TYPE Original Research

PUBLISHED 01 November 2022

DOI 10.3389/fmars.2022.1024988

Application of phycocyanin
OPEN ACCESS from Arthrospira (Spirulina)
EDITED BY
Nitin Trivedi,
Institute of Chemical Technology,
platensis as a hair dye
India

REVIEWED BY Oranit Kraseasintra 1, Yingmanee Tragoolpua 1,

Shailesh Kumar Patidar,
Central University of Rajasthan, India Hataichanok Pandith 1, Ruttiros Khonkarn 2,
Sourish Bhattacharya,
Central Salt & Marine Chemicals
Wasu Pathom-aree 1, Jeeraporn Pekkoh 1,3
Research Institute (CSIR), India and Chayakorn Pumas 3,4*
Jungsoon Lee,
Chungnam National University, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand,
1

South Korea 2
Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University,
Chiang Mai, Thailand, 3 Environmental Science Research Centre, Faculty of Science, Chiang Mai
*CORRESPONDENCE
University, Chiang Mai, Thailand, 4 Research Center in Bioresources for Agriculture, Industry and
Chayakorn Pumas
Medicine, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
[email protected]

SPECIALTY SECTION
This article was submitted to
Marine Biotechnology and
Bioproducts, Almost all of the current hair dye products today contain synthetic chemicals
a section of the journal which may cause allergic reactions in some users. Phycocyanin (PC), a non-
Frontiers in Marine Science
toxic cyanobacterial pigment, has been used in the food and cosmetics sectors.
RECEIVED 22 August 2022 There are however, been a few reports on the application of phycocyanin as a
ACCEPTED 18 October 2022
PUBLISHED 01 November 2022
hair colorant. This study aimed to assess the biological qualities of phycocyanin
for use in natural hair dye product. Phycocyanin was tested for use against anti
CITATION
Kraseasintra O, Tragoolpua Y, skin-pathogen (Staphylococcus aureus ATCC 25923, Staphylococcus
Pandith H, Khonkarn R, Pathom- epidermidis ATCC 14990, Methicillin-resistant Staphylococcus aureus (MRSA)
aree W, Pekkoh J and Pumas C (2022)
Application of phycocyanin from
DMST 20625, Propionibacterium acnes DMST 14916, Candida albicans DMST
Arthrospira (Spirulina) platensis 21424, and Malassezia furfur M21), cytotoxicity of human immortalized
as a hair dye. keratinocyte (HaCaT) cells and tested for color fastness when used as a
Front. Mar. Sci. 9:1024988.
doi: 10.3389/fmars.2022.1024988 shampoo wash. According to the findings, Arthrospira (Spirulina) platensis
COPYRIGHT
phycocyanin has not shown the potential for use against anti-skin
© 2022 Kraseasintra, Tragoolpua, pathogenic microorganisms. While testing phycocyanin at the maximum
Pandith, Khonkarn, Pathom-aree, doses of 2.5 mg/mL, the cytotoxicity test revealed that it is not hazardous to
Pekkoh and Pumas. This is an open-
access article distributed under the HaCaT cells. Bleached hair was dyed with a mixture of phycocyanin, natural
terms of the Creative Commons developers, and mordants. A chroma meter was used to monitor color changes
Attribution License (CC BY). The use,
after shampoo washing. The findings revealed that phycocyanin has dyeability
distribution or reproduction in other
forums is permitted, provided the potential. 50% of the dyed hair color remained after 5 shampoo washes. The
original author(s) and the copyright stability and color degradation of phycocyanin in hair dye powder formulation
owner(s) are credited and that the
original publication in this journal is demonstrated good physical stability along with four cycles of heating/cooling.
cited, in accordance with accepted As a result, we can see that this pigment has the potential to be used as an
academic practice. No use,
active ingredient in natural hair dyes.
distribution or reproduction is
permitted which does not comply with
these terms. KEYWORDS

pigment, phycobiliprotein, mordant, skin-pathogen, cyanobacteria

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Kraseasintra et al. 10.3389/fmars.2022.1024988

