ISSN 2601-8705 (Print) European Journal of January – June 2022

ISSN 2601-8691 (Online) Natural Sciences and Medicine Volume 5, Issue 1

Antioxidant and antibacterial potential of Ulva lactuca

species from Romanian Black Sea Coast

Emin Cadar
Ovidius University of Constanta, Faculty of Pharmacy, Capitan Aviator Al. Serbanescu
Street, No. 6, Campus, Corp C, Constanta, 900470, Romania
Ticuta Negreanu-Pirjol
Ovidius University of Constanta, Faculty of Pharmacy, Capitan Aviator Al. Serbanescu
Street, No. 6, Campus, Corp C, Constanta, 900470, Romania
Bogdan-Stefan Negreanu-Pirjol
Ovidius University of Constanta, Faculty of Pharmacy, Capitan Aviator Al. Serbanescu
Street, No. 6, Campus, Corp C, Constanta, 900470, Romania

Abstract
Green algae biomass represents an important natural resource found in
marine environments which could offer new applications in the
pharmaceutical and cosmeceutical industries. The paper aim was to
characterize some fluid extracts of Ulva lactuca L. green macroalgae species,
in the Ulvaceae family, from Romanian Black Sea Coast, through physical,
chemical and biological methods. For biomedical applications it is important
to study how the difference between the marine environment specific
conditions, from which they are harvested, can influence the chemical
composition of macroalgae. Another important aspect which could affect the
bioactive green macroalgae extracts composition, is the influence of applied
extraction method. In this way, the less distructive cold maceration method in
different solvent concentrations was chosen, according with literature data.
The extracts have been studied in order to determine total polyphenols
content, total antioxidant capacity by photochemiluminescence method and
antibacterial activity. The results emphasized an antioxidant and antibacterial
activity of the studied macroalgae extracts, which can be improved in mixture
with other bioactive natural substances, in order to obtain new
pharmaceutical preparations for topical applications. The valuable obtained
results offer the possibility to propose this natural resource for next studies
as biomaterial in tissue engineering, wound dressing and drug delivery
systems. We consider that Ulva lactuca L. green macroalgae species of
Romanian Black Sea Coast, could be an important source of marine bioactive
compounds with various uses in the biomedical field.

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Keywords: Ulva lactuca L., bioactive compounds, antioxidant capacity, antibacterial

activity
Introduction
Marine algae serve as a large part of the phytoplankton found in various marine
environments of the world. Macroalgae represent, macroscopic algae with increased
growth rate, which can be found in various areas and have relatively low use of fresh
water [1]. Interest in algal biotechnology has been on the rise in recent times,
especially on the production of various macroalgae species, used in the food, medical,
cosmetic and pharmaceutical industries. Of significant importance is the increased
global production of seaweeds in the last few decades [2]. In data literature, there are
multiple studies conducted on Ulva lactuca L., which grows in the waters of seas and
oceans [3], [4].

(a) (b)
(b)
Fig. 1 (a, b). Ulva lactuca L. green macroalgae species
Ulva lactuca L., popular known as sea lettuce, see Fig. 1, represents a green macroalgae
which belongs to the Chlorophyta Phylum, as described by Linnaeus in the 17th
century [5]. It can be found connected, sessile or floating, in various marine
environments and it is widespread on the Romanian Black Sea shore. Ulva lactuca L.
presents polymorphism which led to the assumption, that different species exist
depending on the environment. Linnaeus was the first one to discover that Ulva
lactuca L. presents different phenotypes with a tubular form tail. Taxonomists from
the 19th century believed that green algae with tubular tail represent a completely
different genus called Enteromorpha. In time though, different studies of genetic
analysis showed that diverse phenotypes were not linked to genetic variations, so
there is no real evidence to support the existence of species other than Ulva lactuca L.
[6]. Literature still describes distinct Ulva species, but reproduction among these
species has been demonstrated and therefore they can only be described from now
on as Ulva lactuca L. variants.

