Content uploaded by Mohsen Akbaribazm
Author content
All content in this area was uploaded by Mohsen Akbaribazm on Jul 30, 2021
Content may be subject to copyright.
TOPIC
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
Online Submissions: http://ww w.journaltcm.com J Tradit Chin Med 2021 August 15; 41(4): 642-649
info@journaltcm.com ISSN 0255-2922
© 2021 JTCM. All rights reserved.
REVIEW
Pharmacological and therapeutic properties of the Red Clover (Trifo-
lium pratense L.): an overview of the new findings
Mohsen Akbaribazm, Fatemeh Khazaei, Leila Naseri, Mona Pazhouhi, Mohammad Zamanian, Mozafar Khazaei
aa
Mohsen Akbaribazm, Fatemeh Khazaei, Leila Naseri, Stu-
dent Research Committee, Kermanshah University of Medi-
cal Sciences, Kermanshah 6714869914, Iran
Mona Pazhouhi, Mozafar Khazaei, Fertility and infertility
research center, Health Technology Institute, Kermanshah
University of Medical Sciences, Kermanshah 6714869914,
Iran
Mohammad Zamanian, Seed and Plant Improvement Insti-
tute, Agricultural Research, Education, Extension Organiza-
tion, Karaj 3135933151, Iran
Supported by the Kermanshah University of Medical Sci-
ence (KUMS) (No. 980292)
Correspondence to: Mozafar Khazaei, Fertility and Infertil-
ity Research Center, Health Technology Institute, Medical
School, University Ave, Shahid Shirodi Blvd, Kermanshah
6714869914, Iran
Telephone: +98-918-3360835
DOI: 10.19852/j.cnki.jtcm.20210604.001
Accepted: April 24, 2020
Abstract
OBJECTIVE: To review beneficial effects of red clo-
ver (Trifolium pratense L.) on biological processes.
METHODS: A systematic review was carried out ac-
cording tothe PRISMA checklist. Using MeSH key-
words, 385 articles were extracted from national
and international databases (PubMed, Science Di-
rect, SID, and Google Scholar search engine) with-
out time limit. After removing duplicates, 80 arti-
cles were reviewed.
RESULTS: Our findings revealed that the composi-
tions of red clover promote anti-oxidant and an-
ti-cancer effects. Furthermore, they exude benefi-
cial effects on cardiovascular functionand improve-
symptoms of menopause. Also, these compounds
can regulate blood glucose and lipid markers. The
effects of the herb on have also been investigated
on various tissues including endometrium, breast,
skin and reproductive system.
CONCLUSION: In recent studies, the anti-cancer ef-
fects of clover (Trifolium) extract co-administrated
with standard drugs have been reportedin differ-
ent cancers.
© 2021 JTCM. All rights reserved.
Keywords: plants, medicinal; trifolium; isoflavo-
noid; antioxidants; anti-cancer
INTRODUCTION
Clover (Trifolium) is one of the most important legu-
minous plants in temperate and humid areas. The plan-
tis very valuable for feeding livestock. As a three-leafed
plant, its scientific name is derived from two Latin
words of "Tria" and "Folia" meaning"three" and "leaf-
lets", respectively. Considering isoflavone content, clo-
ver is the second most important dicotyledonous for-
age after alfalfa in the world.1,2 It had been cultivated in
Europe in the 3rd and 4th centuries and used in fami-
lies' food chain by Germans even after the first world
war.1The first studies on the estrogenic effects of this
plant took place in Australiain 1946 when the ewes fed
with plant were spontaneously aborted.3
There are about 300 species of clover in the world
among which 25 species have agricultural importance.
The clover is most widely distributed in the Mediterra-
nean area. Around 150-160 species are found in Eur-
asia, 65-60 in the United States, and 30-25 in Africa.4
According to the Gene Bank Research and the Heri-
tage Research Institute of Seed and Plant Improvement
Research, the number of known species of clover in
Iran is about 54 of which two (T. resupinatum and T.
642
M Akbaribazm et al. / Review
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
alexandrinum) are widely cultivated.5In 2012, a new
cultivar of clover known as T. pratense cv Nasim was
introduced in Iran. The recent is cultivated in different
seasons of year and is resistant to fungal diseases, leafy
pest and cold-weather.6
In Turkey, clover is used as an anti-septic, analgesic and
sedative compound.7In Pakistan, T. pratense and T. re-
pens are used to treat oral and throat injuries, fever,
and meningitis.8In parts of Europe, including Albania,
red clover is used to make beverages to treat stomach
disorders and diarrhea.9Native Americans also use red
clover to treat skin lesions, as well as pulmonary, neuro-
pathic and reproductive disorders.10
METHODS
Overall, 80 related articles were selected for this review
(Figure 1). Articles assessing the role of herb extracts in
animal models, duplicate studies, and those that were
not in line with the purpose of this study were exclud-
ed. Furthermore, studies on cultivation practices and
methods of the plant preservation were not considered.
The phytochemical contents, anti-oxidant, anti-inflam-
matory and anti-cancer properties, and the effects of T.
pratenese on central nervous system, cardiovascular sys-
tem, breast and endometrial tissues, and blood levels of
glucose and lipid factors were evaluated (Figure 2).
Phytochemical constituents
Flavonoids, isoflavonoids, chalcones and coumarins:
while flavonoids are found throughout plants, isofla-
vones are restricted to certain families including Papilo-
noideae subfamily of the Leguminosae family.11 Six pre-
dominant polyphenolic compounds including bio-
chanin A, formononetin, irilone, quercetin, genistein,
ononin, daidzein and maackain have been identified in
different studies.
