Journal of Ethnopharmacology 143 (2012) 14–23

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Review

Trifolium species-derived substances and extracts—Biological activity

and prospects for medicinal applications
Joanna Kolodziejczyk-Czepas n
Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/3, 90-236 Lodz, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Background: Despite of the fact that clovers (family: Fabaceae; genus: Trifolium) have been known for
Received 4 April 2012 many centuries as important forage plants and valuable herbs in folk medicine, their phytochemical
Received in revised form characteristics and biological activity remain only partly established.
21 June 2012
Aim of the study: The presentation of the current knowledge of physiological effects, therapeutic action,
Accepted 21 June 2012
Available online 6 July 2012
new trends in the investigation of Trifolium plants and suggestions for the future applications of these
herbs in therapy of various disorders.
Keywords: Methods: A critical review of literature on the biological activity of Trifolium plants, with the indication
Trifolium on important gaps, was performed. The compilation of existing information on physiological effects and
Clover
medicinal value of clovers, derived from both traditional medicine recommendations and scientific
Phytoestrogens
reports, is presented.
Medicine
Results: The available data indicate on the abundance of biologically active substances in Trifolium
plants, including numerous flavonoids, saponins, clovamides and phenolic acids. The best known clover –
Trifolium pratense L. (red clover) – is used for the production of herbal medicines, an alternative to the
conventional hormonal replacement therapy. The biological activity and potential therapeutic effects of
other Trifolium species have gained a considerable scientific interest; extracts obtained from various clovers
have been shown to possess antioxidative and antiinflammatory activities, inhibiting angiogenesis and
displaying anti-cancer properties.
Conclusions: Clovers other than T. pratense also seem to be a promising source of valuable phytochemicals,
such as isoflavones and various flavonoids. However, the therapeutic use of these Trifolium species is
significantly limited by the lack of clinical evidence; thus further studies are needed.
& 2012 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2. The use of Trifolium plants in traditional medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3. Phytochemical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4. Phytoestrogenic activity of clovers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5. Trifolium plants in the therapy of menopause-related disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
6. Antioxidative properties of Trifolium species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
7. Anti-inflammatory and antiplatelet action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
8. Anticancer and antiangiogenic effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
8.1. Antiangiogenic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
8.2. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
8.3. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
9. Possible role in the therapy of the metabolic syndrome and cardiovascular disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
10. Other therapeutic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

