Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2016, Article ID 7631864, 13 pages
http://dx.doi.org/10.1155/2016/7631864

Research Article
Quantification of Phytochemicals from Commercial Spirulina
Products and Their Antioxidant Activities

Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu

Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science,
King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

Correspondence should be addressed to Naif Abdullah Al-Dhabi; naldhabi@ksu.edu.sa

and Mariadhas Valan Arasu; mvalanarasu@gmail.com

Received 21 July 2015; Revised 20 November 2015; Accepted 25 November 2015

Academic Editor: G. K. Jayaprakasha

Copyright © 2016 N. A. Al-Dhabi and M. Valan Arasu. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The present study aimed to profile the polyunsaturated fatty acids, sugars, free amino acids, and polyphenols in 37 varieties of
Spirulina commonly available in the market using gas chromatography and high performance liquid chromatography. In addition,
the biological potentials of the Spirulina samples were evaluated by analysing the in vitro antioxidant activities using various
analytical techniques. The analyses revealed the presence of 13 polyunsaturated fatty acids, 18 amino acids, 7 sugars, and polyphenols.
The polyunsaturated fatty acids contents were varied between Spirulina samples. The total polyunsaturated fatty acids amount was
4.25 mg/100 g, and the average among of sapienic acid detected was 2.25 mg/100 g, which was followed by linoleic acid (16.7%)
and 𝛾-linolenic acid (14%). Among the 7 sugars, the hexose levels were the highest (73.85%). The total amino acids contents
ranged from 11.49 to 56.14 mg/100 g, and the individual essential amino acids accounted for 17% to 39.18%. The “natural” tablets
exhibited the highest polyphenols levels (24 mg/g). All of the Spirulina samples expressed dose-dependent antioxidant activities. The
polyunsaturated fatty acids, sugars, free amino acids, and polyphenols contents varied widely, and the variations in these compounds
between the Spirulina samples were significant.

1. Introduction chemicals and pharmaceutical metabolites [1]. Each year,

more than three thousand tons of Spirulina are cultivated
Cyanobacteria also include unicellular organisms and all around the world for human nutrition and the production of
of them are not spiral shaped (Spirulina is spiral shaped). other fine commodity chemicals [2]. Several similar studies
They grow naturally in the water of warm climates and are with market samples of Spirulina have been carried out
primarily cultivated in ponds and small lakes. In contrast earlier and data are available [3]. In recent years, people have
to other living organisms, microalgae or Spirulina is one of been interested in consuming Spirulina in tablet and powder
the microalgae and is not the only microalga which do not forms due to its relatively high contents of protein (58%),
require organic, inorganic, nutrient, and other carbon sources carbohydrates (30%), fat (8%), dietary fibres (3%), sugars
for growth and can survive in higher alkaline pHs and in (3%), vitamins (<1%), and phytochemicals [4, 5]. Spirulina
greater bicarbonate and carbonate concentrations. Beginning contains fatty acids such as linoleic acid, docosahexaenoic
in the sixteenth century, cyanobacteria have been used as acid, eicosapentaenoic acid, arachidonic acid, and stearidonic
a traditional food source for African and Mexican people. acid, respectively. Spirulina also contains moderate amounts
Among the microalgae, Spirulina maxima (Arthrospira max- of vitamins such as vitamin A, vitamin C, vitamin E, vitamin
ima), Spirulina platensis (Arthrospira platensis), and Spirulina B12, thiamine, nicotinamide, pyridoxine, riboflavin, and folic
fusiformis (Arthrospira fusiformis) are the most widely culti- acid and beneficial pigments, such as chlorophyll-a, zeax-
vated species around the world and are widely used as health anthin, diatoxanthin, 3󸀠 -hydroxyechinenone, echinenone,
foods, food additives, and potential sources of high value beta-carotene, xanthophyll, canthaxanthin, c-phycocyanin,
2 Evidence-Based Complementary and Alternative Medicine

beta-cryptoxanthin, myxoxanthophyll, oscillaxanthin, phy- tube. After proper mixing, 10.0 mL 8.3 M HCl was added into
cobiliproteins, and allophycocyanin [5, 6]. However, the the tube, which was then incubated in a shaking water bath
nutritional contents of Spirulina depend on the cultivation at 70–80∘ C for 2 h. During the incubation, the contents of the
conditions and the processing methods. The nutritional tubes were intermittently mixed to release the fat from the
components and other phytochemicals in Spirulina primarily walls of the tubes. After incubation, the samples were allowed
exhibit anti-inflammatory, antioxidant, antidiabetic, neuro- to cool at room temperature and mixed with 15 mL diethyl
protective, hepatoprotective, and anticancer activities [7]. ether (DE). The DE layer was then separated and filtered
The regular consumption of Spirulina ameliorates the symp- in the column using Na2 SO4 and petroleum ether (PE).
toms of premenstrual cycles in women and the symptoms Subsequently, the collected PE layers were slowly evaporated
of amyotrophic lateral sclerosis. Spirulina prevents allergic using a nitrogen stream and used for the extraction of the fatty
reactions and aids in the removal of metals from the body. A acids.
recent study suggested that Spirulina helps to bind radioactive
elements and is useful for protecting the human body from 2.3. Extraction and Quantification of the Fatty Acids. The
exposure to radiation therapy. The phenolic compounds total lipids in the Spirulina were extracted according the
present in the Spirulina are primarily involved in the redox method of Mossoba et al. (2003) with modifications [12].
mechanism and act as hydrogen donors, reducing agents, Briefly, the extracted lipids were saponified with 0.5 N NaOH
metal chelator singlets, and oxygen quenchers [8]. Therefore, in methanol (1.5 mL) for 5 min at 100∘ C and cooled at room
phenolic compounds can prevent the formation of ROS temperature. After cooling, the samples were treated with
and reactive nitrogen species, which include free radicals, 2 mL of BF3-methanol and incubated at 100∘ C for 10 min
such as hydroxyl and superoxide anions and nitric oxide, and allowed to cool at room temperature. The samples were
and nonfree radical species, such as hydrogen peroxide and then thoroughly vortexed with 2 mL of isooctane and 1 mL of
nitrous acid. The development of phenolic compounds as saturated NaCl solution for 10 min. Next, the upper isooctane
antioxidants for the treatment of various human diseases layer was carefully transferred into tubes and injected into
has increased. Therefore, there is an urgent need to identify a Hewlett-Packard 6890 series gas chromatograph (GC)
novel antioxidant molecules with fewer side effects and sig- equipped with an autoinjector and a flame-ionization detec-
nificant hepatoprotective effects [9]. To overcome disorders, tor (Agilent Technologies, Little Falls, Del., USA). The fatty
the regular consumption of natural health-promoting foods, acids were separated in a fused-silica capillary column (SP-
such as Spirulina tablets or powders, is advised. In vitro 2560, 100 m × 0.25 nm × 0.2 𝜇m film thickness, Supelco,
studies demonstrated that the Spirulina and Nestoc species USA). The GC oven was heated to 100∘ C and held for 4 min
have several therapeutic properties, due to their ability to and then further increased to 240∘ C at a rate of 3∘ C/min
scavenge superoxide and hydroxyl radicals and inhibit lipid and held at 240∘ C for 15 min. The injector and detector
peroxidation [10, 11]. Therefore, the present study aimed to temperatures were set at 225∘ C and 285∘ C, respectively. The
investigate the metabolite profiles and antioxidant properties mobile gas (helium) applied at a flow rate of 0.75 mL/min. The
of 37 commercially available Spirulina samples. concentrations of the individual fatty acids were calculated
based on the relative retention times of the standard mixtures.
2. Materials and Methods The conversion of FAMEs to corresponding fatty acids are
shown in Table 2.
2.1. Chemicals and Solvents. Standard methyl esters of The response factor (𝑅𝑖 ) of each fatty acid was calculated
fatty acids were obtained from Supelco (37 Component as follows:
FAME Mix). Triglyceride (IS, C11:0 triundecanoin) was
𝑃𝑠𝑖 𝑊
purchased from Nu-Chek Prep (Elysian, MN, USA). BF3- 𝑅𝑖 = × C11:0 , (1)
methanol (10%, w/w) was procured from Supelco (Bellefonte, 𝑃𝑠C11:0 𝑊𝑖
PA, USA). Analytical grade diethyl ether (DE), pyrogal- where 𝑃𝑠𝑖 is peak area of individual fatty acid in mixed
lol, petroleum ether (PE), chloroform, and ethanol were FAMEs standard solution; 𝑃𝑠C11:0 is peak area of C11:0 fatty
purchased from Sigma-Aldrich Chemical Co. (St. Louis, acid in mixed FAMEs standard solution; 𝑊C11:0 is weight of
Mo., U.S.A.). Thirty-seven Spirulina samples with different internal standard in mixed FAMEs standard solution; and
countries of origin in the forms of tablets and capsules were 𝑊𝑖 is weight of individual FAME in mixed FAMEs standard
procured from specialist shops (Table 1). All the studied solution.
Spirulina samples were procured from different markets in The amounts of the individual compounds in the test
the world. The details of the samples were mentioned in samples were calculated as follows:
Table 1.
𝑃𝑡𝑖 × 𝑊𝑡C11:0 × 1.0067
2.2. Extraction of Lipids from the Spirulina Samples. The total 𝑊FAME𝑖 = , (2)
𝑃𝑡C11:0 × 𝑅𝑖
lipids in the Spirulina samples were extracted according to
the following modified method of Mossoba et al. (2003) where 𝑃𝑡𝑖 is peak area of the fatty acid 𝑖 in the test portion;
[12]. Briefly, one gram of finely powdered sample with 2 mL 𝑊𝑡C11:0 is weight of C11:0 in the internal standard added to
pyrogallol solution (in ethanol 95%, 50 mg/mL) and 1 mL test portion, g; 1.0067 is conversion of the internal standard
triglyceride internal standard solution (IS: C11:0 triunde- from triglyceride to FAME; and 𝑃𝑡C11:0 is peak area of C11:0
canoin 5 mg/mL in iso-octane) was transferred into a 50 mL in the internal standards in the test portion.
 Table 1: Spirulina products lists and their country of origin.
S. number Product type Manufacturing company Country of origin Web address
1 Tablets TAAU Australia Pvt Ltd., NT Australia http://www.australianspirulina.com.au/
2 Capsules General Nutrition Corp., Pittsburgh USA http://www.gnc.com/
3 Capsules Nature’s Way Products, Inc., Springville, Utah USA http://www.naturesway.com
4 Tablets Good ‘N Natural, New York USA http://www.goodandnaturalstore.com
5 Tablets Now Foods, Bloomingdale USA http://www.nowfoods.com
6 Tablets Nature Pure, Inc., Larkspur, California USA
7 Tablets Source Naturals, Inc., Santa Cruz, California USA http://www.sourcenaturals.com
8 Tablets Jarrow Formulas, Los Angeles, CA USA http://www.jarrow.com
9 Tablets Earthrise Nutritionals LLC, Irvine, CA USA http://earthrise.com/
10 Tablets Nutrex Hawaii Inc., Kailua-Kona, Hawaii USA http://www.nutrex-hawaii.com
11 Capsules Pure Planet Products, Inc., Long Beach, CA USA https://www.pureplanet.com
12 Tablets Puritan’s Pride, Inc., Oakdale, New York USA http://www.puritan.com
13 Capsules 21st Century HealthCare, Inc., Arizona USA http://www.21stcenturyvitamins.com
14 Tablets Japan Algae Co., Ltd., Tokyo Japan http://www.sp100.com/
15 Tablets All Seasons Health, Hampshire United Kingdom http://www.carehome.co.uk/
Evidence-Based Complementary and Alternative Medicine

