Dyes and Pigments 97 (2013) 9e18

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Dyes and Pigments

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

Investigation on the dyeing power of some organic natural compounds for a green
approach to hair dyeing
Carla Boga a, *, Camilla Delpivo a, Barbara Ballarin b, Marco Morigi c, Samanda Galli c, Gabriele Micheletti a,
Silvia Tozzi a
a
Dipartimento di Chimica Organica ‘A. Mangini’, Alma Mater Studiorum, Università di Bologna, Viale del Risorgimento 4, 40136 Bologna, Italy
b
Dipartimento di Chimica Fisica e Inorganica, Viale del Risorgimento 4, 40136 Bologna, Italy
c
Ilios s.r.l., via L. Basso 170, 47522 Cesena, Italy

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

Article history: Organic compounds present in plants have been used in various experimental conditions for dyeing tests
Received 29 August 2012 aimed to develop safe and environmentally friendly temporary and semipermanent hair dyes. Yak hairs
Received in revised form were used as a model for the colorimetric evaluation of red, yellow, blue, and brown shades conferred to
16 November 2012
hair by selected natural compounds. Two different sources for red, yellow, blue and brown shades were
Accepted 20 November 2012
Available online 29 November 2012
tested. Anthocyanins from mulberry fruits and alizarin emerged as promising candidates for red shades,
anthocyanin-blue and curcumin for blue and yellow, respectively, and p-benzoquinone and juglone for
browns. The influence of pH, dye concentration, soaking time, and medium in which the dyes have been
Keywords:
Vegetal dyes
dissolved or dispersed has been studied.
Hair Ó 2012 Elsevier Ltd. All rights reserved.
Anthocyanins
Yak
Colorimetry
CIELAB

1. Introduction a permanent change to hair color. Semi-permanent hair dyes

The production and the use of compounds both safe and envi-
ronmentally friendly are receiving growing attention in many areas,
and a renaissance of the interest toward the natural pool as valu-
able source of available and renewable row materials is occurring.
Since ancient times plants have been used for dyeing purposes and
even now they play a key role in food, inks, textile, and cosmetic
fields [1]. In this context, the interest in developing efficient and
not-toxic formulations for hair dyes never weakened.
Hair color formulations can be classified [2] as temporary,
semipermanent and permanent (the two formers are based into
a non-oxidation mechanism, whereas the last is based mainly on
oxidation reactions) depending on their persistence on the hair.
Temporary dyes are compounds of low molecular mass which does
not penetrate into the cortex and are deposited on the surface of
the hair. They allow to obtain a slight change or a higher gloss of
hair color that lasts until the first wash. Semipermanent dyes are
able to produce changes in the shade of the natural hair color that
can be fade slowly and progressively, and permanent dyes confer
* Corresponding author. Tel.: þ39 051 2093616; fax: þ39 051 2093654.
E-mail address: carla.boga@unibo.it (C. Boga).
include organic compounds with low molecular weight that allow
them not only to pass through the hair cuticle but also to diffuse
throughout the cortex, providing a longer-lasting coloration, able to
intensify natural hues and to cover the first white hair. They are
usually called ‘direct’ dyestuffs because are ready for use and their
employ is growing owing to the vast availability of formulations
and the easiness of application which permits also household uses.
The mostly used temporary and semi-permanent hair dyes are
of synthetic origin and can be classified in numerous ways, one of
these is the classification as azo compounds, anthraquinones,
triphenylmethanes, nitrophenylenediamines, nitroaminophenols
and aminoanthraquinones [3]. These coloring agents permit to
achieve a wide range of color, with shades strongly dependent on
the nature of the moiety bound to the aromatic ring (e.g. presence
of acid or basic groups) and on the pH of application. When they are
non-ionic and contain no water solubilizing groups, they are called
‘disperse dyes’ [4a]. Their sparing solubility in water can be
increased by adding to the dye bath an organic solvent (2e3%)
partially soluble in water or a surface-active agent. This latter
usually improves the dyestuff solubility increasing its uptake by the
hair and simplifying its diffusion through the hair cuticle by
swelling the same [4b,c].

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http://dx.doi.org/10.1016/j.dyepig.2012.11.020
10 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18

Table 1
Dyeing tests on yak hair using anthocyanins from mulberries fruits.

Entry pH Time (h) % (w/V) Medium L* a* b* DE

Yaka e e e e 69.7  0.4 1.2  0.2 12.5  0.5 e
1b 2.7 12 0.1 Water 48.8  0.6 13.8  0.6 2.5  0.1 28.6  0.7
2b 2.7 12 0.1 Ac 34.6  0.9 17.1  0.7 4.3  0.1 42.1  0.9
3d 2.7 12 0.1 Ac 51  2 7.8  0.8 3.2  0.1 25  2
a
Starting yak hair parameters.
b
Data from extract-1 (see Section 2.4).
c
Water/2-propanol/benzyl alcohol (75/20/5).
d
Data from isolated pigment.

