marine drugs

Article
Cosmetic Potential of Pigments Extracts from the Marine
Cyanobacterium Cyanobium sp.
Fernando Pagels 1,2 , Cíntia Almeida 3 , Vitor Vasconcelos 1,2, * and A. Catarina Guedes 1,3

1 CIIMAR/CIMAR-LA—Interdisciplinary Centre of Marine and Environmental Research, University of Porto,

Novo Edifício do Terminal de Cruzeiros de Leixões, Avenida General Norton de Matos, s/n,
4450-208 Matosinhos, Portugal; [email protected] (F.P.); [email protected] (A.C.G.)
2 FCUP—Faculty of Science, University of Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal
3 ISS—Ínclita Seaweed Solutions, Novo Edifício do Terminal de Cruzeiros de Leixões, Avenida General Norton
de Matos, s/n, 4450-208 Matosinhos, Portugal; [email protected]
* Correspondence: [email protected]

Abstract: The current mindset in the cosmetics market about sustainable ingredients had increased
the search for new sources of natural active ingredients. Cyanobacteria are a great source of func-
tional ingredients for cosmetics, as a producer of pigments with described bioactive potential
(carotenoids and phycobiliproteins). This work aimed to evaluate the cosmetic potential of ma-
rine cyanobacterium Cyanobium sp. pigment-targeted extracts (carotenoids and phycobiliproteins),
evaluating their in vitro safety through cytotoxicity assays, cosmetic-related enzyme inhibition,
ingredient stability, and putative product (serum formulation). Results showed no cytotoxicity
from the extracts in skin-related cell lines. Carotenoid extract showed anti-hyaluronidase capac-
ity (IC50 = 108.74 ± 5.74 mg mL−1 ) and phycobiliprotein extract showed anti-hyaluronidase and
anti-collagenase capacity (IC50 = 67.25 ± 1.18 and 582.82 ± 56.99 mg mL−1 , respectively). Regarding
ingredient and serum stability, both ingredients showed higher stability at low-temperature condi-
tions, and it was possible to maintain the pigment content and bioactive capacity stable during the
Citation: Pagels, F.; Almeida, C.; tested period, although in higher temperatures the product was degraded in a week. As a major
Vasconcelos, V.; Guedes, A.C. conclusion, both extracts can be potential natural and sustainable ingredients for cosmetic uses, with
Cosmetic Potential of Pigments relatively simple formulation and storage, and can be promising natural anti-aging ingredients due
Extracts from the Marine
to their bioactive capacity.
Cyanobacterium Cyanobium sp. Mar.
Drugs 2022, 20, 481. https://doi.org/
Keywords: carotenoids; phycobiliproteins; antioxidant; hyaluronidase; accelerated stability; serum
10.3390/md20080481
formulation
Academic Editor: Daniela Giordano

Received: 5 July 2022

Accepted: 25 July 2022
1. Introduction
Published: 27 July 2022
The use of natural ingredients for aesthetic and health-enhancing applications in
Publisher’s Note: MDPI stays neutral popular and homemade recipes and without scientific bases existed long before today’s
with regard to jurisdictional claims in
concept of cosmetics. Thus, for a long time, cosmetics were made from mineral materials
published maps and institutional affil-
and herbal pastes and oils [1]. Nowadays, the movement toward green packaging, recycling,
iations.
and environmental literacy has contributed to a revolution in the demands of the cosmetics
industry and the rescue of already established suppliers of bioactive products, plants, and
algae [2]. From the formulation to the packaging material, the cosmetic industry has been
Copyright: © 2022 by the authors.
moving toward adaptation to greener manufacturing [3].
Licensee MDPI, Basel, Switzerland.
This dynamic industry is now looking for new opportunities and new goods with fea-
This article is an open access article tures other than beauty; a new segment of the industry, called cosmeceuticals, started with
distributed under the terms and the use of cosmetics for health purposes [2]. Although natural origin does not necessarily
conditions of the Creative Commons mean “healthy”, it is nevertheless true that the compounds and extracts used in natural
Attribution (CC BY) license (https:// cosmetics are also potentially beneficial to health [2].
creativecommons.org/licenses/by/ The use of cyanobacteria as a source of ingredients achieves even greater potential
4.0/). with the idea of cosmeceuticals. These organisms can be a source of compounds with nar-

Mar. Drugs 2022, 20, 481. https://doi.org/10.3390/md20080481 https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2022, 20, 481 2 of 25

rowly defined potential for health applications, such as fatty acids, polyphenols, peptides,
polysaccharides, and pigments [4]. When it comes to cyanobacterial pigments in special
carotenoids and phycobiliproteins, it is possible to combine their bioactive capacity, varied
colors, and cosmetic enhancement characteristics (e.g., moisturizer, stabilizing agents) and
draw interest from the natural cosmetic industry [5,6]. The use of extracts overcomes one of
the biggest concerns of the industry, which is product stability. As these complex matrices
contain other compounds besides pigments, they often increase not only the stability but
also the useful life of the pigments referred to above (e.g., polyphenols) [6].
Carotenoids can be included in sunscreen, anti-ageing, and antioxidant formulations
due to their high-efficiency antioxidant capacity. These compounds, inside cyanobacteria
cells, are responsible for capturing exciding energy from the photosynthetic metabolism,
mitigating harmful effects and cell damage [7]. Once extracted, these compounds can be
used for a similar purpose in human skin, where prolonged exposure to sunlight (UV
radiation and high light intensity) contributes to potential damage to cells [8,9].
A similar thought can be applied to phycobiliproteins, as they can act as functional
additives, since they have been associated with a variety of bioactivities, such as antioxi-
dants, antivirals, and antimicrobials and antitumors, among others [10]. Moreover, these
compounds can be used as natural dyes to mitigate toxicity and damage to the skin, as well
as allergy to synthetic dyes [11]. These blue hydrophilic pigments can be used in skincare
products, particularly creams and lotions, due to their water solubility.
While there is a great diversity of cyanobacteria species and their biochemical pro-
file, Arthrospira platensis (spirulina) is still the main choice for cosmetic usage. Yet, other
cyanobacteria species have been suggested in fundamental research. In addition, most
studies concerning the potential of cyanobacteria in cosmetics are focused on chemical
characterization and bioactivity prospection, whereas the real applicability as an ingredient
in a final product has been kept in the industry and rarely described in academic research.
This research focused on marine cyanobacterium Cyanobium sp. LEGE 06113, which
appears to be an effective strain for industrial use due to its morphological characteristics
and chemical composition. This strain is a fast-growing unicellular organism with a highly
adaptable metabolism and response to light and with great production of pigments, both
carotenoids and phycobiliproteins [12]. Moreover, the extracts from Cyanobium sp. have
been proposed as a great potential source of antioxidant and anti-inflammatory capacity and
a sustainable option as a cyanobacteria-based bioprocess, as from the same biomass both a
carotenoid- and a phycobiliprotein-rich extract can be obtained in a successive extraction
method [13] following the principle of environmental and economic sustainability.
Therefore, this work aims to evaluate the cosmetic potential of Cyanobium sp. pigment-
targeted extracts, evaluating their in vitro safety through cytotoxicity assays and cosmetic-
related enzymes inhibition. Furthermore, to assess the real applicability as a cosmetic
ingredient, the formulation of an ingredient and its application in a product (serum) was
performed, considering stability as the main parameter for the assessment.

2. Results
The cosmetic potential of Cyanobium sp. pigments was evaluated following a struc-
tured line of thought that starts from the assessment of the potential of the extract as a
cosmetic ingredient. Then the extracts were proposed as an active part of a cosmetic ingredi-
ent, being evaluated in terms of bioactive capacity and stability. Finally, as proof of concept,
the ingredient was included in an end product (skin serum) to assess the compatibility,
bioactive capacity, and stability of the ingredient with a more complex formulation.

2.1. Extract Cytotoxicity

Considering a cosmeceutical approach for the extracts from Cyanobium sp., their
cytotoxicity was evaluated in three skin-related cell lines, keratinocytes (HaCat), fibroblasts
(3T3L1), and endothelial (hCMEC/D3) cells. The results are shown in Figure 1 for ethanolic
extract and Figure 2 for water extract. Both ethanolic and water extracts showed no
 Mar. Drugs 2022, 20, 481 3 of 25
Mar. Drugs 2022, 20, x 3 o

cytotoxic effects on the evaluated cell lines, with no significant differences from the control
2.1.>Extract
(p both 24 and 48 h of exposition in concentrations up to 1000 µg mL−1 , meaning
0.05), forCytotoxicity
that the extracts can be safe for cosmetic use, with no toxicity to skin-related cell lines.
Considering a cosmeceutical approach for the extracts from Cyanobium sp., th
2.2. Enzymatic was
cytotoxicity Activity
evaluated in three skin‐related cell lines, keratinocytes (HaC
Skin aging is one
fibroblasts (3T3L1), and of theendothelial
most meaningful processes that
(hCMEC/D3) cosmetics
cells. attempt
The results aretoshown
address.in Figur
Theethanolic
for most noticeable changes
extract and are skin dryness,
Figure decreased
2 for water elasticity,
extract. Bothfine wrinkles,and
ethanolic and skin
water extra
changes that result in expression lines [14]. In cosmetics, evaluation of enzymatic activity
showed no cytotoxic effects on the evaluated cell lines, with no significant differen
can add value and increase the potential of the ingredient. The inhibition of enzymes such
from the controlcollagenase,
as hyaluronidase, (p > 0.05), for both 24and
tyrosinase, and 48 h of
elastase is aexposition
goal for newin concentrations
products regarding up to 1
μg
themL −1 , meaning
anti-aging that the
application. extracts
The canofbe
inhibition safe for cosmetic
hyaluronidase, use,
elastase, andwith no toxicity
collagenase is to sk
related cell lines.
mainly related to a decrease in wrinkles and enhancement of skin elasticity, whereas the
inhibition of tyrosinase is related to skin whitening and anti-melanogenesis treatments.

