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Bakthavachalam Babu
Research Center for Biodiversity, Academia Sinica, Nankang, Taipei 115, Taiwan
Butylated hydroxytoluene (BHT) is one of the syn- oxygen species; SOD, superoxide dismutase;
thetic antioxidant agents commonly used for food TBHQ, t-butylhydroquinone; TLC, thin layer
additives. In the present study, we determined that chromatography
four freshwater phytoplankton, including a green
alga (Botryococcus braunii Kütz.) and three cyanobac-
teria [Cylindrospermopsis raciborskii (Wolłosz.) Seen- Reactive oxygen species (ROS), such as the super-
aya et Sabba Raju, Microcystis aeruginosa (Kütz.) and oxide anion, hydroxyl radicals, and hydrogen perox-
Oscillatoria sp.] were capable of producing this com- ide, cause lipid oxidation and thereby decrease the
pound. Hexane extracts from all the studied species nutritional value and appearance of food. In addi-
exhibited various degrees of antioxidative properties tion, ROS are the major cause of qualitative decay
when they were tested with the b-carotene-linoleate of food, which leads to rancidity, toxicity, and
(b-CL) assay and the 2,2-diphenyl-1-picrylhydrazyl destruction of major biochemical components that
(DPPH) free-radical-scavenging assay. The highest are important in metabolism (Park et al. 2005).
antioxidant activity was observed in the crude Breimer (1990) stated that oxidative stress and ROS
extracts of M. aeruginosa and B. braunii, which dis- play an important role in the etiology and progres-
played a similar activity to synthetic BHT. Gas chro- sion of major human degenerative diseases. Free-
matography ⁄ mass spectroscopy (GC-MS) analysis of radical-mediated modifications of DNA, proteins,
the purified fractions revealed that the active com- lipids, and small cellular molecules are associated
pound was identical to synthetic BHT. Culturing with a number of pathological processes, including
under various irradiances gave rise to different mag- atherosclerosis, arthritis, diabetes, cataractogenesis,
nitudes of BHT production in cyanobacterial cells, muscular dystrophy, pulmonary dysfunction, inflam-
showing that more BHT was produced in the cells matory disorders and ischemia-reperfusion tissue
irradiated with a higher light intensity, and its pro- damage, and neurological disorders such as Alzhei-
duction was irradiance dependent. Moreover, the mer’s disease (Frlich and Riederer 1995). Food
quantity of cellular BHT displayed a positive correla- manufacturers add many commercially available syn-
tion with the antioxidative activity of the tested spe- thetic antioxidant agents, such as butylated hydroxy-
cies. The present study confirms the production of anisole (BHA), BHT, t-butylhydroquinone (TBHQ),
BHT in all four of the studied freshwater phyto- and propyl gallate (PG) to food products to prevent
plankton and suggests that these species constitute a lipid oxidation (Wanita and Lorenz 1996). In addi-
potential source for producing natural BHT. tion, ingestion of these synthetic antioxidants may
Key index words: butylated hydroxytoluene; cyano- decrease the risk of developing the chronic diseases
bacteria; green alga; irradiance; natural antioxidant mentioned above (Gale et al. 1995, Hoffman and
Garewal 1995). However, the U.S. Food and Drug
Abbreviations: b-CL, b-carotene-linoleate; BHA, Administration (FDA) has restricted the use of
butylated hydroxyanisole; BHT, butylated hy- synthetic antioxidants in food products because
droxytoluene; DM, dicyclopentenyloxyethyl meth- of their suspected carcinogenicity (Ito et al. 1986,
acrylate; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EI, Wattenberg 1986, Madhavi and Salunkhe 1995,
Electron Impact; FDA, Food and Drug Adminis- Hettiarachy et al. 1996). Therefore, the search for
tration; GC-MS, gas chromatography-mass spec- natural antioxidants as a substitute for synthetic
trometry; NIST, National Institute of Standards ones has become an important goal for the food
and Technology; PG, propyl gallate; ROS, reactive technology industry in recent years.
Both algae and cyanobacteria are considered to
1
Received 6 December 2007. Accepted 21 May 2008. be commercially potential sources of antioxidants
2
Author for correspondence: e-mail jtwu@gate.sinica.edu.tw.