Besides synthetically-derived dyes there are direct dyes avail-
able in nature and known since ancient times when they were used
not only as skin and hair dyes, but mainly as textile dyes [5]. Among
them, henna leaf and walnut husk were the most efficient natural
hair dyes and at present henna (Lawsonia inermis L.) is still the most
commonly used natural plant for this purpose.
In principle, a wide range of vegetables might be used as starting
material for “green” hair dye compositions but the exploitation of
natural pigments for hair dyeing is more complicated than that on
the textiles that can use longer exposition times, higher tempera-
tures and presence of mordants [2b,c]. This is due to many factors
such as the poor stability of the dyes in solution, their tendency to
undergo oxidation and browning phenomena, the pH-dependent
color shift and the degradation by UV light [2a]. In addition,
usually plants contain low concentration of pigments that are often
impure and poorly soluble [2e]. Notwithstanding, in the light of
several studies about the toxicity of synthetic permanent and
semipermanent hair dyes [6] the interest toward natural dyes has
recently increased (even if this not admit to assume that ‘natural’
means safe) and grown in popularity thus increasing their impor-
tance in the market of natural products.
Due to these reasons, searches aimed to the replacement of
synthetic hair dyes with natural colorants have recently intensified
but only in a few cases the results have been reported in terms of
CIE (International Commission of Illumination) dyeing parameters
[7]. This prompted us to turn our previous interest [8] on hair dyes
toward some vegetal sources in order to explore their potentialities
as hair colorants. Herein we report the results obtained on white
yak hair, used through this work as test fiber.
2. Materials and methods
2.1. Materials
Benzyl alcohol, 2-propanol, ethanol, methanol, HCl 37%,
n-hexane, light petroleum, diethyl ether, ethyl acetate, dichloro-
methane, citric acid, 2-amino-2-methyl-propanol, iron (II) oxalate,
p-benzoquinone, 5-hydroxy-1,4-naphtoquinone (juglone), 1,2-
Dihydroxyanthraquinone (alizarin), 2-(3,4-dihydroxyphenyl)-
3,5,7-trihydroxy-4H-1-benzopyran-4-one, 3,30 ,40 ,5,6-pentahydrox-
yflavone (quercetin), and guaiazulene were purchased from
SigmaeAldrich (Milan, Italy). 1,7-Bis(4-hydroxy-3-methoxyp-
henyl)-1,6-heptadiene-3,5-dione (curcumin) was purchased from
Fluka. The shampoo used for washing dyed yak hair was from Marie
Danielle Linea Hotel, Milan, Italy (pH 5.5, composition: aqua,
sodium laureth sulfate, cocamide Dea, disodium laureth sulfo-
succinatelauramidopropyl betaine, sodium chloride, Triticum vul-
gare gluten, parfum, propylene glycol, methylisothiazolinone,
benzyl alcohol, phenoxyethanol, methylparaben, ethylparaben,
propylparaben, butylparaben, citric acid, CI42051/19140). Paper
used to filter was from Bibby Sterilin Italia cod 250-60. Blue
chamomile oil was a gift of Dr. Lorenzo Micele.
White Yak hair samples, used as substrate for dyeing experi-
ments, were purchased from Socap (Napoli, Italy).
The 1H spectra were recorded at 600 MHz on a Varian Inova 600
Instrument. Chemical shifts were measured in d (ppm) and
referenced to the solvent (3.6 ppm for 1H NMR in CD3OD). J values are
given in Hz. GCeMS analyses were performed on a gas
chromatograph Agilent 6890 equipped with a (5%-phenyl)-methyl-
polysiloxane column (30 m length, 0.250 mm i.d., 0.25 mm thickness),
interfaced to a quadrupole mass detector Agilent 5973 N. Flash chro-
matography (FC) was performed on silica gel (0.040e0.063 mm).
Extraction of anthocyanins fraction from mulberry’s fruits was
carried out using Waters Sep-Pak Vac 12 mL C18 e 2 g cartridges. pH
Values are determined using an AMEL Instruments Mod. 2335
pHMETER with combined electrode Hamilton 3 M KCl.
2.2. Dyeing procedure
The dyeing experiments were carried out, as specified in related
Tables, using different baths, prepared as follows.
Water bath: in this case dyeing experiments was made dis-
solving or dispersing different amount (% w/V are given in
Tables 1e6) of dye in 10 mL of water. For each dye bath the pH was
adjusted to the required value using a 1 M aqueous solution of citric
acid in water or 2-amino-2-methyl-propanol.
Bath in medium A: the dyeing experiment was made dissolving
or dispersing the dye in a solution prepared by mixing 75% of water,
20% of benzyl alcohol, and 5% of 2-propanol (V/V). For each mixture
the pH was adjusted to the required value using a 1 M aqueous
solution of citric acid or 2-amino-2-methyl-propanol.
Bath in Medium B (used as described in Supporting
Information): the dyeing experiment was made using a benzyl
alcohol 1% v/v aqueous solution.
White yak hair samples were divided in locks of 0.150 g weight
and 4e5 cm length and were immersed in the 10 mL of a freshly

Table 2
Dyeing tests using alizarin.a

Entry pH Time (h) % (w/V) Medium L* a* b* DE

Yaka e e e e 69.7  0.4 1.2  0.2 12.5  0.5 e
1 8.0 0.5 0.1 Water 55  1 9.0  0.5 1.5  0.1 20  1
2 8.0 0.5 0.1 Ab 50  1 13.7  0.9 2.3  0.6 27.8  0.9
3 8.0 0.5 0.5 Ab 41  2 15.4  0.4 5.9  0.2 37  1
4 8.0 0.5 1.0 Ab 46  1 16.2  0.5 4.4  0.2 33  1
a
Starting yak hair parameters.
b
Water/2-propanol/benzyl alcohol (75/20/5).
 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18 11

Table 3
Dyeing tests using curcumin.

Entry pH Time (h) % (w/V) Medium L* a* b* DE

Yaka e e e 79.7  0.3 1.6  0.1 14.8  0.1
1 2.7 0.5 0.1 Water 84.4  0.1 0.12  0.02 27.9  0.1 14.0  0.2
2 2.7 0.5 0.5 Water 84.09  0.02 0.4  0.1 30.3  0.1 16.2  0.3
3 2.7 0.5 1 Water 84.3  0.1 0.1  0.2 32.2  0.1 18.1  0.2
4 5.7 0.5 0.1 Water 83.6  0.2 1.69  0.04 19.2  0.3 5.9  0.2
5 5.7 0.5 0.5 Water 86.04  0.04 0.4  0.1 23.3  0.1 10.7  0.3
6 5.7 0.5 1 Water 84.5  0.2 0.93  0.03 21.9  0.4 8.6  0.3
7 8.0 0.5 0.1 Water 82.9  0.1 1.17  0.02 23.4  0.1 9.2  0.2
8 8.0 0.5 0.5 Water 83.7  0.1 1.20  0.03 30.4  0.1 16.2  0.2
9 8.0 0.5 1 Water 84.3  0.1 1.09  0.03 22.0  0.4 8.7  0.4
10 2.7 0.5 0.5 Ab 90.8  0.4 5.5  0.1 56.4  0.6 43.2  0.5
11 6.5 0.5 0.5 Ab 92.1  0.1 3.2  0.1 49.7  0.3 37.0  0.3
a
Starting yak parameters.
b
Water/2-propanol/benzyl alcohol (75/20/5).

