Industrial Crops & Products 108 (2017) 470479

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Industrial Crops & Products

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Evaluation of in vitro and in vivo safety of the by-product of Agave sisalana as MARK
a new cosmetic raw material: Development and clinical evaluation of a
nanoemulsion to improve skin moisturizing
Stella Maria Andrade Gomes Barretoa, Mayara Saatmn Maiaa, Alvaro Moreira Benica,
Hugo Raphael Bezerra Silva de Assisa, Vnia Rodrigues Leite-Silvab, Pedro Alves da Rocha-Filhoc,
Marlia Medeiros Fernandes de Negreirosd, Hugo Alexandre de Oliveira Rochad,
Elissa Arantes Ostroskya, Patricia Santos Lopesb, Valeria Soraya de Farias Salesa,

Raquel Brandt Giordania, Mrcio Ferraria,
a
College of Pharmacy, Federal University of Rio Grande do Norte UFRN, Rua Gustavo Cordeiro de Farias, s/n, Petrpolis, Natal, RN, 59012-570, Brazil
b
Departamento de Cincias Exatas e da Terra, Federal University of So Paulo UNIFESP, Rua So Nicolau, 210, Centro, Diadema, SP, 09913-030, Brazil
c
Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeiro Preto, University of So Paulo, Avenida do Caf, s/n, Monte Alegre, Ribeiro
Preto, SP, 14040-903, Brazil
d
Department of Biochemistry, Federal University of Rio Grande do Norte UFRN, Avenida Salgado Filho s/n, Natal, RN, 59078-970, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Agave sisalana (sisal) is the important global source of hard ber, which is widely used in the production of wires,
Agave sisalana by-product ropes, and handicrafts. The decortication process of the A. sisalana leaf produces large amounts of by-product
Cosmetic that is discarded and can cause environmental damage. Studies have shown the potential of the by-product of A.
Safety sisalana in dierent applications. The aim of this study was to obtain a polysaccharide-enriched fraction (EF)
Nanoemulsion
from the by-product of A. sisalana, assess its safety in vitro and in vivo, to develop a cosmetic nanoemulsion and to
Moisturizing
evaluate its moisturizing clinical ecacy. EF was obtained and total sugar, total phenolic, and protein content
were quantied. The safety of EF was determined using in vitro and in vivo assays. Nanoemulsions were de-
veloped and their stability evaluated for 90 days at dierent temperature conditions. The clinical moisturizing
ecacy was evaluated by biophysical techniques using capacitance measurement and transepidermal water loss.
The fraction exhibited high concentrations of sugar (65.49 0.51%) and the presence of phenolic compounds
(2.53 0.04%) as well as protein (0.04 0.01%). The EF did not exhibit potential cytotoxic or phototoxic
eects and did not present the potential to induce skin irritant reaction in clinical tests. Nanoemulsion con-
taining 40% oil phase, 50% aqueous phase and 10% surfactants, added fraction or not (vehicle), was stable for
90 days. The nanoemulsion containing 0.5% of the fraction increased the water content of stratum corneum by
10.13% vs. vehicle and by 19.28% vs. baseline values and maintained skin barrier function after 5 h of a single
application. The EF obtained from the industrial by-product of A. sisalana demonstrated a promising prole as a
moisturizing cosmetic raw material. This is important because it shows the possibility to increase the commercial
value to the industrial by-product of sisal, and thus reduce the environmental impact caused by the disposal of
this material.

1. Introduction Mexico and adapted to the semi-arid region. In Brazil, this species was
widespread in Caatinga biome, and it is mainly cultivated to obtain
Agave sisalana Perrine, popularly known as sisal, is an herbaceous bers for the production of wires, ropes, and handicrafts (Gutirrez
plant that belongs to the Agavaceae family. This plant originated from et al., 2008; Maran and Priya, 2014). About 5% of the plants weight is

Corresponding author.
E-mail addresses: smagbarreto@gmail.com (S.M.A.G. Barreto), maysaatman@hotmail.com (M.S. Maia), alvarobenica@hotmail.com (A.M. Benic),
hugorbassis@gmail.com (H.R.B.S. de Assis), vania.leite@unifesp.br (V.R. Leite-Silva), pedranjo@fcfrp.usp.br (P.A. da Rocha-Filho),
marilia_negreiros16@yahoo.com.br (M.M.F. de Negreiros), hugo@cb.ufrn.br (H.A. de Oliveira Rocha), aranteselissa@gmail.com (E.A. Ostrosky), patricia.lopes@unifesp.br (P.S. Lopes),
vsales10@gmail.com (V.S. de Farias Sales), raquebg@hotmail.com (R.B. Giordani), ferrarimarcio@uol.com.br (M. Ferrari).