16 Capsules Fushi Wellbeing Ltd., London United Kingdom http://www.fushi.co.uk

17 Tablets Biovea, London United Kingdom http://www.biovea.com
18 Capsules Parry Nutraceuticals, Chennai India http://www.parrynutraceuticals.com
19 Tablets Lifestream International Ltd., Northcote, Auckland New Zealand http://www.lifestream.co.nz
20 Tablets Green Health, Auckland New Zealand http://www.greenhealth.co.nz
21 Tablets RBC Life Sciences, Inc., Burnaby, British Columbia (BC) Canada http://www.rbclifesciences.com
22 Tablets Swiss Herbal Remedies Ltd., Richmond Hill, Ontario Canada http://www.swissnatural.com
23 Capsules Herbal Select, Guelph, Ontario Canada http://www.herbalselect.ca
24 Capsules Gourmet Nutrition F.B. Inc., STE-Julie (Quebec) Canada http://www.gourmetfb.com
25 Capsules Terra Vita Fine Whole Herbs, Brampton, Ontario Canada http://dsld.nlm.nih.gov/
26 Capsules DXN marketing Malaysia http://www.dxnmalaysia.com
27 Capsules Hydrolina Biotech Pvt., India India http://www.hydrolinabiotech.com
28 Capsules Prime Health Laboratories Ltd. Australia Not available
29 Tablets Laurel Herbal products India Not available
30 Tablets IMO Control Pvt Ltd. India http://www.imocontrol.in
31 Capsules Acumen Pharmaceuticals Pvt India http://www.acumenpharm.com
32 Capsules Bio-Life Organic Spirulina Malaysia https://healthreview2u.wordpress.com/2010/09/15/biolifespirulina/
33 Tablets Dharain Pharmaceuticals India http://www.mihnati.com/
34 Tablets 21st Century HealthCare, Inc., Arizona USA http:/www.21stcenturyvitamins.com/
35 Tablets Zuellig Bharma SDW. BHD Malaysia http://www.zuelligpharma.com
36 Tablets Kordels Spirulina India http://www.rakuten.com
37 Tablets Elken Chewable Malaysia https://elken4mrt.wordpress.com
3
4 Evidence-Based Complementary and Alternative Medicine

Table 2: Fatty acids contents (mg/100 g in different Spirulina).

FA
14:00 16:00 16:1 18:0 18:1t
18:1(n-9)c 18:1(n-7)c 18:2t 18:2(n-6)c 20:00 18:3(n-6) 20:02 20:3n6
Sample Total
𝑓FA𝑖
0.9421 0.9481 0.9477 0.9530 0.9527 0.9524 0.95201 0.9517 0.9524 0.9570 0.952 0.9565 0.9557
1 0.01 1.88 0.22 0.29 0.00 0.09 0.02 0.01 0.65 0.01 0.67 0.01 0.01 2.5
2 0.00 2.07 0.10 0.09 0.00 0.09 0.02 0.01 0.32 0.01 0.16 0.01 0.01 2.36
3 0.01 1.97 0.11 0.34 0.01 0.11 0.05 0.04 0.75 0.01 0.67 0.00 0.00 2.55
4 0.01 2.55 0.27 0.06 0.00 0.05 0.01 0.01 0.85 0.00 0.73 0.01 0.01 2.94
5 0.01 1.89 0.17 0.30 0.01 0.07 0.02 0.01 0.62 0.00 0.53 0.00 0.01 2.44
6 0.01 1.90 0.31 0.13 0.01 0.09 0.04 0.01 0.73 0.01 0.70 0.01 0.02 2.45
7 0.01 2.38 0.17 0.79 0.00 0.06 0.02 0.01 0.55 0.01 0.46 0.01 0.01 3.42
8 0.02 2.81 0.14 0.85 0.00 0.15 0.02 0.01 0.80 0.01 0.50 0.01 0.01 3.98
9 0.01 2.00 0.18 0.11 0.00 0.14 0.07 0.01 0.65 0.01 0.40 0.01 0.01 2.45
10 0.01 2.80 0.14 0.45 0.00 0.09 0.01 0.01 0.87 0.01 0.73 0.01 0.01 3.5
11 0.01 2.10 0.23 0.14 0.00 0.16 0.06 0.01 0.60 0.01 0.37 0.01 0.01 2.65
12 0.01 2.12 0.25 0.06 0.00 0.14 0.07 0.01 0.78 0.01 0.52 0.01 0.01 2.59
13 0.01 2.36 0.27 0.40 0.00 0.11 0.02 0.01 0.80 0.01 0.82 0.01 0.01 3.16
14 0.01 2.48 0.24 0.05 0.00 0.13 0.02 0.02 0.99 0.01 0.82 0.01 0.01 2.92
15 0.01 1.78 0.28 0.04 0.00 0.14 0.01 0.01 0.62 0.01 0.58 0.00 0.00 2.26
16 0.01 2.95 0.15 2.13 0.01 0.11 0.03 0.02 0.67 0.02 0.55 0.00 0.00 5.37
17 0.02 1.91 0.10 0.06 0.01 0.15 0.03 0.03 0.70 0.01 0.57 0.00 0.01 2.25
18 0.02 1.95 0.15 0.17 0.00 0.08 0.03 0.03 0.64 0.01 0.60 0.00 0.00 2.38
19 0.02 2.04 0.12 0.16 0.01 0.14 0.03 0.03 0.68 0.01 0.51 0.00 0.01 2.49
20 0.02 2.12 0.12 0.25 0.01 0.12 0.04 0.04 0.71 0.01 0.66 0.00 0.01 2.64
21 0.01 1.81 0.26 0.21 0.01 0.05 0.02 0.01 0.65 0.01 0.62 0.01 0.01 2.35
22 0.00 1.43 0.22 0.04 0.00 0.12 0.02 0.01 0.54 0.00 0.53 0.01 0.01 1.81
23 0.01 3.27 0.14 1.11 0.00 0.18 0.07 0.01 0.76 0.02 0.44 0.01 0.01 4.73
24 0.01 2.58 0.28 0.06 0.00 0.07 0.02 0.02 0.74 0.01 0.59 0.01 0.01 3.01
25 0.01 2.49 0.26 0.06 0.00 0.10 0.03 0.01 0.72 0.01 0.58 0.01 0.01 2.93
26 0.01 1.73 0.23 0.05 0.00 0.10 0.03 0.01 0.44 0.00 0.39 0.00 0.01 2.12
27 0.02 2.17 0.25 0.06 0.01 0.12 0.06 0.01 0.75 0.01 0.60 0.01 0.01 2.63
28 0.01 2.98 0.23 1.35 0.00 0.13 0.03 0.02 0.90 0.02 0.52 0.01 0.01 4.72
29 0.01 1.58 0.11 0.07 0.00 0.17 0.04 0.01 0.53 0.01 0.49 0.01 0.01 1.95
30 0.02 2.36 0.14 0.05 0.01 0.17 0.03 0.03 0.83 0.01 0.65 0.00 0.00 2.75
31 0.01 1.88 0.15 0.05 0.00 0.19 0.03 0.01 0.54 0.00 0.40 0.01 0.01 2.28
32 0.01 1.75 0.19 0.07 0.00 0.08 0.01 0.01 0.59 0.00 0.49 0.01 0.01 2.1
33 0.01 2.29 0.33 0.06 0.00 0.10 0.03 0.01 0.78 0.01 0.70 0.01 0.01 2.8
34 0.04 3.95 0.21 0.15 0.02 0.23 0.08 0.07 1.37 0.01 1.24 0.01 0.01 4.59
35 0.02 2.10 0.31 0.08 0.00 0.16 0.12 0.01 0.55 0.01 0.60 0.02 0.01 2.68
36 0.01 2.62 0.15 0.08 0.00 0.18 0.04 0.03 1.04 0.01 0.58 0.01 0.01 3.05
37 0.02 2.14 0.14 0.05 0.01 0.11 0.04 0.04 0.77 0.00 0.77 0.00 0.01 2.46
Numbers 1 to 37 were the sample names (Table 1). Total fatty acids were considered only for PUFA. 14:00: myristic acid, 16:00: sapienic acid, 16:1: palmitoleic
acid, 18:0: stearic acid, 18:1t: elaidic acid, 18:1(n-9)c: oleic acid, 18:1(n-7)c: vaccenic acid, 18:2t: linolelaidic acid, 18:2(n-6)c: linoleic acid, 20:00: eicosenoic acid,
18:3(n-6): 𝛾-linolenic acid, 20:02: eicosadienoic acid, and 20:3n6: dihomo-gamma-linolenic acid.