1 Introduction include an oxidizing agent composition that includes at least

one chemical oxidizing agent, particularly hydrogen peroxide.
Hair dyeing products are currently in high demand; they are However, it has been shown that some hair dyes can cause
mostly used to conceal gray hair or modify the color of hair increased skin irritation when used with hydrogen peroxide
(Guerra-Tapia and Gonzalez-Guerra, 2014). However, chemical (Park et al., 2018).
oxidation dyeing generally requires the use of a strong base and Weak color and challenges with long-term deposition are
oxidants, which can cause serious hair damage. Worldwide, difficulties when hair dyeing using natural dyes. This is because
highly sensitizing chemicals like para-phenylenediamine (PPD) natural dyes are unable to permeate the hair deeply enough to
and its derivatives are the most common causes of allergic protect the dyed hair from washing or fading (Inman et al.,
contact dermatitis (Guerra-Tapia and Gonzalez-Guerra, 2014; 2020). Some additives such as developer and mordant, can
Antelmi et al., 2017). In a study of 3,000 patients from 12 enhance the dyeability of hair. The dye molecules can
dermatological clinics in Denmark, it was discovered that 4.5% penetrate deeper into the hair shaft and form bonds with hair
of patients tested positive for PPD, 2.8% for toluene-2, 5- proteins because the developer can dissolve chemical bonds and
diamine (PTD), 1.8% for p-aminophenol, 1% for m- open the hair cuticle (Burkinshaw and Kumar, 2009). Hydrogen
aminophenol, and 0.1% for resorcinol (Antelmi et al., 2017). peroxide, the most frequently used developer or oxidizing
The primary patch test screening substance for contact allergies ingredient for hair dye, might cause an allergic reaction
to hair dye is p-phenylenediamine. PPD contact allergy is very (Harrison and Sinclair, 2003). Ascorbic acid, lime juice, and
common on all continents. In the European population, PPD lemon juice have all been traditionally used as natural developers
patch test positive rates ranged from 12.7% to 25.4%. Clinical in hair products for purposes such as permanent waving and
relevance between hair dye use and allergy was about 23–73% in hair bleaching and dyeing. Likewise, mordanting promotes color
Asia, which was higher than in Europe (Krasteva et al., 2009). fastness and fixes dye in hair fibers. The most popular metal salts
More research has revealed that certain hair dye components are used as mordant agents are ferrous sulfate, copper sulfate, and
more likely to include possibly carcinogenic ingredients and may alum, although these substances are considered metal pollution
induce contact allergies (Perveen et al., 2017), whereas natural sources (Beiki et al., 2018). Oxalic acid and tea water extract
dyes are more environmentally friendly, biodegradable, less (tannin), which have the added benefit of being safe for human
allergic, and less toxic (Ali et al., 2009). Consumers are health, have also been employed as natural mordants for dyeing.
becoming more interested in using natural hair dyeing Cyanobacterial pigments such as carotenoids, chlorophylls,
products as a result of a growing global awareness of the and phycobiliproteins have recently been applied in the
importance of using environmentally friendly products manufacture of healthy produc ts because of their
(Boonsong et al., 2012). Therefore, the development of new pharmaceutical and fluorescent properties. Arthrospira
and novel natural based hair dyes is one of commercial interest. (Spirulina) platensis is a helicoidal, filamentous blue-green
Previous reports have described the use of plant pigment cyanobacterium in the Oscillatoriaceae family. Arthrospira
extracts in natural hair dye applications. For instance, Lawsonia (Spirulina) platensis has long been used as a supplement in
inermis (Henna), Indigofera tinctoria (Indigo), Emblica officialis human and animal food, either as a health drink or in tablet
(Amla), Hibiscus rosasinensis (Shoe flower), and Juglans nigra form, due to its nutritional content (Flores et al., 2003).
(walnut hull) have also been documented in literature for use as Arthrospira (Spirulina) platensis can be found in both
natural hair dye sources (Shin and Lee, 2006; Komboonchoo and freshwater and marine environments (Vonshak, 1997).
Bechtold, 2009). Thai plants, including Sappan tree, False daisy, Although Arthrospira (Spirulina) platensis cannot grow
Thao yanang, Kae lae, and turmeric extracts are yellowish, naturally in salt water, earlier reports indicate that with an
brownish, and greenish color. These extracts were applied for adequate supply of bicarbonate, nitrogen, and phosphorus, as
hair dye when mixed with natural developer and metal as a well as an appropriate pH and salinity, Arthrospira (Spirulina)
mordant agent. The appearance color gave a dark reddish-brown platensis species can be produced in seawater (Materassi et al.,
to orangish-brown color (Boonsong et al., 2012). Black beans, 1984). Growing Arthrospira (Spirulina) platensis in natural
one of the highly anthocyanin contents, could be used in food seawater has economic benefits such as lower production costs
and consumer products as well as hair dye colorant when due to reduced use of expensive potable water, which in turn
extracted with hydrochloric ethanol and the color of dyed hair could lower costs on an industrial scale for commercialization.
was maintained through four washings (Inman et al., 2020). Phycocyanin (PC) can be extracted from Arthrospira
Moreover, the fruit skin waste of blackcurrant from the pressing (Spirulina) platensis, a water-soluble pigment protein complex
industry has potential in hair dye after acidified extraction and from the light harvesting phycobiliprotein family, which is
purification with a solid stage. The results of blackcurrant hair ideally suited as a fluorescent reagent for immunological
dye were stable to multiple washes (Rose et al., 2018). There is a analysis due to its broad excitation spectrum (Kuddus et al.,
patent for the use of spirulina extract as a colorant in hair dye 2013). Phycocyanin is an antenna pigment found in
applications (Zirwan and Kleen, 2009). These formulations cyanobacteria and eukaryotic algae, consist with apoprotein