http://dx.doi.org/10.1016/j.indcrop.2017.06.064
Received 2 March 2017; Received in revised form 27 June 2017; Accepted 29 June 2017
0926-6690/ 2017 Elsevier B.V. All rights reserved.
S.M.A.G. Barreto et al.
used to produce bers, and the by-product discarded from production
can become an environmental problem (Maran and Priya, 2014).
Studies have demonstrated the potential of A. sisalana by-product as
a raw material for application in the pharmaceutical and cosmetic in-
dustries. Literature reports the presence of dierent metabolites classes
in chemical composition of this by-product, particularly carbohydrates
(Zhang et al., 2014), avonoids (Chen et al., 2009) and saponin
(Ribeiro et al., 2015a).
Polysaccharides, mainly of natural or biotechnological origin, have
been used as moisturizing agents (Bont, 2011). Dierent placebo-
controlled studies have attributed moisturizing activity to these com-
pounds (Damasceno et al., 2016; Ribeiro et al., 2015b). Zhang et al.
(2013, 2014) attributed antioxidant activities to these compounds in
the vegetal extracts.
The bioactivity of vegetal ingredients combined with the trend of
using plant extracts have improved scientic eorts in the search for
new raw materials for cosmetic products. However, natural products
are not necessarily safe and eective, as these can cause damage to
health, such as irritation, sensitization, and/or photoreactions
(Nohynek et al., 2010). Therefore, the safety and ecacy should be
evaluated to ensure the best conditions of use, and in vitro and in vivo
studies are needed to elucidate the benets of A. sisalana by-product in
skin care.
Initially, safety can be evaluated through preclinical testing in vitro,
an alternative to the use of animals, in order to ensure safety prior to
clinical trials (Felippi et al., 2012). In vivo studies of skin compatibility,
such as the semi-occlusive contact test or occlusal (patch test), re-
present the rst contact of a substance or product with the skin. These
are performed under maximized conditions, with control of the area of
application and quantity applied being an important tool used in de-
termining the absence or presence of allergens or potential irritatives
(Anvisa, 2012; Antignac et al., 2011). Thus, after the in vitro and in vivo
tests of a raw material, together with the evidence of absence of risk,
the extract can be used for the development of formulations and/or
subjected to specic ecacy testing in vivo.
To overcome the disadvantages of the use of cosmetics containing
botanical ingredients, as its lower penetration and high compound in-
stability (Ganesan and Choi, 2016), nanoemulsions can be used (Gianeti
et al., 2012; Peshkovsky et al., 2013).
Scientic and technological advances in the development of re-
search tools for the biological functions of the skin changed the un-
derstanding of the eects caused by cosmetic products, enabling the
interpretation of results obtained in actual conditions of use and thus
enabling the evaluation of ecacy in vivo. Bioengineering methods
enable to investigate the dierent responses of the skin to the multiple
stimuli caused, including the top layer of skin hydration and its barrier
eect (Byrne, 2010; Dreno et al., 2014).
Thus, with the prospect of a promising prole from the by-product
of the processing of A. sisalana as a cosmetic raw material, together
with the possibility of adding commercial value to an industrial residue,
as well as the reduction of damages to the environment, and the con-
tribution to sustainable development, the objective of this work was to
develop a safe and eective moisturizing cosmetic nanoemulsion con-
taining polysaccharide-enriched fraction of the by-product from the
industrial processing of A. sisalana.
2. Materials and methods
2.1. Materials
The following materials were used for the preparation of the for-
mulations: Caprylic/CapricTriglyceride (Crodamol GTCC),
Polysorbate 80 (Tween 80), Sorbitan Monooleate (Span 80),
Ethylhexyl Palmitate (Crodamol OP) donated by Croda of Brazil Ltda
(Brazil), Isostearyl Neopentanoate (Eschercemol 185) provided by
Lubrizol of Brazil Ltda (Brazil), Phenoxyethanol, Caprylyl Glycol
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Industrial Crops & Products 108 (2017) 470479
(Optiphen) provided by Ashland Inc. (Brazil), Disodium EDTA (DEG
Importao de Produtos Qumicos, Brazil), Butylatedhydroxytoluene
BHT (Galena qumica e farmacutica LTDA, Brazil) and distilled water.
For the in vitro safety assessment: Dulbeccos Modied Eagles Medium
(DMEM), EDTA, penicillin, streptomycin, amphotericin, glutamine and
fetal bovine serum were purchased from Vitrocell (Brazil). Neutral Red
(Sigma-Aldrich, Brazil), trypsin solution, phosphate buer at pH 7.4.
Occlusive patch test device of 1 cm2 (IPI ASAC Brazil, Brazil) was used
for the in vivo safety evaluation.
2.2. Crude extract and polysaccharide-enriched fraction
The sisal by-product used in this study was supplied by Sisaltec
Industry (Natal, Brazil). A voucher specimen and the by-product were
collected from a sisal farm in the city of Pedra Grande, Rio Grande do
Norte State, Brazil (50756.3S 355431.7W) on August 8, 2015. The
by-product was collected directly from a decortication machine. The
voucher specimen was deposited at the Herbarium of the Federal
University of Rio Grande do Norte under number UFRN- 20544.
The natural ber (sisal) was mechanically removed from the A. si-
salana leaves and the residual material from this process (by-product)
was pressed aording a liquid portion (we named crude-extract CE)
and a solid portion (mainly residual ber that was discarded). The
crude extract was added to ethanol 95% to promote precipitation ac-
cording to Santos et al. (2013). This process was carried out with 95GL
ethanol at 1:3 (v/v) residue/solvent ratio and stored at 4 C for 24 h;
subsequently, two samples were obtained: a precipitate and a super-
natant. The supernatant was ltrated, concentrated, and lyophilized
(Christ, Germany) aording a sample named polysaccharide-enriched
fraction (EF) (Fig. 1). The yield of EF was calculated from the initial
amount of liquid portion (CE): Yield (%) = (Mass of polysaccharide-
enriched fraction/Mass of liquid portion) 100 (Santos et al., 2013).
2.3. Quantication of total sugars
The quantication of total sugars in CE and EF was performed using
the Phenol/sulfuric acid method (Dubois et al., 1956). The assays were
performed at room temperature (25 2 C) using D-galactose as ca-
libration standard for the construction of the calibration curve. The
spectrometric readings were recorded at 490 nm.
2.4. Quantication of phenolic compounds
The amount of total phenolic compounds in CE and EF was de-