Figure 1. Cytotoxicity of Cyanobium sp. ethanolic extract in skin-related cell lines: endothelial
Figure 1. Cytotoxicity of Cyanobium sp. ethanolic extract in skin‐related cell lines: endothe
(hCMEC/D3), epithelial (HaCat), and fibroblast (3T3L1) exposed during 24 and 48 h (average ± stan-
(hCMEC/D3),
dard deviation, nepithelial (HaCat),
= 3). Different and
symbols fibroblast
in the (3T3L1)
same graphic exposed
for each exposureduring 24 significant
time mean and 48 h (averag
standard
differencesdeviation,
between the n extract
= 3). Different symbols
concentration innegative
and the the same graphic
control with for
DMSOeach
1%exposure
(p < 0.05). time m
significant differences between the extract
DMSO 20% is the positive control for the assay. concentration and the negative control with DMSO
(p < 0.05). DMSO 20% is the positive control for the assay.
Mar. Drugs 2022, 20, x 4 o
Mar. Drugs 2022, 20, 481 4 of 25

Figure 2. Cytotoxicity of Cyanobium sp. water extract in skin-related cell lines: endothelial
Figure 2. Cytotoxicity of Cyanobium sp. water extract in skin‐related cell lines: endoth
(hCMEC/D3), epithelial (HaCat), and fibroblast (3T3L1) exposed during 24 and 48 h (aver-
(hCMEC/D3), epithelial (HaCat), and fibroblast (3T3L1) exposed during 24 and 48 h (averag
age ± standard deviation, n = 3). Different symbols in the same graphic for each exposure time
standard deviation, n = 3). Different symbols in the same graphic for each exposure time m
mean significant differences between the extract concentration and the negative control with DMSO
significant differences
1% (p < 0.05). DMSO 20%between the extract
is the positive concentration
control for the assay. and the negative control with DMSO
(p < 0.05). DMSO 20% is the positive control for the assay.
Here both extracts were evaluated to be used as inhibitors of such enzymes. Ethano-
lic extract
2.2. Enzymaticonly Activity
was able to inhibit hyaluronidase, with an inhibitory concentration of
108.74 ± 5.74 µg mL−1 (IC50 ), whereas the water extract was able to inhibit both hyaluroni-
daseSkin aging is onewith
and collagenase, of the most
an IC meaningful processes that cosmetics attempt to addr
50 of 67.25 ± 1.18 and 582.82 ± 56.9, respectively. Note-
The most noticeable changes are
worthy is that the water extract was 1.6-fold skin dryness, decreased
better than the elasticity, fine wrinkles,
ethanolic extract in the and s
changes that result
hyaluronidase in expression
inhibition. Finally, no lines [14]. Inofcosmetics,
inhibition evaluation
tyrosinase and elastase of
was enzymatic
found activ
in concentrations up to 1000 µg mL −1 in any of the two extracts. Due to the higher potential
can add value and increase the potential of the ingredient. The inhibition of enzymes s
as anti-hyaluronidase
as components, in tyrosinase,
hyaluronidase, collagenase, comparison toand the potential
elastaseas is
anti-collagenase,
a goal for only new produ
this enzymatic inhibition activity was considered for further evaluation. As a proof of
regarding the anti‐aging application. The inhibition of hyaluronidase, elastase, a
concept, a water extract from Camellia sinensis (green tea) was obtained using the same
collagenase is mainlysp.related
setup from Cyanobium to a decrease
first extraction, as it is in wrinkles
a known and enhancement
ingredient for anti-aging of skin elastic
formu-
whereas the inhibition of tyrosinase is related to skin whitening
lations [15]. The IC50 of green tea for hyaluronidase was 122.19 ± 7.64 µg mL , with no and − anti‐melanogen
1

treatments.
statistical differences to the ethanolic extract of Cyanobium sp. and 1.8-fold higher than the
waterHere
extract.
both extracts were evaluated to be used as inhibitors of such enzymes. Ethan
extract only was able to inhibit hyaluronidase, with an inhibitory concentration of 108
± 5.74 μg mL−1 (IC50), whereas the water extract was able to inhibit both hyaluronidase a
collagenase, with an IC50 of 67.25 ± 1.18 and 582.82 ± 56.9, respectively. Noteworthy is t
the water extract was 1.6‐fold better than the ethanolic extract in the hyaluronid
inhibition. Finally, no inhibition of tyrosinase and elastase was found in concentrati
Mar. Drugs 2022, 20, 481 5 of 25

2.3. Cyanobium sp. Cosmetic Ingredients

For better incorporation in cosmetic formulations, two ingredients composed of the
active extract and a liquid vehicle (glycerol for the water extract and linseed oil for the
ethanolic extract) were proposed. The ingredients were then evaluated in terms of their
physical–chemical characterization, bioactive capacity, and stability. Moreover, many
commercial ingredients include a preservative (antioxidant) to prevent degradation of
the active ingredient (extract). Taking that into account, this study also evaluated the
compatibility of the ingredient with several antioxidants to increase its stability. The results
of these assessments are described in the following sections.

2.3.1. Ingredient Characterization

The general composition of a cosmetic includes an active ingredient and excipients
(vehicle, thickening agents, and additives) [11]. The active ingredients are the main com-
ponents of a cosmetic product that are responsible for its function, in this case, pigment
extracts. Furthermore, excipients are all ingredients that do not serve a specific function as
an active ingredient, such as the active ingredient’s vehicle, thickening agents, and addi-
tives (preservatives, colorants, and perfumes). To increase the stability of the extracts and
make their incorporation in different formulations possible, the ethanolic extract was added
to linseed oil (ethanolic ingredient) and the water extract was added to 80% glycerol (water
ingredient). The physical and chemical characteristics of the ingredients are presented in
Table 1. The bioactive capacity (antioxidant and anti-hyaluronidase) and the content of the
total pigment were also measured to control the potential of the ingredients before and
after the stability assays.

Table 1. Physical and chemical characterization of Cyanobium sp. ingredients.

Parameter Ethanolic Ingredient Water Ingredient

pH 4.71 ± 0.06 7.78 ± 0.05
Phases 1 1
Viscosity (cst) 32.85 ± 1.27 74.55 ± 4.88
Density (g cm−3 ) 0.81 ± 0.01 0.98 ± 0.02
Conductivity (µS cm−1 ) 0.41 ± 0.08 7.36 ± 0.06
Color +
L* 35.98 ± 2.12 36.93 ± 1.09
a* −29.33 ± 1.18 −22.72 ± 1.32
b* 45.30 ± 3.16 −8.58 ± 0.85
Optical correspondence Green Blue
Antioxidant capacity (ABTS•+ IC50 , mg mL−1 ) 140.69 ± 6.31 180.93 ± 7.91
Anti-hyaluronidase activity (IC50 , mg mL−1 ) 115.37 ± 10.33 79.41 ± 6.43
Total pigments * (mg g−1 ) 181.83 ± 8.17 505.13 ± 8.18
* Carotenoids for the ethanolic ingredient and phycobiliproteins for the water ingredient. + L*: brightness;
a*: redness; b*: yellowness.

2.3.2. Hot–Cold Ingredient Stability

The first evaluation of ingredient stability was performed in hot–cold cycles for 14 days
in −20 ↔ 20 ◦ C and 4 ↔ 40 ◦ C cycles. The extracts were evaluated in terms of total
pigments, color change (∆E), and bioactive capacity. Moreover, the supplementation with
commercial antioxidants (α-tocopherol, BHT, and a mixture of both (1:1) for the ethanolic
ingredient and ascorbic acid, gallic acid, and a mixture of both (1:1) for the water ingredient)
was tested for compatibility and antioxidant effects in the thermic treatments. Results for
the ethanolic ingredient are shown in Figure 3 and for the water ingredient in Figure 4.
Moreover, due to the influence of antioxidant supplements in the bioactive capacity assays,
only the control condition was evaluated for both extracts (Figure 5).
 Results for the ethanolic ingredient are shown in Figure 3 and for the water ingredient in
Figure 4. Moreover, due to the influence of antioxidant supplements in the bioactive
capacity assays, only the control condition was evaluated for both extracts (Figure 5).
Regarding the ethanolic ingredient, both the content of carotenoids and color were
kept stable in both −20 °C ↔ 20 °C and 4 °C ↔ 40 °C treatments, with no statistical differ‐
Mar. Drugs 2022, 20, 481 ences regardless of the antioxidant supplementation or the number of cycles, as observed 6 of 25
by the homogeneous color gradient present in Figure 3.

A B
Vehicle Vehicle

Control Control

4  40 °C
4  40 °C

α-T α-T

BHT BHT

MIX MIX

Vehicle Vehicle

Control Control

-20  20 °C
-20  20 °C

α-T α-T

BHT BHT

MIX MIX

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Cycles Cycles

0 40 80 120 160 0 10 20 30 40 50

T otal C aroten oid s (m g g -1 ) C olou rch an g e E)

Figure3.3. Effect
Figure Effect of
ofhot–cold
hot–coldcycles
cycles °C◦↔
(4 (4 C↔40 °C ◦ C and
40 and −20
−20 °C ↔◦20
C↔ in ◦(A)
°C)20 C) total
in (A)carotenoids and
total carotenoids
(B) color change (ΔE) of ethanolic ingredients supplemented with commercial antioxidants:
and (B) color change (∆E) of ethanolic ingredients supplemented with commercial antioxidants: α‐to‐
copherol (α‐T), BHT, or a mixture of both (1:1; MIX). The vehicle represents linseed oil and control
α-tocopherol (α-T), BHT, or a mixture of both (1:1; MIX). The vehicle represents linseed oil and
is the ingredient without supplement.
control is the ingredient without supplement.
When it came to the water ingredient, the content of phycobiliproteins was kept sta‐
$ %
9HKLFOH ble in the −20 °C ↔ 20 °C treatment, with no statistical differences regardless of the anti‐
9HKLFOH

oxidant supplementation or the number of cycles. With the 4 °C ↔ 40 °C treatment, a

&RQWURO
reduction was observed from cycle 5 in &RQWURO
the control condition without antioxidant supple‐
lƒ&

lƒ&

mentation and in cycle 7 in the ingredient supplemented with ascorbic acid.

$$ $$
Regarding the color, in the −20 °C ↔ 20 °C treatment no perceptible color change was
*$
observed in the seven cycles (ΔE < 2.0), whereas
*$
in the 4 °C ↔ 40 °C treatment, a linear
change was observed. From cycle 2, the color had a perceptible change (ΔE > 2.0), with
0,; less accentuated change in the ingredients0,; supplemented with gallic acid and the mixture
of antioxidants. The maximum color change was found in cycles 6 and 7, with an ΔE ≈ 7.0,
9HKLFOH which is still an acceptable color change9HKLFOH
in stability assays.

