Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 40-44
http://dx.doi.org/10.4236/jcdsa.2013.33A1006 Published Online September 2013 (http://www.scirp.org/journal/jcdsa)
Hair Protective Effect of Argan Oil (Argania spinosa
Kernel Oil) and Cupuassu Butter (Theobroma
grandiflorum Seed Butter) Post Treatment with Hair Dye
Pamella Mello Faria1, Luciana Neves Camargo1, Regina Siqueira Haddad Carvalho1,
Luis Antonio Paludetti1, Maria Valéria Robles Velasco2, Robson Miranda da Gama1,3*
1Pharmacy School, University of Santo Amaro, São Paulo, SP, Brazil; 2School of Pharmaceutical Sciences of University of São
Paulo, São Paulo, Brazil; 3School of Pharmaceutical Sciences of Faculty of Medicine ABC, Santo André, Brazil.
Received May 30th, 2013; revised June 28th, 2013; accepted July 5th, 2013
Copyright © 2013 Pamella Mello Faria et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Hair coloring is widely used by women and men either to change their natural hair color or to delay the onset of gray
hair. Oxidative dyes may damage the hair, since chemical and physical procedures are involved to alter the structure
hair and consequently, alterations in its mechanical and of surface properties. One benefit of hair conditioners is to pre-
vent flyaway hair, make the hair “shine”, and protect the hair from further damage. In this research we analyzed the hair
protective effect conditioner agents Argania spinosa kernel oil and/or Theobroma grandiflorum seed butter in hair care
on Caucasian hair post treatment with hair dye. The hairs were submitted by quantifying protein loss. The samples were
classified as: hair untreated (I); hair treated with a commercial oxidative ultra-blond hair dye (II); hair post treatment II
and F1: Base hair care formulation (III), hair post treatment II and F2: Base hair care formulation containing 1.0% (w/w)
Argania spinosa kernel oil (IV), hair post treatment II and F3: Base hair care formulation containing 1.0% (w/w) Theo-
broma grandiflorum seed butter (V) and hair post treatment II and F4: Base hair care formulation containing 0.5% (w/w)
Argania spinosa kernel oil and 0.5% (w/w) Theobroma grandiflorum seed butter (VI). For the protein loss, the results
were: IIA = IIIA > IB = IVB = VB = VIB. Results classified with different letters present statistically significant differents,
for α = 5, p ≤ 0.05, n = 6. Based on the results, the incorporation of conditioners agents Argania spinosa kernel oil
and/or Theobroma grandiflorum seed butter in base hair care formulation applied in Caucasian hair post treatment with
hair dye decreased the damage caused to hair by the coloring process.
Keywords: Protein Loss; Damage Hair; Argania spinosa Kernel; Theobroma grandiflorum
1. Introduction
The human hair is composed of protein, lipid, water,
melanin and trace elements. The main constituents of
hair are of α-keratin, a group of proteins which account
for 65% - 95% of hair weight. It is responsible for con-
ferring mechanical properties such as elasticity, shape,
strength and functionality [1].
The human hair presents three principal components:
cuticle, cortex and medull which are spectively from out-
side to inside. The cuticle is composed of protein mate-
rial and amorphous, and it is located in the outer portion
of the hair fiber and consists of enucleate cells, translu-
cent and flattened. Morphologically, the cuticle is com-
posed of 6 to 8 cell layers overlapped in the longitudinal
direction of the fiber. The overlapping cell adherence
provides the physical properties of hair with reflection
light and reduces the friction between the fibers being
responsible for the properties of gloss and combing, re-
spectively. Cosmetic treatments, such as conditioners,
hair sprays, mousses and gels, alter the properties men-
tioned above because they are deposited on the cuticle
layer. However dyes and straightening products due to
the alkaline pH of the cuticle open up the layers for the
active principles or dyes penetrate and act in the cortex,
reducing the size or altering the color of hair. The cortex
is a major constituent of the hair fiber (75%). Cortical
cells are subdivided into macrofibrils formed per material
interfilamentar amorphous rich sulfur and microfibrils
*Corresponding author.

