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Synechocystis sp.:Spirulina sp.:Porphyridium purpureum:

Microalgal Extracts asFunctional Food Supplements:

Experimental Approaches for the

Evaluation of Antioxidant ActivityBackground of the study

Clinical trials and epidemiological studies habe establishedan inverse correlation between the intake of fruits andvegetables and the occurrence of diseases such asinflammation, cardiovascular disease, cancer, and aging-related disorders [Willet, 2001]. One hypothesis that has beenadvanced is that the protection provided by fruits, vegetables,and drinks such as tea or red wine, can be attributed to a largeclass of antioxidant phytochemicals contained in these foodsand drinks [Prior, 1999]. Beside fruits and vegetables alsomicroalgae such as Chlorella or Spirulinaare a known sourceof antioxidants [Wu, 2005]. Their selective cultivation underhighly standardized conditions in photobioreactors representsa major technological advantage.

Antioxidants

A biologically relevant definition of antioxidants is syntheticor natural substances added to products to prevent or delaytheir deterioration by action of oxygen in air. In biochemistryand medicine, antioxidants are organic substances, , thatare capable of counteracting the damaging effects of oxidationin animal tissues [Huang, 2005]. Physically, they can beclassified by their solubility into (i) hydrophilic antioxidantssuch as vitamin C and the majority of polyphenolic compoundsand (ii) lipophilic antioxidants, mainly including vitamin E andcarotenoids. Both types of antioxidants play an important rolein a wide spectrum of biochemical and physiological processes.Of primary interest is their o ptimal antioxidant activity in vitroand in vivo. Lipophilic antioxidants penetrate the lipoproteincell membrane more easily and therefore can reach a highlevel of bioavailability. In contrast, hydrophilic antioxidants donot accumulate in the body but are excreted rapidly [Huang,2002]. However, they are important as functional additives inbeverage industry.

Antioxidant activity assays

There are a number of established methods available formeasuring antioxidant activity in vitro, such as the ABTS, ORAC,and DPPH assay [Huang, 2005]. However, it is not a trivial taskto accurately measure antioxidant activity since crude extractsof microalgae generally present a complex composition ofdifferent compounds acting as antioxidants. Depending on thereactions involved, antioxidant activity assays can roughly beclassified into two different types: assays based on hydrogenatom transfer (HAT) and those based on electron transfer (ET).Moreover, some assays are conducted in aqueous systems,others in organic solvents. In view of this, the application of

just a single antioxi dant a ctivity assay using one chemicalreaction and solvent seems to be rather unrealistic and maygive rise to misleading results.

Objectives of this study

to establish a battery of (bio-)chemical antioxidant assayscovering different reaction mechanisms and being applicableto extracts in water as well as organic solvents

to establish an integrative data analysis scheme summarizingthe results of the different assays

to test the feasibility of this approach by employing it for:i. the selection of microalgae species with superior

antioxidant activityii. testing culture conditions favoring high antioxidant activity

Materials & Methods

Microalgae extracts:Dried algal powder was resuspended in different solventsdepending on the assay and homogenized with an T-25UltraTurrax on ice for 30 sec. Crude extracts were centrifugedfor 10 min at 4000 g. The supernatants were filtered (0,2 mPTFE filter), stored on ice and used for testing on the sameday.

Biochemical antioxidant activity assays:The ABTS assay was conducted according to Reet al., 1999 andWu et al., 2005 with some minor modifications. The DPPH assaywas conducted according to Milleret al., 2001 and Wu et al.,2005 with some minor modifications. The ORAC assay wasconducted according to Prior et al., 2003 with some minormodifications. All tests were performed in 96-well plates.

Voltammetry (VM):Voltammetry has first been introduced in 2001 by Buratti andcoworkers as a novel electrochemical method to evaluate theantioxidant activity of lipophilic compounds in crude vegetableextracts. Up to now, this method has not bee n widely used forthis application. Nevertheless, unlike nearly all other methodsfor measuring antioxidant activity, it does not employ aspectrometric endpoint and, therefore, works well with turbid

and coloured samples. In the study presented here, aComputrace 797 VA instrument from Metrohm was usedaccording to the manufacturers instructions with a glassycarbon working electrode, a Pt counter electrode and a Ag/AgClreference electrode. Voltage range was set to 0.3 1.2 Voltsin order to exclude the influence of metals present in themicroalgal extracts.

Data Analysis:An A rea Under Curve approach was used in all four assays.Results obtained with different microalgae species or cultureconditions respectively were ranked according to theircorrespondingArea Under Curvevalue. Finally, rankings wereplotted as spider charts on axes starting from the same origin.

Results

Fig. 1:Antioxidant activity in the DPPH assay (mechanisticallyET based) of methanolic extracts from Anabaena sp., Isochrysisgalbana, Nannochloropsis salina, Phaeodactylum tr icornutum,

Porphyri dium purp ureum, Spirulina sp. and Synechocy stis sp.

Fig. 2:Antioxidant activity in the ABTS assay (HAT based) ofaqueous extracts from Isochrysis galbana, Nannochlo ropsissalina, Phaeodactylum tricornutum, Porphyr idium p urpureum,

Spirul ina sp. andSynechocysti s sp.

Fig. 3:Antioxidant activity in the ORAC assay (HAT based) ofextracts (acetone/water, 1+1) from An abaena sp., I sochrysisgalbana, Nannochloropsis salina, Phaeodactylum tr icornutum,

Porphyri dium purp ureum, Spirulina sp. andSynechocysti s sp.

Fig. 4:Voltammograms of extracts (ethanol/toluol/H2SO4) from1: Haematococcus pluvi alis, 2: Synechocy stis sp., 3: An abaenasp., 4: Spirulina sp.,5: Nannochlo ropsis salina, 6: Porphyridiumpurpureum.

Fig. 5:Antioxidant activity of extracts from 3 different micro-algae species cultivated in full medium (F medium [Guillardand Ryther, 1962]) obtained with the ABTS, DPPH, ORAC assayand the voltammetric approach (VM). Synechocystis(right) andSpirul ina both displayed a significantly higher antioxidantactivity in all assays as compared to Porphyridium(left).

Fig. 6:Antioxidant activity of extracts from Nannochloropsissalina cultivated in F medium (left) and F medium containingonly 1/4 of regular phosphate (mid) a nd F medium containingonly 1/8 of regular nitrate concentration (right) respectively.Obviously, a reduction in nitrate or phospate leads to a dropof overall antioxidant activity in Nannochloropsis.

Conclusions

applying a battery of antioxidant assays covering differentreaction mechanisms and solvents seems to be a morereliable approach

individual results can clearly be summarized in spiderdiagrams for comparative evaluations of different algaespecies or culture conditions

compared to the other assays investigated, the ORAC assayshowed a rather narrow dynamic range in its response tosamples differing in antioxidant activity. However, this might

be overcome by further improvements. the voltammetric approach seems to be rather promising,since it gave results similar to the standard assays withoutany interference with turbid or coloured algal extracts.

Authors:Marion Justen, Michael Schrder,Dieter Pollet,University of Applied Sciences,Darmstadt;Maren Hoffmann,Research and Technology Centre,Kiel University, Bsum;Kai Marxen,Sebastian Lippemeier,BlueBioTech GmbH, Bsum

Contact:Prof. Dr. D. PolletUniversity of Applied Sciences DarmstadtDepartment of Chemical Engineeringand BiotechnologySchnittspahnstrasse 12D-64287 DarmstadtGermanyPhone +49 (0)6151 168226Fax +49 (0)6151 168404Email: pollet@h-da.de