&RQWURO &RQWURO
lƒ&
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*$ *$

0,; 0,;

               
&\FOHV &\FOHV

           

7 RWDO3K \FRE LOLS URWHLQ V P J J  & RORX UFK DQ J H '(

Figure 4. Effect of hot–cold cycles (4 ◦ C ↔ 40 ◦ C and −20 ◦ C ↔ 20 ◦ C) in (A) total phycobiliproteins

and (B) color change (∆E) of water ingredients supplemented with commercial antioxidants: ascorbic
acid (AA), gallic acid (GA), or a mixture of both (1:1; MIX). The vehicle represents glycerol (80%) and
control is the ingredient without supplement.
Mar. Drugs 2022, 20, 481 7 of 25

Figure 5. Effect of hot–cold cycles (4 ◦ C ↔ 40 ◦ C and −20 ◦ C ↔ 20 ◦ C) on the (A) ABTS•+ scaveng-
ing ability and (B) hyaluronidase inhibition capacity (% inhibition; average ± standard deviation,
n = 3) of the ethanolic (E) and water (W) ingredient. Colors of lines indicate different conditions:
E 4 ◦ C ↔ 40 ◦ C ( ); E −20 ◦ C ↔ 20 ◦ C ( ); W 4 ◦ C ↔ 40 ◦ C ( ); W −20 ◦ C ↔ 20 ◦ C ( ).

Regarding the ethanolic ingredient, both the content of carotenoids and color were kept
stable in both −20 ◦ C ↔ 20 ◦ C and 4 ◦ C ↔ 40 ◦ C treatments, with no statistical differences
regardless of the antioxidant supplementation or the number of cycles, as observed by the
homogeneous color gradient present in Figure 3.
When it came to the water ingredient, the content of phycobiliproteins was kept stable
in the −20 ◦ C ↔ 20 ◦ C treatment, with no statistical differences regardless of the antioxidant
supplementation or the number of cycles. With the 4 ◦ C ↔ 40 ◦ C treatment, a reduction
was observed from cycle 5 in the control condition without antioxidant supplementation
and in cycle 7 in the ingredient supplemented with ascorbic acid.
Regarding the color, in the −20 ◦ C ↔ 20 ◦ C treatment no perceptible color change was
observed in the seven cycles (∆E < 2.0), whereas in the 4 ◦ C ↔ 40 ◦ C treatment, a linear
change was observed. From cycle 2, the color had a perceptible change (∆E > 2.0), with less
accentuated change in the ingredients supplemented with gallic acid and the mixture of
antioxidants. The maximum color change was found in cycles 6 and 7, with an ∆E ≈ 7.0,
which is still an acceptable color change in stability assays.
Finally, regarding the bioactive capacity (antioxidant and anti-hyaluronidase) of the
ingredients (Figure 5), both of them were stable in the −20 ◦ C ↔ 20 ◦ C treatment and the
anti-hyaluronidase activity was stable even under the 4 ◦ C ↔ 40 ◦ C treatment. However,
in the 4 ◦ C ↔ 40 ◦ C treatment, both ingredients had a loss of antioxidant capacity, translated
by a decrease in the inhibition of ABTS•+ from the third cycle by 0.2-fold in the ethanolic
ingredient and by 0.3-fold in the water ingredient. On the other hand, the antioxidant
capacity remained present, with an inhibition higher than 50%.

2.3.3. Accelerated Ingredient Stability

The second evaluation of the ingredient stability involved an accelerated stability
assay for 12 weeks, which represented long-exposure effects in the ingredient. Such tests
are demanded by quality-control agencies, such as ISO/TR 18811/2018 and European
Regulation EC 1223/2009. The ingredients were monitored over time for pigment content,
color change, and bioactive capacity. Results are shown in Figure 6 for the ethanolic
ingredient and Figure 7 for the water ingredient. Due to the influence of antioxidant
supplements in the bioactive capacity assays, only the control condition was evaluated for
both ingredients (Figure 8).
Mar. Drugs 2022, 20, x 9 of 26
Mar. Drugs 2022, 20, 481 8 of 25

A B
Vehicle Vehicle

Control Control
4 °C

4 °C
α-T α-T

BHT BHT

Mix Mix

Vehicle Vehicle

Control Control

20 °C
20 °C

α-T α-T

BHT BHT

Mix Mix

Vehicle Vehicle

Control Control

α-T α-T
40 °C

40 °C

BHT BHT

Mix Mix

0 1 2 3 4 6 8 12 0 1 2 3 4 6 8 12
Time (weeks) Time (weeks)

0 40 80 120 160 0 10 20 30 40 50 60
T otal C aroten oid s (m g g -1 ) C olou rch an g e E)

Figure6.
Figure Effect of accelerated
6. Effect conditions(4(4◦ C,
acceleratedconditions ◦ C, and 40 ◦ C) in (A) total carotenoids and (B) color
°C,2020 °C, and 40 °C) in (A) total carotenoids and (B)
color change
change (∆E) (ΔE) of ethanolic
of ethanolic ingredients
ingredients supplemented
supplemented with
with commercial
commercial antioxidants:
antioxidants: α‐tocoph‐
α-tocopherol
erol (α‐T),
(α-T), BHT,BHT,
or aormixture
a mixture
of of both
both (1:1;
(1:1; MIX).The
MIX). Thevehicle
vehiclerepresents
representslinseed
linseed oil
oil and
and control
control is
isthe
the
ingredient without supplement.
ingredient without supplement.

When it came
Regarding toethanolic
the the water ingredient, the thecontent
contentofofphycobiliproteins
carotenoids was was keptalso stable
stable for
during
12 weekstheunder
12 weeks 4 ◦ Catand
4 °C20 ◦ C20
and °C. At 40 °C,
treatments, withthenocontent decreased
statistical depending
differences on the
regardless of
antioxidant supplementation.
the antioxidant supplementation From week
or the 1, the of
number control
cycles.ingredient
On the otherandhand,
the one 40 ◦ C
supple‐
in the
mented with
treatment, ascorbic
both controlacidandstarted to degrade.
α-tocopherol From weekingredients
supplemented 4, all ingredients started tofrom
were degraded de‐
grade,
week 2,equally
whereasreaching an average
the ingredients of ca. 150 mg
supplemented g BHT
with−1 in week
and 12,
a mixrepresenting a reduction
of antioxidants resisted
two
of 70%weeks
of thelonger.
pigmentFrom week 4, all conditions were degraded, reaching the minimum of
content.
ca. 50
A mg g−1 pattern
similar of carotenoid,
was found3.6-fold lesscolor
in the thanevaluation.
the initial content.
No changes were found at 4 °C
and 20 When it comes
°C, with an ΔE to <the
1.0color,
untilall temperatures
week 6 and 1.0 led
< ΔE to <changes
2.0 from inuntil
the ingredient
week 12. Atcolor. The
40 °C,
4 ◦ C week
from treatment
1 to 3,led
thetocontrol
a colorandchange regardless
ascorbic of antioxidant
acid‐supplemented supplementation.
ingredients For the
had perceptible
first month,
color changesnoΔE perceptible changeingredients
> 10.0, whereas was found supplemented
(∆E < 2.0); fromwith week 6 toacid
gallic 12, the
andcolor
a mixwent
of
from bright green to dark yellow with a ∆E ≈ 14.0. At 20 ◦ C a greater color change was
antioxidants had a less accentuated change (ΔE ≈ 5.0). From week 4, all ingredients had
observed from
perceptible week 1 (∆E
differences, ≈ 14.0),
as well as atstabilizing
week 12 (ΔE with ∆E ≈ 26.0 in 6 weeks, regardless of the
≈ 26.0).
antioxidant supplementation. The optical equivalence changed from bright green to yellow,
probably meaning a chlorophyll bleaching, with no change in carotenoid content. At 40 ◦ C,
the color completely changed from green to yellow and then to brown in less than a month,
resulting in a ∆E > 40.0.
Mar. Drugs 2022, 20, x 10 of 26
Mar. Drugs 2022, 20, 481 9 of 25

A B
Vehicle Vehicle

Control Control
4 ºC

4 °C
AA AA

GA GA

MIX MIX

Vehicle Vehicle

Control Control

20 °C
20 °C

AA AA

GA GA

MIX MIX

Vehicle Vehicle

Control Control
40 °C

40 °C

AA AA

GA GA

MIX MIX

0 1 2 3 4 6 8 12 0 1 2 3 4 6 8 12
Time (weeks) Time (weeks)

0 125 250 375 500 0 10 20 30 40 50 60 70

T otal Ph ycob ilip rotein s (m g g -1 ) C olourch an g e E)

Figure Effectof
Figure7.7.Effect ofaccelerated
acceleratedconditions
conditions(4 ◦ C,20
(4 °C, ◦ C,and
20°C, and40 ◦ C)in
40°C) in(A)
(A)total
totalphycobiliproteins
phycobiliproteinsand
and
(B)
(B) color
color change
change (ΔE)
(∆E) of
of water
water ingredients
ingredients supplemented
supplemented with with commercial
commercial antioxidants:
antioxidants: ascorbic
ascorbic
acid
acid (AA),
(AA), gallic acid (GA),
gallic acid (GA), or
oraamixture
mixtureofofboth
both(1:1;
(1:1; MIX).
MIX). TheThe vehicle
vehicle represents
represents glycerol
glycerol (80%)
(80%) and
and control is the ingredient without supplement.
control is the ingredient without supplement.

