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Ph ton
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The Journal of Ethnobiology and Traditional Medicine. Photon 126 (2016) 1197-1209
https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-ethnobiology-and-traditional-medicine
Review. ISJN: 6642-3194: Impact Index: 6.38
The Journal of Ethnobiology and Traditional Medicine Ph ton
Medicinal Uses of Formononetin- A review
Mr. Vaithiyalingam Jagannathan Vishnuvathan*
a
, Dr. Karunanidhi. Santhana Lakshmi
a
, Dr. Ammayappan
Rajam Srividya
b
a
Department of Pharmacy, SRM college of Pharmacy SRM University Kattankulathur campus Kanchipuram
603203 Tamilnadu India
b
Department of pharmaceutical biotechnology JSS college of Pharmacy, Rocklands Ooty Tamilnadu 643001
Article history:
Received: 14 April, 2016
Accepted: 19 April, 2016
Available online: 01 July, 2016
Keywords:
Formononetin, antimicrobial, antioxidant, Alzheimer’s disease,
antilipidemic, antidiabetic, antitumor
Abbreviations:
AP-1 DNA - AP-1 transcription factor- DNA binding site, HT-
29 human colorectal adenocarcinoma cell line, PPAR-α -
Peroxisome Proliferator—Activated Receptor, AST - Aspartate
aminotransferase, Bcl-2 and Bcl-x B-cell lymphoma-extra large
proteins, NAG-1 - Non-steroidal anti-inflammatory drug-
activated gene-1, ER-α – Estrogen receptor alpha, GPER - G
protein coupled estrogen receptor, PI3K/Akt serine/threonine
kinase Akt (also known as protein kinase B or PKB),
cAMP/PKA.CREB Cyclic adenosine 3',5'-monophosphate
(cAMP) cAMP-dependent protein kinase (PKA) element-
binding protein, CREB, LNCaP- human prostatic
adenocarcinoma cell line, MAPK to mitogen activated protein
kinase, NSCLC- non- small cell lung cancer, bFGF- basic
fibroblast growth factors, TGF-β- transforming growth factors-
β, PDGF- platelets derived growth factors. APOA5 gene region
on plasma HDL-C, apolipoprotein A-I (apoA-I) ACO - acyl-
CoA oxidase. CPT-1- Carnitine palmitoyltransferase I . Fas- an
important cell surface receptor protein of the TNF receptor
family known also as CD95, that induces apoptosis on binding
Fas ligand. (SREBF1) - Sterol regulatory element-binding
transcription factor 1
Corresponding Author:
Mr.Vishnuvathan V.J.*
Research Scholar
Email: Vishnuvj24 ( at ) gmail ( dot ) com
Dr. Lakshmi K.S.
Dean
Dr. Srividya A.R.
Assistant Professor
Abstract
Formononetin is a naturally occurring isoflavone, which
can be found in low concentrations in many dietary
products, which belongs to the family Fabaceae.
Chemical name of formononetin is Biochanin B; and its
IUPAC name is 7-Hydroxy-4'-methoxyisoflavone; 7-
hydroxy-3-(4- methoxyphenyl)-4H-chromen-4-one. Its
molecular formula is C
16
H
12
O
4
. Its molecular weight is
268.26408 g/mol. Formononetin has been proven to
show antimicrobial activity towards Giardia lamblia. It
acts as an antioxidant and as a neuroprotective agent in
Alzheimer’s disease. It also possess early fracture
healing potential, cardio protective activity, antilipidemic
properties, antidiabetic, antitumor and Neuroprotective
activity, It possess anticancer activity in lung cancer,
colorectal cancer and prostate cancer. Even though
formononetin shows different pharmacological actions,
for many studies, mechanism of action has not yet been
established. Especially in case of different anticancer
activity. Mechanism of action of formononetin in various
diseased conditions will help us to find the drug target
which in turn helps us to design the formulations to reach
the target effectively.
Citation:
Vishnuvathan V.J.*, Lakshmi K.S., Srividya A.R., 2016.
Medicinal Uses of Formononetin- A review. The Journal of
Ethnobiology and Traditional Medicine. Photon 126, 1197-1209
All Rights Reserved with Photon.