Biochanin A [10.22 mg/g in dry matter (DM) of T.
pratense], formononetin (9.65 mg/g DM), and glycit-
ein (1.03 mg/g DM) are predominant isoflavones in in
the leaves of all cultivars. Seven minor aglycones (1 >
mg/g DM) include daidzein, genistein, irilone, orobol,
pratensein, pseudo aptigenin and prunetin. In clover
stems, only formononetin (9.57 mg/g DM) and bio-
chanin A (2.37 mg/g DM) were found asdominant
and prunetin, glycitein, pseudo aptigenin, pratensein,
genistein, daidzein, irilone, and orobol as minor (1 >
mg/g DM) compounds.12 The main isoflavonoids iso-
lated from the aerial parts of clover are prunetin, genis-
tein, prunetin-4-glucopyranoside, prunetin-4-glucopy-
ranoside, genistein-7-galactopyranoside and pini-
tol -6-methoxycyclohexane-pentaol.13
Lipids and fatty acids: T. pretense seed has a low con-
tent of essential oil (0.018%).14 The main diglyceride
(>50%) is palmitic acid whereas linoleic and linole-
nic acids constitute main sterol estersand triglycer-
ides.15 The acetone-insoluble fraction of red clover
leaves is comprised of phosphatidycholine (37% ),
phosphatidylglycerol (23% ), phosphatidyl-ethanol-
amine (15% ), uncharacterized acidic compounds
(13% ), phosphatidyl-inositol (2% ) and unknown
compounds (10%).16
Volatile compounds: Kamm et al 17 used hexane extract
ion and identified volatile components (relative% ) of
T. pretense root asorganic alcohols and fatty aldehyde
[2-methylbutanol (3% ), hexanol (19% ), dodecanal
(0.1%) 2-hexenal (4%), tridecanal (2%), tetradecanal
(4%) and pentadecanal (32%)], aliphatic ketones and
esters [octan-3-one, acetoina (7% ), ethyl-tetradecano-
ate (1% ) and ethyl-hexadecanoate (10% )], aromatic
compounds [methyl-benzoate (0.6% ), ethyl benzoate
(3%), Estragole (4-allylmethoxybenzene) (15%), ben-
zylalcohol (1%), 2-phenylethanol (6%) and methyleu-
genol (3.4-dimethoxyallylbenzene (30% )] and other
volatile compounds [1-undecene (25% ), aceticacid
(10%) and hexanoic acid (5%)].17
Other compounds of T. pratense: in a qualitative study,
crystalline saponins were isolated from red clover (T.
pratense) roots. Acid hydrolysis of these saponins yield-
ed soyasapogenols B, C, D, E, and F.18 Crude protein
(20.9% ), non-starch polysaccharides (26.6% ), starch
(3.3%), glucose (10.3%), uronic acids (6.8%), arabi-
nose (2.9% ), galactose (1.85% ), rhamnose (0.7% ),
mannose (0.7% ) and ribose (0.2% ) have also been
found in the plant. The amount of ash in the herbage
of clover has been reported as 8.4%. The amounts of
microelements in alcoholic soxhlet leaf extracts of red
clover determined by scanning electron microscope-en-
ergy dispersive X- ray spectroscopy (SEM-EDS) re-
vealed the presence (ppm) in order of C (780.124) > O
(214.215) > K (110.161) > Cl (50.14) > Ge (42.09) >
Mg (44.12) > Al (31.8) > Si (29.09) > Zn (6.09) > Ni
(3.91) > Cu (2.044) > Co (1.061) > Fe (0.098).19
Figure 1 Flowchart of the entrance steps of the article infor-
mation
In vivo
In vitro
643
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
M Akbaribazm et al. / Review
Isoflavones interactions with estrogen receptors (ERs)
Many plants produce compounds that possess estrogen-
ic activitiesin animals (i.e. phytoestrogens). Two estro-
gen receptors (ER-βand ER-α) identified to date me-
diate physiological responses to estrogen in specific tis-
sues. The ERs are members of the nuclear hormone re-
ceptor family and act as ligand-activated nuclear tran-
scription factors 20 which are most likely to have higher
affinity for the ER-β. Although these derivatives bind
more strongly to ER-β, the concentration required for
stimulation of both αan βreceptors is almost the
same and much higher than what expected from the
binding affinity. Methylation and glucosidation inhibit
the estrogenic activity of isoflavone compounds.21
Furthermore, isoflavones are feedback regulated by se-
rum level of estradiol. On the other hand, genistein
(5-10 μg/mL) has been noted to inhibit tyrosine-specif-
ic protein kinases in A-431 cells which are involved in
cellular proliferation, transformation, and differentia-
tion. Studies on Jurkat T-leukemia cells and prostate
cancer cell lines (PC-3 and LNCaP) suggested that ge-
nistein (5-10 μg/mL) could suppress tumor cell growth
through arresting the cell-cycle (G2/M) and inducing
apoptosis.22- 24
Effects of red clover estrogenic compounds on
menopause
In the treatment of hormonal disorders by hormone re-
placement therapy (HRT), the use of phytoestrogens is
preferred to synthetic estrogenic compounds. In wom-
en, low level of estrogen may cause symptoms such as
hot flashes, irritability and dryness of the vagina. Red
clover contains estrogenic compounds that can simu-
late the secretion of sexual hormones in the female's
body. In line with this, womenwho take supplementary
red clover after menopause may experience less severe
and frequent menopausal symptoms.25, 26
In accordance, isoflavones reduced the level of uric acid
menopause associated bone loss without inducing any
changes in osteogenic indices such as alkaline phospha-
tase and osteocalcin.27 In a randomized and place-
bo-controlled trial, 190 postmenopausal women were
treated with red clover commercial product (Rimostil:
28.6 mg/kg) for 90 d. This intervention reduced symp-
toms of depression and anxiety and improved hair loss
and skin characteristics; however, the changes were not
statistically significant.28 In another randomized dou-
ble-blind and placebo-controlled trial, the ethanolic ex-
tract of red clover aerial parts (398 mg/d standardized
to 120 mg isoflavones) significantly decreased hot flash-
