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

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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 15

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1. Introduction are popular herbs in Pakistan, useful in the treatment of sore

throat, fever, pneumonia, meningitis and feverish feeling (Khan
The Trifolium genus (Fabaceae) comprises of about 240 species and Khatoon, 2008). The recent ethnobotanical research of
of clovers (Zoric et al., 2012), occurring in temperate and Mustafa et al. (2012), conducted in the Albanian Alps in Kosovo
subtropical regions of both hemispheres. The Mediterranean provided additional evidence of the traditional use of T. pratense L.
basin, western North America, and the highlands of eastern Africa and T. repens L. In this region of Europe, a juice obtained from
are the three geographic regions with the greatest diversity of squeezed leaves of T. pratense L. is one of the folk medicines for
Trifolium species, while no species are native to southeastern Asia stomach disorders. T. repens L. decoctions have been applied as
and Australia (Ellison et al., 2006). Some of Trifolium species have anti-diarrhoeal remedy (Mustafa et al., 2012). Native Americans
been known for many centuries as forage plants (T. pratense L., have used T. pratense L. to cure external skin problems, lung
T. repens L., T. resupinatum L., T. incarnatum L., T. hybridum L., illnesses, as well as some disorders of nervous and reproductive
T. pannonicum Jacq., T. subterraneum L., T. fragiferum L., and system (Sabudak and Guler, 2009). T. pannonicum Jacq. is one of
T. medium L.), and valuable herbs in folk medicine of various widespread, wild growing medicinal plants in high mountain
cultures. Nowadays, the therapeutic use of Trifolium plants is still region of Montenegro, used by local people for wound healing
based mainly on traditional medicine recommendations, but the (Menkovic et al., 2011). Barros et al. (2010) report that in
number of scientific data on the biological activity of clovers and Portugal, T. angustifolium L. decoctions are a folk medicine for
their possible therapeutic effects has been growing (Table 1). The stomachache and diarrhea. Traditional administration of decoc-
majority of the studies on the biological properties of clovers tions from aerial parts of this clover to cure diarrhea and
concerns T. pratense L. (red clover) and is focused on its phytoes- swellings has been also shown in the ethnobotanical study,
trogenic action, being a result of isoflavone content. Interactions performed in the Natural Park of ‘‘Serra de Sa~ o Mamede’’
of isoflavones with estrogen receptors are possible due to their (Portugal) (Camejo-Rodrigues et al., 2003). T. repens L. is a
structural similarity to 17b-estradiol, a steroid hormone. For that deworming remedy in the traditional medicine of the Naga tribes
reason, red clover is a source of dietary supplements and herbal of India. This activity of white clover has been confirmed by
medicines administered as an alternative to the conventional in vivo study on animals (Tangpu et al., 2004). The seeds of
hormonal replacement therapy (Engelmann et al., 2009). How- T. alexandrinum L. have been administered in Egypt as an anti-
ever, it should be emphasized that the beneficial effects of red diabetic remedy (Khaled et al., 2000).
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 3. Phytochemical data
ethnopharmacological knowledge of medicinal relevance of Tri-
folium genus. Contrary to the existing evidence of therapeutic The chemical profile of clovers is partly recognized. It is known
properties of T. pratense, obtained from in vivo and clinical that besides isoflavones, Trifolium plants synthesize a wide range
examinations, the curative applications of other Trifolium species of phenolic and polyphenolic compounds such as flavonoids,
are supported mainly by basic investigations or traditional med- saponins, clovamides (caffeic acid esters), phenolic acids and
icine. Evaluation of the novel therapeutic potencies of Trifolium- other substances (Oleszek and Stochmal, 2002; Oleszek et al.,
derived extracts seems to be a promising trend in phytopharma- 2007). Until now, chemical analysis of clovers in the greatest
cological research for a variety of reasons, including the diversity extent have been performed by Oleszek et al. (2007). The aerial
of chemical components and widespread occurrence of clovers, as parts of 57 Trifolium species were analysed in terms of the content
well as low costs of cultivation. The present article critically of flavonoids (mainly isoflavones), phenolic acids, and clovamides,
reviews the available information on physiological activity and and then grouped into five clusters. Cluster 1—species with the
expected medicinal importance of Trifolium plants, and indicate highest concentrations of isoflavones (51–97 mg/g of dry matter),
the future prospects for the use of these herbs as therapeutics in comprises of T. lappaceum L., T. phleoides Willd., T. hirtum All.,
various disorders. T. alpestre L., T. medium L., T. subterraneum L., T. heldreichianum
Evidence of the physiological effects of Trifolium plants derives Hausskn., and T. scabrum L. Cluster 2 includes T. angustifolium L.,
from in vitro and in vivo studies. The review includes data (to May T. bocconei Savi, T. nigrescens Viv., T. pannonicum Jacq., T. desvauxii
2012) obtained from journals indexed in international databases Loisel., and T. leucanthum M.Bieb., particularly rich in flavonoids
(Medline/Pubmed, Scopus, Science Direct/Elsevier, Springer Link/ (16 to 32 mg/g of dry matter). Cluster 3 includes T. resupinatum
ICM), as well as local periodicals, not indexed in international var. majus Boiss., T. arvense L., T. stellatum L., T. incarnatum L.,
scientific databases. In numerous cases, references included to T. xerocephalum Fenzl, T. isthmocarpum Brot., T. glomeratum L.,
those articles (reviews, in particular) were also verified for T. occidentale Coombe, T. pratense, ssp. expansum Jáv., T. ambiguum
suitability. 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
2. The use of Trifolium plants in traditional medicine cluster 4 (T. ligusticum Loisel., T. ochroleucon Huds., T. resupinatum
var. resupinatum L., T. cernuum Brot., T. hybridum L., T. medium var.