The weight of the fatty acid (𝑊𝑖 ) was determined as 2.4. Extraction and Quantification of Sugars Using HPLC.
follows: The carbohydrates present in the Spirulina samples were
quantified according to the following the standard method
𝑊𝑖 = 𝑊FAME𝑖 × 𝑓FA𝑖 , (3) [14]. Briefly, a 100 mg portion of the powdered sample was
mixed with 10 mL sterile distilled water and boiled at 100∘ C
where 𝑓FA𝑖 is conversion factors for the conversion of the for one hour. After heating, the debris was separated by
FAMEs to their corresponding fatty acids. centrifugation at 10,000 rpm for 10 min. Next, the debris-free
Evidence-Based Complementary and Alternative Medicine 5

solution was mixed with 10% 5% trichloroacetic acid (TCA) 2.7. In Vitro Antioxidant Assays
and incubated at room temperature for 10 min. Subsequently,
2.7.1. Preparation of the Spirulina. One gram of the fine
the samples were centrifuged at 10,000 rpm for 10 min and
powder sample was mixed with 5 mL of ethanol in a screw-
then filtered through a 0.45 𝜇m PTFE syringe filter for HPLC cap tube by vortexing for 5 min and then kept in an orbital
analysis (HP1100 Agilent Co., USA). The filtered samples shaker at 150 rpm for 24 h at room temperature for thorough
were separated through a 300 × 7.8 mm Aminex HPX- extraction. After incubation, the samples were centrifuged at
87H (Bio-Rad; Hercules, CA, USA) column at 60∘ C using 13,000 rpm for 15 min at 4∘ C. The resulting supernatant was
a 5.0 mM H2 SO4 monophasic solvent system with a flow vacuum evaporated at 30∘ C, and the resulting extract was
rate of 0.5 mL/min and a column wavelength of 220 nm. used for the antioxidant assays.
The injection sling was 10 𝜇L. The quantifications of the
individual sugars were based on the peak areas and calculated 2.7.2. Reducing Power Activity Assay. The reducing power
as equivalents of standard compounds. assay was performed according to the method of Oyaizu
(1986) [17]. Volume of 100 𝜇L of various concentrations
2.5. Extraction and Quantification of the Free Amino Acids (20–100 𝜇g/mL) of the samples was mixed with phosphate
Using HPLC. Individual free amino acids were extracted buffer (2.5 mL) and 1% potassium ferricyanide (2.5 mL) and
and quantified according to the method of Park et al. incubated at 50∘ C for 20 minutes. After incubation, 2.5 mL
(2014) with modifications [15]. Briefly, a 100 mg portion of 10% trichloroacetic acid was added, and the samples were
of a fine powdered sample was mixed with 1.2 mL of 5% centrifuged at 3000 rpm for 10 min. The upper layer of the
trichloroacetic acid (TCA) in a 2 mL Eppendorf tube and solution (2.5 mL) was mixed with distilled water (2.5 mL)
vigorously shaken for 5 min. The slurry sample was incubated and a freshly prepared 0.1% ferric chloride solution (0.5 mL)
at room temperature for 60 min, and the upper layer was and measured at an absorbance at 700 nm. The control was
then separated by centrifugation. The collected samples prepared in a similar manner, but the sample was excluded.
were diluted with 0.1 M HCl and then filtered through a Vitamin C at various concentrations was used as a standard.
0.45 𝜇m PTFE syringe filter. The filtrate was then analysed Increases in the absorbance of the reaction mixture indicated
by HPLC (Agilent Technologies, Palo Alto, CA). The HPLC increases in reducing power.
analyses of free amino acids were conducted according to the
“rapid, accurate, sensitive, and reproducible HPLC analysis 2.7.3. DPPH Radical Scavenging Activity Assay. The DPPH
of amino acids analysis” method with Zorbax Eclipse-AAA radical scavenging assay performed according to the method
columns using an Agilent 1100 HPLC system. Briefly, the of Hatano et al. (1988) [18]. Briefly, 100 𝜇L of the sample
separation of the free amino acids was performed on a Zorbax and vitamin C (concentration 100–500 𝜇g/mL) was mixed
Eclipse AAA analytical column. The oven temperature of the with 200 𝜇L of freshly prepared DPPH solution (1 mg/mL in
column was set at 40∘ C, and the detection wavelength was methanol) and incubated at room temperature in the dark
set a 338 nm. The injection volume was 10 𝜇L. The mobile for 30 minutes. The controls included only deionized water
phase consisted of a mixture of 40 mM NaH2 PO4 (pH 7.8, and the DPPH solution. The absorbances of the resulting
solvent A), and solvent B (ACN, MeOH, and water at a solutions were measured in triplicate at 517 nm following
45 : 45 : 10 v/v/v ratio) was passed at a rate of 2.0 mL/min. centrifugation at 12000 rpm for 10 min.
The HPLC separation parameters were as follows: 0 min, The scavenging activity was calculated as follows:
0% B; 0–1.9 min, 0% B; 1.9–21.1 min, 57% B; 21.1–21.6 min,
100% B; 21.6–25 min, 100% B; 25–25.1 min, 0% B; and 25.1– (𝐴 0 − 𝐴 1 )
scavenging activity (%) = [1 − ] × 100, (4)
30 min, 0% B. A sample with an amino acid content of 𝐴2
50 pmoL/𝜇L was used as the standard. The quantifications of
the different amino acids were based on the peak areas and where 𝐴 0 is absorbance of sample, 𝐴 1 is absorbance of blank,
were calculated as equivalents of the standard compounds. and 𝐴 2 is absorbance of control.
All contents are expressed as milligrams ∗ gram/fresh weight
(FW). 2.7.4. Hydroxyl Radical-Scavenging Activity Assay. The hy-
droxyl radical-scavenging assay was performed according
2.6. Total Polyphenol Analysis. The total polyphenols were to the method of Elizabeth and Rao (1990) with slight
estimated according to the method of Folin-Ciocalteu (mod- modification [19]. The reagents for the assay were freshly pre-
ified from Lin, and Tang, 2006) [16]. Briefly, 10 mg of the pared. Briefly, one millilitre samples of the working solutions
sample was dissolved in 1 mL of methanol with 2 mL of Folin- that consisted of different ratios of the extract were mixed
Denis reagent and 35% sodium carbonate (Na2 CO3 ). The with 100 mL of 28 mM 2-deoxy-2-ribose in phosphate buffer
mixture was stored at room temperature for 30 min. The (pH 7.4), EDTA (1.04 mM, 1 : 1, v/v), 100 mL H2 O2 (1 mM),
absorbance was measured with an UV-Vis spectrophotometer 200 mL of FeCl3 (200 mM), and 100 mL ascorbic acid (1 mM).
at 750 nm. The total polyphenols were calculated as gallic The resulting solutions were mixed evenly, and the reaction
acid equivalents based on a calibration curve for gallic acid mixtures were incubated at 37∘ C for 1 h. The degradation of
(0, 25, 50, and 100 𝜇g/mL) using the following equation that deoxyribose was determined by reading the absorbance at
was based on the calibration curve: 𝑦 = −0.9706𝑥 + 3.8935 532 nm against the blank solution using a microplate reader
(𝑅2 = 0.9992). (BioRad). Vitamin C was used as a positive control. The
6 Evidence-Based Complementary and Alternative Medicine

