Journal of Environmental Protection and Ecology 13, No 3A, 1744–1750 (2012)

Marine ecology – antioxidants

Comparative Studies Regarding the Antioxidative

Activity of Some Therapeutic Marine Algae
Species along the Romanian Black Sea Coast

T. Negreanu-Pirjola*, B. Negreanu-Pirjola, R. Sirbua,

G. M. Paraschivb, A. Megheac
a
Faculty of Pharmacy, Ovidius University of Constanta, 1 Aleea Universitatii,
Campus, Corp. B, Constanta, Romania
E-mail: ticuta_np@yahoo.com; bogdannegreanupirjol@yahoo.com
b
Faculty of Natural Science and Agriculture, Ovidius University of Constanta,
1 Aleea Universitatii, Campus, Corp B, Constanta, Romania
E-mail: gmparaschiv@gmail.com
c
Faculty of Applied Chemistry and Materials Science, Politehnica University of
Bucharest, 1–7 Polizu Street, Bucharest, Romania
E-mail: a.meghea@gmail.com

Abstract. The superior valorisation of the marine biomass represents a very important resource
for the pharmaceutical industry, supplying raw material for the extraction of bioactive substances
(vitamins, sterols and collagen). The main macrophyte algae, specific for the Romanian Black Sea
coast, belong to Chlorophyta sp., Pheophyta sp. and Rhodophyta sp. In this paper we present a
study on antioxidant capacity of some green algae extracts Ulva rigida Ag. (syn. Ulva lactuca L.),
brown algae – Cystoseira barbata (G o o d. et W o o d.) Ag. and red algae – Ceramium rubrum
(H u d s.) with concentrations of 20 mg dry vegetal product/ml solvent. Also, the paper presents the
correlation regarding the antioxidative capacity and the heavy metals content (Zn, Cu, Pb and Cd)
of the marine algae extracts. The heavy metals content in the macro-algae samples was determined
by flame atomic absorption spectrometry (FAAS) and the antioxidant capacity was evaluated by
photo-chemiluminescence method based on the multiple acceleration of a natural reaction leading
to generation of a superoxide anion radical. The measured antioxidative capacity was quantified
by comparison with a standard substance used for calibration, Trolox, as tocopherol derivative (in
ACL method) using a Photochem, Analytik Jena apparatus. The comparative studies on 3 algae
extracts confirm the antioxidant capacity of the analysed species. The results relieve the possibility
to enlarge the options to use these natural vegetal resources from the Black Sea coast in different
degenerative diseases therapy.
Keywords: marine green algae, brown algae, red algae, antioxidative activity.

*
For correspondence.

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aims and background
The biological role of macrophytes algae is well-known in all water basins, which con-
tribute in maintaining the biological balance and represents the primary productivity
in these ponds. This paper presents comparative studies on heavy metal content and
antioxidant activity of 3 species of algae, relevant for the Romanian Black Sea coast:
Cystoseira barbata, brown algae, Ceramium rubrum, red algae and Ulva rigi­da A g.
(syn. Ulva lactuca L.), green algae. These are vegetal organisms, which meet the
requirements to be used in toxicological monitoring programs, as bioaccumulative.
Cystoseira barbata (G o o d et W o o d) A g., is part of the brown microalgae, phy-
lum Phaeophyta, class Cyclosporeae, order Fucales, family Cystoseiraceae, genus
Cystoseira, which represents the group of macroscopic algae, multicellular, most
are marine and ocean (Fig. 1). The species is widespread in the Black Sea where
it forms a hard substrate perennial pool, very valuable from an environmental
perspective. Currently the association is very low, on the one side due to frost in
some prior periods and, on the other because of pollution, increased water turbid-
ity and substrate clogging.

