of conjugated dienes was lowest in the oil with sage and sweet grass extracts. Conjugated trienes, which can be measured spectrophotometrically at 268 nm, as.
Assessment of Antioxidant Activity of Plant Extracts by Different Methods P.R. Venskutonis and D. Gruzdienė Department of Food Technology Kaunas University of Technology Lithuania
D. Tirzite and G. Tirzitis Latvian Institute of Organic Synthesis Riga Latvia
Keywords: radical scavenging, xanthine oxidase inhibition, reduction of ferric ions, rapeseed oil oxidation Abstract The aim of this study was to assess the radical scavenging and antioxidant activity (AA) of several aromatic and medicinal plant extracts by different methods: measurement of free radical scavenging activity (RSA) [reactions with 1,1-diphenyl2-picrylhydrazyl radical (DPPH•) and 2,2’-azinobis(3-ethylbenzo-thiazo-line-6sulfonate) (ABTS•+)]; assessment of the influence of extracts on the enzyme xanthine oxidase (XO), their effects in carotene-methyl linoleate cooxidation system and Fe3+ reducing ability; evaluation of the effect on oilseed oil stabilization (peroxide value, binding of oxygen, UV absorption). It was demonstrated that the use of different methods is very advisable. This approach opens the possibility for more comprehensive characterization of extract role in the complicated oxidation processes. For instance, the comparison of RSA obtained in DPPH and ABTS test systems did not show linear relationship. Rosemary, lemon balm and peril extracts possessed well-expressed RSA with DPPH, but they were not so active in ABTS test system; savory extract had insignificant RSA with DPPH, but in ABTS test system was more active. The relationship between RSA and extract AA in oil was also rather complicated. Some extracts were quite good radical scavengers (similar to rosemary and sage), whereas their effect on oil stabilization was low or medium. XO is one of the most important enzymes producing O2-. in vivo, therefore, its inhibitors in some cases could be beneficial. The most effective inhibitors of XO were extracts from sage, oregano, Roman chamomile, tansy, rosemary, costmary, lemon balm, thyme, birch, hyssop, nettle and peril. Plant extracts also were able to reduce Fe3+. INTRODUCTION One of the natural mechanisms used by all aerobic organisms, including humans, to counteract autoxidation/peroxidation is utilisation of a series of antioxidant defence systems, which can be non-enzymatic and enzymatic (Halliwell and Gutteridge, 1999). In food science, antioxidants are usually comprehended as “naturally present or added substances that retard the onset or slow down the rate of oxidation of oil or food lipids” (Reische et al., 1998). Unfortunately, often only 1-2 methods are used for the determination of antioxidant and/or radical scavenging activity. Such approach sometimes brings to inaccurate results. It is necessary to underline that the radical scavenging activity reflects the activity in one radical generation system but antioxidant activity reflects the inhibiting activity in the complex autoxidation system where the sum of reactions take place. The antioxidants depending on their origin and the way of their preparation can be synthetic and natural. Plant kingdom in general and aromatic, medicinal herbs and spices in particular are an important source of natural antioxidants. Plants biosynthesise a great number of antioxidant compounds which are present in different concentrations and which possess various chemical and physical properties. Therefore, assessment of natural antioxidants is rather complicated task. Various methods can be used for this purpose, in vitro and in vivo, using model and real systems as oxidation substrates. In the present study antioxidant activity of herb and some other plant extracts was assessed by using several methods, both in model systems and edible oils. Such approach
Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry Eds. K.H.C. Başer, G. Franz, S. Cañigueral, F. Demirci, L.E. Craker and Z.E. Gardner Acta Hort. 677, ISHS 2005
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provides more comprehensive information on the antioxidant properties of plant extracts. MATERIALS AND METHODS Dried plants were obtained from Lithuanian Institute of Horticulture and Kaunas Botanical Garden. The majority of dried plants were extracted with acetone resulting in acetone oleoresins (AO) that contain both essential oil and non-volatile compounds. In some cases deodorised extracts (DAE, DE) were prepared by extracting the residue of herbs remaining after distillation from it the volatile oil. Extraction procedures are described in detail elsewhere (Bandoniene et al., 2000, 2002). Several methods were applied in this study for the assessment of radical scavenging/antioxidant properties. Short descriptions of these methods follow. For measuring radical scavenging activity (RSA) against the stable radical N,Ndiphenyl-N’-picrylhydrazyl (DPPH), 0.1 ml of plant extract was added to 2.9 ml of DPPH 