experiments were conducted in triplicate. The scavenging FID1 A, (FC140730\SIG10006.D)

n-6)c
18:2(n-6)c
(n-6)c
18:3(n-6)c
66))
6)
6)
111 0
111:0

16:0
60
116

18:2(n
((n
2(n
18:3(n
3(n
activities were calculated according to (4). 35

2(
3(
18
18
30

18:0
3. Results and Discussion 25

(pA)

16:1
20
3.1. Variations in the Fatty Acid Contents. The total lipid

18:1(n-9)c
contents were extracted from the Spirulina samples, and the 15

(n-7)c
18:1(n-7)c
18:1t 18:

20:4(n-6)
18:3(n-3)
8 ( -3))

20:3n6
18:1(n11(n

18:3(n
18:2t
18:3t
1(
220 0
20:0
14:0

20:2
118:1
individual fatty acid compositions of the samples were anal-

12:0
10

4:0

2200
18
1

118
1
ysed with gas chromatography. A gas chromatograph coupled
20 30 40 50 60 70 80
with a flame ionization detector guided the identification
of the following 10 unsaturated and fatty acids (sapienic Figure 1: Gas chromatograms of the fatty acids identified in the
acid, palmitoleic acid, elaidic acid, oleic acid, vaccenic acid, Spirulina samples. The peaks numbers refer to the individual fatty
linolelaidic acid, linoleic acid, eicosenoic acid, 𝛾-linolenic acids listed in Table 2.
acid, and dihomo-gamma-linolenic acid) and three saturated
and fatty acids (myristic acid, stearic acid, and eicosadienoic
acid) (Figure 1 and Table 2). The individual PUFA contents extracted from the 37 Spirulina samples and clearly base-
were quantified by comparing the standard fatty acids with line eluted by HPLC. The quantitative results revealed that
their indices. The PUFA contents in the Spirulina samples glucose, fructose, and sucrose were present in the greatest
ranged from 3.01 (DXN Marketing, capsules) to 7.41 g/100 g amounts followed by xylose, ribose, galactose, and rhamnose.
(21st Century HealthCare, Inc., Arizona, tablets; Table 4). The total sugar contents of the Spirulina samples ranged from
Interestingly, there were comparatively lower amounts of 𝛾- 309 to 1221.67 mg/100 g (Table 4). Together, glucose, fructose,
linolenic acid in the studied Spirulina samples; this acid galactose, and rhamnose accounted for an average of 73.85%
accounted for an average of 14% of the total PUFAs. The of the total sugar contents. Among the major individual
amounts of 𝛾-linolenic acid ranged from 0.16 g/100 g (Gen- sugars, glucose accounted for an average of 351 mg/100 g and
eral Nutrition Corp., Pittsburgh, capsules) to 1.24 g/100 g 52% of the total sugar contents. Similarly, Chaiklahan et al.
(21st Century HealthCare, Inc., Arizona, tablets). However, (2013) reported that rhamnose and glucose account for 53%
Mühling et al. (2005) reported palmitic acid (C16:0) noted as and 13% of the total sugars, respectively [28]. The rhamnose
the dominant fatty acid in wild Spirulina samples [20]. In our contents varied from 8 to 58 mg/100 g of the total sugars,
study sapienic acid been noted as the major fatty acids. Many accounting for an average of 6.5% [29]. The results indicated
in vitro studies have confirmed that 𝛾-linolenic acid can be that, among the pentose sugars, xylose (average 9.08%) and
used to effectively lower cholesterol and treat atopic eczema, ribose (average 4.75%) were the major components in the
breast cancer, and premenstrual disorder [21–25]. Recently, Spirulina samples. The final outcome of this study is that
Sajilata et al. (2008) extracted and purified S. platensis the variations in the individual sugar contents between the
active components via lipid fractionation, silica gel column Spirulina samples were acceptable due to the processing con-
purification, and thin-layer chromatographic methods [26]. ditions of the each commercial Spirulina product. Moreover,
It has been reported that 𝛼-linolenic acid and 𝛾-linolenic acid a literature stated that the extraction of total polysaccharides
are required for the survival of animals and humans, Patil and other monosaccharides from Spirulina followed by the
et al. (2007) [27]. Patil et al. (2007) profiled the individual quantification of the individual sugar molecules identified
PUFAs from Bacillariophyceae, Cyanophyceae, Rhodophyceae, rhamnose as the predominant sugar followed by glucose and
Xanthophyceae, Cryptophyceae, Prymnesiophyceae, Eustig- fructose [29].
matophyceae, and Chlorophyceae microalgae and suggested
that the cultivation conditions, particularly light intensity, 3.3. Quantifications of the Individual Amino Acids by HPLC.
and other nutritional components exert important effects HPLC analyses were used to quantify 22 free amino acids,
on the PUFA compositions [27]. Many companies process including aspartate, asparagine, serine, glutamine, histidine,
glycine, threonine, arginine, alanine, 𝛾-aminobutyric acid
microalgae and supply the results in the forms of capsules
(GABA), tyrosine, valine, cystine, methionine, tryptophan,
and tables to the market. Recently, the interest in the use of
phenylalanine, isoleucine, leucine, and lysine, in the Spirulina
Spirulina tablets as energy foods has been renewed due to the samples, but the separation profiles revealed that only 18 free
relatively high contents of protein, phytochemicals, and other amino acids were detectable in the samples, but the other
nutrients in such tablets. Efforts should be made to analyse free amino acids did not detected which may be not present
the metabolite profiles of the commercially available Spirulina in the samples (Table 5 and Figure 2). Since the samples
products because, in a previous study, we confirmed that were marketed by the trademark of different companies and
trace amounts of heavy metals that could cause serious health the nutrient profile of each sample would be varied, these
problems for consumers are present in some commercially results revealed that the amounts of total free amino acids
available Spirulina samples (Table 3) [13]. in the 37 Spirulina samples ranged from 11.49 mg/100 g to
56.14 mg/100 g. The essential amino acid content averages
3.2. Quantification of Individual Sugars by HPLC. Hexose ranged from 2.06 to 31.72 mg/100 g and contributed averages
(i.e., glucose, fructose, galactose, and rhamnose), pentose that ranged from 17.0 to 39.18% of the total amino acids.
(i.e., xylose and ribose), and disaccharide sugars were Among the essential amino acids, leucine was identified
Evidence-Based Complementary and Alternative Medicine 7

Table 3: Content of heavy metals in the Spirulina samples available in the market.