Photon Ignitor: ISJN
66423194D836001072016
1. Introduction
1.1 Source, properties and characteristic of
formononetin
One of the main components of red clover plants is
formononetin and it is considered as a
typicalphytoestrogen. In the roots of Astragalus
embranaceus, Trifolium pretense, Glycyrrhiza
glabra and Pueraria lobata formononetin occurs
naturally as isoflavones. Formononetin is one of
the predominant isoflavones in Trifolium pratense
L. The content of formononetin varies in different
parts, among which the high content is in the leaf
and the low content in the flower (Booth et al.,
2006). Legumes contain large quantity of
Biochanin A and formononetin. Human liver
microsomes could able to convert proestrogenic
isoflavones to more potent phytoestrogens
genistein through demethylation process and
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formononetin resulted in the production of
additional metabolites such as 6,7-dihydroxy-4‘-
methoxyisoflavone, 7,8-dihydroxy-4‘-
methoxyisoflavone, and 7,3‘- dihydroxy-4‘-
methoxyisoflavone a three new hydoxylated
formononetin. Due to its structural similarity to
17_-estradiol, it can mimic estradiol’s effect and
therefore is considered as a “phytoestrogen.”..
(www. Pubchem. Ncbi.nim.gov). Its molecular
formula is C16H12O4. Its molecular weight is
268.26408 g/mol. (www. Molbase.com). This
paper consolidated the pharmacological activities
of formononetin at various diseased conditions.
Formononetin is a naturally occurring isoflavone,
which can be found in low concentrations in many
dietary products,.
2. Pharmacological action of Formononetin
The commonly used herbs in China, Japan and the
western world are Astragalus membranaceus,
Glycyrrhiza glabra and Glycyrrhiza uralensis.
They contains a major isoflavonoid, called as
formononetin which has been demonstrated for
diverse pharmacological actions. (Vaya et al.,
1997). In the activated T- cells, formononetin up
regulates interleukin-4 production via increased
AP-1 DNA binding activity ( Park et al 2005). By
suppressing the arachidonic acid release in HT-29
human colon cancer cell lines, formononetin
produces anti inflammatory activity. (Jun et al
2005). Formononetin reduces the cardiovascular
risk by reducing blood pressure and central arterial
stiffness (Nestel et al., 2007). By activating PPAR-
α and PPAR-γ receptors Formononetin acts as a
potent antidiabetic agent in regulating the
metabolism of lipid (Huo et al., 2007). In an acetic
acid induced abdominal constriction in mouse
model formononetin demonstrated analgesic
activity (Dong et al., 2006) and it also induced
proliferation of osteoblast (Auyeung et al., 2010).
In human colon cancer cells formononetin induced
growth inhibition and promoted caspases-
dependent apoptosis similar to AST. Inhibition of
anti-apoptotic proteins such as Bcl-2 and Bcl-x and
activation of novel proapoptotic protein NAG-1
was produced by formononetin (Auyeung et al.,
2010).
Formononetin showed antimicrobial activity
towards Giardia lamblia. It also acted as an
antioxidant (Lauwaet, et al., 2010) and
neuroprotective agent in Alzheimer’s disease (Mu
et al., 2010). This isoflavonoid has also been
reported to have positive effect on osteoarthritis
(Huh et al., 2010) and promote early fracture
healing through stimulating angiogenesis (Huh et
al., 2009). In cardio vascular diseases
formononetin can be used as vaso relaxant (Wu et
al., 2010). In the prevention of myocardial
infarction formononetin can be used due to its
cardio protective activity (Zhang et al., 2011).
Formononetin also possesses hyperlipidemic
properties, mammary gland proliferation function,
and antitumor effect in colon and breast cancer
(Auyeung and Ko, 2010; Chen et al., 2011;
Kanadaswami, et al., 2005; Xing et al., 2010).
Estrogenic effect of formononetin has also been
reported. This isoflavone has structural similarity
with 17-estradiol; thus, it can bind to estrogen
receptors (Ha et al., 2010; Mu et al., 2009: Zhang,
et al., 2011).
3. Scavenging of free radicals by Formononetin
In aerobic organisms, reactive oxygen species such
as hydrogen peroxide and superoxide anions which
are produced as bye products has its own impact in
oxidative damage to various cellular macro
molecules. (Farber., 1994). In in-vivo, the
important defense systems in clearing up the
detrimental ROS are the antioxidant enzymes such
as super oxide dismutase (SOD), catalase (CAT)
and glutathione peroxidase (GSH). Even though all
organisms possess antioxidant defense and
repairing system which helps them to protect
themselves against the oxidative damage,
sometimes these enzymes may not be sufficient to
prevent the damage completely. In order to prevent
or reduce the damage produced by the free radicals,
it is very much important to evaluate the
antioxidant properties of specific chemical
scavengers. Because of the unsafe and toxicity,
several synthetic antioxidants such as butylated
hydroxyanisole (BHA) and butylated hydroxyl
toluene (BHT) are not used widely. Hence
considerable attention has been given towards the
identification of plant derived antioxidant from
natural sources which might be suitable for human
consumption. (Madhavi and Salunkhe., 1995).
