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Pharmacological and therapeutic properties of the Red Clover (Trifo- lium pratense L.): an overview of the new findings

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Abstract

OBJECTIVE: To review beneficial effects of red clover (Trifolium pratense L.) on biological processes. METHODS: A systematic review was carried out according tothe PRISMA checklist. Using MeSH keywords , 385 articles were extracted from national and international databases (PubMed, Science Direct , SID, and Google Scholar search engine) without time limit. After removing duplicates, 80 articles were reviewed. RESULTS: Our findings revealed that the compositions of red clover promote anti-oxidant and an-ti-cancer effects. Furthermore, they exude beneficial 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 effects of clover (Trifolium) extract co-administrated with standard drugs have been reportedin different cancers.
TOPIC
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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.
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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
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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.
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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
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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·kg1
·d1) 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 solarsimulated 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·kg1
·d1), 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-
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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.
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649
... pratense, T. hybridum, T. repens) Life 2022, 12, 1975 2 of 26 or as a green manure plant to enhance soil fertility [9]. Due to their high content of secondary metabolites, such as isoflavonoids, some species are also being studied for potential pharmacological use [10][11][12]. Soil enrichment with nitrogen via growing plants utilizing BNF, such as clover, is more sustainable than using synthetic nitrogen-based fertilizers. However, not all genotypes within the fixation-capable species have the same nitrogen fixation efficiency [13]. ...
... In this way, each gene expressed in the nodule was inspected for possible duplication events and each gene was classified according to its own duplication mode (whole genome duplication [WGD], tandem, proximal, transposed, dispersed, non-duplicated). The distribution of different Life 2022, 12,1975 6 of 26 duplication modes was checked among DEGs, and this distribution was compared with the global distribution of duplicate modes among all genes expressed in nodules. Statistical significance was calculated using Pearson's chi-squared test for global distribution and Fisher's exact test for particular duplicate modes in R while using RStudio [88]. ...
... After mapping sequencing reads to the reference genome, alignment control was performed to evaluate the mapping rate and quality of the sequencing reads. In general, the Life 2022, 12,1975 8 of 26 number of uniquely mapped reads was about 80%, as shown in Supplementary Figure S1, and gene-body coverage showed a general abundance of reads across transcript bodies to be equal, with a median of around 50 and thus showing no possible 3 or 5 biases. ...
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Commonly studied in the context of legume–rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant–bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover’s root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.
... The active ingredients isolated from red clover differ from plant parts. For example, components isolated from the leaves of red clover are biochanin A, daidzein, genistein, Iris germanica L, pretense, and prunus isoflavones while in stems are biochanin A, genistein, and daidzein (33). Therefore, daidzein, genistein, and biochanin A are the main isoflavones in red clover with good pharmacological activities, which have been used in antiinflammatory and antioxidant. ...
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... The extracts showed a high potential against Gram-negative bacteria and induced a modest antioxidant effect on the experimental inflammation model in Wistar rats. A review of research by Mohsen et al. [18] revealed that recent studies have focused on the effects of red clover exhibiting antioxidant and anticancer effects. In addition, they exert beneficial effects on cardiovascular function and improve menopausal symptoms. ...
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Red clover is a raw material of interest primarily due to its isoflavone content. However, other groups of compounds may affect the pleiotropic biological effects of this raw material. It is used to alleviate menopausal symptoms, but the fact that there are many varieties of this plant that can be grown makes it necessary to compare the biological activity and phytochemical composition of this plant. Also of interest are the differences between the leaves and flowers of the plant. The aim of this study was to evaluate the properties of the leaves and flowers of six clover varieties-'Tenia', 'Atlantis', 'Milena', 'Magellan', 'Lemmon' and 'Lucrum'-with respect to their ability to inhibit α-glucosidase, lipase, collagenase and antioxidant activity. Therefore, the contents of polyphenols and the four main isoflavones-genistein, daidzein, biochanin and formononetin-were assessed. The study was complemented by testing for permeability through a model membrane system (PAMPA). Principal component analysis (PCA) identified a relationship between activity and the content of active compounds. It was concluded that antioxidant activity, inhibition of glucosidase, collagenase and lipase are not correlated with isoflavone content. A higher content of total polyphenols (TPC) was determined in the flowers of red clover while a higher content of isoflavones was determined in the leaves of almost every variety. The exception is the 'Lemmon' variety, characterized by high isoflavone content and high activity in the tests conducted.