Clovers have been used by Oriental and European cultures for sarosiense (Hazsl.) Simonk., T. hirtum All., T. rubens L., T. repens L.,
the treatment of eczema and psoriasis. In Turkish traditional T. cherleri L., T. tomentosum L., T. michelianum Savi, T. michelianum
medicine, T. repens L., T. arvense L. and T. pratense L. have been ssp. balansae (Boiss.) Azn., T. spumosum L., and T. montanum L.)
administered as expectorant, antiseptic, analgesic, sedative and possess a high concentration of phenolic acids, at the range of
tonic mixtures (Sabudak et al., 2008). T. pratense L. and T. repens L. 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,  Estrogenic effect (Booth et al., Menopausal complaints; potentially
analgesic herb; treatment of sore 2006, Pfitscher et al., 2008, Nissan useful in anti-cancer therapy and
throat, fever, pneumonia and et al., 2007, Coon (2007) (Rev.), cardiovascular diseases prevention
meningitis; skin problems, lung Yatkin and Daglioglu, 2011,
illnesses as well as some disorders of Adaikan et al., 2009, Lipovac et al.
nervous and reproductive system (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) Forage plant; analgesic medicine for  Anticestodal properties (Tangpu et
rheumatic disorder; deworming al., 2004)
remedy; treatment of sore throat, fever,
pneumonia, meningitis
T. resupinatum L. (Persian clover) Forage plant  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. 9–10 acids; 7 of them were identified: p-hydroxybenzoic, salicylic,
clypeatum L., T. squarrosum L., T. echinatum M.Bieb., T. pratense ssp. protocatechuic, gentisic, p-coumaric, caffeic and ferulic. In flowers
sativum Ponert, T. pratense ssp. sativum f. albiflorum Puskal, T. of both clovers, p-hydroxybenzoic and salicylic acids were the
miegeanum Maire, T. pratense L., T. isodon Murb., and T. pallidum main compounds. In extracts from leaves of T. repens L., the main
Waldst. & Kit. All these clovers contained clovamides (from 4 to acidic component was caffeic acid, while salicilic and caffeic acids
13 mg/g of dry matter), high concentrations of phenolic acids and predominated in extract from T. pratense L. leaves (Kicel and
flavonoids. Wolbis, 2006). Recently, more details about the phytochemical
The qualitative analyses of phenolic acids present in flowers profile of T. repens L. (white clover) have been given. Flowers and
and leaves of T. repens L. and T. pratense L. revealed the presence of 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 essential role in the regulation of body temperature, mood,
lower. Furthermore, there is no information about studies on the hormonal levels and actions, according to the authors, these
estrogenic activity of other clovers. In comparison to soybeans, findings may explain in part the beneficial effect of T. pratense-
where daidzein, genistein and glycitein are present, red clover derived phytoestrogens in alleviating menopausal symptoms.
