Abstract and Figures

Increasing the consumption of natural substances has increased the demand for biological sources such as Spirulina platensis. The study quantitatively investigates the antioxidant potential and phytonutrient contents in aqueous and ethanol extracts of spirulina. The spirulina was collected from a local farm near Pondicherry and mass cultured in our research laboratory. The spirulina biomass was evaluated for antioxidant potential viz. catalase, SOD, GPx, Vitamin C, Vitamin E, and reduced GSH; phytonutrients contents like total phenol, flavonoid, tannin, carbohydrates, and proteins in both aqueous and ethanolic extracts of spirulina. Significant enzymatic antioxidant activity was observed for ethanolic extract. However, aqueous extracts were higher for catalase, SOD, and GPx activity. The same trend was observed for non-enzymatic activities. Total phenol, flavonoid, and tannin content were observed and high in aqueous extract. However, protein and carbohydrate content were higher in ethanolic extract. We observed a significant change in antioxidant activity and phytonutrient content in ethanolic extract than in aqueous extracts. The strong antioxidant property and higher phytonutrient contents of spirulina can play a vital role in the dietary supplement and combating malnutrition.
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Energy Nexus 6 (2022) 100070
Contents lists available at ScienceDirect
Energy Nexus
journal homepage: www.elsevier.com/locate/nexus
Antioxidant and phytonutrient activities of Spirulina platensis
Agam Kumar
a ,
, Duraisamy Ramamoorthy
a
, Daneshver Kumar Verma
a
, Arvind Kumar
a
,
Naveen Kumar
a
, Kanak Raj Kanak
b
, Binny Mary Marwein
a
, Kalai Mohan
a
a
Department of Ecology and Environmental Sciences, Pondicherry University (A Central University), Puducherry 605014, India
b
Department of Microbiology, Pondicherry University (A Central University), Puducherry 605014, India
Keywords:
Antioxidant
Protein
Vitamin
Extract
Tannin
Flavonoid
Spirulina
Increasing the consumption of natural substances has increased the demand for biological sources such as Spirulina
platensis . The study quantitatively investigates the antioxidant potential and phytonutrient contents in aqueous
and ethanol extracts of spirulina. The spirulina was collected from a local farm of Pondicherry and mass cultured
in our research laboratory. The spirulina biomass was evaluated for antioxidant potential viz. catalase, SOD,
GPx, Vitamin C, Vitamin E, and reduced GSH; phytonutrients contents like total phenol, avonoid, tannin, car-
bohydrates, and proteins in both aqueous and ethanolic extracts of spirulina. Signicant enzymatic antioxidant
activity was observed for ethanolic extract. However, aqueous extracts were higher for catalase, SOD, and GPx
activity. The same trend was observed for non-enzymatic activities. Total phenol, avonoid, and tannin content
were observed and high in aqueous extract. However, protein and carbohydrate content were higher in ethanolic
extract. We observed a signicant change in antioxidant activity and phytonutrient content in ethanolic extract
than in aqueous extracts. The strong antioxidant property and higher phytonutrient contents of spirulina can play
a vital role in the dietary supplement and combating malnutrition.
Introduction
As Arthrospira, Spirulina is a free-oating symbiotic microalga that
displays spiral characteristics of its laments [1–5] . It is also known as
blue-green algae [6] . It possesses photosynthetic ability [7–10] ; Due to
its ability to photosynthesize and the abundance of plant pigments, spir-
ulina was originally classied in the plant kingdom [11–13] . It belongs
to the phylum of cyanobacteria, the family Spirulinaceae or Pseudonabae-
naceae [14] . Later, it was classied in the bacterial kingdom [15,16] .
Spirulina naturally grows on high-salt, alkaline water in tropical and
subtropical regions [ 17 , 18 ]. Three species of edible spirulina are stud-
ied for their high nutritional value and potential therapeutic proper-
ties, including Spirulina platensis ( Arthrospira platensis ) and Spirulina max-
ima ( Arthrospira maxima ), and Spirulina fusiformis ( Arthrospira fusiformis )
[18–21] .
For almost 400 years ago, the Mayans, Toltecs, and Kanembu com-
munity in Mexico took spirulina during the Aztec civilization; re-
searchers have studied the nutritional value of spirulina as a food source
[ 22 , 23 ]. Spirulina was harvested, dried, and used to make spirulina
cakes in Lake Texcoco, Central Africa; the Chadians have consumed spir-
ulina for over a century. Spirulina harvested from Lake Kossorom (Chad)
is used to make cakes or broths and sold in the local market [24] .