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conjugated with a linear tetrapyrrole (phycocyanobilin) by 20625, Propionibacterium acnes DMST 14916, Candida
covalent bond (Padyana et al., 2001), that absorbs light energy albicans DMST 21424, and Malassezia furfur M21 in
and effectively transmits it to a reaction center containing agreement with the agar well diffusion method described by
chlorophyll to improve photosynthesis efficiency by collecting (Balouiri et al., 2016 and Osman et al., 2015) with some
light energy at wavelengths that chlorophyll absorbs poorly modifications. The tested microorganisms were obtained from
(Glazer, 1982). Presently, phycocyanin is a natural dye that the SCB 2711 Microbiology Laboratory, Department of Biology,
has replaced many synthetic dyes in food and cosmetics as it Faculty of Science, Chiang Mai University excluding Malassezia
contains numerous bioactive compounds which have the furfur M21 which was obtained from the Department of
potential to be used as some medicinal additives. Based on a Microbiology, Faculty of Medicine, Chiang Mai University.
number of experimental findings, PC is used as an adjuvant Each species of bacteria was inoculated with a single colony
because of its several beneficial properties, including reactive that was transferred into a 10 mL of sterile culture broth
oxygen species (ROS) inhibitory effects and it’s antimicrobial (Mueller Hinton broth (MHB) for S. aureus ATCC 25923, S.
properties (Romay and Gonzalez, 2000). Furthermore, it has the epidermidis ATCC 14990, and MRSA DMST 20625; Brain Heart
capacity to slow down inflammatory processes (Reddy et al., Infusion Broth (BHI) for P. acnes DMST 14916; Sabouraud
2000), including a decrease in nuclear factor activation in RAW Dextrose broth (SDB) for C. albicans DMST 21424; and Dixon
264.7 macrophages, which attenuated lipopolysaccharide- broth for M. furfur M21) using a sterile loop. The broth cultures
induced iNOS expression and TNF production. In vitro were incubated at 37°C for 24 h excluding P. acnes which was
evidence of the suppression of COX-2 activity (Cherng incubated at 37°C for 72 h in an anaerobic jar. The grown
et al., 2007). bacteria were adjusted to 0.5 McFarland standard turbidity and
However, the application of PC on hair dyeing colorants is spread on agar plates using sterile cotton swabs then 5 wells were
yet to been fully investigated. Thus, the research aimed to study made using sterilized micropipette tips. In each well, 50 µL of 25
the feasibility of using phycocyanin as an alternative hair dye. mg/mL phycocyanin (1.25 mg/well) was loaded including
For this, phycocyanin was evaluated for anti skin-pathogenic positive and negative control. After incubation at suitable
activities, as well as toxicity test of human immortalized conditions for each microorganism, a clear zone of inhibition
keratinocyte cells (HaCaT), and the formulation of the hair (diameter) was measured in millimeters (mm). Treatments using
dye using natural developers and mordants. Additionally, the sterilized distilled water as a negative control and antibiotic as a
dyeability of dyed hair after washing with shampoo positive control (1 mg/mL Gentamycin for S. aureus ATCC
was examined. 25923, S. epidermidis ATCC 14990, and P. acnes DMST 14916;
1mg/mL Doxycycline for MRSA DMST 20625; 0.5 mg/mL
Nystatin for C. albicans; and 10 mg/mL Ketoconazole for M.
2 Materials and methods furfur M21) were performed. Each experiment was performed
three times.
2.1 Extraction of phycocyanin

Phycocyanin extraction was carried out by using a slightly 2.3 Cytotoxicity

modified version of the freezing and thawing method (Takano
et al., 1973). First, two grams of dried Arthrospira (Spirulina) The MTT assay was used to assess the cytotoxicity of
platensis biomass powder was suspended in 40 mL of sterilized phycocyanin on human immortalized keratinocyte (HaCaT) as
distilled water. The cells were lysed freezing at -20°C for 4 h and described by (Lee et al., 2013 and Imbimbo et al., 2019) with
thawing at 37°C for 30 min. This process was repeated 25 times. slight modification. HaCaT cells (CLS Dell Lines Service,
Cell debris was removed by centrifugation at 2800 rpm and 4°C 300493) were maintained in Dulbecco’s modified Eagle’s
for 10 min. After that, the solvent was removed from pigment medium (DMEM) (GibcoTM) with 10% fetal bovine serum
extracts using a rotary evaporator and dehydration with (GibcoTM), in a 5% CO2 humidified atmosphere at 37°C.
a lyophilizer. HaCaT cells were seeded in 96 well plates with a density of 2
× 104 cells/well and incubated for 1 day in a 5% CO2 incubator at
37°C. Approximately 24 h after seeding, phycocyanin in
2.2 Determination of bioactivity against deionized water was diluted with serum-free DMEM media
skin pathogenic microorganisms (0.005-2.5 mg/mL) and then treated in HaCaT cells. The
control was treated with serum-free DMEM media. Three days
The phycocyanin was tested for antibacterial activity against later, 30 µL of MTT (2 mg/mL in PBS, pH 7.4) (Bio Basic Inc.,
six skin pathogenic microorganisms; Staphylococcus aureus Markham, ON, Canada) was added to each well and the mixture
ATCC 25923, Staphylococcus epidermidis ATCC 14990, was further incubated for 4 h. The formazan crystals were then
Methicillin-resistant Staphylococcus aureus (MRSA) DMST solubilized with dimethyl sulfoxide (DMSO) (PanReac