termined by spectrophotometric analysis in the visible region using the
Folin-Ciocalteu method (Athukorala et al., 2006). The assays were
performed at room temperature (25 2 C) with protection from the
light and using Galic acid as calibration standard for the construction of
the calibration curve. The spectrometric readings were recorded at
765 nm.
2.5. Protein content assessment
Protein content in CE and EF was assessed by Bradford method
(1976) by spectrophotometric analysis. The assays were performed at
room temperature (25 2 C) using bovine serum albumin as cali-
bration standard for the construction of the calibration curve. The
spectrometric readings were recorded at 595 nm.
2.6. Raw materials safety evaluation
2.6.1. Safety evaluation in vitro
2.6.1.1. Cell culture. BALB/3T3 clone A31 (ATCC CCL163) the
permanent murine broblast cell line was used in cytotoxicity and
phototoxicity assays. Cells were cultured in Dulbeccos Modied Eagles
S.M.A.G. Barreto et al.
Medium (DMEM) supplemented with fetal bovine serum (10% v/v),
glutamine (4 mM) and antibiotics and then maintained in standard
conditions (5% CO2, 37 C, 97% UM for 24 h). The cell detachment
process was performed using trypsin solution 0.05% (w/v)/0.02%
EDTA (w/v) in phosphate buer pH 7.2; the detached cells were
counted and plated on 96-well plates (P96w) with 20,000 cells per well
(ISO 10993-5, 2009; OECD 432, 2004).
2.6.1.2. Cytotoxicity study. The cytotoxic eect of CE and EF was
evaluated using 3T3 neutral red uptake assay. After 2 h of incubation,
absorbance was measured at 540 nm (ICCVAM, 2006; OECD 129,
2010). CE and EF are water soluble and were evaluated at dierent
concentrations (0.15, 0.3, 0.6, 1.25, 2.5, 5.0, 10.0 and 20.0 mg/mL).
The values of IC10 and IC50 were calculated. Cell viability was
calculated according to Eq. (1):
ODsample
VC = 1 100
ODcontrol (1)
In which: VC is cell viability, OD sample is an optical density of the
extract, OD control is an optical density of the test control cells.
2.6.1.3. Phototoxicity study. The phototoxic eect of CE and EF was
evaluated using 3T3 neutral red uptake assay (OECD TG 432, 2004) at
concentrations determined by cytotoxicity assay. The samples were
exposed to radiation (UVA+) during 1 h and 50 min to reach a dose of
5 J/cm2. Moreover, the samples were also evaluated at the same
concentrations in the absence of light (UVA-). After this period, the
cells were subjected to neutral red uptake assay, and the result was
measured at 540 nm. The data were analyzed by Phototox, and the
Photo Irradiation Factor (PIF) and Mean Phototoxic Eect (MPE)
(OECD TG 432, 2004) were then determined.
2.6.2. Safety evaluation in vivo
Cutaneous compatibility of EF was assessed according to
International Contact Dermatitis Research Group guidelines. It was
evaluated by a single application to 18 volunteers of ages from 22 to 46
years after receiving their written informed consent. The volunteers
involved in the study were with phototype skin types II to IV. The vo-
lunteers were selected according to the inclusion and exclusion criteria
for the study. The study was carried out with the approval of the
Research Ethics Committee of the University of Cuiab (195.680/
2013). In order to evaluate the safety, 3 patch test devices of 1 cm2
embedded with 20 L of the 0.5%, 1.0% of EF and water (negative
control) were applied on the normal skin of the upper back. The patches
were removed after 48 h of application. The skin irritation responses
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Industrial Crops & Products 108 (2017) 470479
Fig. 1. Scheme of extraction and obtainment of
crude extract and polysaccharide-enriched fraction
from Agave sisalanaAgave sisalana by-product.
were evaluated at 30 min and 24 h and 48 h after patch removal
(Felippi et al., 2012; Kligman and Wooding, 1967; Wilkinson et al.,
1970).
2.7. Development of formulations
2.7.1. Pseudo-ternary phase diagram (PTPD)
The formulations were developed according to the ternary diagram
method (Lo et al., 1976). A blend of Polysorbate 80 and Sorbitan
Monooleate (9:1 w/w) with a hydrophilic lipophilic balance (HLB) of
14.0 as surfactants in order to obtain oil-in-water (O/W) emulsions was
used (Mahdi et al., 2011; Silva et al., 2016). The pseudo-ternary phase
diagram consisting of oily and aqueous phases and surfactants was
structured (Rocha-Filho et al., 2016) by varying the component con-
centrations by 10% (w/w). EF (0.5%) was added into the aqueous
phase. Energy was supplied to the system by an ultrasonic probe (So-
nics, USA) for 4 min (30 W). Only the region of the phase diagram with
a concentration of 10.0% of surfactants and an oil concentration of
10.0% to 40.0% was studied.
2.7.2. Preliminary stability study
The samples were stored at 45 2 C and 75% 5% RH (Nova
Etica, Brazil) for 5 days. After 24 h of manipulation the centrifugation
test and macroscopic analyses were performed. Zeta potential, pH
value, particle size and polydispersity index (PI) were measured at 0
and 5th day.
2.7.2.1. Centrifugation test. Centrifugation was performed on 24 h-old
preparations at 3000 rpm (Fanen, Brazil) for 30 min at 25 C (Ribeiro
et al., 2015b). After that, centrifugation appearance, homogeneity and
organoleptic characteristics were evaluated macroscopically.
2.7.2.2. Macroscopic aspect. Organoleptic changes (color and odor) as
well as the presence of nanoemulsion instability phenomena (creaming
or phase separation) were observed visually.
2.7.2.3. pH analysis. The formulations pH values (Hanna Instruments,
mod. HI 21, Brazil) was determined into an aqueous dilution 1:10 (w/
w) by inserting the electrode directly in the sample (Ribeiro et al.,
2015b).
2.7.2.4. Droplet size and polydispersity index measurements. The droplet
diameter and droplet size distribution (evaluated by the polydispersity
index PDI) were measured by Dynamic Light Scattering (DLS)
(Malvern, United Kingdom) at 25 C. The scattered angle was xed at
S.M.A.G. Barreto et al.
Table 1
Quantication of total sugars, protein and phenolic compounds in CE and EF from Agave
sisalana by-product.
Samples Total sugars (%) Protein (%) Phenolic compounds (%)
CE 41.31 2.41 0.12 0.01 1.27 0.13
EF 65.49 0.51 0.04 0.01 2.53 0.04
In which: CE = crude extract and EF = polysaccharide-enriched fraction. p-value <
0.05 for students t-test (unpaired).
173. The formulations were diluted with distilled water by 200-fold
before measurements (Ribeiro et al., 2015b). The experiments were
carried out in triplicate.
2.7.2.5. Zeta potential measurements. Zeta potential of nanoemulsions
were measured by phase analysis light scattering, (Malvern, United
Kingdom) at 25 C. The formulations were diluted with distilled water
