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Int Aquat Res (2022) 14:51-62
https://doi.org/10.22034/IAR.2022.1946410.1217
Phytochemical composition, antioxidant, and antibacterial
activity of the Philippine marine green alga (Ulva pertusa)
Rolando Pakingking Jr . Roselyn Usero . Evelyn Grace de Jesus-Ayson . Dan Joseph Logronio .
Christopher Marlowe Caipang
Received: 03 December 2021 / Accepted: 03 March 2022 / Published online: 09 March 2022
© The Author(s) 2022
Rolando Pakingking Jr ( )
Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD), Tigbauan 5021, Iloilo, Philippines
e-mail: rpakingking@seafdec.org.phe-mail: rpakingking@seafdec.org.ph
Roselyn Usero
Negros Prawn Producers Marketing Cooperative, Inc., Bacolod City 6100, Negros Occidental, Philippines
Evelyn Grace de Jesus-Ayson
Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD), Tigbauan 5021, Iloilo, Philippines and College of Arts
and Sciences, Central Philippine University, Jaro, Iloilo City 5000, Iloilo, Philippines
Dan Joseph Logronio
Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD), Tigbauan 5021, Iloilo, Philippines
Christopher Marlowe Caipang
College of Liberal Arts, Sciences, and Education and the Center for Chemical Biology and Biotechnology, University of San Agustin, General Luna
St., Iloilo City, 5000, Philippines
ORIGINAL RESEARCH
Abstract The phytochemical constituents, total phenolic and avonoid contents, antioxidant and
antimicrobial activities of the ethanolic extract of the marine green alga Ulva pertusa collected from
oating sh net cages o the coast of Guimaras island, Philippines, were investigated. Qualitative
phytochemical analysis revealed the presence of bioactive compounds including alkaloids, avonoids,
phenols, saponins, tannins, and terpenoids. Quantitatively, total phenolic and avonoid contents of the
extract were 20.54±2.08 mg gallic acid equivalent (GAE) g-1 and 539.07±6.36 mg rutin equivalent (RE)
g-1, respectively. The antioxidant activity of the extract using 1,1-diphenyl-1-picrylhydrazyl (DPPH)
assay exhibited a concentration-dependent radical scavenging capacity. Additionally, the ethanolic extract
inhibited the growth of Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Streptococcus
agalactiae, Aeromonas hydrophila, A. sobria, and Vibrio harveyi in a concentration-dependent manner
with strong inhibitory activity at 100 mg mL-1 concentration. Taken together, U. pertusa contains bioactive
compounds that possess potent antibacterial activity and remarkable antioxidant capacities. These substances
are promising candidates that may be utilized in the synthesis of novel drugs.
Keywords Ulva pertusa . Antimicrobial activity . Antioxidant . Streptococcus agalactiae . Aeromonas
species
Introduction
Being one of the primary producers in the ocean, the role that seaweeds play has been regarded crucial
in the marine ecosystem. Seaweeds are potential renewable living resources that are not only utilized as
human food but as well as animal feed and plant fertilizer in many parts of the world (Ferdouse et al.
2018). About 6000 species of seaweeds have by far been identied and accordingly grouped into 3 dierent
classes, i.e. green (Chlorophytes), brown (Phaeophytes), and red (Rhodophytes) algae (Chandini et al.
Int Aquat Res (2022) 14:51-62
52
2008). The global seaweed production had doubled from 14.6 million tonnes in 2005 to 30.4 million tonnes
in 2015, with 29.4 million tonnes and 1.1 million tonnes generated from the aquaculture and capture sectors,
respectively (Ferdouse et al. 2018). There has been a growing interest with regard to mass propagation of
seaweeds because majority of the maricultured seaweeds have been found to possess medicinal activities
(Pérez et al. 2016). Several bioactive substances isolated from green, brown, and red marine seaweeds
including among others, polysaccharides, proteins, fatty acids, phenolics, and terpenes are important as
they have been documented to have signicant antibacterial, antiviral, anticancer, anti-inammatory, and
anti-oxidative properties (Devi et al. 2008; Satpati and Pal 2011; Pérez et al. 2016; Thanigaivel et al. 2016;
Li et al. 2018b).
Microorganisms have developed novel strategies to evade the action of antibiotics, leading to the
emergence of multiple drug-resistant bacterial strains. As sh inevitably becomes susceptible to various
bacterial infectious diseases particularly when reared in high stocking densities, occurrence of unwarranted
bacterial disease outbreaks has been recognized as one of the major contributors responsible for elevated
mortality rates, reduced productivity eciency, and resultant high economic losses to the sh farmers. The
application of antibiotics to treat bacterial infections has by far been intensively practiced by sh farmers
as mitigating measure aimed at controlling if not eradicating the disease. However, over the past several
years, the indiscriminate use of antibiotics has unfortunately led to the emergence of drug-resistant bacteria.
