International Journal of

Molecular Sciences

Review
Utilization of Isoflavones in Soybeans for Women with
Menopausal Syndrome: An Overview
Li-Ru Chen 1,2 and Kuo-Hu Chen 3,4, *

1 Department of Physical Medicine and Rehabilitation, Mackay Memorial Hospital, Taipei 10049, Taiwan;
[email protected]
2 Department of Mechanical Engineering, National YangMing ChiaoTung University, Hsinchu 30010, Taiwan
3 Department of Obstetrics and Gynecology, Taipei Tzu-Chi Hospital, The Buddhist Tzu-Chi Medical
Foundation, Taipei 231, Taiwan
4 School of Medicine, Tzu-Chi University, Hualien 970, Taiwan
* Correspondence: [email protected]; Tel.: +886-2-66289779

Abstract: Based on their nutrient composition, soybeans and related foods have been considered to
be nutritious and healthy for humans. Particularly, the biological activity and subsequent benefits of
soy products may be associated with the presence of isoflavone in soybeans. As an alternative treat-
ment for menopause-related symptoms, isoflavone has gained much popularity for postmenopausal
women who have concerns related to undergoing hormone replacement therapy. However, current
research has still not reached a consensus on the effects of isoflavone on humans. This overview is a
summary of the current literature about the processing of soybeans and isoflavone types (daidzein,
genistein, and S-equol) and supplements and their extraction and analysis as well as information



about the utilization of isoflavones in soybeans. The processes of preparation (cleaning, drying,


crushing and dehulling) and extraction of soybeans are implemented to produce refined soy oil, soy
Citation: Chen, L.-R.; Chen, K.-H.
lecithin, free fatty acids, glycerol and soybean meal. The remaining components consist of inorganic
Utilization of Isoflavones in Soybeans
constituents (minerals) and the minor components of biologically interesting small molecules. Re-
for Women with Menopausal
garding the preventive effects on diseases or cancers, a higher intake of isoflavones is associated
Syndrome: An Overview. Int. J. Mol.
Sci. 2021, 22, 3212. https://doi.org/
with a moderately lower risk of developing coronary heart disease. It may also reduce the risks
10.3390/ijms22063212 of breast and colorectal cancer as well as the incidence of breast cancer recurrence. Consumption
of isoflavones or soy foods is associated with reduced risks of endometrial and bladder cancer.
Academic Editors: Regarding the therapeutic effects on menopausal syndrome or other diseases, isoflavones have been
Luis Cisneros-Zevallos and found to alleviate vasomotor syndromes even after considering placebo effects, reduce bone loss
Marcello Iriti in the spine and ameliorate hypertension and in vitro glycemic control. They may also alleviate
depressive symptoms during pregnancy. On the other hand, isoflavones have not shown definitive
Received: 10 February 2021 effects regarding improving cognition and urogenital symptoms. Because of lacking standardization
Accepted: 20 March 2021
in the study designs, such as the ingredients and doses of isoflavones and the durations and outcomes
Published: 22 March 2021
of trials, it currently remains difficult to draw overall conclusions for all aspects of isoflavones. These
limitations warrant further investigations of isoflavone use for women’s health.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Keywords: menopause; soybeans; isoflavones
published maps and institutional affil-
iations.

1. Introduction

Copyright: © 2021 by the authors.

As a food crop, soybeans have been used in Eastern countries for over 5000 years and
Licensee MDPI, Basel, Switzerland.
serve as an important source of proteins. In recent centuries, soybeans were cultivated
This article is an open access article
and processed for export and became popular in Europe and the United States [1–3]. They
distributed under the terms and grow best in tropical, subtropical and temperate regions. Furthermore, they can be grown
conditions of the Creative Commons throughout the year in the tropics and subtropics if water supply is adequate [1–3]. The
Attribution (CC BY) license (https:// growing time of soybeans ranges from 50 to 200 days, depending on the species, latitude
creativecommons.org/licenses/by/ and weather. After soybeans are harvested, threshing consisting of removing the beans
4.0/). from the pods must be done carefully to prevent breakage of the hulls and beans, which