h
2
2
2 i1=2
prepared dyes bath without stirring. The dyeing experiments were
DE ¼ DL* þ Da* þ Db* (3)
performed at room temperature (25  1  C) for a fixed time (usually
30 min) then the yak hair was removed, rinsed well with distilled
water, washed one time with the shampoo, rinsed again and dried 2.4. Extraction of anthocyanins from mulberry fruits
with a phon. It could be useful to consider that in experiments
reported herein we used only water or water/alcohol as medium in Ripe mulberry fruits (Morus nigra L., Moracee) were collected in
order to have information on the affinity of the dye toward hair. the park of the Faculty of Industrial Chemistry, Viale del Risorgi-
Commercial formulations (information from coauthors of Ilios srl) mento, 4, Alma Mater Studiorum e University of Bologna and
often are gels or creams; about 50 g of this paste is used for 500 g of a specimen of the plant was identified by Dott. Annalisa Managlia of
hair and the amount of dye ranges from 0.1% for light colorations Erbario of Department of Botany of Alma Mater Studiorum e Uni-
until 1.2e1.5% for dark colorations. versità di Bologna, Italy, where a voucher specimen was deposited
(no. BOLO507551). After sampling, the fruits were frozen and
2.3. Color measurements stored at 18  C. 16 g of frozen mulberry fruits were pounded with
a pestle into a beaker and treated with a solution prepared by
Colorimetric analyses on solid yak hair using curcumin, p-ben- mixing 25 mL of methanol with 1.5 mL of aq. HCl 1%. The mixture
zoquinone, and juglone were conducted using a Spectrophotometer was stirred to ensure a uniform extraction and after 30 min the
Jasco V-650 whereas in all other cases, colorimetric analyses were solution was decanted and the supernatant filtered through paper
carried out using a Lambda 35 UVeVis-Nir Perkin Elmer instrument. to avoid solid residues in the solution. This procedure was repeated
Both instruments were equipped with a Labsphere RSA-PE-20 three times and at the end of the extraction procedure the color of
reflectance and transmittance integrative sphere and a software mulberry fruits was changed from purplish black to pale pink. The
color method (CIELAB scale L*, a*, b*, using CIE D65/10 Illuminant/ methanolic extract (called Me-ext) thus obtained was used for
observer condition). A yak hair sample prior to its dyeing was used further processing.
as a blank. For color measurement, yak hair were placed in the For dyeing tests with the extract, the methanolic pigment
apposite Teflon support in such a way that the fibers were aligned solution (Me-ext) was subjected to successive extractions with
and maximum overall smoothness ensured. Three measurement of n-hexane and light petroleum, then the residual water/methanol
the same sample was registered each way changing the fiber phase was concentrated with a rotary evaporator at 40  C to obtain
disposition to ensure that any change in the reflectance of the the aqueous residue. This aqueous solution was further extracted
sample were not simply due to a non-uniformity of the yak hair with ethyl acetate and concentrated in vacuo at 40  C to eliminate
sample preparation assembly, the data reported represent the traces of organic solvent. The aqueous residue (called ‘extract-1’)
average value of these measurements [8b]. Cab * , hab, and DE values
Table 5
are obtained using the following equations (1)e(3) [7]:
Dyeing tests with p-benzoquinone in water (unless otherwise specified).

1=2
DE
Cab * ¼ a*2 þ b*2 (1) Entry pH Time Conc.% L* a* b*
(h) (w/V)
Yaka e e 79.7  0.3 1.6  0.1 14.8  0.1 e
hab ¼ arctg b* =a* (2) 1b,c 2.7 0.5 0.1 77.0  0.7 5.0  0.1 22.3  0.1 8.6  0.4
2b,d 2.7 0.5 0.5 62.1  0.1 14.4  0.1 29.7  0.2 26.4  0.2
3b,e 2.7 0.5 1 41.4  0.3 18.3  0.1 23.8  0.1 42.8  0.1
4b,c 5.7 0.5 0.1 76.6  0.2 6.28  0.03 23.9  0.1 10.7  0.1
Table 4 5b,d 5.7 0.5 0.5 55.9  0.4 17.6  0.1 29.2  0.1 32.1  0.1
Dyeing tests with mulberry’s fruits extract in the presence of iron salts in medium 6b,e 5.7 0.5 1 45.8  0.1 18.6  0.1 26.3  0.1 39.7  0.2
A.a 7b,c 8.0 0.5 0.1 73.2  0.3 8.2  0.2 24.8  0.1 13.6  0.1
8b,d 8.0 0.5 0.5 53.5  0.2 17.52  0.03 28.6  0.1 33.6  0.1
Entry pH Time (h) % (w/V) L* a* b* DE 9b,e 8.0 0.5 1 47.8  0.2 18.7  0.2 26.9  0.3 38.1  0.2
Yakb e e 69.7  0.4 1.2  0.2 12.5  0.5 e 10b,d,f 2.7 0.5 0.5 75.1  0.2 7.7  0.1 25.4  0.2 13.1  0.1
1 3.7 12 0.1c, 0.05d 37  1 1.7  0.1 7.3  0.1 38.0  0.8 a
Starting yak parameters.
2 3.7 0.5 0.1c, 0.05d 48.2  0.8 0.9  0.1 7.8  0.3 29.6  0.5 b
Colour from naked eye evaluation.
a c
Mixture water/2-propanol/benzyl alcohol (75/20/5). Pale brown.
b d
Starting yak parameters. Light brown.
c e
Amount of extract from mulberry’s fruits. Brown.
d f
Amount of iron(II) oxalate. Medium A: water/2-propanol/benzyl alcohol (75/20/5).
12 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18