1447
1448 BAKTHAVACHALAM BABU AND JIUNN-TZONG WU
(Fujimoto and Kaneda 1984, Cahyana et al. 1992, 1983) in the laboratory at 25 ± 1C under continuous illumi-
Pinero Estrada et al. 2001). Although most photosyn- nation with 75 lmol photons Æ m)2 Æ s)1 and aeration with
thetic organisms are exposed to a combination of compressed air.
Culture conditions. Before the experiments, the strains were
light and high oxygen concentrations, which leads to grown exponentially by repeated (at least two times) inocula-
the formation of free radicals and other strong tion into fresh medium. During the exponential phase of each
oxidizing agents, they seldom suffer from serious strain, 50 mL inoculums were transferred to 2 L medium
photodynamic and DNA damage in vivo, suggesting (in triplicates) and incubated under constant illumination
that their cells contain protective antioxidative (by daylight fluorescent lamps) at three different irradiances:
mechanisms and compounds (Sukenik et al. 1993, 25, 75, and 150 lmol photons Æ m)2 Æ s)1. The cell mass was
harvested by centrifugation (himac-CR21G centrifuge, Hitachi
Matsukawa et al. 1997, Sinha et al. 2003, Park et al. High-Technologies Co. Ltd., Minato-Ku, Tokyo, Japan) at
2005, Sigaud-Kutner et al. 2005). Recently, research- 10,000g for 15 min. All of the collected cell masses were
ers have isolated various types of antioxidant com- lyophilized and stored at )70C for further use.
pounds from different algal species, including Extraction of algal samples. One gram of each of the dried
fucoxanthin in Hijikia fusiformis (Yan et al. 1999); algal samples was extracted with hexane at room temperature
phycocyanin and phycocyanobilin in Spirulina and kept in a shaker. The extraction was repeated until the
platensis and Aphanizomenon flos-aquae (Bhat and Mad- sample became colorless. The extracts were condensed in a
vacuum rotary evaporator (Rotavapor-R, Buchi Labortechnik
yastha 2000, 2001, Hirata et al. 2000, Romay and AG, Postfach, Flawil, Switzerland) and dried using a stream of
Gonzalez 2000, Benedetti et al. 2004); phenolic acids, nitrogen gas.
tocopherols, and b-carotene in Spirulina maxima Partial purification of extracts by TLC. Hexane extracts of
(Miranda et al. 1998); fatty acids, polyphenols, and samples were used for partial purification through silica gel
phlorotannin in Sargassum kjellamanianum, S. siliqua- TLC. The dried crude extracts were dissolved in a small volume
strum, Rhodomela confervoides, Symphjocladia latiuscula, of chloroform:methanol (1:1 v ⁄ v), applied to the TLC plates,
and chromatographed using acetone:methanol:water (40:20:1
and Kappaphycus alvarezzi (Yan et al. 1996, Lim et al. v ⁄ v ⁄ v) as the developing solvents. Bands with different Rf values
2002, Huang and Wang 2004, Fayaz et al. 2005); were scraped out separately and extracted according to Bligh
lutein in Botryococcus braunii (Rao et al. 2006); and and Dyer (1959) and centrifuged. The desired ratio of solvents
carotenoids in Dunaliella salina (Ben-Amotz and was obtained by adding the appropriate volume of chloroform
Avron 1988, Neuman et al. 1999, Levy et al. 2000, and water to arrive at a final ratio of chloroform:metha-
Herrero et al. 2006). Our preliminary tests found nol:water (1:1:0.9 v ⁄ v ⁄ v). The chloroform extracts were evap-
orated to dryness. The partially purified dried fractions of all
that certain bloom-forming algae or cyanobacteria samples were tested for antioxidant activity and analyzed for
exhibited strong antioxidation activity, possibly chemical constituents by GC-MS.
because they have to survive in strong light and GC-MS analysis. The Agilent GC-MS system, which consists
oxidative conditions when they float on the water of a GC-6890N equipped with a 5975 MSD and installed with
surface (data not shown). In the present study, we an HP-5 MS ultra low-bleed 5%-phenyl column (30 m ·
further assayed the antioxidant capability of four 0.32 mm · 25 lm; J&W Scientific, Folsom, CA, USA), was used
bloom-forming species, namely, B. braunii, C. racibor- for quantitative and qualitative analyses in this study. The oven
temperature was programmed to rise from 70C (2 min
skii, M. aeruginosa, and Oscillatoria sp., by means of isothermal) to 250C at a ramp of 10C per min and held at
the b-CL model assay and the DPPH free-radical- 250C for 5 min. The injector temperature was kept at 250C.