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 5. Trifolium plants in the therapy of menopause-related
isoflavone extract, rich in formononetin (formononetin/biochanin disorders
ratio of 2.17) led to the higher plasma concentrations of the
demethylated metabolites: daidzein and genistein (0.39 and Extracts of red clover are commercially available as dietary
0.06 mM, respectively). Howes et al. (2002) described the results supplements. Despite of the fact that the biological activity of
of a study on long-term pharmacokinetics of isoflavones from isoflavones may possess therapeutic value in the treatment of
T. pratense L., administered in supplement with a low formonone- hormone-related diseases, such as cardiovascular diseases, can-
tin content (formononetin:biochanin ratio was 0.65). The exam- cer, osteoporosis and menopausal complaints (Tham et al., 1998),
ination involved 14 participants, consuming a low-isoflavone diet the effectiveness of medicinal use of red clover still raises
for 2 weeks as an oral daily dose of two isoflavone tablets (one numerous concerns. After the systematic review and meta-ana-
tablet contained 24.5 mg of biochanin, 1.5 mg of genistein, 16 mg lysis of literature, Coon (2007) concluded that there is evidence of
of formononetin, and 1.5 mg of daidzein). The supplementation a marginally significant effect of T. pratense L. isoflavones in the
with isoflavones resulted in 0.25 micromolar concentration of treatment of hot flushes in menopausal women. Additionally,
daidzein and 0.42 micromolar concentration of genistein in blood these authors found neither apparent evidence of side effects in
plasma. Isoflavones present in red clover in minor amounts such as short-term application of red clover isoflavones, nor data on the
irilone, prunetin and pseudobaptigenin (Fig. 2), are also bioavail- safety of long-term administration. Yatkin and Daglioglu (2011)
able. Irilone concentrations in commercial red clover supplements have paid the attention on the lack of experimental studies based
are sufficient to lead to physiologically relevant plasma concentra- on physiological isoflavone concentrations (including T. pratense
tions. According to Maul and Kulling (2010), the methylenedioxy L. isoflavones), evaluating the effects of phytoestrogens at differ-
bridge attached to the A ring of the irilone skeleton acts as a ent periods of life. Thus, further studies seem to be necessary for
protective group against the degradation of irilone by the human the evaluation of the risk of long-term use of phytoestrogens.
microbiota. Moreover, after the intake of red clover supplement Some new information on the safety of T. pratense L. and soy
both prunetin and pseudobaptigenin were detected. (Glycine max L.) isoflavones in vitro, has been provided recently by
The molecular mechanisms of the physiological and therapeu- Reiter et al. (2011). Plant extracts and isolated isoflavones were
tic effects of clover-derived extracts and isoflavones are only examined in proliferation assays on 11 human cancer cell lines
partly elucidated. In vitro studies of Booth et al. (2006) on the (cancers of the colon, prostate, breast, cervix, liver, pancreas,
chemical and biological profile of a clinical phase II red clover stomach and ovaries) and a fibroblast line to assess their cytotoxic
extract, composed of 35.54% isoflavones (daidzein, genistein, activity. The tested compounds and extracts did not promote the
formononetin, biochanin A), 1.11% flavonoids, 0.06% pterocar- growth of human cancer cells, but reduced cell proliferation,
pans, r0.03% coumarins, and r0.03% tyramine, confirmed its increased apoptosis and cell cycle arrest.
estrogenic action. It has been also found that all of these Contrary to critical conclusions of Coon (2007), results of
isoflavones (except formononetin) bind to one or both of estrogen studies on animals are more optimistic. A prospective, vehicle-
receptors (a and b). Since isoflavones undergo various metabolic controlled study on animal model of menopause, demonstrated
transformations after the dietary intake, the phytoestrogenic that 12-week treatment with red clover isoflavones (daily dose:
actions of their metabolites were also investigated (Pfitscher 100 mg of daidzein per kg of body weight or 6.68 mg of red clover
et al., 2008). The binding and transactivating properties of red extract/kg b.w.—an equivalent of 100 mg of daidzein/kg b.w.) led
clover isoflavones and their metabolites may be assessed by the to significant improvements in bone density, tissue integrity, and
analysis of estrogen receptors a and b, as well as androgen and vaginal blood flow with minimal effect on uterine weight, and
progesterone receptors. The study of Pfitscher et al. (2008), with might be an alternative to conventional treatment with synthetic
the use of yeast as an experimental system, showed that the estrogens (Adaikan et al., 2009). Results of experiments on rats