Amount mg/Kg dry weight

Sample name
Nickel Zinc Mercury Platinum Magnesium Manganese Total
1 0.211 0.533 0.002 0.001 0.002 0.076 0.825
2 4.672 5.627 0.028 0.01 0.03 0.587 10.954
3 2.016 2.397 0.017 0.008 0.018 0.603 5.059
4 2.147 1.628 0.02 0.011 0.026 0.436 4.268
5 2.199 1.235 0.017 0.008 0.014 1.17 4.643
6 3.726 6.225 0.022 0.009 0.028 0.007 10.017
7 2.601 2.817 0.018 0.011 0.028 0.007 5.482
8 3.577 1.871 0.023 0.012 0.031 0.309 5.823
9 3.519 3.267 0.021 0.011 0.03 0.008 6.856
10 2.442 2.041 0.017 0.008 0.021 0.137 4.666
11 3.785 3.007 0.019 0.008 0.028 0.008 6.855
12 3.597 2.859 0.019 0.008 0.024 0.006 6.513
13 2.857 2.901 0.019 0.008 0.026 1.643 7.454
14 2.852 2.114 0.026 0.008 0.042 0.011 5.053
15 2.437 1.568 0.02 0.008 0.028 0.008 4.069
16 2.712 2.434 0.019 0.009 0.033 1.777 6.984
17 2.948 2.513 0.021 0.009 0.028 1.8 7.319
18 2.633 1.876 0.016 0.009 0.023 1.328 5.885
19 3.731 3.184 0.017 0.009 0.036 2.248 9.225
20 2.225 1.548 0.008 0.009 0.019 1.132 4.941
21 1.618 1.478 0.014 0.006 0.02 0.005 3.141
22 1.589 4.626 0.016 0.007 0.023 0.009 6.27
23 3.272 4.428 0.02 0.008 0.018 0.008 7.754
24 3.558 3.733 0.017 0.009 0.034 1.433 8.784
25 2.319 2.586 0.017 0.008 0.024 0.006 4.96
Numbers 1 to 37 were the sample names (Table 1). Samples 26–37 were not analyzed [13].

14 22 contents. However, the brand name products exhibited values

2 9
10
11 that were comparatively lower than the maximum observed
8
Intensity (mAU)

1 3
4 56 13 values. Vitamin U, methionine, norvaline, and tryptophan
1.5 12 1516 19 21
20 were not observed in the samples. The essential amino
7 17 18
1 2 acid compositions of the microalgae were very similar to
0.5
the reported protein contents [30]. Clément et al. (1967)
determined the individual and total amino acid contents
0 in S. maxima [31]. This study reported that aspartate was
dominant in the Spirulina samples and that histidine, cystine,
0 2 4 6 8 10 12 14 16 18
tryptophan, and methionine were observed at the lowest
Retention time (min)
levels. This report found a level of aspartate that was similar
Figure 2: HPLC chromatogram of the standard free amino acids. to that of another report. In general, many companies market
The peaks numbers refer to the free amino acids listed in Table 5. Spirulina samples as nutraceutical food; however, there is an
Peaks numbers 1, aspartate; 2, glutamate; 3, asparagine; 4, serine; 5, S- urgent need to know the nutritional compositions of each of
methylmethionine (vitamin U); 6, glutamine; 7, histidine; 8, glycine; these Spirulina products [32]. This study confirmed that the
9, threonine; 10, arginine; 11, alanine; 12, gamma-aminobutyric amino acid compositions of Spirulina samples varied with the
acid (GABA); 13, tyrosine; 14, cystine; 15, valine; 16, methionine; companies that produced them. Therefore, amino acid-rich
17, norvaline; 18, tryptophan; 19, phenylalanine; 20, isoleucine; 21, samples should be consumed by humans to maintain their
leucine; 22, lysine. health.

3.4. Determination of the Total Phenolic Compounds. Figure 3

as predominant (0.53 to 7.59 mg/100 g) and accounted for shows the total phenolic compounds calculated as equivalents
more than 30% of the essential amino acids. The Spirulina to gallic acid. The results revealed that the distributions of the
tablets marketed in India under the brand name “Dharain total phenolic compounds varied between the commercial
Pharmaceuticals” exhibited the greatest essential amino acid products. The products ranged from 2.4 mg/g (21st Century
8 Evidence-Based Complementary and Alternative Medicine

Table 4: Individual sugar contents (mg/100 g) in different Spirulina.

Sugar contents (mg/100 g)

Sample
Glucose Fructose Xylose Galactose Ribose Sucrose Rhamnose Total
1 172 ± 14 38 ± 3.45 25 ± 1.50 15.5 ± 0.56 18 ± 1.25 54 ± 5.6 25 ± 0.0 342.5 ± 4.33
2 71 ± 13.89 44 ± 1.5 88 ± 4.22 78 ± 3.12 5 ± 0.00 65 ± 0.24 18 ± 0.23 363 ± 5.19
3 229.33 ± 27.59 59 ± 5.05 55 ± 1.60 7 ± 0.71 45 ± 3.00 54 ± 3.45 35 ± 0.89 466.11 ± 15.77
4 51.33 ± 4.16 68 ± 5.01 21 ± 1.11 54 ± 2.01 7 ± 0.03 29 ± 2.03 19 ± 1.1 249.77 ± 0.38
5 347.33 ± 6.43 15 ± 1.24 46 ± 3.4 8 ± 0.23 25 ± 0.25 84 ± 00 58 ± 3.2 581.11 ± 1.92
6 26 ± 3.46 26 ± 2.8 32 ± 1 9 ± 0.17 16 ± 0.00 72 ± 1.30 45 ± 1 222 ± 3.46
7 148.33 ± 10.41 34 ± 1.78 37 ± 4.03 45 ± 3.12 18 ± 1.87 80 ± 9.15 25 ± 0.28 366.44 ± 18.08
8 156.33 ± 16.29 15 ± 12.1 39 ± 5.45 11 ± 1.02 31 ± 1.64 45 ± 7.45 14 ± 0.02 333.11 ± 18.86
9 253 ± 5.20 29 ± 5.98 65 ± 8.4 24 ± 2.0 22 ± 0.03 62 ± 6.05 35 ± 0.96 463.33 ± 23.09
10 255.33 ± 10.02 78 ± 7.4 98 ± 1.2 NA NA 34 ± 5.10 NA 528.44 ± 14.62
11 128.33 ± 2.89 51 ± 0.89 45 ± 7.1 23 ± 1.85 24 ± 0.07 86 ± 6.88 18 ± 1.12 377.11 ± 1.54
12 361.66 ± 6.6 49 ± 1.04 25 ± 0.90 14 ± 2.2 16 ± 0.08 95 ± .00 8 ± 0.32 589.55 ± 18.0
13 482.33 ± 5.8 35 ± 2.08 45 ± 5.03 18 ± 1.03 54 ± 1.09 NA 11 ± 0.78 730.77 ± 1.34
14 592.33 ± 6.15 26 ± 1.0 85 ± 5.23 44 ± 2.9 22 ± 1.07 29 ± 2.15 16 ± 0.47 811.44 ± 2.49
15 248.66 ± 8 59 ± 6.8 48 ± 4.9 11 ± 2.2 15 ± 0.3 67 ± 1.68 24 ± 4.9 470.89 ± 1.54
16 210.33 ± 9.5 72 ± 5.6 95 ± 0.12 10 ± 0.62 24 ± 1.3 49 ± 9.0 17 ± 0.63 472.44 ± 4.23
17 517 ± 24 93 ± 4.05 25 ± .52 12 ± 0.62 26 ± 4.2 52 ± 0.51 28 ± 1.03 754.33 ± 1.15
18 884.66 ± 26.5 15 ± 0.25 22 ± 0.7 18 ± 2.15 28 ± 4.12 58 ± 1.52 38 ± 1.08 1059.89 ± 3.27
19 338.33 ± 12.5 44 ± 1.14 14 ± 0.9 27 ± 3.12 11 ± 0.00 49 ± 3.02 26 ± 0.0 509.77 ± 0.38
20 752 ± 5.26 51 ± 4.6 16 ± 0.12 9 ± 0.25 22 ± 0.8 67 ± 3.07 45 ± .28 987.33 ± 21.93
21 968.66 ± 27.3 50 ± 2.54 18 ± 0.59 58 ± 8.12 24 ± 0.14 84 ± 10.1 19 ± 5.1 1207.22 ± 12.52
22 260 ± 22.9 43 ± 6.3 94 ± 1.26 65 ± 4.74 26 ± 2.95 55 ± 0.78 54 ± 2.90 599 ± 1.73
23 107.33 ± 7.09 24 ± 4.0 25 ± 0.45 22 ± 0.12 25 ± 0.05 78 ± 1.84 28 ± 1.05 303.11 ± 5.38
24 151.66 ± 7.6 52 ± 2.65 63 ± 0.40 45 ± 2.94 11 ± 0.32 37 ± 0.00 47 ± 0.01 402.22 ± 3.85
25 102.66 ± 16 68 ± 4.3 24 ± 1.45 48 ± 2 7 ± 0.05 81 ± 0.28 35 ± 0.25 361.89 ± 3.27
26 79.33 ± 11.15 94 ± 5.0 25 ± 2.09 56 ± 6.23 45 ± 1.10 55 ± 6.12 45 ± 1.7 399.77 ± 0.38
27 604.66 ± 5.3 12 ± 1.37 26 ± 4.23 44 ± 2.56 24 ± 0.00 84 ± 0.71 37 ± 0.32 830.55 ± 0.96
28 901.66 ± 7.6 15 ± 0.7 45 ± .23 25 ± 0.45 10 ± 0.07 56 ± 1.83 34 ± 0.00 1095.55 ± 7.6
29 798 ± 13.11 45 ± 5.24 58 ± 1.12 14 ± 1.57 32 ± 1.18 101 ± 11.02 36 ± 1.88 1101.33 ± 15.0
30 144.16 ± 6.23 48 ± 5.68 48 ± 5.25 60 ± 2.12 22 ± 0.05 28 ± 5.20 28 ± 0.25 392.72 ± 12.62
31 155 ± 8.3 25 ± 2.62 25 ± 2.36 18 ± 4.5 21 ± 0.07 79 ± 6.23 46 ± 0.69 389.66 ± 17.89
32 472.33 ± 13.5 18 ± 2.0 65 ± 4.25 9 ± 0.12 45 ± 1.08 56 ± 1.02 55 ± 5.68 734.77 ± 12.51
33 534 ± 31 44 ± 1.54 77 ± 3.23 28 ± 2.04 24 ± 1.02 48 ± 0.13 28 ± 0.47 794.33 ± 9.81
34 158 ± 3.46 58 ± 4.03 49 ± 1.40 34 ± 3.50 58 ± 0.04 21 ± 2.32 47 ± 5.12 445.66 ± 17.89
35 353 ± 25.6 27 ± 2.45 58 ± 1.0 25 ± 3.26 21 ± 1.79 55 ± 00 55 ± 0.11 598 ± 3.46
36 798 ± 13.11 49 ± 3.07 55 ± 0.9 45 ± 0.18 40 ± 6.4 48 ± 0.36 41 ± 0.02 1092 ± 13.85
37 171.33 ± 15 78 ± 5.0 24 ± 0.45 11 ± 0.01 14 ± 0.34 75 ± 0.45 25 ± 2.05 399.44 ± 1.0
Numbers 1 to 37 were the sample names (Table 1).