Most of the antioxidant studies were carried out in
vitro. Formononetin which has been extracted from
Trifolium pratense L showed weak antioxidant
activity. (Yu et al., 2005; Corinna and Sabine.,
2006). Few in vivo studies have been carried out in
sheep for the antioxidant activity of formononetin.
In sheep, formononetin got metabolized to equol
and it has been reported that 81 % administered
radio labeled formononetin got excreted within 48
hours and most of which was found to be equol
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(Davies et al., 1989). It was also reported that
equol inhibits super oxide anion, formation of
Hydrogen peroxide and lipid peroxidation. (Hwang
et. al.,2000; Desiree et al.,1999)
3.1 Effect of antioxidant activities of formononetin
in ovariectomized adult female Kunming mice
Oxidative process that has been initiated by
superoxide anion would be counteracted by SOD
which is an intra cellular compound. In the
homogenized liver, heart as well as in serum,
formononetin significantly increased SOD
activities but not in kidney. Catalase activity was
found to increase in liver, heart, kidney and serum
but the results were not found to be significant.
Malonoaldehyde formation is the main indication
of lipid peroxidation. In the liver the production
level of malonoaldehyde was found to be
significantly inhibited and it was found to be
weakest in the kidney. It showed prominent effect
in the reduction of formononetin in serum.
Formononetin significantly increased the GSH-PX
activity in liver, heart and kidney homogenates as
well as in serum there by converting the toxic H
2
O
2
to H
2
O and O
2
. Free radicals could able to target
membranes and lipids (Rahaman., 2003).
By the abstraction of a hydrogen atom from a poly
unsaturated fatty acid chain, lipid peroxidation is
initiated. By means of inactivating the membrane
enzymes, receptors, depolymerization of
polysaccharides, cross linking of proteins and
fragmentations, lipid peroxidation produces cell
injury there by forming cytotoxic products such as
MDA which could be a measured by TBA method.
MDA is considered to be the biomarker for lipid
peroxidation. The antioxidant study which has been
carried out in mice showed successful inhibition of
lipid peroxidation that is reflected by the reduction
of MDA production. (Sarkar and Sommer., 1989).
4. Effect of formononetin in osteoporosis
Estrogen plays an important role in the prevention
of osteoporosis in women. Being an antiresorptive
agent, it reacts with estrogen receptors α and β with
osteoblasts and inhibits osteoclatogenesis and
prevent bone loss, when the estrogen level
decreases as in menopausal conditions, which
could lead to fractures (Ardakani and
Mirmohamadi., 2009; Khurana and Fitzpatrick.,
2009; Gallagher and Sai., 2010; Seeman., 2004).
Hormone replacement therapy is being used in
some patients to reduce the menopausal symptoms
as well as osteoporosis even though it produces
side effects such as breast cancer, abnormal uterus
bleeding and cardiovascular diseases (Bowring and
Francis., 2011; Rossouw, et al., 2002).
Phytoestrogens, a promising substance is an
alternative to overcome the adverse effects of
hormone therapy in women (Zhu et al., 2012;
Atkinson., 2004).
5. Effect of estrogen in bone
An intensified body mass gain, decreased uterus
mass and increased thymus mass is the
characteristic of estrogen deficiency in
ovariesectomized rats. Bone’s mechanical
properties were also found to deteriorate in
ovariesectomy rats. Ovariesectomized rats
produced all the symptoms which are similar to
those of post menopausal women. So
ovariesectomized rats are preferred for such types
of study ( Kalu., 1991) Raloxifene an estrogen
receptor modulators revealed estrogenic activity in
bone cells and it also inhibited osteoporosis
development in post menopausal women without
uterotropic and mammographic effect (Delmas et
al., 1997; Gautam et al., 2011, Cauley et al.,
2011, Johnell et al., 2002).
Formononetin at the dose of 1-10 mg/kg increased
trabecula area in tibia and lumbar vertebra when it
is injected intra peritoneally (Ha et al ., 2010).
Formononetin showed the improvement of bone
micro architecture which could be identified by
µCT method. It also increased the thickness of tibia
and femur bone but distance between them found
to decrease. Chemical composition of bones has
influence on its mechanical properties apart from
bone micro architecture. (Pru., 2012; Tyagi., 2012)
Most dangerous symptoms of osteoporosis are the
biomechanical features of bones such as
susceptibility to fractures (Ruppel., 2008). Bones in
ovariesectomized rats are less resistant to fractures
and more susceptible to deformations.
