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JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 19 (2006) 531–537
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Critical Review

Spice antioxidants isolation and their antiradical activity: a review

Milan Suhaj
Food Research Institute, Priemyselná 4, 824 75 Bratislava, Slovakia
Received 19 February 2004; received in revised form 8 July 2004; accepted 29 November 2004

Abstract

Antioxidant compounds in food play important roles as health-protecting factors. Antioxidants are also widely used as additives in
fats and oils and in food processing to prevent or delay spoilage of foods. Spices and some herbs have received increased attention as
sources of many effective antioxidants. This review presents some information about the most common and most-used spice
antioxidants, methods of their preparation and describes their antioxidant/antiradical properties.
r 2005 Elsevier Inc. All rights reserved.

Keywords: Spices; Herbs; Antioxidants; Isolation; Antioxidant activity

1. Introduction various methyl and ethyl esters of these substances in

The number of contributions to isolation methods,
techniques and activity testing of plant-origin antioxidants
has significantly increased in recent years. Oxidation is one
of the major causes of chemical spoilage, resulting in
rancidity and/or deterioration of the nutritional quality,
colour, flavour, texture and safety of foods (Antolovich
et al., 2002). There is at present increasing interest both in
the industry and in scientific research for spices and
aromatic herbs because of their strong antioxidant and
antimicrobial properties, which exceed many currently
used natural and synthetic antioxidants. These properties
are due to many substances, including some vitamins,
flavonoids, terpenoids, carotenoids, phytoestrogens, miner-
als, etc. and render spices and some herbs or their
antioxidant components as preservative agents in food
(Calucci et al., 2003).
Except basic plant antioxidants some specific ones are
characteristic for some important aromatic herbs and
spices. Some examples of specific antioxidants are pimentol
from allspice; gallates, biflorin, its isomer eugenol and
eugenyl acetate in clove (Anon, 1997; Lee and Shibamato,
2001; Peter, 2000); carnosol, carnosic acid, rosmanol,
rosmaridiphenol, rosmadial and rosmariquinone, and
Tel.:+421 2 50237146.
E-mail address: milan.suhaj@vup.sk.
rosemary and sage (Bandoniené et al., 2002a,b; Pizzale
et al., 2002); diarylheptanoids, gingerol and zingerone in
ginger (Kikuzaki and Nakatani, 1993; Peter, 2000);
curcumin and tetradehydrocurcumin in turmeric
(Relajakshmi and Narasimhan, 1996); flavonides, ferulic
acid, piperine, phenolic amide feruperine in black pepper
(Peter, 2000; Shahidi and Wanasundara, 1992; Nakatani
et al., 1986); thymol and carvacrol in essential oils from
Algerian origanum (Ruberto et al., 2002); derivatives of
phenolic acids, flavonoids, tocopherols, rosmarinic acid
and carvacrol in oregano (Peter, 2000; Pizzale et al., 2002);
etc. According to a phytochemical database (USDA, 2003),
the number of different antioxidants in some plants can
reach up to 40 (soybean 42, tea 36, fennel 35, oregano 34,
onion 32, thyme 32, etc). In this database, plants with the
highest contents of antioxidants are walnut, betel nut,
guava, coconut, and other less known plants. A list of some
known substances with antioxidant activity in some very
common spices is reported in Table 1. More information is
available for examples on USDA food antioxidant
database (USDA, 2003).
2. Methods of antioxidant isolation from spices
Spices can be added to foods in several forms: as
whole spices, as ground spices, or as isolates from their
extracts. Spices are aromatic and pungent food ingredients,

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doi:10.1016/j.jfca.2004.11.005