Fig. 1. Cystoseira barbata

Fig. 2. Ceramium rubrum

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 Ceramium rubrum (H u d s.) A g., is part of the red microalgae, phylum
Rhodophyta, class Rodophyceae, order Ceramiales, family Ceramiaceae, genus
Ceramium including unicellular algae, or multicellular that lives in aquatic, mostly
marine, few species are found only in freshwater (Fig. 2). Algae is widespread in
the Atlantic and Pacific oceans, the Mediterranean and Black Sea. It is an annual
species of large, sometimes heavily colonised rocky substrates of mid- and infra-
littoral in areas exposed to wave agitation. In the Black Sea it is met along the
entire coastal areas, on rocks, at depths from 0.5 to 4–5 m throughout the year, with
higher spring and summer development. Literature data showed that Ceramium
rubrum species produces a quality agar and from many species of red alga, Ce-
ramium rubrum is the only one with strong antibiotic properties on gram-positive
bacteria, testing being done on Bacillus subtilis, algal inhibitory properties do not
manifest on gram-negative bactera.
Ulva rigida A g. (syn. Ulva lactuca L.) – also known by the commoon name
sea salad, is part of the green microalgae, phylum Chlorophyta, class Ulvophyc-
eae, order Ulvales, family Ulvaceae, genus Ulva. Talus of this alga looks like a
foliacee blade irregularly shaped, sometimes with many other breaks in the middle
of a relatively firm consistency caught by the substrate through a restraint system
consisting of dark rhizoids (Fig. 3a,b).

Fig. 3. Ulvae Lactuca L.: macroscopic appearance (a), microscopic appearance, talus section (b)

It is an Atlanto-tropical species (lower boreal) found in north-east Atlantic

and north-west Mediterranean, Pacific and Black Sea (Romanian seaside in the
south between Costinesti and Mangalia). This species was found in polluted areas,
sometimes abundant in waters rich in nutrients1–5.

EXPERIMENTAL
Manual harvesting of algae was used in the Romanian Black Sea coastal area,
between Navodari and Mangalia, during March–April 2010. Algal material was
sorted, washed and weighed. For the determination of heavy metals, samples
were mineralised by dry mineraliastion method according to standardised meth-

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ods4. Following amounts were used: sample 1 – Cystoseira barbata = 10.9201 g;
sample 2 – Ceramium rubrum = 11.2702 g; sample 3 – Ulva lactuca = 9.7628 g.
The determination of heavy metals in algae samples was done by flame atomic
absorption spectrometric (FAAS). The determined metals were: Zn (λ = 213 nm),
Cu (λ = 324 nm), Pb (λ = 217 nm) and Cd (λ = 228 nm).
To determine antioxidant capacity of the biological samples6–9, well-dried
algae were ground, triturated to fine powder, then a cold extract of each species
of algae was got, using 20 mg of dried plant product dissolved in 1 ml ethyl alco-
hol p.a. Merck. Extraction was performed for 24 and 72 h, respectively, at room
temperature in amber glass bottles, sealed. Mixtures were shaken regularly and
separation of extracts was done by decanting, without filtering. For each algal
extract, antioxidant capacity was determined using the standardised method ACL
(antioxidant capacity of lipid soluble substances), Analytik Jena10 by comparison
with standard substance Trolox® (Hoffman – La Roche trade name – 6-hydroxy-2,
5,7,8-tetramethylchroman-2-carboxylic acid), which is vitamin E derivative, used
for calibration curve, and was quantified as equivalent unit of standard substance.
For the calibration curve, standard reagent kit, Analytik Jena Germany Standard
was used: R1 (dilution solvent), R2 (buffer reagent), R3 (photosensitive reagent),
R4 (reagent sized). The calibration curve was constructed by measuring a series of
standard solutions containing 0.5, 1.0, 2.0, 3.0 nmol Trolox (suitable for 5–30 μl
R4), as in Fig. 4.

Fig. 4. Calibration curve for standard Trolox (ACL method, Analytik Jena)

Exposure to external radiation from a Hg lamp lined with phosphorus

that provides the maximum energy at λ = 351 nm, photosensitive reagent,
produces free radicals in the sample for analysis, resulting a photochemi-
cal reaction. The free radicals (superoxide anion radicals), produced by optical
excitation of a photosensitised substance added in standardised volumes, are

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partially eliminated by reaction with the antioxidants present in the sample. The
residual radicals cause the detector substance to luminescence, which is then
exactly determined in a separate cell by means of a photomultiplier tube. The
measuring signal produced by the luminescence is traced over a selectable period
(1–3 min). Depending on the measuring system used, the measuring curves show a
varied behaviour. The total antioxidant capacity of the sample is measured by
converting the electrical signal which is then converted into concentration
values and is quantified by comparison with the standard Trolox® in equivalent
units of standard.
Apparatus. High-resolution continuum source atomic absorption spectrophotom-
eter ContraA 700, Analytik Jena AG, Germany, 2008; photochemiluminometer
PHOTOCHEM Analytik Jena, Germany, 2008.