Bone chemistry such as increased water and
organic content, decreased mineral fractions
especially calcium occurs due to the lack of
estrogen (Kaczmarczyk-Sedlak et al., 2005;
Kaczmarczyk-Sedlak et al., 2009; Folwarczna et
al., 2009; Cegieła et al., 2012). Intraperitonial
administration of formononetin at the dose of 0.1,
1.0 and 10 mg/Kg did not affect the body mass
gain and uterus mass. No uterotropic effect was
observed ( Ha et al ., 2010; Gautam et al., 2013)
but at the dose of 50 to 500 mg/kg formononetin
produced uterotropic effect in ovariesectomized
mice(Mu et al., 2009).
Young’s modulus is a parameter which shows the
elasticity and internal rigidity of bones (Turner.,
2006). In formononetin treated ovariesectomized
rat s, femoral diaphysis was found to be higher
when compared to animals treated with estradiol.
Biomechanical parameters such as maximum
fracture load was found to decrease in control
animal group when compared to animals treated
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with estradiol., but formononetin did not affect
displacements maximum fracture loads.
Administration of formononetin improved the
biochemical features in femoral diaphysis and tibial
metaphysic and showed no beneficial effect on
mechanical properties in the femoral neck. These
results confirmed the lower affinity of
formononetin towards estrogenic receptors when
compared to that of estrogen.
In in-vivo study carried out by the administration of
formononetin showed the positive effect of bone
mineralization by using histo morphometric
method. In that study formononetin showed
significant increase in mineralizing surface of
femur, organic fraction content in femur, tibia and
L-4 vertebra in ovariesectomized rats. Both in
vitro and in vivo studies on osteoblast in rat fracture
model showed the higher expression of collagen
markers. Inhibition of bone turnover rate is the
main cause of osteoprotective effect of
formononetin on skeletal system in
ovariesectomized rats which could be observed by
decreased level of serum osteocalcin and urinary
Carboxy-terminal collagen crosslink (CTx) in
ovariesectomized rats. Due to its antioxidant
activity, formononetin might have revealed
beneficial effect on the bone tissue altered by
osteoporosis (Lean et al., 2003; Lee et al., 2005)
as well as in the improvement of bone micro
architecture (Muhammad et al., 2012).
Consumption of phytoestrogens is considered to be
the safe therapy. In spite of its advantages it might
produce some adverse effects from mild scarce to
the development of prostate cancer. For example
equol which is considered to be the most potent
phytoestrogens, when it is consumed in higher
doses it produces certain pathological changes in
uterine weight as well as mammotropic effects in
women (Racho´n et al., 2007; Racho´n et al.,
2008). Lignans which belongs to another class of
phytoestrogens develops prostate cancer in men
and colorectal cancer in women when it consumed
at higher doses (Ward et al ., 2010). All these
adverse reactions of phytoestrogens is due to its
lack of selectivity of action; i.e it can interacts with
other receptors also.
6. Neuro protection activity of formononetin
Paralyses, speech disorder, loss of consciousness
are the symptoms of stroke either due to ischemia
or hemorrhage. Worldwide stroke is considered to
be one of the reasons for death and disability. Even
though some intravascular techniques and
thrombolytic agents are used in these conditions
which may decrease the functional deficits, still
advances in the treatment for stroke related brain
damage is needed (Donnan et al., 2008). In animal
models, estrogens proved to have neuroprotective
effects in a variety of cerebral ischemia. (Lebesgue
et al., 2009; Toran-Allerand, 2004; Schreihofer.,v
2013). At the same time estrogen has its own side
effects of increasing the chances of developing
breast and endometrial cancers. In order to make a
substitute of estrogen, considerable attention was
focused on phytoestrogens which resemble like
estrogen (Ososki., 2003). Apart from the structural
similarity phytoestrogens have the ability to bind
estrogen receptors selectively (Zhao., 2002). In
recent years, many researches proved that
phytoestrogens could able to act against many
disease (Kim, 2008; Patisaul et al., 2010
) including
focal and global cerebral ischemia (Donzelli et al.,
2010; Liang et al., 2008: Lovekamp-Swan.,
2007)
Both in vitro and in vivo studies proved the
proliferation inhibition and pro-apoptotic effects in
cancer cells (Chen et al., 2013; Tian et al., 2011;
Chen and Sun, 2012; Chen et al., 2011;). By
binding with the classical estrogen receptors,
estradiol and phytoestrogens exerts neuroprotective
effects in cerebral focal and global ischemia. As a
typical phytoestrogens, formononetin proved to
increase the brain function and injured areas in the
cerebral (I/R rats). At the molecular level
formononetin showed increase in ER-α and p-Akt,
the same time it showed down regulation in the
ratio of Bax/Bcl-2. Formononetin also showed less
effect in p-ERK1/2 protein expression. Modulation
of the expression of genes involved in the control
of cell death and apoptosis which includes anti-
apoptotic bcl-2 group family proteins which is the
potential mechanism in the estrodiol induced neuro
protection. The usage of phytoestrogens which is a
natural alternative to estrogen has increased in
menopause women after the clinical studies which
proved the negative health consequences of
hormone therapy. In cardio vascular
neuroprotection, phytoestrogens have emerged as a
promising alternative. By interacting with estrogen
alpha (ERα) and estrogen receptor β (ERβ),
phytoestrogens regulates gene expression which
gets expressed in the brain (Shughrue et al., 1977).