Over the past, great eort and extensive research have been dedi-
cated to developing nutraceuticals and functional foods to prevent or
Corresponding author.
control various diseases and provide energy alternatives [25] . Spirulina
is considered one of the healthiest food because of its high protein con-
tent (60-70% by dry weight) and its richness in vitamins (B12 & E), min-
erals, essential fatty acids, carotenoids, antioxidants, and phycocyanin
[ 19 , 26 ]. Spirulina has been promoted as a high dietary food by the In-
tergovernmental Institution for Micro-algae Spirulina Against Malnutri-
tion (IIMSAM), an intergovernmental organization since the mid-1970s
to combat hunger and malnutrition in maternal women and new born
children [27] . As a nutraceutical food, spirulina has proven eective
for many diseases. It has been reported that spirulina as a diet supple-
ment can prevent and control hypercholesterolemia, certain inamma-
tory diseases, allergies, cancer, toxicities caused by environmental tox-
icants and drugs, and cardiovascular diseases and diabetes [ 21 , 28 ]. In
addition, due to its concentrated macro and micronutrients, spirulina
has been recommended as the best and most sustainable space food (in
long-term space missions) by both the National Aeronautics and Space
Administration (NASA) and the European Space Agency (ESA) [29] .
Food and drug administration (FDA) and dietary supplements infor-
mation expert committee (DSI-EC) categorize spirulina is safe for hu-
man consumption [30] . Spirulina recently got more attention as a func-
tional food because of its cholesterol-lowering properties, immune sys-
tem modulation, and antioxidant [31] . Various antioxidant compounds
in edible green plants were reported to reduce or prevent oxidative dam-
age caused by free radicals like superoxide radicals, hydroxyl radicals,
https://doi.org/10.1016/j.nexus.2022.100070
Received 26 December 2021; Received in revised form 6 April 2022; Accepted 16 April 2022
Available online 21 April 2022
2772-4271/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ )
A. Kumar, D. Ramamoorthy, D.K. Verma et al. Energy Nexus 6 (2022) 100070
and non-free radical species such as H
2
O
2
, which can cause cellular or
metabolic damage, accelerated aging, or cause cancer [ 32 , 33 ].
Spirulina also attracted scientic attention earlier because of its
health benets and nutritional components [10] . As a whole protein
source, spirulina and its features, positively aect various human health
issues, ranging from malnutrition to antioxidant potential [34] . Its ex-
tracts have been shown to prevent cancer in controlled studies, and
this anti-apoptotic activity has been correlated to its antioxidant poten-
tial [35] . Bioactive antioxidants deactivate free radicals and unstable
molecules with various adverse eects on organisms [36] . During an
oxidative burst, antioxidants inhibit reactive oxygen species (ROS). It
is well-established that antioxidants protect cellular components from
oxidative stress [37] . Since, It contains natural pigments such as chloro-
phyll, beta-carotene, phycoerythrin, and phycocyanin, S. platensis has
demonstrated antioxidant activity and free radical scavenging prop-
erties [38] . It includes phycobilisomes as a light-harvesting protein
pigment complexes [37] and contains two essential phycobiliproteins,
i.e., allophycocyanin and phytocyanin. Both proteins are members of
a chromophore family. In addition to phycocyanin, spirulina contains
carotenoids and xanthophylls molecules, which have numerous double
bonds and respond to free radicals and modulate inammatory pro-
cesses [ 35 , 39 ].
A non-traditional and new antioxidant source is required as an alter-
native, such as spirulina, promising an opportunity for this issue. Spir-
ulina is a good alternative to conventional food crops because it does
not compete with conventional food crops, grows faster, and can be har-
vested faster for production to provide a higher yield. As a result, choos-
ing suitable species as an antioxidant source for industrial use is crucial.
Cellular damage caused by oxidative stress can currently induce can-
cer, cardiovascular disease, aging, and declining immune function [28] .
Through lipid oxidation, free radicals cause food deterioration, aecting
organoleptic properties and edibility [40] . To combat oxidative stress,
the consumption of antioxidants as synthetic food additives that contain
preservatives is high [41] . However, most antioxidant sources to date
compete with conventional foods and crops. However, fast production
and applications for industrial and medicinal purposes are complicated
and hard to achieve intact growth [ 42 , 43 ].
Since synthetic antioxidants have been reported to be dangerous for
humans for long-term use, natural antioxidants got more and more at-
tention [ 44 , 45 ]. The objective of the present study was to evaluate the
antioxidant and phytonutrients properties of S. platensis .
Material and Methods
Procurement of raw materials
S. platensis was obtained from the culture collection of Aurospirulin,
Puducherry, India. By horizontal towing of phytoplankton net (No. 10,
bolting silk cloth, 48 µm) during early morning as described by Kumar
et al., [46] . The collected samples were washed with ltered seawater to
remove the associated debris and transferred to the laboratory in aseptic
settings. Then the phytoplankton soup was prepared and rinsed with
ltered seawater using a net cloth and seeded in Zarrouk medium to
maintain mixed culture [47] .