... In parts of Europe, including Albania, T. pratense is used to make beverages to treat diarrhea and stomach disorders. In addition, Native Americans also use red clover to treat neuropathic, pulmonary, and reproductive disorders, as well as skin lesions (Akbaribazm et al., 2021). ...
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The most well-known phytoestrogens (flavonoids, isoflavonoids, lignans, coumestans, stilbenes, and prenylflavonoids) are isoflavonoids, which are important active ingredients in medicinal and food plants. They are highly abundant in the Fabaceae family. More than 1,000 types of isoflavonoids have been isolated from nearly 300 kinds of plants, and more are being discovered through modern analytical methods. Glycosides O and C of isoflavonoids are poorly absorbed in the intestine. They are converted by bacterial esterases and/or β-glycosidase enzymes to aglycones, which are absorbed more efficiently. Their bioavailability shows significant differences due to variation in the intestinal microflora of various races. The compounds formed during their biotransformation are structurally similar to estrogens. In Traditional Chinese medicine, several herbs rich in phytoestrogens are used to prevent and cure various diseases, such as osteoporosis, cardiovascular diseases, diabetes mellitus, hypertension, hyperlipidemia, tumors, and inflammation; additionally, 185 herbs are used to treat menopausal symptoms. Some of these herbs can be used to alleviate the unpleasant symptoms of menopause and treat breast and prostate cancer. From a nutritional physiology perspective, the consumption of Glycine max and Vigna unguiculata should be emphasized. Soy has been consumed in China for about 5,000 years while it was introduced to Europe nearly 300 years ago. Soybean cultivation in Hungary dates back only 100 years. The assessment of the efficacy of phytoestrogens is unclear. Although several experimental and molecular biology studies have shown favorable results, studies on humans have not shown prominent therapeutic benefits. However, comparing and interpreting the findings of modern studies might elucidate the therapeutic utility of phytoestrogens.
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Red clover (RC) extract is rich in isoflavones (formononetin and biochanin A) that have various biological functions. However, its low water solubility limits its bioavailability. In this study, an RC extract/β-cyclodextrin (RC/β-CD) dispersion was prepared by ball milling to enhance its water solubility and biological availability. The water solubility of formononetin and biochanin A was 34.45 and 13.65 μg/mL (increased to 3.11 and 2.14 times higher than that of RC alone), respectively. The alleviating effects of the dispersion on lipid accumulation and gut microbiota were evaluated in mice. The RC/β-CD dispersion showed a better effect on inhibiting lipid accumulation, especially on total triglycerides. The dispersion group had a higher relative abundance of Akkermansia, Muribaculaceae, and Bacteroides than RC alone, along with a higher level of acetic and butyric acid. The study provides a feasible way for improving the bioaccessibility and bioactivity of RC isoflavones in red clover.
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Background: Red clover (Trifolium pratense L.) is a diploid perennial temperate legume with 14 chromosomes (2n = 14) native to Europe and West Asia, with high nutritional and economic value. It is a very important forage grass and is widely grown in marine climates, such as the United States and Sweden. Genetic research and molecular breeding are limited by the lack of high-quality reference genomes. In this study, we used Illumina, PacBio HiFi, and Hi-C to obtain a high-quality chromosome-scale red clover genome and used genome annotation results to analyze evolutionary relationships among related species. Results: The red clover genome obtained by PacBio HiFi assembly sequencing was 423 M. The assembly quality was the highest among legume genome assemblies published to date. The contig N50 was 13 Mb, scaffold N50 was 55 Mb, and BUSCO completeness was 97.9%, accounting for 92.8% of the predicted genome. Genome annotation revealed 44,588 gene models with high confidence and 52.81% repetitive elements in red clover genome. Based on a comparison of genome annotation results, red clover was closely related to Trifolium medium and distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum among legumes. Analyses of gene family expansions and contractions and forward gene selection revealed gene families and genes related to environmental stress resistance and energy metabolism. Conclusions: We report a high-quality de novo genome assembly for the red clover at the chromosome level, with a substantial improvement in assembly quality over those of previously published red clover genomes. These annotated gene models can provide an important resource for molecular genetic breeding and legume evolution studies. Furthermore, we analyzed the evolutionary relationships among red clover and closely related species, providing a basis for evolutionary studies of clover leaf and legumes, genomics analyses of forage grass, the improvement of agronomic traits.