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An increased number of studies enphasize marine habitats and other factors such as
water temperature or marine pollutants can influence the chemical composition of
marine algae. The study of macroalgae increased in interest due to their role of
bioindicators [7], [8]. They represent markers that can highlight negative changes in
marine ecosystems due to the accumulation of different pollutant agents in their
thallus [9]. Ulva lactuca L. predominantly invades and blooms near beaches, where it
can affect tourism and damage marine ecosystems. The acidic vapors and
biodegradation can induce animal deaths and possibly human deaths, due to Ulva
lactuca L. large quantities degradation [10]. Ulva lactuca L. blooms have been
reported all over the world, but the biggest event in the world so far was the green
tide from the Yellow Sea for ten consecutive years which covers approximately 10%
of the whole Yellow Sea surface [11]. In Romania, the South-east coast of the Black
Sea, near Constanta and Mangalia cities reported the biggest Ulva lactuca L. blooms.
The Romanian Black Sea coast offers great possibilities for harvesting Ulva lactuca L.,
which can be found in significant numbers near the shore and at low depths. Ulva
lactuca L. is an edible green seaweed with no threats to human health, traditionally
consumed in various Asian countries but also approved in Europe [12].
Ulva lactuca L. consists of various natural compounds that can be utilized in many
areas like pharmaceutical, cosmetical, chemical, energy or food sector. Seaweed
composition includes soluble and insoluble dietary fibers together with minerals,
polysaccharides, vitamins, chlorophylls, proteins. The integral use of the plant can
represent a more profitable approach in order to utilize all the different constituents
that Ulva lactuca L. possesses [13], [14].
In the last thirty years, medicinal plants practice has become a major trend
worldwide, as the analysis of algae, plant extracts and natural products has shown
that green algae represent a potential source of antibacterial activity. Traditional
compounds face crises due to the rapidly growing resistance of bacteria, e.g.
Staphylococcus aureus. Unlike chemicals, herbs are considered non-toxic due to their
natural origin and long-term use as folk medicine. In this paper we present the results
obtained regarding the antioxidant and antibacterial activity evaluation of this
common green macroalgae species present along Romanian Black Sea Coast.
Materials and methods
The analyzed samples consist of green algae species Ulva lactuca L., class
Ulvophyceae, order Ulvales, family Ulvaceae. The vegetal material was obtained from
Romanian Black Sea shore, from four different zones: Vadu, Constanta (the highest
macrophyte seaweed area), Mamaia and Navodari, Constanta County, Romania, in the
August - October 2021 period. The marine algae collected samples were processed by
washing thoroughly with tap water and drying the harvested vegetal material, at
room temperature, on metal sieves, 25 ± 2 ˚C, for 24 hours, followed by drying at 40
˚C in oven with natural convection and grinded in an electric grinder to a fine powder.

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Analysis methods were used in accordance to phytochemical, physico-chemical and

pharmacognostical studies in literature data. Studies and experiments were made in
the laboratories of Ovidius University of Constanta, Faculty of Pharmacy.
The chemical composition was explored in order to identify and classify the classes of
compounds that support the physical-chemical and biological characteristics of the
samples in comparison with the literature data [3].
Three types of extracts were obtained on using distinct solvents and different
reactions for identifying the presence of important active principles of
pharmaceutical interest, as Table 1.
For chemical compounds identification of the three extracts, they were analysed
separately, using methods fit for the physical and chemical properties for each group
of active principles [3].
Table 1. Type of methods and specific reactions used in farmacognostic study [3]
Type of extractive solution Reaction/Method utilised

Etheric extractive solution Fluorescent UV (λ=365nm) and

(A) Lieberman-Burchard
Alcoholic extractive solution Fehling reaction, Borntrager
(B) reaction and UV method (λ =
365 nm), Iron Chlorure reaction,
Liebermann Bourchard reaction
Water extractive solution (C) Fehling reaction, reaction with H2SO4
conc. and tymol, Foaming reaction
and reaction with FeCl3
Total phenolic content determination and pigments chemical composition
Total phenolic content determination (TPC): Total phenols present in Ulva lactuca L.
samples were determined UV-Vis spectrophotometrically, applying Folin-Ciocalteu
reagent as stated by Singleton et al. [15]. Absorbance was determined at 760 nm in
comparison to the calibration curve of gallic acid.
Total chlorophylls determination (T-Ch): We homogenized three grams of Ulva lactuca
samples in acetone (30 mL, 80%) and incubated over the night, to protect it from
sunlight, at 4 °C in order to obtain a complete extraction followed by centrifugation at
10,000 xg for 5 min. Total chlorophyll (T-Chl), chlorophyll a (Chl a) and chlorophyll b
(Chl b) found inside the supernatant were measured UV-Vis spectrophotometrically
at 664 nm wavelength, in accordance to Lichtenthaler and Buschmann method [16].