scavenging assay. The crude extracts were partially The Electron Impact (EI) mass spectra were obtained with
purified through thin-layer chromatography (TLC) 70 eV electron energy, and the MS spectra were scanned in the
and then subsequently by GC-MS to identify the anti- mass range of m ⁄ z from 50 to 550. Helium gas was used as
oxidant compounds. Synthetic compounds were used the carrier gas at a flow rate of 1 mL Æ min)1. One microliter of
the algal crude extract and the partially purified TLC fraction
for comparison. sample were injected into the injector using the splitless mode.
DM was used as an internal standard. Identification of
MATERIALS AND METHODS compounds was based on comparison of their retention
Chemicals. Linoleic acid, Tween 40 (polyoxyethylene sorbi- indices and mass spectra with those of the Wiley and National
tan monopalmitate), BHT, DPPH, dicyclopentenyloxyethyl Institute of Standards and Technology (NIST) mass spectral
methacrylate (DM), and b-carotene were purchased from databases and a synthetic BHT standard under the same
Sigma Chemicals Co. (St. Louis, MO, USA). All the other chromatographic conditions.
organic chemicals and the silica gel TLC plate (60 F254, Antioxidant assay. The antioxidant activity of the crude
20 · 20 cm) used in this study were of analytical grade from extracts and the partially purified fractions of the algal and
Merck (Darmstadt, Germany). cyanobacterial samples was determined using the DPPH free-
Samples for tests. Four freshwater algal strains were used for radical-scavenging assay and the b-CL assay. A synthetic BHT
the present studies: a green alga, B. braunii, and three species standard was used as the control.
of cyanobacteria, C. raciborskii, M. aeruginosa, and Oscillatoria sp. b-CL assay. The antioxidant activity of the studied samples
The cell mass of B. braunii (Trebouxiophyceae) was collected was evaluated by a b-CL assay model with a slight modification
by a plankton net (pore size 55 lm) in Li-Yu Lake (Jayaprakash et al. 2001). A mixture of 0.2 mg of b-carotene,
(2355¢45¢¢N, 12130¢33¢¢E), Taiwan, when it bloomed in the 20 mg of linolenic acid, and 200 mg of Tween 40 (polyoxy-
spring of 2006. The cyanobacteria species were collected from ethylene sorbitan monopalmitate) was mixed with 0.5 mL
water reservoirs in Taiwan when they formed water blooms in of chloroform. The chloroform was then removed at 40ºC with
the summer of 2004. After isolation, the three cyanobacteria a vacuum rotary evaporator. After the removal of chloroform,
were mass-cultured in an inorganic medium (Jüttner et al. the residue was immediately diluted with 10 mL of distilled
A N T I O X I D A N T F R O M FR E S H W A T E R P H Y T O P L A N K T O N 1449
and BHT content, suggesting the dependence of of unsaturated lipids (Kaur and Perkins 1991), and
the production of BHT on irradiance. antioxidant compounds are believed to intercept
To further elucidate the role of cellular BHT in the free-radical chain of oxidation and to donate
the antioxidative capability of organisms, the BHT hydrogen from the phenolic hydroxyl groups,
content in all of the studied species was plotted thereby forming a stable end product that does not
against the antioxidative activity assayed either by initiate further oxidation of the lipid (Sherwin
the b-CL method or by the DPPH method. Figure 5 1978). The results of the present study suggest
shows that the species that produced more BHT that the extracts of all four freshwater algae are
displayed a higher antioxidative activity, indepen- free-radical inhibitors and primary antioxidants that
dently of the assay methods. Apparently, a positive can react with free radicals. Of the four species, the
correlation exists between antioxidative activity and extracts of M. aeruginosa and B. braunii exhibited
the quantity of BHT produced in all of the four higher antioxidant activity than those of C. raciborskii
studied species. and Oscillatoria sp. The antioxidant activity was
observed to increase with an elevated test dose.