532
Table 1
Antioxidant active chemicals isolated from some of the most common and used spices (USDA, 2003)
Spices (vernacular and scientific
names)
Black pepper (Piper nigrum)
Caraway (Carum carvi)
Chilli pepper (Capsicum frutescens)
Coriander (Coriandrum sativum L.)
Dill (Anethum graveolens)
Ginger (Zingiber officinale)
Marjoram (Origanum majorana)
Nutmeg (Myristica fragrans)
Oregano (Origanum Vulgare)
Red (sweet) pepper (Capsicum
annuum)
Rosemary (Rosemarinus officinalis)
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M. Suhaj / Journal of Food Composition and Analysis 19 (2006) 531–537
Antioxidants (name, part of plant, quantity in ppm)
Ascorbic-acid fruit 0–10, beta-carotene fruit 0.114–0.128, camphene fruit, carvacrol fruit, eugenol fruit, gamma-
terpinene fruit, lauric-acid fruit 400–447, linalyl-acetate fruit, methyl-eugenol fruit, myrcene fruit, myristic-acid
fruit 700–782, myristicin fruit, palmitic-acid fruit 12,200–13,633, piperine fruit 17,000–90,000, terpinen-4-ol fruit,
ubiquinone fruit
Beta-carotene fruit 0–2, camphene fruit 1–900, camphene plant 0–30, carvacrol plant, gamma-terpinene fruit
12–14,160, gamma-terpinene plant 0–270, lauric-acid fruit 100–110, myrcene fruit 180–1560, myrcene plant 0–150,
myristic-acid fruit 400–444, myristicin fruit 60–420, myristicin plant 0–120, palmitic-acid fruit 2000–6658,
quercetin fruit, tannin plant, terpinen-4-ol plant 0–120
Alanine fruit 820–6691, ascorbic-acid fruit 350–19,992, beta-carotene fruit 1–38, caffeic-acid fruit 0–32,
campesterol fruit, capsaicin fruit 100–17,900, capsanthin fruit, chlorogenic-acid stem, hesperidin fruit, histidine
fruit 410–3346, kaempferol anther, lauric-acid resin, exudate, sap, lutein fruit, methionine fruit 240–1958, myrcene
fruit, myristic-acid fruit 10–82, myristic-acid seed, p-coumaric-acid fruit 0–540, palmitic-acid fruit 150–1224,
palmitic-acid seed, pentadecanoic-acid fruit, quercetin fruit 0–63, scopoletin fruit, stigmasterol fruit, terpinen-4-ol
fruit, tocopherol fruit 0–24, tryptophan fruit 260–2122
Apigenin fruit, ascorbic-acid leaf 780–6290, beta-carotene leaf 29–228, beta-carotene seed, beta-sitosterol fruit,
caffeic-acid fruit, caffeic-acid leaf, camphene fruit 2–155, chlorogenic-acid plant 305–320, gamma-terpinene fruit
762–2626, isoquercitrin fruit, myrcene fruit 13–169, myristic-acid fruit 200–219, myristicin fruit, p-hydroxy-
benzoic-acid fruit 0–960, p-hydroxy-benzoic-acid plant 252–333, palmitic-acid fruit 5000–16,800, protocatechuic-
acid fruit 0–760, protocatechuic-acid plant 167–179, quercetin fruit, rhamnetin fruit, rutin fruit, scopoletin fruit,
tannin fruit, terpinen-4-ol fruit 6–80, trans-anethole fruit 1–2, vanillic-acid fruit 0–960, vanillic-acid plant 221–347
Alpha-tocopherol leaf 16–147, anethole plant, ascorbic-acid plant 0–1440, beta-sitosterol plant, caffeic-acid fruit,
camphene plant, carvacrol plant, chlorogenic-acid fruit, eugenol plant, ferulic-acid fruit, gamma-terpinene fruit
12–77, histidine fruit 3200–3466, isoeugenol plant, isorhamnetin plant, kaempferol fruit, lauric-acid fruit 100–108,
methionine fruit 1430–1549, myrcene fruit 84–924, myristic-acid fruit 100–108, myristicin fruit, myristicin root,
palmitic-acid fruit 5800–6281, quercetin plant, scopoletin fruit, selenium plant 0.001–0.012, stigmasterol plant,