Hair Protective Effect of Argan Oil (Argania spinosa Kernel Oil) and Cupuassu Butter
(Theobroma grandiflorum Seed Butter) Post Treatment with Hair Dye
41
arranged in α-helix, consisting of four protofibrils, and
these two protofilaments, dimers possessing two α-kera-
tin subunits. The α-keratin presented in the microfibrils
determines the mechanical properties of fiber, such as
strength and elasticity. In the same way as the cuticle, it
has cells filled by cross links of cystine and others cells
separated by the cell membrane complex (CMC). The
medulla is a thin cylindrical layer at the center of the hair
thread may or may not be present; it is presented only in
terminal hair and its role is not clearly defined [1-3].
Hair coloring is widely used by women and men either
to change their natural hair color, and to delay the onset
of gray hair, or to donate new pigments to gray hair [3].
The oxidative dyes are formed by two components, in
cosmetic base as emulsions or gels, which are mixed and
provide the coloration by chemical reactions in alkaline
and/or oxidant medium, in the cuticle and cortex of the
hair fiber. Oxidative dyes may damage the hair, since
chemical and physical (exposure of cortex because of
damage of cuticle) procedures are involved to alter the
hair color [4,5].
When exposed, the hair fiber, with the adverse envi-
ronmental conditions as solar radiation, wind, wet, pollu-
tion and the daily care routines and/or cosmetic treat-
ments including permanents and dyes among others can
present damages in its structure and, consequently, al-
terations are in its mechanical and of surface properties.
Damaged hair can appear cloudy, dry, rough, fragile and/
or dull [5].
The primary function of hair conditioners is to make
the hair easier to comb due to reduction in antistatic pro-
perty to the hair. Secondary benefits such as preventing
flyaway hair, making the hair “shine”, and protecting the
hair from further damage are also important functions to
hair conditioners. Substances that perform this function
are usually silicones, polyquats, cationic surfactant, hy-
drolyzed proteins, fatty alcohols, fatty esters, vegetable
oils, mineral oils, or humectants [6,7].
Rele and Mohile [8] established the superiority of the
protective effect of coconut oil on hair damage in groom-
ing processes when it is used as a pre-wash conditioner
as compared to mineral oil and other vegetable oils such
as sunflower oil. It not only has a protective effect on un-
damaged hair but also on chemically treated hair, UV-
treated hair, and hair treated with boiling water (i.e., hair
in water at 100˚C for 2 hr). The ability of coconut oil to
penetrate into hair cuticle and cortex seems to be respon-
sible for this effect. In general, saturated and monoun-
saturated oils penetrate into the hair because of a com-
pact molecular structure and the polar head group of the
triglyceride molecules that constitute these oils [9].
Argan oil is prepared from the fruits of argan trees
(Argania spinosa (L.) Skeels) following a multistep proc-
ess. The argan tree (Argania spinosa (L.) Skeels; Sapota-
ceae) is a slow-growing tree exclusively endemic to the
barren lands of southwest Morocco. The argan oil is con-
stitute of acylglycerols, including 95% of triacylglycerols,
constitute 99% of extract. The remaining 4% are com-
posed of monoacylglycerols (0.27% - 0.65%), diacyl-
glycerols (0.68 - 1.53), and free fatty acids (1.1% - 2.0%)
[10].
Fat from seeds of Theobroma grandiflorum (cupuassu)
has been particularly investigated because of its increas-
ing demand as a new fruit crop. There is an increasing
market for natural products, and the Brazilian Theo-
broma species could be used as alternative source of spe-
cial fats. The percentage composition of fatty acids in the
fat of cupuassu is 58.13%, 39.19% and 2.61% respec-
tively for saturated, monounsaturated and polyunsatu-
rated fatty acids [11].
In this research we analyzed the hair protective effect
conditioner agents Argania spinosa kernel oil and/or
Theobroma grandiflorum seed butter in hair care on Cau-
casian hair post treatment with hair dye. The hairs were
submitted by quantifying protein loss.
2. Materials and Methods
2.1. Hair Samples
Caucasian virgin dark brown hair tresses of 20.0 cm in
length, purchased from Bella Hair® (Brazil), were used.
