Horseradish (Armoracia rusticana), a neglected medical and condiment species with a relevant glucosinolate profile: A review

Abstract

Armoracia rusticana (horseradish), a member of the Brassicaceae family, has been known since ancient times as a folk medicinal herb and as a plant of nutritional value and culinary interest. Currently horseradish is cultivated for its thick, fleshy and white roots which have a delicious intense pungency and for its tender leaves which are frequently used for salad mixed to other vegetables. The traditions to use horseradish plant for medicinal purpose are still applied in many countries. Horseradish is a rich source of a number of bioactive compounds such as glucosinolates (GLSs) and their breakdown products. Sinigrin is the dominant glucosinolate in both leaves and roots. Recent studies have shown that crude plant extracts have a complex profile of naturally occurring GLSs, with particular regard to sprouts. The increasing interest in these secondary metabolites, associated to the long and diffuse tradition of using horseradish in food preservation and as condiment in many parts of the world, is generating new applications of this plant in several agro-industrial and pharmaceutical sectors and is encouraging the use of its roots and leaves in functional food and medicine for human health. A bibliography review is discussed on ethnobotanical aspects and uses of this plant, as well as knowledge about its flavour compounds and GLS content and composition. This study summarizes also the updated information concerning the influence of the genotype and environment on GLS profile in horseradish.

Full text

REVIEW PAPER
Horseradish (Armoracia rusticana), a neglected medical
and condiment species with a relevant glucosinolate
profile: a review
Rosa Agneta •Christian Mo
¨llers •
Anna Rita Rivelli
Received: 20 March 2013 / Accepted: 28 May 2013 / Published online: 14 August 2013
ÓSpringer Science+Business Media Dordrecht 2013
Abstract Armoracia rusticana (horseradish), a
member of the Brassicaceae family, has been known
since ancient times as a folk medicinal herb and as a
plant of nutritional value and culinary interest. Cur-
rently horseradish is cultivated for its thick, fleshy and
white roots which have a delicious intense pungency
and for its tender leaves which are frequently used for
salad mixed to other vegetables. The traditions to use
horseradish plant for medicinal purpose are still
applied in many countries. Horseradish is a rich
source of a number of bioactive compounds such as
glucosinolates (GLSs) and their breakdown products.
Sinigrin is the dominant glucosinolate in both leaves
and roots. Recent studies have shown that crude plant
extracts have a complex profile of naturally occurring
GLSs, with particular regard to sprouts. The increasing
interest in these secondary metabolites, associated to
the long and diffuse tradition of using horseradish in
food preservation and as condiment in many parts of
the world, is generating new applications of this plant
in several agro-industrial and pharmaceutical sectors
and is encouraging the use of its roots and leaves in
functional food and medicine for human health. A
bibliography review is discussed on ethnobotanical
aspects and uses of this plant, as well as knowledge
about its flavour compounds and GLS content and
composition. This study summarizes also the updated
information concerning the influence of the genotype
and environment on GLS profile in horseradish.
Keywords Brassicaceae Ethnobotany 
Ethnopharmacognosy Glucosinolates 
Horseradish Isothiocyanates
Introduction
Horseradish, Armoracia rusticana G. Gaertn., B. Mey.
et Scherb., is an extremely hardy perennial plant,
member of the Brassicaeae family (Weber 1949;
Shehata et al. 2009). Its root and leaves have been used
in antiquity as both a medicinal herb and a condiment
(Rosengarten 1969), and the latter use is the principal
nowadays. It is currently cultivated for its thick, fleshy
and white roots that have a mix of delicious intense
pungency and cooling taste which is caused by sulfur
compounds, namely glucosinolates (GLSs) (Weber
1949; Balasinska et al. 2005; Walters and Wahle 2010).
R. Agneta (&)
Doctoral School of Crop Systems, Forestry and
Environmental Sciences, University of Basilicata,
Via dell’Ateneo Lucano, 85100 Potenza, PZ, Italy
e-mail: rosa.agneta@unibas.it
C. Mo
¨llers
Department of Crop Sciences, Georg-August-Universita
¨t
Go
¨ttingen, Von Siebold-Str. 8, D-37075 Go
¨ttingen,
Germany
A. R. Rivelli
School of Agricultural, Forest, Food and Environmental
Sciences, University of Basilicata, Via dell’Ateneo
Lucano, 85100 Potenza, PZ, Italy
123
Genet Resour Crop Evol (2013) 60:1923–1943
DOI 10.1007/s10722-013-0010-4

The species has become naturalized in many parts
of the world. Horseradish can be found in various
environments including fields, home gardens, weedy
areas, farmland, roadsides, ditches and disturbed areas
throughout Europe (Al-Shehbaz 1988; Hammer et al.
1992; Luczaj and Szymanski 2007; Sampliner and
Miller 2009). In open fields, horseradish is cultivated
as an annual or perennial crop. As annual crop it
requires a long growing season with high temperatures
during the summer and fall to enhance root develop-
ment; as a perennial it may stay productive for
10–20 years or more, thus requiring a careful field
management (Walters and Wahle 2010).
The main areas of commercial cultivation are found
in Europe and in North America (California, Illinois,
and Wisconsin) (Sampliner and Miller 2009). In
European countries, horseradish is especially pro-
duced in Germany, Hungary, and Poland (Shehata
et al. 2009). Particularly, large scale productions can
be found in certain regions of Germany, like Upper
Franconia, Thuringia and the Spreewald, firstly doc-
umented in 1569 (Hanelt 2001). In the Serbian
province of Vojvodina, the collection of wild horse-
radish began a long time ago, and the particular
ecotype collected, called ‘‘Novosadski’’, was highly
sought after by the Austro-Hungarian Empire court
thanks to its exceptional quality (Perlaki and Djurovka
2009). In Italy this plant grows in the Northern part
and in the Lazio and Basilicata regions (Hammer et al.
1990,1992; La Rocca and Chisci 2005; Guarrera
2006; Sarli et al. 2012). Although the hypothesis needs
to be confirmed by more detailed historic-botanical
studies (Pieroni et al. 2005), it is probable that
horseradish arrived in villages of inland Lucania
(viz. Basilicata) via migrants from Swabia during the
thirteenth century. Hammer et al. (2011) reported that
it was possibly introduced as a traditional spice plant
from Albania with Albanian immigrants to South
Italy, where today is particularly diffused in the
Arbe
¨reshe
¨areas of Basilicata, far away from North
Italy. A. rusticana is cultivated also in Russia, the
Caucasus, Asia and in some regions 1,000 m above
sea level in tropical countries (Sampliner and Miller
2009). In some regions of Russia, it may still be
possible to find horseradish landraces (Vetela
¨inen
et al. 2009). Recently, China has also begun commer-
cial production of horseradish, although much of the
crop is produced by small farmers (Shehata et al.
2009).
In this review we first present a botanical descrip-
tion of A. rusticana and its traditional uses in food and
medical fields due to its peculiar chemical composi-
tion. Afterwards, we review knowledge on flavour
constituents and profile, concentration and chemical
form of GLSs and their breakdown products in roots
and leaves. This review also stresses the influence of
the genetic variability and agronomic management on
GLS content and composition of horseradish plants.
Botanical description and origin
Horseradish has well defined botanical characteristics
which clearly separate the species from other
Brassicaceae. Various Latin names have been used
in the course of time by several authors for this
species. Taxonomically, horseradish has been placed
in various genera by different botanists (Weber 1949).
In 1969, Courter and Rhodes have recorded the names
for classification of horseradish during three historical
periods, as shown in Table 1. It was classified as
Nasturtium armoracia Fries, Roripa armoracia
Hitchc., Radicula armoracia Robinson, Cochlearia
armoracia L., Armoracia armoracia Britton, and A.
rusticana Gaertn. (Weber 1949). Specific epithets
used were rivini,rusticana,armoracia or lapathifolia.
Although A. lapathifolia is favored by some taxono-
mists, Lawrence (1953) and Fosberg (1965) have
pointed out that A. rusticana should have preference
(Courter and Rhodes 1969). The first valid published
scientific name of horseradish which resulted from the
generic name Armoracia was A. rusticana in 1800 by
Gaertner, Meyer, and Scherbius: this binomial is used
today (Shehata et al. 2009). Even more complex is the
variety of common names used to designate this
species whose polynomial is due to different lan-
guages and dialectical differentiation in different
territories. Horseradish has its gene center in Eastern
Europe and the south western parts of Russia
(Wedelsba
¨ck Bladh and Olsson 2011). In fact, early
records indicate that horseradish is a native of the
temperate regions of Eastern Europe and western Asia,
where wild types are found growing from Finland and
Poland to the regions around the Caspian Sea and the
desert of Cumania (now Romania) and Turkey. From
here, horseradish spread to Western Europe and across
the Atlantic to the New World by early settlers. Today,
horseradish has become naturalized in many parts of
1924 Genet Resour Crop Evol (2013) 60:1923–1943
123