Finally,
When itregarding
came to the
the bioactive capacity ofthe
water ingredient, thecontent
ingredients (Figure 8), both had
of phycobiliproteins wasa sta‐
also
ble antioxidant
stable during the capacity
12 weeks at 44◦°C
in the and 20
C and 20◦°C At 40 ◦ C, theHowever,
C. treatments. at 40 °C, both
content decreased ingre‐
depending
dients
on thehad a loss ofsupplementation.
antioxidant antioxidant capacity,From translated
week 1, theby acontrol
decrease in the inhibition
ingredient and the one of
ABTS •+ from week 1, with a continuous decrease until week 4. After this, no significative
supplemented with ascorbic acid started to degrade. From week 4, all ingredients started
inhibition
to degrade, was observed,
equally meaning
reaching a loss of
an average of the 150 mg g−1capacity
ca. antioxidant in weekof12,the ingredients.a
representing
When it came
reduction to the
of 70% anti‐hyaluronidase
of the pigment content.activity, a similar pattern was found at 4 °C and
20 °CAtreatments, wherewas
similar pattern both ingredients
found wereevaluation.
in the color stable, whereas at 40 °C,
No changes the found
were ethanolic ◦C
at 4in‐
and 20 C,◦ an ∆E < ∆E < 2.0 ◦
gredient lostwith
activity by<week
1.0 until week
4, and the 6water
and 1.0ingredient hadfrom until week
a reduction from 12.80Atto40
15% C,
from
of week 1 to 3,inhibition
hyaluronidase the controlbyand ascorbic
week 4. acid-supplemented ingredients had perceptible
color changes ∆E > 10.0, whereas ingredients supplemented with gallic acid and a mix of
antioxidants had a less accentuated change (∆E ≈ 5.0). From week 4, all ingredients had
perceptible differences, as well as at week 12 (∆E ≈ 26.0).
Mar.
Mar.Drugs
Drugs2022,
2022,20,
20,x 481 11 10
of of2625

80
A

Antioxidant capacity
60

(% Inhibition)
40

20

0
0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812

Weeks
B
100
Anti-hyaluronidase activity

80
(% Inhibition)

60

40

20

0
0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812

Weeks

Figure 8. Effect of accelerated conditions (4 ◦ C, 20 ◦ C, and 40 ◦ C) on the (A) ABTS•+ scavenging

Figure 8. Effect of accelerated conditions (4 °C, 20 °C, and 40 °C) on the (A) ABTS•+ scavenging
ability and (B) hyaluronidase inhibition activity (% inhibition; average ± standard deviation, n = 3)
ability and (B) hyaluronidase inhibition activity (% inhibition; average ± standard deviation, n◦ = 3)
of ethanolic (E) and water (W) ingredients. Colors of lines indicates different conditions: E 4 C ( );
of ethanolic (E) and water (W) ingredients. Colors of lines indicates different conditions: E 4 °C (
◦ C ( ); E 40 ◦ C ( ); W 4 ◦ C ( ); W 20 ◦ C ( ); W 40 ◦ C ( ).
); EE 20
20 °C ( ); E 40 °C ( ); W 4 °C ( ); W 20 °C ( ); W 40 °C ( ).

Finally, regarding the bioactive capacity of the ingredients (Figure 8), both had a stable
2.4. Serum Formulation
antioxidant capacity in the 4 ◦ C and 20 ◦ C treatments. However, at 40 ◦ C, both ingredients
2.4.1.
had aFormulation Characterization
loss of antioxidant capacity, translated by a decrease in the inhibition of ABTS•+ from
week The1, major
with agoal
continuous
of applyingdecrease until week
a natural extract4.inAfter this, noissignificative
cosmetics to develop ainhibition
formulation was
observed,
using meaning
the active a loss of
ingredient. the antioxidant
Here capacity of was
a putative formulation the ingredients.
an attempt basedWhenon it came
a skinto
the anti-hyaluronidase
serum activity, aetsimilar
described by Chowjarean al. [16]pattern
with thewas found of
addition 4 ◦ Cconcentrated
at the and 20 ◦ C treatments,
ingredi‐
where ◦
ent: 3% both ingredients
of vehicle and a were stable, whereas
final extract at 40 of
concentration C, 5the
mg ethanolic
g−1. Theingredient lost activity
ethanolic ingredient
by week
was 4, andinto
introduced thethe
water ingredient
ethanolic serum had
anda the
reduction from 80 tointo
water ingredient 15%theof hyaluronidase
water serum.
inhibition by week 4.
The supplementation of antioxidants was also performed as before: α‐tocopherol, BHT,
or a mixture of both (1:1) for the ethanolic serum and ascorbic acid, gallic acid, or a mixture
of2.4.
bothSerum
(1:1)Formulation
for the water serum. The physical and chemical characteristics of the formu‐
2.4.1. Formulation
lated serum are presented Characterization
in Table 2. The bioactive capacity and the content of the total
The major goal of applying
pigment were also measured as the a natural
controlextract in cosmetics
of the serum beforeisandto develop
after theastability
formulation
as‐
using the active ingredient. Here a putative formulation was
says. Noteworthy is that the physical–chemical characteristics were similar in bothan attempt based on a skin
se‐
serum
rums, described
although by Chowjarean
there was a phaseet al. [16] with
separation the ethanolic
in the addition of the concentrated
serum subjected to ingredient:
a centrif‐
3% of vehicle
ugation process.andThisa phase
final extract concentration
separation mg g−1 . Theby
of 5homogenized
can be easily ethanolic
manual ingredient
shaking,
was introduced
which is similar tointo the ethanolic
bi‐phasic productsserum and theIn
in cosmetics. water ingredient
addition, the finalinto theof
color water serum.
the serum
The supplementation of antioxidants was also performed as before:
was very similar to the one found in the ingredient, with a similar pigment content and α-tocopherol, BHT,
or a mixture of
antioxidant capacity. both (1:1) for the ethanolic serum and ascorbic acid, gallic acid, or a
mixture of both (1:1) for the water serum. The physical and chemical characteristics of
the formulated serum are presented in Table 2. The bioactive capacity and the content
of the total pigment were also measured as the control of the serum before and after the
stability assays. Noteworthy is that the physical–chemical characteristics were similar in
Mar. Drugs 2022, 20, 481 11 of 25

both serums, although there was a phase separation in the ethanolic serum subjected to
a centrifugation process. This phase separation can be easily homogenized by manual
shaking, which is similar to bi-phasic products in cosmetics. In addition, the final color
of the serum was very similar to the one found in the ingredient, with a similar pigment
content and antioxidant capacity.

Table 2. Physical and chemical characterization of Cyanobium sp. serum formulations.

Parameter Ethanolic Serum Water Serum

pH 7.42 ± 0.06 7.47 ± 0.05
Phases 2 1
Viscosity (cst) 28.46 ± 1.45 26.95 ± 1.21
Density (g cm−3 ) 1.09 ± 0.02 0.96 ± 0.01
Conductivity (µS cm−1 ) 1714 ± 41 1613 ± 55
Color +
L* 35.11 ± 2.02 39.93 ± 2.17
a* −30.47 ± 2.94 −25.77 ± 2.10
b* 51.82 ± 4.39 −9.11 ± 0.54
Optical correspondence Green Blue
Antioxidant capacity (ABTS•+ IC50 , mg mL−1 ) 186.12 ± 10.42 205.18 ± 13.16
Anti-hyaluronidase activity (IC50 , mg mL−1 ) 135.53 ± 11.23 85.43 ± 3.40
Total pigments * (mg g−1 ) 175.34 ± 12.72 497.38 ± 10.82
* Carotenoids for the ethanolic serum and phycobiliproteins for the water serum. + L*: brightness; a*: redness;
b*: yellowness.

2.4.2. Serum Hot–Cold Stability

In a similar method to that performed with the ingredient, the two formulated serums
were subjected to stability tests using hot–cold cycles and temperature-accelerated stability
for 12 weeks. Results for the hot–cold stability assay for the ethanolic serum are shown in
Figure 9 and for the water serum are shown in Figure 10. Again, due to the influence of
antioxidant supplements on the bioactive capacity assays, only the control condition was
evaluated for both serums (Figure 11).
The ethanolic serum subjected to −20 ◦ C ↔ 20 ◦ C had a slight reduction in carotenoid
content from cycle 1 of about 20%. In the one supplemented with a mixture of antioxidants,
a more accentuated loss was observed (ca. 30%). When subjected to 4 ◦ C ↔ 40 ◦ C cycles,
the content was slightly reduced in cycle 3 by 25% in the control serum and the serums
supplemented with α-tocopherol and a mixture of antioxidants. The loss reached 40%
by cycle 7 in the control and α-tocopherol serums, and 50% in the serums supplemented
with the mixture of antioxidants. BHT supplementation protected the degradation, with a
reduction of only 25% by cycle 7.
Regarding the color, no statistical differences were found in the serums subjected to
−20 ◦ C ↔ 20 ◦ C, whereas a big color change was found in the ones subjected to 4 ◦ C ↔ 40 ◦ C
cycles. In the 4 ◦ C ↔ 40 ◦ C treatment, a ∆E ≈ 35.0 was observed from cycle 1 regardless of
the antioxidant supplementation, reaching ∆E > 40.0 from cycle 2. Optimal equivalence
went from bright green to brown.
No statistical differences were observed in the pigment content of the water serums
subjected to −20 ◦ C ↔ 20 ◦ C cycles for all seven evaluated cycles. On the other hand,
the ones subjected to 4 ◦ C ↔ 40 ◦ C had a huge reduction in the content of about 50% from
cycle 1, and reached a loss of 90% of pigments in cycle 5. In a similar trend, no changes
were observed in the color of the water serums in the −20 ◦ C ↔ 20 ◦ C cycles, whereas a
greater difference was found in the 4 ◦ C ↔ 40 ◦ C cycles. From cycle 1, the control serum
and the one supplemented with ascorbic acid had a ∆E ≈ 20.0, whereas the ones with gallic
acid and the mixture of antioxidants had a ∆E ≈ 30.0. From cycles 4 to 7, the color was
stable but very different from the original—the control serum and the one with ascorbic
acid had a ∆E ≈ 35.0, and the ones with gallic acid and the mixture of antioxidants had a
∆E ≈ 30.0. The optical equivalence went from blue to grey.
Mar. Drugs 2022, 20, 481 12 of 25

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Figure 9. Effect of hot–cold cycles (4 ◦ C ↔ 40 ◦ C and −20 ◦ C ↔ 20 ◦ C) in (A) total carotenoids

and (B) color change (∆E) of the ethanolic serum supplemented with commercial antioxidants:
α-tocopherol (α-T), BHT, or a mixture of both (1:1; MIX). The vehicle represents a serum with linseed
oil and control is the serum without supplements.