Each hair strand was washed for 30 s with 15.0% (w/v)
sodium lauryl sulphate to remove impurities. All were
wetted with warm distilled water (37.0˚C ± 2.0˚C) con-
stant flow of 240.0 mL·min−1 for 1 min and the excess of
water was first removed by passing the tresses three
times between the fingers and then they were dried on
paper towel, for 12 h dried at room temperature (22.0˚C
± 1.0˚C) and relative humidity (RH 60% ± 5%) prior to
the analysis [12].
After drying, the tresses were treated with a comer-
cial oxidative ultra-blond hair dye (Niely®), color 10.0,
composed by: aqua, cetearyl alcohol, propylene glycol,
deceth-3, laureth-12, ammonium hydroxide, oleth-30,
hexadimetrine chloride, lauric acid, glycol distearate,
polyquaternium-22, ethanolamine, silica dimethyl saly-
late, CI 77881, 2,4-diamophenoxyethanol HCl, p-amino-
phenol, m-aminophenol, ascorbic acid, sodium metabis-
sulfite, p-phenilenodiamine, pentasodiumpentetate, car-
bomer, dimethicone, resorcinol and parfum (fragrance).
The dye was mixed with hydrogen peroxide (30 vol) in a
ratio of 1:1 (w/w) and applied to hair tresses in a ratio of
1:1 (w/w). The reaction occurred for 40 min. After this
period, the tresses were washed according to the meth-
odology described, and left to dry at room temperature.

Hair Protective Effect of Argan Oil (Argania spinosa Kernel Oil) and Cupuassu Butter
(Theobroma grandiflorum Seed Butter) Post Treatment with Hair Dye
42
2.2. Hair Care Formulations Development
A hair care formulation was prepared according to the
composition in Table 1.
Conditioner agents were incorporated into the formu-
lation, according to Table 2. The pH was adjusted to a
4.5 value. Hair care formulations (Table 2) were applied
to the hair tresses, in a 1:0.5 (hair:formulation) ratio, be-
fore each assay for protein quantification. The applying
procedure was performed with gentle movements assur-
ing that the product distribution was uniform.
The emulsion base hair care formulation was prepared
for conventional method in which oil and aqueous phases
are heated at 70.0˚C - 75.0˚C and the aqueous phase are
Table 1. Qualitative and quantitative composition of base
hair care formulation.
INCIa component Proportion
(% w/w)
Oil phase
Cetearyl alcohol (Mapric®) 5.00
Cetearyl alcohol (and) behentrimonium methosulfate
(Mapric®) 2.00
Cocoamide DEA (Mapric®) 1.00
BHT(Mapric®) 0.05
Aqueous phase
Propylene glycol(Mapric®) 1.00
PEG-12 dimethicone (Dow Corning®) 1.00
Cetrimonium chloride (Mapric®) 5.00
Phenoxyethanol (and) methylparaben (and)
ethylparaben (and) butylparaben (and)
isobutylparaben (Croda®)
0.30
Dissodium EDTA (Mapric®) 0.05
Citric acid (Mapric®) q.s. pH 4.5
Aqua 84.60
Legend: INCIa: International Nomenclature of Cosmetic Ingredient.
Table 2. Proportion of conditioner agents Argania spinosa
kernel oil and/or Theobroma grandiflorum seed butter in
hair care formulations.
Proportion of components
incorporated (% w/w)
INCIa component
F1 F2 F3 F4
Argania spinosa kernel oil
(Beraca®) - 1.0 - 0.5
Theobroma grandiflorum
seed butter (Croda®) - - 1.0 0.5
Legend: F1: Base hair care formulation; F2: Base formulation containing
1.0% (w/w) Argania spinosa kernel oil; F3: Base formulation containing
1.0% (w/w) Theobroma grandiflorum seed butter; F4: Base formulation
containing 0.5% (w/w) Argania spinosa kernel oil and 0.5% (w/w) Theo-
broma grandiflorum seed butter; (–) not added. INCIa: International No-
menclature of Cosmetic Ingredient.
added to the oil phase, gradually and continuously, with
stirred until 45˚C, when the conditioning agents were
added in the base hair care formulation in accordance
with Table 2.