terpinen-4-ol fruit 12–77, trans-anethole fruit 12–539, vicenin fruit
6-Gingerol rhizome 130–7138, 6-shogaol rhizome 40–330, alanine rhizome 310–1793, ascorbic-acid rhizome 0–317,
beta-carotene rhizome 0–4, beta-sitosterol plant, caffeic-acid rhizome, camphene rhizome 28–6300, capsaicin plant,
chlorogenic-acid plant, curcumin plant, delphinidin plant, ferulic-acid plant, gamma-terpinene rhizome 0.4–25,
rhizome 300–1738, kaempferol plant, lauric-acid rhizome 390–3630, methionine rhizome 130–737, myrcene 2–950,
myricetin plant, myristic-acid rhizome 180–1650, p-coumaric-acid rhizome 0–19, p-hydroxy-benzoic-acid plant,
palmitic-acid rhizome 1200–11,220, quercetin plant, selenium rhizome 10, shikimic-acid leaf, sucrose rhizome,
terpinen-4-ol rhizome, tryptophan rhizome 120–693, vanillic-acid plant, vanillin plant
Ascorbic-acid plant 514–555, beta-carotene plant 48–52, beta-sitosterol plant, caffeic-acid plant, carvacrol plant
1092–6261, eugenol plant 200–1152, hydroquinone plant, linalyl-acetate plant 3–17, myrcene plant 18–103,
oleanolic-acid plant 0–4700, phenol plant 1431–8204, rosmarinic-acid plant 0–33,000, tannin plant, terpinen-4-ol
plant 1365–7826, trans-anethole plant 8–43, ursolic-acid plant 500–2100
Camphene seed 80–640, cyanidin plant, eugenol seed 40–320, gamma-terpinene seed 580–4640, isoeugenol seed
140–320, kaempferol plant, lauric-acid seed 375–1600, methyl-eugenol seed 20–900, myrcene seed 740–5920,
myristic-acid seed 60–304,000, myristicin leaf 410–620, myristicin seed 800–12,800, oleanolic-acid seed, palmitic-
acid seed 25,000–128,000, quercetin plant, terpinen-4-ol seed 600–4800
Camphene plant 0–1, carvacrol plant 0–12, gamma-terpinene plant 0–13, linalyl-acetate plant 0–50, myrcene plant
0–5, terpinen-4-ol plant 0–220, thymol plant
Alanine fruit 350–4774, alpha-tocopherol fruit 22–284, ascorbic-acid fruit 230–20,982, beta-carotene fruit 0–462,
beta-sitosterol plant, caffeic-acid fruit 0–11, campesterol fruit, camphene fruit, capsaicin fruit 100–4000,
capsanthin fruit, chlorogenic-acid fruit, eugenol fruit, gamma-terpinene fruit, hesperidin fruit, histidine fruit
170–2319, lupeol seed, lutein fruit, methionine fruit 100–1364, myrcene fruit, myristic-acid fruit 10–136, p-
coumaric-acid fruit 0–79, palmitic-acid fruit 500–6820, palmitic-acid seed, pentadecanoic-acid fruit, scopoletin
fruit, selenium fruit 0.001–0.002, stigmasterol fruit, terpinen-4-ol fruit, tocopherol fruit 0–24, tryptophan fruit
110–1500
Apigenin plant, ascorbic-acid plant 612–673, beta-carotene plant 19–21, beta-sitosterol plant, caffeic-acid plant,
camphene leaf 0–23, camphene leaf 0–145, camphene plant 23–2350, camphene shoot 355–1435, camphene shoot
620–1260, camphene shoot 1035–2280, carnosic-acid plant, carnosol leaf 530–9803, carvacrol leaf 0–5, carvacrol
leaf 0–6, carvacrol leaf 5–6, carvacrol plant, chlorogenic-acid plant, gamma-terpinene leaf 0–4, gamma-terpinene
plant 4–400, gamma-terpinene shoot 25–50, gamma-terpinene shoot 37–225, gamma-terpinene shoot 105–300,
hesperidin leaf, hispidulin plant, isorosmanol flower 0–17, labiatic-acid plant, luteolin leaf, luteolin plant,
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Table 1 (continued )