In the nucleus of the cell as well as in cytoplasm
and membrane of the cells ERα and ERβ are
localized. They demonstrated to mediate their
action through nuclear receptor signaling as well as
by non- genomic extra nuclear signaling
pathways.(Raz et al., 2008; Levin., 2005). Because
of the activation of ER in the nucleus reduces pro-
death and apoptotic genes by increasing the pro-
survival and anti-apoptotic genes. To exert
neuroprotective effects, activation of extra nuclear
ER regulates the correlative kinases and post
translation ally modified proteins (Brann et al.,
2012). In mediating neuro protective action of
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estrogen and estrogen like compounds ER α plays
an important role in cerebral ischemia (Dubal et al.,
2001; Merchenthaler et al., 2003). Growth factors,
apoptotic and apoptotic mediators and the
expression of diverse genes that are involved in
neuro protection are regulated by estradiol because
ERR acts as a ligans dependent nuclear
transcription factors.
The membrane associated ER GPER ( G protein
coupled estrogen receptor) as well as several
signaling pathways includes PI3K/Akt,
MAPK/SRC, cAMP/PKA.CREB and other growth
factors are the extensive range of target where
estradiol could target which in turn regulates the
activation or inactivation of cell survival and death.
Neuronal cell survival or cell death is linked with
P13K/Akt and ERK1/2 which has been proved
through research in the field of stroke. The activity
of Akt by phosphorylation has been increased by
the receptor tyrosine kinase which in turn activated
by growth factors. Akt is known to block apoptosis
via the phosphorylation of multiple down
streaming signaling molecules as well as in
activation of Bcl-2 family member proteins and
inhibition of cell death pathway enzyme caspases-9
( Franke et al., 1997; Datta et al., 1997; Cardone et
al., 1998). Formononetin reduced the neuronal
damage primarily via Akt pathway rather than ERK
pathway by inducing phosphorylation of Akt which
has been confirmed by in vivo studies.
7. Anticancer activity of formononetin
7.1 Action of formononetin in prostate cancer
In men prostate cancer is the second leading cause
of cancer related death after the lung cancer in
western countries, which has been commonly
diagnosed as malignant cancer (kiJemal et al.,
2007). In Asian countries due to changes in diet
and life style the incidence rate of prostate cancer
has increased in last few years (Center et al., 2012).
Phytoestrogens could able to inhibit the growth of
both androgen dependent and independent prostate
cancer cells which has been identified by in vitro
studies (Zhao et al., 2009).
Formononetin caused apoptosis in human prostate
cancer cells LNCaP by inactivating ERK1/2
mitogen activated protein kinas (Ye et al., 2012).
Mitogen activated protein kinase such as ERK1/2,
Jun No-terminal stress activated protein kinase ½,
JNK ½ and p 38 MAPK plays an important role in
cell proliferation and apoptosis (Fan et al., 2007).
Formononetin inhibited the proliferation of DU-
145 cells dramatically and also could able to
induce early apoptosis and its action was found to
be dose dependent.
In the status of prostate cancer it will be a complex
to know the status of sex hormones (Smith et al.,
2008). Both estrogen and its receptor (ER) play an
important role (Hara et al., 2008) in prostrate
carcinogenesis because proliferation is stimulated
by estrogen and it is inhibited by phytoestrogens
which appeared to be related to estrogen receptors.