10-4 M solution in ethanol and the absorbance (A) was measured at 517 nm after 30 min incubation at 30°C (Brand-Williams et al., 1995). RSA was calculated in percent by the following formulae: RSA = (Acontr. - Asample / Acontr. - Ablank) × 100. In ABTS radical cation method, 300 µl of 0.07 mM solution of methemoglobin in deionised water, 16 µl of extract solution in ethanol and 489 µl of 5 mM PSB (pH=7.4) were placed in 1 ml spectrophotometer cell and afterwards 167 µl of 0.1 M H2O2 added (Rice-Evans and Miller, 1994). The mixture was incubated at 37°C and absorbance was measured at 734 nm after 2 min. Trolox was used as a reference antioxidant. The RSA was expressed in trolox equivalents: RSA = (Acontrol - Asample / Acontrol - Atrolox) × 100. Inhibition of xanthine oxidase was expressed as decreasing of uric acid generation (Noro et al., 1983). The mixture of 2.6 ml of 0.225 M xanthine solution in 0.65 M PBS (pH=7.4) with 0.1 ml of plant extract (30 mg ml-1) in ethanol (control – 0.1 ml ethanol) was incubated 5 min at 37°C. Afterwards 0.2 ml of XO (0.15 U ml-1) in 0.65 M PBS (pH=7.4) was added and absorbance (A) at 290 nm was measured after 5 min. The inhibition was calculated in percent by the formulae: IE = 100 × (Asample - Acontrol)/Asample. Antioxidant activity (AOA) of plant extracts was also measured in widely used for this purpose carotene-methyl linoleate cooxidation system (Dapkevicius et al., 1997). For the measurements of the reduction of ferric ions a mixture of 1.5 ml 6 mM solution of 1,10-phenantroline in ethanol, 1.5 ml of 0.3 M ferric sulphate hexahydrate solution in water and 1.5 ml of test substance was incubated at 37°C in spectrophotometer cell; the absorbance was read at 520 nm after 30 min. (Tirzite et al., 1999). Other analyses were performed in commercial rapeseed oil using standard methods for the determination of peroxide value (PV), p-anisidine value (p-An) and spectrophotometrical purity (IUPAC, 1987). TOTOX values (2PV+p-An) were calculated for the complex assessment of primary and secondary oxidation products. Oxidation process and the effect of some plant extracts also were assessed during oil storage by measuring weight gain which occur due to the binding of atmosphere oxygen (Pokorny et al., 1997). RESULTS AND DISCUSSION Testing of Plant Extracts in Model Systems Radical scavenging activity determined in DPPH reaction system reflects the activity towards the week free radicals, while the reaction in ABTS system provides data on the interaction with more active positively charged radicals. The ability of the substance to reduce ferric ions into ferrous ones shows that it could be an additional source of active peroxidation driving ferrous radicals. Inhibition of XO (or reactivity with superoxide generated by this enzyme) shows that active substance could be promising for the studies in vivo. The results obtained in the above-mentioned systems, their linking and comparison can provide important information for further development of plant extracts and assessment of their antioxidant properties. Fig. 1 clearly demonstrates variations in antioxidant activity of various extracts
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assessed by different methods. Rosemary, sage, oregano, birch leaf and lemon balm extracts were the most effective DPPH scavengers. In the other system, containing ABTS, some other extracts, such as thyme and sweet grass were more effective comparing to rosemary and sage. Birch leaf extract was almost equally efficient in the reactions with both compounds, while rosemary was more effective in the reaction with DPPH as compared to ABTS more than two times, lemon balm more than three times. Abovementioned extracts were also effective in XO reaction. Rosemary, sage, oregano and thyme extracts were effective antioxidants in carotene-methyl linoleate cooxidation system. However, in some cases disagreement between DPPH RSA and XO inhibition can be observed. For instance, peril extract was good DPPH scavenger and poor XO inhibitor; on the contrary, Roman chamomile extract effectively inhibited XO but was weak DPPH scavenger. It is well known that ferrous ions (Fe2+) are very effective peroxidation drivers in lipid systems, including edible oils (Halliwell and Gutteridge, 1999). Ferric ions (Fe3+) are less active. Antioxidants as a rule possess good reducing ability and could be additional reducing power converting Fe3+ into Fe2+. We compared reducing ability of some extracts in the reaction Fe3+ + e (from antioxidants) → Fe2+, monitored by measuring absorbency of 1,10-phenantroline-Fe2+ complex (Fig. 2). Oregano extract was the most effective in reduction of ferric ions followed by lemon balm extract. Antioxidant Activity in Plant Oil Plant oils contain unsaturated fatty acids, which are most sensitive to oxidative degradation. Therefore, plant oils are widely used as a substrate for testing antioxidant activity. The effect of various plant extracts was measured on several important oxidation