Spices (vernacular and scientific Antioxidants (name, part of plant, quantity in ppm)
names)

luteolin-30 -o-(300 -o-acetyl)-beta-D-glucuronide leaf, luteolin-30 -o-(400 -o-acetyl)-beta-D-glucuronide leaf, methyl-eugenol

Sage (Salvia officinalis)
Thyme (Thymus vulgaris)
Turmeric (Curcuma domestica)
plant, myrcene leaf 0–7, myrcene leaf 0–215, myrcene plant 25–5605, myrcene shoot 6–13, myrcene shoot 50–100,
oleanolic-acid plant 0–10,500, oleanolic-acid shoot 0–20, rosmadial leaf 0–30, rosmanol leaf 0–92, rosmaridiphenol
plant, rosmarinic-acid leaf 0–3500, rosmarinic-acid plant 0–25,000, rosmarinic-acid shoot 0–13,500, rosmarinic-acid
tissue culture 0–38,957, rosmariquinone plant, squalene plant, tannin plant, terpinen-4-ol leaf 0–40, terpinen-4-ol leaf
0–85, terpinen-4-ol plant 4–521, terpinen-4-ol shoot 25–240, terpinen-4-ol shoot 55–110, terpinen-4-ol shoot 60–190,
thymol plant, trans-anethole plant, ursolic-acid plant 28,000–41,000, ursolic-acid shoot 0–20
Alanine plant, apigenin plant, ascorbic-acid leaf 320–350, beta-carotene leaf 35–38, beta-sitosterol leaf 5–2450,
beta-sitosterol stem 0–1214, caffeic-acid shoot, campesterol fruit, camphene leaf 20–18,592, carnosic-acid leaf,
carnosol leaf, carnosol plant, carnosolic-acid leaf 0–2100, catechin plant, chlorogenic-acid shoot, cholesterol fruit,
chrysoeriol plant, ferulic-acid plant, fumaric-acid plant, gallic-acid plant, gamma-terpinene leaf 15–140, hispidulin
leaf, labiatic-acid leaf, linalyl-acetate plant 0–6048, luteolin shoot, myrcene plant 0–336, oleanolic-acid leaf
140–786, oleanolic-acid stem 0–400, p-coumaric-acid plant, palmitic-acid seed, rosmanol plant, rosmarinic-acid
plant 0–30,000, rosmarinic-acid shoot 2000–5800, salicylic-acid plant, selenium leaf, stigmasterol leaf 0–5, tannin
plant 20,000–80,000, terpinen-4-ol leaf 10–1120, thymol essential oil, ursolic-acid leaf 1255–1300, ursolic-acid stem
0–200, uvaol plant, vanillic-acid plant
4-Terpineol plant 73–8320, alanine plant, anethole essential oil, apigenin plant, ascorbic-acid leaf, beta-carotene
plant 24–25, caffeic-acid leaf 0–16,900, camphene plant 15–375, carvacrol plant 8–18,720, chlorogenic-acid plant,
chrysoeriol plant, eriodictyol plant, eugenol plant, ferulic-acid plant, gallic-acid plant, gamma-terpinene plant
36–5460, isochlorogenic-acid leaf, isoeugenol plant, isothymonin plant, kaempferol plant, labiatic-acid leaf, lauric-
acid plant 2300–2484, linalyl-acetate plant 15–4680, luteolin plant, methionine plant 1370–1980, myrcene plant
36–676, myristic-acid plant 1500–1620, naringenin plant, oleanolic-acid plant 0–6300 p-coumaric-acid leaf 0–420,
p-hydroxy-benzoic-acid plant, palmitic-acid plant 17,200–18,576, rosmarinic-acid plant 0–26,000, rosmarinic-acid
shoot 5000–13,500, selenium leaf 0–16, tannin plant 80,000–100,000, thymol plant 15–24,100, tryptophan plant
1860–2009, ursolic-acid plant 15,000–18,800, vanillic-acid plant
Ascorbic-acid rhizome 0–293, beta-carotene rhizome, caffeic-acid rhizome 0–5, curcumin rhizome 9–38,888,
eugenol essential oil 0–2100, p-coumaric-acid rhizome 0–345, protocatechuic-acid leaf, syringic-acid leaf, vanillic-
acid leaf