Table 6
Dyeing test with juglone in water (unless otherwise specified).a

Entry pH Time (h) Conc.% (w/V) L* a* b* DE

Yaka e e e 79.7  0.3 1.6  0.1 14.8  0.1 e
1 2.7 0.5 0.1 77.3  0.1 6.0  0.2 38.2  0.5 23.9  0.5
2 2.7 0.5 0.5 77.7  0.1 5.6  0.1 37.3  0.3 17.6  0.3
3 2.7 0.5 1 76.5  0.1 7.1  0.1 37.7  0.2 23.8  0.2
4 5.7 0.5 0.1 76.87  0.01 4.94  0.07 36.2  0.1 21.8  0.3
5 5.7 0.5 0.5 77.3  0.1 4.2  0.1 36.6  0.1 22.1  0.2
6 5.7 0.5 1 77.75  0.04 4.2  0.1 35.5  0.3 21.0  0.3
7 8.0 0.5 0.1 72  0.6 4.6  0.2 31.9  0.1 17.6  0.3
8 8.0 0.5 0.5 76.3  0.4 4.6  0.1 30.8  0.1 16.6  0.1
9 8.0 0.5 1 74.9  0.2 5.33  0.04 33.1  0.2 19.3  0.2
10b,c 2.7 0.5 0.5 75.9  0.3 7.9  0.2 44.1  0.3 30.2  0.2
11b,c 6.5 0.5 0.5 72.7  0.3 7.8  0.2 40.5  0.4 27.3  0.3
12d,e,f 7.00 12 1d 28 2 15 1 14 2 44 2
a
Starting yak parameters.
b
Light brown, evaluated by naked eye.
c
In medium A.
d
Redebrown, evaluated by naked eye.
e
Yak parameters as in Table 1.
f
Juglone isolated from green walnuts husks.

was used for dyeing tests both as red-coloring source (Table 1,
entries 1 and 2), and as blue-coloring source after addition of iron
oxalate (Table 4, entries 1 and 2). In this latter case, preliminar
experiments were made by soaking yak hair for 30 min into
a mixture containing ‘extract-1’ in medium B, and iron oxalate at
various percentages, from 1 to 10% w/V of medium B. The dye was
evaluated by naked eye and we noted that on increasing the
concentration of the iron salt the hair color shifted from light silver
(1% of iron salt) to light blue (from 3 to 8% of iron salt) until to reach
an intense blue color with 10% of iron salt (see Table SI-3).
For dyeing experiments with only anthocyanin fraction, the
methanolic extract (Me-ext) was concentrated with rotary evapo-
rator at 40  C until to obtain a volume of 5 mL of pigment solution.
2 mL of this solution were loaded onto an SPE column previously
conditioned eluting first with 5 mL of aqueous HCl 0.01% in
methanol and then with 5 mL of HCl 0.01% in water. After the
loading on SPE, the extract was washed eluting with 4 mL of HCl
0.01% in water, then with 4 mL of ethyl acetate. Finally the antho-
cyanin fraction was eluted with 3 mL of HCl 0.01% (v/v) in methanol
and collected. The solvent was removed under nitrogen stream
giving 0.020 g of a shiny dark solid with yellow-green reflexes. The
1
H NMR spectrum in CD3OD of this residue (Fig. 1) showed pres-
ence of two main compounds in a 64/36 relative ratio whose
spectral data agree with those reported in the literature [9] for
cyanidin-3-O-rutenoside (keracyanin) and cyanidin 3-O-glucoside
(kuromanin). This mixture of anthocyanins was used for dyeing
experiments (Table 1, entry 3).
2.5. Extraction of juglone from green walnut husks
Green walnuts fruits of Juglans regia L. were collected before the
hardening of the endocarp in Lizzano in Belvedere (BO) and a spec-
imen of the plant was identified by Dott. Annalisa Managlia of
Erbario of Department of Botany of Alma Mater Studiorume
Università di Bologna, Italy, where a voucher specimen was depos-
ited (no. BOLO507512). After sampling, the fruits were frozen and
stored at 18  C. Frozen green walnut husks (w80 g) were sliced into
small pieces and extracted into a beaker with 100 mL of dichloro-
methane for three times and stirring the solution to ensure
a uniform extraction. For each extraction, after 60 min, the stirring
was stopped, the solution was decanted and the supernatant filtered
through paper to avoid solid residue in the solution. The dichloro-
methane extract thus obtained was concentrated with rotary
evaporator at 40  C to obtain 0.219 g of solid residue. This residue
was purified by FC on silica gel (dichloromethane:n-hexane ¼ 2:3)
obtaining 0.137 g of a solid (orange needles) which 1H NMR and GCe
MS spectral data agreed with those of an authentic commercial
sample of juglone. This solid was suspended in 15 mL of water (1% w/
V) and used as soaking bath for the long-time (12 h) dyeing test.
3. Results and discussion
Since a desired dyeing can be achieved using formulations
containing dyes that can be considered combinations of the three
primary colors (red, yellow, and blue), we decided to focus our
investigation on a selection of pigments easily available from
vegetal sources and potentially able to confer red, yellow, and blue
shades to white yak hair chosen as test fiber. Subsequently, the
investigation was extended to some brown-coloring pigments. In
all cases the color assumed by the yak hair has been evaluated using
the colorimetric CIE La*b* Space system [7] which main parameters
are: lightness (L*) whose values run from 0 (black) to 100 (white),
a* (on the aea’ axis, positive values indicate amounts of red while
negative values indicate amounts of green), and b* (on the beb’
axis, yellow is positive and blue is negative). For both axes, zero
is neutral gray. Other parameters belonging to CIE L*C*h polar color
space system [7] have been calculated, in particular chroma
[Cab * , saturation or ‘color purity’, ranging from 0 for a sample
completely unsaturated (i.e. a neutral gray, black or white) to 100 or
more] and hue angle (hab, which units are in degrees, ranging from