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Figure 10. Effect of hot–cold cycles (4 ◦ C ↔ 40 ◦ C and −20 ◦ C ↔ 20 ◦ C) in (A) total phycobiliproteins
and (B) color change (∆E) of the water serum supplemented with commercial antioxidants: ascorbic
acid (AA), gallic acid (GA) or a mixture of both (1:1; MIX). The vehicle represents a serum with
glycerol (80%) and control is the serum without supplements.
Mar. Drugs 2022, 20, 481 13 of 25

Figure 11. Effect of hot–cold cycles (4 ◦ C ↔ 40 ◦ C and −20 ◦ C ↔ 20 ◦ C) on the (A) ABTS•+ scavenging
ability and (B) hyaluronidase inhibition activity (% inhibition; average ± standard deviation, n = 3)
of the ethanolic serum (E) and water serum (W) extract ingredient. Colors of lines indicate different
conditions: E 4 ◦ C ↔ 40 ◦ C ( ); E −20 ◦ C ↔ 20 ◦ C ( ); W 4 ◦ C ↔ 40 ◦ C ( ); W −20 ◦ C ↔ 20 ◦ C ( ).

Finally, regarding the bioactive capacity of the serums (Figure 11), both were kept
stable in the −20 ◦ C ↔ 20 ◦ C cycles. Under the 4 ◦ C ↔ 40 ◦ C cycles, the ethanolic serum
had a reduction in the antioxidant capacity, with a decrease of inhibition power by 1.3-fold
in cycle 3, whereas the water serum showed no significant antioxidant capacity (<5%) from
cycle 1. Regarding the anti-hyaluronidase activity, the same accentuated reduction in cycle
3 was observed: The ethanolic serum had a reduction of 2.0-fold and the water serum lost
the anti-hyaluronidase activity by cycle 2 (<7%).

2.4.3. Serum Accelerated Stability

Regarding the accelerated stability of serum pigments, the formulated serums were
also subjected to different temperatures for 12 weeks and the pigment content, color change,
and bioactive capacity were monitored. Results are shown in Figure 12 for ethanolic
serum and Figure 13 for water serum. Due to the influence of antioxidant supplements in
the bioactive capacity assays, only the control condition was evaluated for both serums
(Figure 14).
The ethanolic serums subjected to 4 ◦ C kept the carotenoid content with no statistical
differences regardless of time and antioxidant supplementation. The ones subjected to
20 ◦ C had a reduction in carotenoid content of about 30%, observed from weeks 2 to 12,
regardless of the antioxidant supplementation; the supplementation of α-tocopherol and
the mixture of antioxidants had a slower degradation. Finally, the serums subjected to
40 ◦ C had a big reduction in carotenoid content from week 1 of about 50% and week 2 of
about 50 mg g−1 , representing a reduction of 75%.
When it came to color, the ethanolic serums subjected to 4 ◦ C had no perceptible
differences within the 12 weeks. The ones subjected to 20 ◦ C had a big change in color from
week 1 (∆E ≈ 27.0), and even bigger from week 2 to 12 (∆E ≈ 40.0). The optical correspon-
dence goes from green to dark yellow, again due to the bleaching of chlorophyll. Finally,
the serums subjected to 40 ◦ C suffered a change in the color from week 1, with an ∆E > 40.0,
corresponding to a change from bright green to brown and then to a muddy color.
 When it came to color, the ethanolic serums subjected to 4 °C had no perceptible dif‐
ferences within the 12 weeks. The ones subjected to 20 °C had a big change in color from
week 1 (ΔE ≈ 27.0), and even bigger from week 2 to 12 (ΔE ≈ 40.0). The optical correspond‐
ence goes from green to dark yellow, again due to the bleaching of chlorophyll. Finally
Mar. Drugs 2022, 20, 481 the serums subjected to 40 °C suffered a change in the color from week 1, with
14 of 25an ΔE >
40.0, corresponding to a change from bright green to brown and then to a muddy color.

A B
Vehicle Vehicle

Control Control
4 °C

4 °C
α-T α-T

BHT BHT

MIX MIX

Vehicle Vehicle

Control Control

20 °C
20 °C

α-T α-T

BHT BHT

MIX MIX

Vehicle Vehicle

Control Control

α-T α-T
40 °C

40 °C

BHT BHT

MIX MIX

0 1 2 3 4 6 8 12 0 1 2 3 4 6 8 12
Time (weeks) Time (weeks)

0 40 80 120 160 0 10 20 30 40 50 60
-1
T otal C aroten oid s (m g g ) C olou rch an g e E)

Figure 12. Effect of accelerated conditions (4 ◦ C, 20 ◦ C, and 40 ◦ C) in (A) total carotenoids

and (B) color change (∆E) of the ethanolic serum supplemented with commercial antioxidants:
α-tocopherol (α-T), BHT, or a mixture of both (1:1; MIX). The vehicle represents a serum with linseed
oil and control is the serum without supplements.

Regarding the water serum, a similar pattern was found. The content was kept the
same during the 12 weeks at 4 ◦ C regardless of the antioxidant supplementation. At 20 ◦ C
all the serums had a reduction of 20% from week 4, with no further reduction. Thus,
at 40 ◦ C a linear decrease was observed in all serums from week 1 to week 4, with a
reduction of 90% of total phycobiliprotein content. The changes directly affected the color:
At 4 ◦ C, no perceptible changes were found until week 4, when the ones supplemented
with ascorbic acid had an ∆E = 10.0. The serums subjected to 20 ◦ C had a linear color
change with an increase in ∆E from week 1 (∆E < 10.0), being more accentuated in week 4
(∆E ≈ 30.0). The optical color equivalence changed from blue to greenish. Finally, the ones
subjected to 40 ◦ C had a big color change (blue to yellow/brown) from week 1 (∆E ≈ 30.0),
reaching ∆E > 50.0 by week 4.
Mar. Drugs 2022, 20, 481 15 of 25

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Figure 13. Effect of accelerated conditions (4 ◦ C, 20 ◦ C, and 40 ◦ C) in (A) total phycobiliproteins and
(B) color change (∆E) of the water serum supplemented with commercial antioxidants: ascorbic acid
(AA), gallic acid (GA), or a mixture of both (1:1; MIX). The vehicle represents a serum with glycerol
(80%) and control is the serum without supplements.

Last, the bioactive capacity of the serum pigments (Figure 14) followed the trend
found in total pigments and color. For the carotenoid serum, the antioxidant capacity was
stable at 4 ◦ C, had a modest decrease at 20 ◦ C (1.2-fold), and lost the antioxidant capacity
continuously at 40 ◦ C, reaching only 21.75% of inhibition of ABTS•+ by 12 weeks. For the
phycobiliprotein serum, the antioxidant capacity was stable at 4 ◦ C and 20 ◦ C, whereas at
40 ◦ C it lost the antioxidant capacity by week 1 (<5%). Regarding the anti-hyaluronidase
activity, both serums had a stable capacity at 4 ◦ C and 20 ◦ C. Both serums had an abrupt
loss of bioactive capacity in the first month at 40 ◦ C, losing their anti-hyaluronidase activity.
 stable at 4 °C, had a modest decrease at 20 °C (1.2‐fold), and lost the antioxidant capacity
continuously at 40 °C, reaching only 21.75% of inhibition of ABTS•+ by 12 weeks. For the
phycobiliprotein serum, the antioxidant capacity was stable at 4 °C and 20 °C, whereas at
40 °C it lost the antioxidant capacity by week 1 (<5%). Regarding the anti‐hyaluronidase
activity, both serums had a stable capacity at 4 °C and 20 °C. Both serums had an abrupt
Mar. Drugs 2022, 20, 481 16 of 25
loss of bioactive capacity in the first month at 40 °C, losing their anti‐hyaluronidase activ‐
ity.

80
A

Antioxidant capacity
60

(% Inhibition)
40

20

0
0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812

Weeks
B
100
Anti-hyaluronidase activity

80
(% Inhibition)

60

40

20

0
0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812 0 1 2 3 4 6 812

Weeks

Figure 14. Effect of accelerated conditions (4 ◦ C, 20 ◦ C, and 40 ◦ C) on the (A) ABTS•+ scavenging
Figure 14. Effect of accelerated conditions (4 °C, 20 °C, and 40 °C) on the (A) ABTS•+ scavenging
ability and (B) hyaluronidase inhibition activity (% inhibition; average ± standard deviation, n = 3)
ability and (B) hyaluronidase inhibition activity (% inhibition; average ± standard deviation, n◦= 3)
ofof ethanolicserum
ethanolic serum(E)(E)and
andwater
waterserum
serum(W).
(W).Colors
Colorsofof lines
lines indicates
indicates different
different conditions:
conditions: EE 44 °C
C (( );
E); 20 ◦ ◦ ◦ ◦ ◦
E 20C°C
( );
( E);40E 40
C (°C);( 4 );C4( °C
); (W 20
); WC20
( °C
); W
( 40); WC 40
( ).°C ( ).