Species isolation
The microalgae S. platensis were isolated through the serial dilution
protocol described by Kavisri et al. [48] . The samples were transferred to
the containers. The asks were examined for algal growth using an op-
tical microscope every two days, with serial dilutions made in Zarrouk’s
medium from asks showing growth. The maintenance of subcultures
was performed in the test tubes, a frequent plating technique conrmed
culture purity, and regular observation was done under the microscopic
identication.
Mass culture of S. platensis
The growing S. platensis in Zarrouk’s medium (100 ml in a 250 ml
conical ask) was used for trials. The culture was grown at 30°C at a pH
between 8.5 and 11 with approximately 12:12 light/dark cycle of light
intensity. The algal culture attained a density of 2 ×105 cells/ml, ensur-
ing a cell density of at least 100 cells/ml by the end of the treatment.
Preparation of spirulina extract
A mixture of forty grams of Spirulina powder in 200 mL of ethanol
and water was stirred for two hours, and the supernatants were col-
lected. Solvents were retained in the rotary evaporator, and residues
were stored at 4°C for further analysis [49] . Analytical-grade chemicals
were used in this experiment.
Enzymatic and non-enzymatic antioxidant activity
Superoxide dismutase (SOD) activity
The SOD activity was estimated by Beauchamp & Fridovich [50] .
Using a Polytron homogenizer, spirulina extract was homogenized in
50 mmol/L phosphate buer (pH 7.8), and the homogenate was cen-
trifuged at 1,600 rpm for 15 minutes. To various concentrations of the
extract supernatants, we added 20 ml of pyrogallol solution containing
10mmol/L, and the rate of autoxidation was measured spectrophotomet-
rically at 420 nm. In terms of SOD activity, SOD/mg protein represents
the enzyme’s ability to inhibit the 50% autoxidation of pyrogallol.
Catalase (CAT) activity
The catalase activity was assayed after by Kar & Mishra [51] with
certain modications. Isotonic buer (pH 7.4) was used to homogenize
the spirulina extracts. The homogenate was centrifuged for 10 minutes
at 1,000 rpm. To 20 µl of 100-fold diluted extracts, the supernatant
was applied to 900 µl of 10mmol/L H
2
O
2
, 50 µl of Tris HCI buer (pH
8.0), and 30 µl of puried water in a 980 µl assay mixture. At 240 nm,
the rate of H
2
O
2
decomposition was measured spectrophotometrically.
CAT operation is measured in units of k/mg protein, with k denoting
the rst-order rate constant.
Glutathione Peroxidase (GPx) Activity
Mills’ procedure [52] was updated to examine GPx behaviour with
certain modications. In the presence of GSH, GPx degrades H
2
O
2
, de-
pleting it. Using 5,5 -dithiobis 2-nitrobenzoic acid, the remaining GSH
is then calculated (DTNB). 80mM of sodium phosphate buer (pH 7.0),
80mM of EDTA, 1mM of NaN
3
, 0.4mM of GSH, 0.25mM of H
2
O
2
, and ex-
tracts were used in the incubation mixture at 37°C. This solution was ex-
tracted after 3 minutes and treated with a metaphosphoric acid precipi-
tation solution. Using 0.4M Na
2
HPO
4
and 1mM DTNB in a 1% trisodium
citrate, the GSH in the protein-free ltrate was determined. That solu-
tion’s absorbance was measured at 412 nm. Since non-enzymatic GSH
oxidation by H
2
O
2
occurs during incubation, a blank was performed si-
multaneously with the samples. One gram of GSH ingested per minute
equalled one GPx enzyme activity unit [53] .
Reduced glutathione (GSH) activity
GSH content of algal buer extracts was measured by reaction with
5,50-dithiobis-2-nitrobenzoic (DTNB) by Silber et al. [54] . The algal
extract was homogenized in a 50 mmol/L Tris HCI buer (pH 7.4).
The homogenate was centrifuged for 20 minutes at 10,000 rpm, then
for 60 minutes at 100,00 rpm. 1.5 ml of 0.2 mol/L Tris HCI buer
(20 mmol/L EDTA, pH 8.2), 0.1 ml of 0.01 mol/L 5,5 -dithiobis-(2-
nitrobenzoic acid), and 7.9 ml of methanol were applied to 0.5 ml of ma-
terial supernatant. For 30 minutes, the mixture was incubated at 37°C
with intermittent stirring. This incubated mixture was centrifuged for
15 minutes at 3,000 rpm, and the supernatant’s absorbance was mea-
sured at 412 nm. The GSH concentrations were calculated using a reg-
ular curve made with known quantities of GSH.