0 (red) through 90 (yellow), 180 (green), 270 (blue) and back to
0 ); they will be reported in the text only when they are considered
useful for the discussion. The last parameter considered has been
the total color difference DE between the L*, a* and b* values of the
sample and those of the reference (starting yak hair). For the sake of
simplicity, the results obtained will be separately presented and
discussed in sub-headings, as follows.
3.1. Red-coloring sources
Although it is known that a natural red hue is hard to obtain
from vegetal sources, we tried to investigate the dyeing behavior
toward yak hair of some pigments belonging to anthocyanins and
anthraquinones classes.
3.1.1. Anthocyanins
Anthocyanins are a group of reddish-blue, water soluble
pigments responsible of the color of many flowers, fruits and
 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18
Fig. 1. 1H NMR spectrum (600 MHz, CD3OD, 25  C) with related expanded view of anthocyanin fraction extracted from mulberry’s fruits (keracyanin).
vegetables and are mostly known for their properties as antioxi-
dants [10a,b], and natural additives for food industry [11]. Their use
as colorants is limited since their color and stability are dependent
from several factors, such as anthocyanin structure, temperature,
light exposure, presence of other compounds (pigments, metal
ions, ascorbic acid, oxygen, etc.) and pH [12].
Nevertheless, while until a few years ago there was very scarce
literature about the use of anthocyanins in hair treatment, recently
some patent applications on this subject appeared [13]. This sug-
gested us to investigate the dyeing behavior of these compounds on
yak hair fiber. For dyeing purposes it has to be considered that in
aqueous solution the color of anthocyanins depends from the
coexistence of several pH-dependent forms [14]. The four main
species are the quinoidal base A, the flavylium cation AHD, the
carbinol (or pseudobase) B, and the chalcone C (Scheme 1). Through
these forms, anthocyanins display huge color variations in the pH
range 1e14. In particular, at very low pH values (below pH 2) the
predominant form is the red flavylium cation (AHD form). In weakly
acidic solutions the flavylium cation is hydrated into the colorless
carbinol form that, after a further increasing of the pH, undergoes
ring opening producing the chalcone form (C). Contemporaneously,
the small residual amount of AHD begin to lose protons giving
weakly purple non-ionized quinoidal base (A) that, at higher pH
values, shifts toward his deep blue ionized form AL.
As a consequence of these equilibria, the red form AHD is the
only species in solution at pH values around 0.5. When this solution
is made less acidic (4 < pH < 6) the purple color of quinoidal base A
appears and a further increase of pH value (pH > 7) causes a blue
coloration that may fade over time. Since the percentage of qui-
noidal base is very small at any pH value, there is very little amount
or percentage of colored species in anthocyanin solutions when the
pH is increased beyond pH 4 [15].
13
From these data one can evince that a pH value around 0.5 might
be suitable to obtain a red coloration, but in these conditions the
hair fibers are positively charged (for unaltered human hair the
keratin isoelectric point is w6.0) [16] and repulsive chargeecharge
interactions with the dyeing molecule in AHD form might take
place; moreover a very low pH value is inappropriate for dyeing
hair. Keeping this in mind, we planned to try the dyeing of yak hair
at pH value of w3, hoping that this could limit ionic unfavorable
interactions between the red flavylium cationic form and keratin.
As vegetal source of anthocyanins we chose mulberry’s fruits
and as first approach we carried out some preliminary dyeing
experiments by soaking white yak hair in an aqueous extract (see
Experimental) at different pH values in the range 1.2e8.8 for
different exposure times (from 0.5 to 12 h). As expected, due to the
presence of leuco forms, no significant dyeing was observed at pH
values higher than 2.7. In more acidic medium (pH 1.21 and 2.7),
after 1 h a very feeble pink dyeing was observed and only after 1
night pink colored hairs were obtained.
Afterward, we carried out further experiments at pH 2.7. Firstly,
yak hair were soaked for 12 h in two baths containing, respectively,
the extract of mulberry’s fruits dissolved in water (0.1% w/V) and in
a mixture water/2-propanol/benzyl alcohol (75/20/5 by volume),
hereafter called medium A and used with the aim to improve the
diffusion of the dye owing to the swelling of the hair fiber [8b]. A
third experiment was carried out with the aim to verify whether
the components different from anthocyanins present in the extract
of mulberry might influence the dyeing uptake on the hair. For this
purpose after a work-up and a treatment on SPE column of a crude
mulberry’s fruits extract (see Experimental Section) we isolated
a residue which 1H NMR spectrum (Fig. 1) showed presence of two
main anthocyanins. A comparison with literature data [17] allowed
their identification as keracyanin (cyanidin-3-O-rutenoside) and
14 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18

