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590 FARMACIA, 2009, Vol.

57, 5

REGENERATIVE PROPERTIES OF
ALOE VERA JUICE ON HUMAN
KERATINOCYTE CELL CULTURE
ANDRA TUDOSE1,2, CHRISTIAN CELIA1, FRANCESCO
CARDAMONE1, MARGHERITA VONO1,3, ROBERTO MOLINARO1,3,
DONATELLA PAOLINO3,*
1
Department of Pharmacobiological Sciences, Faculty of Pharmacy,
University “Magna Græcia” of Catanzaro, Campus Universitario “S.
Venuta” - Building of BioSciences, Viale S. Venuta, I-88100 Germaneto
(CZ), Italy
2
Department of Applied Mathematics and Biostatistics, Faculty of
Pharmacy, University of Medicine and Pharmacy “Carol Davila “
Bucharest, Romania
3
Department of Experimental and Clinical Medicine, Faculty of
Medicine, University “Magna Græcia” of Catanzaro, Campus
Universitario “S. Venuta” - Building of BioSciences, Viale S. Venuta, I-
88100 Germaneto (CZ), Italy
*
corresponding author: paolino@unicz.it

Abstract
Aloe Vera, a medicinal plant widely used in food supplements, beverages,
pharmaceuticals and cosmetics, was used as an effective natural remedy for its healing properties
and positive influence in different inflammatory skin disorders. In particular, regenerative
properties of Aloe Vera juice are investigated on human keratinocyte cells that are used as an in
vitro skin model system. The therapeutic efficacy of the plant extract was evaluated using trypan
blue assay and the counting cell determination. The obtained results showed that Aloe Vera juice
could represent a natural therapeutic strategy through the topical route.
Rezumat
Aloe Vera, planta medicinală regasită adesea în suplimente alimentare, băuturi,
produse farmaceutice şi cosmetice, este utilizată ca şi remediu natural eficace, datorită
proprietăţilor regenerative şi a influenţei pozitive asupra diferitelor afecţiuni inflamatorii ale pielii.
Proprietăţile regenerative ale extractului de Aloe Vera sunt investigate in vitro
folosind culturi celulare de keratinocite umane.
Eficacitatea terapeutică a extractului a fost evaluată folosind metoda excluderii
cu albastru de tripan.
Rezultatele obţinute demonstrează că extractul de Aloe Vera poate fi considerat o
strategie terapeutică naturală viabilă pentru admininstrare topică.
Keywords: Aloe Vera; viability activity; human keratinocyte cells

Introduction
Skin health is increasingly becoming an important aspect of
primary health care among many communities. Many preventive and
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treatment options are used according to the various international guide lines
and therapeutic and cosmetic approaches to achieve varying degrees of
success, especially in the case of patients having some degree of skin
damage [9, 16]. A novel and alternative approach for possible benefits
concerning skin damage is represented by various plant-based treatments
[13, 14, 19]. In fact, plant constituents such as carotenoids and flavonoids
are involved in protection against excess light and contribute to the
prevention of UV damage in humans [15]. At the same time, a wide range
of chemical components isolated from the Fraxinus species, such as
coumarins, secoiridoids, phenylethanoids, flavonoids and lignans are
recently being investigated for their anti-inflammatory activity and skin-
regenerating effects [8, 18]. Moreover, Prunella vulgaris L. (Labiatae) and
its main phenolic acid component, rosmarinic acid (RA) have shown a
remarkable efficacy as photoprotectants against harmful effects of UV
radiation [12]. Among various natural therapeutic remedies, Aloe Vera, a
medical plant widely used in food supplements, beverages, pharmaceutical
and cosmetics [4], has been recognized as an effective natural remedy for its
healing properties and positive effects in various inflammatory skin
disorders [7, 20, 21]. Aloe Vera contains 75 potentially active constituents
such as vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic
acids, and amino acids and carries on biological and toxicological activities
[1]. Aloenin, magnesium lactate, aloe-emodin, barbaloin, cinnamic and
succinic acids, mannose-6-phosphate, polysaccharides and flavonoids were
found in the Aloe Vera juice and play an important role in the anti-
inflammatory and antioxidant activity of the plant [5, 6], and they are also
responsible of its pharmacological properties [2, 10, 11, 17].
The aim of this study was the evaluation of the regenerative
properties of Aloe Vera juice on human keratinocyte cell culture, namely
NCTC2544 human cell line. It was used as in vitro skin based-model system
and regenerative properties of plant extracts have been evaluated using
trypan blue assay. A suitable effect on cutis regeneration is carried out and
curative activities of plant components have been examined as a function of
viability percentages of juice.