the world and can be found both cultivated and
growing wild (De Candolle 1890; Shehata et al. 2009).
According to De Candolle (1890) the most probable
place of origin of A. rusticana was the temperate
region of Eastern Europe because of the word chren,
common in Slavic languages which was the most
primitive name for horseradish. Chren was introduced
into German and French dialects in the forms of kren,
kreen,cran and cranson.Meerretig,meer-radys and
meridi, which literally mean sea-radish, are other
words for horseradish that are common in several
western European languages, but these words are less
primitive than the name chren. The names horseradish
in English, raifort in French, and pepperrot in Swedish
are also of more recent origin than chren (Courter and
Rhodes 1969). The first use of the term horse radish
was made by John Gerard in his famous English herbal
(1597) that contains a lengthy entry with a woodcut
and clear description of the plant. Some believe that
the English called the plant horseradish in reference to
its propensity to rapidly spread in a ‘galloping’
behavior (i.e., it is difficult to control once it has been
introduced in an area) (Shehata et al. 2009). It is also
possible that it was called harsh radish because it was
so bitter on the tongue (McCann 2004). The word
armoracia has been commonly used as a generic name
or specific epithet: Armoracia is formed from the
Celtic: ar ‘near’, mor ‘the sea’, rich ‘against’, viz., a
plant growing near the sea (Courter and Rhodes 1969).
Armoracia belongs to the Cardamineae, which
comprise 10 genera and more than 340 species that
grow in moist habitats. The three species within the
genus (A. macrocarpa,A. rusticana,A. sisymbrioides)
have an affinity for wet habitats and commonly spread
through rhizomes (Al-Shehbaz et al. 2006; Sampliner
and Miller 2009). Brzezinski (1909) believes that
horseradish is not a natural species but a hybrid
(Weber 1949). Sampliner and Miller (2009) hypoth-
esized that A. rusticana is a species known only from
cultivation, and that cultivated A. rusticana popula-
tions were derived from natural populations of either
A. macrocarpa or A. sisymbrioides.
Horseradish is a large-leaved, hardy, and glabrous
perennial herb that grows to a height of up to 120 cm
(Fig. 1). Cauline leaves vary in shape and margin
depending on the habitat in which they are growing.
The leaves are long-petioled, oblong-ovate, cordate at
the base, unevenly crenate, and they grow to a length
of 30 cm up to 100 cm. (Sampliner and Miller 2009;
Shehata et al. 2009). Moreover, cultivars with an acute
leaf-base have smooth leaves, while those with a
cordate leaf-base have crinkled leaves; some geno-
types have intermediate leaf-types (Courter and
Rhodes 1969). The leaves on stalk are smaller in size,
have narrow bases and mostly sessile, are alternate,
lanceolate, and unevenly serrate to entire-margined. It
is interesting to note, as reported by Shehata et al.
(2008), that horseradish leaf morphology varies
through the season from entire (laminate) in the
summer to divided (pinnate or fern leaf) in the fall;
intermediate types of leaves are visible throughout the
season. The same authors (Shehata et al. 2008) have
shown in their work a photo with the different types of
leaves. The unstable leaf morphology of horseradish is
of considerable interest to botanists. In general, the
change from entire to pinnate in most obvious in the
fall as plants approach dormancy, suggesting environ-
mental influence (Shehata et al. 2008).
Table 1 Classification of horseradish during three periods of
history
Name Source
Ancient
Persicum sinapi Dioscorides
Sinapi persicum Dioscorides
Thlaspi cratevae Dioscorides
Persicon napy Pliny
Renaissance
Thlaspi alterum Turner
Raphanus vulgaris Matthioli
Raphanus magna Dodoens
Raphanus rusticanus Gerard
Raphanus sylvestris Bauhin J.
Linnaeus to modern
Armoracia rivini Ruppins
Cochlearia armoracia Linnaeus
Cochlearia rusticana Lamarck
Armoracia lapathifolia Gilibert
Armoracia rusticana Gaertner, Meyer
and Scherbius
Nasturtium armoracia Fries
Roripa rusticana Gren. & Godr.
Roripa armoracia Hitchoock
Radicula armoracia Robinson
Armoracia armoracia Britton
Table from Courter and Rhodes (1969) reproduced with
permission
Genet Resour Crop Evol (2013) 60:1923–1943 1925
123

The plant may or may not form a flower stalk
depending on environmental conditions (Weber
1949). In natural habitats, horseradish blooms very
profusely and for a long period of time, until mid-
August (Winiarczyk and Bednara 2008). Shehata et al.
(2009) reported that A. rusticana produces numerous
fragrant flowers with 5–7 cm long upright pedicels
borne on racemes that have four sepals, four petals,
and six tetradynamous stamens: their sepals are
2.5–3 mm long, broadly ovate and have membranous
white margins; petals are white, 5–7 mm long, and
broadly obovate; the inner stamens are 2.5 mm long
and the outer ones are 1.5 mm long; the stigma is
broad, round, and gently two-lobed. Horseradish bears
4–6 mm long, globose to obovate siliques with
persistent style on 20 mm long, upright-spreading
stems (Shehata et al. 2009). A. rusticana produces few
if any seeds, usually none, at most 6 seeds per pod
(Sampliner and Miller 2009). The seeds are smooth
and brown when mature (Shehata et al. 2009).
Winiarczyk and Bednara (2008) observed that, a
dozen or so days after the fall of the perianth of the
inflorescence, siliques started to form (Fig. 2): most of
the flowers were withered, but on every inflorescence,
there were a few dozen fruits, the majority of which,
however, were shriveled and deformed; in every
silique, most of the ovules were aborted, some were
much smaller and deformed, but sporadically there
were also large, properly developed young seeds. As a
matter of fact, many early botanists reported that
horseradish rarely produced viable seed and, until the
early part of the twentieth century, the plant was
considered to be highly sterile (Courter and Rhodes
1969). Weber (1949) observed partial pairing of
chromosomes and other irregularities during both
microsporogenesis and megasporogenesis. Wini-
arczyk and Bednara (2008) noted on the stigma of A.
rusticana morphological symptoms of the reaction of
self-incompatibility, typical for some species of the
Brassicacea family. This could explain the fact that
most A. rusticana plants are incapable of producing
viable seeds. In addition, the plants growing in a given
area, i.e. constituting a certain population, are often
Fig. 1 Armoracia rusticana plant grown in Basilicata Region
(South Italy). Personal photo
Fig. 2 Typical silique of a horseradish plant 2-year-old:
aFruits-silique started to form at the end of the vegetative
period; bsingle silique; cdevelopment of seeds in silique.
Images captured with stereo microscope (Carl Zeiss) at 96.3 by
a camera Olympus 8 Megapixel. The bar in Fig. 2bis2mm
1926 Genet Resour Crop Evol (2013) 60:1923–1943
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genetically identical with the parent plant, as a
consequence of their vegetative reproduction (Wini-
arczyk and Bednara 2008). In fact, horseradish is
asexually propagated by planting root sections col-
lected from the previous year’s crop (Walters and
Wahle 2010) through the formation of adventitious
buds (Kamada et al. 1995). In particular, lateral roots
are separated from the healthy taproots for next year’s
planting stock (Shehata et al. 2009). The root system
of horseradish consists of a long, white, cylindrical or
tapering main root that can grow to about 60 cm in
loose soils; several thin lateral roots are formed around
the main root and near the collar of the crown;
undisturbed, the root system can reach a depth of
3–4 m with a lateral spread of about 1 m (Shehata
et al. 2009). Horseradish roots exhibit distinct polarity
with a proximal end (or point of attachment to the
main root that sprouts producing new vegetation) and
distal end (Walters and Wahle 2010).
According to Sampliner and Miller (2009) the
clonal propagation of A. rusticana may have resulted
in increased sterility due to (1) the accumulation of
deleterious mutations in gene regions associated with
seed production; (2) reproduction of sterile individuals
derived from an interspecific hybridization; or (3) self-
incompatibility mechanisms precluding fertilization
between widespread, genetically-identical plants. Fur-
thermore, research aimed at obtaining horseradish
plants capable of sexual reproduction could result in
better adaptation to various environmental conditions
and pest resistance (Winiarczyk et al. 2007).
Traditional uses as a spice and as folk medicinal
plant
The use of horseradish as food or folk medicinal plant
has spread from the Eastern and Mediterranean areas
both north- and westward during the Middle Ages (De
Candolle 1890; Wedelsba
¨ck Bladh and Olsson 2011).
It is believed that horseradish became popular as a
condiment in old Europe because there was no
refrigeration and its sharp spiciness covered the taste
of tainted meat (Shehata et al. 2009). In Bulgaria,
Romania and Russia, the whole root, grated root, or
piece of cut root are still used to flavour, help ferment,
aerate pickling liquid, and conserve cabbage for the
winter; leaves were also cut and used to conserve cut
vegetables for the winter (Sampliner and Miller 2009).
Horseradish was used by European Jews as a
symbolic food in the ritual meal as bitter herb of
Passover because its bitterness remembered the
suffering of their ancestors of the exodus from Egypt
(Schaffer 1981). By the late 1600s the English people
added horseradish to beef and oysters, and particularly
in spring, the tender leaves frequently were used for
greens mixed with other wild plants (Shehata et al.
2009); for Germans, horseradish roots were an excel-
lent addition to meat and fish when cut in very small
pieces, then crushed and mixed with salt and vinegar
(Courter and Rhodes 1969). These latter authors wrote
that horseradish was also mixed in catsup used to
flavour ground beets or added to mustard as a
seasoning. As reported by Shehata et al. (2009),
horseradish was often consumed boiled as a pot herb;
it was boiled, the water was drained, and it was boiled
a second time to eliminate bitter or harmful substances
before consumption.
The main portions of this plant are still used today
to prepare traditional dishes in many countries around
the world. In Poland, roots and leaves are used for
seasoning or as preservatives: the roots, usually
collected from the wild plants, were utilized as a
condiment, with pickled cucumbers, grated with
chopped boiled eggs, soups or meat dishes, often used
at Easter. The leaves placed in the oven under baking
bread, partly to prevent the bread from sticking and
partly to flavour the bread, are still widely used
(Łuczaj and Szyman
´ski, 2007). In Bulgaria, Romania
and Russia, grated roots are used as a condiment for
any cooked meat (e.g., lamb, pig, and chicken); they
are often combined with vinegar, salt, oil, cream, or
yogurt (Sampliner and Miller 2009). Particularly, in
Romania, grated roots, with or without oil, are eaten
with potatoes or polenta, or are grilled and eaten with
cream accompanying lamb and chicken; a mix of
grated horseradish, apples, salt, sugar, and vinegar is a
garnish used for meals; small pieces of cut A.
rusticana root are eaten in soups; leaves can also be
put into bread dough, which is then grilled (Sampliner
and Miller 2009). In South Italy there are many
traditional culinary uses, particularly, in the Basilicata
area this plant is typical base for preparing dishes
during the carnival period (Sarli et al. 2012): horse-
radish is grated as condiment for pasta or home-made
noodles (‘ferriciedd’), and it is used in fried eggs paste
to prepare a traditional dish (‘rafanat’) during the
carnival period; pieces of roots are also used for
Genet Resour Crop Evol (2013) 60:1923–1943 1927
123