es and vasomotor symptoms in postmenopausal wom-
en after 12-month.29
Likewise, administration of 57 and 85.5 mg/kg of Ri-
mostil over a 6-month period increased the bone min-
eral densities of proximal sections of the radius and ul-
na by 1.4% and 3% , respectively in postmenopausal
women.30 Nevertheless, treatment with neither 43.5, 50,
nor 82 mg/kg of isoflavones over 3, 12 and 30 months
alter bone marrow markers; urinary N-thulopapid and
serum osteocalcin, in postmenopausal women.31-33
Cardiovascular effects
Flavonoids have a variety of effects including anti-in-
flammatory, anti-anesthetic and anti-allergic ones. The
NF-κB plays a role in inducing the expression of in-
flammatory mediators such as cytokines, surface cell re-
ceptors, adhesive molecules, and acute phase proteins.34
Treatment with genistein (0.3 mg/kg), a constituent of
the red clover, over 8 weeks inhibited the development
of atherosclerosis via inhibiting NF-κB, thrombin,
TNF-α, and vascular cell adhesion molecule-1
(VCAM-1) expressions in LDL-R knockout mice.35
These factors (i.e. NF-κB, TNF-α, and VCAM-1) are
important contributors to atherosclerosis by inducing
accumulation and promoting adhesion of monocytes
to the vessel wall at sites that are prone to form athero-
sclerotic lesions.36 In addition, the Fas/Fas ligand sys-
tem has been identified to be under the control of es-
trogen receptor in monocytes. This suggests a link be-
tween estrogen and many disorders including athero-
sclerosis, vascular inflammation (vasculitis), and rheu-
matoid arthritis.37
Figure 2 Comparison of the plant studies, the most studies done on the antioxidant properties and menopausal symptoms ef-
fects of herb all in vivo (animal models and clinical studies) and in vitro researches are considered.
644
M Akbaribazm et al. / Review
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
The risk of cardiovascular diseasesis increased after
menopause. In a 20-year follow-up of 2873 women
less than 55 years old in the initial Framingham exami-
nation, the annual incidences of cardiovascular diseases
(coronary heart disease, stroke, and congestive heart
failure) were reported as 0.6 and 5 in 1000 population
in premenopausal women aged < 40 and 50-54 years,
respectively.38 In a randomized double-blind place-
bo-controlled trial by Archer et al. (2005), administra-
tion of drospirenone (2 mg) plus β-estradiol (E2)
(1 mg) over a 13-month period significantly decreased
blood pressure and endometrial bleeding in 1142 post-
menopausal women.39 The most plausible explanation
for these observations is the protective effects of endog-
enous female sex steroids, particularly estrogens, dur-
ing premenopausal years.40 In various studies, the ef-
fects of crude extracts and isolated compounds of clo-
ver have been tested on diseases of the heart and blood
vessels. The use of commercial compounds containing
active ingredients of red clover including biochanin A
and formononetin increased vascular vasoconstriction,
SAC, and pulmonary arterial velocity. The reason for
this process can be linked to the release of vasoconstric-
tor compounds including NO, prostaglandins (PGs),
and endothelium-derived hyperpolarizing factor. Ca2+ is
essential for contraction of vascular wall smooth mus-
cles; however, formononetin inhibits intracellular Ca2 +
influx into vascular smooth muscle cells.41
Effects of red clover isoflavoneson serum lipids
The Promensil and Rimostil commercial tablets con-
taining red clover isoflavones have been described to af-
fect serum lipid profile.42 In a double-blind random-
ized placebo-controlled trial, supplementation of
50 mg of Rimostil for 2 years reduced the triglycerides
and LDL and also increased HDL levels in 189 meno-
pausal women.43 In another randomized double-blind
placebo-controlled prospective trial on 37 postmeno-
pausal women with symptoms of estrogen deficiency,
treatment with 40 mg/kg Promensilover a 12-week pe-
riod significantly decreased the levels of triglycerides
and LDL and also increased HDL compared to the pla-
cebo group.44
In a randomized double-blind placebo-controlled trial
on 19 patients with the symptoms of premenstrual syn-
drome, administration of 40 mg biochanin A over a
12-week period decreased the levels of triglycerides and
LDL and increased HDL which led to a significant im-
provement in the premenstrual syndrome symptoms
such as fatigue and swelling.45
Histological effects on the breast tissue
Phytoestrogens affect the sensitivity of breast tumor
cells to vitamin D3analogues. Mammographic breast
density (MBD) is considered as an indicator of breast
cancer progressionor treatment failure.46 This is a mark-
er related to the radiopaque appearance of connective,
epithelial, and radiolucent fat tissues of the breast. Diet
has been shown to influence MBD, potentially
through affecting endogenous estrogen levels. For ex-
ample, a low-fat and high-carbohydrate diet containing
isoflavones has been reported to reduce MBD.47 Al-
though anti-estrogenic drugs such as tamoxifen has
been shown to reduce MBD, HRT increases this pa-
rameter.48 On the other hand, insulin-like growth fac-
tor -I (IGF-I) has been noted to increase the risk of de-
veloping breast cancer. In a randomized placebo-con-
trolled double-blinded crossover trial in four hospitals
in the Netherlands, using Promensil (tablets per day cor-
responding to 86 mg/day total isoflavones) for 2 months
reduced IGF-Iin 23 postmenopausal women.49
A double-blind randomized placebo-controlled trial re-