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Total carotenoids determination (TCAR): Total carotenoids were determined UV-Vis

spectrophotometrically at 450 nm wavelength, in accordance to AOAC [17]. β-
carotene was utilised as a standard.
Antioxidant activity
Algae fluid extracts were obtained using two different methods, cold maceration and
Soxhlet method, see Fig. 2.
Sample preparation - The vegetal product has been made into fluid extracts using as
solvents, ethyl alcohol 50% and 70% concentration, using two common extraction
methods:
Cold maceration for variable concentrations of vegetal product, 2.5 g, 5.0 g,
respectively 10 g in 100 mL, both ethyl alcohol 50% and 70% concentration, for 12
days at darkness, room temperature, periodic stirring.
Soxhlet extraction for three hours refluxing for variable concentrations of vegetal
product, 2.5 g, 5.0 g, respectively 10 g in 150 mL ethyl alcohol 70% concentration.
All the hydroalcoholic extracts obtained were filtered at normal pressure through
quantitative Whatman filter paper and present different colors, from pale green to
green-brown and dark green.
For determining total antioxidant capacity (TEAC), 10 μL volume of algae extracts
were taken for analysis. Each determination lasted 120s. Analyses employed the
photo-chemiluminescence method by ACL (Antioxidative Capacity in Lipid Soluble
Substances) procedure Analytik Jena, using Photochem apparatus Analytik Jena AG,
Germany, see Fig. 2. Triplicate samples of hydroalcoholic extracts were quantified by
comparison with the standard substance Trolox®, Hoffman-LaRoche's trade name
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). For calibration, the
standard kit of reagents, Analytik Jena, Germany, was used: R1 (dilution solvent), R2
(buffer reagent), R3 (photosensitive reagent), R4 (reagent sized). For the calibration
curve standard solutions containing 0.5, 1.0, 2.0, 3.0 nmol Trolox were measured
(suitable for 10 μL R4), as Trolox calibration curve, Fig. 3.

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Fig. 2. Ulva lactuca L. hydroalcoholic Fig. 3. Trolox standard calibration

extracts samples obtained by cold curve
maceration and Soxhlet extraction and
Photochem apparatus Analytik Jena AG,
Germany

Antibacterial activity
In order to demonstrate the antibacterial activities, we measured areas of inhibition
for the different microorganisms that were classified under standardized conditions:
from the gram positive bacteria we had Staphylococcus aureus (MTCC 737) and
Staphylococcus epidermidis (MTCC 3615). We also had the gram negative bacteria -
Escherichia coli (MTCC 1687) and Pseudomonas aeruginosa (MTCC 424). Ampicillin
was used as reference substance, the standard drug.
Solvents chloroform, n-hexane and ethyl alcohol – water (1:1) were used to extract
the material previously powdered (300 g), using the maceration process for 72 hours,
ocassionally stirring from time to time.
Each of the three extracts were concentrated after it was brought together with the
filtrate and we kept them in the desiccator for future experiments. The n-hexane
extract, chloroform extract and ethanol: water (1:1) extract were concentrated after
combining the filtrate and kept in desiccator for further investigation.
Results and Discussions
The marine algae samples were first treated with a non-polar solvent such as benzen
or dyethil ether, then with a medium polarity solvent such as methyl alcohol or ethyl
alcohol and finally with water. Each extract has been afterwards analysed in order to
identify the presence of active principles of pharmaceutical interest. The extracts
were analysed individually using the specific methods for each group of active
principles.

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Table 2. Active principles identified in green algae Ulva lactuca L.