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The aim of this report was to determine the impact of flaxseed, soy and red clover, and their bioactive substances on the lipid profile in postmenopausal women in cardiovascular diseases prevention. We used the following databases: MEDLINE (PubMed), EMBASE and the Cochrane Library. Meta-analysis indicates that the intake of flaxseed by postmenopausal women is associated with a statistically significant reduction in total cholesterol (TC) levels (weighted-mean difference (WMD) = −0.26; 95% confidence interval (95% CI): −0.38 to −0.13; p = 0.0001), low-density lipoprotein cholesterol (LDL-C) levels (WMD = −0.19; 95% CI: −0.30 to −0.08; p = 0.0006), and high-density lipoprotein cholesterol (HDL-C) levels (WMD = −0.06; 95% CI: −0.11 to −0.01; p = 0.0150). The effect of soy protein on the lipid profile showed a significant decrease in TC levels: WMD = −0.15; 95% CI: −0.25–0.05; p = 0.0048, LDL-C levels: WMD = −0.15; 95% CI: −0.25–0.05; p = 0.0067, as well as a significant increase in HDL-C levels: WMD = 0.05; 95% CI: 0.02–0.08; p = 0.0034. Changes in the lipid profile showed a significant reduction in TC levels after the use of red clover (WMD = −0.11; 95% CI: −0.18–−0.04; p = 0.0017) and a significant increase in HDL-C levels (WMD = 0.04; 95% CI: 0.01 to 0.07; p = 0.0165). This meta-analysis provides evidence that consuming flaxseed, soy and red clover can have a beneficial effect on lipids in postmenopausal women and suggest a favorable effect in preventing cardiovascular diseases.
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Objective Trifolium pratense has many healing properties, including fewer complications of menopause, cancer cell suppression, reducing blood glucose and lipids, as well as cardiovascular beneficial effects. The purpose of this study was to identify the phytochemical and mineral composition of T. pratense. Methods Plant aerial parts were harvested and dried, and then hydroalcoholic and alcoholic extracts were prepared. Gas chromatography–mass spectrometry (GC-MS) analytical method was used to identify volatile compounds then liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used to identify polyphenols and the mineral elements were identify by inductively coupled plasma atomic emission spectrometer/ICP-AES and scanning electron microscope-energy-dispersive X-ray spectroscopy (SEM-EDS) methods. Total phenolic content (TPC) was determined based on colorimetric method, and total flavonoid content (TFC) was established based on the folin-chiocalteau reagent. Furthermore, two assays (DPPH and FRAP) were used to measure the antioxidant capacity of T. pratense ethanolic extract. Results A total of 37 polyphenol and 107 peaks were identified by LC-ESI-MS analysis, and the GC/MS method also detected 21 volatile compounds, the most important of which were methylcyclopentane, dimethylpentanal and hexadecanol. A total of 18 mineral elements, including K, Mg, Al, Si, Zn, Ni, Cu, Se, Co, Fe, Mn, and Ca in the plant, were identified ICP-AES and SEM-EDS analysis. Conclusion T. pratense has many therapeutic compounds such as polyphenol (isoflavone and flavonoids), volatile compounds, and essential mineral elements, which can be formulated purely and used in the pharmaceutical and traditional medicine industries.
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Red clover (Trifolium pratense L.) is the second most important perennial forage legume, after alfalfa. It is a significant source of proteins, which are essential in the diet of domestic animals, especially ruminants. The objective of this study was to determine the most important characteristics of dry matter quality (crude proteins, structural carbohydrates, fats and minerals) in three varieties of red clover (K-17, Kolubara and Una). The characteristics were monitored, per individual cuts, during one production cycle of red clover, i.e., in the period 2004-2006. Highest contents of crude protein in all three test years were obtained in the second cut. Significantly increased contents of crude proteins (18.32%) and potassium (K-2.53%) were found in the red clover cultivar Una developed in Novi Sad. Significantly increased values of both cellulose fiber fractions (NDF, ADF) were registered in the second year of growing. The highest content of minerals was determined at the beginning of the growing season in the first and second year (8.68% and 8.52%, respectively). Here it should be mentioned that the contents of basic biogenous elements, phosphorus and potassium, were within the expected optimum range.