vealed that receiving 40 and 80 mg/day Promensil tab-
lets alleviated breast pain by 44% and 31% respectively
in 18 premenopausal women during three menstrual
periods.50 In another study, ovariectomized rats treated
with, red clover extract (250, 500 and 750 mg/kg) plus
β-estradiol (50 μg·kg-1
·d-1) for 21 d showed signifi-
cantly increased uterus weight and the degree of vagi-
nal cells differentiation; nevertheless, no significant in-
crease was observed in the proliferation of mammary
gland cells.51
Effects of red clover constituents on the endometrium
Phytoestrogens which are present in different parts of
clover can affect reproductive tissues and the estrogen
receptor. In a randomized double-blind placebo-con-
trolled trial, 30 postmenopausal women were treated
with Promensil (corresponding to 80 mg of red clover
isoflavones). The results showed no significant changes
neither in plasma levels of estradiol, FSH, and LH hor-
mones nor in the endometrial thickness; however, the
incidence of hot flushes significantly decreased in 44%
of the subjects.52 While ER-αis expressed in breast,
uterine and ovarian tissues, the ER-αis expressed in
bone and blood vessels.53 Furthermore, genistein (375
and 750 μg/g) treatment for 21 d increased the uterus
weight and decreased bone loss and osteoporosis.54
In asystematic review (1985-2001), it was reported
that isoflavones such as daidzein and genistein in-
creased the proliferation of endometrial gland cells, in-
creased the expression of ER-β, and decreased the ex-
pression of ER-α. In normal condition, endometrial
cells express higher levels of ER-αthan ER-β.55 In an
in vitro study, endometrial glandular cells were isolated
from premenopausal and non-pregnant women's endo-
metrium in proliferative phase and incubated with ge-
nistein (1.15 μmol/mL) and daidzein (2.4 μmol/mL)
which significantly decreased the ER-αand increased
the ER-βmRNA expressions and also suppressed the
secretion of cytokines such as TNF-αand IL-1α. Ac-
cording to this finding, we can say that ER-α/βexpres-
sions at both mRNA and protein levels in endometrial
glandular cells may be regulated by phytoestrogens.56
Effects of red clover components on thyroid
Thyroid cancer is known to be linked to estrogen and
645
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
M Akbaribazm et al. / Review
is more common in women than men. Accordingly,
17-βestradiol (E2) was shown to activate the PI3K
pathway, inhibit p27, and regulate the transcription of
thyroid-specific genes (i.e. DUOX1, TPO, and NIS).57,
58 In a multiethnic population-based case-control study
on 817 volunteers (72% of the participants were under
the age of 50, and 70% of women were at premeno-
pausal period) in San Francisco between 1998 and
1995, the risk of thyroid cancer was lower in individu-
als taking 1 g/d total phytosterogens (containing Genis-
tein 51% , daidzein 42% , coumestrol 4% , and other
isoflavones 1% ).59 The C-cells of the thyroid have a
role in the synthesis and secretion of calcitonin which
promotes calcium precipitation in bones. These cells al-
so express estrogen receptors and therefore are sensitive
to estrogen.60
In addition, daidzein (30 mg·kg-1
·d-1) administrated
subcutaneously for 3 weeks stimulated the thyroid
C-cells, increased bone mineral density, and ultimately
prevented osteoporosis in orchidectomized rats (mod-
els of osteoporosis). It has been suggested that daidzein
may predominantly act on thyroid C- cells by binding
to ER-β.61 In another study on ovariectomized ewes,
they were fed 3.5 kg of 100% red clover silage (contain-
ing 1.01 g formononetin, 0.03 g daidzein, 0.61 g bio-
chanin-A, and 0.08 g genistein per kg) for two weeks.
In the recent study, total T3and free T3were significant-
ly increased, and thyroid follicles showed larger dimen-
sions in histological examinations.62 In addition, feed-
ing a diet containing red clover to ovariectomized ewes
(1.27 total isoflavones per kg) for 60 d increased total
and free T3levels but did not show a significant effect
on total and free T4levels.63
Daidzein (10 mg/kg) and genistein (10 mg/kg) deriva-
tives of red clover increased TSH level and C cell vol-
ume and decreased T3and T4levels in 60-month-old
orchidectomized Wistar rats treated with the both com-
pounds. Regarding these results, phytoestrogens can
stimulate the production of pituitary TSH by regulat-
ing the pituitary-thyroid axis. In the recent study, the
effects of daidzein were more pronounced in compari-
son with genistein.64 Genistein (500 ppm in diet/day
for 4 weeks) was noted as an inhibitor of thyroid proxi-
dase (TPO) in pregnant female rats.65
Anti-inflammatory, anti-oxidant and anti-platelet
effects
Many studies have been carried out to investigate the
scavenging effects of red clover isoflavones against free
radicals and inflammatory metabolites. According to
one report, topical application of the plant compounds
(20 μM lotions) protected against oxidative damage in-
duced by moderate doses of solar‐simulated UV radi-
ation in hairless mice through reducing inflammato-
ry-induced edema and contact hypersensitivity.65 In the
study of Mu et al 66 on the antioxidant effects of for-
mononetinin ovariectomized mice, it was found that
this isoflavone at the doses of 0.05, 0.2 and 0.5 g/kg
for 3 d increased the activities of superoxide dismutase
(SOD), glutathione peroxidase, and catalase (CAT)
while decreased lipid peroxidation. Furthermore, for-
mononetin treatment (2-10 mg/kg) for 6
hours'dose-dependently decreased the expressions of
IL-6 and TNF-αinflammatory factors in the bron-