n
Tel./fax: þ 48 42 635 44 84.
E-mail address: joannak@biol.uni.lodz.pl

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.06.048
 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1. Introduction
The Trifolium genus (Fabaceae) comprises of about 240 species
of clovers (Zoric et al., 2012), occurring in temperate and
subtropical regions of both hemispheres. The Mediterranean
basin, western North America, and the highlands of eastern Africa
are the three geographic regions with the greatest diversity of
Trifolium species, while no species are native to southeastern Asia
and Australia (Ellison et al., 2006). Some of Trifolium species have
been known for many centuries as forage plants (T. pratense L.,
T. repens L., T. resupinatum L., T. incarnatum L., T. hybridum L.,
T. pannonicum Jacq., T. subterraneum L., T. fragiferum L., and
T. medium L.), and valuable herbs in folk medicine of various
cultures. Nowadays, the therapeutic use of Trifolium plants is still
based mainly on traditional medicine recommendations, but the
number of scientific data on the biological activity of clovers and
their possible therapeutic effects has been growing (Table 1). The
majority of the studies on the biological properties of clovers
concerns T. pratense L. (red clover) and is focused on its phytoes-
trogenic action, being a result of isoflavone content. Interactions
of isoflavones with estrogen receptors are possible due to their
structural similarity to 17b-estradiol, a steroid hormone. For that
reason, red clover is a source of dietary supplements and herbal
medicines administered as an alternative to the conventional
hormonal replacement therapy (Engelmann et al., 2009). How-
ever, it should be emphasized that the beneficial effects of red
clover as well as other clovers may be dependent on the action of
various biologically active substances occurring in these
herbs—not only isoflavones. There are significant gaps in the
ethnopharmacological knowledge of medicinal relevance of Tri-
folium genus. Contrary to the existing evidence of therapeutic
properties of T. pratense, obtained from in vivo and clinical
examinations, the curative applications of other Trifolium species
are supported mainly by basic investigations or traditional med-
icine. Evaluation of the novel therapeutic potencies of Trifolium-
derived extracts seems to be a promising trend in phytopharma-
cological research for a variety of reasons, including the diversity
of chemical components and widespread occurrence of clovers, as
well as low costs of cultivation. The present article critically
reviews the available information on physiological activity and
expected medicinal importance of Trifolium plants, and indicate
the future prospects for the use of these herbs as therapeutics in
various disorders.
Evidence of the physiological effects of Trifolium plants derives
from in vitro and in vivo studies. The review includes data (to May
2012) obtained from journals indexed in international databases
(Medline/Pubmed, Scopus, Science Direct/Elsevier, Springer Link/
ICM), as well as local periodicals, not indexed in international
scientific databases. In numerous cases, references included to
those articles (reviews, in particular) were also verified for
suitability.
2. The use of Trifolium plants in traditional medicine
Clovers have been used by Oriental and European cultures for
the treatment of eczema and psoriasis. In Turkish traditional
medicine, T. repens L., T. arvense L. and T. pratense L. have been
administered as expectorant, antiseptic, analgesic, sedative and
tonic mixtures (Sabudak et al., 2008). T. pratense L. and T. repens L.
15
are popular herbs in Pakistan, useful in the treatment of sore
throat, fever, pneumonia, meningitis and feverish feeling (Khan
and Khatoon, 2008). The recent ethnobotanical research of
Mustafa et al. (2012), conducted in the Albanian Alps in Kosovo
provided additional evidence of the traditional use of T. pratense L.
and T. repens L. In this region of Europe, a juice obtained from
squeezed leaves of T. pratense L. is one of the folk medicines for
stomach disorders. T. repens L. decoctions have been applied as
anti-diarrhoeal remedy (Mustafa et al., 2012). Native Americans
have used T. pratense L. to cure external skin problems, lung
illnesses, as well as some disorders of nervous and reproductive
system (Sabudak and Guler, 2009). T. pannonicum Jacq. is one of
widespread, wild growing medicinal plants in high mountain
region of Montenegro, used by local people for wound healing
(Menkovic et al., 2011). Barros et al. (2010) report that in
Portugal, T. angustifolium L. decoctions are a folk medicine for
stomachache and diarrhea. Traditional administration of decoc-
tions from aerial parts of this clover to cure diarrhea and
swellings has been also shown in the ethnobotanical study,
performed in the Natural Park of ‘‘Serra de Sa~ o Mamede’’
(Portugal) (Camejo-Rodrigues et al., 2003). T. repens L. is a
deworming remedy in the traditional medicine of the Naga tribes
of India. This activity of white clover has been confirmed by
in vivo study on animals (Tangpu et al., 2004). The seeds of
T. alexandrinum L. have been administered in Egypt as an anti-
diabetic remedy (Khaled et al., 2000).
3. Phytochemical data
The chemical profile of clovers is partly recognized. It is known
that besides isoflavones, Trifolium plants synthesize a wide range
of phenolic and polyphenolic compounds such as flavonoids,
saponins, clovamides (caffeic acid esters), phenolic acids and
other substances (Oleszek and Stochmal, 2002; Oleszek et al.,
2007). Until now, chemical analysis of clovers in the greatest
extent have been performed by Oleszek et al. (2007). The aerial
parts of 57 Trifolium species were analysed in terms of the content
of flavonoids (mainly isoflavones), phenolic acids, and clovamides,
and then grouped into five clusters. Cluster 1—species with the
highest concentrations of isoflavones (51–97 mg/g of dry matter),
comprises of T. lappaceum L., T. phleoides Willd., T. hirtum All.,
T. alpestre L., T. medium L., T. subterraneum L., T. heldreichianum
Hausskn., and T. scabrum L. Cluster 2 includes T. angustifolium L.,
T. bocconei Savi, T. nigrescens Viv., T. pannonicum Jacq., T. desvauxii
Loisel., and T. leucanthum M.Bieb., particularly rich in flavonoids
(16 to 32 mg/g of dry matter). Cluster 3 includes T. resupinatum
var. majus Boiss., T. arvense L., T. stellatum L., T. incarnatum L.,
T. xerocephalum Fenzl, T. isthmocarpum Brot., T. glomeratum L.,
T. occidentale Coombe, T. pratense, ssp. expansum Jáv., T. ambiguum
M.Bieb., T. carmeli Boiss., T. fragiferum L., T. dubium Sibth.,
T. striatum L., T. fragiferum ssp. bonnani C.Presl, and T. campestre
Schreb., that have low phenolic content. Species classified to
cluster 4 (T. ligusticum Loisel., T. ochroleucon Huds., T. resupinatum
var. resupinatum L., T. cernuum Brot., T. hybridum L., T. medium var.
sarosiense (Hazsl.) Simonk., T. hirtum All., T. rubens L., T. repens L.,
T. cherleri L., T. tomentosum L., T. michelianum Savi, T. michelianum
ssp. balansae (Boiss.) Azn., T. spumosum L., and T. montanum L.)
possess a high concentration of phenolic acids, at the range of
1–1.8% (of dry matter). Cluster 5 includes species with high
16 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23

Table 1
Agricultural uses, biological activity and medicinal applications of various clovers (compilation of the available data).

Species Agricultural uses and/or traditional Results of studies on biological Contemporary medicine
medicine application activity—beneficial effects administration and potential
future applications

T. alexandrinumL. (Berseem clover)
Forage plant; antidiabetic herb
 Antioxidative properties (Sabudak
et al., 2009)
 Hepatoprotective effect (Al-Rawi,
2007);
 Antibacterial action (Khan et al.,
2012)

Trifolium angustifolium L. (Narrow Stomachache and diarrhea  Weak antioxidative properties

clover) (Barros et al., 2010)

T. arvense L. (Rabbitfoot clover) Eczema and psoriasis therapy

T. balansae, T. michelianum (Boiss.) Forage plant
Azn. (Balansa clover)
T. fragiferum L. (Strawberry clover) Forage plant  Tyrosinase inhibition (Sabudak Potentially useful in the treatment of
et al., 2006) melanin biosynthesis-related skin
diseases
T. hybridum L. (Alsike clover) Forage plant
T. incarnatum L. (Crimson clover) Forage plant
T. medium L. (Zigzag clover) Forage plant
T. nigrescens subsp. petrisavii Forage plant  Antioxidative action(Sabudak et al.,
(Clementi) Holmboe 2009)

T. pallidum Waldst. & Kit.  Antioxidative action (Kolodziejczyk Potentially useful in the anti-platelet
et al., 2011); therapy
 Antiplatelet effect (Kolodziejczyk-
Czepas et al., 2012)