metabolic transformations of isoflavones led to the changes in with surgically-induced menopause, suggest that red clover pre-
both their affinity to estrogen receptors and transactivation paration in dosages amenable to clinical practice may improve
potential. The demethylation of formononetin and biochanin A ovariectomy-induced osteoporosis. Moreover, mild metabolic
to daidzein and genistein, respectively, resulted in the evident alkalosis might further synergize some therapeutic aspects. The
alterations in receptor affinity, and significantly enhanced poten- examination was performed with the use of a quality-controlled
cies against estrogen receptor b. The enzymatic conversion of red clover extract, containing 40% of isoflavones (genistein,
biochanin A, formononetin, daidzein and genistein to their daidzein, biochanin A, and hydrolyzed aglycones of formonone-
reduced metabolites dihydrobiochanin A, dihydroformononetin, tin). Animals were randomised into four groups: A (sham-oper-
dihydrogenistein and dihydrodaidzein, respectively, did not cause ated rats), and three other groups (ovariectomised), being fed for
any significant effects on the receptor affinity to either estrogen three months as follows: standard food (group B), 6 mg/kg/day
receptor. The possible beneficial health effects of isoflavones food mixed with red clover extract (group C), or given 6 mg/kg/
might be attributed to equol (daidzein metabolite), which pos- day of red clover extract with a modified alkaline supplementa-
sesses a higher receptor transactivation potential than its tion through a nasogastric tube at a dose of 16 mg (group D)
precursor—daidzein. The formation of equol resulted in a 30 (Kawakita et al., 2009). In other study on animal model of
times higher potency, but its reduced metabolite dihydrodaidzein osteoporosis, T. pratense isoflavones (oral dose of 20 and 40 mg
displayed about 100 times lower transactivation potential. Bind- of total isoflavones daily, for 14 weeks) considerably increased
ing of components of a clinically administered T. pratense extract bone mineral content, mechanical strength of the tibia, femoral
(0.3–300 mg/ml), to the m- and d-opiate receptors, has been weight and density, as well as effectively prevented the rise of
shown in experiments on cell lines expressing human opiate serum alkaline phosphatase level. The number of osteoclasts was
receptors (Nissan et al., 2007). Since the opioid system plays an also significantly reduced, compared to the ovariectomised
 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23 19

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-
Fig. 3. Clovamide structure.
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 oxidative effects of isoflavones were not recorded (Hodgson et al.,
marker - urinary deoxypyridinoline, but did not affect bone 1999; Hsu et al., 2001). A pilot clinical trial, described by Campbell
formation markers (serum bone alkaline phosphatase and serum et al. (2004) indicated on a little or no effects of 1-month isoflavone
osteocalcin) in menopausal women. Weaver et al. (2009) (86 mg/day red clover-derived isoflavones) supplementation on
described results of a randomised, crossover, blinded trial in 11 markers of antioxidant status. This randomised, placebo-controlled
healthy postmenopausal women, designed to assess the antire- and crossover examination involved 16 healthy pre- and 7 post-
sorptive action of isoflavones on bone. In those study, the effects menopausal women. In contrast to these observations, study on
of four commercial sources of isoflavones from soy cotyledon, soy animals (Mu et al., 2009) revealed antioxidative and estrogenic
germ, kudzu, and red clover, as well as a positive control (1 mg actions of T. pratense-derived formononetin, administered for
estradiol combined with 2.5 mg medroxyprogesterone or 5 mg/ 6 months. The examination was conducted on mice divided into
day risedronate (Actonels)) were examined. Only isoflavones 5 groups: sham-operated group and 4 ovariectomised groups,
from soy cotyledon and germ significantly decreased net bone control group, stilbestrol replacement therapy group (0.20 mg/kg
resorption (by 9% and 5%, respectively). Based on those findings, b. w. daily), low-dose formononetin group (0.05 g/kg b. w. daily)
the authors concluded that genistein-like isoflavones displayed a and high-dose formononetin group (0.5 g/kg b. w. daily). The
significant, but modest ability to suppress bone resorption in intake of formononetin significantly increased the activities of
postmenopausal women. superoxide dismutase, glutathione peroxidase, catalase, and
Studies on the effects of isoflavones on bone metabolism reduced lipid peroxidation in animal body; the estrogenic effect
provided controversial results, but simultaneously, the effective- was not dosage-related.