by 200-fold before measurements (Ribeiro et al., 2015b). The
experiments were carried out in triplicate.
2.7.3. Accelerated stability study
The nanoemulsions considered stable after the preliminary tests
were packed in hermetically sealed glass bottles and subjected to ac-
celerated stability tests. The formulations were evaluated initially
(t0 = 24 h) and 90 days after exposure to dierent temperature con-
ditions: 4 2 C (Consul, Brazil), room temperature (25 2 C) and
45 2 C (Odontobrs, Brazil) (Ribeiro et al., 2015b). The macro-
scopic analyses (appearance, homogeneity and organoleptic char-
acteristics) (2.7.2.2), droplet size and polydispersity index measure-
ments (2.7.2.4) and zeta potential measurements (2.7.2.5) were used as
evaluation parameters of stability.
2.8. In vivo evaluation of skin hydration
The study was carried out with the approval of the Research Ethics
Committee of the University of Cuiab (195.680/2013) where 18 vo-
lunteers were selected, aged between 22 and 35 years of both genders,
who declared no history of skin diseases. Injuries were not visible at the
time of the tests, in which the goals and methods of the survey were
made clear, and the volunteers agreed to participate after reading and
signing the informed consent. These volunteers were banned to use any
cosmetic products for two weeks before and on the day of the experi-
ment, except for cleaning products such as soaps. The study was mono-
blind, randomized, and placebo-controlled (Ribeiro et al., 2015b).
Prior to the start of the tests, the volunteers remained in the room
for at least 30 min in order to allow the skin adaptation to the rooms
temperature (21 2 C) and relative humidity (55 5%) (Felippi
et al., 2012).
Stratum corneum aqueous content was determined by noninvasive
biometrical measurements using a skin capacitance meter (Corneometer
CM 825, Courage & Khazaka, Electronic GmbH, Khln, Germany). Four
sites (9 cm2 each) on volunteers forearm skin were randomly selected
(A1 to A4): one (A1) as the control where only measurements were
taken, and the others (A2 to A4), to which dierent formulations without
fraction (vehicle), formulation containing 0.5% polysaccharide-enriched
fraction (A3) and a positive control-commercial formulation (A4) were
applied respectively. All formulations were applied to the surface of the
skin at 2 mg/cm2 with a light massage of approximately 10 s.
Transepidermal water loss (TEWL) from the skin was assessed by an
evaporimeter (Tewameter TM 300, Courage & Khazaka, Electronic
GmbH, Germany). TEWL values were registered for 2 min following a
30-s period of equilibration of the probe on the skin. One reading in
each eld was taken for TEWL evaluation, (Maia Campos et al., 2012;
Ribeiro et al., 2015b).
Skin capacitance and TEWL were determined before (baseline), and
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Industrial Crops & Products 108 (2017) 470479
at 1, 2, 3, 4 and 5 h after a single application of the formulations to
elds A2-A4 and to the control eld without formulation (A1).
2.9. Statistical analysis
Statistical analyses were performed using the software Graph Pad
Prism 6.01. Analyses between the two groups were performed by the
Students t-test. Statistical signicance for two or more groups were
evaluated by the variance analysis (ANOVA) followed by the Tukey test
for multiple means comparisons. p < 0.05 was considered statistically
signicant.
3. Results and discussion
The CE and EF presented a greenish and greenish-yellow color, re-
spectively, with an odor characteristic of the plant. The polysaccharide-
enriched fraction yield was 7.08%.
Characterization of botanic bioactives by the determination of total
sugar, phenols and protein is an important step in the development of
cosmetics. The chemical composition may indicate its use and also
predict the potential eects on the ecacy of the nal product
(Garbossa and Maia Campos, 2016).
By analyzing the results showed in Table 1, it can be seen that the
fraction (EF) showed higher concentration of sugar than CE (p-
value < 0.05). The quantication of total sugars methodology used
(Dubois et al., 1956) is applied to the analysis of any mixtures of sugars
and has proven of value for the analysis of hydrolysates of oligo-
saccharides, dierent polysaccharides, plant gums and hemicelluloses
(Dubois et al., 1956). To obtain the fraction assessed, we used an ex-
traction methodology (Santos et al., 2013) commonly used to obtain
polysaccharides. Zhang et al. (2014) used a similar methodology for
extraction of polysaccharides of A. sisalana by-product. In this way, we
extrapolated and denominated the fraction of polysaccharide-enriched
fraction.
Polysaccharides are used as moisturizing molecules in cosmetics
products because of their capacity of promoting regulation of skin
water content, being able to retain humidity in the skin or forming a
soft moisture lm (Bont, 2011). Polysaccharides can act as emulsion
stabilizers, which is an interesting feature in the development of new
products (Shao et al., 2017). These compounds can also exhibit anti-
oxidant properties, activity which is usually related to phenolics com-
pounds (Garbossa and Maia Campos, 2016; Stojiljkovi et al., 2016).
Furthermore, benets of proteins in skin care products were ob-
served in silk protein studies performed by Daithankar et al. (2005);
these proteins promoted an increase at skin water content. Protein
moisturizing action was observed in studies using protein extracts of
wheat (Triticum vulgare) and soy (Glycine soya) (Gianeti and Maia
Campos, 2012).
Thus, polysaccharide-enriched fraction containing phenolic com-
pounds and protein can be used a new multifunctional raw material for
cosmetic industry. Therefore, clinical tests must be performed to con-
rm in vivo biological activity of this compounds.
3.1. Safety evaluation in vitro
According to Nohynek et al. (2010), mixtures of components present
in plant extracts can produce local reactions when applied to the skin,
and this can be a critical factor for use as cosmetic ingredients. Cos-
metic products containing these ingredients may be used frequently and
may be in contact with the skin for long periods. Polysaccharides are
usually considered non-toxic (Zhang et al., 2014), although the irrita-
tive potential of these components should be evaluated.
The results for the cytotoxicity test are shown in Fig. 2. At con-
centrations of 0.128 and 0.45 mg/mL, CE and EF, respectively, showed
90% cell viability for BALB/c 3T3 cells. CE showed an IC50 value of
0.252 mg/mL, suggesting that the use of higher concentrations can
S.M.A.G. Barreto et al. Industrial Crops & Products 108 (2017) 470479