Materials and methods


Materials
Human keratinocyte cells (NCTC2544) are obtained from Istituto
Zooprofilattico della Lombardia e dell’Emilio Romagna (Brescia, Italy).
DMEM (Dulbeco’s Modified Eagle Medium) culture medium, fetal bovine
serum (FBS), penicillin-streptomycin solution and Trypsin/EDTA (1x)
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solution are purchased from GIBCO (Invitrogen Corporation, Giuliano


Milanese (Mi), Italy). Amphotericin B solution (250 μg/ml), trypan blue
solution (0.4 % v/v), phosphate buffer solution (PBS) are obtained from
Sigma Chemicals Co. (St. Louis, USA). Aloe Vera is provided from the
botanical garden of University “Magna Græcia” of Catanzaro (Italy). All
other materials and organic solvents are of analytical grade (Carlo Erba,
Milan, Italy).
Extraction of juice
Aloe Vera juice is mechanically extracted from plant leaves. They
are cut from the trunk and roots of the plant and the terminal tip and lateral
thorns are eliminated. The gelatinous substance inside the leaves is
separated from the protective external envelop in a dark room to prevent the
oxidation of the plant extract. The obtained substance is then divided into
different pieces and homogenized (15000 rpm) for 1 min by means of an
Ultra-Turrax T 25 equipped with an S25N-8G homogenizing probe
(IKAWERKE) under continuous cooling. The triturated mixture is filtered
in the dark through 0.45 μm polystyrene filter, divided into 2 ml volume
tubes and stored for 3 days at -20°C before being used for the in vitro
experiments. Every tube used in the experiment was completely wrapped in
alluminium foil to prevent any possible degradation of the juice.
NCTC 2544 cell cultures
Human keratinocyte cells (NCTC 2544) were transferred from
cryovial tubes to plastic culture dishes (100 mm diameter) and seeded at 5
% CO2 for three days at 37°C by using DMEM supplemented with
penicillin (100 UI/ml), streptomycin (100 μg/ml), amphotericin B solution
(1 % v/v) and FBS solution (10 % v/v) as culture medium throughout all in
vitro experiments. After the incubation time, NCTC 2544 cells (80 % of
confluence) were harvested with 2 ml of trypsin solution, washed with a
phosphate buffer solution (2 ml) and transferred into 10 ml plastic
centrifuge tubes. Then, culture medium (4 ml) was added to human
keratinocytes, thus achieving a final volume of 8 ml. Cells were centrifuged
using a Megafuge 1.0 (Heraeus Sepatech) at 1200 rpm for 10 min at room
temperature. Pellets were separated from the supernatant and re-suspended
with 6 ml of culture medium to obtain 1×106 cells/ml. The cell suspension
was diluted up to a final concentration of 5×104 cells/ml and 1 ml of diluted
cellular suspension was seeded into 12 well plastic culture dishes before the
in vitro investigations.
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Viability study
The regenerative property of Aloe Vera extract was evaluated on
human keratinocyte cells (NCTC2544) by determining the viability activity
of its juice using trypan blue dye exclusion assay. NCTC 2544 cells were
seeded at a density of 5×104 cells/ml into 12-well plastic culture dishes.
After 48 h of incubation, the culture medium was replaced for each well
with a solution of fresh culture medium and Aloe Vera juice was added (1
ml) at final scalar dilutions (10, 30 and 50 % v/v of plant extract). Viability
experiments were carried out at different incubation times, i.e. 3, 6, 12, 24,
48 h. Untreated NCTC2544 cells were used as control in the various
experiments. To perform the trypan blue dye exclusion assay, NCTC2544
were harvested using trypsin/EDTA (1×) solution (2 ml), washed twice with
phosphate buffer solution (2 ml) and transferred into 5 ml plastic centrifuge
tubes. Culture medium (4 ml) was added to plastic centrifuge tubes up to a
final volume of 8 ml. Samples were then centrifuged with a Megafuge 1.0
(Heraeus Sepatech) at 1200 rpm at room temperature for 10 min. The
supernatant was withdrawn and the pellet suspended in 100 μl of trypan blue
buffer. The amount of viable cells was observed with a hematocytometer
chamber using an optical microscope (Labophot-2, Nikon, Japan). The
percentage of cell proliferation was calculated using the following equation:
% Viability = (CV/CT) × 100
where CV is the number of viable cells treated with different Aloe Vera juice
concentrations and CT the total number of viable control cells. Values of
cell viability are expressed as the mean of six different experiments ±
standard deviation.
NCTC2544 cell growth determination
NCTC 2544 cells were seeded at a density of 5×104 cells/ml into
12-well plastic culture dishes. After 24 h the cell culture medium was
removed and cells were washed with PBS buffer solution (1 ml) and
replaced with fresh culture cell medium containing Aloe Vera juice at
different concentrations (10 %, 30 % and 50 % v/v). In all experiments,
untreated NCTC2544 cells were used as control. The growth of human
keratinocyte cells was determined at different incubation times, i.e. 3, 6, 12,
24 and 48 h. The improvement of cell number was carried out by counting
the viable cells. NCTC2544 cells were harvested using trypsin/EDTA (1×)
solution (2 ml), washed twice with PBS (2 ml) and transferred into 5 ml
plastic centrifuge tubes. Culture medium (4 ml) was added to the cells to
achieve a final volume of 8 ml of cell suspension. The obtained samples
were centrifuged with a Megafuge 1.0 (Heraeus Sepatech) at 1200 rpm at
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room temperature for 10 min. The supernatant was withdrawn and the pellet
suspended into 100 μl of cell culture medium. The number of NCTC2544
cells was counted with a hematocytometer chamber using an optical
microscope (Labophot-2, Nikon, Japan). Each determination represented the
mean value of six different experiments ± standard deviation.
Statistical analysis
The statistical analysis of the data was carried out using ANOVA t-
test. A p value <0.05 was considered statistically significant. All values are
reported as the average ± standard deviation.