3. Discussion
3. Discussion
As a vastly known producer of secondary metabolites such as mycosporine-like amino
As alkaloids,
acids, a vastly known
amides,producer
fatty acids,ofand
secondary
peptides,metabolites such
cyanobacteria areasanmycosporine‐like
excellent source of
amino acids, alkaloids, amides, fatty acids, and peptides, cyanobacteria
natural products [17]. Moreover, pigments from cyanobacteria have been are proposed
an excellent
as a
source
highly bioactive group of compounds [9,10]. Because of their color and bioactivebeen
of natural products [17]. Moreover, pigments from cyanobacteria have pro‐
properties,
posed
theseaspigments
a highly are
bioactive group of
well known forcompounds [9,10].appealing
their extremely Because ofqualities
their color
for and bioac‐
commercial
tive properties, these pigments are well known for their extremely appealing
application in food, feed, medicines, nutraceuticals, and cosmetics. Thus, when compared qualities for
commercial application
to carbohydrates, in food,
proteins, feed, obtained
and lipids medicines, from nutraceuticals,
cyanobacteria,and cosmetics.
pigments Thus,as
emerged
when compared to carbohydrates, proteins, and lipids obtained from cyanobacteria,
the components with the highest market pricing, serving as the primary source of revenue pig‐
ments emerged as the components with the highest market pricing, serving
for businesses [18]. Cyanobacterial pigments can be used either as pure compounds or asas the primary
source of revenue
raw extracts. for businesses
In the [18].the
present study, Cyanobacterial pigments can
potential of Cyanobium sp. be used either
Extracts as pure
has reinforced
compounds or as raw extracts. In the present study, the potential of Cyanobium
that those extracts can be a functional source of bioactive compounds. Purification of sp. Extracts
compounds can account for up to 80% of the cost of production, and the use of extracts can
be advantageous due to lower production costs and greater stability [19].
In this study, the cosmetic potential of Cyanobium sp. followed a continuous line
of thought from the extract to a cosmetic ingredient and finally to an end product (skin
serum). First, the two obtained pigment-target extracts were evaluated in terms of in vitro
safety and cosmetic bioactive potential. As already mentioned, cyanobacteria are a unique
and complex group of microorganisms that live in a wide range of environments. Their
adaptation to these environments is linked to their ability to change their metabolisms and
frequently produce secondary metabolites, which can be either an advantage as a bioactive
ingredient or a disadvantage with toxic compounds. Cyanotoxins are thus a common
group of compounds found in cyanobacteria [20]. These toxic compounds require careful
Mar. Drugs 2022, 20, 481 17 of 25

consideration for both environmental impacts (blooms) and human health. Any food or
ingredient derived from cyanobacterial biomass that is intended for human consumption
must be thoroughly tested for the presence of these toxins. The results presented here
suggest that Cyanobium sp. pigment-targeted extracts can be safe for cosmetic application.
The strain has been also evaluated by Morone et al. [21], who found no cytotoxicity of
ethanol (70%) in skin-related cell lines, although the authors tested relatively low concen-
trations (up to 100 µg mL−1 ). Moreover, Pagels et al. [13] also showed that ethanolic and
successive water extracts had no cytotoxicity in HepG2 cells (liver) in concentrations up to
750 µg mL−1 , although acetonic extracts showed cytotoxic effects in this cell line.
Regarding the bioactive potential, Cyanobium sp. extracts have been proposed as
antioxidant and anti-inflammatory [13], and here, results showed the extracts were able to
inhibit cosmetic-related enzymes: The phycobiliprotein-targeted extract was able to inhibit
hyaluronidase and collagenase and the carotenoid-targeted extract was able to inhibit
hyaluronidase. The anti-hyaluronidase capacity has been reported before in cyanobacteria
extracts and purified compounds [22]. Yamaguchi et al. [23] showed that polysaccharide-
targeted extracts from Nostoc spp. have high inhibitory potential with IC50 for hyaluronidase
from 14.4 to 56.2 µg mL−1 , depending on the species, whereas Yamaguchi and Koketsu [24]
showed that a purified polysaccharide from Nostochopsis lobatus had an IC50 of 7.2 µg mL−1 .
Fujitani et al. [25] showed an ethanol-insoluble fraction from a water extract of Arthrospira
platensis showed an IC50 of 150 µg mL−1 . Furthermore, Montalvo et al. [26] showed that
isolated peptides from Arthrospira platensis showed an IC50 from 920 to 1660 µg mL−1 .
Morone et al. [21] showed that ethanolic (70%) extract from Tychonema sp. and from an-
other strain of Cyanobium sp. (LEGE 07175) had an IC50 for hyaluronidase of 182.7 and
208.4 µg mL−1 , respectively. Moreover, a common ingredient in anti-aging products is
green tea [15]. Here, as proof of concept, a water extract was prepared from Camellia sinensis,
with an anti-hyaluronidase IC50 of 122.19 µg mL−1 . The Cyanobium sp. extracts evalu-
ated in this study showed an IC50 of 108.7 and 67.2 µg mL−1 for carotenoid-targeted and
phycobiliprotein-targeted extracts, respectively, representing a powerful ingredient for
anti-aging products.
When it came to collagenase inhibition, only Cyanobium sp. phycobiliprotein-targeted
extract showed an IC50 of 582.8 µg mL−1 . Collagenase inhibition has been studied in
cyanobacteria to a smaller extent and focused on isolated compounds, with the exam-
ples of Montalvo et al. [26], who studied isolated peptides from Arthrospira platensis that
showed an IC50 of 32.5 to 96.7 µg mL−1 , and Tarasuntisuk et al. [27], who studied isolated
mycosporine-2-glycine from Aphanothece halophytica that showed an IC50 for collagenase of
ca. 115 µg mL−1 .
The evaluation of Cyanobium sp. pigment-targeted extracts indicated that these extracts
could be used as cosmetic ingredients; therefore, introducing these extracts into a vehicle
would facilitate further product formulation. Linseed oil and glycerol were chosen as
vehicles due to the current and approved used cosmetic ingredients, the compatibility with
the extracts, and their green solvent label. The ingredients were subjected to hot–cold cycles
and accelerated thermal stability. Overall, temperature similarly affected both ingredients.
Lower temperatures (−20 ◦ C ↔ 20 ◦ C cycles and 4 ◦ C and 20 ◦ C treatments) preserved
the pigment content, color, and antioxidant capacity during the study, except for the color
change (from green to yellow) in the ethanolic ingredient (carotenoid-targeted) subjected
to 20 ◦ C for 12 weeks due to chlorophyll bleaching. However, in warmer temperatures
(4 ◦ C ↔ 40 ◦ C cycles and 40 ◦ C treatment), both ingredients were degraded, although it
is notable that antioxidant supplements delayed that degradation—BHT and the antiox-
idant mixture for the ethanolic ingredient (carotenoid-targeted), and gallic acid and the
antioxidant mixture for the water ingredient (phycobiliprotein-targeted). Pigments are
thermo-sensitive compounds, and the stability can be reduced in higher temperatures; in
addition, the vehicle itself can be less stable in such conditions. The stability of cyanobac-
terial pigments in cosmetic products has not been described, although a few studies on
pigments have been found for food processing. Szterk et al. [28] showed a reduction in
Mar. Drugs 2022, 20, 481 18 of 25

carotenoid content of 26.8% using linseed oil for a β-carotene beverage storage at 2 ◦ C
for 12 weeks, and the authors linked the loss of carotenoid content to the oxidation of
the pigment and the oil. Noteworthy, the auto-oxidation of β-carotene at 30 ◦ C can occur
in about 30 h, and the supplementation of antioxidants such as BHT and α-tocopherol
is required [29]. In this study, although BHT and the mixture of BHT and α-tocopherol
delayed the effect of degradation, the degradation at higher temperatures was not avoided.
Regarding phycobiliproteins, these natural pigments are sensitive to temperature, pH, hu-
midity, and light. Galetović and Dufossé [30] evaluated the use of phycobiliproteins from
Nostoc sp. as a colorant for dairy beverages and found that the isolated pigment was only
stable in temperatures up to 21 ◦ C (evaluated from 0 to 83 ◦ C for 3 days), although it was
stable during skim milk processing (138 ◦ C for 4 s). In the food industry, preservatives such
as citric acid, sodium chloride, calcium chloride, ascorbic acid, and benzoic acid are used to
avoid the degradation of the products [30]. In this study, the addition of gallic acid was
more advantageous than the addition of ascorbic acid, although no antioxidant was able
to avoid degradation after one month at 40 ◦ C. Mishra et al. [31] evaluated phycoerythrin
stability at 0 and 35 ◦ C for 45 days with the addition of commercial preservatives; at 0 ◦ C
the control treatment had a loss of 70% of phycoerythrin content, and at 35 ◦ C a loss of 90%.
The best preservative was citric acid, which led to a reduction of 50% at both temperatures.
Overall, the stability of Cyanobium sp. ingredients was satisfactory, and these ingre-
dients were then included in a serum formulation. The chosen formulation was based on
Chowjarean et al. [16], who had promising results in clinical trials of serum containing
an extract of Grammatophyllum speciosum (vascular plant; orchid). Chowjarean et al. [16]
evaluated the serum stability in 4 ◦ C ↔ 40 ◦ C cycles and 40 ◦ C treatment, with positive
results in terms of the bioactive compound stability (gastrodin; phenolic glycoside). Here,
the results for Cyanobium sp. pigment serums were not as positive, but the stability in lower
temperatures was indeed satisfactory. Similar to the equivalent ingredient, both serums
were stable under lower temperatures (−20 ◦ C ↔ 20 ◦ C cycles and 4 ◦ C treatment); how-
ever, at 20 ◦ C, a small reduction in pigment content was observed (30% in carotenoids and
20% in phycobiliproteins), with a color change only in the ethanolic serum due to chloro-
phyll bleaching. Moreover, the abrupt degradation at 40 ◦ C, which reduced the carotenoid
content by 75% in two weeks and the phycobiliprotein content by 90% in one week, may
require a lower shelf life or improvements in the formulation before commercialization.
For example, the use of nanoparticles, as reviewed by Souto et al. [ref], who observed
evidence of efficient delivery of natural extracts to cosmetic products when applied together
with liposomes, chitosan/tripolyphosphate nanoparticles, and gold nanoparticles, among
others, has already been applied in some brands/products (e.g., Chantecaille-Nano Gold
Energizing Cream, NanosomesTM ).
Therefore, the requirement of low-temperature storage in skin serum is frequent in
cosmetics, as it is common to find cosmetic fridges on sale (e.g., Skincare Mini Fridge,
Cooluli, Brooklyn, NY, USA).