Fig. 2. Cytotoxicity evaluation of the crude extract and polysaccharide-enriched fraction from Agave sisalana by-product.
In Which: (A) Cytotoxicity evaluation of the crude extract from Agave sisalana by-product; (B) Cytotoxicity evaluation of polysaccharide-enriched fraction from Agave sisalana by-product.

cause cytotoxic eects on the skin. Thus, from these results, the crude
extract was not evaluated in the subsequent tests.
The approach using an IC50 value from an in vitro basal cytotoxicity
test to determine the concentration of compounding promoting 50%
cell viability is widely use (OECD 129, 2010). The 0% viability is
considered the maximum toxicity and 100% viability is considered non-
cytotoxic. At all tested concentrations evaluated, it was not possible to
calculate the IC50 value for EF, indicating that this is a non-cytotoxic
fraction as the maximum concentration did not inhibit 50% of the cells
used in the assay. In other words, the EF highest concentration tested,
do not presented cell toxicity (20 mg/mL) and it is much higher than
the proposed concentration (5 mg/mL) for the development of the
cosmetic moisturizing nanoemulsion. Thus, the polysaccharide-en-
riched fraction from A. sisalana by-product can be considered safe for
cosmetic application, and it was then possible to conduct the safety in
vivo tests.
Phototoxicity is an acute response generated after applying an ac-
tive substance to the cells, with subsequent exposure to ultraviolet light
and an absence of cytotoxicity. Thus, the cells were treated with dif-
ferent concentrations and exposed or not exposed to UVA light. Fig. 3
shows the results obtained for CE and EF.
A large number of cases of irritation or sensitization induced by
exposure to UVA radiation, after human skin contact with plants or
botanical ingredients, have been reported in the literature (Antignac
et al., 2011). A common feature of the substances that induce photo-
toxic eects is their ability to absorb the ultraviolet radiation (Freitas
and Gaspar, 2016).
The phototoxic potential of three dierent compounds (apigenin,
chrysin e beta-carotene) was evaluated by Freitas and Gaspar (2016).
Chrysin and beta-carotene presented a phototoxic prole when the PIF
values were greater than 1.0 and MPE superior to 0.15; these com-
pounds were then considered phototoxic. OECD TG 432 (2004) de-
scribes the parameters for the prediction of PIF and MPE, based on the
validation study: a substance with PIF < 2 or MPE < 0.1 predicts: no
phototoxicity. PIF values > 2 and < 5 or MPE > 0.1 and < 0.15 pre-
dicts:" likely phototoxicity and PIF > 5 or MPE > 0.15 predicts:"
phototoxicity.
The possibility of exposure to solar radiation when using cosmetic
products for skin care, indicate the importance of the evaluation of this
new eld for topical use.
3.2. Safety evaluation in vivo
Because of their complex chemical structure, plant ingredients may
contain potentially allergic or irritative substances. The skin compat-
ibility assessment provides a preview of possible irritative and or al-
lergenic mechanisms; these are main concerns when assessing the
safety of new ingredients for personal care (Antignac et al., 2011). In
the evaluated concentrations, none of the volunteers showed signs of
erythema, edema or papules on the skin; therefore, it is considered safe
for use in products for topical use. However, to obtain more data, it is
necessary to perform skin compatibility and sensitization tests by re-
petitive application and to conduct tests with a larger number of vo-
lunteers.