T. pannonicum Jacq. (Hungarian Forage plant; wound healing

clover)
T. pratense L. (Red clover) Forage plant; expectorant, antiseptic,
analgesic herb; treatment of sore
throat, fever, pneumonia and
meningitis; skin problems, lung
illnesses as well as some disorders of
nervous and reproductive system
 Estrogenic effect (Booth et al., Menopausal complaints; potentially
2006, Pfitscher et al., 2008, Nissan useful in anti-cancer therapy and
et al., 2007, Coon (2007) (Rev.), cardiovascular diseases prevention
Yatkin and Daglioglu, 2011,
Adaikan et al., 2009, Lipovac et al.
(2010)., 2011, Del Giorno et al.,
2010);
 Antioxidative (Campbell et al.,
2004, Mu et al., 2009) and anti-
platelet activity (Simoncini et al.,
2005);
 Antiangiogenic action (Krenn and
Paper, 2009);
 Anticancer properties (Liu et al.,
2011, Jarred et al., 2002)

T. repens L. (White clover)
T. resupinatum L. (Persian clover)
Forage plant; analgesic medicine for
rheumatic disorder; deworming
remedy; treatment of sore throat, fever,
pneumonia, meningitis
Forage plant
 Anticestodal properties (Tangpu et
al., 2004)
 Antiinflammatory properties
(Sabudak et al., 2008, 2009)
 Antioxidative action (Sabudak
et al., 2008)

T. subterraneum L. (Subterranean Forage plant

clover)
T. scabrum L. (Rough clover)  Antiplatelet activity Potentially useful in the prevention of
(Kolodziejczyk-Czepas et al., 2012) cardiovascular diseases

content of total phenolics: T. apertum Bobrov, T. alexandrinum L., T.
clypeatum L., T. squarrosum L., T. echinatum M.Bieb., T. pratense ssp.
sativum Ponert, T. pratense ssp. sativum f. albiflorum Puskal, T.
miegeanum Maire, T. pratense L., T. isodon Murb., and T. pallidum
Waldst. & Kit. All these clovers contained clovamides (from 4 to
13 mg/g of dry matter), high concentrations of phenolic acids and
flavonoids.
The qualitative analyses of phenolic acids present in flowers
and leaves of T. repens L. and T. pratense L. revealed the presence of
9–10 acids; 7 of them were identified: p-hydroxybenzoic, salicylic,
protocatechuic, gentisic, p-coumaric, caffeic and ferulic. In flowers
of both clovers, p-hydroxybenzoic and salicylic acids were the
main compounds. In extracts from leaves of T. repens L., the main
acidic component was caffeic acid, while salicilic and caffeic acids
predominated in extract from T. pratense L. leaves (Kicel and
Wolbis, 2006). Recently, more details about the phytochemical
profile of T. repens L. (white clover) have been given. Flowers and
leaves of this plant contain 12 flavonoids, identified as quercetin and
 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23 17

kaempferol 3-O-(600 -a-rhamnopyranosyl-200 -b-xylopyranosyl)-b- endogenous estrogens, and in consequence affect hormonal
galactopyranosides, kaempferol 3-O-(200 ,600 -a-dirhamnopyranosyl)- signalling (Tham et al., 1998). The available evidence of physio-
b-galactopyranoside, mauritianin, quercetin and kaempferol logical effects of phytoestrogens in humans has been obtained
3-O-(200 -b-xylopyranosyl)-b-galactopyranosides, kaempferol and chiefly from studies with soy (Glycine max L.) isoflavones (e.g.,
quercetin 3-O-b-(600 -O-acetyl)-galactopyranosides, trifolin, hypero- Wuttke et al., 2007; Szkutnik-Fiedler et al., 2010; Castelo-Branco
side, myricetin 3-O-b-galactopyranoside, quercetin, ononin, medi- and Cancelo Hidalgo, 2011; Bolaños et al., 2010; Andres et al.,
carpin 3-O-b-glucopyranoside and methyl caffeate (Kicel and 2011). The number of clinical studies on humans treated with
Wolbis, 2011). In roots of T. pratense L., the presence of formono-
netin as well as isoflavonoid glycosides: formononetin-7-O-b-D-
galactopyranoside and inermin-3-O-b-D-galactopyranoside, was
confirmed (Drenin et al., 2008). In the further phytochemical
analyses of T. pratense L. aerial parts, formononetin, prunetin,
genistein, prunetin-40 -O-b-D-glucopyranoside, genistein-7-O-b-D-
galactopyranoside, and two new compounds: prunetin-40 -O-a-D-
glucopyranoside and (þ )-pinitol, were identified (Drenin et al.,
2010). Janda et al. (2009) confirmed the presence of chlorogenic
acids, four quercetin and two kaempferol glycosides, and formo-
nonetin-7-glucoside in aerial parts of T. resupinatum L., while
chemical analyses of seeds of this plant, described by Sabudak
et al. (2008), showed a diversity of structural variants of triterpene
saponins (aglycones) such as soyasaponin I, II and III, and flavonoid
compounds (e.g., quercetin).

4. Phytoestrogenic activity of clovers

The treatment with plant-derived substances and extracts

containing phytoestrogens is commonly believed to be more safe
than conventional hormonal replacement therapy. Isoflavones
(Figs. 1 and 2) are thought to be the most important cluster of
phytoestrogens, however the estrogenic properties of other plant-
derived compounds such as lignans, coumestans, and stilbenes,
have been also found (Pilsakova et al., 2010). Isoflavones possess
mild estrogenic activity, but in humans, these compounds can be
accumulated even at 100-fold higher concentrations than Fig. 2. Structure of irilone, prunetin and pseudobaptigenin.