2
A. Kumar, D. Ramamoorthy, D.K. Verma et al. Energy Nexus 6 (2022) 100070
Vitamin C Estimation
Roe & Kuether [55] used the procedure to calculate ascorbic acid.
10% of 1.5 ml ice-cold TCA was applied to 0.5 g of spirulina extract
and centrifuged for 10 minutes at 1800 rpm. 0.1 ml of thiourea-copper
sulfate reagent (DTC) was used with 0.5 ml of the supernatant and thor-
oughly combined. The tubes were incubated at 37°C for 3 hours, then
0.75 ml of ice-cold 65% H
2
SO
4 was applied and allowed to cool for
another 30 minutes. A series of requirements containing 10-50 ml of
ascorbic acid is prepared to a concentration of 0.5 ml and treated in the
same way as a blank containing 0.5 ml of 10% TCA. A 520 nm wave-
length was used to measure the colour developed. Ascorbic acid was
expressed as lmol/mg protein in materials.
Vitamin E Estimation
Albert et al. [56] method measured vitamin E 0.5 g of extracts, 1.5
ml ethanol, and 2.0 ml petroleum ether were mixed and centrifuged
for 5 minutes at 1000 rpm. at 80°C, the supernatant was evaporated
to dryness. The intense red colour produced was measured at 520 nm
after 0.2 ml of butanol was applied to UV visible spectrophotometer
(UV-Visible spectrophotometer UV 2450 B, LabTech). As a control, 𝛼-
tocopherol was used. The volume of 𝛼-tocopherol in the extract was
measured in l mol/mg protein.
Quantitative Phytochemical Analysis
Total phenolics content Estimation
Total soluble phenolics of spirulina extracts were determined using
the method described by Swain & Hill [57] . The sample was immersed
in 10 ml ethanol 80% and kept in the dark bottle for 24 h at 0°C. After
re-extraction three times, the claried extract was completed to 50 ml
with ethanol 80%. After mixing the extract with the Folin-Denis reagent
and thoroughly shaking the test tube, 1 ml of the extract was added. The
solution was then cooled, kept overnight at room temperature, and l-
tered. 1 ml of saturated Na
2
CO
3
(33 g of anhydrous salt) was dissolved
in 100 ml dH
2
O and boiled for 3 minutes. A spectrophotometer (UV-
Visible spectrophotometer UV 2450 B, LabTech) measured absorbance
at 725 nm. The concentration of total soluble phenols was determined
with the help of a gallic acid standard curve. Total phenols were ex-
pressed as gallic acid equivalents per gram dry weight of the sample.
Total flavonoids Estimation
Quettier-deleu et al. [58] determined the total avonoid concen-
tration using a colorimetric assay. This mixture contained one millil-
itre of spirulina extract, 4.3 millilitres of 80% ethanol, 0.1 millilitres
of 10% aluminium nitrate, and 0.1 millilitres of aqueous potassium ac-
etate (1M). We measured the absorbance at 415 nm after maintaining
the samples at room temperature for 40 minutes. The total avonoids
were calculated by comparing them to the rutin equivalents in the ex-
tract (RE mg/g).
Tannin Estimation
With few modications, tannins estimation uses the Reynolds et al.
[59] method. The extract solution was mixed with 1 ml of distilled wa-
ter, and 1 to 2 drops of ferric chloride solution were added, observed
for blue or green-black coloration.
Carbohydrates Estimation
Masuko et al. [60] Phenol-sulfuric acid method was followed for car-
bohydrate analysis. The sample was dried by air and hydrolysed in boil-
ing water for 6 hours with 1N HCl. A nal volume of 100 ml of distilled
water was added after the ltering and neutralizing the resultant solu-
tion. Using a UV-VIS spectrophotometer, the total reducing sugars were
determined by calorimetry in 1 ml of a sample using an alkaline potas-
sium ferricyanide reagent at 420 nm.
Protein Estimation
Using the Coomassie brilliant blue dye (G 250) binding method by
Bradford [61] and bovine serum albumin as the standard curve, we de-
termined the protein concentration of spirulina extract at 595 nm.
Statistical analysis
The results were expressed as mean ± SE with ve independent mea-
surements for each sample by IBM SPSS 25.0 version software. Student
t-tests analysed dierences between aqueous and ethanolic extract, and
the dierence was considered statically signicant when p 0.05.
Results and discussion
Antioxidants act as radical scavengers, hydrogen donors, electron
donors, peroxide decomposers, singlet oxygen quenchers, enzyme in-
hibitors, synergists, and metal-chelating agents. Both enzymatic and
non-enzymatic antioxidants exist in the intracellular and extracellular
environment to detoxify ROS [62] .