Fig. 3. Phototoxicity evaluation of the crude extract and polysaccharide-enriched fraction from Agave sisalana by-product.
In Which: (A) Phototoxicity evaluation of crude extract; (B) Phototoxicity evaluation of polysaccharide-enriched fraction from Agave sisalana by-product.

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S.M.A.G. Barreto et al. Industrial Crops & Products 108 (2017) 470479

Table 2
Nanoemulsion compositions (%, w/w).

COMPOUNDS N1 N2 N3 N4 N1v N2v N3v N4v

Caprylic/Capric 16.0 12.0 8.0 4.0 16.0 12.0 8.0 4.0

Triglyceride
EthylhexylPalmitate 16.0 12.0 8.0 4.0 16.0 12.0 8.0 4.0
IsostearylNeopentanoate 8.0 6.0 4.0 2.0 8.0 6.0 4.0 2.0
Phenoxyetanol (and) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Caprylic glicol
BHT 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Sorbitan Monooleate 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9
Polyssorbate 80 9.1 9.1 9.1 9.1 9.1 9.1 9.1 9.1
Dissodium EDTA 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Enriched fraction (EF) 0.5 0.5 0.5 0.5
Distilled Water 48.3 58.3 68.3 78.3 48.8 58.8 68.8 78.8

formulation, nanoemulsion were subjected to centrifugation 24 h after

Fig. 4. Pseudo-ternary phase diagram region showing formulations with nanoemulsion
characteristics.
3.3. Pseudo-ternary phase diagram
An important point in the development of the nanoemulsion is the
choice of its components. The composition of the formulations was
determined according to the nanoemulsions obtained by Ribeiro et al.
(2015b). A blend of emollients was selected to improve the perfor-
mance of the sensory of the formulations. The selection of mixed sur-
factants, Polysorbate 80 and Sorbitan Monooleate, was used following
the results achieved when these surfactants were used to obtain the O/
W emulsions with dierent EHL values (Ribeiro et al., 2015b; Mahdi
et al., 2011; Silva et al., 2016). The type of emulsion (O/W or water-in-
oil W/O) is based on the surfactant system used in the process of
emulsication; the concentrations of each phase (aqueous and oily)
inuence the process of formation of nanoemulsions (Ribeiro et al.,
2015b). The pseudo-ternary phase diagram of formulations is a useful
tool in the development of dispersed systems, because it is possible to
obtain formulations with improved performance, which are able to
produce dierent systems such as gels, emulsions, microemulsions and
nanoemulsions among others (Amaral-Machado et al., 2016). A na-
noemulsion region was obtained in the pseudo-ternary phase diagram
(Fig. 4).
Nanoemulsions are translucent, transparent or opaque systems, with
a droplet size of between 50 and 500 nm. They are formulated pre-
dominantly with an aqueous phase and consequently present low
viscosity, which can be obtained at surfactant concentrations in a range
of 510% (Gianeti et al., 2012; Izquierdo et al., 2002; Mahdi et al.,
2011; Jafari et al., 2006; Tadros et al., 2004). Formulations with a
surfactant concentration of 10%, oily phase concentrations between
10% and 40%, and 50% of aqueous phase were uid and an opaque
aspect. Rocha-Filho et al. (2016) used systems containing sunower oil
and mineral oil (7.5: 2.5, ratio w/w), obtained by the phase inversion
method, observed in the same pseudo-ternary phase diagram region O/
W emulsions with lamellar liquid crystalline phase. Amaral-Machado
et al. (2016) obtained a stable nanoemulsion in an oil concentration of
12% (bullfrog oil), 8% surfactants (Span 20 and Tween 80), and 80%
water. The composition of selected formulations, and their respective
vehicle formulations, are shown in Table 2.
3.4. Stability study
3.4.1. Preliminary stability
Initially, the nanoemulsions containing 0.5% of EF were light
yellow, homogeneous and uid. The nanoemulsions vehicle was white,
homogeneous and uid.
The centrifugation test was performed to obtain information on
possible instability processes (Ribeiro et al., 2015b). To choose the best
of preparation. Except for formulation N3 v, the samples showed no
change in their initial characteristics and stability.
3.4.1.1. Droplet size, polydispersity-index, and zeta-potential measure-
ments. The droplet size is an important parameter for evaluating the
stability of nanoemulsions. Due to the smaller droplet sizes, they
present greater stability against instability phenomena, considering
that small droplets reduce gravitational force enabling the Brownian
movement to overcome them (Kabri et al., 2011; McClements and Rao,
2011). However, the Ostwald ripening phenomenon appears as the
main instability mechanism of these systems (McClements, 2012).
Ostwald ripening causes increase in the droplet size through the
absorption of smaller droplets; thus, this phenomenon occurs due to
dierences in the Laplace pressure as well as the dierence in solubility
of the droplets. Thus, the droplet size increases, aecting the stability of
the system and can result in phase separation (Gianeti et al., 2012;
McClements and Rao, 2011). Nanoemulsions can be obtained by using
low energy methods based on their physical or chemical characteristics
or high energy methods. The high pressure methods are considered
superior because they provide nanoemulsions of smaller droplet sizes
and polydispersity, associated with better stability (Peshkovsky et al.,
2013; Tadros et al., 2004). The results of the preliminary stability tests
are shown in Table 3.
After 24 h of the preparation (t0), the samples showed the mean
droplet size from 83 nm to 155 nm. The formulations N1 and N1v did
not present signicant increase in droplet size. For N3, N4 and N4v, we
can observe the signicant reduction (p > 0.05) in the PDI values and
a signicant increase in droplet size, indicates relatively instability in
formulations. The solubility of the dispersed phase in the continuous
phase is a critical factor in the formation of Ostwald ripening (Gupta
et al., 2016). Wooster et al. (2008) evaluated the inuence of the oily
composition on the formation of Ostwald ripening and stated that the
insolubility of the oils provides a kinetic barrier capable of preventing
the appearance of this phenomenon of nanoemulsion instability, and
suggested that there were proportions of at least 50% of insoluble oils in
the phase. McClements and Rao (2011) observed similar results and
reported that the addition of 10% of insoluble oils in their formulations
was sucient to maintain the stability of the nanoemulsions.
The zeta potential is a parameter used to evaluate the nanoemulsion
stability (Mahdi et al., 2011; Ribeiro et al., 2015b; Tichota et al., 2014).
It indicates the force of repulsion between adjacent equally charged
droplets, and thus demonstrates the important role of the surface layer
of the droplets in the process of stabilization. High values of zeta po-
tential (> 30 mV) indicate resistance to particle aggregation and
consequently a greater stability (Bali et al., 2010). However, in addition
to electrostatic stabilization, the addition of nonionic stabilizers to
colloidal systems can promote steric stabilization achieved by the sol-
vation eect (Wu et al., 2011).
The formulations presented negative zeta potential values and less

475
S.M.A.G. Barreto et al. Industrial Crops & Products 108 (2017) 470479

Table 3
Mean droplet diameter (Z-average) polydispersity index (PDI) and zeta potential (ZP) values of preliminary stability tests of nanoemulsions with and without the polysaccharide-enriched
fraction.

Formulation Z-average (d.nm) PDI Zeta Potencial (mV) pH

(t0) (t5) (t0) (t5) (t0) (t5) (t0) (t5)

N1v 155.80 3.99 158.60 3.33 0.09 0.02 0.07 0.00 * 20.41 2.58 16.01 0.43 5.01 0.05 5.17 0.06
N2v 130.0 1.99 134.0 1.92 * 0.12 0.02 0.09 0.01 17.33 1.51 14.23 1.13* 4.88 0.32 5.24 0.11
N3v PS PS PS PS PS PS PS PS
N4v 84.69 1.64 160.01 15.81* 0.24 0.00 0.05 0.03 * 13.57 1.30 12.70 0.75 5.21 0.04 5.33 0.06 *
N1 155.0 1.29 158.4 2.91 0.10 0.01 0.09 0.01 17.67 0.40 12.27 1.88 4.47 0.09 4.48 0.01
N2 124.3 4.50 PS 0.12 0.01 PS 14.67 1.24 PS 4.43 0.02 PS
N3 113.5 1.24 133.2 7.30 * 0.17 0.00 0.09 0.01* 13.02 1.8 11.30 0.44 4.42 0.02 4.45 0.03
N4 83.57 2.35 188.07 7.30 * 0.23 0.00 0.05 0.00 * 10.64 1.73 10.67 0.67 4.42 0.02 4.44 0.01

In Which: * = p-value < 0.05 for students t-test (paired). PS = Phase separation.