aromatization of hand-made sausages; it is used raw
grated as condiment for cooked pork meat or for
lamb and chicken where the pieces of roots are
often combined with oil, salt and vinegar; the leaves
are used for salad, mixed to other vegetable species
(Pieroni et al. 2005; Sarli et al. 2012). Grated roots
are often combined with oil and used all year round,
also smeared on grilled bread (Sarli et al. 2012).
The root is still popular in Europe and North
America and is appreciated freshly grated, mixed
with vinegar or in a sauce to form a condiment often
used with meats or fish and as a flavour in other
recipes in salads and soups, on sandwiches, and also
in drinks such as the Bloody Mary (Wedelsba
¨ck
Bladh and Olsson 2011).
In addition to be used as food and condiment,
horseradish has a long history of utilization for
medicinal purposes in the traditional medicine (Sheh-
ata et al. 2009). In a recent review, Wedelsba
¨ck Bladh
and Olsson (2011) have well documented the histor-
ical uses of horseradish as a medicinal plant in
different parts of the world, and have shown also a
table with the different uses in various historical
periods. Since the ancient Greeks horseradish was
used as an aphrodisiac and as a rub to alleviate pain in
the back. Material medica’s of ancient physician and
the herbal of early botanists record many medical
virtues of horseradish (Courter and Rhodes 1969).
Early records, believed to be about horseradish are
found in ‘‘Naturalis Historia’’ by the Roman naturalist
and philosopher Gajus Plinius Secundus (AD 23-79)
that recommended the plant freshly grated for the
digestion after a heavy meal, and in ‘‘De Materia
Medica’’ by the Greek physician Pedanius Dioscorides
(AD 40-90) that described the plant as a diuretic and
very hot herb (Bostock and Riley 1856; Courter and
Rhodes 1969; Wedelsba
¨ck Bladh and Olsson 2011).
Hildegard of Bingen (1098–1179), a German abbess,
in her book about medicinal plants, recommended
horseradish mixed with warm vine or water as a
treatment for lung diseases; to cure heartache or heart
diseases, dry and pulverized horseradish could be
mixed with Alpinia galanga, a Chinese herb often used
as a stimulant and drug (Wedelsba
¨ck Bladh and
Olsson 2011). In 1597, the botanist and herbalist John
Gerard claimed that horseradish reduced pain from
sciatica, relieved colic, increased urination, and killed
worms in children (Courter and Rhodes 1969). It was
stated to be an expectorant, soothing for respiratory
problems, and may help to relieve rheumatism by
stimulating blood flow to inflamed joints (Shehata
et al. 2009). The most common use of horseradish was
as a remedy for scurvy, as also recommended by
herbalist William Coles (1626–1662), especially for
seamen on long voyages that had not access to fresh
vegetables or fruit, so the lack of vitamin C in the diet
often caused scurvy outbreaks on board (Wedelsba
¨ck
Bladh and Olsson 2011). In 1880, Bentley and Trimen
reported that grated horseradish root was used mixed
with honey and warm water for influenza, and it could
be used as poultice by adding cornstarch to fresh
horseradish and applying it to the affected areas in a
gauze bandage. Native Americans used horseradish to
treat toothaches, as a urinary aid for gravel (kidney
stones), as a diuretic, as a gastrointestinal aid to
improve digestion, and as a respiratory aid to treat
asthma, cough, and bronchitis (Moerman 1998;
McCann 2004; Shehata et al. 2009). The tradition to
use the plant for medicinal purpose is still applied in
countries like Bulgaria, Romania, and some part of
Russia: grated roots or leaves are put in a cloth
(sometimes with alcohol or vinegar) and applied to
the skin to ease pain, or a paste made from the root is
applied to the throat to ease breathing problems.
Mixtures with honey from Robinia pseudoacacia are
used for coughs and bronchitis, while in combination
with vinegar, salt, and sugar it is used for lowering
blood pressure (Sampliner and Miller 2009). Further-
more, in an ethnobotanical study about a community
of Russlanddeutsche (Germans from Russia) living in
Ku
¨nzelsau (Germany), it was reported that Russian
sauerkraut, used as an important home medicine for
treating flu and liver disease, contained among other
herbs, also horseradish leaves (Pieroni and Gray
2008). In the Basilicata region (South Italy), horse-
radish leaves and roots have been traditionally used
as a remedy for rheumatism, headaches, sinuses,
coughs and bronchitis. Added to water, vinegar, salt,
sugar, or aromatised with Anethum graveolens and
Laurus nobilis leaves, this plant is also used as a
remedy for healing drunkenness. Particularly, in
Castelmezzano (Basilicata), the Ukrainian women
have introduced the functional use of pickled toma-
toes (with dill, bay and horseradish leaves), con-
sumed to recover from a drunken state. Leaves, added
to dog food, are used because of their antimicrobial
activity (Pieroni et al. 2004; Pieroni and Quave 2005;
Sarli et al. 2012).
1928 Genet Resour Crop Evol (2013) 60:1923–1943
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Also, according to Wedelsba
¨ck Bladh and Olsson
(2011), the traditional knowledge of using horseradish
in food preservation at home is generating new
applications for the plant as a natural anti-bacterial
component in food (e.g., to reduce coli-bacteria or
other microbial growth) and the new knowledge of
bioactive components such as GLSs and their break-
down products, combined with traditional knowledge
of medicinal properties, is encouraging the use of
horseradish roots and leaves in functional food and
medicine to inhibit different cancer forms or gastric
lesions.
Glucosinolates, the main flavour constituents
of horseradish
Horseradish tissues, when unbroken, are inodorous.
The intense pungent aroma and the lachrymatory
odour of horseradish results from crushing, grinding,
or chewing the cells (Courter and Rhodes 1969; Jiang
et al. 2006) and therefore it is specially cultivated to
supply a hot spicy flavour (Sultana et al. 2003). The
characteristic taste and odour is due to compounds
such as GLSs and their mostly volatile breakdown
products (Mucete et al. 2006; Redovnikovic et al.
2008a). GLSs are hydrolysed by the enzyme com-
monly known as myrosinase to a variety of com-
pounds as isothiocyanates, nitriles, thiocyanates,
epithionitriles, and oxazolidines. The most common
products are isothiocyanates (Bones and Rossiter
2006). GLSs are compounds mainly found in plants
belonging to the order of Capparales, with particular
reference to several species of the Brassicacee family
(radish, cabbage, cauliflower, broccoli, mustard, tur-
nip, oilseed rape) including horseradish (Fahey et al.
2001; Blazevic and Mastelic 2009). These secondary
metabolites are characterized by a core sulfated
isothiocyanate group, which is conjugated to thioglu-
cose, and a further R-group derived from amino acids
(Clarke 2010). GLSs can be divided into three classes
based on the structure of different amino acid precur-
sors: (1) aliphatic GLSs derived from methionine,
isoleucine, leucine or valine; (2) aromatic GLSs
derived from phenylalanine or tyrosine; and (3) indole
GLSs derived from tryptophan (Wittstock and Halkier
2002; Redovnikovic et al. 2008b). The number of
reported GLSs is now approaching 200 (Clarke 2010).
A number of plants contain only a single GLS, the
majority contain 2–5, while 34 individual GLSs are
reported in the seeds and leaves of a collection of
ecotypes of Arabidopsis thaliana (Kliebenstein et al.
2001; Clarke 2010). GLS concentrations in plants,
although highly variable, are around 1 % of dry weight
in some Brassica vegetables (Rosa et al. 1997; Clarke
2010). The GLS content is higher in black mustard
seed (Brassica nigra) and horseradish roots (over
10 % by dry weight) than in the other constituent parts
of the Brassicaceae (Li and Kushad 2004; Mucete
et al. 2006). However, very little is known about the
concentration and biochemical composition of horse-
radish, especially the distribution of GLSs in the
different organs of the plants (Li and Kushad 2004).
A first study about the identification of GLSs of
horseradish roots by GC–MS (gas chromatography-
mass spectrometry) was published by Grob and Matile
(1980). They identified 30 GLSs (Table 2) which
exceeds the number of GLSs detected before in any
other species. The authors reported that a close
examination of the chromatograms may indicate that
the number of structures present in horseradish may be
even larger, although today it is necessary to investi-
gate and confirm all individual molecules listed by
Grob and Matile (1980). Several methods have been
developed to detect either intact GLSs or their desul-
foglucosinolates (Lee et al. 2006; Cataldi et al. 2007).
The structural identity of GLSs is known and
described only for those which can be isolated and
identified by traditional methods. With increasing
complexity of GLS composition and decreasing con-
centration of individual GLSs in the sample, more
sensitive and selective analytical methods are required
for their identification (Agneta et al. 2012; Lelario et al.
2012). Hence, for their accurate structural identifica-
tion, in addition to traditional methods, sensitive and
selective analytical methods are required, especially
when GLSs are present in trace amounts only. Mevy
et al. (1997) investigated the occurrence of GLSs in
various differentiated horseradish tissues (Table 3)by
HPLC analysis of desulphoglucosinolates. Particu-
larly, the individual GLSs present in regenerated
plantlets were compared with those of embryoids,
suspension cells and calli in order to investigate
whether the distribution of these compounds resulted
from cellular differentiation. Moreover, the authors
revealed the presence of two GLSs, i.e. 2-phenylethyl-
and 2-propenyl-, which appeared to be confined to
roots and leaves, respectively, while the highest
Genet Resour Crop Evol (2013) 60:1923–1943 1929
123