4. Materials and Methods

4.1. Cyanobacterial Biomass Source
Cyanobium sp. LEGE 06113 was obtained from Blue Biotechnology and Ecotoxicology
Culture Collection (LEGE-CC). The cyanobacterium was grown in previously optimized
conditions [12] for 14 days (10 days in white LED plus 4 days in red LED, aiming for maxi-
mum pigment content) with a light intensity of 200 µmolphotons m−2 s−1 and a light:dark
cycle of 16:8 h. Blue Green medium (BG11) (Allen, 1968) was used as culture medium,
with the addition of NaCl (10 g L−1 ), NaNO3 (3 g L−1 ), NaHCO3 (0.1 g L−1 ), and K2 HPO4
(0.1 g L−1 ), and with pH set at 9.0 and kept constant with CHES-buffer (2 g L−1 ). Con-
stant airflow was also assured at 0.75 Lair L−1 min−1 . Biomass was harvested through
centrifugation (10 min, 4000× g) and freeze-dried.
Mar. Drugs 2022, 20, 481 19 of 25

4.2. Pigment-Targeted Extracts

As previously optimized for Cyanobium sp. [13], the successive extraction using ethanol
and then water led to two promising extracts rich in carotenoids and phycobiliproteins,
being the selected extraction methodology for the present study. Two extracts were obtained
from the freeze-dried biomass, an ethanolic one (carotenoid-targeted) and a water one
(phycobiliprotein-targeted) [13]. For the ethanolic extract, cells were crushed using a
Precellys Homogenizer bead beater (Bertin, France), using 250 mg of biomass in 3 cycles
with 5 mL of ethanol (≥99.8%; 6 series of 8000 rpm for 30 s with 45 s of pause) and 670 mg
of 0.1 mm beads to maximize cell disruption. Extracts were centrifuged (10 min 2000× g)
and the supernatant was dried in a rotavapor. The remaining biomass was resuspended
in 15 mL of water (phycobiliprotein-targeted extract), homogenized using a vortex, and
centrifuged (10 min 2000× g). The supernatant was then freeze-dried. The quality of each
extract was confirmed following the pigment content (30.7 ± 1.9 mg g−1 of carotenoids
and 108.1 ± 7.9 mg g−1 of phycobiliproteins) as previously reported [13]. Both extracts
were stored in low humidity (desiccator) in the dark until further analyses.

4.3. Extract Cytotoxicity

The cytotoxicity of both extracts was evaluated using 3-(4,5-dimethylthiazole-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay in skin-related cell lines: a ker-
atinocyte cell line (HaCat, ATCC), fibroblast cell line (3T3L1, ATCC), and endothelial cell
line (hCMEC/D3) [21]. Cells were grown in DMEM Glutamax medium (Gibco, Waltham,
MA, USA), supplemented with 10% (v/v) fetal bovine serum (Biochrom, Berlin, Germany),
1% Pen–Strep (Biochrom, Berlin, Germany) and 0.1% Amphotericin B (GE Healthcare, Chal-
font St Giles, UK). Before extract exposure, cells were seeded in 96-well plates at densities
of 2.5 × 104 cells mL−1 (HaCat), 3.3 × 104 cells mL−1 (3T3L1), and 1.0 × 105 cells mL−1
(hCMEC/D3) and incubated for 24 h. Serial dilutions of the extracts (1.9 to 1000.0 µg mL−1 )
were prepared in DMEM with 1% DMSO, established as the maximum DMSO concentra-
tion not interfering with the assay. DMSO at 1% and 20% were used as the negative and
positive control, respectively. Cells were exposed for 24 and 48 h. After each incubation
time, 20 µL of 1 mg mL−1 MTT (Sigma-Aldrich) was added to each well and incubated
for 3 h. Following incubation, the purple-colored formazan salts were dissolved in DMSO
and the absorbance was read at 550 nm. The assay was performed in quadruplicate and
independently repeated three times.

4.4. Enzymatic Activities

The cosmetic potential of extracts was evaluated by enzymatic assays: hyaluronidase,
tyrosinase, collagenase, and elastase.

4.4.1. Hyaluronidase
Hyaluronidase inhibition assay was determined as reported by Ferreres et al. [32].
First, 25 µL of extract dilutions (31.2 to 1000.0 µg mL−1 ) plus 175 µL of hyaluronic acid
solution (0.7 mg mL−1 in water:buffer, 5:2 v/v, kept at 37 ◦ C) were added to the reaction
tube. The reaction was started by adding 25 µL of hyaluronidase (900 U/mL in NaCl
0.9%) and kept at 37 ◦ C for 30 min. The reaction was stopped with 25 µL of disodium
tetraborate (0.8 M), followed by subsequent heating for 3 min at 100 ◦ C. After cooling to
room temperature, 375 µL of DMBA solution (0.67 M) was added. The tubes were then
incubated at 37 ◦ C for 20 min and the absorbance of the colored product was measured
at 560 nm. The enzymatic inhibition was calculated based on the values of 100% activity
(using DMSO 10% instead of the extract) and 0% activity (using NaCl 0.9% instead of the
enzyme). The assay was performed in triplicate.

4.4.2. Tyrosinase
Tyrosinase inhibition assay was determined as reported by Adhikari et al. [33]. First,
10 µL of extract dilutions (31.2 to 1000.0 µg mL−1 ) plus 20 µL of tyrosinase (50 U mL−1 )
Mar. Drugs 2022, 20, 481 20 of 25

and 70 µL of phosphate buffer (50 mM, pH 6.5) were added to a 96-well plate and kept at
25 ◦ C during 5 min. The reaction was started by adding 70 µL of the substrate (L-DOPA,
2.5 mM). Kojic acid was used as the positive control. The absorbance was measured at 0
and 15 min at 475 nm. The enzymatic inhibition was calculated based on the values of 100%
activity (using DMSO 10% instead of the extract) and 0% activity (using buffer instead of
the enzyme). The assay was performed in triplicate.

4.4.3. Elastase
Elastase inhibition assay was determined as reported by Mota et al. [34]. First,
50 µL of extract dilutions (31.2 to 1000.0 µg mL−1 ) plus 87.5 µL of HEPES buffer (0.1 M
with NaCl 0.5 M, pH 7.5), 10 µL of the substrate (N-succinyl-Ala-Ala-Ala p-nitroanilide,
1.12 mg mL−1 ), 70 µL of acetate buffer (200 mM, pH 5.5) and 2.5 µL of DMSO were added
to a 96-well plate. The reaction was started by adding 30 µL of elastase (1 U mL−1 ) and
kept at 37 ◦ C for 10 min. The absorbance was then measured at 405 nm. The enzymatic
inhibition was calculated based on the values of 100% activity (using DMSO 10% instead of
the extract) and 0% activity (using buffer instead of the enzyme). The assay was performed
in triplicate.

4.4.4. Collagenase
Collagenase inhibition assay was determined as reported by Van Wart and Stein-
brink [35] and modified by Andrade et al. [36]. First, 30 µL of extract dilutions (31.2 to
1000.0 µg mL−1 ) and 30 µL of collagenase (1 U mL−1 ) were added to a 96-well plate and
kept at 37 ◦ C for 15 min. The reaction was started by adding 120 µL of the substrate
(FALGPA, 0.4 mM). The absorbance was read for 10 min at 345 nm. The enzymatic inhibi-
tion was calculated based on the values of 100% activity (using DMSO 10% instead of the
extract) and 0% activity (using buffer instead of the enzyme). The assay was performed
in triplicate.

4.5. Cyanobium sp. Cosmetic Ingredients

4.5.1. Ingredient Vehicle
For better incorporation into cosmetic formulations, two ingredients (vehicle plus
extract) were proposed; the ethanolic ingredient contained the ethanolic extract resus-
pended in linseed oil and the water ingredient contained the water extract resuspended in
80% glycerol. Both extracts were kept in a concentration of 5 mg g−1 .

4.5.2. Ingredient Characterization

The Cyanobium sp. ingredients were characterized following ISO/TR 18811/2018
and European Regulation EC 1223/2009 in terms of color, pH, phase separation, viscos-
ity, density, conductivity, total pigments, and bioactive capacity (antioxidant and anti-
hyaluronidase). The characterization assay was performed in triplicate batches of 50 mL of
each ingredient (without antioxidant supplementation) and all parameters were analyzed
in triplicate for each ingredient replicate.

4.5.3. Color
Color measurements were performed using a CR-400 colorimeter (Konica Minolta,
Japan) with an aperture of 8 mm at standard illuminate D65 using the CIE 1976 (L*, bright-
ness; a*, redness; b*, yellowness). The CIE system uses a three-dimensional colorimetric
measurement system: L* values represent the color’s brightness, a* values represent the
red–green content, and b* values represent the yellow–blue content. Color changes (∆E)
determine the three-dimensional color space and are calculated as:

∆E = [(∆L*)2 + (∆a*)2 + (∆b*)2 ]1/2 (1)

The human perception chart was followed as not perceptible (∆E ≤ 1.0), perceptible
through close observation (1.0 < ∆E ≤ 2.0), perceptible at a glance (2.0 < ∆E ≤ 10.0), colors
Mar. Drugs 2022, 20, 481 21 of 25

are more similar than different (10.0 < ∆E ≤ 50.0), and colors are completely different
(∆E > 50.0) [37].