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AppliChem) and the absorbance at 540 nm and 630 nm were a clean container and kept at -20°C until ready for use
determined using a microplate spectrophotometer (Biochrom, (Packianathan and Karumbayaram, 2010).
UK). Each experimental value was the mean value from three The formulation of hair dye is designed followed by previous
wells. Cell survival was expressed as the percentage of viable cells of natural hair dye (Boonsong et al., 2012) with some
in the presence of the phycocyanin concentration compared modifications. Phycocyanin’s specific volume is based on its
with the control. maximal solubility in water. In order to establish a developer for
a new formula, phycocyanin hair dye containing the ingredients
mentioned in Table 1 was initially produced.
Then, the hair dye mixtures were applied to the bleached
2.4 Phycocyanin hair dye formulation
hair for 1.30 h before being thoroughly cleansed with water and
and color fastness with shampoo wash
shampoo. The samples were air dried at 70 ± 5% RH. The color
2.4.1 Hair sample preparation appearance of dyed hair was observed by the naked eyes and
tested for the color coordinate values measured by a Chroma
Black human hair for dye testing was purchased from a local
salon in Chiang Mai, Thailand. Bleaching was done to lighten Meter (MINOLTA CR-300, JAPAN) as follows.
the hair so that color changes could be observed more clearly qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
∗
while using natural dyes. The hair was shampooed, air-dried, DEab = ðDL ∗Þ2 +ðDa ∗Þ2 +ðDb ∗Þ2
and then bleached using 6%(v/v) hydrogen peroxide (H2O2) Where DL* is difference in lightness/darkness value (0-100;
mixed with 30% (w/v) potassium persulfate (K2S2O8) for 10 min.
black to white), Da* is differenced on red/green axis (-60 to +60;
The proportion of hair sample to bleaching agent was 1:25 (w/v). green to red), Db* is differenced on yellow/blue axis (-60 to +60;
The bleached black hair turned blonde. After that, the bleached
blue to yellow), and DE*ab is total color difference values.
hair was bundled the rubber, approximately 2 cm long and 0.2 g The best formula for developer identification was used to
for each sample. It was then kept at 25°C ± 3 with a relative
identify the mordant list in Table 2. After that, the hair dye
humidity of 70 ± 5% (Boonsong et al., 2012). The bleached hair mixtures were applied. The technique was completed as
was color assessed and used as a reference before being dyed
described previously. The concentration of mordant was listed
with the new formula of phycocyanin hair dye. The color of the in Table 3. Finally, the color fastness of shampoo wash was
hair dyed sample was recorded in terms of CIELAB color system, measured by comparing the total color difference values after
which are internationally recognized as the standard method repeating the shampoo wash at the first, fifth, and tenth time.
(Lozano et al., 2017).

2.4.2 Ingredients preparation 2.5 Stability testing and color

Hair dye consists of three components: colorant, developer, degradation of phycocyanin in
and mordants. Phycocyanin extracted from Arthrospira hair dye formulation
(Spirulina) platensis was using as a coloring agent. Hydrogen
peroxide (H2O2) (The United Drug (1996) Co., Ltd.), ascorbic 2.5.1 Stability test
acid (Northeast Pharmaceutical Group Co., Ltd.), and 5% The stability of the phycocyanin hair dye formulation was
Chamomile tea (dried Chamomile flowers from a local market investigated in liquid formulations (ready to use) and powder
in Chiang Mai, Thailand) were used as developers. Alum (Ward formulations (fresh mixing before use). They were kept in
william), 5% Assum tea (dried Assum tea leaves from a local microcentrifuge tube and collected each formulation by
market in Chiang Mai, Thailand), Arabic gum (Union Science covered with aluminium foil and non-covered with aluminium
Co., Ltd.), and Aloe vera extract (fresh Aloe vera leaves from a foil. After that incubated at room temperature and accelerated
local market in Chiang Mai, Thailand) were used as mordants. stability testing. The test was also performed in four cycles of the
5% Chamomile tea and 5% Assum tea were freshly prepared. heating/cooling method (45°C, 48 h alternated with 4°C, 48 h for
Briefly, 5 g of dried Chamomile flowers or dried Assum tea 1 cycle). The formulation’s appearance, including any variations
leaves were stirred and boiled with distilled water for 30 min, in pH, color and phase separation, was examined
then cooled down at room temperature and the extracted was (Whangsomnuek et al., 2019).
poured through a filter cloth (Boonsong et al., 2012). The Aloe
vera extract was prepared by washing thoroughly and the outer 2.5.2 Color degradation
green surface was peeled off and rinsed the gel twice to three The color degradation of phycocyanin present in the hair
times. Filtration was performed on 100 g of the Aloe vera gel was dye formulation (liquid/powder) during storage was estimated
crushed to a semi-solid consistency which was subjected to in terms of the percentage of phycocyanin. The water soluble
filtration. The filtrate was subjected to evaporation to 1/10th of phycocyanin was collected in same condition and estimated the
its volume under a controlled temperature (60°C), and stored in percentage of phycocyanin remaining to demonstrate the trend

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TABLE 1 Formulation of hair dye solution (net content 15 mL).

Formula of hair dye Hair dye ingredients

PC (g) H2O2 (mL) Ascorbic acid (g) 5% Chamomile tea (mL) Water (mL)

(T1) PC + water 0.15 – – – 15

(T2) PC + H2O2 0.15 0.3 – – 14.7
(T3) PC + Asc 0.15 – 0.75 – 15
(T4) PC + Cha 0.15 – – 5 10