For example, emergence of multiple drug-resistant bacterial strains implicated in diseases of various pond-
cultured sh species like tilapias and milksh has been reported in Streptococcus iniae, S. agalactiae,
Aeromonas hydrophila, A. sobria, A. salmonicida, Edwardsiella tarda, E. ictaluri, Vibrio harveyi, and V.
anguillarum among others (Martinez et al. 2009; Cabello et al. 2013; Lo et al. 2014). It should be noted
that these sh bacterial pathogens have been implicated in human diseases (food-borne or zoonotic) thereby
making the aquaculture products as potential risk to the consumers (Cabello et al. 2013). Decreased ecacy
and resistance of bacterial sh pathogens to commonly used antibiotics necessitates the development of
new alternatives.
In recent years, seaweed extracts have been widely utilized for the prevention of bacterial and viral
diseases in aquatic animals (Bansemir et al. 2006; Li et al. 2018a). In particular, seaweed extracts have
been found to exhibit marked antibacterial activity against sh pathogenic bacteria (Bansemir et al. 2006;
Cavallo et al. 2013; Thanigaivel et al. 2016). The green alga, Ulva pertussa, a light green seaweed typically
composed of foliaceus blade with papery thin undulating margin, is a fast-growing seaweed that is typically
opportunistic for space and nutrient uptake (Hurtado et al. 2006). Ulva is commonly found in the intertidal
zones and labeled as nuisance species as they could over proliferate producing blooms or ‘green tide’
(Largo et al. 2004). Nevertheless, Ulva is not grown commercially outside Asia because there is no market
for it (Bolton et al. 2009). However, it should be noted that U. pertusa is abundant in vitamins, trace
elements, dietary bers, and nutritiously low in calorie (Benjama and Payap 2011). Additionally, various
bioactive compounds extracted from U. pertusa have been reported to have potent antibacterial (Choi et al.
2014), antiviral (Sun et al. 2018), and antioxidant (Choi et al. 2011) properties. For example, the sulfated
polysaccharide extracted from U. pertusa has been shown to have powerful antioxidant activities, including
scavenging ability of the superoxide and hydroxyl radicals, chelating ability of iron ion, and reducing
power (Qi et al. 2005). While various studies delving on antioxidant, antibacterial, and antiviral properties
of the dierent classes of seaweeds and concomitantly their wide application in food, pharmaceutical,
and aquaculture industry have been comprehensively conducted in some seaweed-producing countries
around the world, many types of wild and cultured seaweeds, particularly the green seaweed U. pertusa,
in the Philippines have by far been unexplored. It is in this light that the current study was conducted to
evaluate the phytochemical composition, antioxidant, and antibacterial activity of the ethanolic extract of
the green alga (U. pertusa) collected from the oating net cages used to rear pompano (Trachinotus blochii)
in Igang Marine Station (IMS), Aquaculture Department of the Southeast Asian Fisheries Development
Center (SEAFDEC/AQD) located in Guimaras island, Philippines. Resolute results generated from this
study would plausibly strengthen the potentiality of U. pertusa extract as a novel and cost-eective
antioxidant and antibacterial agent against pathogenic bacteria, particularly those involved in zoonosis,
and undoubtedly, would be a substantial springboard that could spur further investigations on the isolation,
purication, and identication of some of its yet unidentied bioactive compounds.
Int Aquat Res (2022) 14:51-62 53
Materials and methods
Chemicals used
2,2-Diphenyl-1-picrylhydrazyly (DPPH), ascorbic acid, gallic acid, sodium carbonate (Na2CO3), aluminum
chloride (AlCl3), and rutin were purchased from Sigma Aldrich (Philippines). Folin-Ciocalteu’s phenol
reagent and hydrogen peroxide, methanol, and dimethylsulfoxide (DMSO) were purchased from Merck
(Philippines). All other solvents and chemicals used were of analytical grade.
Seaweed material
The green seaweed (U. pertusa) growing on the nets of grow-out oating cages used to rear pompano
(T. blochii) broodstocks in SEAFDEC/AQD’s Igang Marine Station (IMS), were collected in April and
May wherein the ambient daytime water temperature ranged from 29-32 ºC. The collected seaweed
samples used in the dierent experiments were authenticated by SEAFDEC/AQD’s seaweed expert. The
seaweed samples collected were thoroughly washed with sterile seawater followed by sterile distilled
water. The washed samples were then stored in sterile plastic bags and transported in an iced condition to
SEAFDEC/AQD’s Fish Health Laboratory in Tigbauan, Iloilo within 1 hour after sampling. The seaweed
samples were shade dried (38±2 ºC) in a drier for about 24 to 36 h, cut into small pieces and powdered
using a mixer grinder. The powdered samples were collected in sterile amber bottles and stored at –20ºC
until used.