indicators. All plant extracts, except for tarragon, retarded peroxide formation at 80°C the most effective being sage and sweet grass extracts (Fig. 3). Antioxidant activity of sage was widely investigated previously and reported in numerous studies, while sweet grass antioxidant power and main constituents were discovered recently (Pukalskas et al., 2002). The same extracts were effective inhibitors of peroxide formation at 40°C (Fig. 4). Peril extract being medium strength DPPH scavenger (Fig. 1) had a rather little effect on peroxide formation. Conjugated dienes are formed during fatty acid peroxidation. These species can be measured spectrophotometrically at 234 nm. In general, the results of measurement of conjugated dienes (Fig. 5) were in a good agreement with PV measurements. The amount of conjugated dienes was lowest in the oil with sage and sweet grass extracts. Conjugated trienes, which can be measured spectrophotometrically at 268 nm, as well as p-anisidine value, are related to the formation of secondary oxidation products. The curves representing these indicators in rapeseed oil are quite similar for various extracts (Fig. 6 and 7). Again, sage and sweet grass extracts most effectively retarded oxidation of rapeseed oil. TOTOX value represents formation of both primary (PV) and secondary (p-AnV) oxidation products (Fig. 8). This indicator was the best for the oil with sage and sweet grass extracts. These extracts at the applied concentration were more effective antioxidants compared to synthetic compound BHT. Finally, the effect of the extracts was measured on the rapeseed oil weight increase during its storage at 80°C (Fig. 9). As in previous experiments with rapeseed oil sage and sweet grass extracts were very effective antioxidants. It is interesting to note that in this test deodorised savory extract (DAE) was even more effective antioxidant than sage AO. Sweet grass DAE was also more effective comparing to the extract obtained before deodorisation (AO). It is likely, that during distillation the hydrolysis of some glycosides can take place resulting in the release of antioxidatively stronger aglycones. It was reported that sweet grass contains very strong antioxidant 5,8-dihydroxybenzopyranone and its glycoside; the latter was less powerful radical scavenger comparing to aglycone (Pukalskas et al., 2002).
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CONCLUSIONS It was demonstrated that the use of several test methods for radical scavenging and antioxidant activity provides valuable data for better understanding of purposeful application of the plant extracts. The extracts possessing good radical scavenging activity would be promising for the development as ingredients of functional foods and nutraceuticals, for example with cancer preventing and/or anti ageing properties. The extracts retarding oil oxidation could be used as substitutes for synthetic antioxidants, also improving some consumer-attractive quality attributes, e.g. such as “naturalness”, “absence of synthetic additives”. ACKNOWLEDGEMENTS We wish to thank Dr. P. Viskelis from Lithuanian Institute of Horticulture and Dr. O. Ragazinskiene from Kaunas Botanical Garden for providing plant materials. Literature Cited Bandoniene, D., Pukalskas, A., Venskutonis, P.R. and Gruzdiene, D. 2000. Preliminary screening of antioxidant activity of some plant extracts in rapeseed oil. Food Res. Int. 33:785-791. Bandoniene, D., Venskutonis, P.R., Gruzdiene, D. and Murkovic, M. 2002. Antioxidative activity of sage (Salvia officinalis L.), savory (Satureja hortensis L.) and borage (Borago officinalis L.) extracts in rapeseed oil. Eur. J. Lipid Sci. Technol. 104:286292. Brand-Williams, W., Cuvelier, M.E. and Berset, C. 1995. Use of free radical method to evaluate antioxidant activity. Food Sci. Technol. 28:25-30. Dapkevicius, A., Venskutonis, R., van Beek, T.A. and Linssen, J.P.H. 1998. Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania. J. Sci. Food Agric. 77:140-146. Halliwell, B. and Gutteridge, J.M.C. 1999. Free Radicals in Biology and Medicine. Oxford Univesrsity Press, Oxford. IUPAC. 1987. Standard Methods for the Analysis of Oils, Fats and Derivatives. 7th Revised and Enlarged Ed., Blackwell Scientific Publications, Oxford. Noro, T., Oda ,Y., Miyase, T., Ueno, A. and Fukushima, S. 1983. Inhibitors of xanthine oxidase from the flowers and buds of Daphne genkwa. Chem. Pharm. Bull. 31:39843987. Pokorny, J., Nquyen, H.T.T. and Karczak, J. 1997. Antioxidant activity of rosemary and sage extracts in sunflower oil. Nahrung. 41:176-177. Reische, D.W., Lillard, D.A. and Eitenmiller, R.R. 1998. Antioxidants. p.423-448. In: C.C. Akoh and D.B. Min (eds.), Food Lipids, Marcel Dekker, New York. Rice-Evans, C.A. and Miller, N.J. 1994. Total antioxidant status in plasma and body fluids. Method Enzymol. 234:279-293. Tirzite, D., Tirzitis, G. and Antipova, D. 1999. Reductive ability of 1,4-dihydropyridine derivatives in relation to ions of trivalent iron. Chem. Heterocycl. Comp. 35:592-594.