than |30|Mv (Table 3). N2v showed signicant reduction (p > 0.05) in
the zeta potential values after storage at 45 C. Negative zeta potential
values can be related to the adsorption of hydroxyl ions from the
polyoxyethylene chains present in the surfactants used (Polysorbate 80
and Sorbitan Monooleate) (Mahdi et al., 2011).
Topical formulations must have a pH value compatible with skin
(4.56.0) (Ribeiro et al., 2015b). Except for formulation N4v, no change
was observed in the pH values after storage at 45 2 C; this indicates
that there were no modications in the components of the formulations.
N1 e N1v formulations were considered stable under conditions of
preliminary stability tests and were therefore evaluated by the ac-
celerated stability tests.
3.4.2. Accelerated stability assessment
Table 4 presents the results for the mean diameter (Z-average),
polydispersity index (PDI), and zeta potential (ZP) values of the na-
noemulsions after 24 h of production and after 90 days of storage in
dierent conditions.
No signicant variations in mean diameter and polydispersity index
values were observed at temperatures of 4 C and 25 C indicating
stability. In contrast, a variation in Z-average values was observed with
nanoemulsions stored at 45 C. The increase in droplet size may cause
instability in terms of phase separation (Saberi et al., 2013). According
to Komaiko and McClements (2014), these changes in droplet size may
be associated with the sensitivity of nonionic surfactants to tempera-
ture. Similar to that observed in our studies, Luo et al. (2017) observed
changes in droplet size in nanoemulsions due to exposure to higher
temperatures. These authors obtained stable nanoemulsions with
changes in droplet size when stored at temperatures of 55 C for a
period of 14 days, with a small droplet aggregation not observed for the
samples stored at 4 C and 25 C.
Carpenter and Saharan (2017) used the same methodology (ultra-
sonication) of obtaining and the same surfactants, they observed a
proportional increase in the droplet size of nanoemulsions, when ex-
posed to the temperatures of 40 C, 60 C and 80 C for 90 days. When
these authors increased the sonication time of the samples from 15 to
30 min, the formulations showed no change in the droplet size in these
temperature conditions. Thus, we can suggest that an increase in the
time of preparation of the our nanoemulsions could inhibit the changes
caused by the exposure of nanoemulsions to higher temperatures.
During storage time, there was no macroscopic evidence of instability
phenomena such as cremation, coalescence or occulation, attributed
to the droplet size remaining in the manometer scale during the eva-
luation period.
Despite the signicant increase in droplet size at 45 C, the PDI
values were not signicantly altered and remained at around 0.1.
Ribeiro et al. (2015b) and Saberi et al. (2013) observed changes in
droplet size without signicant changes in polydispersity index values.
Thus, it can be stated that because nanoemulsions do not present such
changes, and when compared to their initial characteristics, can be
considered stable (Ribeiro et al., 2015b).
The droplet size distribution by intensity evaluation showed an
occurrence of a high intensity peak, monomodal type (Fig. 5). In ad-
dition, a lower PDI values of the formulations indicated the stability of
systems and the eciency of the ultrasound method to obtain stable
nanoemulsions.
After 90 days of storage at 4 C, 25 C, and 45 C, the formulations
showed no change in the zeta potential values, indicating stability for
the nanoemulsions. The lower values of zeta potential can be due the
steric stabilization mechanism (Wu et al., 2011).
In this context we can consider that both the vehicle (N1v) and the
formulation containing 0.5% polysaccharide-enriched fraction (N1)
were stable.
In the rst stage of this research, we obtained the raw material and
evaluated the safety of the polysaccharide-enriched fraction from A.
sisalana by-product. We then conducted the development of the stable
nanoemulsions. Finally, formulations added or not added with poly-
saccharide-enriched fraction were subjected to clinical tests to evaluate
their moisturizing ecacy.

Table 4
Results of accelerated stability tests after 24 h and 90 days. Mean diameter (Z-average) polydispersity index (PDI) and zeta potential (ZP) values of the nanoemulsion.

Evaluated Initial values (24 h) Nanoemulsion N1 Initial values (24 h) Nanoemulsion N1v
parameters
After 90 days After 90 days

4 C 25 C 45 C 4 C 25 C 45 C

Z-average 171. 23 3.49 165.0 4.27 174.86 2.29 237. 84 3.84* 174. 81 5.37 172.71 10.6 175.97 6.19 215.79 11.11
(d.nm)
PDI 0.12 0.02 0.09 0.02 0.09 0.01 0.08 0.03 0.11 0.02 0.09 0.03 0.10 0.02 0.09 0.04
ZetaPotential 15. 77 2.20 18. 02 1.78 16. 10 2.73 18. 91 3.14 23. 03 1.94 20.78 2.19 22. 78 3.04 19.04 2.31
(mV)

In which: * = p < 0.05 for ANOVA (one way); v = vehicle.

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S.M.A.G. Barreto et al. Industrial Crops & Products 108 (2017) 470479

Fig. 5. Droplet size distribution (intensity vs droplet diameter) obtained by Dynamic Light Scattering.