Preparation of U. pertusa extract
The powdered seaweed was extracted with ethanol following a modied method adapted from (Li et al.
2018a). Briey, 150 g of the powdered dried seaweed was soaked in 450 ml of 80% ethanol and allowed
to stand at room temperature (28ºC) for 72 hours. The mixture was shaken at regular intervals during the
course of the experiment. The extract was then ltered with sterile Whatman lter paper No. 1 (320 mm, 11
µm). The extraction procedure was repeated once more for another 72 hours, after which the two ltrates
were combined and concentrated under reduced pressure at 45ºC using a rotary evaporator. The dried
extracts were stored at –20ºC until needed for various analysis.
Phytochemical screening
The ethanolic extract of U. pertusa was subjected to various tests to detect the presence of phytochemical
compounds following standard procedures as described by Harborne (1998) and Evans (2009). The
qualitative detection of alkaloids present in U. pertusa ethanolic extract was carried out using Mayer’s
and Wagner’s test. Additionally, the presence avonoids (Shinoda test), glycosides (Keller-Killiani test),
phenols and tannins (ferric chloride test), saponin (foam test), and terpenoids (Salkowski test), were
likewise qualitatively examined.
Total phenolic content
The total soluble phenolic compounds in the U. pertusa extract were estimated with Folin-Ciocalteu reagent
using gallic acid as standard following the method of Singleton et al. (1999) as described by Stankovic et
al. (2011). The ethanolic solution of the extract in the concentration of 1mg mL-1 was used in the analysis.
Briey, 0.5 mL of the ethanolic extract was mixed with 2.5 mL of Folin-Ciocalteu reagent (previously
diluted 10-fold with distilled water) and 2 mL of 7.5% NaHCO3. The reaction mixture was mixed thoroughly
and allowed to stand for 45 min at 45°C after which, the absorbance was measured at 765 nm versus blank
sample using a spectrophotometer (Shimadzu, Japan). Samples were prepared in triplicate for each analysis
and the mean value of the absorbance was accordingly obtained. The total phenolic content was determined
using a standard curve with gallic acid and the result was expressed as mg gallic acid equivalent per gram
(mg GAE g-1) of extract.
Int Aquat Res (2022) 14:51-62
54
Total avonoid content
The total avonoid content of U. pertusa extract was determined spectrophotometrically following a
standard method (Quettier-Deleu et al. 2000). Briey, 1 mL of 2% AlCl3 reagent was mixed with the same
volume of the U. pertusa ethanolic extract (1 mg mL-1). After allowing the mixture to be incubated for 1
hour at room temperature (28ºC), absorption readings at 415 nm were recorded against a blank (ethanol)
sample. The samples were prepared in triplicate for each analysis and the mean value of the absorbance was
generated. The total avonoid content was determined using a standard curve with rutin and the result was
expressed as mg rutin equivalent per gram (mg RE g-1) of extract.
DPPH radical scavenging activity
The ability of U. pertusa extract to scavenge 1,1-diphenyl-1-picrylhydrazyl (DPPH) free radicals was
performed following the method described by Cho et al. (2010) with slight modications. Briey, the stock
solution of extracts was prepared in ethanol to obtain concentrations of 3.0, 2.5, 2.0, 1.0, 1.50, 0.75, 0.50,
and 0.25 mg mL-1. One hundred microliter of each diluted solution was mixed with 100 µL of 0.1 mM
DPPH prepared with ethanol. The solution was then incubated in the dark at room temperature (28ºC) for
30 minutes, after which the absorbance was measured at 515 nm using a microplate reader. Ethanol was
used as blank while 100 µL ethanol mixed with 100 µL DPPH served as control. Additionally, ascorbic
acid (20 µg mL-1) (Vitamin C) standard was used as positive reference (Cho et al. 2010). The percentage of
scavenged DPPH was calculated using the following equation:
DPPH Scavenging activity (%) = ([ACon – ATest]/Acon) × 100
Where ACon is the absorbance of the control and ATest is the absorbance of the sample. The half-maximal
inhibitory concentration (IC50) was calculated by linear regression analysis and expressed as mean of three
determinations.
Antibacterial assay
Bacterial strains
Streptococcus agalactiae isolated from the kidney (isolate code: TKG510208) and spleen (TMD10206)
of tilapia with streptococcal infection (Pakingking unpublished study), Aeromonas hydrophila isolated
from the gills (TI201301) (Pakingking et al. 2020) and kidney (P1261308) (Albances 2015) of tilapia
(Oreochromis niloticus) with aeromonad septicemia, A. sobria (TI2013025) isolated from the gills of
O. niloticus (Pakingking et al. 2020), and Vibrio harveyi (TbE090) isolated from diseased pompano (T.
blochii) (Pakingking et al. 2018) were used in the antibacterial assay. Additionally, Staphylococcus aureus
ATCC25923 (gram-positive) and Escherichia coli ATCC25922 (gram-negative) were used as reference
strains. All microorganisms used in the assay were maintained in trypticase soy broth (TSB; Merck)
supplemented with 15% glycerol at –80ºC.