ness of phytoestrogenic therapy on other menopause-related The antioxidative activity of some Trifolium species may be also
disorders has been confirmed by Lipovac et al. (2010). This a result of the abundance of flavonoids and other phenolic
randomised clinical investigation was designed to evaluate the compounds, such as catechins, saponins, clovamides and phenolic
effects of a non-prescription red clover extract (MF11RCE) on acids, present in clovers. The in vitro study on the clovamide-rich
mood disorders. Participants (190 postmenopausal women) were extract of T. pallidum Waldst. & Kit. (at the final concentration
randomly assigned to one of two groups: treated with two range of 12.5–100 mg/ml) revealed antioxidative action of all used
capsules of either MF11RCE (80 mg of red clover isoflavones) or concentrations, in the protection of proteins and lipids of blood
placebo of equal form for a 90-day period. After a 7-day washout platelets and plasma against the oxidative stress-induced damage,
period, subjects received the opposite treatment for another 90 in comparison to the samples exposed to oxidative stress in the
day. The medication with red clover isoflavones resulted in a absence of the extract (Kolodziejczyk et al., 2011). Antioxidative
significant decrease of depressive and anxiety symptoms among action of clovamide (Fig. 3), a derivative of caffeic acid, is
postmenopausal women. In other research of these authors, the attributed mainly to its ability to prevent lipid peroxidation (Ley
treatment with the same dose of red clover isoflavones (80 mg/ and Bertram, 2003). The in vitro study of Sanbongi et al. (1998)
day) caused a visible improvement of scalp hair and skin status as showed that in the protection of lipids, clovamide might be more
well as libido, mood, sleep, and tiredness in postmenopausal effective antioxidant than epicatechin or quercetin. Sabudak et al.
women. The examination involved 109 participants, randomly (2009) have found the antioxidative activity in vitro of hexane
assigned to one of two groups: receiving capsules of red clover extracts, obtained from T. balansae Boiss., T. stellatum L., T.
isoflavones or receiving placebo of equal appearance for a 90-day nigrescens subsp. petrisavii (Clementi) Holmboe, T. constantinopo-
period. Then, after a washout period of 7 day, medication was litanum Ser. and T. resupinatum L. Moreover, these effects corre-
crossed over and continued for 90 day more (Lipovac et al., 2011). lated with the content of unsaturated fatty acid in the tested
On the other hand, according to Del Giorno et al. (2010), 12- extracts. Contrary to the mentioned reports suggesting the high
month treatment with T. pratense extract does not yield a anti-oxidative activity of Trifolium plants, the ethnobotanical study
considerable improvement in menopausal symptoms and sexual of Barros et al. (2010) demonstrated relatively weak antioxidant
satisfaction. The described results were obtained in a prospective, properties of T. angustifolium L. in vitro, most likely being a result
randomised, double-blind, placebo-controlled study on 120 of a low content of phenolics and flavonoids (95.61 and 26.78 mg
women aged 45–65, that received 40 mg of red clover extract of gallic acid equivalents (GAC) per g of extract, respectively). The
(one capsule daily). 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)
6. Antioxidative properties of Trifolium species level, and the inhibition of b-carotene bleaching.

The overproduction of reactive oxygen and nitrogen species

(ROS/RNS) is involved in the etiology and pathophysiology of 7. Anti-inflammatory and antiplatelet action
inflammation and cancer, as well as autoimmune, neurodegenera-
tive, cardiovascular and other disorders (Ronson et al., 1999; Blood platelet activation and their accumulation at the site of
Ohshima et al., 2003; Sorg, 2004). A growing number of in vitro vascular injury is the crucial event in physiological haemostasis.
and in vivo studies has showed the antioxidative properties of Platelets are also responsible for the formation of pathogenic
isoflavones (Kapiotis et al., 1997; Lissin and Cooke, 2000; Wiseman thrombi. Both the activated platelets and platelet-leukocyte
et al., 2000; Jenkins et al., 2000), but in some investigations, anti- 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- angiogenesis such as IL-8, matrix metalloproteinase 13, and
eases, including atherosclerosis, myocardial infarction and fibronectin. Moreover, the synthesis of antiangiogenic factors:
ischemic cerebral stroke (Smyth et al., 2009; Angiolillo et al., plasminogen activator inhibitor-1 (PAI-1), angiostatin and throm-
2010). Until recently, very few studies have been designed to bospondin may also undergo the isoflavone-mediated upregula-
evaluate the effects of Trifolium plants on the haemostatic system. tion. Evaluation of the antiangiogenic action of red clover extracts
Red clover isoflavones were found to activate an antiplatelet (with the use of the chorioallantoic membrane assay, at a dosage
factor—nitric oxide (NO) synthesis in endothelial cells by stimu- of 250 mg of extract per pellet) demonstrated a considerable
lation of transcriptional pathways. These experiments were inhibition of angiogenesis. Non-methylated isoflavones: daidzein
designed to examine the influence of various combinations of and genistein were found to possess stronger antiangiogenic
isoflavone amounts on activity of endothelial nitric oxide activity than the methylated compounds: formononetin and
synthase (eNOS). The investigated content of isoflavones was biochanin A (Krenn and Paper, 2009).