Int. J. Mol. Sci. 2021, 22, 3212. https://doi.org/10.3390/ijms22063212 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 3212 2 of 23

can impair the quality [1–3]. There are a lot of nonfermented soy foods including soymilk,
tofu, soya sprouts, soya film, vegetable or roasted soybeans, and fermented oriental soya
foods such as miso, tempeh, soya sauce and natto [1–4].
Soybeans have an exceptional nutritional and functional food profile. Because soy-
beans contain practically no starch, they are an important part of a diabetic diet. Soybeans
are rich in very high levels of protein, carbohydrate conjugates, fatty acids, soybean oil and
amino acids. In addition, soybeans contain several biologically interesting phytochemicals
as minor components, consisting of organic and inorganic constituents (minerals). All of
the components in soybeans have health impacts on humans, and they can be extracted
with different techniques [5,6]. Based on their nutrient composition, soybeans and relative
foods have been considered to be nutritious and healthy for humans [1]. There is increas-
ing evidence reporting that consumption of soy foods can significantly affect health [7].
Phytoestrogen-rich soybeans, which are common in Asian diets, are related to a lower
incidence of menopausal symptoms, osteoporosis, cardiovascular diseases and hormone-
dependent cancers [1]. In addition, there is a significant connection between increased
soybean consumption and a decreased cancer risk [4]. Furthermore, a report confirmed that
soy proteins could lower blood cholesterol concentrations [4]. Particularly, the biological
activity and subsequent benefits of soy products may be associated with the presence of
isoflavones in soybeans [3,7]. The US Food and Drug Administration (FDA) approved soy
products as an official cholesterol-lowering food along with other heart and health benefits
due to the evidence that soy product intake was correlated with significant decreases in
serum cholesterol, low-density lipoprotein (LDL) and triglycerides. Nevertheless, it seems
to be somewhat difficult to support soy product benefits by in vitro studies using individu-
ally isolated phytochemicals (a wide variety of compounds produced by plants) or crude
products prepared from soybeans. It is important to note that all phytochemicals isolated
from soybeans show an array of weak biological activities; thus, normal consumption of
foods that contain these phytochemicals may not provide sufficient amounts to elicit a
visible physiological response in humans in short-time clinical research.
Menopause is an aging process that can carry troublesome symptoms including hot
flush, sweating and mood changes, and it can also elevate the risk of mortality because of
osteoporosis, fractures and decreased physical activity. The most effective way to alleviating
menopausal syndrome is hormone replacement therapy (HRT). Nonetheless, a study by
the Women’s Health Initiative clearly demonstrated that in postmenopausal women, HRT
increased the risks for coronary heart disease, stroke and breast cancer [8]. Since that time,
postmenopausal women and healthcare workers have been searching for an alternative
therapy. A Canadian study has shown that many postmenopausal women consider the use
of complementary or alternative medicine (CAM) for alleviating menopausal syndrome
yet have concerns regarding its cost and efficacy [9]. Most women using CAM chose not to
inform their physicians because they felt physicians were not good enough for judging the
effects of CAM [10].
In postmenopausal women, vasomotor symptoms (VMS) including hot flush, sweating
and palpitations are quite common [11]. Approved by the U.S. Food and Drug Administra-
tion (FDA), estrogen-based therapy remains the most effective for reducing VMS [12]. The
indications of HRT include relieving VMS, treating menopause-associated atrophic vulvo-
vaginitis and preventing osteoporosis. Furthermore, HRT elevates the mineral densities of
bones and lowers the occurrence of fractures due to osteoporosis [13]. In consideration of the
aforementioned harmful effects of HRT, it is recommended by the FDA to use the approved
non-estrogen treatments carefully before using HRT solely for osteoporosis prevention [14].
Recently, nutraceuticals including phytoestrogens and herbal derivatives have drawn
public attention because of their possible ability to alleviate menopausal symptoms [15].
For example, Actaea racemosa alleviates menopausal symptoms including hot flush, sweat-
ing, insomnia, irritability and musculoskeletal pain; Valerian officinal is effective for hot
flush, anxiety and sleep disorders; Panax ginseng appears effective on treating depression,
insomnia and improving sexual function; Ginkgo biloba can be used to improve atten-
Int. J. Mol. Sci. 2021, 22, 3212 3 of 23