cho-alveolar lavage fluid in an animal model of the lu-
pus insensitive lipoprotein (LPS) lung injury.67 In a ran-
domized double-blind placebo-controlled study on six-
ty postmenopausal women aged > 40 years old,
long-term use of tablets containing red clover (consti-
tuting of 0.6 g genistein, 9.6 g biochanin A, 2.3 g daid-
zein, and 27.5 g formononetin) activated thenitric ox-
ide synthases (NOSs) enzyme through inducing the es-
trogen receptor signaling pathway.68 In one study on
rats, caffeic acid (10 μmol·kg-1
·d-1), a constituent of
red clover, increased the activities of SOD, CAT, and
glutathione peroxidase (GPx) in cardiomyocytes and
protected them against the peroxidation of membrane
lipids.69
An in-vitro study on RAW 264.7 and HT-29 cell lines
also described the anti-inflammatory and anti-prolifera-
tive activities of biochanin A. In this study, biochanin
A inhibited lipopolysacharide (LPS)-induced nitric ox-
ide (NO) production in RAW 264.7 macrophage cell
line and dose-dependently inhibited inducible nitric
oxide synthase (iNOS) expression in HT-29 cells. Be-
sides, LPS-induced phosphorylation of the inhibitor of
NF-κB (IκBα) protein and p38 MAPK and produc-
tion of IL-6, IL-1βand TNF-αin RAW264.7 cells
were blocked by biochanin A.70 In the mouse model of
LPS/GalN-induced liver injury, biochanin A (25 and
50 mg/kg) dose-dependently up-regulated the expres-
sions of Nrf2and HO-1 and inhibited hepatic expres-
sions of IL-1βand TNF-α. These functions protected
liver by activating the Nrf2pathway and inhibiting NL-
RP3 inflammasome activity.71
Anti-cancer effects of red clover isoflavones
Red clover isoflavones (biochanin A and formonone-
tin) inhibit cytochrome P-450 enzymes which can in-
crease the metabolism of drugs (including chemothera-
peutic agents) and reduce their toxic effects.72, 73 Bio-
chanin A (25 μM) inhibits the function and expression
of the P-450 (CYP 19) enzyme in MCF-7 and
SK-BR-3 cell lines in which the enzyme acts as an aro-
matase to transform androgen to estrogen. Considering
the estrogenic activity of this isoflavone, it can contrib-
ute to hormone therapy in patients with breast can-
cer.74 In vitro studies have shown that genistein inhibits
the growth of both ER-and ER+breast cancer cell lines
(i.e. MDA-MB-231, MDA-MB-435, and MCF-7).75
Genistein (10 nM) also reduced the activity of CYP24
enzyme which is responsible for the storage of vitamin
D (1, 25-dihydroxyvitamin D3) metabolites and re-
quired for suppressing mitosis in prostate and colorec-
tal cancer cells. In other words, increased activity of the
CYP24 enzyme reduces the level of 1, 25-D-hydroxyvi-
646
M Akbaribazm et al. / Review
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
tamin D3in prostate cancer cells.76, 77 Nonetheless, bio-
chanin A (10-100 nM) had no significant effects on
the expression and activity of the CYP19 enzyme in hu-
man granulose-luteal cells.78
We have already shown that the hydroalcoholic extract
of clover (50-400 μg/mL) significantly reduced the sur-
vival of the glioblastoma cell line (U87MG) indose-
and time-dependent mannersby up-regulating the ex-
pressions of ATG-7, p53, Bax, caspase-3, and Beclin-1
genes. In addition, the synergistic effect of this extract
with temozolomide (TMZ) (300-2400 μM) increased
the cytotoxicity of TMZ.79 Furthermore, formononetin
(40 and 80 μM) inhibited the proliferation of LNCaP
and PC-3 prostate cancer cell lines through inducing
the ERK1,2/MAPK-Bax and inhibiting the extracellu-
lar signal-regulated kinase 1/2 (ERK1/2)/mitogen acti-
vating protein kinase (MAPK) pathways as well as en-
hancing the expression of the Bcl-2 apoptotic factor in
cancerous cells.80
In conclusion, Clover has a special role in traditional
medicine to control and treat various diseases. Isofla-
vones, as well as phenolic and polyphenolic com-
pounds of this plant are used to relieve cough, reduce
muscle aches and menopausal symptoms, treat asthma,
alleviate joint diseases, heal wounds, prevent cancer,
and finally improve reproductive disorders. In addition
to the traditional uses of clover and its derivatives, an-
ti-cancer properties of this plant have widely been in-
vestigated today. Nevertheless, most of these studies
have been carried out in vitro, and there is a need for
conducting in vivo studies along with clinical trials
to verify biological properties of the plant and its
constituents.
REFERENCES
1Sabudak T, Guler N. Trifolium L. A review on its phyto-
chemical and pharmacological profile. Phytother Res
2009; 23(3): 439-446.
2Vasiljević S, Ćupina B, Krstic Đ, Pataki I, Katanski S, Mi-
lošević B. Seasonal changes of proteins, structural carbohy-
drates, fats and minerals in herbage dry matter of red clo-
ver (Trifolium pratense L.). Biotechnol Anim Husb 2011;
27: 1543-1550.
3Bennetts HW, Uuderwood EJ, Shier FL. A specific breed-
ing problem of sheep on subterranean clover pastures in
Western Australia. Aust Vet J 1946; 22: 2-12.
4Ellison NW, Liston A, Steiner JJ, Williams WM, Taylor
NL. Molecular phylogenetics of the clover genus (Trifoli-
um—Leguminosae). Mol Phylogenet Evol 2006; 39(3):
688-705.
5Abbasi MR. Evaluation of genetic diversity in red clover
collection at National Plant Gene Bank of Iran. 2008; 15
(4): 324-335.
6Zamanian M, Akbari RM, Rahjou V, et al. Release of new
cultivar: Nasim, a new red clover cultivar. J Appl Seed
Prod 2012; 28 (3): 381-383.
7Sabudak T, Dokmeci D, Ozyigit F, Isik E, Aydogdu N.
Antiinflammatory and antioxidant activities of Trifolium
resupinatum var. microcephalum extracts. Asian J Chem
2008; 20(2):1491.