relevant to amounts found after the intake of a standardized red
clover extracts preparation in one standard tablet (containing: 8.2. Prostate cancer
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 Data concerning the anticancer properties of red clover derive
human endothelial cells to red clover extracts enhanced the mainly from examinations of prostate cancer. Under in vitro
expression and the activity of eNOS. Molecular mechanisms of conditions, red clover isoflavones (100 nM) may partly suppress
these effects may involve the recruitment of estrogen receptor b. the proinflammatory effects of transforming growth factor b1 in
It has been also shown that clover-derived isoflavones synergize human primary prostate cancer-derived stromal cells (Liu et al.,
with 17b-estradiol in increasing eNOS activity and expression 2011). A non-randomised and non-blinded trial on 38 patients,
(Simoncini et al., 2005). demonstrated that dietary red-clover isoflavones (daily dose:
Lam et al. (2004) demonstrated that isoflavones obtained from 160 mg) may halt the progression of prostate cancer by the
red clover might suppress inflammation. In experiments of these induction of apoptosis in low to moderate-grade tumors (Jarred
authors, red clover-derived isoflavones effectively inhibited et al., 2002). The inhibitory effect of formononetin on the
cyclooxygenase activity in the macrophage cell line RAW 264.7 proliferation of both LNCaP and PC-3 prostate cancer cell lines,
and human monocytes, as estimated by the reduction of prosta- was also found. The molecular mechanisms of formononetin
glandin E2 and/or thromboxane B2 synthesis. The antiinflamma- action involve the inhibition of extracellular signal-regulated
tory and antioxidative properties were also shown for T. kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK)
resupinatum L. var. microcephalum Zoh. Sabudak et al. (2008) signalling pathway, leading to the increased expression of BCL2-
revealed, for the first time, the antioxidative action of this clover associated X (Bax) mRNA and protein, and apoptosis induction
on animal model of arthritis. The administration of T. resupinatum (Ye et al., 2012). On the other hand, in a case-controlled study on
L. var. microcephalum Zoh. extract (1.35 and 13.5 mg/kg b. w.) plasma concentrations of phytoestrogens in relation to risk of
significantly reduced the paw edema induced by the complete subsequent prostate cancer, no statistically significant associa-
Freund’s adjuvant. Furthermore, a significant inhibition of lipid tions were recorded for circulating concentrations of daidzein,
peroxidation was found. According to the authors, effects of this equol, enterolactone or enterodiol in relation to overall risk for
extract might be a result of the presence of flavonoids and their prostate cancer. In addition, no evidence for a protective associa-
free radical scavenging activities. tion with plasma lignans was found. Only higher plasma concen-
It has been established, that some of Trifolium species (e.g., T. trations of genistein were associated with lower risk of prostate
pallidum Waldst. & Kit.) are particularly rich in clovamides, a cancer. The study was performed on 950 men with incident
caffeic acid esters, displaying antioxidative properties (Arlorio prostate cancer and 1042 control subjects, participating in the
et al., 2008). Clovamides may be an interesting group of plant- European Prospective Investigation into Cancer and Nutrition
derived phenolics with expected beneficial effects, however, until (EPIC) (Travis et al., 2009).
now their physiological activity has been inadequately recog-
nized. The most of studies on biological effects of clovamides was 8.3. Breast cancer
performed with compounds from cocoa beans (Theobroma cacao
L.). In addition to antioxidative action, the antiplatelet properties In contrast to the increase of mammographic breast density
of clovamides have been also observed. Results from animal occurring after conventional hormone replacement therapy, study
study, showed that N-caffeoyldopamine (50 and 100 mg/35 g b. of Atkinson et al. (2004) on the safety of red clover isoflavones
w.) effectively reduced the P-selectin expression and platelet- showed that the isoflavone supplementation (with one tablet/day,
leukocyte interactions by 31 to 45% and 34 to 43%, respectively providing 26 mg of biochanin A, 16 mg of formononetin, 1 mg of
(Park and Schoene, 2006). Recently, the antiplatelet actions of T. genistein and 0.5 mg of daidzein) did not influence breast density.
pallidum Waldst. & Kit. and T. scabrum L. extracts (used at the final Furthermore, in these double-blind, randomised, placebo-con-
concentration range of 12.5–50 mg/ml), have been found in vitro. trolled trial, no effects on estradiol, gonadotrophins, lymphocyte
The examined extracts: from T. pallidum Waldst. & Kit. (phenolic tyrosine kinase activity, or menopausal symptoms were observed.