*** PC, Phycocyanin; H2O2, Hydrogen peroxide; Asc, Ascorbic acid; Cha, 5% Chamomile tea.

of color degradation in neutral solution. The phycocyanin 3.2 Determination of bioactivity against
concentration in the hair dye formulation was calculated using skin pathogenic microorganisms
absorbance at 615 nm and 625 nm using a microplate
spectrophotometer (Biochrom, UK) with the simultaneous Phycocyanin did not show a clear zone on the surface of
equations of Bennett and Bogorad (1973). culture plates when tested for skin pathogenic microorganisms,
as shown in Table 4.
A615 − 0:474(A652)
½PC = (mg=mL)
5:34
The remaining phycocyanin quantity was calculated and 3.3 Cytotoxicity
compared with that of the initial concentration.
The in vitro cytotoxicity of phycocyanin was investigated in
HaCaT cells at various concentrations from 2.5 mg/mL then two-
2.6 Statistical analysis fold dilution was performed 4 times. As demonstrated in Figure 2,
phycocyanin was shown to be highly safe and non-toxic to the
Three replicate analyses were performed, and the average HaCaT cells at concentration that used from 0.156-2.5 mg/mL.
results (mean standard deviation) were reported. Difference in
significance values between treatments were assessed by one-way
ANOVA using Duncan’s multiple range tests (p< 0.05). The 3.4 Phycocyanin hair dye formulation
SPSS statistical program (SPSS version 28.0 for Windows, SPSS and color fastness with shampoo wash
Inc., Chicago, IL, https://www.ibm.com/products/spss-statistics)
was used for the statistical study. As shown in Figure 3, the color coordinate values (L*, a*, and
b*) were significantly affected by the interaction of the
phycocyanin hair dye compared with the control. T1; The
3 Results phycocyanin powder was dissolved in water. It was discovered
that the phycocyanin could make the hair slightly darker and
3.1 Extraction of phycocyanin increase the greenness and blueness of the dyed hair, as shown in
Figure 4B. T2; 2%(v/v) of H2O2 was used as a dissolving agent and
The appearance of phycocyanin after lyophilization shows a chemical developing agent in this formula. The color coordinate
bright blue color and fine powder that resembles flour (Figure 1). values of T2 demonstrated that this formula could increase the

TABLE 2 Formulations of hair dye solution (net content 15 mL).

Formula of hair dye Hair dye ingredients

PC Ascorbic acid Alum 5% Assamtea Arabic gum Aloevera extracted Water

(g) (g) (g) (mL) (g) (mL) (mL)

(T3) PC + Asc 0.15 0.75 – – – – 15

(T5) PC + Asc + Alu 0.15 0.75 0.75 – – – 15
(T6) PC + Asc + Ass 0.15 0.75 – 5 – – 10
(T7) PC + Asc + Ara 0.15 0.75 – – 0.75 – 15
(T8) PC + Asc + Aloe 0.15 0.75 – – – 5 10

*** PC, Phycocyanin; Asc, Ascorbic acid; Alu, Alum; Ass, 5% Assum tea; Ara, Arabic gum; Aloe, Aloe vera.

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TABLE 3 Formulation of hair dye solution (net content 15 mL).

Formula of hair dye Hair dye ingredients

PC(g) Ascorbic acid (g) Arabic gum (g) Water (mL)

PC + Asc + Ara0.2 0.15 0.75 3 15

PC + Asc + Ara0.4 0.15 0.75 6 15
PC + Asc + Ara0.6 0.15 0.75 9 15
PC + Asc + Ara0.8 0.15 0.75 12 15
PC + Asc + Ara1.0 0.15 0.75 15 15

*** PC, Phycocyanin; Asc, Ascorbic acid; Ara, Arabic gum.

lightness, greenness, and blueness of dyed hair, as illustrated in on the redness, but also slightly increased in the blueness value,
Figure 4C. T3; 5%(w/v) of ascorbic acid was used as a dissolving as shown in Figure 6B. T6; 1.6%(W/V) of Assum tea could
and natural developing agent. This formula could make the hair a darkened the hue and enhanced greenness, blueness, and the
little darker than the control, with a considerable increase in effects were similar in formulas T7; 5%(w/v) of Arabic gum, and
greenness and blueness, as well as the highest difference in T8; 1.6%(W/V) of Aloe vera, which is shown in Figures 6C–E.
appearance as shown in Figure 4D. T4; Chamomile tea at 5%(v/ However, when observing a higher Db*, which represents the
v) was used as a dissolving and developing agent. The color difference on the yellow/blue axis, formula T7 showed increased
coordinate values demonstrated that it could lighten the hair and blueness. As a result, Arabic gum was chosen and optimized for
also increased the value of greenness and blueness, Figure 4E. a concentration in the next experiment.
However, the total color difference was not significantly different Figure 7 shows the results of various Arabic gum
from the formulas T1, T2. concentrations. The color difference is demonstrated in terms
The color coordinate value and the color appearance of hair of DE*ab. The strongest color, which appeared on dyed hair in
of the hair dyed with formula T3 show the highest significance the first shampoo wash was equal to 42 when 0.2 g/mL of Arabic
compared to other formulas. To improve the phycocyanin hair gum was added, and slightly dropped to 40 when added 0.4 g/
dye, ascorbic acid was selected as the mordant in the mL, but moiety decreased when 0.6 g/mL of Arabic gum was
next experiment. added and gradually decreased as the concentration of Arabic
Figure 5 shows the results of mordant when phycocyanin gum was increased.
was used as a dye and ascorbic acid was used as a developing The different color box in Figure 7 represents how the dyed
agent in the hair dye formula. T5; 5%(w/v) of alum was used as hair changes color after the first, fifth, and tenth shampoo
the mordant, which lightened the color and had a minor effect washes. The purpose of this test was to examine the color

FIGURE 1
Visual characteristics of lyophilized phycocyanin powder.

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TABLE 4 Antibacterial activity of phycocyanin by the agar well diffusion method.