8Khan SW, Khatoon SU. Ethnobotanical studies on some
useful herbs of Haramosh and Bugrote valleys in Gilgit,
northern areas of Pakistan. Pak J Bot 2008; 40(1): 43.
9Mustafa B, Hajdari A, Krasniqi F, et al. Medical ethnobot-
any of the Albanian Alps in Kosovo. J Ethnobiol Eth-
nomed 2012; 8(1): 1-4.
10 Nissan HP, Lu J, Booth NL, Yamamura HI, Farnsworth
NR, Wang ZJ. A red clover (Trifolium pratense) phase Ⅱ
clinical extract possesses opiate activity. J Ethnopharmacol
2007; 30(112): 207-210.
11 Dixon RA, Sumner LW. Legume natural products: under-
standing and manipulating complex pathways for human
and animal health. Plant Physiol 2003; 131(3): 878-885.
12 Tsao R, Papadopoulos Y, Yang R, Young JC, McRae K.
Isoflavone profiles of red clovers and their distribution in
different parts harvested at different growing stages. J Agr
Food Chem 2006; 54(16): 5797-5805.
13 Lim TK. Trifolium pratense. InEdible medicinal and
non-medicinal plants. Springer 2014; 925-948.
14 Tava A, Ramella D, Grecchi M, Aceto P, Paoletti R, Piano
E. Volatile constituents of Trifolium pratense and T. repens
from NE Italian alpine pastures. Nat Prod Commun
2009; 4(6): 1934578X0900400619.
15 Weenink RO. Minor constituents of the acetone-soluble
lipids of red-clover (Trifolium pratense) leaves. Biochem J
1962; 82(3): 523.
16 Weenink RO. Acetone-soluble lipids of grasses and other
forage plants. I.galactolipids of red clover (Trifolium
pratense) leaves. J Sci Food Agric 1961; 12(1): 34-38.
17 Kamm JA, Butterys RG. Root volatile components of red
clover: identification and bioassay with the clover root bor-
er (Coleoptera: Scolytidae). Environ Entomol 1984; 13
(5): 1427-1430.
18 Oleszek W, Jurzysta M. Isolation, chemical characteriza-
tion and biological activity of red clover (Trifolium
pratense L.) root saponins. Acta Soc Bot Pol 1986; 55(2):
247-252.
19 Akbaribazm M, Khazaei MR, Khazaei M. Phytochemi-
cals and antioxidant activity of alcoholic/hydroalcoholic ex-
tract of Trifolium pratense. Chin Herb Med 2020; 12(3):
326-335.
20 Valsecchi AE, Franchi S, Panerai AE, Sacerdote P, Trovato
AE, Colleoni M. Genistein, a natural phytoestrogen from
soy, relieves neuropathic pain following chronic constric-
tion sciatic nerve injury in mice: anti-inflammatory and
antioxidant activity. J Neurochem 2008; 107(1): 230-240.
21 Mueller M, Hobiger S, Jungbauer A. Red clover extract: a
source for substances that activate peroxisome prolifera-
tor-activated receptor αand ameliorate the cytokine secre-
tion profile of lipopolysaccharide-stimulated macrophages.
Menopause 2010; 17(2): 379-387.
22 Morito K, Hirose T, Kinjo J, et al. Interaction of phytoes-
trogens with estrogen receptors αand β. Biol Pharm Bull
2001; 24(4): 351-356.
23 Akiyama T, Ishida J, Nakagawa S, et al. Genistein, a spe-
cific inhibitor of tyrosine-specific protein kinases. J Biol
Chem 1987; 262(12): 5592-5595.
647
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
M Akbaribazm et al. / Review
24 Spinozzi F, Pagliacci MC, Migliorati G, et al. The natural
tyrosine kinase inhibitor genistein produces cell cycle ar-
rest and apoptosis in Jurkat T-leukemia cells. Leukemia
Res 1994; 18(6): 431-439.
25 Davis JN, Kucuk O, Sarkar FH. Genistein inhibits
NF-kB activation in prostate cancer cells. Nutr Cancer
1999; 35(2): 167-174.
26 Hidalgo LA, Chedraui PA, Morocho N, Ross S, San
Miguel G. The effect of red clover isoflavones on meno-
pausal symptoms, lipids and vaginal cytology in menopaus-
al women: a randomized, double-blind, placebo-controlled
study. Gynecol Endocrinol 2005; 21(5): 257-264.
27 Salimpour F, Mostafavi G, Sharifnia F. Micromorpholog-
ic study of the seed of the Genus Trifolium, Section Lotoi-
dea, in Iran. Pak J Biol Sci 2007; 10(3): 378-382.
28 Chen X, Anderson JJ. Isoflavones and bone: animal and
human evidence of efficacy. J Musculoskelet Neuronal In-
teract 2002; 2(4): 352-359.
29 Tice JA, Ettinger B, Ensrud K, Wallace R, Blackwell T,
Cummings SR. Phytoestrogen supplements for the treat-
ment of hot flashes: the Isoflavone Clover Extract (ICE)
study: a randomized controlled trial. JAMA 2003; 290(2):
207-214.
30 Geller SE, Shulman LP, Van Breemen RB, et al. Safety
and efficacy of black cohosh and red clover for the manage-
ment of vasomotor symptoms: a randomized controlled tri-
al. Menopause 2009; 16(6): 1156.
31 Clifton-Bligh PB, Baber RJ, Fulcher GR, Nery ML,
Moreton T. The effect of isoflavones extracted from red
clover (Rimostil) on lipid and bone metabolism. Meno-
pause 2001; 8(4): 259-265.
32 Hale GE, Hughes CL, Robboy SJ, Agarwal SK, Bievre M.
A double-blind randomized study on the effects of red clo-
ver isoflavones on the endometrium. Menopause 2001; 8
(5): 338-346.