tion disorders of postmenopausal women [15]. Unlike phytoestrogens, herbal derivatives

such as Actea racemosa, Valerian officinal and Ginkgo biloba act via different estrogen-
independent pathways to ameliorate menopausal symptoms.
Epidemiological research has explored a connection between the consumption of
soybeans and reduced VMS [16]. The intake of soybeans has been estimated to be far
greater in Asian countries including China, Japan, Taiwan and Korea than in Western
countries. Relevantly, postmenopausal Asian women have significantly lower incidences
(10–25%) of hot flush in comparison with those in Western countries (60–90%) [17].
Isoflavones can be extracted from soybeans. They can exert estrogen-like actions and
thus alleviate postmenopausal syndrome. Two types of estrogen receptors include ERα,
which is distributed predominantly in the uterus and breast, and ERβ, which is distributed
predominantly in bone, the urogenital system and the cardiovascular system [10]. Gener-
ally, the binding affinity of isoflavones is higher to ERβ than to ERα. Using in vitro human
cell culture for estimating the estrogenic effects of isoflavones, research has revealed the po-
tencies as follows: estradiol (E2) 100, daidzein 0.013, genistein 0.084, and S-equol 0.061 [18].
Nevertheless, isoflavones may reach 10,000 times the E2 concentration and have significant
stimulation due to their abundance [19]. An example showed the effect of isoflavones on
female mice that were aromatase-knockout and estrogen-deficient. Isoflavone can act as
estrogen and improve the ovarian morphology [20].
A variety of studies have reported the different effects of isoflavones and not reached
an overall consensus yet. The possible explanations are that related trials vary in study
designs and in the ingredients and doses of isoflavones and that trials are confined to
small sample sizes and high rates of dropout [21]. Moreover, there is a high variability
in age and onset of menopause for the recruited women. On the basis of prior studies
discussing processing of soybeans, the nature of isoflavones and their mechanisms of
action, the overview presents a summary of the current literature about processing of
soybeans, isoflavone types (daidzein, genistein, and S-equol), supplements, extraction and
analysis as well as information about the utilization of isoflavones in soybeans.
In this review, all of the reference articles were retrieved from the databases Med-
line and PubMed using the search term “isoflavone” for the topic. For further screening
and selection, only full-text articles were considered to be included for a further anal-
ysis. A literature search was conducted on the Medline and PubMed databases up to
31 December 2020 to identify all potential articles in the data sources. In the screening
process, duplicate articles and research before 1980 were excluded. After initial processing,
two experts in the field independently reviewed potentially eligible studies for exclusion
and inclusion by inspecting the sources including year, authors, research design and clinical
outcomes. Articles with poor study design or unmatched outcomes were deemed not
eligible for the study. During the period of study selection, disagreements between the two
reviewers were resolved by mutual discussion until a consensus was reached. Using the
search term and searching strategy (database identification, article screening, consideration
of eligibility and final inclusion), all eligible articles were selected for the topic.

2. Processing of Isoflavones in Soybeans

2.1. Processing of Soybeans
Processing of soybeans refers to continuous steps to prepare soy products for use in
aspects of food, industry and animal feed. Advanced processing can produce refined soy
oil, glycerol, free fatty acids, lecithins (soy gums) and protein-rich soymeal containing soy
protein concentrates and isolated soy proteins [1–3,22]. In addition, soybeans also contain a
wide range of micronutrients and phytochemicals including minerals, vitamins, sterols and
isoflavones. Isoflavones can be also extracted in the process for further use. The processing,
preparation and extraction of soybeans are showed in Figure 1.
 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 24

Int. J. Mol. Sci. 2021, 22, 3212 4 of 23

sterols and isoflavones. Isoflavones can be also extracted in the process for further use.
The processing, preparation and extraction of soybeans are showed in Figure 1.

Figure 1. The processing, components and functions of soybeans.

Figure 1. The processing, components and functions of soybeans.