content of indole-3-methyl-GLS (glucobrassicin) was
found in calli. Their data clearly demonstrated that the
occurrence of indole GLSs in embryoids, suspensions
cells and calli relates to their age. It has also been
reported that younger and growing tissues synthesize
more indole GLSs than older and senescent tissue, so
their results raise the question of the role of GLSs in
healthy plants (Mevy et al. 1997). Mevy et al. (1999),
by comparison of immobilized and suspension cells of
horseradish, showed that GLS production may be
improved by cell immobilization technique. More-
over, the chromatograms obtained revealed that gluco-
brassicin occurred earlier than its hydroxylated form in
cells, therefore their data suggest that hydroxygluco-
brassicin might result directly from modification of the
side chain of glucobrassicin.
Table 2 Glucosinolates of
horseradish roots as
identified by their
corresponding mustard oils
(Table modified from Grob
and Matile 1980) and odour
description (reported by
Sultana et al. 2003)
The common names are
those generally accepted
(Fahey et al. 2001;
Bellostas et al. 2007a;
Clarke 2010)
a
Concentration is
expressed as lg/g fresh
weight
b
Odour description of the
corresponding
isothiocyanates
GLS common name GLS systematic name lg/g
a
Odour description
b
Glucoviorylin 2-Methylthioethyl- 2
Glucoputranjivin Iso-propyl- 1 Chemical, weak mustard
like
Sinigrin Allyl- 240 Strongly pungent, mustard-
like, lachrymatory, bitter
Glucoarabidopsithalin 2-Hydroxypropyl- 3
Glucoiberverin 3-Methylthiopropyl- 5 Strongly radish-like, pungent
Glucoiberin 3-Methylsulfinylpropyl- 3
Glucocheirolin 3-Methylsulfonylpropyl- 3
n-butyl- 5
sec-butyl- 22 Chemical, weak mustard like
Glucoconringianin iso-butyl- 6 Sweet, chemical
Gluconapin 3-Butenyl- 14 Green, pungent, aroma
Glucocappariflexin 3-Hydroxybutyl- 0.5
Glucoerucin 4-Methylthiobutyl- 0.5
Glucokohlrabiin n-pentyl- 1
3-Methylbutyl- 8
2-Methylbutyl- 0.5
Glucobrassicanapin 4-Pentenyl- 20 Green, pungent, acrid,
fragrant leaf
Glucomoracialapathin 2-Hydroxypentyl- 0.1
3-Hydroxy-4-pentenyl- 0.8
Glucoberteroin 5-Methylthiopentyl- 0.2 Radish-like, pickle-like
3-Hydroxy-5-
methylthiopentyl-
0.2
Iso-hexyl- structure
unknown
0.4
Glucowasabiamin 5-Hexenyl- 2 Green, pungent, fatty
Glucolesquerellin 6-Methylthiohexyl- 0.1 Radish-like, sweet, fatty
Iso-heptyl- structure
unknown
0.05
Glucotropaeolin Benzyl- 4 Strongly radish-like, pungent,
strong watercress aroma,
tingling sensation
Gluconasturtiin 2-Phenylethyl- 55
Glucoarmoracialapicin 3-Phenylpropyl- 0.1
Glucoarmoracialafolicin 4-Phenylbutyl- 0.5
Methoxybenzyl- 0.5
1930 Genet Resour Crop Evol (2013) 60:1923–1943
123

Redovnikovic et al. (2008a) analyzed horseradish
plantlets leaves by reversed-phase high-performance
liquid chromatography (RP-HPLC) analysis of des-
ulphoglucosinolates and showed that the aliphatic
GLS 2-propenyl-GLS (sinigrin) accounted for more
than 80 % of total GLSs. Lower amounts of phenyl-
ethyl-GLS (gluconasturtiin) and of the three indole
compounds 3-indolylmethyl-GLS (glucobrassicin),
4-methoxy-3-indolylmethyl-GLS (4-methoxygluco-
brassicin) and 4-hydroxy-3-indolylmethyl-GLS (4-
hydroxyglucobrassicin) were found. Li and Kushad
(2004) evaluated roots from 27 horseradish accessions
and leaves from 9 accessions for their GLS content by
RP-HPLC as desulphoglucosinolates. In roots, total
GLS concentration ranged from 2 to 296 lmol g
-1
of
dry weight (DW), with sinigrin representing on
average 83 % of the total GLSs, followed by glu-
conasturtiin with 11 %, and glucobrassicin with 1 %.
In some of the accessions, other GLSs as progoitrin,
gluconapin, 4-hydroxyglucobrassicin, and 4-meth-
oxyglucobrassicin, were detected in minor concentra-
tions (\1 %). According to the same authors (Li and
Kushad 2004), the origin of plant did not seem to have
an effect on total GLSs level; furthermore, the average
total GLS concentration in most horseradish roots
accessions was similar to the value for brown and
oriental mustard seeds, but significantly higher than
what the authors had previously reported for broccoli
(13 lmol g
-1
of DW), brussels sprouts (25 lmol g
-1
of DW), cabbage (11 lmol g
-1
of DW), cauliflower
(15 lmol g
-1
of DW), or kale (15 lmol g
-1
of DW).
The total GLS levels in the leaves, instead, ranged
from 34 to 201 lmol g
-1
of DW. Similar to the roots,
sinigrin was the dominant GLSs in the leaves,
representing on average 92 % of the total. Leaves
also contained gluconasturtiin, but at a much lower
concentration (0.2 lmol g
-1
of DW) than in roots
(10.3 lmol g
-1
of DW). Conversely, leaf tissues
contained neoglucobrassicin (2.5 % of total) instead
of glucobrassicin.
Recently, Alnsour et al. (2012), referring to the
GLS content, confirmed previous results of Redov-
nikovic et al. (2008a) and Mevy et al. (1997) showing
that the overall GLS concentration (maximum
40 lmol g
-1
of DW) in the in vitro cultivated
Armoracia plants is much lower than that found by
Li and Kushad (2004) in soil-grown ones (in average
100 lmol g
-1
of DW). Surprisingly, in contrast to soil
grown plants, where the GLS concentration in roots
and leaves were quite similar, in the in vitro plantlets
the concentration of GLS in the root was much lower
than in the leaves (Alnsour et al. 2012).
The distribution of the GLSs varies among plant
organs, with both quantitative and qualitative differ-
ences between roots, leaves, stems and seeds; in any
case the plant age is a major determinant of the
qualitative and quantitative GLS composition in the
plants (Fahey et al. 2001; Velasco et al. 2007;
Cleemput and Becker 2011). Li and Kushad (2004)
reported differences in GLS content between young
Table 3 Glucosinolate contents of various horseradish tissue
Materials Medium
a
Glucosinolate contents (lg/g dry wt.)
b
Total
contents
IMG OH-IMG PhG PrG
Callus (28 days) MS 4.95 (0.30)
c
0.32 (0.03) – – 5.27
Suspension cells (21 days) MS 0.09 (0.02) – – – 0.09
Suspension cells (21 days) D-MS 0.31 (0.04) – – – 0.31
Embryoids (28 days) D-MS 0.71 (0.02) – – – 0.71
Root of r. p.
d
(3 months) H-MS – – 2.35 (0.40) – 2.35
Small leaves of r. p. (3 months) H-MS – – – 16.4 (4.00) 16.4
Large leaves of r. p. (3 months) H-MS – – – 28.3 (5.00) 28.3
Table modified from Mevy et al. (1997)
a
Murashige and Skoog basal salts with organic substances, MS; 2,4-D-free-MS, D-MS; hormone-free-MS, H-MS
b
Indole-3-methylglucosinolte, IMG; 4-hydroxy-indole-3-methylglucosinolate, OH-IMG; 2-phenylethylglucosinolate, PhG;
2-propenylglucosinolate, PrG
c
Mean values of three independent extractions ±(SD)
d
r. p. regenerated plantlets
Genet Resour Crop Evol (2013) 60:1923–1943 1931
123