Fig. 1. The structures of main isoflavones naturally occurring in various Trifolium species (Panel A) and isoflavone metabolites (Panel B).
18 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23
isoflavones or extracts derived from red clover is significantly
lower. Furthermore, there is no information about studies on the
estrogenic activity of other clovers. In comparison to soybeans,
where daidzein, genistein and glycitein are present, red clover
contains at least seven additional isoflavones (Wu et al., 2003). In
a pilot study (non-randomised, with 7 volunteers) of Maul and
Kulling (2010), a single bolus intake of about 40 mg red of clover
isoflavone extract, rich in formononetin (formononetin/biochanin
ratio of 2.17) led to the higher plasma concentrations of the
demethylated metabolites: daidzein and genistein (0.39 and
0.06 mM, respectively). Howes et al. (2002) described the results
of a study on long-term pharmacokinetics of isoflavones from
T. pratense L., administered in supplement with a low formonone-
tin content (formononetin:biochanin ratio was 0.65). The exam-
ination involved 14 participants, consuming a low-isoflavone diet
for 2 weeks as an oral daily dose of two isoflavone tablets (one
tablet contained 24.5 mg of biochanin, 1.5 mg of genistein, 16 mg
of formononetin, and 1.5 mg of daidzein). The supplementation
with isoflavones resulted in 0.25 micromolar concentration of
daidzein and 0.42 micromolar concentration of genistein in blood
plasma. Isoflavones present in red clover in minor amounts such as
irilone, prunetin and pseudobaptigenin (Fig. 2), are also bioavail-
able. Irilone concentrations in commercial red clover supplements
are sufficient to lead to physiologically relevant plasma concentra-
tions. According to Maul and Kulling (2010), the methylenedioxy
bridge attached to the A ring of the irilone skeleton acts as a
protective group against the degradation of irilone by the human
microbiota. Moreover, after the intake of red clover supplement
both prunetin and pseudobaptigenin were detected.
The molecular mechanisms of the physiological and therapeu-
tic effects of clover-derived extracts and isoflavones are only
partly elucidated. In vitro studies of Booth et al. (2006) on the
chemical and biological profile of a clinical phase II red clover
extract, composed of 35.54% isoflavones (daidzein, genistein,
formononetin, biochanin A), 1.11% flavonoids, 0.06% pterocar-
pans, r0.03% coumarins, and r0.03% tyramine, confirmed its
estrogenic action. It has been also found that all of these
isoflavones (except formononetin) bind to one or both of estrogen
receptors (a and b). Since isoflavones undergo various metabolic
transformations after the dietary intake, the phytoestrogenic
actions of their metabolites were also investigated (Pfitscher
et al., 2008). The binding and transactivating properties of red
clover isoflavones and their metabolites may be assessed by the
analysis of estrogen receptors a and b, as well as androgen and
progesterone receptors. The study of Pfitscher et al. (2008), with
the use of yeast as an experimental system, showed that the
metabolic transformations of isoflavones led to the changes in
both their affinity to estrogen receptors and transactivation
potential. The demethylation of formononetin and biochanin A
to daidzein and genistein, respectively, resulted in the evident
alterations in receptor affinity, and significantly enhanced poten-
cies against estrogen receptor b. The enzymatic conversion of
biochanin A, formononetin, daidzein and genistein to their
reduced metabolites dihydrobiochanin A, dihydroformononetin,
dihydrogenistein and dihydrodaidzein, respectively, did not cause
any significant effects on the receptor affinity to either estrogen
receptor. The possible beneficial health effects of isoflavones
might be attributed to equol (daidzein metabolite), which pos-
sesses a higher receptor transactivation potential than its
precursor—daidzein. The formation of equol resulted in a 30
times higher potency, but its reduced metabolite dihydrodaidzein
displayed about 100 times lower transactivation potential. Bind-
ing of components of a clinically administered T. pratense extract
(0.3–300 mg/ml), to the m- and d-opiate receptors, has been
shown in experiments on cell lines expressing human opiate
receptors (Nissan et al., 2007). Since the opioid system plays an
essential role in the regulation of body temperature, mood,
hormonal levels and actions, according to the authors, these
findings may explain in part the beneficial effect of T. pratense-
derived phytoestrogens in alleviating menopausal symptoms.
5. Trifolium plants in the therapy of menopause-related
disorders
Extracts of red clover are commercially available as dietary
supplements. Despite of the fact that the biological activity of
isoflavones may possess therapeutic value in the treatment of
hormone-related diseases, such as cardiovascular diseases, can-
cer, osteoporosis and menopausal complaints (Tham et al., 1998),
the effectiveness of medicinal use of red clover still raises
numerous concerns. After the systematic review and meta-ana-
lysis of literature, Coon (2007) concluded that there is evidence of
a marginally significant effect of T. pratense L. isoflavones in the
treatment of hot flushes in menopausal women. Additionally,
these authors found neither apparent evidence of side effects in
short-term application of red clover isoflavones, nor data on the
safety of long-term administration. Yatkin and Daglioglu (2011)
have paid the attention on the lack of experimental studies based
on physiological isoflavone concentrations (including T. pratense
L. isoflavones), evaluating the effects of phytoestrogens at differ-
ent periods of life. Thus, further studies seem to be necessary for
the evaluation of the risk of long-term use of phytoestrogens.
Some new information on the safety of T. pratense L. and soy
(Glycine max L.) isoflavones in vitro, has been provided recently by
Reiter et al. (2011). Plant extracts and isolated isoflavones were
examined in proliferation assays on 11 human cancer cell lines
(cancers of the colon, prostate, breast, cervix, liver, pancreas,
stomach and ovaries) and a fibroblast line to assess their cytotoxic