Antioxidant potential of S. platensis
An antioxidant is a molecule stable enough to donate an electron to
a rampaging free radical and neutralize it, thus reducing free radical
damages. These antioxidants delay or inhibit cellular damage mainly
through their free radical scavenging property. Recently, much attention
has been paid to spirulina’s antioxidant potential, and several studies re-
ported that spirulina signicantly reduces oxidative stress. The antiox-
idant protective eects are mediated by phycocyanin, 𝛽-carotene, and
other vitamins and minerals found in spirulina [ 63 , 64 ]. Reactive oxygen
species (ROS) attack and damages DNA, RNA, proteins, and lipids, re-
sulting in metabolic disorders, tissue injury, and cell death [64] . Oxida-
tive stress and ROS play an important role in many diseases, including
hypertension, diabetes mellitus, atherosclerosis, ischemic disease, and
malignancy. The lipid peroxides (LPOs), malondialdehyde (MDA), and
4-hydroxynonenal are essential markers of oxidative stress [65] .
This study estimated spirulina’s enzymatic and non-enzymatic an-
tioxidant potentials in the aqueous and ethanolic extract. The activity
of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase
(GPx), reduced glutathione (GSH), vitamin C and vitamin E of aqueous
and ethanolic extract of S. platensis are presented in Table (1-2) and
illustrated in Figure 1 - 2 .
SOD, CAT, and GPx are important antioxidant enzymes in the algal
cell that defends against peroxidation activity and maintain the cell’s re-
dox state [66] . The SOD is widely regarded as the rst cellular defence
during stress conditions [67] , which breakdown the harmful superox-
ide anion (O2
) into hydrogen peroxide and oxygen molecules, which
prevents the peroxynitrite production in the cells [68] , and the levels of
hydrogen peroxide in the cells are neutralized by antioxidant enzyme
activities.
Various stress conditions cause enhanced production of ROS, for ex-
ample, high light intensity and carbon depletion [69] . CAT is an impor-
tant enzyme in proximal H
2
O
2 scavenging in plants and animals. The
production of such antioxidant enzymes could be a defensive response
of the organism exposed to an unfavourable environment [70] . The prin-
cipal H
2
O
2
scavenging enzyme CAT showed higher activity in aqueous
extract than in comparative ethanolic extract, as shown in Figure 1 (A).
CAT remove H
2
O
2
only at high cellular concentrations and is insucient
at removing H
2
O
2
at low concentration [71] . Further, CAT activity is a
key enzyme of several defence systems during pathogenic infection and
stress conditions. The enhanced activity of CAT prevents the accumula-
tion of ROS, as evidenced by lesser total hydrogen peroxide [72] .
The SOD activity of both extracts was compared and found signif-
icant, as shown in Figure 1 (B); the higher SOD activity was obtained
with the value of 4.735 U/mg from the aqueous extract, and lower SOD
activity of 2.009 U/mg was observed in the ethanolic extract. There has
3
A. Kumar, D. Ramamoorthy, D.K. Verma et al. Energy Nexus 6 (2022) 100070
Figure 1. Graphical representation of Enzymatic antioxidant (A. Catalase, B. Superoxide dismutase, and C. Glutathione peroxidase) activity of aqueous and ethanolic
extract of S. platensis .
Figure 2. Graphical representation of Non-Enzymatic antioxidant (A. Reduced glutathione, B. Vitamin C, and C. Vitamin E, and) activity of aqueous and ethanolic
extract of S. platensis .
4
A. Kumar, D. Ramamoorthy, D.K. Verma et al. Energy Nexus 6 (2022) 100070
Table 1
Enzymatic antioxidant potential of S. platensis in aqueous
and ethanolic extract.
Aqueous Extract Ethanolic Extract
Catalase
a
(U/g) 120.827 ± 0.672 79.907 ± 0.447
SOD
b
(U/g) 4.735 ± 0.179 2.009 ± 0.305
GPx
c
(U/g) 211.098 ± 0.563 157.878 ± 0.790
a = µmole of H
2
O
2
decomposed / min/g
b = amount of enzyme required for 50% inhibition of
nitro blue tetrazolium (NBT) reduction.
c = µg of glutathione utilized / min/g.