and fully developed roots and leaves of horseradish
(Table 4), noting that the major differences were
observed regarding indole GLSs, in fact the younger
tissue of roots and leaves tended to accumulate more
glucobrassicin and neoglucobrassicin, respectively,
than fully developed organs ([60-fold higher in young
leaves than fully developed leaves). Due to the
complexity of this vegetal matrix, further studies are
required to better understand the accumulation of
various GLSs during different growth stages also by
comparing of different genotypes. For the model plant
Arabidopsis thaliana, a member of the Brassicaceae, it
was reported that GLS accumulation varies signifi-
cantly among different organs and developmental
stages, with regards to both composition and concen-
tration (Brown et al. 2003; Yang and Quiros 2010).
Particularly, dormant and germinating seeds had the
highest concentration (2.5–3.3 % by dry weight);
inflorescences and siliques had the next highest levels
(0.6–1.2 %) followed by roots, stems and cauline
leaves, and rosette leaves (0.3–1.0 %) (Fahey et al.
2001; Brown et al. 2003). The GLSs are plant defense
compounds that accumulate preferentially in the
reproductive organs as seeds, flowers, fruits and young
sprouts (Fahey et al. 2001; Brown et al. 2003;
Bellostas et al. 2007b). The low GLS concentration
in the leaves and stems in comparison with the seeds
could be attributed to the dilution of GLSs during plant
growth (Clossais-Besnard and Larher 1991; Cleemput
and Becker 2011). Recently, Agneta et al. (2012) gave
a complex profile of naturally occurring intact GLSs in
sprouts and roots of a horseradish accession largely
diffused in southern Italy by using reversed-phase
liquid chromatography coupled with electrospray
ionization and a hybrid quadrupole linear ion trap
and Fourier transform ion cyclotron resonance mass
spectrometry (LC-ESI-FTICR MS). In sprouts 16
and in roots 11 GLSs were identified (Table 5).
The authors confirmed the presence of sinigrin, 4-hy-
droxyglucobrassicin, glucobrassicin, gluconasturtiin,
and 4-methoxyglucobrassicin and identified glucoiber-
in, gluconapin, glucocochlearin, glucoconringianin,
glucosativin, glucoibarin, 5-hydroxyglucobrassicin,
glucocapparilinearisin or glucobrassicanapin, glucotro-
paeolin, and glucoarabishirsutain, not previously char-
acterized in horseradish. Particularly notable was the
presence of the putative 2-methylsulfonyl-oxo-ethyl-
GLS, not reported before. In addition, horseradish
Table 4 Change in total, individual and residual glucosinolates in two accession of horseradish roots and leaves as a function of
growth stage
Accession Growth stage Sinigrin Glucobrassicin Gluconasturtiin Total GLSs
a
Residual GLSs
b
lmol g
-1
of DW
Roots
810-A Small 58.0b
c
13.4b 0.2c 74.7b 3.1c
810-A Mature 258.0a 2.8c 20.1a 295.8a 14.9a
1573 Small 77.3b 33.9a 0.2c 119.1b 7.7ab
1573 Mature 47.5b 0.7c 2.0b 55.5c 5.3bc
Accession Growth stage Sinigrin Neoglucobrassicin Gluconasturtiin Total GLSs Residual GLSs
lmol g
-1
of DW
Leaves
810-A Small 77.6ab 66.2a 0.5a 150.2a 2.3a
810-A Mature 114.8a 1.2b 0.1a 126.0a 1.1a
1573 Small 60.3b 69.7a 0.3a 129.2a 2.4a
1573 Mature 48.6c 1.1b 0.4a 52.7b 1.2a
Table modified from Li and Kushad (2004)
a
Total glucosinolates in roots and leaves represent the sum of sinigrin, glucobrassicin, and gluconasturtiin plus residual
glucosinolates and the sum of sinigrin, neoglucobrassicin and gluconasturtiin plus residual glucosinolates, respectively
b
Residual glucosinolates is the sum of at least four additional glucosinolates
c
Values represent the means of four replicates per accession. Mean separation within each column by Duncan’s multiple-range test,
P=0.05
1932 Genet Resour Crop Evol (2013) 60:1923–1943
123

sprouts were found to be richer in GLSs than roots.
Further studies are required to quantify the content of
these molecules that are responsible for the typical
flavour of this plant.
Normally, the components that cause the intense
pungency are physically separated from one another
(Shehata et al. 2009). GLSs are stored in the vacuole
(Grob and Matile 1979; Helmlinger et al. 1983;
Koroleva et al. 2000) while the myrosinase enzyme
that causes their degradation (Mucete et al. 2006)is
confined within specialized cells known as myrosin
cells (Kissen et al. 2009; Borgen et al. 2010). Upon
crushing horseradish roots or leaves allyl and 2-phen-
ylethyl isothiocyanate are produced from sinigrin
and 2-phenylethyl-GLS. Allyl isothiocyanate is
reported to be a lachrymatory compound, bitter in
taste with a strong, pungent smell, while 2-phenylethyl
isothiocyanate had no pungency and lachrymatory role
at all, like allyl isothiocyanate (Sultana et al. 2003;
Shehata et al. 2009). Both isothiocyanates are largely
responsible for the typical, characteristic flavour and
pungency in mustard as well as in horseradish (Mithen
et al. 2000; Sultana et al. 2003; Kosson and Horbowicz
2008).
Recently, eighteen compounds that contribute to
the flavour of horseradish were identified in roots
immediately after harvesting (Table 6) by D’Auria
et al. (2004), using SPME–GC–MS (solid phase
microextraction coupled with gas chromatography
mass spectrometry). The main compounds were allyl
isothiocyanate, 4-isothiocyanato-1-butene, and 2
phenylethyl isothiocyanate. Allyl isothiocyanate was
the largest peak in the chromatogram and accounted
for 81 % of the overall integrated peak area. It is
noteworthy that methyl, ethyl, and isopropyl isothio-
cyanate were not found. By the same authors (D’Auria
et al. 2004) the different volatile organic compounds
listed in Table 6were identified in cut horseradish
kept at 5 °C for 12 h. The concentration of most of the
volatile compounds declined rapidly and 2-phenyl-
ethyl isothiocyanate compounds became the most
abundant compound. The fact that the flavour com-
position of cut horseradish samples changes is relevant
when specifying the treatment of horseradish to obtain
mustard.
To preserve the pungency and quality, the ground
product should be consumed quickly or refrigerated
to minimize loss of volatile flavour compounds.
Table 5 Glucosinolates identified in different portions of horseradish plants by LC-ESI-FTICR MS: common name, retention time
(t
R
), molecular formulae, monoisotopic value as [M-H]
-
ion (m/z), mass error (ppm)
GLS common name t
R
(min) Molecular
formulae
Monoisotopic calculated
value as [M-H]
-
ion (m/z)
Mass error
(ppm)
a
Glucoiberin 4.3 C
11
H
21
NO
10
S
3
422.02549 -1.30
Sinigrin 4.4 C
10
H
17
NO
9
S
2
358.02720 -0.59
2-Methylsulfonyl-oxo-ethyl-GLS 4.6 C
10
H
17
NO
12
S
3
437.98402 -1.21
Gluconapin 5.5 C
11
H
19
NO
9
S
2
372.04285 -1.30
Glucocochlearin 6.2 C
11
H
21
NO
9
S
2
374.05850 1.00
Glucoconringianin 6.4 C
11
H
21
NO
9
S
2
374.05850 -0.70
Glucosativin 6.5 C
11
H
21
NO
9
S
3
406.03057 1.35
Glucoibarin 7.3 C
15
H
29
NO
10
S
3
478.08808 -0.10
4-Hydroxyglucobrassicin 7.4 C
16
H
20
N
2
O
10
S
2
463.04866 -1.50
5-Hydroxyglucobrassicin 7.5 C
16
H
20
N
2
O
10
S
2
463.04866 -1.70
Glucocapparilinearisin or
Glucobrassicanapin
7.8 C
12
H
21
NO
9
S
2
386.05850 -0.20
Glucotropaeolin 8.5 C
14
H
19
NO
9
S
2
408.04285 -1.10
Glucobrassicin 10.3 C
16
H
20
N
2
O
9
S
2
447.05375 -1.50
Gluconasturtiin 12.1 C
15
H
21
NO
9
S
2
422.05850 -1.20
4-Methoxyglucobrassicin 12.7 C
17
H
22
N
2
O
10
S
2
477.06431 -1.20
Glucoarabishirsutain 16.7 C
15
H
29
NO
9
S
3
462.09317 -1.20
Table modified from Agneta et al. (2012)
a
Mass error in part per million, ppm =10
6
9(accurate mass-exact mass)/exact mass
Genet Resour Crop Evol (2013) 60:1923–1943 1933
123