After estrogen binds to the estrogen receptor,
transcriptional activation following ligans receptor
binding could be supplemented by the cytoplasmic
signaling pathway (Driggers et al., 2002). MAPK
pathway gets activated by the membrane associated
receptor tyrosine kinase which in turn activates Ras
followed by the activation of protein kinase Raf
which then mediate signal transduction to mitogen
activated protein kinase (MAPK) and downstream
ERK pathway and p38 pathway ( Di et al., 2008;
Lau et al., 2009; Bapat et al., 2001).
7.2 Action of formononetin in breast cancer
Via regulating steroid receptor (AR and ER) and
their expression (Fritz et al., 2002; Shapiro et
al.,1999) inhibition of proliferation of cells and
DNA topoisomerase II (Constantinou et al., 1995).
Formononetin promoted apoptosis in cancer cell.
Formononetin induced cell cycle arrest at the
G0/G1 phase by inactivating IGF1/P13K/ AKT
pathways in both in-vitro and in- vivo studies in
human breast cancer (Chen et al., 2012)
In breast cancer carcinogenesis, formononetin
induced the apoptosis by means of activating
Ras/p38 mitogen activated protein kinase ( Chen et
al., 2012). In human prostate cancer cells
formononetin induced apoptosis in dose dependent
manner, through ERK/2 mitogen activated protein
kinase. All these results suggested that inhibitory
effect of formononetin on proliferation of human
prostate cancer cells through to Ras-Raf- MAPK
pathway.
In ArT-20 pituitary cell line, dexamethasone was
found to be an inducible agent for the activator of
G- protein signaling which belongs to the RAS
super family of small GTPases (Kemppainen and
Behrend et al., 1998). In normal human tissues
including brain, heart, liver, kidney and bone
marrow RASD I which is otherwise called as
AGS1, m RNA and protein is generally expressed
but in various human cancer cell line such as HL-
60, K-562, SW-480, A549, G-361 and MCF-7 it is
not detected (Kemppainen et al., 2003)
RASD1 regulates the various aspects of cell
growth, differentiation and mutations in the
prevention of aberrant cell growth in different types
of tumors. Transfection with RASD1 in NIH- 3T3
murine fibroblast cells, MCF-7 human breast
cancer cell line and A 549 diminishes the number
of G418 resistant colonies in clonogenic studies.
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Cells infected with adenovirus (engineered cells)
inhibited log phase growth but increased the
percentage cells undergoing apoptosis in in- vitro
studies carried out in A549 cells. RASD1
expression is inhibited in in-vivo studies conducted
in nude mice model. Injection of AGS1/RASD1-
expressing cells (Ad AGS1), the animals remained
tumor free even after 60 days, proved that Ad
AGS1 completely prevented the formation of
tumors (Vaidyanathan et al., 2004).
Anti apoptotic members such as Bcl-2 , Bcl-xl and
pro-apoptotic members such as Bax, Bad and Bak
belongs to Bcl-2 family proteins.. By getting
translocation from the cytosol to the mitochondria,
Bax protein exerts pro-apoptotic activity where it
induces cytochrome c release ((Li et al., 2011).
Weidinger et al., 2010 suggested that death signals
might have induced a conformational change in
Bax, leading to its mitochondrial translocation,
oligomerization or cluster formation and
cytochrome c release. It was also noticed that after
the treatment with formononetin, there was an
increase in the activated Bax based on dose, while
the expression level of Bcl-2protein was found to
decrease.
7.3 Effect of formononetin in Lung cancer
In worldwide, lung cancer is the leading cause of
cancer- related death. Among lung cancer, 80 %
are non- small cell lung cancer (NSCLC) which is
found to have poor therapeutic efficacy (Yi Yang
et al., 2014 ; Jun et al., 2005). Due to low survival
rate (5 years), even though there are significant
developments in clinical treatment, its prognosis is
still unsatisfactory. Better therapies are being
obtained from many traditional Chinese medicines
to treat human NSCLC (Auyeung and Ko, 2010;
Kondo et al., 2010).
In activated T- cells, formononetin exhibits a
metabolic effect by up regulating interleukin-4, by
increasing AP-1 DNA binding activity
(Sukhthankar et al., 2008). By inhibiting
arachidonic acid release in HT-29 human colon
cancer cells, anti-inflammatory action of
formononetin has been demonstrated (Carusi ,
2000). Studies that has been carried by various
researchers also proved that anticancer activity of
formononetin against breast cancer (Harris, 2003),
prostate cancer (Fukumura et al., 1998) and
cervical cancer (Hanahan et al., 1996).