The samples were classified as: hair untreated (I); hair
treated with a commercial oxidative ultra-blond hair dye
(II); hair post treatment II and F1: Base hair care formu-
lation (III), hair post treatment II and F2: Base hair care
formulation containing 1.0% (w/w) Argania spinosa ker-
nel oil (IV), hair post treatment II and F3: Base hair care
formulation containing 1.0% (w/w) Theobroma grandi-
florum seed butter (V) and hair post treatment II and F4:
Base hair care formulation containing 0.5% (w/w) Ar-
gania spinosa kernel oil and 0.5% (w/w) Theobroma
grandiflorum seed butter (VI).
2.3. Assay for Protein Quantification
This assay was based on the reduction of the Folin re-
agent by protein previously treated by copper in alka-
linemedium. A copper atom bonds to four residuals of
amino acid. This complex reduces the Folin reagent, be-
coming the solution blue. In this work, the Lowry me-
thod modified by Peterson was used [13,14].
Samples of 0.10 g, in 15.0 mL of distilled water, were
sonicated (Ultrasonic Clean® 1600 unique) for 40 min
[15]. Protein loss was determined from 2.0 mL super-
natant aliquots, which were added to 2.0 mL of Re-
agent A, and waited for 10 minutes for the reaction
occurs. After this time it was added 1.0 mL of Reagent B,
waited for 30 minutes, in the dark conditions in order to
complete the reaction [16,17].
In a first step, the hair protein and the secondary stan-
dard Bovine Serum Albumin (BSA) one react with Re-
agent A withcupric ions (Cu2+), in alkaline medium with
sodium hydroxide and sodium carbonate (buffer with pH
about 10.0). In this stage, sodium potassium tartarate is
also used to avoid copper precipitation and, this way, in-
creasing the solution stability. It is believed that the com-
plexation of cupric ions (Cu2+) with peptide bonds leads
it to the reduction to cuprous ions (Cu+). Ten minutes are
waited for the reaction processing. The production of cu-
prous ions is followed by the reduction of Folin reagent
(Reagent B) in the second stage of the assay, which turns
the solution blue. Wait for 30 minutes in order for the
reaction occurs [13,14].
Quantification was performed in Micronal® B-542UV-
Visible spectrophotometer with a 1 cm quartz cuvette at
750.0 nm. Analytical curve was obtained with BSA, and
used distilled water as blank. The calculation equation of
the analytical curve was made by linear regression using
the least squares method and calculating the linear corre-
lation coefficient. The Equation (1) used to calculate the
results. This method previously validated by Gama [18].

Hair Protective Effect of Argan Oil (Argania spinosa Kernel Oil) and Cupuassu Butter
(Theobroma grandiflorum Seed Butter) Post Treatment with Hair Dye
43
2
y0.0028x0.0402 with R0.992  (1)
2.4. Statistical Analyses
Possible significant differences in the results were ana-
lyzed by Kruskal-Wallis and the differences between
treatments were identified by Student-Newman-Keulstest
(α = 0.05).
3. Results
Protein loss albumin equivalent of hair samples for: un-
treated (I); treated with a commercial oxidative ultra-
blond hair dye (II); post treatment II and F1: Base hair
care formulation (III), post treatment II and F2: Base
hair care formulation containing 1.0% (w/w) Argania
spinosa kernel oil (IV), post treatment II and F3: Base
hair care formulation containing 1.0% (w/w) Theobro-
ma grandiflorum seed butter (V) and post treatment II
and F4: Base hair care formulation containing 0.5% (w/w)
Argania spinosa kernel oil and 0.5% (w/w) Theobroma
grandiflorum seed butter (VI) are shown in Figure 1.
4. Discussion
During the coloration process, the hair dyes provide the
opening of the cuticle, and optimizing the absorption of
the colorants into the cortex. This mechanism reduces the
Figure 1. Protein loss albumin equivalent from Caucasian
hair tresses before and after application of the oxidative dye
hair emulsions either with or without conditioners agents
(Protein loss was expressed in μg protein albumin equi-
valent/g hair). Legend: untreated hair (I); hair treated with
a commercial oxidative ultra-blond hair dye (II); hair post
treatment II and F1: Base hair care formulation (III), hair
post treatment II and F2: Base hair care formulation con-
taining 1.0% (w/w) Argania spinosa kernel oil (IV), hair
post treatment II and F3: Base hair care formulation con-
taining 1.0% (w/w) Theobroma grandiflorum seed butter (V)
and hair post treatment II and F4: Base hair care formula-
tion containing 0.5% (w/w) Argania spinosa kernel oil and
0.5% (w/w) Theobroma grandiflorum seed butter (VI). Re-
sults classified with different letters presents statistically
significant differents, for α = 5, p < 0.05, n = 6.
softness, brightness and difficult to comb of hair are the
attributes of healthy hair [4].