However, ground horseradish slowly losses its pun-
gency, becomes dark, and develops off flavours even
under refrigeration; this quality loss can be slowed by
adding a fat or oil, such as cream (Courter and Rhodes
1969). Kosson and Horbowicz (2009) showed that
higher storage temperature (2, 8 and 18 °C were
tested) caused faster decline of isothiocyanates con-
centration in horseradish cream. Therefore, to main-
tain the human health-promoting compounds in
processed horseradish, the authors suggested storing
horseradish cream under cold conditions. Further-
more, also dehydrated horseradish in diced, flaked or
powder form has a great potential as a flavour
ingredient in sauces, dressings, and seasoning formu-
lations (Sahasrabudhe and Mullin 1980). Saha-
srabudhe and Mullin (1980) recommended that in
processing horseradish to a dried product, one must
process the cleaned roots immediately after dicing or
crushing and maintain the temperature below 65 °C, to
retain viability of the myrosinase to obtain a product
with the desired flavour intensity and acceptable color.
In addition, Sahasrabudhe and Mullin (1980) evalu-
ated the odour intensity of the powders by sniffing,
after adding water: they noted that freeze dried
Table 6 Volatile organic
compounds found in the
fresh horseradish root and
in the cut of roots kept at
5°C for 12 h
Table modified from
D’Auria et al. (2004)
Compound r.t. (min) Fresh 5 °C for 12 h
ppm ppm
Thiobismethane 1.76 0.8 ±0.2 1.4 ±0.1
Carbon disulphide 1.81 0.8 ±0.2 –
3-Butenenitrile 2.38 7.8 ±0.1 –
3-Methylbutanal 2.73 0.4 ±0.2 0.6 ±0.1
2-Ethylfuran 2.78 1.2 ±0.1 5.8 ±0.3
3-Methyl-1-butanol 3.16 – 1.4 ±0.1
Toluene 3.69 – 0.8 ±0.1
Hexanal 4.32 4.5 ±0.1 3 ±0.3
E-2-hexenal 5.42 1.6 ±0.1 3 ±0.2
Heptanal 6.45 – 0.3 ±0.1
Allyl isothiocyanate 6.64 3,300 ±2.0 175 ±2.0
Benzaldehyde 7.78 – 0.5 ±0.1
Isobutyl isothiocyanate 7.85 15 ±0.3 –
4-Isothiocyanato-1-butene 8.45 63 ±1.0 –
2-Pentylfuran 8.45 – 3 ±0.3
Butyl isothiocyanate 8.74 13 ±0.3 –
2-Ethyl-1-hexanol 9.24 – 1.8 ±0.2
Phenylacetaldehyde 9.60 – 0.3 ±0.1
3-Methylbutyl isothiocyanate 9.98 8.6 ±0.2 –
Pentyl isothiocyanate 10.78 3.7 ±0.2 –
Nonanal 10.85 1.6 ±0.2 1.4 ±0.2
4-Ethylbenzaldehyde 12.01 – 0.4 ±0.1
4-Methylpentyl isothiocyanate 12.02 0.4 ±0.1 –
L-(-)-Menthol 12.20 – 0.2 ±0.1
Naphthalene 12.43 – 0.2 ±0.1
Decanal 12.76 – 0.5 ±0.1
Trans,trans-nona-2,4-dienal 12.95 – 0.2 ±0.1
Benzenepropanenitrile 13.49 0.8 ±0.1 0.2 ±0.1
Benzyl isothiocyanate 15.67 14 ±0.2 8.8 ±2.0
Junipene 16.43 – 0.2 ±0.1
Italicene 17.24 – 0.8 ±0.1
2-Phenylethyl isothiocyanate 17.44 400 ±1.0 432 ±3.0
1934 Genet Resour Crop Evol (2013) 60:1923–1943
123

samples and samples processed at 65 °C had the same
odor intensity as fresh samples while samples pro-
cessed at 90 °C lacked odor, as a consequence of
deactivation of myrosinase at temperatures higher
than 70 °C.
Further investigations are required to quantify the
content of the molecules that are responsible for the
typical flavour, which is one of the main characteris-
tics for making a high-quality product and to achieve a
high marketable yield, as requested by the agro-
industrial and pharmaceutical sectors.
Biological activities of Armoracia rusticana: human
health, antimicrobial and insecticidal effects
When plant tissue is disrupted or crushed, myrosinase
hydrolyzes GLSs to variety of molecules. These
products, especially isothiocyanates, are reported to
have biological activities including anticarcinogenic
activity (Verhoeven et al. 1997; Murillo and Mehta
2001; Balasinska et al. 2005). GLSs and their break-
down products may act as inhibitors of microbial
growth (Tierens et al. 2001), deterrents for herbivores,
and as attractants of specialist insects (Redovnikovic
et al. 2008b); moreover, these secondary metabolites
suppress soil-borne organisms such as bacteria, fungi,
viruses, nematodes and weeds (Tedeschi et al. 2011).
Particularly, the activity of isothiocyanates such as
sulforaphane against numerous human pathogens (e.g.
Escherichia coli,Salmonella typhimurium,Candida
sp.) could contribute to the medicinal properties
ascribed to Brassicaceae vegetables, such as cabbage
and mustard, which have been used as wound poul-
tices and antitumor agents for centuries (Hartwell
1982; Fahey et al. 2001). More recent interest has been
focussed on the potential anticarcinogenic activity of
GLS degradation products (Mithen 2001). The reason
of this increasing interest is due to the strong
correlation found between the consumption of Brass-
icaceae vegetables and the decreased risk for pancreas,
lung, stomach, prostate, and breast cancer, as recently
reported by Velasco et al. (2010). Particularly, several
studies have shown that allyl isothiocyanate may
inhibit different kinds of human prostate cancer
(Srivastava et al. 2003) or could contribute to the
lower incidence of bladder cancer (Munday 2002).
Horseradish contains more than 10 times as much
GLSs as broccoli. The high levels of sinigrin in
horseradish (Balasinska et al. 2005), easily hydrolyzed
by myrosinase to allyl isothiocyanate when the roots
are crushed (Matsuda et al. 2007), makes this root
interesting as a possible cancer-protecting component
in the diet (Talalay and Fahey 2001; Wedelsba
¨ck
Bladh and Olsson 2011). As reported by Shehata et al.
(2009), some German studies have investigated the
effects of horseradish on nonspecific urinary tract
infections and the antibacterial action of its essential
oils. A preparation with horseradish containing sini-
grin or allyl isothiocyanate was patented in 1995 for
topical application as a spray or applied with a swab
onto the affected mucosa, for treatment of nasal and
sinus dysfunction (Friedman 1995). Horseradish has
been approved in Germany for the treatment of
infections of the respiratory tract and as supportive
treatment in urinary tract infections. In the United
States, the root is the active ingredient of Rasapen
Ò
,a
urinary antiseptic drug (Shehata et al. 2009).
Recently, reported health benefits of horseradish
and its constituents include plasma cholesterol reduc-
tion in mice (Balasinska et al. 2005; Hara et al. 2008),
tumor cell proliferation and cyclooxygenase activity
prevention (Weil et al. 2005; Hara et al. 2008), as well
as an antimicrobial effect on Vibrio parahaemolyticus
by horseradish extract (Yano et al. 2006; Hara et al.
2008). Matsuda et al. (2007) showed that allyl
isothiocyanate provided significant protection against
ethanol-induced gastric lesions. Majewska et al.
(2004) have evaluated some antioxidant properties of
leaf and root extracts and mustard oil from four
different types of horseradish growing under different
environmental conditions: although leaf and root
extracts did not exhibit strong antioxidant properties,
the different environmental conditions affected these
properties significantly; also, volatile oil obtained
from horseradish roots revealed stronger antioxidant
properties than pure allyl isothiocyanate.
According to Mari et al. (2003) allyl isothiocyanate,
a naturally occurring flavour compound in mustard
and horseradish, has a well-documented antimicrobial
activity, therefore this volatile substance can be
employed successfully in modified atmosphere pack-
aging or as a gaseous treatment before storage. Also,
horseradish distillate is a potentially useful antimicro-
bial adjunct in packaged, refrigerated precooked
meats, in fact, essential oils of Brassicaceae plants,
including horseradish, have long been known to
possess antibacterial activity versus pathogenic
Genet Resour Crop Evol (2013) 60:1923–1943 1935
123