Based on the GC-MS analysis, the antioxidant
DISCUSSION
compound observed in the algal species is identical
Blooms of Microcystis, Cylindrospermopsis, and to that of a synthetic BHT, as it not only shared the
Oscillatoria are commonly associated with toxic events same retention time in the total ion chromatogram
in the aquatic environment due to production of but also had similar mass spectral data. The present
compounds such as anatoxins, cylindrospermopsins, study thus revealed that all the four algal species
and microcystins (Falconer 2001). These toxins cause produce a natural BHT that exhibits antioxidant
mortality in animals as well as human illness (Carmi- activity similar to that of synthetic BHT. Thus, these
chael and Falconer 1993, Ueno et al. 1996). Oxida- algal species have the potential to be used as an
tive damage might be involved in the toxic effects of alternative commercial source for BHT production.
microcystins, because increased lipid peroxidation Khotimchenko and Yakovleva (2004) reported the
and oxidative stress generation have been observed production of ROS in algae under high irradiances.
in animals or cells exposed to microcystins (Ding In general, algae show two major types of defense
et al. 2000, Botha et al. 2004, Bouaicha and Maatouk mechanism against ROS, the enzymatic and the non-
2004, Li et al. 2005). The antioxidant potential is enzymatic antioxidant systems. The enzymatic mecha-
assumed to be attributable to their defense mecha- nism studied in Anabaena, Microcystis, and Nodularia
nism acquired against the toxic substances, or in pre- showed an increase in superoxide dismutase (SOD)
venting photodynamic and DNA damage caused by and ascorbate peroxidase activity at high irradiance
exposure to varying light intensities and high oxygen (Canini et al. 1991, 1996, 1998, 2001). The nonenzy-
concentrations (Sukenik et al. 1993, Matsukawa et al. matic antioxidant mechanism observed in Ulva fene-
1997, Sinha et al. 2003, Sigaud-Kutner et al. 2005). strata shows variations in the lipid composition at
The b-CL assay was applied to determine the different irradiances, which protect the cell from
antioxidation activity of B. braunii, C. raciborskii, photooxidation (Khotimchenko and Yakovleva
M. aeruginosa, and Oscillatoria sp. The b-CL assay 2004). Tedesco and Duerr (1989) found that high
illustrated that the extracts of all four tested species irradiance (1,411 lmol photons Æ m)2 Æ s)1) increased
inhibited the discoloration of b-carotene, hence total lipids and the percent composition of polyunsat-
displaying positive antioxidation activity. The dis- urated fatty acids, such as b-linolenic acid, in Spirulina
coloration of b-carotene is a free-radical-mediated platensis. Walsh et al. (1997) reported that the
process resulting from the hydroperoxide donated by variation in the fatty acid composition in Microcystis
linoleic acid. The free radicals donated by linoleic aeruginosa was due to high levels of irradiance, which
acid strike the highly unsaturated b-carotene mole- occurred as a cellular response to reduce the suscep-
cules to cause loss of its double bonds via oxida- tibility of the algal membranes to photooxidation.
tion. As a result, b-carotene molecules lose their In the present study, high irradiance lowered the
chromophore and their characteristic orange color, biomass production but induced a higher production
which can be monitored spectrophotometrically of BHT. Apparently, the amount of BHT produced
(Jayaprakash et al. 2001). The presence of an antioxi- by the cells is related to irradiance, rather than to
dative compound, however, can hinder b-carotene growth rate or biomass. Thus, the production of BHT
bleaching by neutralizing, to some degree, the linole- exhibits a light dependency and is considered to
ate free radical and other free radicals formed in this protect the cells from photooxidation.
system: the slower the discoloration, the stronger the In conclusion, the extracts of the four species
antioxidant activity for a test species. studied exhibited moderate to strong antioxidant
The DPPH assay exhibited radical-scavenging activities. The variation in activity was related to the
activity in all the four algal species. The hydrogen- quantity of BHT produced in the cells. Both
donating ability of the extracts is responsible for the M. aeruginosa and B. braunii contained similar
scavenging activity (Shimada et al. 1992). Free amounts of this compound and exhibited an anti-
radicals in food are known to induce auto-oxidation oxidant activity comparable to that of synthetic
A N T I O X I D A N T F R O M FR E S H W A T E R P H Y T O P L A N K T O N 1453
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