The results are shown as the mean ± SE. The signicant
dierence is indicated by
(P 0.005).
been an inverse proportion between protein amount and specic SOD
activity when volumetric SOD activities and percentage inhibitions are
similar [73] . Several authors have carried out experiments to evaluate
the SOD activity. Al Zoubi [74] reported that the percentage inhibition
of S. maxima extract was 2.30 after seven days of cultivation. For tem-
perate phytoplankton cultures, the inhibition was more variable. In the
case of Nannochloropsis, SOD inhibition was 32% at 1°C and 5% at 30°C
[73] , whereas Synechococcus showed 99% of NBT formazan inhibition
the high activity of SOD. It is known that anoxygenic photosynthetic
bacteria contain SOD, as demonstrated for Chromatium and Chlorobium
[75] . Kanematsu & Asada [70] suggested that the low concentration of
dioxygen in the atmosphere before cyanobacteria appeared even then
a problem for these anaerobic bacteria and necessitated the eective
scavenging of SOD. In Darbyshire & Henry [76] report, SOD levels in
cyanobacteria, Anabaena cylindrica, were lower than in the vegetable
cells. They correlated this with the lower O
2
content in cyanobacteria, a
fact known from another organism where this enzyme is always associ-
ated with ambient O
2
. The photosynthetic cyanobacterium, S. platensis ,
is an ideal model organism to study the environmental regulation of
oxidative stress in both bacteria and plants [77] .
S. platensis maintained the activity of cellular antioxidant enzymes
total GPx, GPx-Se, and GR) and increased the levels of reduced glu-
tathione in cells. Intriguingly, the antioxidant capacity of S. platensis
could be enhanced when exposed to additional environmental stress
[78] . For example, Hussein et al. [3] examined the possibility of in-
creasing the amounts of some bioactive compounds in S. platensis by
cultivating cells in a medium supplemented with varying concentrations
of hydrogen peroxide (H
2
O
2
). The present study found increased GPx
in the aqueous extract presented in Figure 1 (C) and Table 1 . The GPx
enzyme has an antioxidant role in spirulina against oxidative stress, and
NADPH-dependent GPx activity was reported to protect the membrane
from lipid peroxidation in a cyanobacterium [41] . In addition, increas-
ing H
2
O
2
levels led to a signicant linear increase in the activities of an-
tioxidant enzymes in S. platensis , including CAT, peroxidase (PX), SOD,
and ascorbate peroxidase (APx).
Kurutas [79] found a positive correlation between increasing H
2
O
2
concentrations and increasing amounts of cellular lipophilic antioxi-
dants (total carotenoids and 𝛼-tocopherol) and hydrophilic antioxidants
(glutathione and ascorbic acid). Aside from ascorbate peroxidase, glu-
tathione peroxidase (GPx) serves as the main enzyme for scavenging
H
2
O
2
. GPx can also reduce alkyl and lipid hydroperoxides, and their
catalytic residue, selenocysteine, is conserved in mammalian GPx. Glu-
tathione peroxidases are enzymes that oxidize glutathione and reduce
peroxides. As a result, glutathione reductase acts as an electron donor
to reduce the oxidized form of glutathione [80] . Higher plants contain
multiple homologs of GPx. A cysteine residue instead of a selenocys-
teine residue has been found in their structure, which results in a lower
activity than mammalian GPx [81] .
S. platensis is known for its rich nutritional contents, such as pro-
teins, fatty acids, minerals, and vitamins, used as supplementary nutri-
ents for humans, poultry, and aquaculture [82] . The results reported in
Table 2
Non-enzymatic antioxidant potential of S. platensis in aqueous
and ethanolic extract.
Aqueous Extract Ethanolic Extract
Vitamin C (mg/g) 0.128 ± 0.005 0.111 ± 0.003
Vitamin E (mg/g) 0.152 ± 0.010 0.264 ± 0.163
Reduced GSH (nm/g) 122.758 ± 0.793 42.081 ± 0.913
The results are shown as the mean ± SE. The signicant dier-
ence is indicated by
(P 0.005).
Table 3
Phytonutrient contents of S. platensis in aqueous and ethano-
lic extract.
Aqueous Extract Ethanolic Extract
T. Phenol (mg/g) 9.919 ± 0.449 3.476 ± 0.362
Flavonoid (mg/g) 1.047 ± 0.004 0.585 ± 0.054
Tannin (mg/g) 0.792 ± 0.006 0.568 ± 0.061
Carbohydrates (g/g) 0.153 ± 0.008 0.248 ± 0.009
Protein (g/g) 0.707 ± 0.046 0.775 ± 0.047
The results are shown as the mean ± SE. The signicant dier-
ence is indicated by
(P 0.005).
Table 2 shows that the vitamin contents (C and E) of S. platensis were
signicantly observed in aqueous and ethanolic extracts, which are also
presented in Figure (A and B), with the maximum value of vitamin C
observed in the aqueous extract (0.128 mg/g) and higher value of vita-
min E in ethanol extract (0.264 mg/g) which is signicant with aqueous
extract. Vitamin levels recorded in this study are relatively comparable
with those in other Arthrospira species [ 83,84 ]. In spirulina, vitamins
and coenzymes synthesize haemoglobin and act upon hematopoietic ox-
idative processes in the bone marrow [85] .