activity. The tested compounds and extracts did not promote the
growth of human cancer cells, but reduced cell proliferation,
increased apoptosis and cell cycle arrest.
Contrary to critical conclusions of Coon (2007), results of
studies on animals are more optimistic. A prospective, vehicle-
controlled study on animal model of menopause, demonstrated
that 12-week treatment with red clover isoflavones (daily dose:
100 mg of daidzein per kg of body weight or 6.68 mg of red clover
extract/kg b.w.—an equivalent of 100 mg of daidzein/kg b.w.) led
to significant improvements in bone density, tissue integrity, and
vaginal blood flow with minimal effect on uterine weight, and
might be an alternative to conventional treatment with synthetic
estrogens (Adaikan et al., 2009). Results of experiments on rats
with surgically-induced menopause, suggest that red clover pre-
paration in dosages amenable to clinical practice may improve
ovariectomy-induced osteoporosis. Moreover, mild metabolic
alkalosis might further synergize some therapeutic aspects. The
examination was performed with the use of a quality-controlled
red clover extract, containing 40% of isoflavones (genistein,
daidzein, biochanin A, and hydrolyzed aglycones of formonone-
tin). Animals were randomised into four groups: A (sham-oper-
ated rats), and three other groups (ovariectomised), being fed for
three months as follows: standard food (group B), 6 mg/kg/day
food mixed with red clover extract (group C), or given 6 mg/kg/
day of red clover extract with a modified alkaline supplementa-
tion through a nasogastric tube at a dose of 16 mg (group D)
(Kawakita et al., 2009). In other study on animal model of
osteoporosis, T. pratense isoflavones (oral dose of 20 and 40 mg
of total isoflavones daily, for 14 weeks) considerably increased
bone mineral content, mechanical strength of the tibia, femoral
weight and density, as well as effectively prevented the rise of
serum alkaline phosphatase level. The number of osteoclasts was
also significantly reduced, compared to the ovariectomised
 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23
control rats (Occhiuto et al., 2007). However, it should be
emphasized that animal experimental models do not strictly
reflect the physiology of human body. Therefore, there are
significant gaps in our knowledge of the incidence or lack of
biological effects of isoflavones (particularly Trifolium-derived
phytoestrogens) in humans. The most of studies on physiological
effects of isoflavones in humans concerns soy-derived com-
pounds. The systematic review and meta-analysis of randomised
controlled trials of Taku et al. (2010), indicated that soy isoflavone
supplements moderately decreased the level of bone resorption
marker - urinary deoxypyridinoline, but did not affect bone
formation markers (serum bone alkaline phosphatase and serum
osteocalcin) in menopausal women. Weaver et al. (2009)
described results of a randomised, crossover, blinded trial in 11
healthy postmenopausal women, designed to assess the antire-
sorptive action of isoflavones on bone. In those study, the effects
of four commercial sources of isoflavones from soy cotyledon, soy
germ, kudzu, and red clover, as well as a positive control (1 mg
estradiol combined with 2.5 mg medroxyprogesterone or 5 mg/
day risedronate (Actonels)) were examined. Only isoflavones
from soy cotyledon and germ significantly decreased net bone
resorption (by 9% and 5%, respectively). Based on those findings,
the authors concluded that genistein-like isoflavones displayed a
significant, but modest ability to suppress bone resorption in
postmenopausal women.
Studies on the effects of isoflavones on bone metabolism
provided controversial results, but simultaneously, the effective-
ness of phytoestrogenic therapy on other menopause-related
disorders has been confirmed by Lipovac et al. (2010). This
randomised clinical investigation was designed to evaluate the
effects of a non-prescription red clover extract (MF11RCE) on
mood disorders. Participants (190 postmenopausal women) were
randomly assigned to one of two groups: treated with two
capsules of either MF11RCE (80 mg of red clover isoflavones) or
placebo of equal form for a 90-day period. After a 7-day washout
period, subjects received the opposite treatment for another 90
day. The medication with red clover isoflavones resulted in a
significant decrease of depressive and anxiety symptoms among
postmenopausal women. In other research of these authors, the
treatment with the same dose of red clover isoflavones (80 mg/
day) caused a visible improvement of scalp hair and skin status as
well as libido, mood, sleep, and tiredness in postmenopausal
women. The examination involved 109 participants, randomly
assigned to one of two groups: receiving capsules of red clover
isoflavones or receiving placebo of equal appearance for a 90-day
period. Then, after a washout period of 7 day, medication was
crossed over and continued for 90 day more (Lipovac et al., 2011).
On the other hand, according to Del Giorno et al. (2010), 12-
month treatment with T. pratense extract does not yield a
considerable improvement in menopausal symptoms and sexual
satisfaction. The described results were obtained in a prospective,
randomised, double-blind, placebo-controlled study on 120
women aged 45–65, that received 40 mg of red clover extract
(one capsule daily).
6. Antioxidative properties of Trifolium species
The overproduction of reactive oxygen and nitrogen species
(ROS/RNS) is involved in the etiology and pathophysiology of
inflammation and cancer, as well as autoimmune, neurodegenera-
tive, cardiovascular and other disorders (Ronson et al., 1999;
Ohshima et al., 2003; Sorg, 2004). A growing number of in vitro
and in vivo studies has showed the antioxidative properties of
isoflavones (Kapiotis et al., 1997; Lissin and Cooke, 2000; Wiseman
et al., 2000; Jenkins et al., 2000), but in some investigations, anti-
19
Fig. 3. Clovamide structure.
oxidative effects of isoflavones were not recorded (Hodgson et al.,
1999; Hsu et al., 2001). A pilot clinical trial, described by Campbell
et al. (2004) indicated on a little or no effects of 1-month isoflavone
(86 mg/day red clover-derived isoflavones) supplementation on