R'' R'' OH OH
OH OH OH OH
–
OH Cl– Cl
+ HO O O+
HO O
R''' H2O, - H
+ R''' HO O+ HO

OR' pK ~ 2.6 OR'

O O
OH OH
OH OH OH OH
O O
flavylium cation (AH+) carbinol pseudobase (B)
(red) (colourless)
OH OH
O O OH OH OH

- H+ pK ~ 4.3 pK ~ 3
HO OH
OH
R'' R''
Keracyanin chloride Kuromanin chloride
OH OH
OHO Fig. 2. Structure of keracyanin chloride and kuromanin chloride.
O O HO
R''' R'''

OR' OR' 3.1.2. Anthraquinones

OH OH
chalcone (C)
quinoidal base (A)
(purple) (pale yellow)
- H+ pK ~ 7
R''
O–
O O
R'''
OR'
OH
ionized quinoidal base
(blue)
Scheme 1. pH-dependent equilibria in aqueous solution of anthocyanins.
kuromanin (cyanidin 3-O-glucoside) chloride (Fig. 2) in 64/36
relative ratio, in agreement with reported data [9,18].
The evaluation of the hair color was expressed using the color-
imetric CIE La*b* Space system which main parameters, together
those of the starting yak hair are reported in Table 1.
As it can be seen, the color of the yak hair after the soaking
shifted toward red (Da between dyed and virgin hair >0 along the
CIELAB a axis) and toward blue (Db < 0 along the b axis). This trend
is evident also in terms of hue angle (hab in the CIELCh polar
coordinate system) that shifts from 84 (corresponding to the pale
yellow color of starting yak hair) to values corresponding to the red
region (350 , 346 , and 338 for entries 1, 2, and 3, respectively). In
addition, a comparison of data of entries 1 and 2 evidenced that the
use of A as medium improves the darkness of the hair fibers (L*
decreases from 48.8 to 34.6). The dyeing of the yak hair obtained
with the pigment isolated (Table 1, entry 3) is less satisfactory than
that obtained with the extract in medium A (entry 2) and roughly
comparable with that obtained in aqueous solution (entry 1). This
suggests that the vegetal matrix of the extract plays an important
Madder plant (Rubia tinctoria L.) and plants of the Rubiaceae
family have been used since prehistoric times as natural source of red
pigments [19]. Its roots contain several anthraquinone derivatives
whose main aglycones are alizarin (1,2-dihydroxyanthraquinone,
Fig. 3), purpurin, ruberythric acid, pseudopurpurin and lucidin-
primeveroside [20].
Alizarin is sparingly soluble in water (1.32  103 mol L1 at
303 K) [21] but its solubility increases in aqueous alkaline solutions
in the pH range 7.82e12.87. Its color, as previously seen for
anthocyanins, is pH-dependent: below 5.2 the main form is the
yellow-colored undissociated molecule while at pH values between
6.8 and 10.1 alizarin is mostly present as red monovalent anion. At
pH above 12.1 alizarin dissociates into divalent anions showing
a blueeviolet color [22]. Being interested to the red form of alizarin,
we decided to carry out some dyeing experiments at a pH value of
8.0 using commercial alizarin. Under these conditions the result
could not be easily predicted since at this pH adverse ionic inter-
actions between dyes and negative charges on keratin might take
place but, conversely, the alkaline pH significantly favors the
swelling of the fiber owing to the ionization of diacidic amino acid
residues [2c]. In Table 2 are shown the results obtained.
Alizarin was dispersed both in water and in medium A (Table 2,
entries 1 and 2, respectively) and this variation caused the shift of
the color of the yak hair from pale pink (entry 1) to a more deep
reddish color (entry 2). This is quantified by the CIELCh parameters
that show, for the dyeing in medium A with respect to water,
a decrease of L* value from 55 to 50, and a parallel increase of the
vividness (chroma value shifted from 9.2 to 13.9) and of the total
difference DE with respect to the original hair color. Concerning the
effect of the amount of dye, the results obtained suspending
different amounts of alizarin in medium A (Table 2, entries 2e4)
gave not rise significant color changes probably due to the large
excess of dye with a consequent situation of saturation.
O OH