bacteria such as Staphylococcus aureus,Escherichia
coli and Serratia grimesii (Ward et al. 1998).
Khomvilai et al. (2006) observed the fungicidal
activities of allyl isothiocyanate against Saprolegnia
parasitica, a fish-pathogen oomycete, and determined
both minimum inhibitory concentration for mycelia
growth (68 mg/l with 60 min exposure) and zoospore
germination (42.5 mg/l with 5 min exposure).
Recently, first results of antifungal tests demonstrated
that horseradish ethanol extracts also present a fungi-
static activity against Sclerotium rolfsii Sacc., Fusar-
ium oxysporum Schlecht and Fusarium culmorum
(Wm. G. Sm.) Sacc. (Tedeschi et al. 2011).
Moreover, Park et al. (2006) have tested essential
oils from 40 plant species for their insecticidal
activities against larvae of Lycoriella ingenue (Du-
four) using a fumigation bioassay: horseradish showed
the most potent insecticidal activity among the plant
essential oils (only 1.25 ll/l caused 100 % mortality)
the most toxic compound to L. ingenue was allyl
isothiocyanate. In addition, also the results obtained
by Wu et al. (2009) suggest good insecticidal efficacy
of allyl isothiocyanate obtained from A. rusticana as
alternative fumigant against four major pest species of
stored products, maize weevil Sitophilus zeamais
(Motsch.), lesser grain borer Rhizopertha dominica
(F.), Tribolium ferrugineum (F.) and book louse
Liposcelis entomophila (Enderlein). Furthermore,
interesting and significant insecticidal activity against
larvae of Aedes albopictus (Skuse) (LC
50
value of
2.34 g/l) was obtained by using horseradish extract
prepared from fresh plants in a solution of ethanol
80 % (Tedeschi et al. 2011). Recently, Chen et al.
(2011) have assessed the fumigant toxicity of essential
oil from horseradish against Plodia interpunctella
(Hu
¨bner) and Sitophilus zeamais Motschulsky. The
essential oil of A. rusticana showed fumigant bioac-
tivity against all stages of P. interpunctella and adults
of S. zeamais. It caused high mortality of all stages of
P. interpunctella when exposed to 32 ll/l gas vapour
of A. rusticana. The oil appeared not to have much
impact on the adults of S. zeamais when the gas vapour
was lower than 16 ll/l, however, at 32 ll/l, the
percentage mortality was 100 %. GC–MS showed
that the main component of the essential oil from A.
rusticana was allyl isothiocyanate (98 %).
Armoracia rusticana oil or its major constituents
could be efficient fumigants and also could be
integrated with other pest management procedures
(Chen et al. 2011). For the practical use of horseradish
oil and their constituents as novel fumigants, further
study is necessary on the safety of these materials to
humans and on development of formulation to
improve efficacy and stability, and to reduce cost
(Park et al. 2006). Although higher concentrations of
these natural anti-fungal and insecticidal products
should be used, their intrinsically low toxicity towards
humans makes them an interesting alternative to
current chemicals. All these researches demonstrated
that horseradish extracts could be promising alterna-
tives to synthetic agrochemical products although
more work is needed (Tedeschi et al. 2011).
Another suggested use for horseradish includes
weed suppression via the allelopathic response of
GLSs in soil. GLSs and their byproducts can act as
biofumigants when used as green manure, thus
showing potential for weed control in organic pro-
duction (Shehata et al. 2009).
Genetic variability and influence of agronomic
management on glucosinolate content
The GLS content and composition in planta can be
strongly influenced by genotype and environment
(Rosa et al. 1997,2005; Sarikamis¸ et al. 2009;
Kabouw et al. 2010).
The genetic variability may play a primary role in
determining the amount of functional metabolites,
such as GLSs (Brown et al. 2002; De Pascale et al.
2007). Only few studies have investigated GLS
variation within different horseradish genotypes.
Among these, Li and Kushad (2004) have evaluated
the GLS levels in root and leaf samples of horseradish
accessions of a germplasm collection of the Vegetable
Research Farm, University of Illinois, Urbana-Cham-
paign, IL. They found that GLS content varied widely
among all accessions. According to Faltusova
´et al.
(2011), genotypes with high GLS contents will be
further utilized as a potential genetic source for
breeding. To date, information on genetic diversity
between different accessions of horseradish, are
scarce. Although molecular marker analyses are
successfully used for characterization of genetic
resources of many species, bio-agronomic character-
ization is the first step for the description and
classification of germplasm (Lotti et al. 2008; Sarli
et al. 2012).
1936 Genet Resour Crop Evol (2013) 60:1923–1943
123

Rhodes et al. (1969) have classified 20 genotypes of
horseradish, grown at the University of Illinois
Horticultural Farm, chosen from a gene pool contain-
ing over 400 genotypes cultivars to represent the
extreme and intermediate forms of morphological
variability found in horseradish. Two methods of
classification were compared. One classification was
based on 2 highly diagnostic characters (basal angle
and rugose index) that showed the extreme and
intermediate limits of the germplasm in form of a
scatter plot. The other classification was based on 40
characters. For this last classification, methods of
numerical taxonomy were used to show the germ-
plasm diversity by scatter diagrams and phenograms.
The two classifications appeared to be equal in
defining the extreme limits of the genetic variability,
although minor differences were found among the
relative positions of some genotypes. Sarli et al.
(2012) have recorded and statistically analyzed data
regarding the variation of 26 morphological descrip-
tors by using modified UPOV (International Union for
the Protection of New Varieties of Plants) guidelines
for performing tests for distinctness, uniformity and
stability for horseradish. In this study morphological
traits of 30 horseradish accessions from Basilicata
region (South Italy) were characterized and compared
to two European genotypes: the resulting cluster
displayed four major groups of genotypes, on the base
of the characteristics of rhizome and leaves. The
results showed a significant degree of variability
among accessions. Also, the results of univariate and
multivariate analysis showed that the investigated
accessions cannot be clearly separated according to
their place of origin, assuming that a distribution of an
accession is facilitated by territorial continuity and
similarity. According to Sarli et al. (2012), this
preliminary screening of a horseradish germplasm
collection is quite useful to perform future programs of
conservation in situ and ex situ, in order to expand and
improve the cultivation of A. rusticana. In any case,
there is a lack of basic biological and genetic
information about horseradish (Shehata et al. 2009).
The first studies, as reported above, could be a good
starting point to develop methods to measure genetic
diversity in different accessions of horseradish. The
genetic relationships among cultivars, breeding lines,
and wild accessions need to be determined, so that the
greatest possible gain can be made in specific crosses
(Shehata et al. 2009). Until today, only few studies
were performed on the characterization of genetic
diversity of horseradish. Various molecular marker
systems applied in plants belonging to the Brassica-
ceae family (Margale
´et al. 1995; Farnham 1996;
Rabbani et al. 1998; Das et al. 1999, Negi et al. 2004;
Faltusova
´et al. 2011) may be useful for a first
detection of polymorphisms in this species. However,
the morphological and genetic variation that seems to
exist among different horseradish accessions could be
compared with the GLS content of various horseradish
accessions for developing improved cultivars. In fact,
there is a growing interest in the metabolic engineering
of plants for the production of high-value bioactive
compounds, because the availability of phytochemi-
cals in their natural sources is often limited (Møldrup
et al. 2011). Therefore the information on genetic
diversity could be utilized in order to identify geno-
types with desirable traits for future breeding
programs.
There are even less studies on the effects of
environment and cultivation techniques on yield and
GLS content of this plant. Some researchers have
demonstrated that GLS profile can be manipulated by
cultural management (Rosen et al. 2005). About the
crop management, fertilization have been shown to
significantly affect GLS concentration in plant, partic-
ularly sulphur (S) and nitrogen (N) (Kim et al. 2002;
Rosen et al. 2005), but some controversy still exists on
such effects (Aires et al. 2006). Horseradish is a crop
that needs a lot of nutrients. Its powerful root system
drains large amounts of soil moisture and nutrient
reserves (Perlaki and Djurovka 2009). However, until
today, there is little information about the GLS content
in relation to S and N fertilization, with particular
regard to sprouts, young leaves and roots. S and N are
necessary for the synthesis of amino acids, proteins,
and various other cellular components, including thiol
compounds and the so-called secondary sulphur com-
pounds, which have a significant bearing on protection
of plants against stress and pests (Anjum et al. 2012).
GLS content is highly correlated with the S content
(Koroleva et al. 2000), therefore, there is a strong
potential for modifying GLS accumulation in crop
plants by altering S nutrition (Falk et al. 2007). Several
reports indicated that S fertilization may affect the GLS
level more than N fertilization (Rosen et al. 2005;De
Pascale et al. 2007). In fact, each GLS molecule
contains at least two sulfur atoms. One is part of the
sulfate group originating from 30-phosphoadenosine
Genet Resour Crop Evol (2013) 60:1923–1943 1937
123