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Background: Although postmenopausal combined hormone replacement therapy reduces the risk of hip fracture, long-term use may be associated with an increased risk of breast cancer, and in women more than 10 years after menopause it is associated with an increased risk of cardiovascular disease. Isoflavones, because of preferential binding to estrogen receptor beta, may retain the beneficial effects on bone but lessen the adverse effects on the breast. Objective: The objective of this study was to study the effects of an isoflavone obtained from red clover (Rimostil) on bone mineral density, and on low-density lipoprotein (LDL) cholesterol. Design: In a double-blind, randomized, placebo-controlled trial, 50 mg of Rimostil was given to women who were menopausal for at least 1 year. Bone mineral density of the spine, femoral neck and forearm and serum LDL cholesterol were measured at baseline and at 6-month intervals. The duration of follow-up was 2 years. Results: There was no beneficial effect of Rimostil on bone density at any site. There was a 12% fall in serum LDL cholesterol in the Rimostil-treated arm, which was significantly greater than the 2% drop seen in the control arm (P=0.005).
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Objective: Glioblastoma multiforme is the most malignant form of brain tumors. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. Here we have investigated the effects of T. pratense extract on glioblastoma multiforme cell line (U87MG). Materials and methods: In this experimental study, the effect of T. pratense extract on cell viability was investigated using trypan blue staining, MTT assay, and lactate dehydrogenase activity measurement. Apoptosis and autophagy cell death were detected by fluorescent staining. Nitric oxide (No) production was measured using Griess reaction. Expression levels of some apoptotic and autophagic-related genes were detected using real-time polymerase chain reaction (PCR). The combination effects of T. pratense extract and temozolomide (TMZ) were evaluated by calculating the combination index and dose reduction index values. Results: After treatment with T. pratense extract, the cell viability was significantly reduced in a time- and dosedependent manner (P<0.05). Apoptosis and autophagy of U87MG cells were significantly increased (P<0.05). Also, T. pratense extract significantly decreased NO production (P<0.05) by U87MG cells. Combination of TMZ and T. pratense extract had a synergistic cytotoxic effect. Conclusion: T. pratense showed anti-cancer properties via induction of apoptosis and autophagy cell death.
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Background: Isoflavone phytoestrogen therapy has been proposed as a natural alternative to hormone replacement therapy (HRT). HRT has a beneficial effect on bone, but few trials in humans have investigated the effects of isoflavones on bone. Objective: The objective of the study was to determine the effect on bone density of a red clover-derived isoflavone supplement that provided a daily dose of 26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein for 1 y. Effects on biochemical markers of bone turnover and body composition were also studied. Design: Women aged 49-65 y (n = 205) were enrolled in a double-blind, randomized, placebo-controlled trial; 177 completed the trial. Bone density, body composition, bone turnover markers, and diet were measured at baseline and after 12 mo. Results: Loss of lumbar spine bone mineral content and bone mineral density was significantly (P = 0.04 and P = 0.03, respectively) lower in the women taking the isoflavone supplement than in those taking the placebo. There were no significant treatment effects on hip bone mineral content or bone mineral density, markers of bone resorption, or body composition, but bone formation markers were significantly increased (P = 0.04 and P = 0.01 for bone-specific alkaline phosphatase and N-propeptide of collagen type I, respectively) in the intervention group compared with placebo in postmenopausal women. Interactions between treatment group and menopausal status with respect to changes in other outcomes were not significant. Conclusion: These data suggest that, through attenuation of bone loss, isoflavones have a potentially protective effect on the lumbar spine in women.
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The antiinflammatory and antioxidant activities of the ethanol extract of Trifolium resupinatum L. var. microcephalum (Leguminosae) have been assessed on arthritic rats. The extract (1.35 and 13.5 mg/kg, i.p.) significantly reduced the paw edema induced by the complete Freund's adjuvant. Furthermore, it also inhibited lipid peroxidation. These effects of T. resupinatum extract may be directly linked to the content in triterpene saponins and flavonoid compounds and consequently to their free radical scavenging activities.
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Papaya is indigenous to tropical Mexico, Central America and northern South America. Papaya has been distributed throughout the tropics and subtropics, where it is extensively cultivated, and as far north and south as 32°latitude.