Table 2 Type of extractive solution Class of active principles
Etheric extractive solution (A) steroles
triterpenes
cumarines
Alcoholic extractive solution (B) catehic tannins
reducing compounds
antracenozides
cumarines
Water extractive solution (C) reducing compounds
saponosides
catehic tannins
highlights the active principles identified in green algae Ulva lactuca L. It is interesting
that in all three extractive solutions studied different classes of bioactive compounds
have been identified that are responsible for the antioxidant and antimicrobial
activity that we studied in the paper.
Total phenolic content determination and pigments chemical composition

The antioxidant capacity and antibacterial potential of the marine algae samples were
highlighted by identifying and determining different natural compounds and their
active principles, recognized in literature data for having the potential to give
antioxidant and antibacterial activity. In the above table we showed our results for
the determination of total phenolic contents, total chlorophylls (a +b) and total
carotenoids. Table 3 presents the results obtained for the compositions of the studied
marine algae. There is a good consistency with the literature data of other researchers
who have worked on green seaweed. Phenolic compounds can be defined by their
aromatic rings and their hydroxyl groups. A phenolic compound which contains an
aromatic ring with one or two groups of hydroxyls are named simple phenols, for
example benzoic acid or hydroxycinnamic acid which can be found in marine algae
structure. Polyphenols represent a large family comprised by multiple phenol units
[18]. From this method we can conclude that the most stable extracts and the highest
inhibition are the Ulva lactuca L. extracts made with the Soxhlet extraction, from
which it can highlight Ulva lactuca L. extract 10 g/100 mL ethanol 70%, with an
increased antioxidant activity. From the cold maceration, the best results were

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obtained from Ulva lactuca L. extract 5 g/100 mL ethanol 50% and Ulva lactuca L.
extract 2.5 g/100 mL ethanol 70%.
Table 3. Total phenolic content, total chlorophylls and total carotenoids contents from
extracts of green macroalgae Ulva lactuca L. from the Romanian Black Sea shore
Sample Total phenolic Total Total Total
TPC chlorophylls chlorophylls carotenoids
(TCAR):
(mg. GAE/100g T-Chl (a) T-Chl (b)
d.w.)
mg/g d.w mg/g d.w
Ulva lactuca L. 286.1 ± 0.6 21.08 ± 2.69 3.77±0.36 10.57±0.85

All values show mean of three replicates, ± standard deviation

The unique chemical, physical and biological structure of polyphenols can divide
them into different classed like phloroglucinols and phlorotannins. In recent times,
various studies showed multiple beneficial therapeutical actions for phenolic
compounds found in marine algae, such as antioxidant, antibacterial, anticancer and
anti-inflammatory activities.
Marine algae organisms can synthesize different pigments such as carotenoids,
phycobiliproteins and chlorophylls [19]. The green pigment for Ulva lactuca L. is given
by the presence of clorophylls a and b [19].
Antioxidant activity
Samples were prepared and the results were expressed as nmol Trolox
equivalents/sample volume, according to Table 4.
Table 4. Maximum inhibition of free radicals and total antioxidant capacity of Ulva
lactuca L. extracts
Extract samples Max. Inhibition Total antioxidant capacity
of free radicals (nmol equiv. Trolox/sample
volume)
Ulva lactuca extract 2.5g/100mL 0.213 0.109
ethanol 50%, cold maceration
Ulva lactuca extract 5g/100mL 0.257 0.161
ethanol 50%, cold maceration
Ulva lactuca extract 10g/100mL 0.178 0.023
ethanol 50%, cold maceration
Ulva lactuca extract 2.5g/100mL 0.381 0.285
ethanol 70%, cold maceration

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Ulva lactuca extract 5g/100mL 0.245 0.138

ethanol 70%, cold maceration
Ulva lactuca extract 10g/100mL 0.024 0.022
ethanol 70%, cold maceration
Ulva lactuca extract 2.5g/100mL 0.140 0.035
ethanol 70%, Soxhlet extraction
Ulva lactuca extract 5g/100mL 0.231 0.111
ethanol 70%, Soxhlet extraction
Ulva lactuca extract 10g/100mL 0.311 0.272
ethanol 70%, Soxhlet extraction