fraction and clovamide fraction) and T. scabrum L. (phenolic Similar results have been described by Powles et al. (2008), in a
fraction), modulated platelet adhesion to different proteins (col- report from three-year clinical study on the effects of a commer-
lagen and fibrinogen) (Kolodziejczyk-Czepas et al., 2012). 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
8. Anticancer and antiangiogenic effects differences in breast density, endometrial thickness, serum cho-
lesterol, follicle stimulating hormone levels and bone mineral
8.1. Antiangiogenic properties density were recorded. A systematic review and meta-analysis of
randomised controlled studies on the effects of isoflavones on
Phytoestrogens may influence angiogenesis and act as chemo- breast density in pre- and postmenopausal women (Hooper et al.,
preventive agents. Isoflavones, including genistein and daidzein, 2010), showed that isoflavone intake did not alter breast density
downregulate genes and mRNA levels of proteins involved in in post-menopausal women, but might cause a small increase in
 J. Kolodziejczyk-Czepas / Journal of Ethnopharmacology 143 (2012) 14–23 21

breast density in premenopausal women. Since the meta-analysis et al. (2003), a double-blind, randomised, parallel study on the
was performed on eight randomised controlled studies (1287 effects of purified red clover isoflavones (first menstrual cycle
women) with placebo, conducted for 6 months to 3 years, the placebo, for the next three menstrual cycles a daily dose of 86 mg
authors concluded, that larger and long-term trials are required to of isoflavones were administered) in 25 premenopausal women,
determine, if these effects are clinically relevant. did not show significant effect on cholesterol homeostasis or
insulin resistance, in subjects with low risk of coronary heart
disease.
9. Possible role in the therapy of the metabolic syndrome and
cardiovascular disorders
10. Other therapeutic effects
The range of medicinal applications of T. pratense may include
amelioration of the metabolic syndrome. It has been found that Results of experiments on mesencephalic neuron-glia cultures
red clover extracts affects functions of peroxisome proliferator- and microglia-enriched cultures, suggest that biochanin A,
activated receptor (PPAR) g, an important regulator of adipocyte obtained from red clover (97% purity) may possess neuroprotec-
differentiation and insulin sensitization. Red clover extracts and tive action. At the concentration range of 0.25–2.5 mM, biochanin
isoflavones such as genistein and biochanin A act as ligands and A protected dopaminergic neurons against lipopolysaccharide-
activators of PPAR g. Furthermore, some isoflavone metabolites induced damage by the inhibition of microglia activation and
displayed higher binding affinities or transactivational activities proinflammatory factors generation (Chena et al., 2007). Results
than their precursor molecules in vitro. For example, 6-hydro- from a randomised in vivo study with the use of animal model of
xydaidzein had a more than 100-fold higher binding affinity in streptozotocin-induced diabetes, described by Al-Rawi (2007),
comparison to its precursor—daidzein. The observed maximal indicate on the protective effect of T. alexandrinum L. on liver. In
transactivational activity of 6-hydroxydaidzein and 30 -hydroxy- these experiments a Trifolium flower-derived extract was used,
genistein exceeded the action of rosiglitazone, a PPAR g agonist contrary to the most studies carried out on substances or
(Mueller et al., 2008). Besides the influence on PPAR g functions, mixtures obtained from leaves. The treatment with 50 mg/kg of
the blood pressure lowering effects of red clover isoflavones were body weight (hexane extract), or 100 mg/kg b. w. (ethanol and
also reported. In a randomised double-blind crossover trial with aqueous extracts) resulted in a remarkable improvement in
placebo, dietary supplementation with isoflavones from red clo- histological structure of liver of diabetic animals. Furthermore,
ver to postmenopausal women with type 2 diabetes (approxi- the water extract was more effective than hexane and ethanol
mately 50 mg/day isoflavones for four weeks) was found to extracts. Recently (2012), the antibacterial action of this plant has
significantly lower mean daytime systolic and diastolic blood been also demonstrated. The non-polar and polar extracts from
pressures (Howes et al., 2003). On the other hand, it should be leaves of T. alexandrinum L. inhibited the growth of seven Gram-
underlined that the reports on the effects of isoflavones, including positive (Staphylococcus aureus, Staphylococcus aureus ATCC
red clover-derived isoflavones on blood lipid profile are incon- 25953, Staphylococcus albus, Streptococcus haemolyticus Group-A,
sistent. Furthermore, differences in the physiological effect of Streptococcus haemolyticus Group-B, Streptococcus faecalis, Bacillus