markers of antioxidant status. This randomised, placebo-controlled
and crossover examination involved 16 healthy pre- and 7 post-
menopausal women. In contrast to these observations, study on
animals (Mu et al., 2009) revealed antioxidative and estrogenic
actions of T. pratense-derived formononetin, administered for
6 months. The examination was conducted on mice divided into
5 groups: sham-operated group and 4 ovariectomised groups,
control group, stilbestrol replacement therapy group (0.20 mg/kg
b. w. daily), low-dose formononetin group (0.05 g/kg b. w. daily)
and high-dose formononetin group (0.5 g/kg b. w. daily). The
intake of formononetin significantly increased the activities of
superoxide dismutase, glutathione peroxidase, catalase, and
reduced lipid peroxidation in animal body; the estrogenic effect
was not dosage-related.
The antioxidative activity of some Trifolium species may be also
a result of the abundance of flavonoids and other phenolic
compounds, such as catechins, saponins, clovamides and phenolic
acids, present in clovers. The in vitro study on the clovamide-rich
extract of T. pallidum Waldst. & Kit. (at the final concentration
range of 12.5–100 mg/ml) revealed antioxidative action of all used
concentrations, in the protection of proteins and lipids of blood
platelets and plasma against the oxidative stress-induced damage,
in comparison to the samples exposed to oxidative stress in the
absence of the extract (Kolodziejczyk et al., 2011). Antioxidative
action of clovamide (Fig. 3), a derivative of caffeic acid, is
attributed mainly to its ability to prevent lipid peroxidation (Ley
and Bertram, 2003). The in vitro study of Sanbongi et al. (1998)
showed that in the protection of lipids, clovamide might be more
effective antioxidant than epicatechin or quercetin. Sabudak et al.
(2009) have found the antioxidative activity in vitro of hexane
extracts, obtained from T. balansae Boiss., T. stellatum L., T.
nigrescens subsp. petrisavii (Clementi) Holmboe, T. constantinopo-
litanum Ser. and T. resupinatum L. Moreover, these effects corre-
lated with the content of unsaturated fatty acid in the tested
extracts. Contrary to the mentioned reports suggesting the high
anti-oxidative activity of Trifolium plants, the ethnobotanical study
of Barros et al. (2010) demonstrated relatively weak antioxidant
properties of T. angustifolium L. in vitro, most likely being a result
of a low content of phenolics and flavonoids (95.61 and 26.78 mg
of gallic acid equivalents (GAC) per g of extract, respectively). The
antioxidative action of T. angustifolium L. at different concentra-
tions (0.03–4 mg/ml) was measured by DPPH scavenging activity,
reducing power, thiobarbituric acid-reactive substances (TBARS)
level, and the inhibition of b-carotene bleaching.
7. Anti-inflammatory and antiplatelet action
Blood platelet activation and their accumulation at the site of
vascular injury is the crucial event in physiological haemostasis.
Platelets are also responsible for the formation of pathogenic
thrombi. Both the activated platelets and platelet-leukocyte
interactions are involved in inflammatory processes, as well as
20 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23
in the pathogenesis and progress of various cardiovascular dis-
eases, including atherosclerosis, myocardial infarction and
ischemic cerebral stroke (Smyth et al., 2009; Angiolillo et al.,
2010). Until recently, very few studies have been designed to
evaluate the effects of Trifolium plants on the haemostatic system.
Red clover isoflavones were found to activate an antiplatelet
factor—nitric oxide (NO) synthesis in endothelial cells by stimu-
lation of transcriptional pathways. These experiments were
designed to examine the influence of various combinations of
isoflavone amounts on activity of endothelial nitric oxide
synthase (eNOS). The investigated content of isoflavones was
relevant to amounts found after the intake of a standardized red
clover extracts preparation in one standard tablet (containing:
0.6 mg of genistein, 9.6 mg of biochanin A, 2.3 mg of daidzein, and
27.5 mg of formononetin). The prolonged exposure of cultured
human endothelial cells to red clover extracts enhanced the
expression and the activity of eNOS. Molecular mechanisms of
these effects may involve the recruitment of estrogen receptor b.
It has been also shown that clover-derived isoflavones synergize
with 17b-estradiol in increasing eNOS activity and expression
(Simoncini et al., 2005).
Lam et al. (2004) demonstrated that isoflavones obtained from
red clover might suppress inflammation. In experiments of these
authors, red clover-derived isoflavones effectively inhibited
cyclooxygenase activity in the macrophage cell line RAW 264.7
and human monocytes, as estimated by the reduction of prosta-
glandin E2 and/or thromboxane B2 synthesis. The antiinflamma-
tory and antioxidative properties were also shown for T.
resupinatum L. var. microcephalum Zoh. Sabudak et al. (2008)
revealed, for the first time, the antioxidative action of this clover
on animal model of arthritis. The administration of T. resupinatum
L. var. microcephalum Zoh. extract (1.35 and 13.5 mg/kg b. w.)
significantly reduced the paw edema induced by the complete
Freund’s adjuvant. Furthermore, a significant inhibition of lipid
peroxidation was found. According to the authors, effects of this
extract might be a result of the presence of flavonoids and their
free radical scavenging activities.
It has been established, that some of Trifolium species (e.g., T.
pallidum Waldst. & Kit.) are particularly rich in clovamides, a
caffeic acid esters, displaying antioxidative properties (Arlorio
et al., 2008). Clovamides may be an interesting group of plant-
derived phenolics with expected beneficial effects, however, until
now their physiological activity has been inadequately recog-
nized. The most of studies on biological effects of clovamides was
performed with compounds from cocoa beans (Theobroma cacao
L.). In addition to antioxidative action, the antiplatelet properties
of clovamides have been also observed. Results from animal
study, showed that N-caffeoyldopamine (50 and 100 mg/35 g b.
w.) effectively reduced the P-selectin expression and platelet-
leukocyte interactions by 31 to 45% and 34 to 43%, respectively