Antibacterial screening
The antibacterial activity of the seaweed extract was examined using the modied agar well diusion method
of Perez et al. (1990) as described by Mattana et al. (2010). Briey, the seaweed extract was dissolved in
3% DMSO to obtain an initial concentration of 1000 mg mL-1 and sterilized by ltration through 0.45 µm
membrane lter (Millipore). All tests were conducted in triplicate using dierent concentrations of the seaweed
extracts diluted in 3% DMSO. Amoxicillin (0.02 mg mL-1) was used as the standard antimicrobial agent.
Agar well diusion method
Agar cultures of the bacteria used in the antibacterial assay were prepared as described by Pakingking
Int Aquat Res (2022) 14:51-62 55
et al. (2015). Briey, the bacterial isolates were inoculated in TSB and incubated for 18-24 h at 35ºC. The
concentration of the cultures was standardized by matching the turbidity with 0.5 McFarland standard using
sterile normal saline solution (NSS) to obtain approximately 1 × 108 colony forming units per mL (CFU
mL-1). The prepared bacterial suspensions were then swabbed on the surface of Mueller-Hinton agar (MHA;
Merck) plates, i.e. 25 mL of solidied MHA per plate, and subsequently punched with 7 mm diameter wells
at appropriate distances apart using sterile cork borer. Each well was lled with 100 µL of the seaweed extract
with varying concentrations ranging from 3.125 to 100 mg mL-1 (Table 3). Concomitantly, wells lled with 100
µL of amoxicillin (0.02 mg mL-1) and 3% DMSO served as positive and negative controls, respectively. Plates
were incubated at 35ºC for 24 h. After incubation, the plates were retrieved and the zones of growth inhibition
surrounding the wells were recorded. Clear zones around the wells indicated the presence of antibacterial
activity. All experiments were conducted in triplicate and one-way ANOVA was employed to compare the
mean values of each treatment. Signicant dierences among the bacterial isolates tested per treatment or
concentration of U. pertusa extract were compared by Duncan test (P < 0.05).
Results
Yield of U. pertusa extract
The quantity of the crude U. pertusa ethanolic extract obtained in the current study was 20.5±0.7 g which is
equivalent to a yield of 13.7±0.3 %. This result comparatively concurs with the previous report of Li et al.
(2018a) wherein these authors obtained a yield of approximately 12.9% from a 30 g powdered U. pertusa
using 85% ethanol as extractant.
Phytochemical screening
As shown in Table 1, qualitative phytochemical analysis of the dierent aliquots of the crude U. pertusa
ethanolic extract revealed the presence of dierent bioactive compounds including alkaloids, avonoids,
phenols, saponins, tannins, and terpenoids. However, all the representative aliquots examined were negative
for the detection of glycosides.
Total phenolic content
As shown in Table 2, the total phenolic compounds quantied in the three aliquots of U. pertusa ethanolic
extract ranged from 19.33±0.29 to 22.95±0.40 mg GAE g-1 with a mean of 20.54±2.08 mg GAE g-1.
Total avonoid content
The total avonoid content of the crude U. pertusa ethanolic extract was determined with reference to the
standard rutin and expressed as its equivalent (mg RE g-1). The total avonoids present in the three aliquots
of U. pertusa ethanolic extract examined ranged from 533.69±23.03 to 546.09±6.83 mg RE g-1 with a mean
Table 1 Qualitative phytochemical screening of the Ulva pertusa ethanolic extract
Phytochemical compound
Test Result
Aliquot 1 Aliquot 2 Aliquot 3
Alkaloids
Mayer’s and Wagner’s test
+
+
+
Flavonoids
Shinoda test
+
+
+
Glycosides
Keller-Killiani test
−
−
−
Phenols
Ferric chloride test
+
+
+
Saponin
Foam test
+
+
+
Tannins
Ferric chloride test
+
+
+
Terpenoids
Salkowsk i test
+
+
+
(+) = present; (−) = absent
Table 1 Qualitative phytochemical screening of the Ulva pertusa ethanolic extract
(+) = present; (−) = absent
Int Aquat Res (2022) 14:51-62
56
of 539.07±6.36 mg RE g-1 (Table 2).