To determine the effect of formononetin on
NSCLC cells proliferation, MTT assay was
performed and it was found that the proliferation of
A549 and NCI-H23 cells was found to be inhibited
by formononetin and the results were found to be
dose dependent. These effects were not seen in 16
HBE-T cells. During the cell cycle analysis of
A549 and NCI- H23, cells treated with
formononetin with different concentration, A549
and NCI-H 23 cells showed the increased
proportion of cells in G-1 phase and decreased
number of cells in S phase.
Cell cycle distribution was analyzed by flow
cytometry with the cells line A 549 and NCI –H 23
after treating with different concentration of
formononetin for about a period of 24 hours.
Formononetin promoted the apoptosis in A549 and
NCI-H 23 cells in the dose dependent manner.
Formononetin promoted the apoptosis in NSCLC
cells by inducing G1-phase cell cycle. Expression
of p 21 protein was found to increase with the dose
of formononetin and cell cycle regulatory proteins
such as Cyclin D and cyclin A expression was
found to decrease in G1 phase in a dose dependent
manner. (Yi Yang et al., 2014).
8. Antidiabetic and hyperlipidemic activity of
formononetin
In vivo studies which has been carried out by
Longxin Qiu et al., 2012 demonstrated the
activation of PPAR-γ regulated genes in the liver
which improves the insulin sensitivity that is
responsible for maintaining the glucose level in the
blood. By direct control over the transcriptional
gens involved in the peroxisomal and
mitochondrial β-oxidation pathways. fatty acid
uptake and TG metabolism, PPAR α, an important
metabolic nuclear receptor regulates the lipid
metabolism. Formononetin did not altered the
expression of mRNA of APOA5, ACO and CPT-1
In denovo fatty acid synthesis and in the long term
regulations of lipogenesis. FAS is multi functional
protein plays a central role.. SREBP-1C is the
binding site for the transcription factor in the
promoter region of FAS protein. treatment of
diabetic db/db mice with formononetin down
regulated the m RNA expression of FAS. To act as
a transcription factor, SREBP has to be cleaved by
proteolytic process by site-1 (S1P) and site-2
(S2P). In hepatocytes and other cells proteolytic
release of SREBP stimulates the lipid synthesis.
Agonist activity of formononetin with PPARα/γ
demonstrated the antidiabeti and hypolipidemic
activity type 2 diabetic animals.
Discussion
The capacity of augmenting the activity of SOD
and CAT is the main factor that is present in
antioxidant therapy (Cheeseman and Scater.,1993).
One of the principle causes of toxicity of CCl4 is
the lipid per oxidative degradation of the bio
membrane. (Kaplowitzetal.,1986), which is
evidenced by the elevation of TBARS and
reduction of antioxidant enzyme levels, which
plays an important role in the body defense
Ph ton
1203
mechanism against the harmful effects of the
reactive oxygen species.
Antioxidant Studies which has been carried out by
using formononetin, a phytoestrogens, main active
ingredients of red clover plant, traditional Chinese
medicinal herb, exhibited a strong antioxidant
activity when it is administered in mice.
Administered formononetin prevented the
formation of lipid peroxidation by means of
enhancing the enzymatic and non- enzymatic
antioxidant defense mechanism.
Formononetin improved the neurological deflect by
significantly reducing the infracted volume and
brain water content. Estrogen receptor activation,
up regulation of p- Akt and by down regulation in
Bax/Bcl-2 ration are the main mechanism by which
formononetin exhibits nerve cell proliferation.
In the human colon cancer cells, formononetin
exerts anti – angiogenic effect and the protein
expression of bFGF, VEGF and MMPs were also
assessed. In the HCT-116 cells treated with
formononetin, down regulated both the gene and
protein expression of VEGF. Formononetin found
to inhibit the protein expression of MMP-2 and
MMP-9. Down regulation of pro- angiogenic
factors occurred in a time dependent manner by
formononetin in human colon cancer cells. In
human colon cancer cell lines formononetin has
proved to have strong pro- apoptotic activity (Jun
et al., 2005). On the cell cycle, formononetin does
not appear to exert any direct effect which suggests
that pro- apoptotic effect of formononetin may not
directly correlates with the modulation of mitotic
inhibition (Auyeung and Ko., 2010).