(Park and Schoene, 2006). Recently, the antiplatelet actions of T.
pallidum Waldst. & Kit. and T. scabrum L. extracts (used at the final
concentration range of 12.5–50 mg/ml), have been found in vitro.
The examined extracts: from T. pallidum Waldst. & Kit. (phenolic
fraction and clovamide fraction) and T. scabrum L. (phenolic
fraction), modulated platelet adhesion to different proteins (col-
lagen and fibrinogen) (Kolodziejczyk-Czepas et al., 2012).
8. Anticancer and antiangiogenic effects
8.1. Antiangiogenic properties
Phytoestrogens may influence angiogenesis and act as chemo-
preventive agents. Isoflavones, including genistein and daidzein,
downregulate genes and mRNA levels of proteins involved in
angiogenesis such as IL-8, matrix metalloproteinase 13, and
fibronectin. Moreover, the synthesis of antiangiogenic factors:
plasminogen activator inhibitor-1 (PAI-1), angiostatin and throm-
bospondin may also undergo the isoflavone-mediated upregula-
tion. Evaluation of the antiangiogenic action of red clover extracts
(with the use of the chorioallantoic membrane assay, at a dosage
of 250 mg of extract per pellet) demonstrated a considerable
inhibition of angiogenesis. Non-methylated isoflavones: daidzein
and genistein were found to possess stronger antiangiogenic
activity than the methylated compounds: formononetin and
biochanin A (Krenn and Paper, 2009).
8.2. Prostate cancer
Data concerning the anticancer properties of red clover derive
mainly from examinations of prostate cancer. Under in vitro
conditions, red clover isoflavones (100 nM) may partly suppress
the proinflammatory effects of transforming growth factor b1 in
human primary prostate cancer-derived stromal cells (Liu et al.,
2011). A non-randomised and non-blinded trial on 38 patients,
demonstrated that dietary red-clover isoflavones (daily dose:
160 mg) may halt the progression of prostate cancer by the
induction of apoptosis in low to moderate-grade tumors (Jarred
et al., 2002). The inhibitory effect of formononetin on the
proliferation of both LNCaP and PC-3 prostate cancer cell lines,
was also found. The molecular mechanisms of formononetin
action involve the inhibition of extracellular signal-regulated
kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK)
signalling pathway, leading to the increased expression of BCL2-
associated X (Bax) mRNA and protein, and apoptosis induction
(Ye et al., 2012). On the other hand, in a case-controlled study on
plasma concentrations of phytoestrogens in relation to risk of
subsequent prostate cancer, no statistically significant associa-
tions were recorded for circulating concentrations of daidzein,
equol, enterolactone or enterodiol in relation to overall risk for
prostate cancer. In addition, no evidence for a protective associa-
tion with plasma lignans was found. Only higher plasma concen-
trations of genistein were associated with lower risk of prostate
cancer. The study was performed on 950 men with incident
prostate cancer and 1042 control subjects, participating in the
European Prospective Investigation into Cancer and Nutrition
(EPIC) (Travis et al., 2009).
8.3. Breast cancer
In contrast to the increase of mammographic breast density
occurring after conventional hormone replacement therapy, study
of Atkinson et al. (2004) on the safety of red clover isoflavones
showed that the isoflavone supplementation (with one tablet/day,
providing 26 mg of biochanin A, 16 mg of formononetin, 1 mg of
genistein and 0.5 mg of daidzein) did not influence breast density.
Furthermore, in these double-blind, randomised, placebo-con-
trolled trial, no effects on estradiol, gonadotrophins, lymphocyte
tyrosine kinase activity, or menopausal symptoms were observed.
Similar results have been described by Powles et al. (2008), in a
report from three-year clinical study on the effects of a commer-
cial, standardized red clover extract (40 mg/day) on healthy
women with a family history of breast cancer. The investigated
red clover-derived medicine was well tolerated; no significant
differences in breast density, endometrial thickness, serum cho-
lesterol, follicle stimulating hormone levels and bone mineral
density were recorded. A systematic review and meta-analysis of
randomised controlled studies on the effects of isoflavones on
breast density in pre- and postmenopausal women (Hooper et al.,
2010), showed that isoflavone intake did not alter breast density
in post-menopausal women, but might cause a small increase in
 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23
breast density in premenopausal women. Since the meta-analysis
was performed on eight randomised controlled studies (1287
women) with placebo, conducted for 6 months to 3 years, the
authors concluded, that larger and long-term trials are required to
determine, if these effects are clinically relevant.
9. Possible role in the therapy of the metabolic syndrome and
cardiovascular disorders
The range of medicinal applications of T. pratense may include
amelioration of the metabolic syndrome. It has been found that
red clover extracts affects functions of peroxisome proliferator-
activated receptor (PPAR) g, an important regulator of adipocyte
differentiation and insulin sensitization. Red clover extracts and
isoflavones such as genistein and biochanin A act as ligands and
activators of PPAR g. Furthermore, some isoflavone metabolites
displayed higher binding affinities or transactivational activities
than their precursor molecules in vitro. For example, 6-hydro-
xydaidzein had a more than 100-fold higher binding affinity in
comparison to its precursor—daidzein. The observed maximal
transactivational activity of 6-hydroxydaidzein and 30 -hydroxy-
genistein exceeded the action of rosiglitazone, a PPAR g agonist
(Mueller et al., 2008). Besides the influence on PPAR g functions,
the blood pressure lowering effects of red clover isoflavones were
also reported. In a randomised double-blind crossover trial with
placebo, dietary supplementation with isoflavones from red clo-
ver to postmenopausal women with type 2 diabetes (approxi-
mately 50 mg/day isoflavones for four weeks) was found to
significantly lower mean daytime systolic and diastolic blood
pressures (Howes et al., 2003). On the other hand, it should be