therefore, their direct use as antioxidants is limited. The
extraction procedure is determined by the types of
antioxidant compounds to be extracted. Selection of a
suitable extraction procedure can increase the antioxidant
concentration relative to the plant material. For poly-
phenols and other antioxidants in plant materials three
principal extraction techniques may be used: extraction
using solvents, solid-phase extraction and supercritical
extraction. It is advisable to complete the extraction using
dry, frozen or lyophilized samples since some antioxidants
are unstable or can be degraded by enzyme action in
undried plant material. Ultrasound-assisted extraction is
often used for the extraction of plant material using liquid
solvents. The extraction can be also performed in a Soxhlet
apparatus, thus combining percolation and immersion
techniques. Several extraction techniques have been pa-
tented using solvents with different polarities, such as
petrol ether, toluene, acetone, ethanol, methanol, ethyl
acetate, and water. In addition, supercritical CO2-extrac-
tion and medium-chain triglycerides as carrier in a
mechanical extraction process have been applied (Schwarz
et al., 2001). Extraction using edible oil or fat is a very
simple method. Natural material containing antioxidants,
such as herbs and spices, is mixed with fats and/or oils, and
the mixture is left at a room or moderately increased
temperature for a defined time. The mixture is then filtered
and used (Pokorny et al., 2001).
Methanol is the solvent most commonly employed.
Extraction of anthocyanins is commonly carried out
under cold conditions with methanol containing a small
amount of acid. It has been indicated that methanol is the
best solvent for catechin extraction, whereas a better
yield for procyanidins is obtained with 70% acetone. It has
also been indicated that aqueous methanol, due to its
polarity, extracts polyphenols linked to polar fibrous
matrices more effectively, while acetone/water mixtures
are more useful for extracting polyphenols from proteic
matrices, since they appear to degrade the polyphenol–
protein complexes. Microwave-assisted extraction is a
new extraction technique that combines microwave and
traditional solvent extraction with advantages that include
shorter time, less solvent used, or higher extraction
rate. Pressurized liquid extraction has been recently
introduced for phenolic compound extraction. In this
technique high temperature and high pressure are used to
accelerate the extraction. Very interesting results were
obtained in the study of sage antioxidant activity, where
pressurized hot water extraction was found to be the most
effective extraction procedure, followed by maceration
with 70% ethanol, hydrodistillation, and ultrasonication-
assisted methanol extraction (Ollanketo et al., 2002).
For industrial purposes ethanol would probably be better
than methanol as eventual solvent residues would be less
toxic. A review of some extraction procedures used for
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Table 2
Extraction and isolation procedures of antioxidant chemicals from some of the most common and used spices

Spices Process of extraction References

Basil, black pepper, cinnamon, nutmeg, (1) Trichloacetic acid extract for ascorbate determination, centrifugation Calucci et al. (2003)
oregano, parsley, rosemary, sage 8000 g, filtration
(2) Methanol extract for carotenoids and capsaicinoids determination,
vacuum concentration, filtration
Allspice, clove Methanol extract Anon (1997)
Ginger, turmeric, cayenne pepper, Mechanical medium-chain triglyceride and propylene glycol extracts Schwarz et al. (2001)
rosemary, sage, thyme, oregano, green prepared by hydraulic laboratory press
tea, spice mixture
Clove buds Dichlormethane extract of water steam-distillation, extract reduction by a Lee and Shibamato (2001)
rotary flash evaporator
Sage Crude methanol extract (80% methanol, 20% water) Bandoniené et al. (2002a, b)
Sage Pressurized hot water, maceration with 70% ethanol Ollanketo et al. (2002)
Sage Soxhlet extraction by ethyl acetate, reextraction by petroleum ether Gu and Weng (2001)
Pepper (Capsicum species) Methanol extract (50% methanol, 50% water) Howard et al. (2000)

Ginger, nutmeg, coriander Ethanol extract (96% ethanol, 4% water) Takácsová et al. (1999)
Oregano, sage, thyme Homogenization in phosphate buffer (pH 7), centrifugation Zheng and Wang (2001)

Oregano, sage, thyme SFE under specific operating conditions Nguen et al. (1991)
Sage, rosemary, oregano Soxhlet extraction with pure methanol Ayar et al. (2001)

Basil 80% ethanol extract Juliani and Simon (2002)