Increased expression of VEGF has been shown to
have a strong correlation with tumor development
(Kondo et al., 2000, Sukhthankar et al., 2008). In
the tumor micro environment, cancer cells and
tumor stroma cells are involved in the secretion of
VEGF (Carusi., 2000). In the process of
modulating angiogenesis which is an important
step in cancer growth and metastasis, formononetin
found to down regulated both the gene and protein
expression of VEGF (Harris., 2000). Tumor mass
are triggered by the angiogenic growth factors such
as VEGF, basic fibroblast growth factors (bFGF),
transforming growth factors- β (TGF-β) and
platelets derived growth factors ( PDGF)
(Fukumura, et al., 1998; Hanahan et al., 1996;
Hanahan and Folkman, 1996). The transition from
the a vasculature state into a highly angiogenic
state (angiogenic switch) is due to the imbalance
between pro- angiogenic and anti- angiogenic
signals released by tumor cells and host cells
(Harris., 2003). Tumor becomes vascularized
when pro- angiogenic signal dominates. In
colorectal cancer progression, non vascularization
in tumor plays a critical role. Increase in
angiogenesis in the primary tumor correlates with
poor prognosis and relapse of the disease
(Takebayashi., 1996; Vermeulen et al., 1999)
In colon cancer, the tumor cells can reach the
lymphatic channels or blood vessels by invading
through muscularis mucosa and then gets migrate
into the sub mucosa which is an essential process
for the metastasis to occur .(Portera et al., 1998).
Due to the secretion of basement membrane
degrading enzymes such as MMP’s which can
degrade the extra cellular matrix and basement
membrane, as well as increase in the cell motility,
invasion may increase in colon cancer. But studies
showed that formononetin reduced the expression
of MMP-2 and MMP-9 which have been proposed
to serve as a marker for colorectal cancer
invasiveness, there by tumor cell invasiveness got
suppressed (Gutman M and Fidler., 1995; Radinsky
R and Ellis., 1996). In the suppression of
tumorigenicity, a novel antisense- based inhibitor
of MMP-9 which is also known as gelatinase B has
been found to be capable of attenuating
angiogenesis as well as human prostate cancer cell
division (Devi., 2003). The role of MMP-2 which
is localized at the specific surface protrusions on
the cell membrane founds to promotes invasion,
and MMP-9 helps in tumor cell migration,
invasion, angiogenesis and tumor growth
(Coussens et al., 2002; . Fridman et al., 2003).
The expression of MMP-9 in tumor epithelial cells
is correlated positively with colorectal cancer
progression because of its control over essential
functions that are mediating metastasis (Zuzga et
al., 2008). Many studies indicated that extra
cellular degradation and tumor cell seeding could
be promoted by MMP-9 which is secreted by colon
cancer cells (Lubbe et al., 2006).
The cleavage of specific factors such as collage IV
in the extra cellular matrix by the MMP-9, is the
mainly important for tumor inventions and
neovascularization. (Curran and Murray., 1999).
For controlling the progression of colorectal
cancer, targeting MMp-9 in tumor epithelial cells is
regarding as a key therapeutic strategy (Lubbe et
al., 2009). Only in the invasive tumors expression
of MMP-9 will be mainly found but not in normal
human tissues. Inhibition of MMP-9 expression
leads to ERK- mediated p16 expression resulted in
cell cycle arrest, at the same time inducing
apoptosis. (Rao et al., 2007; Bhoopathi et al.,
2008). Various studies suggested that the impact of
MMP-9 inhibition is not only restricted to the
suppression of tumor angiogenesis and
invasiveness but also helps in the promotion of
apoptosis.
Ph ton
1204
Conclusion
From this review concluded various
pharmacological actions of formononetin. but in
most of the studies only the pharmacological uses
were proved. in majority of the studies mechanism
of action of formononetin at the molecular level
has not yet proved and identified. Anticancer
activity of formononetin concluded that
formononetin is capable of inducing tumor growth
inhibitions as well as anti angiogenic and anti-
invasive effect with minimal drug reactions.
Formononetin and their derivatives could be further
developed as a potential chemotherapeutic/
adjuvant agent in the treatment of various cancers
and as well as in various ailments and their
mechanism of action could also be explored. unless
and until we know the target of formononetin at
various diseased conditions it would be very
difficult to design the formulations for the better
therapy. Our future work would be depended on
the identification of mechanism of action of
formononetin in various diseased conditions.
Author’s Contribution and Competing Interests
As a Ph.D scholar I am formulating a formulation
of formononetin which could be easily absorbed by
the cells especially cancer cells. Before going to the
formulations it is better to understand the medicinal
uses of formononetin so that formulations could be
done in a better manner as per the requirement of
treatment for various diseased conditions. So
various literatures were collected by Dr. K.S.
lakshmi and Dr. A.R. Srividya consolidated as a
review. It is a team work which would be useful for
making different formulations.
Conflict of interest
Authors hereby declare there is no conflict of
interest.
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