Table 5 product were signicantly higher (p < 0.05) than the vehicle for-
Water content values of the stratum corneum (Arbitrary Units) before (baseline) and 1, 2, mulation. This result showed that the moisturizing property is from
3, 4 and 5 h after single application of the formulations. The results are shown in mean
polysaccharide-enriched fraction and not from vehicle components of
values standard error of the mean.
the formulation.
Time A1 A2 A3 A4 The moisturizing capacity of the polysaccharide-enriched fraction
can be related to the compounds present in the chemical constitution
Baseline 39.32 1.86 37.51 1.59 39.05 1.87 37.86 1.86abc
such as polysaccharides (results no shown). Futrakul et al. (2010)
1 37.29 1.84 41.74 1.14 44.37 1.57a 58.66 2.56abc
2 38.66 1.98 46.81 1.09a 50.84 1.22ab 58.64 2.65abc
evaluated the moisturizing capacity of a polysaccharide gel obtained
3 40.73 2.05 49.74 1.40a 53.07 1.46ab 58.44 2.75abc from Durio zebethinus Murr. and demonstrated the ability of these
4 42.74 1.91 47.99 1.70a 52.18 1.51ab 57.80 2.47abc constituents to promote the increase in the water content of the stratum
5 44.30 2.38 47.98 2.03 52.84 1.84ab 58.52 2.89abc corneum. Polysaccharides biotechnologically obtained from Klebsiella
pneumoniae and polysaccharides obtained from Myrtus communis extract
In which: A1 Negative control; A2 Vehicle; A3- Nanoemulsion containing 0.5% of the
polysaccharide-enriched fraction; A4 Positive control (commercial moisturizer);
promoted increased hydration of the skin (Camargo Junior et al., 2012).
a = statistically dierent from A1, b = statistically dierent from A2; c = statistically According to DalBello et al. (2006) the association of polysaccharides
dierent from A3; p-value < 0.05 for ANOVA + Tukey. and amino acids (histidine, arginine, threonine, serine, glycine and
alanine) was able to promote increase of skin hydration. Table 6 shows
3.5. In vivo evaluation of skin hydration the transepidermal water loss values obtained from the treated areas
(A2, A3 and A4) and untreated area (A1).
The evaluation of the water content on the stratum corneum was
assessed by corneometry based on the changes in the skin capacitance
caused by the increase or decrease in its aqueous content. The barrier Table 6
Transepidermal water loss (g/m2/h) values before (baseline) and 1, 2, 3, 4 and 5 h after
function of the skin was evaluated by transepidermal water loss, by
single application of the formulations. The results are shown in mean values standard
measuring the density gradient of the water evaporation from the skin error of the mean.
(Byrne. 2010). Table 5 shows the results obtained for the water content
of the stratum corneum. Time A1 A2 A3 A4
When compared to the negative control (A1), the vehicle (A2) sig-
Baseline 11.33 1.09 12.54 1.05 12.90 1.03 14.17 1.20
nicantly increased (p < 0.05) the aqueous content of the stratum 1 11.61 1.09 12.09 1.12 12.13 1.21 12.97 1.15
corneum, and this eect continued until the fourth hour of the study. 2 12.06 1.11 12.45 1.17 12.92 1.13 13.03 1.06
However, the nanoemulsion containing 0.5% of the polysaccharide- 3 11.77 0.95 12.84 1.02 12.79 0.98 13.32 0.98
enriched fraction (A3) achieved hydration eect of the skin from the 4 11.57 1.05 11.94 1.00 12.33 1.06 13.05 1.13
5 12.22 1.01 12.98 0.93 12.98 1.03 12.94 1.09
rst hour and this property was maintained throughout the study
period (5 h). From the second hour, the hydration values achieved by In which: A1 Negative control; A2 Vehicle; A3- Nanoemulsion containing 0.5% of the
the nanoemulsion containing the fraction from the A. sisalana (EF) by- polysaccharide-enriched fraction; A4 Positive control (commercial moisturizer).

477
S.M.A.G. Barreto et al.
There were no statistically signicant changes in the transepidermal
water loss. Similar results were observed by Ribeiro et al. (2015b)
wherein nanoemulsions containing Opuntia cus-indica hydroglycolic
extract promoted increase in the water content in the supercial layer
of the skin and maintained unchanged the skin barrier condition. Ac-
cording to DalBello et al. (2006), the ability to increase the water levels
in the supercial layer of skin combined with non-modication in water
loss values may be related to the hygroscopic capacity of mono- and
polysaccharides present in extracts.
The hygroscopic characteristic of these compounds combined with
the higher permeability obtained by the nanoemulsions can promote
the permeation of the active compounds by the stratum corneum and,
thus by extension, the retention of water (DalBello et al., 2006; Lu
et al., 2014). However, it should be noted that the type of vehicle can
inuence the mechanism of action by which the moisturizer active can
act (Damasceno et al., 2016).
According to Lden (2012), the reduction of transepidermal water
loss is dependent on the amount of lipid content present in the for-
mulations. Thus, the predominantly aqueous content of nanoemulsions
may be the reason for the non-formation of lm capable of reducing the
loss of skin water. On the other hand, the increase in water content
caused by presence the actives that promote the retention of water in
the stratum corneum may support the increase in the transepidermal
water loss (Draelos, 2000; Ribeiro et al., 2015b).
Finally, through this work, it was possible to nd that EF, a raw
material obtained from the by-product of the sisal industry, can be
considered as a new raw material for the cosmetic industry with
moisturizing ecacy. This work contributes to the productive chain of
A. sisalana, thus proposing other uses for the by-product that can have
an environmental impact. This can also have socioeconomic contribu-
tion, especially for families, who are nancially dependent on sisal
cultivation and production.
4. Conclusion
The industry of sisal (Agave sisalana) produces a large amount of by-
product that is disposed into the environment. This study has con-
tributed to the cosmetic and environmental area by enabling a dis-
carded material to become a marketable raw material. A poly-
saccharide-enriched fraction was obtained from the crude extract of the
by-product of the processing of the leaves of A. sisalana. In vitro and in
vivo tests have demonstrated that the fraction for topical use is safe,
does not show cytotoxicity or phototoxicity, and can be considered non-
irritating. A nanoemulsion formulation of 40% oily phase, 50% aqueous
phase, and 10% surfactant additive with 0.5% of the fraction was ob-
tained and assessed as stable for a period of 90 days. The nanoemul-
sions containing the polysaccharide-enriched fraction showed moist-
urizing properties, improving both the increase in the water content of
the stratum corneum, and it maintained the skin barrier function after
5 h of a single application. Thus, a nanocosmetic product was devel-
oped and it will contribute to biodiversity appreciation and social-
economic development, enabling the use of the industrial residue to be
a potential raw material for skin care.
Acknowledgments
The authors are grateful to Sisaltec Industry, Croda of Brazil Ltda.,
Lubrizol of Brazil Ltda. and International Pharmaceutical Immunology
do Brasil Ltda (IPI ASAC Brasil) for providing free sample materials. The
authors would also like to thank the Laboratrio de Sistemas Dispersos
(LASID-UFRN). The authors are thankful Federal University of Rio
Grande do Norte (UFRN), Conselho Nacional de Desenvolvimento
Cientco e Tecnolgico (CNPq) and Comisso de Aperfeioamento de
Pessoal do Nvel Superior (CAPES) for supporting this work.
478
Industrial Crops & Products 108 (2017) 470479
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