Pathogens Zone of inhibition (mm)

Phycocyanin Positive control Negative control

S. aureus ATCC 25923 NZ 10 ± 3 NZ

S. epidermidis ATCC 14990 NZ 10 ± 2 NZ
MRSA DMST 20625 NZ 7.5 ± 1 NZ
P. acnes DMST 14916 NZ 10 ± 3 NZ
C. albicans DMST 21424 NZ 15 ± 2 NZ
M. furfur M21 NZ OZ NZ

***NZ, No clear zone; OZ, Over clear zone.

fastness of dyed hair. The results of hair dye using the mordant the hair dye formulation did not change after testing. Other
in concentration at 0.2 and 0.4 g/mL shows highly significant appearances, such as the color of liquid formulations,
change in color in first shampoo wash and half elimination after dramatically changed from bright blue to brown. The
the fifth shampoo wash, and a slight decrease after the tenth formulations were separated after heating/cooling cycle 3, as
shampoo wash. The total difference in color after the first shown in Table 5. This result was different from the stability
shampoo wash was less than two concentrations before, and testing of powder formulations, which did not change when
somewhat decreased when shampoo wash was repeated while visually observed.
using higher concentrations of mordant (0.6, 0.8, and 1 g/mL). A The color degradation of phycocyanin present in the hair
photograph showing the most change using the dye in this study dye formulation is presented as a percentage of phycocyanin
after washing is shown in Figure 8. remaining in Figure 9. The phycocyanin in water and
formulation decreased in all storage conditions. The
phycocyanin that was soluble in water tended to decrease
3.5 Stability testing and color linearly when incubated at room temperature and through a
degradation of phycocyanin in heating/cooling cycle. The phycocyanin concentration remained
hair dye formulation about 60% and 40% after four cycles (16 days). The amount of
phycocyanin in liquid formulations declined linearly when
The stability testing of liquid formulations under room incubated at room temperature, although this decreased
temperature and accelerated conditions showed that the pH of considerably after the first heating/cooling cycle and continued

FIGURE 2
a,b,c
MTT cell viability assay of HaCaT cell treated with various concentrations of phycocyanin (PC). Means followed by a different letter are
significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of (%) cell viability.

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FIGURE 3
Different in color coordinate values compared with the bleached hair (control); lightness values (ΔL*), redness (Δa*), yellowness (Δb*), and total
difference (DE*ab) of hair dyed with (T1) PC + water, (T2) PC + 2% H2O2, (T3) PC + 5% ascorbic acid, (T4) PC + 5% Chamomile. a,b,c Means followed by
a different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of lightness values (ΔL*), a*,b*,c* means followed by a
different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of redness (Δa*), A,B,C means followed by a different letter
are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of yellowness (Δb*), and A*,B*,C* means followed by a different letter are
significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of total difference (ΔE*ab).

to decrease to less than 10% after the fourth. The phycocyanin ruckeri, and E. coli (Safari et al., 2020). The phycocyanin
concentration in powder formulations was more stable than extracts used in this investigation were not purified so the
before in all conditions. There was very little change up to the active ingredient compound may not be concentrated enough
second cycle, then gradually decreased, but the phycocyanin to inhibit skin pathogenic microorganisms. The phycocyanin
concentration remained above 75%. extracts have no inhibitory effect on the growth of M. furfur M21
because Malassezia species are lipid-dependent fungi which
require external lipid sources for growth (Juntachai, 2018).
4 Discussion The tested extract is a water-soluble protein (Kuddus et al.,
2013). Therefore, the infection was not inhibited.
According to the research, concentration of phycocyanin at The in vitro cytotoxicity test is a general approach for
25 mg/mL which was equal to the concentration used to assessing the toxicity of chemical and biological compounds in
formulate the hair dye was studied. Crude phycocyanin a laboratory. Moreover, the type of selected cell line depends on
obtained from Arthrospira (Spirulina) platensis has no effect the investigation purpose. HaCaT cells is a spontaneously
on skin pathogenic microorganisms in this study. However, immortalized human keratinocyte cell line that has been
previous studies have been reported the anti-microbial effect of widely used in skin biology and differentiation research
phycocyanin extracted from Arthrospira (Spirulina) platensis. (Wilson, 2013). The biocompatibility of Arthrospira (Spirulina)
Crude phycocyanin extracted from Arthrospira (Spirulina) platensis commercial PC with HaCaT cell line was previously
platensis isolated from the extreme haloalkakine environment documented using a low concentration range of 5-80 µg/mL and
of Lonar Crater Lake has an antibacterial potency against E. coli, it also offered UVB protection, reduced wrinkle formation,
S. aureus, S. typhi, Shigella spp., and P. aeuroginosa. However, restored the physical barrier function of skin, and suppressed
fungi were found to be not affected (Mohite et al., 2015). oxidative stress (Jang and Kim, 2021). Alternatively, the
Arthrospira (Spirulina) platensis phycocyanin extracted by phycocyanin that was extracted from Anabaena cylindrica
enzymatic (lysozyme) digestion process and partial purified by exhibited some toxicity to HaCaT cells and especially evident
ammonium sulphate precipitation showed the antimicrobial in the purified phycocyanin extracts (Macá rio et al., 2022). In
activities against L. monocytogenes, S. aureus, S. iniae, Y. this study, phycocyanin was used as a colorant in the hair dye

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A B C D E

FIGURE 4
Hair samples after dyeing with phycocyanin and some developer. (A) bleached hair, (B) PC + water, (C) PC + 2% H2O2, (D) PC + 5% ascorbic
acid, (E) PC + 5% Chamomile tea.