GSH is a powerful antioxidant that has been commercialized and is
widely used in many elds. It plays an important role in detoxication,
the immune response, and protection against reactive oxygen species
[86] . This study found considerable variation in GSH activity in aque-
ous and ethanolic extract, shown in Table 2 . The higher GSH was found
in the aqueous extract (122.758 nm/g), followed by (42.081nm/g) in
ethanolic extract. GSH is crucial for non-enzymatic antioxidants, while
ascorbic acid also plays a vital role in reducing free radicle. There is
evidence that GSH can remove ROS, such as hydrogen radicals (
OH),
superoxide radical (O
2
), hydrogen peroxide (H
2
O
2
), and singlet oxy-
gen (
1
O
2
), and protect protein thiol groups from oxidizing into denat-
uration [87] . When spirulina is exposed to oxidative stress, glutathione
can be oxidized to oxidized glutathione by ROS, and ascorbate is ox-
idized to monodehydroascorbate (MDA) and dehydroascorbate reduc-
tase (DHAR). Finally, oxidized glutathione, MDA, and dehydroascor-
bate can be reduced to glutathione and ascorbate by the ascorbate-
glutathione cycle and detoxies ROS [88] . Prokaryotic and eukary-
otic cells contain glutathione, the primary low-molecular-weight thiol,
representing the signicant pool of reduced sulphur from non-protein
sources [ 89 , 90 ]. Phytonutrients are primary water-soluble antioxidants
in plant cells.
Quantitative Phytochemical Analysis of S. platensis
In this section, phytonutrients are estimated: tannin, total phenol, to-
tal avonoids, protein, and carbohydrate, and their results are presented
in Table 3 and shown in Figure 3 .
Due to the hydrogen-donating ability of the polyphenols’ hydroxyl
groups and their ability to donate electrons to arrest the formation of
free radicals caused by oxidative stress, polyphenols serve as powerful
antioxidants [ 90 ]. Currently, avonoids are the largest class of polyphe-
nols and have a standard diphenyl propane structure (C6–C3–C6) com-
posed of two aromatic rings linked by three carbons; Flavonoids act
through scavenging or chelating mechanisms [ 91 ].
5
A. Kumar, D. Ramamoorthy, D.K. Verma et al. Energy Nexus 6 (2022) 100070
Figure 3. Graphical representation of Phytonutrient contents (A. Total Phenol, B. Total Flavonoid, and C. Tannin D. Carbohydrate, and E. Protein) of aqueous and
ethanolic extract of S. platensis .
Table 3 illustrates that S. platensis biomass showed considerable
contents of total phenolic compounds (TPC) in aqueous and ethano-
lic extracts (9.919 mg/g and 3.476 mg/g), respectively. According
to the results, the high phenolic compounds in the aqueous extract
(99.919 mg/g) can be correlated with signicant antioxidant activity,
Figure 3 (A). Bolanho et al. [36] found that the content of phenolic com-
pounds in spirulina prepared under dierent conditions ranged from 2.4
to 5 g kg
1
. Interestingly, Kanta et al. [ 92 ] observed a signicant cor-
relation between phenolic content and antioxidant activity, suggesting
that the radical scavenging ability of each extract might be attributed to
its high concentration of phenolic hydroxyl groups. Studies have shown
that polyphenols are highly eective antioxidants and possess redox
properties, such as absorbing and neutralizing free radicals, quench-
ing singlet and triplet oxygen, and decomposing proxygene. From these
studies, Aehle et al. [ 93 ] reported that phenol containing methanolic ex-
tracts of lyophilized spirulina could not only enhance refrigeration life
but also act as an antioxidant in biological systems; the authors also con-
cluded that phenol contains methanol extracts of lyophilized spirulina
could signicantly reduce the amount of peroxidase induced browning
of guaiacol and that the amount of phenolic compounds produced by
spirulina was related to the antioxidant capacity of the extract. Borow-
itzka et al. [18] recorded that phenolic compounds have the antioxidant
capacity and interact with free radicals; they can inhibit lipid peroxida-
tion (LPO) in vitro through their ability to sequester free radicals and
act as metal chelators. Deng & Chow [1] analysed the antioxidant activ-
ity of carotenoids, phenolics, and tocopherols extracted from spirulina
and concluded that phenolic compounds responsible for the antioxidant
actions of spirulina extracts were organic acids (caeine, chlorogenic,
salicylic, synaptic, and trans-cinnamic) that acted both individually and
synergistically.
The total avonoids content was performed using the AlCl
3
method
with quercetin as a standard, and the results are expressed as mg/g
of quercetin equivalent. The aqueous extract showed high avonoids
content (1.047 mg/g), whereas ethanolic extract showed low (0.568
mg/g) content, as shown in Table 3 . Earlier, Prabhakaran et al., Patil
& kalibal [ 94 , 95 ] reported the high concentration of avonoids in dif-
ferent solvents (acetone, ethanol, methanol, and chloroform) extracts of
Tetraselmis sp . and Oscillatoria sp ., respectively. Similarly, Adhoni et al.
