
These differences, in terms of polyphenols composition, may be at-
tributed to several factors such as environmental conditions (Bioclimatic

6
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al.
stage; temperature, solar radiation, water stress, soil factors, viz, type,
chemistry, pH, salinity and nutritional status; altitude; harvest period;
etc.), biological conditions (phases of growth; plant organ, viz, roots,
stems, leaves, flowers, etc.) and technical factors (extraction methods,
solvents used, etc.) [31,35-37]].
In order to assess its effectiveness as topical photoprotective agent,
C.E aqueous extract was incorporated into a cream formulation at dif-
ferent concentrations (2% and 10%). The physicochemical character-
istics (pH, apparent viscosity and spreadability) of the formulations
without (F1) and with C.E extract (F2 and F3) were explored in order
to evaluate the effect of the added plant extract on those properties.
pH determination was preceded by a dilution of the samples to one-
tenth (1:10 dilution) of their original concentration with distilled wa-
ter. As reported in literature, such dilution factor does not affect mea-
7
European Journal of Integrative Medicine 42 (2021) 101286
Fig. 4. Ultraviolet absorption spectra for Carpobro-
tus Edulis L. aqueous leaf extract in UV region (190–
400 nm): (A) UV-C (𝜆 = 190 – 290 nm), (B) UV-B+UV-A
(𝜆 = 290 – 400 nm) absorption bands.
surements [38]. According to the results, pH value of the cream for-
mulation decreased significantly in presence of C.E extract. This varia-
tion may be attributed to the chemical composition of the added aque-
ous extract. However, the obtained value remained within the range
of the physiological skin pH, which is acidic (pH ranging from 4 to 6)
[39].
Viscosity is an important physical property to assess when topical
formulations are prepared since it plays a key role on their use (ease of
application, uniformity of spreading, maintaining on the skin after ap-
plication); on their stability (high viscosity interferes with the migration
of the oil droplets at the top of the emulsion under the influence of the
density difference between the two phases); on the applied dose amount;
and on the delivery of the active ingredient to the skin, its efficiency and
its transdermal diffusion capacity [38,40].
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al.
In this study, the addition of the extract led to a decrease of the vis-
cosity of the cream when compared to the cream base. This finding is in
good accordance with the literature. In fact, previous studies reported
that the addition of plant extract, generally, affect the formulation con-
sistency. This phenomenon can be attributed to its chemical composition
which may interact with the thickener and causes a potential internal
disarrangement of the emulsion or to its viscosity since the addition of
a plant extract to the continuous phase could result in a system dilution
and likely to a destabilization of the emulsion structure [24,41,42].
Spreadability, which indicates the area surface on which a semi-solid
formulation will expand after a specified time, is closely related to the
qualitative and quantitative composition of the formulation and to its
viscosity [43]. This parameter has a determining influence on both user
acceptance and application efficiency of the product since a poor spread-
ability can lead to an uneven distribution of the topical formulation and
be perceived as weakness by the consumer [38,43]. Spreadability was
assessed using the scanned image analysis method [23]. The addition of
C.E extract resulted in a significant increase of the spreadability value of
the formulation in comparison with the cream base. This phenomenon
could be explained by the difference in viscosity between F1 and F2 .
The lower is the viscosity the higher is the spreadability [44]. Hence,
the chemical composition of an added extract needs to be taken into
consideration when formulating a preparation for a topical use to get
the expected physicochemical parameters.
In vitro anti-radical activity of formulated creams (F1 to F3 ) was as-
sessed by DPPH radical scavenging method. DPPH assay is considered
as a simple, rapid, economic and reliable method to estimate antirad-
ical properties of both hydrophilic and hydrophobic compounds since
it may be used in different solvent systems (aqueous and nonpolar or-
ganic solvents). Besides, free radical scavenging activity is determined
at room temperature which avoids thermal degradation of tested com-
pounds [45].
Antioxidant compounds protect against oxidative damages mediated
by free radicals, including, reactive oxygen and nitrogen species (ROS
and RNS, respectively). These latter are products of cellular metabolism
(metabolic processes in biological systems) and environmental oxidative
stress [45-47]. Elevated intracellular levels of reactive species can alter
biomolecules (lipids, proteins and DNA) structure which may lead to
cellular dysfunction. Such deleterious changes are linked to a myriad
of health disorders including immune response alteration, premature
aging, cardiovascular diseases, mutagenesis and carcinogenesis [6,45-
47].
According to the obtained results, F1 (cream base) showed the lowest
DPPH activity which is most probably due to the presence of olive oil in
the formulation. In fact, in a previous study, Valavanidis et al. [48] re-
ported the radical scavenging activity of olive oil which was explained
by the presence of components with high antioxidant potential such as
phenolic compounds. The authors found that the amount of oil sample
that can reduce DPPH activity by 50% (IC50 ) was about 11 ± 0.6 mg/ml
and 17.5 ± 1.2 mg/ml for extra virgin olive oil and commercial olive
oil, respectively [48].
The formulation containing the highest amount of C.E extract (F3 )
presented the greatest activity in comparison to the two other formu-
lations. This may be explained by the fact that an increase in plant ex-
tract concentration led to an entrapment of more free radicals formed
by DPPH and, thus, to a greater anti-radical activity. DPPH radical scav-
enging capacity of C.E may be explained by its richness in phenolics
compounds, particularly catechin and procyanidin B2, which are strong
free radical scavengers that act as electron donors [25,32,49]. The other
identified compounds, in C.E aqueous extract, should not be neglected
since synergy between the phenolic compounds could affect the global
biological activity of plant organs [50].