DPPH radical scavenging activity
The DPPH radical scavenging activity of the U. pertusa ethanolic extract is shown in Fig. 1. The U. pertusa
ethanolic extract exhibited a concentration-dependent DPPH radical scavenging capacities from 0.25 to 3
mg mL-1, such that the scavenging activities recorded correspondingly increased from 17±0.2 to 80±0.4 %,
respectively. Accordingly, the computed IC50 of the U. pertusa ethanolic extract was 1.53±0.012 mg mL-1.
The scavenging activity of the U. pertusa extract was comparable to the scavenging capacity of our positive
control (ascorbic acid).
Antibacterial activity
To better elucidate the antibacterial activity of the U. pertusa ethanolic extract, the agar well diusion
method was employed. All of the 3% DMSO control wells did not produce any zones of inhibition on any of
the bacterial strains examined, while the antibiotic control wells produced zones of inhibition of 29.67±0.58
for S. aureus ATCC25923. As shown in Fig. 2 and Table 3, the U. pertusa ethanolic extract possesses potent
antibacterial activity against S. aureus ATCC25923 as supported by a marked inhibition zone of 30.67±0.58
mm produced after a 24h exposure to 100 mg mL-1 of the extract. Concomitantly, a decreasing trend parallel
to the decreasing concentrations of the extract was evidently noted such that at 6.25 mg mL-1, the zone of
inhibition that was produced was only 10.67±0.58 mm. Notably, the extract exhibited antimicrobial activity
against E. coli, A. hydrophila and A. sobria. The antibacterial activity induced by the extract at 100 mg mL-1
against S. agalactiae and Aeromonas spp. was not signicantly dierent (P <0.05) whereas, a markedly
lower activity against V. harveyi was noted (Table 3).
Discussion
Concordant to the previous phytochemical studies on green seaweed extracts (Cho et al. 2010; Wang et al.
Table 2 Total phenolic and flavonoid contents of the U. pertusa ethanolic extract
Test
Result
Aliquot 1
Aliquot 2
Aliquot 3
Mean
Total phenolics content
(mg gallic acid equivalent [GAE] g-1 of extract)
22.95±0.40
19.35±1.72
19.33±0.29
20.54±2.08
Total flavonoids content
(mg rutin equivalent [RE] g-1 of extract)
533.69±23.03
546.09±6.83
537.42±24.81
539.07±6.36
Data are presented as Mean ± SD
Table 2 Total phenolic and avonoid contents of the U. pertusa ethanolic extract
Data are presented as Mean ± SD
Table 3 Antimicrobial activity of the U. pertusa ethanolic extract against selected bacterial fish pathogens
Microorganism
Inhibition Zone (mm ±SD)
Amoxicillin 0.02 mg mL-1
DMSO
100 mg mL-1
50 mg mL-1
25 mg mL-1
12.5 mg mL-1
6.25 mg mL-1
3.125 mg mL-1
Gram positive
Staphylococcus aureus
ATCC25923
29.67±0.58 – 30.67±0.58a 26.67±0 .58a 24.00±00a 19.33±0.58a 10.67 ±0.58a –
Streptococcus agalactiae
TKG510208
21.67±0.58 – 17.67±0.58c 13.17±0 .29b 10.33±0.58b 8.00±0.00b – –
Streptococcus agalactiae
TMD10206
21.33±0.58 – 18.17±0.29bc 12.83±0 .29b 9.83±0.29b 8.00±0.00b – –
Gram negative
Escherichia coli
ATCC25922
11.33±0.58 – 14.67±0.58d 9.50±0.50c – – – –
Aeromonas hydrophila
TI201301
– – 19.33±0.58b 10.00±0.00c – – – –
Aeromonas hydrophila
P1261308
– – 20.00±0.00b 10.00±0.00c – – – –
Aeromonas sobria
TI2013025
– – 17.67±0.58c 10.33±0.58c – – – –
Vibrio harveyi
TbE0901
– – 9.00±0.00e – – – – –
(–) no zone of inhibition. Data are presented as Mean ± SD. Values with different superscripts a, b, c, d, e within each
column are significantly different as determined by Duncan test (P< 0.05)
Table 3 Antimicrobial activity of the U. pertusa ethanolic extract against selected bacterial sh pathogens
(–) no zone of inhibition. Data are presented as Mean ± SD. Values with dierent superscripts a, b, c, d, e within each column are signi-
cantly dierent as determined by Duncan test (P < 0.05)
Int Aquat Res (2022) 14:51-62 57
2010), our current data likewise revealed the presence of dierent bioactive compounds in the U. pertusa
ethanolic extract including alkaloids, avonoids, phenols, saponins, tannins, and terpenoids. Bioactive
compounds stored in seaweeds possess antioxidant and antibacterial activities that can be utilized as
alternative to treat bacterial infections in sh, particularly those bacterial pathogens implicated in zoonosis
(Vatsos and Rebours 2015; Thanigaivel et al. 2016). These bioactive compounds detected in U. pertusa
extract have been previously documented to confer resistance in opposition to microbial pathogens (Cho
et al. 2010; Choi et al. 2011, 2014) and this could be accountable for the exposition of antioxidant and
antibacterial activities of the extract in the present study. It should be noted that secondary metabolites
stored in plants like terpenoids have been documented to have antiviral, antimalarial, antibacterial, and
anti-inammatory properties (Mahato and Sen 1997). Alkaloids, on the other hand, have been reported to
have a wide range of pharmacological potentials like antiasthma, antimalarial, and anticancer activities
(Thawabteh et al. 2019). Moreover, saponins have been identied to possess antidiabetic, antifungal, and
anti-inammatory properties (Feroz 2018). Additionally, tannins are polyphenolic compounds found to
exhibit antioxidant, antimicrobial, and anti-inammatory properties (Macáková et al. 2014).