6
A. Kumar, D. Ramamoorthy, D.K. Verma et al. Energy Nexus 6 (2022) 100070
[ 96 ] screened the phytochemical prole of C. vulgaris in various polar
and nonpolar solvent extracts and reported 14 dierent phytochemical
constituents; a high concentration of phytochemicals was recorded in
polar solvent extracts. Thus, the earlier ndings of the phytochemical
prole revealed that the phytochemical composition depends on the sol-
vent system.
Qualitative analysis of S. platensis extracts revealed a low tannin con-
tent. Tannins belong to the secondary metabolites synthesized by plants
and microorganisms to accomplish ecological functions. In Table 3 and
Figure 3 (C), tannin concentration was signicant in both extracts viz.
aqueous (0.792 mg/g) and ethanol (0.568 mg/g), respectively. These re-
sults contrasted with the previously reported literature, which revealed
promising tannin content in the genus Arthrospira [ 97 , 98 ].
Spirulina carbohydrates, especially polysaccharides, generate energy
and cellular structure. The carbohydrate content in aqueous and ethano-
lic extract was about 0.153 and 0.248, as sown in Figure 3 (D) and pre-
sented in Table 3 . This value is considerably lower than the carbohy-
drate concentration average of 15 to 25% reported previously for other
spirulina strains [ 99 ]. Besides, the spirulina study showed a carbohy-
drate content typically below average for many microalgae, overall ac-
counting for 10 to 27% dw [ 100 ]. In addition, it was lower than that
reported in edible seaweeds, which ranged from 20 to 68% [ 101–104 ].
The soluble carbohydrates constitute a major part of the total carbohy-
drate content, whereas, in higher plants, insoluble carbohydrates are a
major constituent of total carbohydrates [ 105 , 106 ]. This result agrees
with other reports that showed whole dried microalgae could be used as
food due to their higher carbohydrate digestibility. However, like carbo-
hydrate composition, the protein content of algae was linked to seasonal
variation [ 105 ].
Figure 3 (E) and Table 3 illustrate that S. platensis is a rich source of
protein in both extracts. This value was higher than that of various spir-
ulina species harvested from other countries [ 99 ], with its amino acid
composition of aspartic acid, glutamic acid, serine, glycine, histidine,
arginine, threonine, alanine, proline, valine, isoleucine, leucine, pheny-
lalanine, and lysine shown in [ 106 ]. It showed a fairly higher protein
concentration than other microalgae (6 to 71%) [ 107 , 108 ]. The brown
edible seaweed’s dry matter content was also greater than most red and
green seaweeds (10-47% dw) [ 101 ] and green edible seaweeds (3-15%)
[109] . This result conrms that the protein content was considerably
higher than the source. Furthermore, several studies have demonstrated
that the protein content of microalgae varied according to species, envi-
ronmental conditions, and analytical methods for protein determination
[ 108 ]. Furthermore, to evaluate protein quantity, it is also important
to examine the protein’s quality, which is determined by the quantity,
proportion, and availability of its amino acids [ 110 ]. Since proteins in
spirulina is made up of all essential amino acids, making it a complete
protein source on a qualitative and nutritional point of view [ 111 ].
Conclusion
It can be concluded that S. platensis exhibited considerable antiox-
idant and phytonutrient potential. High antioxidant activity was ob-
served in the aqueous extract, and all other enzyme activities and phy-
tonutrients were higher in the aqueous extract. Vitamins are a good pre-
cursor for the health of an organism. The results showed vitamin C and
vitamin E signicantly higher in ethanolic extract. Vitamins have stim-
ulated prostaglandin synthesis, blood pressure regulation, cholesterol
synthesis, inammation, and cell proliferation. The spirulina products
available in the market are rich in antioxidant and phytonutrient com-
ponents and suitable for regular consumption. Many leading health or-
ganizations have praised spirulina, a ‘wonder food supplement. With
information based on scientic studies and further validation studies,
spirulina is likely to pass the various stages of clinical trials to become
certied as a nutritional and dietary supplement approved by USFDA.
Many herbal cosmetics such as face creams, hair lotion, and biolistics
have been formulated from phycocyanin pigment present in spirulina.
Beta-carotene and other carotenoids might be formulated to prevent
cancer in humans and increase the quality of egg-laying chickens, meat
production, and as a feed additive in aquaculture.
The appreciable antioxidant and phytonutrient contents observed in
spirulina extracts can facilitate its use for commercial exploitation in
pharmaceuticals, food additives, and cosmetic industries. It can also lead
to the identication of novel pharmaceutical compounds to treat various
diseases in the future.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
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