dietary interventions on LDL-cholesterol level between women subtilis) and eleven Gram-negative (Escherichia coli, Edwardsiella
and men are also observed in the response to isoflavone supple- tarda, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris,
mentation. Nestel et al. (2004) described the results of a rando- Pseudomonas aeruginosa, Sallmonella typhi, Shigella boydii, Shigella
mised, placebo-controlled, double-blind trial with two parallel dysenteriae, Shigella flexneri, Plesiomonas shigelloides) hospital
groups (46 middle-aged men and 34 postmenopausal women) isolated human pathogenic bacteria strains, responsible for tropic
treated for 6 weeks with 40 mg/day of red clover extract enriched diseases. All the used extracts (at the concentrations of 1, 2, 5, 10
in one of the two isoflavones (biochanin or formononetin). The and 15 mg/ml) had the antimicrobial activity against at least six
baseline LDL concentrations did not differ significantly between types of microorganisms. Polar (methanol or ethyl acetate)
men and women, or between those randomised to biochanin or extracts displayed considerably stronger antibacterial activity
formononetin. Red clover extract enriched in biochanin (genistein against the tested pathogens, but none of the used extracts was
precursor), but not in formononetin (daidzein precursor) effec- able to inhibit the growth of Pseudomonas aeruginosa (Khan et al.,
tively diminished the LDL level only in male patients. The 2012). In other study, Sabudak et al. (2006) found in vitro that
formononetin-enriched mixture was ineffective in both sexes. In stigmast-5-ene-3-b, 26-diol isolated from T. balansae Boiss. might
a semi-randomised studies of Asgary et al. (2007), performed on be a potential therapeutic agent, useful in the treatment of
the animal model of atherosclerosis, a considerable decrease of melanin biosynthesis-related skin diseases such as hyper- and
the level of C-reactive protein, triglyceride, total cholesterol and hypo-pigmentation.
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 11. Conclusions
might prevent the progression of atherosclerotic lesions and
could be a potent approach in lessening of cardiovascular risk Until recently, studies on medicinal applications of clovers
factors. The beneficial effects of Trifolium pratense-derived iso- have been focused mainly on the effects of red clover-based
flavones on the lipid profile of postmenopausal women with extracts and supplements, administered as an alternative for the
increased body mass index, have been also found (Chedraui conventional hormonal therapy. However, the physiological rele-
et al., 2008). This investigation involved 60 postmenopausal vance of substances and extracts derived from T. pratense still
women, randomly assigned to one of two groups: supplemented raises considerable controversies. Furthermore, there are signifi-
with two capsules of T. pratense extract (80 mg of red clover cant gaps in knowledge of the possible medicinal applications of
isoflavones) daily for 90 day or placebo group. After a 7-day other Trifolium species. Besides T. pratense L., also other clovers
washout period, medication was crossed-over for another 90 day. (e.g., T. repens L., T. resupinatum L., T. pallidum Waldst. & Kit.,
The favorable effects of red clover extracts were evidenced by a T. alexandrinum L.) may be a source of valuable phytochemicals,
significant reduction of total cholesterol, LDL-cholesterol and but the therapeutic use of these herbs is limited by the lack of
lipoprotein A (LpA) levels. According to the report of Blakesmith 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 Howes, J.B., et al., 2003. Effects of dietary supplementation with isoflavones from
particular, clinical investigations are needed. red clover on ambulatory blood pressure and endothelial function in post-
menopausal type 2 diabetes. Diabetes Obesity and Metabolism 5, 325–332.
Hsu, C-S., et al., 2001. Soy isoflavone supplementation in postmenopausal women.
Effects on plasma lipids, antioxidant enzyme activities and bone density.
Journal of Reproductive Medicine 46, 221–226.
Acknowledgements Janda, B., et al., 2009. Phenolics in aerial parts of Persian clover Trifolium
resupinatum. Natural Product Communications 4 (12), 1661–1664.
This work was supported by grant 506/810 and 545/217 from Jarred, R.A., et al., 2002. Induction of apoptosis in low to moderate-grade human
prostate carcinoma by red clover-derived dietary isoflavones. Cancer Epide-
University of Lodz.Special thanks to Professor Barbara Wachowicz miology, Biomarkers and Prevention 11, 1689–1696.
for directions and helpful suggestions. Jenkins, D.J., et al., 2000. Effect of soy-based breakfast cereal on blood lipids and
oxidized low-density lipoprotein. Metabolism 49, 1496–1500.
Kapiotis, S., et al., 1997. Genistein, the dietary derived angiogenesis inhibitor,
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