Beforefurther
Before furtherprocesses
processesareareimplemented,
implemented,the thesoybeans
soybeansneedneedtotobebecleaned,
cleaned,dried
dried
(heated), crushed and then dehulled. The initial processing methods
(heated), crushed and then dehulled. The initial processing methods are as follows [1–3,22]: are as follows
[1–3,22]:
(1) Cleaning: After a threshing operation, plant waste, small gravel, soil, insects, weed
seeds (1)
andCleaning: After aseeds
broken soybean threshing operation, plant
may contaminate waste,The
soybeans. small gravel,procedure
cleaning soil, insects,
is
weed seeds
performed and broken
several times insoybean seeds may
the post-harvest contaminate soybeans. The cleaning proce-
system.
dure(2)is Drying:
performed Theseveral timesinvolves
operation in the post-harvest
drying the system.
soybeans with a cycle of natural or
(2) drying
artificial Drying:until
Thetheoperation involvesisdrying
“safe-moisture” reached.the soybeans with a cycle of natural or
artificial drying until
(3) Crushing: Thethe “safe-moisture”
process consists ofisbreaking
reached. the whole soybeans into pieces to
(3) Crushing:
facilitate dehulling. The process consists of breaking the whole soybeans into pieces to fa-
cilitate dehulling. The process is to remove the outer covering from soybeans or other seeds.
(4) Dehulling:
As
(4)aDehulling:
major source The ofprocess
dietary is
fiber, the hullsthe
to remove (the tough
outer exteriorfrom
covering skinsoybeans
of soybeans) are
or other
processed
seeds. to produce a fiber additive for breads, cereals and snacks. Livestock feed can
also beAsmanufactured
a major source from soybeanfiber,
of dietary hullsthe
[1–3,22].
hulls (the tough exterior skin of soybeans) are
Even though there are continuous advancements
processed to produce a fiber additive for breads, cereals and and
refinements in the technology
snacks. Livestock feed can
ofalso
soybean processing, currently
be manufactured from soybeanthe main
hullsmethod
[1–3,22].used is referred to as the “extraction or
solvent”
Even process.
thoughUsing
thereorganic solventsadvancements
are continuous such as hexane, therefinements
and method recovers
in the 95–98%
technologyof
the
of soy oil and
soybean 95% of thecurrently
processing, soy proteinthe[22].
main Typically,
method aused bushel of soybean
is referred grains
to as (60 pounds)or
the “extraction
can yield about 11 pounds (18.3%) of soy oil and 48 pounds (80%) of protein-rich meal. As
for the rest, hulls make up the remaining 1.7% [22].
Int. J. Mol. Sci. 2021, 22, 3212 5 of 23

After extraction, crude soy oil can be obtained, but it has to be further processed to
produce a refined oil. The additional processing can remove substances which compromise
the quality of the soy oil. Another product of soy oil is soybean lecithin, which mainly
consists of phospholipids and acts as an important natural emulsifying agent for food and
pharmaceuticals [2,3,22]. Extracted from crude soy oil to avoid cloudiness and discoloration
of the refined oil, lecithin can also be used as animal feed and in paints, lubricants and
other industrial applications [1–3,22]. Glycerol and free fatty acids are formed as a result
of soy oil breakdown. Free fatty acids are also used as emulsifying agents. Glycerol is
collected from the oil under a deodorization process. As important nutritional substances,
further products including sterols, sterol esters and the antioxidant tocopherol (vitamin E)
are extracted from the oil as well under a deodorization process [1–3,22].
In addition, protein-rich soybean meal can be manufactured during the process, and
it has been recognized as one source of proteins available for humans and livestock [2,3,22].
Soybean proteins are equivalent to animal proteins in quality, highly digestible and com-
parable to beef, fish, milk and egg proteins in terms of protein quality [22]. The detailed
process of isoflavone extraction from soybeans is described in Section 2.3.