33 Atkinson C, Compston JE, Day NE, Dowsett M, Bing-
ham SA. The effects of phytoestrogen isoflavones on bone
density in women: a double-blind, randomized, place-
bo-controlled trial. Am J Clin Nutr 2004; 79(2): 326-333.
34 Muraoka K, Shimizu K, Sun X, et al. Flavonoids exert di-
verse inhibitory effects on the activation of NF-kappa B.
Transplant Proc 2002; 34(4): 1335.
35 Wang J, Zhang R, Xu Y, Zhou H, Wang B, Li S. Genis-
tein inhibits the development of atherosclerosis via inhibit-
ing NF-κB and VCAM-1 expression in LDLR knockout
mice. Can J Physiol Pharmacol 2008; 86(11): 777-784.
36 Ley K, Huo Y. VCAM-1 is critical in atherosclerosis. J
Clin Invest 2001; 107(10): 1209-1210.
37 Mor G, Sapi E, Abrahams VM, et al. Interaction of the es-
trogen receptors with the Fas ligand promoter in human
monocytes. J Immunol 2003; 170(1): 114-122.
38 Kannel WB, Hjortland MC, McNamara PM, Gordon T.
Menopause and risk of cardiovascular disease: the Framing-
ham study. Ann Intern Med 1976; 85(4): 447-452.
39 Archer DF, Thorneycroft IH, Foegh M, et al. Long-term
safety of drospirenone-estradiol for hormone therapy: a
randomized, double-blind, multicenter trial. Menopause
2005; 12(6): 716-727.
40 Nasr A, Breckwoldt M. Estrogen replacement therapy and
cardiovascular protection: lipid mechanisms are the tip of
an iceberg. Gynecol Endocrinol 1998; 12(1): 43-59.
41 Tao SU, Rui LI, Cao YX. Vasorelaxant and antihyperten-
sive effects of formononetin through endothelium-depen-
dent and-independent mechanisms. Acta Pharmacol Sin
2011; 32(8): 1009.
42 Booth NL, Piersen CE, Banuvar S, Geller SE, Shulman
LP, Farnsworth NR. Clinical studies of red clover (Trifoli-
um pratense) dietary supplements in menopause: a litera-
ture review. Menopause 2006; 13(2): 251-264.
43 Clifton-Bligh PB, Nery ML, Clifton-Bligh RJ, et al. Red
clover isoflavones enriched with formononetin lower se-
rum LDL cholesterol—a randomized, double-blind, place-
bo-controlled study. Eur J Clin Nutr 2015; 69(1): 134.
44 Knight DC, Howes JB, Eden JA.The effect of Promensil™,
an isoflavone extract, on menopausal symptoms. Climac-
teric 1999; 2(2): 79-84.
45 Taher S, Cahill A, Eliahoo J, Calvin M, Rothon C, Panay
N. Randomised placebo controlled pilot study on compar-
ing red clover (p-07) versus placebo for the treatment of
premenstrual syndrome. Maturitas 2009; 63(1): 114-115.
46 Maskarinec G, Williams AE, Carlin L. Mammographic
densities in a one-year isoflavone intervention. Eur J Can-
cer Prev 2003; 12(2): 165-169.
47 Atkinson C, Warren RM, Sala E, et al. Red clover-derived
isoflavones and mammographic breast density: a dou-
ble-blind, randomized, placebo-controlled trial [IS-
RCTN42940165]. Breast Cancer Res 2004; 6(3): 170.
48 Mullooly M, Gierach GL. The potential for mammo-
graphic breast density change as a biosensor of adjuvant
tamoxifen therapy adherence and response. JNCI Cancer
Spectr 2018; 2(4): 72.
49 Campbell MJ, Woodside JV, Honour JW, Morton MS,
Leathem AJ. Effect of red clover-derived isoflavone supple-
mentation on insulin-like growth factor, lipid and antioxi-
dant status in healthy female volunteers: a pilot study. Eur
J Clin Nutr 2004; 58(1): 173.
50 Ingram DM, Hickling C, West L, Mahe LJ, Dunbar PM.
A double-blind randomized controlled trial of isoflavones
in the treatment of cyclical mastalgia. Breast J 2002; 11
(2): 170-174.
51 Burdette JE, Liu J, Lantvit D, et al. Trifolium pratense
(red clover) exhibits estrogenic effects in vivo in ovariecto-
mized Sprague-Dawley rats. J Nutr 2002; 132(1): 27-30.
52 Van de Weijer PH, Barentsen R. Isoflavones from red clo-
ver (Promensil®) significantly reduce menopausal hot flush
symptoms compared with placebo. Maturitas 2002; 42(3):
187-193.
53 Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F.
Estrogen receptors alpha (ERα) and beta (ERβ): sub-
type-selective ligands and clinical potential. Steroids 2014;
90 (15): 13-29.
54 Santell RC, Chang YC, Nair MG, Helferich WG. Dietary
genistein exerts estrogenic effects upon the uterus, mam-
mary gland and the hypothalamic/pituitary axis in rats. J
Nutr 1997; 127(2): 263-269.
55 Albertazzi P. Purified phytoestrogens in postmenopausal
bone health: is there a role for genistein? Climacteric
2002; 5(2): 190-196.
56 Staar S, Richter DU, Makovitzky J, Briese V, Bergemann
C. Stimulation of endometrial glandular cells with genis-
648
M Akbaribazm et al. / Review
JTCM
|
www. journaltcm. com August 15, 2021
|
Volume 41
|
Issue 4
|
tein and daidzein and their effects on ERα-and
ERβ-mRNA and protein expresion. Anticancer Res 2005;
25(3A): 1713-1718.
57 Lian Z, Niwa K, Tagami K, et al. Preventive effects of iso-
flavones, genistein and daidzein, on estradiol-17β-related
endometrial carcinogenesis in mice. Jpn J Cancer Res
2001; 92(7): 726-734.