HealthCare, Inc., Arizona) to 24.4 mg/g (source Naturals, [34]. The polyphenols contained the ideal chemical structures
Inc., Santa Cruz, California). The highest level of total and different bioactivities that included anti-inflammatory,
polyphenol contents (24 mg/g) was observed in the tablets antiviral, antioxidant, antithrombotic, vasodilatory, and anti-
procured from source Naturals, Inc., Santa Cruz, Califor- carcinogenic properties [35]. Wu et al. (2005) demonstrated
nia. Miranda et al. (1998) claimed that the main phenolic that the presence of total phenolic components and other
compounds, namely, chlorogenic acid, synaptic acid, salicylic metabolites are related to antioxidant properties [36].
acid, trans-cinnamic acid, and caffeic acid were commonly
present in Spirulina [33]. The present study also coincides 3.5. Antioxidant Properties. The results revealed that the
with the report of Miranda et al. (1998) [33]. However, the antioxidant properties of the Spirulina samples were dose-
metabolic pathways for the formation of phenolics com- dependent (Figures 4–6). The DPPH assay and hydroxyl
pounds in Spirulina and their importance are still unknown scavenging assay results revealed that all the Spirulina extracts
 Table 5: Free amino acid contents (mg/100 g fresh wt.) in different Spirulina.

(a)
Number Amino acids RT (min) Molecular weight 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 Aspartate 1.48 133.10 1.37 1.37 2.03 2.66 5.24 1.34 3.66 1.24 2.47 0.97 3.55 4.05 3.33 0.89 1.06 1.69 1.70
2 Glutamate 2.77 147.13 ND(a) ND(a) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
3 Asparagine 5.83 132.12 0.25 0.25 ND 0.92 0.39 ND 0.51 4.07 0.32 0.42 0.27 0.17 0.30 0.23 0.28 0.43 0.46
4 Serine 6.03 105.09 1.69 1.69 0.49 1.17 1.14 1.29 1.59 2.02 1.58 1.37 0.92 1.40 0.57 1.61 1.86 2.12 1.18
5 Vitamin U 6.59 199.70 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
6 Glutamine 6.84 146.15 0.64 0.64 ND 1.89 3.25 0.55 1.46 0.66 ND 0.53 2.12 ND 1.09 ND 0.65 ND 0.98
7 Histidine 7.15 155.15 ND ND ND 0.72 ND 0.43 ND ND ND ND ND ND ND ND ND ND ND
8 Glycine 7.36 75.07 2.53 2.53 0.51 3.51 1.28 1.45 1.77 2.30 1.98 3.05 ND 1.59 1.02 2.04 2.36 2.71 2.28
9 Threonine 7.62 119.12 1.54 1.54 0.45 2.12 0.98 0.78 1.71 1.28 1.24 1.45 1.92 1.48 0.84 1.07 1.08 1.63 2.08
10 Arginine 8.47 174.20 2.81 2.81 1.87 1.87 2.82 4.00 3.04 2.20 5.12 4.70 1.55 2.81 1.59 2.31 2.48 5.12 3.02
11 Alanine 8.97 89.09 ND ND ND 11.68 ND ND ND ND ND ND ND 8.12 ND ND 9.01 16.12 12.94
12 GABA 9.26 103.12 0.38 0.38 0.21 1.54 ND 0.32 0.35 1.26 1.00 0.84 ND 0.72 0.46 0.32 0.34 1.05 1.32
13 Tyrosine 10.43 181.19 2.08 2.08 0.46 3.04 0.69 1.70 1.81 1.43 ND 2.54 ND 1.66 ND 1.24 1.50 2.19 1.28
14 Cystine 12.26 240.30 ND ND ND 1.45 ND ND ND ND 1.30 ND 0.45 ND 0.86 ND ND ND 0.52
15 Valine 12.56 117.15 2.13 2.13 0.72 5.34 0.76 1.92 2.51 ND ND 1.07 ND 2.68 ND 1.42 1.20 4.02 3.19
Evidence-Based Complementary and Alternative Medicine

16 Methionine 12.82 149.21 ND ND ND 0.80 ND ND ND ND ND ND 0.93 ND ND ND 0.43 ND ND

17 Norvaline 13.23 117.15 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
18 Tryptophan 13.88 204.33 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
19 Phenylalanine 14.37 165.19 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
20 Isoleucine 14.60 131.17 1.98 1.98 0.44 4.31 0.69 1.69 2.23 1.13 2.01 0.62 0.39 2.07 0.61 0.89 0.77 2.79 2.00
21 Leucine 15.43 131.17 2.77 2.77 0.54 5.96 1.14 2.68 3.33 1.21 2.63 0.88 0.53 2.87 0.74 1.15 1.40 3.83 3.29
22 Lysine 16.05 146.19 1.68 1.68 0.38 4.04 0.79 0.00 2.44 1.63 1.90 2.21 0.71 1.70 0.72 1.38 1.45 2.95 1.63
Total 21.86 21.86 8.09 53.04 19.18 18.16 26.41 20.44 21.56 20.65 13.34 31.32 12.13 14.55 25.87 46.66 37.88
(b)
Number Amino acids RT (min) Molecular weight 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
1 Aspartate 1.48 133.10 2.76 3.21 1.44 2.88 1.45 0.71 2.75 0.46 1.10 1.87 1.98 1.56 0.56 1.21 1.16 1.62 1.28 0.49 1.94 1.02
2 Glutamate 2.77 147.13 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
3 Asparagine 5.83 132.12 0.89 0.96 0.42 0.97 ND ND 0.22 0.39 0.21 ND ND 0.32 ND 0.35 ND 0.24 1.26 ND 0.52 0.37
4 Serine 6.03 105.09 1.12 1.91 0.92 1.04 2.17 1.11 0.56 0.69 1.14 1.12 1.96 1.01 0.62 1.02 1.01 1.11 3.17 0.79 1.74 1.15
5 Vitamin U 6.59 199.70 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
6 Glutamine 6.84 146.15 1.31 1.52 0.98 1.87 ND ND 1.07 ND ND ND ND 0.53 ND ND 0.57 ND ND ND ND ND
7 Histidine 7.15 155.15 0.48 0.56 ND 0.91 ND ND ND ND ND ND ND ND ND 0.30 ND ND 0.53 ND 0.39 0.31
8 Glycine 7.36 75.07 2.65 3.13 1.96 3.95 1.75 1.69 1.08 2.04 2.27 2.19 3.37 1.41 1.46 2.24 2.31 1.51 6.33 1.47 5.37 2.03
9 Threonine 7.62 119.12 2.83 3.63 1.84 2.34 1.30 0.72 0.73 0.58 0.94 1.57 1.61 1.09 0.69 2.10 1.71 1.04 1.74 0.70 1.34 1.19
10 Arginine 8.47 174.20 2.52 3.12 1.98 2.35 5.37 2.47 1.74 1.64 2.19 2.66 3.58 2.66 1.34 2.80 3.01 2.41 8.46 1.88 2.63 2.91
11 Alanine 8.97 89.09 9.27 13.57 8.60 12.80 11.68 9.04 ND 8.03 9.26 9.84 12.89 4.36 7.60 15.30 11.57 6.51 23.85 6.03 16.34 6.24
12 GABA 9.26 103.12 1.26 1.83 0.99 1.35 0.48 1.79 0.20 1.74 1.13 1.63 4.36 0.44 6.72 1.16 0.57 0.44 0.77 0.23 2.35 0.97
13 Tyrosine 10.43 181.19 2.33 1.36 1.21 3.30 2.22 1.44 0.64 1.51 1.84 1.93 3.26 1.36 1.09 1.17 1.71 1.63 4.45 1.13 2.20 1.82
9
 10