2.2. Isoflavones (Daidzin, Genistin, Daidzein, Genistein) and S-Equols

Isoflavones are composed of two benzene rings linked through a heterocyclic pyrane
C-ring at the 3 position, which distinguishes them from flavones. Being malonyl-glucoside
conjugates in chemical structures, the primary isoflavones in soybeans belong to the
daidzein, genistein and glycitein families [3,4,7]. Each family comprises its respective
aglycone, β-glucoside, malonyl-glucoside and acetyl-glucoside [3,4,7]. Malonyl-glucoside
is the predominant form of many isoflavones in unprocessed soybeans. Research has
revealed that malonyl-glucoside is heat sensitive and easily converted to its corresponding
acetyl-glucoside and/or β-glucoside according to the thermal conditions of preparation
and processing [7]. Similarly, a study has indicated that soy flour that had not been
heat-treated mainly contained malonyl-β-glucoside conjugates; by contrast, heated soy
flour consisted of large amounts of acetyl-β-glucoside conjugates, formed by means of
heat-induced decarboxylation of the malonate group to acetate [4]. Analyzed using high-
performance liquid chromatography (HPLC)-mass spectrometry, isolated soy proteins and
textured vegetable proteins were composed of a mixture of all three types of isoflavone
conjugates. Frying or baking of textured vegetable proteins at 190 ◦ C and baking of soy
flour did not change the total content of isoflavones, but there was a stable increase in
β-glucoside conjugates at the expense of malonyl-β-glucoside conjugates [4]. Therefore,
the chemical structures of isoflavones in foods should be considered when evaluating the
availability of isoflavones for absorption from the diet [4]. Different processing conditions
produce soybean products with a variety of isoflavone composition and content. Recently,
research has shown that the chemical structures and abundance of isoflavones in soybean
foods have an important impact on their biological effects and bioavailability [3,7].
A study has assessed isoflavone concentrations of various soybean cultivars with
maturity grade 0 to 5 grown under different environments and analyzed the effects of
other important seed characters [23]. Analyzed using HPLC-mass spectrometry, the com-
ponents of isoflavones had abundant genetic variations in soybean seeds. Furthermore,
individual and total concentrations of isoflavones in soybeans were significantly influ-
enced by cultivar, site, year and maturity grading [23]. A trend existed in the content of
isoflavones, in which isoflavone concentration was lower in earlier than in later matur-
ing soybean cultivars. Nevertheless, a genetic factor still plays the most critical role for
isoflavone concentration [23].
Although the terms phytoestrogen and isoflavone are frequently used interchange-
ably in the literature, they are not exactly the same. More precisely, phytoestrogen is a
plant compound that can exert an estrogen-like effect, and isoflavones, including daidzin
and genistin, belong to bioflavonoids that are available in animals and plants [24]. Some
foods such as soybeans, alfalfa and red clover contain abundant isoflavones. Through the
 maturing soybean cultivars. Nevertheless, a genetic factor still plays the most critical
role for isoflavone concentration [23].
Although the terms phytoestrogen and isoflavone are frequently used inter-
changeably in the literature, they are not exactly the same. More precisely, phytoestrogen
is a plant compound that can exert an estrogen-like effect, and isoflavones, including
Int. J. Mol. Sci. 2021, 22, 3212 6 of 23
daidzin and genistin, belong to bioflavonoids that are available in animals and plants
[24]. Some foods such as soybeans, alfalfa and red clover contain abundant isoflavones.
Through the metabolic actions of gastrointestinal enzymes in the human gut, both of the
isoflavone precursors
metabolic actions daidzin and genistin
of gastrointestinal enzymes areintransformed intoboth
the human gut, daidzein
of the and genistein,
isoflavone pre-
respectively
cursors daidzin[25] and
(Figure 2). The
genistin areprocess of soybean
transformed fermentation
into daidzein concentrates
and genistein, isoflavone,
respectively [25]
while
(Figurethe2).process of removing
The process of soybean thefermentation
fat, color or concentrates
taste predisposes the removal
isoflavone, while theof process
isofla-
vone [26]. More
of removing the than 50% or
fat, color of taste
Asianpredisposes
women have theintestinal
removal ofbacteria that [26].
isoflavone are capable
More than of
converting
50% of Asian daidzein
women and havegenistein
intestinal to S-equol,
bacteria thataare
bioactivator similar to estrogens
capable of converting daidzein and in
structure. However, this is not the case for the women on other continents [27] (Figure
genistein to S-equol, a bioactivator similar to estrogens in structure. However, this is not the 2).
Compared
case for thewith
women isoflavones, S-equol has
on other continents [27]a(Figure
similar2).
preference
Comparedfor binding
with to ERβS-equol
isoflavones, but a
has a similar
higher preference
transcriptional for binding
expression to ERβ but a higher transcriptional expression [26].
[26].