Phenolic compounds are ubiquitous secondary metabolites commonly found in plants and have been
documented to have several biological activities including antioxidant and antibacterial activities (Kuda et
al. 2005). Accordingly, the Folin-Ciocalteu method was used to examine the total phenolic content of the
U. pertusa extract. Folin-Ciocalteu reagent determines the total phenols as evidenced by the production
of a blue color indicative of the reduction of the yellow heteropoly phosphomolydate-tungstate anions
(Stankovic et al. 2011). The total phenolic content of the U. pertusa ethanolic extract in the current study
was signicantly higher compared to the values previously reported for ethanolic extracts of U. pertusa
Fig. 1 DPPH (1,1-Diphenyl-1-picrylhydrazyl) radical scavenging activity of the U. pertusa ethanolic
extract. Ethanol + DPPH (CeDPPH) and ascorbic acid (Asc) were used as negative and positive controls,
respectively.
0
10
20
30
40
50
60
70
80
90
100
CeDPPH 0.25 0.5 0.75 11.5 22.5 3Asc
DPPH scavenging effect (%)
Concentration mg mL-1
Fig. 2 Representative plates for the agar well diffusion assay showing zones of inhibition produced by the
varying concentrations of the U. pertusa ethanolic extract (UpEE) on S. aureus ATCC25923. Negative
control (DMSO) (a); Amoxicillin 0.02 mg mL-1 (b); 100 mg mL-1 UpEE (c); 50 mg mL-1 UpEE (d); 25
mg mL-1 UpEE (e); 12.5 mg mL-1 UpEE (f); 6.25 mg mL-1 UpEE (g); 3.125 mg mL-1 UpEE (h).
a
b
d
c
a
f
b
e
a
b
h
g
Fig. 1 DPPH (1,1-Diphenyl-1-picrylhydrazyl) radical scavenging activity of the U. pertusa ethanolic extract. Ethanol + DPPH
(CeDPPH) and ascorbic acid (Asc) were used as negative and positive controls, respectively.
Fig. 2 Representative plates for the agar well diusion assay showing zones of inhibition produced by the varying concentrations of
the U. pertusa ethanolic extract (UpEE) on S. aureus ATCC25923. Negative control (DMSO) (a); Amoxicillin 0.02 mg mL-1 (b); 100
mg mL-1 UpEE (c); 50 mg mL-1 UpEE (d); 25 mg mL-1 UpEE (e); 12.5 mg mL-1 UpEE (f); 6.25 mg mL-1 UpEE (g); 3.125 mg mL-1
UpEE (h).
Int Aquat Res (2022) 14:51-62
58
and another green seaweed Capsosiphon fulvescens (Cho et al. 2010). However, current values were lower
than those of the ethanolic extracts reported from another green seaweeds Chaetomorpha monilifera and
Enteromorpha compressa (Cho et al. 2010). Although reports have indicated that phenolic content was
found to be higher in ethanolic extracts due to its polarity, the dierences in the total phenolic contents of
the U. pertusa ethanolic extract between our current and previous studies may be attributed to the extraction
protocol employed and viably, inuence of various environmental factors on the phenolic contents (Kuda
et al. 2005; Wang et al. 2010). Flavonoids, a group of secondary metabolites that refers to a class of
naturally occurring polyphenols, are also commonly found in plants. They are typically accountable for
taste, color, disablement of fat oxidation and prevention of enzymes and degradation of vitamins in food.
Flavonoids have been reported to have anti-oxidative activity, free-radical scavenging capacity, coronary
heart disease prevention, anticancer activity, and some have been found to exhibit potential for anti-human
immunodeciency virus functions (Yao et al. 2004). The mean total avonoid content of the U. pertusa
ethanolic extract obtained in the current study was signicantly higher than the previous report of Farasat
et al. (2014) for the methanolic extract of the green seaweeds Ulva clathrata and U. intestinalis. The
dierences noted between the data generated in the current study and the previous report of Farasat et al.