Results and discussion


The therapeutic properties of the various components of Aloe Vera
have gained a certain amount of attention during the last decades as
potential natural remedies in the pharmacological treatment of cutaneous
diseases based on the skin degeneration, thus using the regenerative
properties of this extract [16].
The regenerative properties of Aloe Vera juice were tested on
NCTC2544 cells as a function of both the drug concentration (dose-
dependent proliferate effect) and the incubation time (plant extract
exposition effect). To achieve a clear picture of the real curative effect of
these plant components, the cellular proliferation, induced by Aloe Vera
juice, was evaluated. As shown in Fig. 1, NCTC2544 cells treated with Aloe
Vera juice showed a percentage of proliferation superior to that of untreated
cells (control) for the different concentrations tested (10, 30 and 50% v/v),
during an incubation time of 48 hours. The improvement of cell viability
percentages, in the NCTC2544 cells, was confirmed by counting the number
of cells for the different Aloe Vera juice concentrations (Fig. 2). In
particular, the cellular growth was directly proportional to the concentration
of plant juice used (Fig. 2). This effect is probably correlated to a gradual
increase of type I/type III collagen matrix occurring in the case of
stimulation of NCTC2544 cells with a concentrated solution of Aloe Vera
juice, showing how the different concentrations of plant extract were able to
improve the amount of collagen in the connective matrix [3]. The obtained
results are in agreement with those achieved in the case of NCTC2544 cells
stimulated for 48 h. In this case, it was noteworthy that a gradual increase of
cell number was obtained for the different concentrations when compared to
untreated cells and other incubation times (Fig. 2).
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Figure 1
Viability percentages of NCTC2544 human keratinocyte cells treated with Aloe Vera juice at
different concentrations: (■) 10% v/v; (○) 30% v/v; () 50% v/v. Untreated cells are used as
control (●). The cellular viability has been evaluated as a dose-dependent effect for different
exposition times: 3 h; 6 h; 12 h; 24 h; 48 h. Error bars if not visible are within the symbols.
Each point represents the mean value of six different experiments ± standard deviation

Figure 2
Improvement of NCTC2544 cell number after stimulation with different concentrations of
Aloe Vera juice (10, 30 and 50 % v/v) for various exposition times (3, 6, 12, 24, 48 h).
Untreated cells are used as control. Error bars if not visible are within the symbols. Each
value represents the mean of at least five different experiments ± standard deviation
596 FARMACIA, 2009, Vol. 57, 5

Conclusions
The experimental investigations have shown that plant extracts may
be used as natural agents for the treatment of different skin disorders or
damage. This effect is probably correlated to the different components
present in Aloe Vera juice that are able to promote the regenerative process
of the skin as a function of the dose-dependent concentrations and the
incubation time.
The obtained results show that Aloe Vera juice offers various
opportunities for the development of natural therapeutic strategies through
the topical route.

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Manuscript received: 05.05.2009

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