Oregano Methanol extract Jun et al. (2001)
Marjoram Ethanolic extract Bendini et al. (2002)
Sage, borage, savory Acetone oleoresins and deodorized acetone extract Bandoniené et al. (2002a, b)
Thyme, basil, rosemary, chamomile, Liquid–liquid continuous extraction following steam distillation under Lee and Shibamato, (2002)
lavender, cinnamon reduced pressure
Sage, oregano Methanolic extracts Pizzale et al. (2002)
Rosemary CO2 supercritical fluid extraction Ibanez et al. (2001)
Oregano, sage, savory Ethanol and acetone extracts of the dried material Exarchou et al., (2002)

Chilly, black pepper, ginger Near-critical carbon dioxide, propane, and dimethyl ether Catchpole et al. (2003)
Chilly, black pepper, turmeric Aqueous extract, homogenization, centrifugation, filtration Sharma et al. (2000)
Cinnamon Fat-free cinnamon Soxhlet extraction with 80% methanol, then n-hexane Bozan et al. (2003)
and ethyl acetate
Pepper (Capsicum sp.) 50% methanol Howard et al. (2000)

preparation of some spice and herb antioxidant substances
is given in Table 2.
The extracts obtained using organic solvents may be
further concentrated, for instance, by molecular distilla-
tion. Essential oils present in spice extracts, are responsible
for the characteristic aroma of the spices and can be
removed by steam distillation at normal atmospheric
pressure or in a vacuum, but antioxidant activity may be
partially lost. Commercial antioxidant extracts from spices
are available in powder form or as oily oleoresins (Pokorny
et al., 2001).
3. Antioxidant/antiradical activity of isolated chemicals
from spices
A variety of tests expressing antioxidant potency of food
components has been suggested. These can be categorized into
two groups: assays for radical scavenging ability and assays
that test the ability to inhibit lipid oxidation under accelerated
conditions (Schwarz et al., 2001). The features of an oxidation
are a substrate, an oxidant and an initiator, intermediates and
final products. Measurement of any of one of these can be used
to assess antioxidant activity (Antolovich et al., 2002).
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M. Suhaj / Journal of Food Composition and Analysis 19 (2006) 531–537
Radical scavenging ability tests aim to simulate basic
mechanisms involved in lipid oxidation by measuring either
the reduction of stable radicals or radicals generated by
radiolysis, photolysis, or other reactions. Hydrogen per-
oxide-scavenging activity using the peroxidase-based assay
system was evaluated by Martinéz-Tomé et al. (2001) for
Mediterranean spices in an aqueous medium, in an amino
acid fraction from aged garlic extract (Ryu et al., 2001) and
for methanol extract of oregano (Jun et al., 2001).
Hydroxyl radical scavenging can often be calculated using
the ‘‘deoxyribose assay’’(Sánches-Moreno, 2002). This
system was used to evaluate antioxidant activities of
certain Mediterranean spices (Martinéz-Tomé et al.,
2001), extract from onion skin (Suh et al., 1999), and
garlic (Zang et al., 1994). Piperine was found to act as a
hydroxyl radical scavenger at low concentrations (Mittal
and Gupta, 2000). Some of the spice extracts (basil,
marjoram, hyssop, summer savory, oregano, sage) exhibit
high scavenging activity for the hydroxyl radicals (Madsen
et al., 1996). Scavenging of hypochlorous acid (HOCl) used
by Martinéz-Tomé et al. (2001) for antioxidant activity
characterization of some Mediterranean spices. The
peroxyl radical is a common free radical found in
biological substrates and used in antioxidant assays. In
the oxygen-radical absorbance capacity (ORAC) determi-
nation phycoerytrin is used as a target of free radicals.
Using this system the antioxidant capacities in extracts of
oregano, thyme and sage were determined (Zheng and
Wang, 2001). Zheng and Wang (2001) stated that the herb
oregano had 3–20 times higher antioxidant activity than
the other herbs studied according to ORAC method and
total phenolic contents. Additionally, oregano has 42 times
more antioxidant activity than apples, 30 times more than
potatoes, 12 times more than oranges and 4 times more
than blueberries (AChSNS, 2002). Nakatani et al. (1986)
determined that all the phenolic amides identified from
black pepper possess significant antioxidant activities that