Sunscreen formulations must protect the skin against the harmful
effects of both short and long wavelength UV radiations (UV-B+UV-A)
and, thus, should cover a board-spectrum, viz, between 290 nm and
400 nm. UV-B radiation is 1000 times capable of causing actinic ery-
8
European Journal of Integrative Medicine 42 (2021) 101286
thema than UVA [4]. Several in vivo and in vitro methods were developed
for measurement of the UV protection factor. In vivo SPF measurement
on volunteers involves the exposure to source of radiation which may
present risks to human subjects, are tedious, time consuming process
and present ethical issues. In comparison, in vitro tests are easy-to-use,
quick, simple and economical methods which can be considered as a
useful tool at development stage of a novel product as a supplement of
the measurement of in vivo SPF. They, also, permit to reduce the number
of in vivo experiments [8,51,52].
According to the obtained SPF values, C.E crude extract exhibited a
high photoprotective effect, at the concentration of 1000 μg/ml, corre-
sponding to a percent of UV blockage ranging from 80 to 90% [8]. F3
formulation showed a SPF value near 2 which corresponds to a percent
of UV blockage around 50% [8]. These results suggest that C.E aqueous
leaf extract possesses an interesting photoprotective effect which is di-
rectly proportional to its concentration. This activity may be explained
by its richness in phenolic compounds (mainly flavonoids). In fact, sev-
eral studies highlighted the multiple functions of natural flavonoids in
photoprotection and it has been reported that, generally, the efficiency
of these compounds, as photoprotector, was related to their abundance
in the plant material [4,53-55].
5. Limitations
In this current study, C.E aqueous extract was obtained by a decoc-
tion of a fresh matter for 20 min. It is well known that experimental con-
ditions (methods of extraction, temperature, nature (polar and nonpo-
lar) and concentration of solvent, acidification, etc.) may have an impact
on qualitative and quantitative phytocomposition of an extract. Hence,
a potential limitation of our work is the extraction process. Indeed, in
a previous published study [33], the authors prepared an aqueous ex-
tract of C.E using a dried leaf powder as matter. The extraction was
realized under magnetic stirring for 30 min at 60 °C. They found a total
flavonoid content (TFC) (21.9 ± 1.7 μg of quercetin/mg of dry extract)
higher than that obtained in our case (3.07 ± 0.51 μg of QE / ml). Hence,
further studies are necessary in order to optimize the extraction process.
Another limitation is that the photoprotective activity was assessed by
mean of in vitro methods (UV spectrum analysis and SPF determination).
Although these in-vitro results are promising and highlighted the poten-
tial photoprotective effect of C.E aqueous leaf extract, in-vivo studies
must be undertaken to support this current work.
6. Conclusion
The aim of this present study was to assess the potential photopro-
tective effect of a Tunisian halophyte plant Carpobrotus edulis L. Phy-
tochemical characterization (FT-IR, TPC, TFC and LC-MS) indicated its
richness in phenolic compounds. UV spectrophotometric analyses in the
UV region showed that the plant extract absorbed in both UV-B and UV-
A ranges. C.E crude extract exhibited a great photoprotective activity
with a SPF value of ≈7 at the concentration of 1000 μg/ml correspond-
ing to a percent of UV blockage ranging from 80% to 90%. This plant
extract, when incorporated into an O/W emulsion at a concentration of
10% (m/m), presented a good antioxidant activity (≈70%) and an in
vitro SPF value of ≈1.7 which corresponds to a percent of UV blockage
around 50%. Those results could be explained by the presence of phe-
nolic compounds, mainly flavonoids, in its composition. Overall, this
present work highlighted the potential of Carpobrotus edulis L., as an
interesting photoprotective and antioxidant source. However, on basis
of previously published data, which demonstrated the impact of envi-
ronmental, biological and experimental factors on extraction yield, as
well as on the qualitative and quantitative phytocomposition of an ex-
tract, further studies should be conducted in the aim to explore more
this activity taking into consideration all the factors implied.
M.A. Lassoued, N.E.H. Ben Fatma, M. Haj Romdhane et al.
Authors contribution
Mohamed Ali Lassoued: Conceived the project; designed and per-
formed the experiments; wrote the manuscript.
Nour El Houda Ben Fatma: Contributed to the preparation of the
plant extract and to the phytochemical characterization.
Mariem Haj Romdhane: Carried out the LC-MS analysis; contributed
to the interpretation of the results and to the drafting of the manuscript.
Adel Faidi: Contributed to the interpretation of the results.
Hatem Majdoub: Contributed to the interpretation of the results and
to the correction of the manuscript.
Souad Sfar: Supervised the project and contributed to the correction
of the manuscript.
Financial support
This research received no specific grant from any funding agency in
the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors have no conflicts of interest to disclose.
Acknowledgements
We thank Pr. Evangelia Varella and Dr. Eleni Koliarmou from the
laboratory of Organic chemistry in university of Aristotle of Thessaloniki
(Greece) for providing the necessary equipment for the LC-MS analysis.
Data availability
The study information can be obtained from the author on request.
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