Results and discussion

The results of heavy metals determination in macro-algae samples studied are
presented in Table 1.

Table 1. Heavy metal content of marine macro-algae

Algae type Pb (mg/kg) Cd (mg/kg) Cu (mg/kg) Zn (mg/kg)
Cystoseira barbata 2.15 0.195 3.905   1.82
Ceramium rubrum 4.95 0.206 5.43   5.51
Ulva lactuca 1.98 0.179 3.65 18.25

A higher content of heavy metals is found in red algae Ceramium rubrum

than in brown algae Cystoseira barbata, in particular because of biogeographi-
cal disposition of these algae in the sea. Barbata cystoseira algae lives in coastal
areas, in small depths, with sunlight penetration and, therefore, some of these
metals within the biogeochemical cycle participate in photosynthesis. The red
algae Ceramium rubrum lives below depths where sunlight does not penetrate
and where photosynthesis is not carried out due to specific Black Sea currents
deposited and sediments of heavy metals are formed. Compared with the other 2
species, green algae Ulva lactuca is very resistant in polluted environments, and
presents the lowest content of metals Pb, Cd, Cu, but increased Zn content (the
highest values). It was noted that the concentration limits of heavy metals in algae
treated in this study are comparable with literature data and are in accordance with
current regulations for heavy metal concentrations in seaweed.
Antioxidant capacity of algae samples was reported at the time of extraction
and sample volume used in the works, according to the ACL method quantified by
comparison with standard substance Trolox®. The results are expressed in nmol/
sample, Trolox equivalent units, according to Fig. 5 and Table 2.

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Fig. 5. Antioxidative capacity (nmol) for marine algae extracts
(ACL method, Analytik Jena)

Table 2. Antioxidant capacity compared to Trolox (ACL method)

Sample Extraction Volume of Analysis time Trolox equi- Trolox equi-
(algae type) time (h) sample used (s) valent units valent units
(μl) (nmol/volume (nmol/g dry
sample) sample)
Cystoseira barbata 24 20 120 1.058   52.9
Ceramium rubrum 24 20 120 1.042   52.1
Ulva lactuca 24 20 120 0.681   34.05
Cystoseira barbata 72 20 120 2.83 141.5
Ceramium rubrum 72 20 120 2.64 132
Ulva lactuca 72 20 120 1.24   62

The highest antioxidant capacity was recorded in the alga Cystoseira barbata,
both at 24 and 72 h, as compared with the obtained values for Ceramium rubrum
and Ulva lactuca algae. The Ulva lactuca algae shows the lowest antioxidant capac-
ity both at 24 and 72 h, respectively. This behaviour could be correlated with the
highest Zn content which acts as pro-oxidant, thus counteracting the antioxidant
capacity of the organic matrix.

CONCLUSIONS
A comparative study on heavy metal content of seaweeds and their antioxidant
capacity has been performed. Three species of macrophyte algae from the Ro-
manian Black Sea littoral: brown alga Cystoseira barbata, red algae Ceramium

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rubrum and green algae Ulva lactuca, which satisfy the conditions necessary for
organisms to be used in monitoring programs, were analysed.
The heavy metals contents (Pb, Cd, Cu and Zn) in algae samples studied are
within the limits permitted by the current standards. Of the three species studied,
algae Ceramium rubrum shows the highest content of heavy metals.
All three algae samples show high antioxidant activity, most intensive being
at the species Cystoseira barbata.
The lowest antioxidant activity of Ulva lactuca algae could be correlated with
the highest content in Zn which acts as pro-oxidant agent.
This study could be used for capitalisation of these marine algae species in
the treatment of degenerative diseases.
Acknowledgements. The scientific investigations were funded by Norway Grants Program – Innova-
tion Norway, ‘Norwegian Cooperation Programs for Economic Growth and Sustainable Development
in Bulgaria and Romania’, Project No KNRIN 2008/115241.

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Revised 18 July 2010

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