role in increasing the uptake of the dye. This might be due to the
OH
presence, in the extract, of substances such as carbohydrates that
are removed during the work up to isolate the pigments. A positive
effect in the dye absorption has been already observed in the
semipermanent dyestuff process in the presence of cellulose-based
polymers [8b,c].
It is important to note that results shown in Table 1 have been O
obtained without addition of other components except alcohols
used as swelling agents. Fig. 3. Molecular structure of alizarin.
 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18
It has to be noted that in all cases the dyeing time was 0.5 h;
a rough comparison with the results obtained with anthocyanins
(even if the two dyeing systems are different and work in different
conditions such as pH and solubility) brings to the consideration
that alizarin seems to be a more efficient reddish-dyestuff.
3.2. Yellow-coloring sources
Contrary to the situation of the reds, the sources of yellow
chromophores are enormous and among them we decided to select
two safe and widespread compounds: quercetin (3-hydroxy
flavone), belonging to the class of flavonols and curcumin,
a compound characterized by its unique conjugate structure (Fig. 4).
Quercetin, nowadays known for its antioxidant properties [23]
was a constituent of many historical yellow mordant dyes for
textiles [5c]. It exists in several ionic forms; at pH 5.0 the equilib-
rium between the neutral and the monoanion form predominates
while at pH 7.5 the main forms are the mono and the dianion [24].
We tried to dye yak hair at pH 6 using a suspension of quercetin
both in water and in the above described medium A but the dyeing
was limited to a pale green-yellow coloration, difficult to be eval-
uated by naked eye (for related CIE parameters see Table 1 in SI).
Curcumin is a component of the spice turmeric (Curcuma
longa L.). It is widely used as food ingredient and well known
both in traditional and in modern medicine for its antifungal,
anti-inflammatory and antitumor activity [25]. From thousands of
years curcumin is used as coloring agent [2a] and recently it
has been used to confer also antimicrobial properties to dyed
textiles [26].
Curcumin is a bis-a,b-unsaturated diketone and in solution
exists in equilibrium with the corresponding enol tautomer. Under
acidic and neutral conditions the bis-keto form predominates,
whereas above pH 8 the enol tautomer is favored [27]. Curcumin is
practically insoluble in water at acidic and neutral pH while in alkali
the formation of more soluble anionic species takes place [28]. In
the pH range 1e7 curcumin has a yellow color that changes to
orange above pH 7.5 and over pH 8.4 the predominance of the
monoanionic form makes the solution scarlet. A further increasing
of the pH over 9.9 causes the deepening of the color of the aqueous
solution that becomes red until to being violet at pH values higher
than 10.5. These color changes are related to the pH-dependent
stability of the curcuminoids in aqueous media and to the forma-
tion under basic conditions of degradation products [29].
We carried out a series of dyeing tests at pH values 2.7, 5.7, and
8.0 dispersing different amounts (0.1%e0.5%e1% w/V) of curcumin
in water for an exposure time of 0.5 h. The colorimetric parameters
related to the dyed yak hair obtained in these conditions are
reported in Table 3, entries 1e9. Two tests were made dispersing
the dye (0.5% w/V) in the medium A at pH 2.7 and 6.5.
All the samples assumed a yellow coloration. The value of b*
resulted higher than that of the starting yak hair especially after the
soaking at pH 2.7 in medium A (Table 3, entry 10). In this case the b*
value reached 56.4 producing the greatest shift toward the yellow
OH
OH
O O
HO O O
OH HO
OH O
Quercetin Curcumin in its neutral keto form
Fig. 4. Molecular structure of quercetin and curcumin.
15
(Db ¼ 41.6 between colored and starting non-colored hair). It is
notewhorthy that in all cases the dyed material exibits higher L*
than the undyed material, especially when the dye was dispersed in
medium A (Table 3, entries 10 and 11): this might be due to
a fluorescent effect of curcumin [30]. Analyzing the data for each pH
value, is evident that at pH ¼ 2.7 (entries 1e3) a little increase of the
dyeing by increasing the amount of the suspended dye was ob-
tained. This behavior was observed also for experiments at pH ¼ 5.7
and pH ¼ 8 (Table 3, entries 4, 5 and 7, 8 respectively) on going from
0.1% to 0.5% of dye suspended. However, from an overall view of the
DE data obtained dispersing curcumin in water at different pH
values and with different amount of dye (Table 3, entries 1e9) one
can evince that the results are very similar and the related dyeing is
not very intense. This is probably due to the limited solubility of
curcumin that could made difficult the diffusion of the dye into the
hair. Actually, when curcumin was dispersed in medium A with the
aim to increase the solubility of the dye and its uptake (Table 3,
entries 10 and 11) the hair assumed a bright yellow color, especially
in acidic conditions (Table 3, entry 10).
3.3. Blue-coloring sources
Blue color is not widespread in nature and likewise no
many vegetal sources of blue-coloring pigments are available.
Among the earliest and most popular blue natural sources there
are indigo-giving plants whose coloring principle is used as vat
dye and requires a reduction step in alkaline medium. This means
that indigo can not be used as direct dye for human hair unless
it is synthetically modified, as in the case of indigo carmine
[31]. Besides indigo, there are some other less known blue-coloring
sources as terphenylquinones in mushrooms [32], chamazulene
in chamomile oil [33] or some “anthocyanin blue” in flowers petals.
In this investigation we have focused our attention on two
chemical classes of blue pigments: azulenes and ‘anthocyanin blu’.
3.3.1. Azulenes
Azulenes (Fig. 5) are polycyclic non-benzenoid aromatic
compounds that have long attracted attention of chemists due to
their beautiful colors and their unusual electronic properties [34].
Among them, 7-ethyl-1,4-dimethylazulene (chamazulene) is
a component of chamomile (and other plants, such as Artemisia
arborescens L [33b].) essential oil obtained by steam distillation of
flower heads. This oil contains a complex mixture of compounds,
such as sesquiterpene derivatives and spiroethers. Its characteristic
deep blue color is due to heat decomposition of matricin into
chamazulene during the distillation process. Another blue azulene
derivative is guaiazulene (1,4-dimethyl-7-isopropylazulene, Fig. 5),
present in the essential oil of guaiac wood. Recently, it has become
a popular ingredient in body care products (lotions, emollient
creams, toothpastes, eye drops) owing to its use as a skin condi-
tioning agent in cosmetic (including hair dyes) formulations [35].
Azulene
O
OH
Chamazulene Guaiazulene
Fig. 5. Structure of azulene and its derivatives chamazulene and guaiazulene.
16 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18
The absence of polar functional groups in these compounds
suggests that only very weak interactions with hair might occur.
However, the availability in our lab of a sample of essential oil [36]
obtained by steam distillation of fresh flower heads of Matricaria
chamomilla L. made us curious to try the dyeing of yak hair with this
blue oil. A preliminary rough test was carried out by soaking the yak
hair in a solution of this oil in ethanol. After a permanence for 12 h
in the bath, the color of the yak hair became light blue-green. Then,
we carried out an experiment using a suspension of guaiazulene in
medium A: also in this case the yak hair assumed a light blue-sky
coloration (see related CIELAB parameters reported in Table SI-2)
only after a soaking for 12 h.
3.3.2. Anthocyanin blue
Another natural blue source is the anthocyanin blue. This
color is due to the formation of self-assembled complexes and
requires particular care devoted to the preservation of the supra-
molecular structure [17], strongly dependent from many param-
eters, such as pH and concentration. In the last century,
investigations on many blue anthocyanin pigments revealed that
blue color arises from metal complexation [37], self-associations
[38] and co-pigmentation [39]. Usually, all these mechanisms
take place allowing the color development and stability, as in the
case of corn-flowers [40] blue pigments. The most common metals
in anthocyanin complexes are iron, aluminum, and magnesium
[41a] and only anthocyanins which have more than one free
hydroxyl group in the B-ring are capable of metal chelation
[41b,c].
From the applied point of view, while metal salts are commonly
used as mordants to form complexes used as synthetic dyes in
textile field, in the hair dyeing the use of metal complexes must