(2014) may be plausibly attributed to the method of extraction used and possibly due to marked changes
in the chemical constituents of seaweeds with the change of seasons and environmental conditions such as
variation in physicochemical parameters of the rearing water (Kuda et al. 2005; Wang et al. 2010; Farasat
et al. 2014). Because phenolic compounds including avonoids which are commonly found in plants have
been generally proven to be eective free radical scavengers and antioxidants, we further evaluated the
antioxidant capacity of the U. pertusa ethanolic extract employing the DPPH radical scavenging method.
This method is based on the reduction of an ethanolic DPPH solution in the presence of a hydrogen donating
antioxidant thereby resulting in the formation of the non-radical form DPPH-H molecule upon the uptake of
a hydrogen atom from antioxidant species (Schlesier et al. 2002). DPPH is a stable nitrogen centered free
radical which can be eectively scavenged by antioxidants, hence, it has been widely used for rapid evaluation
of antioxidant activity of plant extracts including seaweeds due to its reliability. The present study showed
that the scavenging eect of the U. pertusa extract typically behaved in a concentration-dependent manner,
i.e. the inhibitory eect increased with increasing concentration of the extract. Moreover, the IC50 value
generated for U. pertusa ethanolic extract in the current study was lower compared with the U. clathrata
extract but considerably higher than U. linza, U. intestinalis, and U. exuosa methanolic extracts previously
reported by Farasat et al. (2014). Several studies have by far elucidated the antioxidant activity in Ulva
species. For example, among the 48 marine algae evaluated for their antioxidant activity, low antioxidant
activity with a relatively high IC50 were documented for U. intestinalis (Zubia et al. 2007). However,
some researchers have otherwise reported high free radical scavenging activity for other Ulva species. For
instance, extracts of U. compressa, U. linza, and U. tubulosa exhibited high antioxidant activity in linoleic
acid system, notably, with an excellent DPPH radical scavenging activity obtained for the methanolic
extract of U. compressa as evidently supported by an IC50 of 1.89 mg mL-1 (Ganesan et al. 2011). Moreover,
Qi et al. (2006) demonstrated that the natural ulvan, a group of sulfated heteropolysaccharides obtained
from Ulva species, and its derivatives have higher scavenging activity on superoxide radical than vitamin
C. Additionally, Hassan et al. (2011) accordingly documented that prolonged consumption of U. lactuca
polysaccharides extract conferred potent antioxidant and hypocholesterolemic eects in experimentally-
induced hypercholesterolemic rats. Corroborative to the published reports on Ulva species (Ganesan et
al. 2011; Farasat et al. 2014), the antioxidant activity of U. pertusa extract in the current study likewise
correlated with the quantity of its phenolic and avonoid contents. As free radicals are formed during
metabolism, i.e. as a result of the mitochondrial metabolism, inammatory responses, phagocytosis, and
physical activities, their production may be accelerated by external factors such as radiation, drugs and
pesticides. As a consequence, overproduction of free radicals in the biological systems may damage all
classes of chemicals including proteins, amino acids, nucleic acids, and carbohydrates in the biological
materials (Sanjeewa et al. 2018). Thus, counteracting oxidative stress in the biological systems through
the use of natural antioxidants such as those coming from seaweed extracts would be a practical strategy of
protecting the organism from oxidative damage (Wang et al. 2010; Sanjeewa et al. 2018).
As shown in Table 3, U. pertusa ethanolic extract exhibited potent antibacterial activity against S. aureus
ATCC25923. Li et al. (2018a) previously demonstrated that 10 mg mL-1 of the U. pertusa ethanolic extract
Int Aquat Res (2022) 14:51-62 59
could induce a mean inhibition zone of 10.00±0.00 mm against S. aureus. Noticeably, such antibacterial
activity is lower compared with the data that we generated in the present study since comparable values were
markedly obtained when the S. aureus was exposed to a lower concentration (6.25 mg ml-1) of the U. pertusa
ethanolic extract. Our current ndings consistently corroborate with the previous reports demonstrating
the sensitivity of gram-positive S. aureus strains to seaweed extracts (Morales et al. 2006; Stirk et al.
2007), particularly against U. pertusa extract (Mtolera and Semesi 1996). Staphylococcus aureus typically
constitutes the natural microbiota of the pond-reared sh and their culture environments (Pakingking et al.
2015). Staphylococcus species are generally not considered as sh pathogens as they have not by far been
implicated in any serious disease outbreaks. However, among the principal foodborne bacteria described as
human pathogens, S. aureus is the leading cause of gastroenteritis due to the consumption of sh products
contaminated with this pathogen and its enterotoxin (Arfatahery et al. 2015). In fact, high population of
these bacteria in harvested sh indicates the degree of spoilage it might have undergone. The extract likewise
induced potent antibacterial activity against S. agalactiae isolates in a concentration-dependent manner.