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Figures
100 90 DPPH
80
ABTS*
XO
AOA
Activity
70 60 50 40 30 20 10
R os
em ar y Sa g Th e y O me re ga no Le B m irc on h ba H lm Sw ys ee sop tg ra R om ss an ca Per m il om ile Ta C n Se os sy tm a bu ar ck y th or C n at ni p N e M t tl ar e jo ra Sa m vo ry
0
Fig. 1. Comparison of antioxidant activity of various plant extracts assessed by different methods (*Trolox RSA=100).
Rosemary Oregano Lemon balm Catnip
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Sage Hyssop Thyme
A (520 nm)
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Fig. 2. Reduction of ferric ions by plant extracts.
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250
Blank Sage AO, 0.1% Sweet grass AO, 0.1% Tarragon AO, 0.1% Lavender AO, 0.1% Borrage AO, 0.1% Lemon balm AO, 0.1% Marjoram AO, 0.1% Lovage AO, 0.1% Costmary AO, 0.1% Savory DE, 0.1% BHT, 0.02%
PV (meq/kg)
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Fig. 3. Effect of extracts obtained from various aromatic herbs on the formation of peroxides in rapeseed oil at 80°C.
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Blank BHT 0.02% Sage AO 0.1% Sage DAE 0.1% Sweet grass 0.1% Borrage 0.1% Lovage 0.1% Costmary 0.1% Tansy 0.1% Peril 0.1% Horehound 0.1%
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PV (meq/kg)
120 100 80 60 40 20 0 0
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20 Time (days)
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Fig. 4. Effect of extracts obtained from various aromatic herbs on the formation of peroxides in rapeseed oil at 40°C.
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17
Blank BHT 0.02% Sage AO 0.1% Sage DAE 0.1% Sweet grass 0.1% Borrage 0.1% Lovage 0.1% Costmary 0.1% Tansy 0.1% Peril 0.1% Horehound 0.1%
16 15 14
Absortion (234nm)
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35
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Time (days)
Fig. 5. Effect of plant extracts on the formation of conjugated dienes (UV absorption at 234 nm) in rapeseed oil.
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Blank Sage AO 0.1% Sweet grass 0.1% Lovage 0.1% Tansy 0.1% Horehound 0.1%
Absortion (268 nm)
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1.3 1.2 1.1 1.0 0.9 0
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30
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40
Time (days)
Fig. 6. Effect of plant extracts on the formation of conjugated trienes (UV absorption at 268 nm) in rapeseed oil.
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15
Blank Sage AO 0.1% Sweet grass 0.1% Lovage 0.1% Tansy 0.1% Horehound 0.1%
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p-AnV
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BHT 0.02% Sage DAE 0.1% Borrage 0.1% Costmary 0.1% Peril 0.1%
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Fig. 7. Effect of plant extracts on p-anisidine value (p-AnV) in rapeseed oil.
300 250
150 100 50
0. 1% gr as s 0. Bo rra 1% ge 0. Lo va 1% ge C 0 os tm .1% ar y 0. Ta 1 ns % y 0. 1% Pe H r il 0 or eh . ou 1% nd 0. 1%
0. 1%
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ee t
e
e
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Fig. 8. Effect of plant extracts on the TOTOX value in rapeseed oil.
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TOTOX
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Control BHT 0.02% Sage AO 0.1% Sage DAE 0.1% Savory AO 0.1% Savory DAE 0.1%
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Time (hrs) Control BHT 0.02% Sage AO 0,1% Sweet grass AO 0.1% Sweet grass DAE 0.1% Costmary AO 0.1% Costmary DAE 0.1%
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101.2 101 100.8 100.6 100.4 100.2 100
0
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Fig. 9. Effect of plant extracts on relative weight gain in rapeseed oil at 80°C temperature.
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