take into account the whole treatment made on the hair. For
example, if hydrogen peroxide is used for bleaching hair to lighten
their color or to overdye them to achieve a different shade,
decomposition by catalytic effect due to the presence of iron,
copper, or manganese salts can occur, causing serious damage to
the hair [42]. Nevertheless, a certain attention has recently been
devoted to this kind of hair treatment [14a] and this suggested us to
use the anthocyanin extract from mulberry’s fruits (containing
keracyanin and kuromanin with two free hydroxyl groups in the
B-ring capable of metal chelation) in the presence of iron (II)
oxalate.
Firstly, yak hair were soaked for 30 min in different baths
prepared dissolving the extract of mulberry fruits in water/benzyl
alcohol (25/75 by volume, medium B) and adding iron oxalate at
various percentages, from 1 to 10% (w/V). We noted that on
increasing the concentration of the iron salt the hair color shifted
from light silver (with 1% of iron salt) to light blue (from 3 to 8%)
until to reach an intense blue color with 10% of iron salt (see
Table SI-3). After these preliminary tests, we decided to dye yak
hair in conditions more compatible with hair treatment using the
mulberry fruits extract in the presence of 0.05% of iron salt in
medium A. Table 4 shows colorimetric parameters obtained.
A comparison of the CIELAB values reported in Table 1 with
those in Table 4 shows that the addition of iron salts to the extract
of mulberry fruits produced a significant shift of the color toward
blue. Actually, in Table 4, Db* values (calculated between b* of dyed
hair and b of undyed sample) are negative indicating a shift toward
blue, confirmed also by the hue values that fall within the blue
region (hab is 283 and 263 for entries 1 and 2, respectively). As
expected, the sample soaked for 12 h is darker (L* ¼ 37, Table 4,
entry 1) with respect to that soaked for 0.5 h (L* ¼ 48, Table 4, entry
2) but the relative difference in chroma (Cab * is 12.5 for undyed
material and 7.5 and 7.8 for entries 1 and 2, respectively) and in DE
is not very huge thus indicating that a satisfactory dyeing result can
be obtained after 0.5 h. This indicates that this dyeing system could
be suitable for more deepen investigations focused to increase the
uptake after having developed a suitable formulation.
3.4. Brown-coloring sources
Vegetal brown dyes are usually derived from flavonoids, tannic
acid and quinone compounds, which allow to obtain brown hues
that may further darkened, depending on the dyeing time, due to
polymerization processes and maybe to chemical reactions
between the dyestuff and the fiber.
As the present investigation is focused on possible vegetal
pigments for dyeing hair, it seemed useful to check also some
brown-coloring sources, even if brown color can be obtained
overlaying primary colors. Usually, quinone dyes contain some
unsaturated groups conjugated to the benzoquinone ring but also
the benzoquinone p-system alone can be used as chromophore. In
this context, we decided to compare the dyeing power on yak hair of
p-benzoquinone and 5-hydroxy-1,4-naphthalenedione (juglone).
3.4.1. 1,4-Benzoquinone
1,4-Benzoquinone is a small organic compound and, in principle,
its low molecular weight makes it suitable for semipermanent hair
dye formulations. It can be considered a natural brown-dyeing
source being it found in young shoots of the pear (genus Pyrus L.),
where it exhibits strong antibacterial activity [43]. The p-benzo-
quinone molecule is slightly soluble in water (1.38% w/w at 25  C)
[44] and makes beige colored its aqueous solutions both in acidic
and alkaline pH. The evaluation of the p-benzoquinone dyeing
power on yak hair was made at different pH values and with
different amounts of dye, as reported in Table 5.
Dyeing tests have been carried out immersing yak hair for
a fixed time of 0.5 h in a series of baths containing different amount
of p-benzoquinone in water (0.1%, 0.5%, and 1% w/v) at different pH
values (2.7, 5.7, and 8.0). Using this direct dye three different
shades of brown that, at the same pH value, darkened increasing
the dye concentration (L* value decreases and DE increases), were
observed. On the contrary, using the same amount of dye in the
bath at different pH values, the change of color of the yak hair was
not evaluable by naked eye, as confirmed by the very similar values
of the CIE parameters. By comparing the results of entries 2 and 10,
obtained using as medium water and A, respectively, a less intense
color of hair was obtained. Taking in consideration the indication
that a satisfactory hair brown shade may be achieved when the DE
value between dyed and virgin hair is at least 30 [45]. From data of
Table 5 one can evince that satisfactory brown shades can be
achieved using a 1% w/v amount of dye in water. From the DE
values obtained at different pH values (Table 5, entries 3, 6, and 9)
the dyeing seems not to be strongly influenced by the pH of
application.
3.4.2. Juglone
Juglone (5-hydroxy-1,4-naphthoquinone) can be extracted from
different parts of nut trees (Juglandaceae). Several herbal prepa-
rations from walnut leaves and green shell of walnuts can be used
for various purposes such as to prepare the traditional walnuts
liqueur, or a coloring sunscreen oil or a dyestuff for many substrates
such as wool, silk, linen, cotton, nylon and polyester [46].
Juglone is an isomer of the more known lawsone (2-Hydroxy-
1,4-naphthoquinone), the coloring agent of Henna and both are
some of the oldest dyes used throughout history for dyeing hair.
They are known to react also with the keratin proteins present in
the skin to form colored compounds [47]. Although juglone is
a weak acid and in the undissociated form has a very low solubility
in water (0.005% w/v) [48] it is known that at pH acidic it confers
 C. Boga et al. / Dyes and Pigments 97 (2013) 9e18
a yellow color to hair more intense that at alkaline pH values. This
behavior has not yet satisfactorily explained and the occurrence of
a complexation of juglone in its anionic form, even if present in low
extent at acidic pH (18% at pH ¼ 3) [48], with positive keratin
groups on the hair has been hypothesized. This is complicated also
by the fact that juglone is rather unstable and tends to polymerize
to brown pigment; as a matter of facts, when in the dye bath the
juglone concentration is increased brown shades are obtained.
Dyeing tests on yak hair, whose results are reported in Table 6,
were carried out dispersing pure juglone in water at different
pH values (2.7, 5.7, and 8.0) and at different concentrations (0.1%,
0.5%, and 1% w/v) for the same exposure time, fixed to 0.5 h. A
further dyeing test carried out for 12 h produced a red-brown color
of the hair.
In has to be noted that, even if the amount of dye was higher
than its solubility, we used these conditions based on the consid-
eration that the suspended excess may provide a reservoir to
replace dissolved juglone complexed by the hair [48].
Results reported in Table 6 show how, using juglone as
disperse dye, very similar yellow shades were obtained (Table 6,
entries 1e9). In fact, changing the pH values or varying the dye
amount in the bath causes only a slight change of color of yak hair.
More intense shades (DEw30) were achieved using A solution with
0.5% w/v of dye at pH values 2.7 and 6.5 (Table 6, entries 10 and 11).
In both cases the yak hair samples resulted in a light brown color,
confirming that the use of the medium A improves the concen-
tration of the dye dissolved or dispersed in the bath thus increasing
the dyestuff uptake on the hair.
4. Conclusions
To obtain information on the potentialities of some pigments of
vegetal origin as temporary or semipermanent hair dyes different
natural compounds have been used for dyeing tests on yak hair,
chosen as model for human hair. Two different sources for red,
yellow, blue and brown shades have been considered. Among red
pigments, alizarin showed to be better with respect to mulberry
fruits extracts mainly because of the minor dyeing time required.
Anthocyanin pigments extracted from mulberry fruits, when used
in the presence of iron salts, gave blue a material more intensely
colored with respect to that treated with azulene derivatives.
Yellow shades were not satisfactorily obtained using quercetin
while curcumin, p-benzoquinone, and juglone conferred to hair
brown shades. In almost all cases, when the dye was dissolved or
dispersed in a mixture water/alcohols, an enhancement of the
uptake of the dye favored by a swelling of the hair was obtained
with respect to the use of water as medium. These results can be
considered useful for further investigations aimed to replace
temporary and semipermanent hair dyes with safe and environ-
mentally friendly compounds.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2012.11.020.
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