50-phosphosulfate (PAPS), while the other, originating
from cysteine, is part of an S-linked glucose residue. In
addition, many GLSs are synthezised from methionine
and so may have a third sulfur atom in their structures
(Falk et al. 2007). S fertilization leads to increases in
GLS content in most cases. In some cases an over
10-fold increase was observed, as reported by Falk
et al. (2007) in their review about the effect of S supply
on GLS content.
Alnsour et al. (2012) showed recently that in vitro
grown horseradish plantlets contained increasing GLS
concentrations when the culture media was supple-
mented with additional sulfate. They concluded that
GLS concentrations in the stalks and leaves of
Armoracia in vitro plants could be modulated 20-fold
by varying the sulfate concentration in the medium,
while the increase of sulfate concentration had nearly
no impact on the GLS concentration in the roots.
The GLS content of a plant is dependent not only on
S nutrition, but also on the N level of the plant.
Particularly, through handling of fertility manage-
ment, the experimental results of Rosen et al. (2005)
clearly demonstrate that GLSs in cabbage were
maximized at low N and high S application rates. As
the ratio of N to S increases, GLS content declines,
probably because vegetative growth outpaces GLS
biosynthesis, diluting the content of these metabolites
(Kim et al. 2002; Rosen et al. 2005; Falk et al. 2007).
To quantify the effects of supply and combination of
these nutrients on GLS composition, Li et al. (2007)
showed as the total GLS concentration in turnip roots
was strongly influenced by N and S supply: with low S
supply (10 and 20 kg ha
-1
), total GLS concentrations
were 0.8–2.3 times higher at 80 kg N ha
-1
compared
to treatments with 160 and 240 kg N ha
-1
; however,
at the high S level (60 kg ha
-1
), increasing N supply
(160, 240, and 320 N ha
-1
) did not affect total GLS
concentration. In broccoli, Omirou et al. (2009) found
that the GLS concentration was low in the 50 kg ha
-1
N treatments in all plant organs, but it did not respond
to N application above 250 kg ha
-1
, while total GLS
concentrations, clearly responded to increasing S
applications within the whole wide range of S
applications (from 10 to 150 kg ha
-1
), indicating that
S is main determinant of the concentration of total
GLSs in the plant organs.
All these studies suggest that manipulating the N
and S supply might be one means of altering GLS level
in plants, thereby providing an opportunity to enhance
organoleptic properties and health benefits of horse-
radish, or its value as biofumigant (Falk et al. 2007;Li
et al. 2007).
To maintain proper nutrition, fertilizer is applied
based on results from chemical soil analysis, soil type,
cultivar, and cropping history (Shehata et al. 2009). As
reported in the review of Walters and Wahle (2010), in
A. rusticana a typical fertilizer program includes about
168 kg ha
-1
nitrogen (N), with phosphorus (P) and
potassium (K) rates based on soil type or soil test
results, with applications generally ranging from 56 to
280 kg ha
-1
. In addition, about 1–2.8 kg ha
-1
boron
and 17–28 kg ha
-1
sulfur is made with the initial N–
P–K broadcast application. Excessive nitrogen rates
should be avoided, as this can result in excessive foliar
growth and highly branched, irregular root formation
(Shehata et al. 2009). For the best response to
fertilizers, a pH range of 6–7.5 is ideal. The horserad-
ish plant thrives in deep loam or sandy soil types, well-
drained; organic matter is often added to maintain a
good soil structure, while shallow soils or those with
hard pans are not suitable as they compromise strong
root development and thus may curtail yield (Shehata
et al. 2009; Walters and Wahle 2010). Fields are
prepared for planting with ridgers to create raised beds
that increases yield of high-quality roots by ensuring
that the soil is loose so that large roots can develop.
Planting is done either by hand or using modified
transplanters that place sets (about 10 cm deep for
hand planting) in the beds at a 45-degree angle; the
spacing adopted is 40–60 cm between plants and
75–90 cm between rows, giving a plant population of
20,000–25,000 plants ha
-1
(Shehata et al. 2009).
Irrigation during drought conditions, especially during
late summer and fall, is sometimes done to improve
marketable yields because most root-sizing occurs
during this period (Walters and Wahle 2010). Usually,
horseradish is harvested in late October–November
once the foliage has been killed by frost, continuing
through the winter and early spring months (Shehata
et al. 2009; Walters and Wahle 2010).
Finally, to preserve the traditional knowledge
related to crop management, it is important to
underline that in regions like Bavaria (Germany) or
Hajdu
´sa
´g (Hungary), some cultivation practices (e.g.
removing sprouts after planting to obtain a root with a
single crown), even today, take place mainly by hand.
More studies are required for a better understanding
particularly of the effects of irrigation, fertilization
1938 Genet Resour Crop Evol (2013) 60:1923–1943
123

and time of harvest on yield and quality of horseradish
to optimize crop management.
Conclusion
Over recent years, the GLSs, a large group of nitrogen-
and sulfur-containing secondary metabolites, particu-
larly abundant in plants belonging to the Brassicaceae
and related families, have attracted the attention of the
scientific community for their fungicidal, bactericidal,
nematicidal, allelopathic and anticancerogenic proper-
ties. Among the Brassicaceae vegetables, horseradish is
particularly interesting because it is an exceptional rich
source of GLSs which could be employed for agro-
industrial and pharmaceutical purposes.
Considering this interest, more studies are needed
on qualitative and quantitative characterization of
GLSs, especially in relation to the variability of the
currently available horseradish germplasm, growing
conditions and crop management, plant phenological
phases and harvest dates.
In addition, it is important to stress that recently the
interest in natural substances has increased and
numerous studies on the biocidal activity of a wide
range of secondary metabolites have been reported, as
for example for the allyl isothiocyanate produced from
sinigrin with a well-documented antimicrobial activ-
ity. As reported by Mari et al. (2003), in Japan,
synthetic allyl isothiocyanate is registered as a food
additive and preliminary data showed that food
preserved with this compound contains only very
low residue of allyl isothiocyanate (Isshiki et al. 1992).
Therefore, natural extracts of horseradish could be
used as alternative to synthetic molecules.
For the great heritage about the gastronomic and
folk medical uses of horseradish, the species should be
valorized to prevent extinction of genotypes of high
nutritional value and specific taste and flavour and
farmers should continue to cultivate landraces in
traditional cultivation areas, maintaining the links
with territory and its history. In fact, the clonal
propagation of A. rusticana endangers its genetic
diversity, so efforts are required to establish well-
documented collections and to analyze genetic diver-
sity in all accessions available. In addition, legal
protection of geographic identity of horseradish land-
races could contribute to conservation of genetic
resources, and at same time, could increase the
viability of little farms. For example, by following a
centuries-old tradition, horseradish is today grown by
farmers in Bavaria and Hajdu
´sa
´g regions as an PGI
(Protected Geographical Indication) and PDO (Pro-
tected Designation of Origin), respectively, to preserve
the historical link with the territory. For small scale
growers, horseradish has potential marketing outlets
through fresh sales to local restaurants, grocery and
specialty food stores, and to specialized food process-
ing companies. They can also make sales at farmers
markets and roadside stands. For large scale produc-
tion, it is common for condiment processors to work on
a contract basis with growers. These markets can be
competitive and difficult to break into (Bratsch 2009).
Today, further investigations on the chemical,
biological and molecular aspects of the GLSs and
the composition and partitioning in the plant are
needed to achieve the goal of efficient biodiversity
conservation to prevent the genetic erosion of local
genotypes of horseradish and the risk to lose traits that
can be useful in future breeding programs.
Acknowledgments The authors are grateful to Mr. Vito
Agneta for assistance in graphic design images and Prof.
Ippolito Camele of the University of Basilicata for providing the
images in Fig. 2. The authors are also thankful to the Editors of
Economic Botany, Phytochemistry and Journal of Agricultural
and Food Chemistry for: (1) the use of the Table 1; (2) the adapt
of the Table 2—Copyright (1980) Elsevier Ltd and Table 3—
Copyright (1979) Elsevier Ireland Ltd; (3) the permission to
adapted Tables 4and 5—Copyright (2004) American Chemical
Society, respectively.
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