are more effective than the naturally occurring antioxidant,
alpha-tocopherol, and feruperine has antioxidant activity
as high as the synthetic antioxidants butylated hydro-
xyanizole and butylated hydroxytoluene. When the Trolox
equivalent antioxidant capacity (TEAC) assay was used to
provide a ranking order of antioxidant activity, the result
in decreasing order of antioxidant capacity was cinnamon
approximately equal to propyl gallate4mint4anise4
BHA4licorice approximately equal to vanilla4ginger4
nutmeg4BHT (Murcia et al., 2004). Using of radical
cation ABTS (2,2-azinobis-(3-ethylbenyothiazoline)-6-sul-
phonate) the antioxidant activity of phenolic compounds
from sage (Wang et al., 1998) and basil (Juliani and Simon,
2002) were determined. Scavenging of the stable radical
2,2-diphenyl-1-picrylhydrazyl (DPPH) is considered a valid
and easy assay to evaluate scavenging activity of antiox-
idants, since the radical compound is stable and does not
have to be generated as in other radical scavenging assays.
This assay has been applied to characterize antioxidant
activities of quercetin from onion skin (Suh et al., 1999),
535
polyphenols from sage (Wang et al., 1998), colorant from
curcumin (Noguchi et al., 1994), extract from cardamom
(Kikuzaki et al., 2001), extract from ginger, turmeric, and
cayenne pepper, a mixture of rosemary, sage, thyme, and
oregano (Schwarz et al., 2001), cinnamon (Bozan et al.,
2003), oregano, sage, satureja (Exarchou et al., 2002),
black pepper (Tewtrakul, 1998). Different colored bell
peppers (Capsicum annuum L.), green, red, and yellow were
examined for their free radical scavenging activity using
DPPH assay and antioxidant activity using b-carotene/
linoleic acid assay. In the methanol extracts red pepper
showed a higher level of DPPH radical scavenging activity
compared to green and yellow peppers, while green pepper
showed the highest in the phenolic extracts (Zhang and
Hamauzu, 2003). Superoxide anion radical scavenging
activities of the crude extracts of oregano (marjoram) were
determined by a lactate dehydrogenase (LDH-NADH)
oxidation system (Jun et al., 2001). Very useful method for
free radical determination is electron paramagnetic reso-
nance (EPR) spectroscopy. This technique allows radicals
to be detected in dry samples. In the case of spices the
method is used mainly for detection of their irradiation.
EPR was used for free radical determination of basil, black
pepper, cinnamon, nutmeg, oregano, parsley, rosemary,
and sage (Calucci et al., 2003), for scavenging of hydroxyl
radicals with a mixture of spices (ginger, turmeric and
cayenne pepper, or rosemary, sage, thyme, and oregano)
(Schwarz et al., 2001).
Test systems that evaluate the ability to inhibit lipid or
other sensitive component oxidation commonly involve
deterioration tests in which oxidation is accelerated some-
times as a result of the action of light or UV radiation, but
more frequently by elevated temperatures. For instance, in
monitoring antioxidant activity in a food, potential
measurements include peroxide value, thiobarbituric acid
value, iodine value, free fatty acid content, polymer
content, viscosity, creation of conjugated dienes and trienes
at 232 and 268 nm, etc. (Antolovich et al., 2002).
Measurements of oxygen consumption of linoleic acid
emulsion and induction period of lard oxidation at 100 1C
(Rancimat assay) were used for evaluation of antioxidant
activities of extracts from ginger, turmeric, and cayenne
pepper, or a mixture of rosemary, sage, thyme, and
oregano (Schwarz et al., 2001, Pizzale et al., 2002).
Antioxidant activity in a linoleic acid system measured by
the Rancimat method in sunflower oil at 100 1C was
investigated for extracts of cinnamon (Bozan et al., 2003).
Antioxidant activities of the volatile extracts isolated from
thyme, basil, rosemary, chamomile, lavender, and cinna-
mon were evaluated by two independent assays: the
aldehyde/carboxylic acid assay and the conjugated diene
assay (Lee and Shibamato, 2002). Pepper isolate (from
Capsicum Species) was tested for its ability to inhibit
oxidation of the beta-carotene-linoleate emulsion through
scavenging of peroxyl radicals (Howard et al., 2000).
Effects of methanolic extracts of sage, rosemary and
oregano on butter stability were investigated using the