formula at high concentration. The cytotoxicity of phycocyanin Hair dyes are classified as temporary, semipermanent, and
at various concentrations, from 0.156 mg/mL to 2.5 mg/mL, was permanent based on their capacity to penetrate the surface of
examined in HaCaT cells. The result demonstrated that the deeper parts of the hair shaft and the longevity of the color after
percentage of HaCaT cell viability did not decrease comparing washing (Guerra-Tapia and Gonzalez-Guerra, 2014).
with the control for all of the phycocyanin concentrations. This Temporary dyes cause less damage to the hair than the other
could mean that certain pigment is not harmful to human skin. two types and do not reach the cortex layer. The intensity of
This is in accordance with the previous studies which these colors is only suitable for 1 shampoo wash (Sargsyan et al.,
demonstrated that the phycocyanin extracted from Arthrospira 2021). Natural dyes function as similarly to temporary hair dye.
(Spirulina) platensis is non-toxic to HaCaT cells (Jang and They have weak color and long-term deposition challenges, so
Kim, 2021). they are unable to penetrate deeply enough into the hair to

FIGURE 5
Different in color coordinate values compared with the bleached hair (control); lightness values (ΔL*), redness (Δa*), yellowness (Δb*), and total
difference (ΔE*ab) of hair dyed with (T3) PC + Asc, (T5) PC + Asc + Alu, (T6) PC + Asc + Ass, (T7) PC + Asc + Ara, (T8) PC + Asc + Aloe. a,b,c Means
followed by a different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of lightness values (ΔL*), a*,b*,c* means
followed by a different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of redness (Δa*), A,B,C means followed
by a different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of yellowness (Δb*), and A*,B*,C* means followed
by a different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of total difference (ΔE*ab).

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A B C D E

FIGURE 6
Hair samples after dyeing with phycocyanin ascorbic acid and some mordant. (A) PC + Asc, (B) PC + Asc + Alu, (C) PC + Asc + Ass, (D) PC +
Asc + Ara, (E) PC + Asc + Aloe.

prevent the dyed hair from washing or fading (Inman et al., phycocyanin that had entered and adhered to the hair fiber due
2020). Hair dyeability can be improved by adding ingredients to some functional groups of phycocyanin could be bonded to a
like a developer which can open the hair cuticle and disrupt cationic polymer with the hair cuticle (Guerra-Tapia and
chemical connections allowing dye molecules to go deeper into Gonzalez-Guerra, 2014). Additionally, the phycocyanin protein
the hair shaft and interact with hair proteins. Furthermore, function might be conjugated with a keratin protein function in
mordant fixes the dye in hair fibers and improves hair color hair, resulting in a fusion insoluble protein (Tinoco et al., 2019).
fastness (Boonsong et al., 2012). It may be argued that phycocyanin protein has the ability to
In this study, phycocyanin dissolved in water made the color function as both a coloring agent and a mordanting agent in this
of the hair slightly darker and increase the greenness and hair dye formula. Hydrogen peroxide, ascorbic acid, and
blueness a bit compared with control (Figure 4A), as shown in Chamomile tea were used as developers. Hair dye mixed with
Figure 4B. It is possible to presume that some molecules of ascorbic acid as a natural developer, had the highest amount of

FIGURE 7
Total different color (ΔE*ab) values of hair dyed compared with the dyed hair before washing. a,b,c Means followed by a different letter are
significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of lightness values (ΔL*), a*,b*,c* means followed by a different
letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of redness (Δa*), A,B,C means followed by a different
letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of yellowness (Δb*), and A*,B*,C* means followed by a
different letter are significantly different in relation to Duncan’s Multiple Range Test (p < 0.05) of total difference (ΔE*ab).

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FIGURE 8
The appearance of the dyed hair according to the Arabic gum concentration.

total color different (DE*ab), as shown in Figure 3, and the improved the appearance of dyed hair that was dyed. Alum,
appearance of dyed hair is presented in Figure 4D. This is metal ion, is able to contact the functional groups of
consistent with the previous studies (Boonsong et al., 2012). phycocyanin protein and precipitate (Boonsong et al., 2012) so
Ascorbic acid could break some hydrogen and ionic bonds in the that the formulation T5 fails to dye, as Figure 6B. Arabic gum
hair structure. In addition, a few disulfide bonds in the hair performed the best in this experiment (Figure 5). Arabic gum or
structure might also be broken in an acidic solution (Rinzler, acacia gum is a polysaccharide that is widely used as an
1982), and reduced to be sulfhydryl groups. As a result, the important naturally occurring oil in water emulsifiers as a
phycocyanin’s reactive functional groups, such as methyl, suspending agent and thickener in food and in pharmaceutical
carboxyl, and hydroxyl, could bind to sulfhydryl groups by industries. The USFDA (U.S. Food and Drug administration)
covalent of ionic bonds with the amino groups of hair keratin classifies it as Generally Recognized as Safe (Barak et al., 2020).
(Burkinshaw and Kumar, 2009). For the reason that apoproteins This gum’s thickening properties boosted dyeability by
in phycocyanin were consequently denatured by the acidic increasing the contact surface area between hair and pigment.
conditions caused by ascorbic acid (Li et al., 2021). The acid- The highly polymerized polysaccharides offered strong affinities
induced denaturation is usually accompanied by changes in its to the phycocyanin molecules through both electrostatic and
blue hue fading, so the color of hair dyed with phycocyanin and hydrophobic interactions, according to characterizations and
ascorbic acid revealed a greenness more than blue, which is the mechanistic studies (Li et al., 2021). However, the color of the
color of phycocyanobilin. dyed hair decreased with the increasing concentration of Arabic
The following mordants for phycocyanin and ascorbic acid gum. This is because the dye molecule infiltration on the hair
were identified as appropriate; alum, Assum tea, Arabic gum, surface was troubled by the over sticky formula.
and Aloe vera as shown in Figure 6. In addition to the As shown in Figure 8, the dyed hair was able to appear vivid
formulation T5, the results showed that nearly all formulations blue. The color fastness was repeated 10 times. Additionally, L*,

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TABLE 5 The stability testing results of phycocyanin hair dye formulation.