This nding is imperative as streptococcal infection among net-caged tilapias conned in open waters
such as lakes have been recently recognized in the Philippines as a persistent problem that has resulted in
serious economic losses (Legario et al. 2020). Fish farmers have resorted to using antibiotics, particularly
amoxicillin (a newer version of penicillin), which is mixed with the feed (Legario et al. 2020). However,
the emergence of multidrug-resistant S. agalactiae strains found in human and sh with high penicillin
non-susceptibility has been recently reported (Li et al. 2020). Thus, the addition of U. pertusa extract in sh
feed as an alternative prophylactic agent against streptococcal infection in cultured sh is worth pursuing.
Of note, the capacity of U. pertusa ethanolic extract to inhibit the growth of A. hydrophila and A. sobria is
an unequivocally important nding considering that these Aeromonas species are opportunistic pathogens
which are ubiquitously found in sh and their rearing environments (Pakingking et al. 2015, 2020).
While there is no standard way to carry out a qualitative evaluation of the antimicrobial activity of a
certain seaweed extract as dierent authors used dierent scales to assess in vitro antimicrobial susceptibility
test (Silva et al. 2020), nevertheless, pertinent discrepancies observed with regard to variations in the
antibacterial activity of the U. pertusa extract obtained in the current study and those previously reported
for U. pertusa and other seaweed extracts feasibly lie on the extraction method employed, source and
composition of macroalgae, and as well as protocols (method, time/ period/ season, etc.) utilized for the
seaweed collection as previously reported (Stirk et al. 2007; Choi et al. 2014; Thanigaivel et al. 2016). For
example, Choi et al. (2014) documented seasonal variations in the antimicrobial activity of the U. pertusa
extracts against Gardnerella vaginalis; the extracts did not show activity during summer and autumn but
notable activity from early winter (December) to middle spring (April). Despite the numerous studies
by far conducted on the antimicrobial activities of the dierent seaweed extracts against sh pathogens,
particularly those with zoonotic potential, there is still limited information on the exact mechanism of action
for the majority of these extracts (Vatsos and Rebours 2015). Nevertheless, antibacterial compounds in
seaweeds that have been considerably documented include fatty acids, lipophilic and phenolic compounds,
lectins, acetogenins, terpenes, alkaloids, polyphenolics, isoprenoid metabolites, tannins, and hydrogen
peroxide as reviewed by Mohamed et al. (2012). Thus, the potent antibacterial activity exhibited by the
U. pertusa ethanolic extract against S. aureus in the current study could be attributed to these bioactive
compounds present in the U. pertusa extract that we examined. Hitherto, pertinent investigations on U.
pertusa ethanolic extract did not point out one individual substance or particular substance class which
could be entirely responsible for its antimicrobial action, clearly indicating that a synergistic action is
essential for the biological activity of the U. pertusa extract to be liberally expressed. Future studies should
therefore isolate and characterize the major bioactive components of this green alga and their corresponding
mode of action against important bacterial sh pathogens. Moreover, the practical therapeutic application
of U. pertusa ethanolic extract against bacterial infections in tilapia and other susceptible high value marine
sh species such as sea bass and groupers via feed supplementation is warranted.
Conclusion
In summary, our current data clearly demonstrated that U. pertusa ethanolic extract possesses potent
antibacterial activity, particularly against infectious bacterial sh pathogens, and some bioactive components
Int Aquat Res (2022) 14:51-62
60
with remarkable antioxidant capacities that may be utilized in the synthesis of novel drugs. With reference
to the many studies by far conducted on the antimicrobial activities of the U. pertusa extracts that we
reviewed, this study is the rst to demonstrate the antioxidant and antibacterial activity of the ethanolic
extract of U. pertusa commonly found in the Philippines and may therefore serve as promising alternatives
for the prevention and control of infectious diseases in aquacultured species.
Authors’ contributions The conception and design of the study were developed by RPJ and EGDJ-A. RPJ, DJL, RU, EGDJ-A, and
CMC contributed to the data acquisition, analyses, and interpretation. RPJ wrote the manuscript with input from all authors. The nal
manuscript was approved by all authors.
Acknowledgements We would like to thank Drs. Teruo Azuma, Takuro Shibuno and Chihaya Nakayasu, former GOJ-TF managers,
and the sta of IMS and NPPMCI for their invaluable assistance during the collection and analyses of our samples, respectively. This
study was funded by the Government of Japan Trust Fund V through the Regional Disease Project (study code: FH01-F2010-T) and
partly by SEAFDEC AQD.
Conicts of interest The authors declare that they have no conict of interest.
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