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determination of peroxide value, free fatty acids and
thiobarbituric acid determinations (Ayar et al., 2001).
Antioxidant activity of oregano ethanol extract was
determined according to inhibitory effect on oxidation
model lipid system made up of refined bleached peanut oil
(Bendini et al., 2002). The antioxidant properties of seven
dessert spices (anise, cinnamon, ginger, licorice, mint,
nutmeg, and vanilla) were compared with those of the
common food antioxidants butylated hydroxyanisole,
butylated hydroxytoluene, and propyl gallate (Murcia
et al., 2004). Mint and cinnamon exhibited a higher
percentage of inhibition of oxidation than the other spices
analyzed and the food antioxidants, as tested by the lipid
peroxidation assay. Nutmeg, anise, and licorice showed the
strongest protection in the deoxyribose assay. Vanilla
exhibited the highest antioxidant activity in the peroxidase-
based assay. Nutmeg, propyl gallate, ginger, and licorice
improved the stability of oils (sunflower, corn, and olive)
and fats (butter and margarine) against oxidation (110 1C,
Rancimat). Cinnamon was a better superoxide radical
scavenger than the other analyzed spices and additives
(Murcia et al., 2004).
The observation that the more polar antioxidants are
more active in pure lipids, and non-polar antioxidants most
active in a polar substrate (e.g., oil-in-water emulsion), and
for which the term ‘‘polar paradox’’ has been introduced,
may at least partially explain the variation of antioxidative
activity for different herbs and spices in different foods
(Frankel et al., 1996). It was also found that there was a
reduced antioxidant activity in extracts prepared from an
equivalent amount of spice as opposed to that prepared
from the whole spice, confirming that a wide range of
compounds acting together are important as antioxidants
in the plant material, which further may act synergistically
(Pokorny et al., 2001). Leafy spices like thyme, marjoram,
basil, sage, summer savory showed pro-oxidative activity
for foods exposed to light, while the same food stored in
the dark confirmed the antioxidative effect of the spices
(Pokorny et al., 2001).
In the reviewed methods most popular in vitro anti-
oxidant measurements were designed primarily for hydro-
philic components of herbs and spices. Recently Huang et
al. (2002) developed a lipophilic ORACFL measurement
method that employed methylated b-cyclodextrin as a
solubility enhancer, which allows the measurement of the
antioxidant capacity of lipophilic and hydrophilic compo-
nents for a given sample separately, but based on the same
peroxyl free radical. For the first time, a database of the
antioxidant capacities of both the lipophilic and hydro-
philic components of foods has been developed using
ORACFL assay and a peroxyl radical generator (Wu et al.,
2004). Using this method only 28 common foods (some
fruit, vegetables, nuts, dried fruits and rice bran, not spices)
were evaluated, other foods will be analyzed and the
completed data will be incorporated into the USDA
Nutrient Database, which will be posted on the USDA
web site (Wu et al., 2004).
4. Conclusion
In view of the diversity of methods used for spice and
herb antioxidant isolation and their activity determination,
there is a great need to standardize them for both these
measurements. The search for more specific assays that
give us chemical information, which could be related
directly to oxidative deterioration of foods and biological
systems, should be the objective of future research. Modern
consumers ask for natural products, free of synthetic
additives. Therefore, the application of natural antiox-
idants will probably continue even the future, and it will be
necessary to study their changes and interactions in more
details. Scientists will look for new effective herbal sources
with potent antioxidants. All these plant extracts, their
mixtures, isolates and concentrates with antioxidant effects
have to meet all the requirements of human health safety.
Acknowledgement
This work was conducted with the support of Slovak State
subprogram of Research No. 2003SP270280E010280E01,
‘‘Food quality and safety’’.
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