58 Antico-Arciuch VG, Dima M, Liao XH, Refetoff S, Di
Cristofano A. Cross-talk between PI3K and estrogen in
the mouse thyroid predisposes to the development of follic-
ular carcinomas with a higher incidence in females. Onco-
gene 2010; 29(42): 5678.
59 Horn-Ross PL, Hoggatt KJ, Lee MM. Phytoestrogens
and thyroid cancer risk: the San Francisco bay area thyroid
cancer study. Cancer Epidemiol Biomarkers Prev 2002; 11
(1): 43-49.
60 Bléchet C, Lecomte P, De Calan L, Beutter P, Guyétant S.
Expression of sex steroid hormone receptors in C cell hy-
perplasia and medullary thyroid carcinoma. Virchows Ar-
chiv 2007; 450(4): 433-439.
61 Filipović B, Šošić-Jurjević B, Ajdžanović V, et al. Daidzein
administration positively affects thyroid C cells and bone
structure in orchidectomized middle-aged rats. Osteoporos
Int 2010; 21(9): 1609-1616.
62 Madej A, Persson E, Lundh T, Ridderstråle Y. Thyroid
gland function in ovariectomized ewes exposed to phytoes-
trogens. J Chromatogr B 2002; 777(1-2): 281-287.
63 Adams NR. Morphological changes in the organs of ewes
grazing oestrogenic subterranean clover. Res Vet Sci 1977;
22(2): 216-221.
64 Šošić-Jurjević B, Filipović B, Ajdžanović V, et al. Suppres-
sive effects of genistein and daidzein on pituitary–thyroid
axis in orchidectomized middle-aged rats. Exp Biol Med
2010; 235(5): 590-598.
65 Widyarini S, Spinks N, Husband AJ, Reeve VE. Isoflavo-
noid compounds from Red Clover (Trifolium pratense)
protect from inflammation and immune suppression in-
duced by UV radiation. Photochem Photobiol 2001; 74
(3): 465-470.
66 Mu H, Bai YH, Wang ST, Zhu ZM, Zhang YW. Research
on antioxidant effects and estrogenic effect of formonone-
tin from Trifolium pratense (red clover). Phytomedicine
2009; 16(4): 314-319.
67 Ma Z, Ji W, Fu Q, Ma S. Formononetin inhibited the in-
flammation of LPS-induced acute lung injury in mice asso-
ciated with induction of PPAR gamma expression. Inflam-
mation 2013; 36(6): 1560-1566.
68 Kolodziejczyk J, Olas B, Wachowicz B, Szajwaj B, Stoch-
mal A, Oleszek W. Clovamide-rich extract from Trifolium
pallidum reduces oxidative stress-induced damage to
blood platelets and plasma. J Physiol Biochem 2011; 67
(3): 391-399.
69 Okutan H, Ozcelik N, Yilmaz HR, Uz E. Effects of caffe-
ic acid phenethyl ester on lipid peroxidation and antioxi-
dant enzymes in diabetic rat heart. Clin Biochem 2005; 38
(2): 191-196.
70 Kole L, Giri B, Manna SK, Pal B, Ghosh S. Biochanin-A,
an isoflavon, showed anti-proliferative and anti-inflamma-
tory activities through the inhibition of iNOS expression,
p38-MAPK and ATF-2 phosphorylation and blocking
NFκB nuclear translocation. Eur J Pharmacol 2011; 653
(1-3): 8-15.
71 Liu X, Wang T, Liu X, et al. Biochanin A protects lipo-
polysaccharide/D-galactosamine-induced acute liver injury
in mice by activating the Nrf2 pathway and inhibiting NL-
RP3 inflammasome activation. Int Immunopharmacol
2016; 38: 324-331.
72 Sarkar FH, Li Y. Soy isoflavones and cancer prevention:
clinical science review. Cancer invest 2003; 21(5):
744-757.
73 Smith M, Boon HS. Counseling cancer patients about
herbal medicine. Patient Educ Couns 1999; 38(2):
109-120.
74 Roberts DW, Doerge DR, Churchwell MI, Gamboa da
Costa G, Marques MM, Tolleson WH. Inhibition of extra-
hepatic human cytochromes P450 1A1 and 1B1 by metab-
olism of isoflavones found in Trifolium pratense (red clo-
ver). J Agr Food Chem 2004; 52(21): 6623-6632.
75 Li Y, Upadhyay S, Bhuiyan M, Sarkar FH. Induction of
apoptosis in breast cancer cells MDA-MB-231 by genis-
tein. Oncogene 1999; 18(20): 3166-3172.
76 Kallay E, Adlercreutz H, Farhan H, et al. Phytoestrogens
regulate vitamin D metabolism in the mouse colon: rele-
vance for colon tumor prevention and therapy. J Nutr
2002; 132(11): 3490S-3493S.
77 Hanchette CL, Schwartz GG. Geographic patterns of
prostate cancer mortality. Evidence for a protective effect
of ultraviolet radiation. Cancer 1992; 70(12): 2861-2869.
78 Rice S, Mason HD, Whitehead SA. Phytoestrogens and
their low dose combinations inhibit mRNA expression
and activity of aromatase in human granulosa-luteal cells.
J Steroid Biochem 2006; 101(4-5): 216-225.
79 Khazaei M, Pazhouhi M, Khazaei S. Evaluation of hy-
dro-alcoholic extract of Trifolium Pratens L. for its an-
ti-cancer potential on U87MG cell line. Cell J 2018; 20
(3): 412-421.
80 Ye Y, Hou R, Chen J, et al. Formononetin-induced apop-
tosis of human prostate cancer cells through ERK1/2 mito-
gen-activated protein kinase inactivation. Horm Metab
Res 2012; 44(4): 263-267.
649