Formulations Stability test Packaging pH Color Separation

Liquid Initial 3.45 ± 0.03 Bright blue X

Cycle 1 RT C Blue X
N Blue X
H/C C Blue green X
N Blue green X
Cycle 2 RT C Blue X
N Blue X
H/C C Green X
N Green X
Cycle 3 RT C Blue green X
N Blue green X
H/C C Green brown ✓
N Green brown ✓
Cycle 4 RT C Blue green ✓
N Blue green ✓
H/C C Brown ✓
N Brown ✓

*** RT, room temperature; H/C, heating/cooling; C, covered with aluminium foil; N, non-covered with aluminium foil.

a*, and b* were recorded and demonstrated in terms of DE*ab formulation is natural, it has little effect on the dyeing process.
before washing, rinsed with water, and washing with shampoo at Moreover, some previous studies (Inman et al., 2020) that used
the first, fifth, and tenth. The color appearance of dyed hair was the color from black bean and took 45 min for dyeing time and
able to persist through shampooing until 5 times when visually (Gargote, 2012) took 2 hours for dyeing hair, the colorant from
observed. The type of hair dye can be divided according to the plant extract, showed more durability time than the less time. It
color durability after the application on hair strands: temporary may be assumed that adhesion of the natural dye to the hair
(the color leaving the hair after the first shampoo wash), semi- enhances the contact time of the extract, enabling penetration of
permanent (washing off thoroughly in 4–12 shampoo washes), the colorant molecules into the hair cuticle and partially
and permanent (lasting for up to 28 shampoo washes) (Da diffusing throughout the cortex.
Franç a et al., 2015). The phycocyanin’s capacity for color The stability and color degradation of phycocyanin hair dye
staining in this developed hair dye formulation could be powder formulation kept at room temperature and covered with
categorized as a semi-permanent hair dye. This result aluminum foil showed the best stability in this study. The
resembled other earlier documented. The use of anthocyanin acidified condition and high temperature were the main
derived from black beans as an alternative hair dye and the wash reasons for phycocyanin denaturation, which cause a fading
fastness shown the monitored color were significantly different rapidly (Zhang et al., 2020). The thermodynamic stability of an
at least four shampooing (Inman et al., 2020). However, the enzyme is significantly influenced by its solution state. Dried
color fastness of dyed hair is less compared to studies utilizing proteins contain a very small amount of water, they are typically
natural dyes from several Thai plants and ferrous sulfate as a more stable and some of them can be kept in dark environments
mordant, which showed slight fading of the color in dyed hair for several months or even over a year without losing their
until fifteen shampoo washings (Boonsong et al., 2012). The activity (Plou et al., 1998). Thus, this study advises that to use
results also demonstrate that hair colored with those extracts had phycocyanin as hair dye application were keeping it at a normal
a darker and more color fastness property. Due to the fact that or low temperature in dark packaging and mixing with water
ferrous sulfate (metal ions) frequently forms a complex with the before use.
hair fiber and the electron-donating chromophores of natural
pigments (Burkinshaw and Kumar, 2009). The dyeing time in
the experiment is 1.30 hours. This may take longer than the 5 Conclusion
average amount of time for hair dyes to be marketed, which
generally suggests dying hair for 45-60 min and may need more Phycocyanin extracted from Arthrospira (Spirulina)
time if you want a brighter effect. Since every ingredient of this platensis has the potential to be used as a hair dye with no

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FIGURE 9
The percentage of phycocyanin remaining in the hair dye formulation during storage.

toxicity to HaCaT cells. The best developer was ascorbic acid and editing. All authors contributed to the article and approved
the best mordant was Arabic gum. The aim of further study will the submitted version.
be aimed to improve the dye ability and stability for
commercial products.
Funding
This research was funded by National Research Council of
Data availability statement Thailand (NRCT) and the Research and Researchers for Industries
(RRi), grant number PHD61I0009 and partially supported by Chiang
The original contributions presented in the study are Mai University, and the Graduate School of Chiang Mai University.
included in the article/supplementary material. Further
inquiries can be directed to the corresponding author.

Acknowledgments
Author contributions I would like to thank Dr. Thida Kaewkod and Miss Kanok-
orn Mayer for their assistance with the cytotoxicity assay. I also
OK: conceptualization, methodology, formal analysis, thank Assoc. Prof. Dr. Itthayakorn Promputtha for providing
resources, investigation, and writing-original draft. YT: microorganisms in this research. Thank you, Mr. Andrew John
resources, writing-review, and editing. HP: writing-review Semansco, for proofreading. We appreciate the funds provided
and editing. RK: writing-review and editing. WP-a: writing- for this effort by the National Research Council of Thailand
review and editing. JP: writing-review and editing. CP: (NRCT) and the Research and Researchers for Industries (RRi),
supervision, conceptualization, project administration, grant number PHD61I0009. This research was partially
funding acquisition, resources, and writing-review and supported by Research Center in Bioresources for Agriculture,

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Industry and Medicine, Chiang Mai University, and the Publisher’s note
Graduate School of Chiang Mai University.
All claims expressed in this article are solely those of the
Conflict of interest authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
The authors declare that the research was conducted in the reviewers. Any product that may be evaluated in this article, or
absence of any commercial or financial relationships that could claim that may be made by its manufacturer, is not guaranteed
be construed as a potential conflict of interest. or endorsed by the publisher.

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