(b) Continued.
Number Amino acids RT (min) Molecular weight 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
14 Cystine 12.26 240.30 1.30 0.80 0.65 1.64 0.38 ND ND ND ND ND ND ND ND 0.76 ND ND 0.57 ND 0.78 ND
15 Valine 12.56 117.15 4.34 5.38 2.43 6.05 2.97 1.49 0.70 1.29 1.69 2.65 3.05 1.19 1.62 3.60 3.42 1.67 16.12 ND 4.41 1.44
16 Methionine 12.82 149.21 0.69 0.35 ND 0.98 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
17 Norvaline 13.23 117.15 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
18 Tryptophan 13.88 204.33 ND ND ND ND ND ND ND ND ND ND ND ND ND 0.43 ND ND 0.64 ND 0.69 ND
19 Phenylalanine 14.37 165.19 2.76 2.53 1.13 3.36 1.42 0.83 ND 0.42 0.46 0.38 0.98 0.48 0.62 1.65 0.87 0.59 2.93 0.62 1.73 0.74
20 Isoleucine 14.60 131.17 2.95 3.43 1.29 3.98 2.07 0.95 0.44 0.92 1.05 1.50 1.70 0.85 1.06 1.92 2.67 1.11 ND 0.68 ND ND
21 Leucine 15.43 131.17 4.74 5.82 2.41 6.59 3.99 1.81 0.71 1.03 1.21 1.47 1.95 1.24 1.61 3.32 3.35 1.62 7.59 0.96 3.14 1.23
22 Lysine 16.05 146.19 2.88 3.01 1.08 4.47 2.48 1.63 0.65 1.62 1.61 1.21 2.70 1.16 0.78 1.68 1.40 1.36 6.27 1.08 2.74 1.59
Total 47.05 56.14 29.35 60.83 39.74 25.68 11.49 22.34 26.10 30.01 43.38 19.65 25.77 41.00 35.33 22.85 85.97 16.08 48.31 23.01
Numbers 1 to 37 were the sample names (Table 1). (a) ND: not detected.
Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 11
equivalent (mg/g)
samples was not directly proportional with respect to the total
30 phenolic compounds results. The regular consumption of
Gallic acid

20
10 antioxidant-containing food additives helps to slow oxidative
0 stress and minimize the spread of oxidative stress-related dis-
eases [37]. The antioxidant compounds, such as phycobilins
1
2
3
4
5
6
7
8
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Spirulina samples and phycocyanins, that are present in Spirulina exert their
actions by scavenging free radicals by acting as hydrogen, per-
Figure 3: Determination of the total phenolic compounds of the 37 oxyl radical, and peroxynitrite acceptors. These antioxidant
varieties (Table 1) of Spirulina (𝑛 = 3).
compounds also inhibit the activities of catalytic enzymes,
such as lipoxygenase and cyclooxygenase, or enhance the
activity of enzymes, such as glutathione peroxidase, catalase,
and superoxide dismutase [38]. Wu et al. (2005) reported that
activity (%)
Antioxidant

100 Spirulina extracts exhibited greater antioxidant properties

80
60 due to the presence of various phenolic compounds [36]. A
40
20 number of cyanobacteria especially the species of Chlorella
0
are believed to be useful as excellent food sources with
1
2
3
4
5
6
7
8
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Spirulina samples antioxidant activities by modern researchers [39]. Due to its
rich vitamin, protein, phenolic compound, polyunsaturated
Figure 4: Antioxidant activities of the 37 varieties (Table 1) of fatty acid, and other microelement contents, Spirulina could
Spirulina by DPPH radical scavenging activity assay (𝑛 = 3). be used as a better nutrient food by consumers.

4. Conclusions
100
activity (%)
Antioxidant

80
60 Thirteen unsaturated fatty acids, 19 free amino acids, 7 sugars,
40
20 and the total polyphenolic components were separated and
0 identified from 37 Spirulina samples using GC and HPLC
1
2
3
4
5
6
7
8
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

Spirulina samples
methods. The contents of each metabolite were quantified,
and remarkable variations in the individual metabolites were
Figure 5: Antioxidant activities of the 37 varieties (Table 1) of observed between the different varieties. Specifically the
Spirulina by reducing power activity assay (𝑛 = 3). Spirulina tablets distributed by 21st Century HealthCare, Inc.,
were relatively suitable due to their abundance of fatty acids,
sugars, amino acids, and polyphenols. The in vitro antioxidant
activity results confirmed that the activities were dose-
125
activity (%)
Antioxidant

100 dependent. The Spirulina products that are available on the

75 market are rich in antioxidant polyphenolic components and
50
25 are suitable choices for regular consumption. The presence
0
of individual phenolic compounds in the different products
1
2
3
4
5
6
7
8
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

Spirulina samples should be studied because these metabolites are used for
the treatment of stress-related diseases and cardiovascular
Figure 6: Antioxidant activities of the 37 varieties (Table 1) of disorders.
Spirulina by hydroxyl radical-scavenging activity assay (𝑛 = 3).

Conflict of Interests
showed the activity in a concentration-dependent manner. The authors declare no conflict of interests.
Numbers of antioxidant metabolites are present in plants and
Spirulina. The scavenging abilities and reductive properties
of the Spirulina samples exhibited dose-dependent activities. Authors’ Contribution
Metabolites especially those having the phenolic functional
Naif Abdullah Al-Dhabi and Mariadhas Valan Arasu con-
group in their chemical structure have been reported to
ceived the study and designed the experiments and also are
show many useful properties, including anti-inflammatory
involved in writing the paper. All authors read and approved
activity, oestrogenic activity, enzyme inhibition, antiallergic
the final version of the paper. Naif Abdullah Al-Dhabi and
activity, antioxidant activity, vascular activity, and cytotoxic
Mariadhas Valan Arasu contributed equally to this work.
antitumour activity. The results from three antioxidant assays
of the 37 samples were not correlated with the total phe-
nolic compounds or the other determined compounds. The Acknowledgment
results indicated that the antioxidant potentials were not
significantly correlated with their total phenolic compounds The Project was full financially supported by King Saud
(data were not shown), because the antioxidant activity of the University, through Vice Deanship of Research Chairs.
12 Evidence-Based Complementary and Alternative Medicine

References their stimulatory effects on mouse splenocyte proliferation,”