In the hydroalcoholic extracts of Ulva lactuca, obtained by cold maceration, a

significant total antioxidant capacity was recorded for Ulva lactuca extract 5 g/100
mL ethanol 50% with 0.161 nmol equiv. Trolox/10 μL sample and for Ulva lactuca
extract 2.5 g/100 mL ethanol 70% with 0.285 nmol equiv. Trolox/10 μL sample.
In the hydroalcoholic extracts of Ulva lactuca, obtained by reflux, Soxhlet extraction,
a significant total antioxidant capacity, compared to extracts obtained by cold
maceration, was recorded for Ulva lactuca extract 10 g/100 mL ethanol 70%, with
0.272 nmol equiv. Trolox/10 μL sample.
The highest antioxidant activity was obtained through cold maceration for Ulva
lactuca extract 2.5 g/100 mL ethanol 70% sample, with a value of 0.285 nmol equiv.
Trolox/10 μL sample.
The results obtained emphasize that extracts obtained in ethanol 70% induce the
highest values of total antioxidant activities for different concentrations of the
samples.
Antioxidants are very important compounds that can stop the production of free
radicals and the chain reactions that may affect important cells in the organism
through oxidation. They are also used in food and pharmaceutical preservation, with
increasing percentage in recent times because of the toxicity and danger to human
health caused by the use of synthetic antioxidants [20]. Seaweeds represent an
important natural source for antioxidants with very little side effects. In human
organism antioxidants are significant in their activity of regulating the oxidative
stress related diseases [20, 21].
Ulva lactuca L. contain a varied compounds array with antioxidant activit such as,
tocopherols, carotenoids and polyphenols. An important compound with antioxidant
potential in green seaweeds is represented by ulvan, a sulfated heteropoly-saccharide
obtained from Ulva lactuca [22].

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Antioxidant potential was measured by scavenging activity of hydroxyl and

superoxide radicals, metal chelating potential and superoxide radicals. Sulfate
contents in ulvan has been altered by using trioxide/N,N-dimethyl formamide. After
the modification all the parameters studied gave better results, higher scavenging
activity, better reducing power and stronger chelating ability [22].
From this study we draw the conclusion that even if sulfates are already in the
composition of ulvan, by increasing the number of sulfate radicals we can enhance the
potential of its antioxidant activity [22].
Antibacterial activity
Many species of marine algae have been studied in order to examine their
antibacterial and antiviral properties. A lot of polysaccharides have been found in the
composition of seaweeds, from which we highlight some that could potentially exhibit
antibacterial and antiviral activity: alginic acid, agar, laminarin, fucoidan, galactans,
carrageenan, and mannans [23]. In this present study we explore the potential
antibacterial activity of green seaweed Ulva lactuca L. Review of literature has
highlighted possible antibacterial activity in different compounds present in the
green algae. In the view of this, an effort was made to verify some of the isolated
compounds from Ulva lactuca L. for their antibacterial activity. The area of inhibition
of microorganisms under standardized conditions was used in order to demonstrate
antibacterial action for the various compounds. For present work, efficacy of eight
compounds were detected against Staphylococcus aureus, Staphylococcus epidermidis,
Pseudomonas aeruginosa and Escherichia coli., see Fig. 4. The concentration used for
the test compound was 1mg/mL. and we took Ampicillin as the standard drug.
14
Zone of Inhibition (mm)

12
10
8
6
4
2
0
1 2 3 4
E 9 10 9 9
C 11 11 11 10
H 10 12 11 12
Ampicillin 11 12 12 12

Test Organisms used

1-S. aureus, 2-S. epidermidis, [Link], 4-P. aeruginosa

Fig. 4. Antibacterial activity of Ulva lactuca L. species

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Against Staphylococcus aureus the chloroform extract showed the highest inhibition
zone (11 mm) comparable to that of the standard ampicillin. In the case of
Staphylococcus epidermides and Pseudomonas aeruginosa, the extract in n-hexane
achieved the largest inhibition zone (12 mm) comparable to that of the ampicillin
standard. In the case of Escherichia coli, the extracts in n-hexane and in chloroform
achieved the largest areas of inhibition (11 mm) but are below the values presented
by the ampicillin standard. From the experiments we concluded that n-hexane and
chloroform extracts had antibacterial activity comparable to the standard drug,
ampicilin.
Conclusions
Green algae Ulva lactuca L. shows promising potential for both antioxidant and
antimicrobial activity, that are due to the existence of known bioactive chemical
compounds that support these characteristics. Ulva lactuca L. is also a source for many
essential bioactive compounds ranging from antioxidants and essential minerals to
the highly signified and complete profiles of amino acids and fatty acids. In the present
study we determined the total phenolic content, total chlorophylls T-Chl (a + b) and
total carotenoids. We managed to demonstrate that various compounds from Ulva
lactuca L. exhibit antioxidant activity which is given by various compounds such as
phenolic constituents, pigments and other active principles and we showed that the
n-hexane and chloroform extracts display good antibacterial activity on various gram
positive and negative bacteria.
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