underlined that the reports on the effects of isoflavones, including
red clover-derived isoflavones on blood lipid profile are incon-
sistent. Furthermore, differences in the physiological effect of
dietary interventions on LDL-cholesterol level between women
and men are also observed in the response to isoflavone supple-
mentation. Nestel et al. (2004) described the results of a rando-
mised, placebo-controlled, double-blind trial with two parallel
groups (46 middle-aged men and 34 postmenopausal women)
treated for 6 weeks with 40 mg/day of red clover extract enriched
in one of the two isoflavones (biochanin or formononetin). The
baseline LDL concentrations did not differ significantly between
men and women, or between those randomised to biochanin or
formononetin. Red clover extract enriched in biochanin (genistein
precursor), but not in formononetin (daidzein precursor) effec-
tively diminished the LDL level only in male patients. The
formononetin-enriched mixture was ineffective in both sexes. In
a semi-randomised studies of Asgary et al. (2007), performed on
the animal model of atherosclerosis, a considerable decrease of
the level of C-reactive protein, triglyceride, total cholesterol and
LDL-cholesterol was found, while HDL-cholesterol level was
significantly increased. According to those authors, the addition
of dried red clover extract (8% of a diet) to the hyperlipidemic diet
might prevent the progression of atherosclerotic lesions and
could be a potent approach in lessening of cardiovascular risk
factors. The beneficial effects of Trifolium pratense-derived iso-
flavones on the lipid profile of postmenopausal women with
increased body mass index, have been also found (Chedraui
et al., 2008). This investigation involved 60 postmenopausal
women, randomly assigned to one of two groups: supplemented
with two capsules of T. pratense extract (80 mg of red clover
isoflavones) daily for 90 day or placebo group. After a 7-day
washout period, medication was crossed-over for another 90 day.
The favorable effects of red clover extracts were evidenced by a
significant reduction of total cholesterol, LDL-cholesterol and
lipoprotein A (LpA) levels. According to the report of Blakesmith
21
et al. (2003), a double-blind, randomised, parallel study on the
effects of purified red clover isoflavones (first menstrual cycle
placebo, for the next three menstrual cycles a daily dose of 86 mg
of isoflavones were administered) in 25 premenopausal women,
did not show significant effect on cholesterol homeostasis or
insulin resistance, in subjects with low risk of coronary heart
disease.
10. Other therapeutic effects
Results of experiments on mesencephalic neuron-glia cultures
and microglia-enriched cultures, suggest that biochanin A,
obtained from red clover (97% purity) may possess neuroprotec-
tive action. At the concentration range of 0.25–2.5 mM, biochanin
A protected dopaminergic neurons against lipopolysaccharide-
induced damage by the inhibition of microglia activation and
proinflammatory factors generation (Chena et al., 2007). Results
from a randomised in vivo study with the use of animal model of
streptozotocin-induced diabetes, described by Al-Rawi (2007),
indicate on the protective effect of T. alexandrinum L. on liver. In
these experiments a Trifolium flower-derived extract was used,
contrary to the most studies carried out on substances or
mixtures obtained from leaves. The treatment with 50 mg/kg of
body weight (hexane extract), or 100 mg/kg b. w. (ethanol and
aqueous extracts) resulted in a remarkable improvement in
histological structure of liver of diabetic animals. Furthermore,
the water extract was more effective than hexane and ethanol
extracts. Recently (2012), the antibacterial action of this plant has
been also demonstrated. The non-polar and polar extracts from
leaves of T. alexandrinum L. inhibited the growth of seven Gram-
positive (Staphylococcus aureus, Staphylococcus aureus ATCC
25953, Staphylococcus albus, Streptococcus haemolyticus Group-A,
Streptococcus haemolyticus Group-B, Streptococcus faecalis, Bacillus
subtilis) and eleven Gram-negative (Escherichia coli, Edwardsiella
tarda, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris,
Pseudomonas aeruginosa, Sallmonella typhi, Shigella boydii, Shigella
dysenteriae, Shigella flexneri, Plesiomonas shigelloides) hospital
isolated human pathogenic bacteria strains, responsible for tropic
diseases. All the used extracts (at the concentrations of 1, 2, 5, 10
and 15 mg/ml) had the antimicrobial activity against at least six
types of microorganisms. Polar (methanol or ethyl acetate)
extracts displayed considerably stronger antibacterial activity
against the tested pathogens, but none of the used extracts was
able to inhibit the growth of Pseudomonas aeruginosa (Khan et al.,
2012). In other study, Sabudak et al. (2006) found in vitro that
stigmast-5-ene-3-b, 26-diol isolated from T. balansae Boiss. might
be a potential therapeutic agent, useful in the treatment of
melanin biosynthesis-related skin diseases such as hyper- and
hypo-pigmentation.
11. Conclusions
Until recently, studies on medicinal applications of clovers
have been focused mainly on the effects of red clover-based
extracts and supplements, administered as an alternative for the
conventional hormonal therapy. However, the physiological rele-
vance of substances and extracts derived from T. pratense still
raises considerable controversies. Furthermore, there are signifi-
cant gaps in knowledge of the possible medicinal applications of
other Trifolium species. Besides T. pratense L., also other clovers
(e.g., T. repens L., T. resupinatum L., T. pallidum Waldst. & Kit.,
T. alexandrinum L.) may be a source of valuable phytochemicals,
but the therapeutic use of these herbs is limited by the lack of
clinical evidence. Therefore, further studies on medicinal
22 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23
applications of various clovers (not only T. pratense L.), and, in
particular, clinical investigations are needed.
Acknowledgements
This work was supported by grant 506/810 and 545/217 from
University of Lodz.Special thanks to Professor Barbara Wachowicz
for directions and helpful suggestions.
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