Food Chemistry, vol. 101, no. 1, pp. 140–147, 2006.
[1] C. Jiménez, B. R. Cossı́o, and F. X. Niell, “Relationship between [17] M. Oyaizu, “Studies on product of browning reaction prepared
physicochemical variables and productivity in open ponds for from glucoseamine,” Japanese Journal of Nutrition, vol. 44, pp.
the production of Spirulina: a predictive model of algal yield,” 307–315, 1986.
Aquaculture, vol. 221, no. 1–4, pp. 331–345, 2003. [18] T. Hatano, H. Kagawa, T. Yasuhara, and T. Okuda, “Two
[2] L. Brennan and P. Owende, “Biofuels from microalgae—a new flavonoids and other constituents in licorice root: their
review of technologies for production, processing, and extrac- relative astringency and radical scavenging effects,” Chemical
tions of biofuels and co-products,” Renewable and Sustainable and Pharmaceutical Bulletin, vol. 36, no. 6, pp. 2090–2097, 1988.
Energy Reviews, vol. 14, no. 2, pp. 557–577, 2010. [19] K. Elizabeth and M. W. A. Rao, “Oxygen radical scavenging
[3] J. J. Ortega-Calvo, C. Mazuelos, B. Hermosin, and C. Saiz- activity of curcumin,” International Journal of Pharmaceutics,
Jimenez, “Chemical composition of Spirulina and eukaryotic vol. 58, pp. 237–240, 1990.
algae food products marketed in Spain,” Journal of Applied [20] M. Mühling, A. Belay, and B. A. Whitton, “Screening
Phycology, vol. 5, no. 4, pp. 425–435, 1993. Arthrospira (Spirulina) strains for heterotrophy,” Journal of
[4] K. Chopra and B. Mahendra, “Antioxidant profile of Spirulina: Applied Phycology, vol. 17, no. 2, pp. 129–135, 2005.
a blue-green microalga,” in Spirulina in Human Nutrition and [21] D. R. Reddy, V. S. S. V. Prassas, and U. N. Das, “Intratumoural
Health, M. E. Gershwin and A. Belay, Eds., pp. 101–119, CRC injection of gamma leinolenic acid in malignant gliomas,”
Press, London, UK, 2008. Journal of Clinical Neuroscience, vol. 5, no. 1, pp. 36–39, 1998.
[5] Ö. Tokuşoglu and M. K. Ünal, “Biomass nutrient profiles of [22] V. A. Ziboh, “Biochemical basis for the anti-inflammatory
three microalgae: Spirulina platensis, Chlorella vulgaris, and action of gamma-linolenic acid,” in Omega-6 Essential Fatty
Isochrisis galbana,” Journal of Food Science, vol. 68, no. 4, pp. Acids: Pathophysiology and Roles in Clinical Medicine, D. F.
1144–1148, 2003. Horrobin, Ed., pp. 187–201, Alan Liss, New York, NY, USA, 1989.
[6] A. S. Babadzhanov, N. Abdusamatova, F. M. Yusupova, N. [23] A. Bordoni, P. L. Biagi, M. Masi et al., “Evening primrose oil
Faizullaeva, L. G. Mezhlumyan, and M. K. Malikova, “Chemical (Efamol) in the treatment of children with atopic eczema,”
composition of Spirulina platensis cultivated in Uzbekistan,” Drugs under Experimental and Clinical Research, vol. 14, no. 4,
Chemistry of Natural Compounds, vol. 40, no. 3, pp. 276–279, pp. 291–297, 1988.
2004. [24] D. F. Horrobin, “The role of essential fatty acids and
[7] A. Ramamoorthy and S. Premakumari, “Effect of supplementa- prostaglandins in the premenstrual syndrome,” The Journal of
tion of Spirulina on hypercholesterolemic patients,” Journal of Reproductive Medicine, vol. 28, pp. 465–468, 1983.
Food Science and Technology, vol. 33, no. 2, pp. 124–128, 1996. [25] T. Ishikawa, Y. Fujiyama, O. Igarashi et al., “Effects of gam-
[8] C. A. Rice-Evans, N. J. Miller, and G. Paganga, “Antioxidant malinolenic acid on plasma lipoproteins and apolipoproteins,”
properties of phenolic compounds,” Trends in Plant Science, vol. Atherosclerosis, vol. 75, no. 2-3, pp. 95–104, 1989.
2, no. 4, pp. 152–159, 1997. [26] M. G. Sajilata, R. S. Singhal, and M. Y. Kamat, “Fractionation of
[9] J. Ferreira, “Effect of butylated hydroxyanisole on electron lipids and purification of 𝛾-linolenic acid (GLA) from Spirulina
transport in rat liver mitochondria,” Biochemical Pharmacology, platensis,” Food Chemistry, vol. 109, no. 3, pp. 580–586, 2008.
vol. 40, no. 4, pp. 677–684, 1990. [27] V. Patil, T. Källqvist, E. Olsen, G. Vogt, and H. R. Gislerød,
[10] M. Khan, J. C. Shobha, I. K. Mohan et al., “Protective effect “Fatty acid composition of 12 microalgae for possible use in
of Spirulina against doxorubicin-induced cardiotoxicity,” Phy- aquaculture feed,” Aquaculture International, vol. 15, no. 1, pp.
totherapy Research, vol. 19, no. 12, pp. 1030–1037, 2005. 1–9, 2007.
[11] D. S. Lee, D. S. Jeon, S. G. Park et al., “Effect of cold storage on [28] R. Chaiklahan, N. Chirasuwan, P. Triratana, V. Loha, S. Tia,
the contents of glucosinolates in Chinese cabbage (Brassica rapa and B. Bunnag, “Polysaccharide extraction from Spirulina sp.
L. ssp. pekinensis),” South Indian Journal of Biological Sciences, and its antioxidant capacity,” International Journal of Biological
vol. 1, no. 1, pp. 38–42, 2015. Macromolecules, vol. 58, pp. 73–78, 2013.
[12] M. Mossoba, J. Kramer, P. Delmonte, M. Yurawecz, and J. Rader, [29] J.-B. Lee, T. Hayashi, K. Hayashi et al., “Further purification and
AOAC Official Method 996.06, Fat (Total, Saturated, and Unsat- structural analysis of calcium spirulan from Spirulina platensis,”
urated in Foods), Hydrolytic Extraction Gas Chromatographic Journal of Natural Products, vol. 61, no. 9, pp. 1101–1104, 1998.
Method, First Action 1996, Revised 2001, AOCS Press, Urbana, [30] M. R. Brown, C. D. Garland, S. W. Jeffrey, I. D. Jameson, and
Ill, USA, 2003. J. M. Leroi, “The gross and amino acid compositions of batch
[13] N. A. Al-Dhabi, “Heavy metal analysis in commercial Spirulina and semi-continuous cultures of Isochrysis sp. (clone TISO),
products for human consumption,” Saudi Journal of Biological Pavlova lutheri and Nannochloropsis oculata,” Journal of Applied
Sciences, vol. 20, no. 4, pp. 383–388, 2013. Phycology, vol. 5, pp. 285–296, 1993.
[14] M. Valan Arasu, M. W. Jung, D. H. Kim et al., “Identification and [31] G. Clément, C. Giddey, and R. Menzi, “Amino acid composition
phylogenetic characterization of novel Lactobacillus plantarum and nutritive value of the alga Spirulina maxima,” Journal of the
species and their metabolite profiles in grass silage,” Annals of Science of Food and Agriculture, vol. 18, no. 11, pp. 497–501, 1967.
Microbiology, vol. 65, no. 1, pp. 15–25, 2015. [32] A. Richmond and E. W. Becker, “Technological aspects of mass
[15] S. Park, M. V. Arasu, M.-K. Lee et al., “Analysis and metabolite cultivation—a general outline,” in Handbook of Microalgal Mass
profiling of glucosinolates, anthocyanins and free amino acids Culture, A. Richmond, Ed., pp. 245–263, CRC Press, Boca
in inbred lines of green and red cabbage (Brassica oleracea L.),” Raton, Fla, USA, 1984.
LWT—Food Science and Technology, vol. 58, no. 1, pp. 203–213, [33] M. S. Miranda, R. G. Cintra, S. B. M. Barros, and J. Mancini-
2014. Filho, “Antioxidant activity of the microalga Spirulina maxima,”
[16] J.-Y. Lin and C.-Y. Tang, “Determination of total phenolic and Brazilian Journal of Medical and Biological Research, vol. 31, no.
flavonoid contents in selected fruits and vegetables, as well as 8, pp. 1075–1079, 1998.
Evidence-Based Complementary and Alternative Medicine 13

[34] L. M. Colla, C. Oliveira Reinehr, C. Reichert, and J. A. V.

Costa, “Production of biomass and nutraceutical compounds
by Spirulina platensis under different temperature and nitrogen
regimes,” Bioresource Technology, vol. 98, no. 7, pp. 1489–1493,
2007.
[35] C. Manach, A. Mazur, and A. Scalbert, “Polyphenols and
prevention of cardiovascular diseases,” Current Opinion in
Lipidology, vol. 16, no. 1, pp. 77–84, 2005.
[36] L.-C. Wu, J.-A. A. Ho, M.-C. Shieh, and I.-W. Lu, “Antioxidant
and antiproliferative activities of Spirulina and chlorella water
extracts,” Journal of Agricultural and Food Chemistry, vol. 53, no.
10, pp. 4207–4212, 2005.
[37] S. Shukla, A. Mehta, V. K. Bajpai, and S. Shukla, “In vitro
antioxidant activity and total phenolic content of ethanolic
leaf extract of Stevia rebaudiana Bert,” Food and Chemical
Toxicology, vol. 47, no. 9, pp. 2338–2343, 2009.
[38] V. B. Bhat and K. M. Madyastha, “Scavenging of peroxynitrite
by phycocyanin and phycocyanobilin from Spirulina platensis:
protection against oxidative damage to DNA,” Biochemical and
Biophysical Research Communications, vol. 285, no. 2, pp. 262–
266, 2001.
[39] E. Christaki, P. Florou-Paneri, and E. Bonos, “Microalgae: a
novel ingredient in nutrition,” International Journal of Food
Sciences and Nutrition, vol. 62, no. 8, pp. 794–799, 2011.
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