Hair fibers are constituted mainly by protein. The
study of the damage hair shaft can evolve the quantifica-
tion of protein loss after the coloring process on hair
tresses and one option is the Lowry method modified by
Peterson. The higher the greater the damage to cuticle
protein loss compared to virgin hair [5].
The analyses of the protein loss from Caucasian hair
tresses before and after application of the oxidative dye
hair emulsions either with or without conditioner agents
(Figure 1) showed difference in total protein loss be-
tween undamaged hair (I treatment) and hair damage
after coloring process with (II treatment). The results
confirmed by Robbins and Crawford [19] described that
oxidative process of hair fibers produce extensive dam-
age throughout several cuticle layers.
In this study, we observed (Figure 1) that using condi-
tioners hair (III, IV, V and VI treatments) has a positive
effect on reducing of protein loss in hair post treated with
a commercial oxidative ultra-blond hair dye. The effec-
tiveness of (III treatment) are expected, since the base
hair care formulation contains a cationic compounds
(cetrimonium chloride and behentrimonium methosul-
fate), which is substantive to hair and adsorbs on hair
surface following a charge-driven mechanism [6,7] and
silicone (PEG-12 dimethicone) that involved their ad-
sorption to the hair fiber, because of its hydrophobic
characteristics that reduce the intermolecular forces and
surface tension leading to the formation of a hydrophobic
film other the cuticle [20].
The addition of Argania spinosa kernel oil and/or
Theobroma grandiflorum seed butter in the base hair care
formulation (IV, V and VI treatments) statistically re-
duced the protein loss when compared to just base for-
mulation (III treatment). This difference in results could
arise from the composition of each of these oils. Keis et
al. [9] have compared the ability of different oils (min-
eral oil, sunflower oil, and coconut oil) to penetrate into
hair fibers, showing that their affinity to hair fiber de-
pends on various factors, such as oil polarity, chain satu-
ration and molecular weight.
Rele and Mohile [8] have compared the ability of dif-
ferent oils (mineral oil, sunflower oil and coconut oil)
which were used as a pre-wash conditioner to prevention
of hair damage measured by protein loss post chemically
treated hair. Coconut oil, being a triglyceride of lauric
acid (principal fatty acid), has a high affinity for hair
proteins and it is able to penetrate inside the hair shaft
because of its low molecular weight and straight linear
chain.
The two main fatty acids found in Argania spinosa
kernel oil acylglycerols are oleic acid (46% - 48%) and

Hair Protective Effect of Argan Oil (Argania spinosa Kernel Oil) and Cupuassu Butter
(Theobroma grandiflorum Seed Butter) Post Treatment with Hair Dye
44
linoleic acid (31% - 35%), a monounsaturated and diun-
saturated fatty acid, respectively [10]. However the major
fatty acids presented in Theobroma grandiflorum seed
butter have: saturatedfatty acid (palmitic acid (11.25%),
stearic acid (38.09%), arachidonic acid (7.97%)), mono-
unsaturated fatty acid (oleic acid (38.79%)), and diun-
saturated fatty acid (linoleic acid (2.39%) [11].
Introduction of this hydrophobic component reduces
the swelling propensity of the cuticle, which limits the
upward curving of the surface cuticle. This reduces the
chipping away of the cuticle cells which reduces protein
loss, as observed in this work.
Considering the assays performed quantification of
protein loss the conditioner agents Argania spinosa ker-
nel oil and Theobroma grandiflorum seed butter allowed
a decrease in protein loss albumin equivalent when added
separately (IV or V treatment) or together (VI treat-
ment) in base hair care formulation applied in Caucasian
hair post treatment with hair dye decreased the damage
caused to hair by the coloring process.
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