4.5.4. pH and Conductivity

pH and conductivity were measured using an HQ40D digital two-channel multimeter
(Hach, Loveland, CO, USA), using an Intellical™ PHC101 pH probe (Hach) and an Intel-
lical™ CDC401 conductivity probe. The noteworthy pH of linseed oil is relative, as the
calibration is performed using aqueous buffers.

4.5.5. Phase Separation

Cyanobium sp. ingredients were centrifuged (15 min 2000× g) to evaluate phase
separation [38].

4.5.6. Viscosity
Viscosity was measured using a Zahn cup viscosimeter with an aperture of 2.74 mm
and a working volume of 44 mL (Baoshishan, China).

4.5.7. Total Pigments

Pigments were quantified spectrophotometrically. For the ethanolic ingredient, total
carotenoids were quantified following Zavřel et al. [39], diluting the ingredient in methanol
in a final concentration of 0.5 mg mL−1 . For the water ingredient, total phycobiliproteins
were quantified following Bennett and Bogobad [40], diluting the ingredient in water in a
final concentration of 0.5 mg mL−1 . The results were expressed in milligrams per gram of
dry extract (mg g−1 ).

4.5.8. Bioactive Capacity

The bioactive capacity of the Cyanobium sp. ingredients was evaluated in terms
of antioxidant capacity and anti-hyaluronidase activity. The antioxidant capacity was
evaluated via the ABTS•+ assay [41] with some modifications—the assay was performed
in triplicate in a 96-well plate. A total of 63 µL of the sample was added to 180 µL of
ABTS reagent and gently shaken. The reaction occurred in the dark for 6 min and the
plate was read at 734 nm; the final concentration of the ingredient was 250 µg mL−1 . The
anti-hyaluronidase activity was evaluated as described before, with a final concentration of
the ingredient of 250 µg mL−1 .

4.5.9. Antioxidant Supplementation and Compatibility

The formulation containing antioxidants was evaluated to keep the color and pig-
ment content in both extracts. The ethanolic ingredient was supplemented with 1 mg g−1
α-tocopherol, BHT (butylated hydroxytoluene), or a mixture of both (1:1, w/w), whereas the
water ingredient was supplemented with 1 mg g−1 ascorbic acid, gallic acid, or a mixture
of both (1:1, w/w). Both ingredients were also evaluated without antioxidant supplement
(control) and the vehicle without extract (80% glycerol and linseed oil).

4.5.10. Ingredient Hot–Cold Stability

To assess the physical stability of the Cyanobium sp. ingredients, a hot–cold stabil-
ity study was carried out, following ISO/TR 18811/2018 and European Regulation EC
1223/2009. The extract ingredients with and without antioxidants were subjected to two
heating–cooling cycle assays in 24-well plates with a working volume of 1.7 mL: (1) 7 cycles
of 24 h at 4 ± 2 ◦ C followed by 24 h at 40 ± 2 ◦ C, and (2) 7 cycles of 24 h at −20 ± 2 ◦ C
followed by 24 h at 20 ± 2 ◦ C. The color, total pigments, and bioactive capacity (antioxidant
and anti-hyaluronidase) were evaluated at the beginning and after each cycle. The stability
assay was performed in triplicate in all parameters.
Mar. Drugs 2022, 20, 481 22 of 25

4.5.11. Accelerated Ingredient Stability

Long-term stability and determination of the period-after-opening (PAO) were as-
sessed by an accelerated stability test following ISO/TR 18811/2018 and European Regu-
lation EC 1223/2009. The Cyanobium sp. ingredients with and without antioxidants were
subjected to three different temperatures (40 ± 2, 4 ± 2, 20 ± 2 ◦ C) for 12 weeks in 24-well
plates with a working volume of 1.7 mL. Color, total pigments, and bioactive capacity
(antioxidant and anti-hyaluronidase) were evaluated at nine timepoints of the assay (W0,
W1, W2, W3, W4, W6, W8, W10, W12). The stability assay was performed in triplicate and
all parameters were analyzed in triplicate for each ingredient replicate.

4.6. Serum Formulation

4.6.1. Formulation
To evaluate the possible application of Cyanobium sp. ingredients, a basal serum
in water was formulated following Chowjarean et al. [16] composed of PEG 400 (12%),
Aristoflex AVC (Clariant, Switzerland) (0.5%), Microcare PHC (1%), and extract vehicle
(3%). The extracts were added to the vehicle for a final concentration in the serum of
5 mg g−1 .

4.6.2. Formulation Characterization

The Cyanobium sp. serum formulations were characterized following ISO/TR 18811/2018
and European Regulation EC 1223/2009 in terms of color, pH, phase separation, viscos-
ity, density, conductivity, total pigments, and bioactive capacity (antioxidant and anti-
hyaluronidase). The characterization assay was performed in triplicate batches of 50 mL
of serum formulation (without antioxidant supplementation) and all parameters were
analyzed in triplicate for each serum replicate.

4.6.3. Serum Hot–Cold Stability

To assess the physical stability of the Cyanobium sp. serum formulations, a hot–cold
stability study was carried out, following ISO/TR 18811/2018 and European Regulation
EC 1223/2009. The formulations were subjected to two heating–cooling cycle assays in
24-well plates with a working volume of 1.7 mL: (1) 7 cycles of 24 h at 4 ± 2 ◦ C followed by
24 h at 40 ± 2 ◦ C, and (2) 7 cycles of 24 h at −20 ± 2 ◦ C followed by 24 h at 20 ± 2 ◦ C. Color,
total pigments, and bioactive capacity (antioxidant and anti-hyaluronidase) were evaluated
at the beginning and after each cycle. The stability assay was performed in triplicate in all
parameters.

4.6.4. Accelerated Serum Stability

Long-term stability and determination of the period-after-opening (PAO) were as-
sessed by an accelerated stability test following ISO/TR 18811/2018 and European Reg-
ulation EC 1223/2009. The Cyanobium sp. serum formulations were subjected to three
different temperatures (40 ± 2, 4 ± 2, 20 ± 2 ◦ C) for 12 weeks in 24-well plates with a
working volume of 1.7 mL. Color, total pigments, and bioactive capacity (antioxidant and
anti-hyaluronidase) were evaluated at nine timepoints of the assay (W0, W1, W2, W3, W4,
W6, W8, W10, W12). The stability assay was performed in triplicate and all parameters
were analyzed in triplicate for each ingredient replicate.

4.7. Statistical Analysis

Statistical analyses were performed using GraphPad Prism v.8 software (GraphPad,
San Diego, CA, USA). IC values for enzymatic activity were calculated through curve spline
interpolation. Each data set’s homoscedasticity was verified by the Cochran test. One-
way ANOVA was used for enzymatic inhibition, cytotoxicity, and physical and chemical
characterization. Whenever significant differences were detected, post hoc multiple com-
parisons were made, for cytotoxicity using Dunnett’s test to identify differences between
control and Tukey´s test for enzymatic inhibition and physical–chemical characterization.
Mar. Drugs 2022, 20, 481 23 of 25

The significance level in all analyses was 95% (p < 0.05). For stability assays, a two-way
ANOVA was performed, and whenever significant differences were detected, post hoc mul-
tiple comparisons were made using Tukey’s test to identify differences for the conditions
and time.

5. Conclusions
The cosmetic potential of pigment-targeted extracts from Cyanobium sp. was proposed
and evaluated in three steps: extract, ingredient, and product. The extract showed no
cytotoxic effects in skin-related cell lines, with a high anti-hyaluronidase capacity in both
extracts and an anti-collagenase effect in the water extract. Moreover, both extracts were
stable as ingredients and products (skin serum) at low temperatures (−20 ◦ C ↔ 20 ◦ C
cycles, and 4 ◦ C and 20 ◦ C treatments) and it was possible to keep the pigment content
and antioxidant capacity stable during the testing period, whereas at higher temperatures
(40 ◦ C) the product degraded in a week. Furthermore, because of their in vitro bioactive
capacity and stability, both extracts can be potential ingredients for cosmetic uses (anti-
aging), with relatively simple formulation and storage. Finally, the approach used in this
study, by evaluating extract, ingredient, and product, gives a much wider overview of the
real applicability of the extracts within the cosmetic industry, highlighting not only the
potential of Cyanobium sp. extracts as a cosmetic ingredient but also the use of other sources
of cyanobacteria apart from the ones already used in the industry.

6. Patents
This work has formed the basis for a patent application—Portuguese Provisional
Patent Application No. 117951—in which the authors are inventors.

Author Contributions: Conceptualization, F.P. and A.C.G.; methodology, F.P.; formal analysis, F.P.
and C.A.; writing—original draft preparation, F.P.; writing—review and editing, C.A., V.V. and A.C.G.;
supervision, V.V. and A.C.G.; funding acquisition, V.V. and A.C.G. All authors have read and agreed
to the published version of the manuscript.
Funding: A PhD fellowship (reference SFRH/BD/136767/2018) for author Fernando Pagels was
granted by Fundação para a Ciência e Tecnologia (FCT, Portugal) under the auspices of Programa
Operacional Capital Humano (POCH), supported by the European Social Fund and Portuguese funds
(MECTES). This work was financially co-supported by strategic funding from FCT UIDB/04423/2020
and UIDP/04423/2020 to CIIMAR and by the CCDR-N—Norte 2020, PORTUGAL2020, ERDF project
ATLANTIDA—Platform for the monitoring of the North Atlantic ocean and tools for the sustainable
exploitation of the marine resources (NORTE-01-0145-FEDER-000040).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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