Figure
Figure2.2.The
Thechemical
chemicalstructures
structures of
of isoflavones
isoflavones including daidzin and
including daidzin and genistin
genistinand
andtheir
theirderivatives
deriva-
tives daidzein,
daidzein, genistein
genistein and S-equol
and S-equol (structurally
(structurally similar
similar to estrogen).
to estrogen).

AA study
study reported
reported that
that even
even though
though non-equol
non-equol producers
producers consumed
consumed an an adequate
adequate
amount
amountof ofdaidzein,
daidzein,the theeffects
effectsofofrelieving
relievingmenopausal
menopausalsymptoms
symptomswere werestill
stilllimited
limited[27].
[27].
AA study enrolling aatotal
study enrolling totalofof364
364 women
women reported
reported thatthat equol
equol producers
producers gained
gained benefits
benefits from
from daidzein
daidzein for alleviating
for alleviating VMSAlthough
VMS [28]. [28]. Although ingestion
ingestion of probiotics
of probiotics did notdid not increase
increase the pro-
duction
the of S-equol
production [29], another
of S-equol trial which
[29], another was conducted
trial which later indicated
was conducted that thethat
later indicated extract
the
of red clover
extract of red plus probiotics
clover successfully
plus probiotics improved
successfully VMS [19].
improved VMS Equol-transforming bacteria
[19]. Equol-transforming
such as Lactococcus
bacteria garvieae were
such as Lactococcus separated
garvieae fromseparated
were the fecal cultures
from the of equol-transforming
fecal cultures of
women [29,30], and women
equol-transforming a randomized
[29,30],controlled trial (RCT)controlled
and a randomized conductedtrial
in Japan
(RCT)revealed that
conducted
a supplement
in Japan revealed with equol
that for non-equol
a supplement withproducers
equol foreffectively
non-equol reduced
producers postmenopausal
effectively re-
emotional
duced symptoms [31].
postmenopausal emotional symptoms [31].

2.3. Extraction of Isoflavones from Soybeans

The concentrations of isoflavones are low in soybeans and related food products;
therefore effective extraction prior to further analyses is quite important [3,7]. Before ex-
traction, the preparation process includes removal of water from the samples and grinding
or homogenization of samples [7].
Soybeans and related products contain high levels of proteins, lipids and carbohy-
drates. As a minor component of complex mixtures, isoflavone must be separated from
Int. J. Mol. Sci. 2021, 22, 3212 7 of 23

the bulk of the matrix constituents prior to further analyses [3,7]. There is a practical
way to extract isoflavones from soybeans in their original forms using available solvents
and laboratory equipment [7]. According to chromatographic behaviors on reversed-
phase columns in the presence of acids in the mobile phase to protonate the glycosidic
isoflavones, the hydrophobicity of the isoflavone conjugates is aglycone > acetyl-glucoside
> malonyl-glucoside > β-glucoside. The ester bonds of acetyl- and malonyl-glucose of
isoflavones are liable to be broken under an elevated temperature and changed acidic or
basic conditions [7].
The solvents used for extraction of isoflavones from soybeans and related foods
can be chosen on the basis of the solubility of isoflavones and the soyfood matrix. Be-
cause of the diversity of soybean isoflavones in polarity, the extraction requires the com-
bined use of organic solvents and water. The solvents, including acetone, acetonitrile,
methanol and ethanol, have been used to extract isoflavones from soybeans and re-
lated foods [7]. Isoflavones are relatively steady compounds but can degrade under
certain circumstances. Genistein and daidzein are light-sensitive [7]; malonyl-glycosides of
isoflavones are heat-labile [4,7].
The extraction techniques used for bioactive components (isoflavones and other
polyphenols) [6] and the summary (Table 1) are listed below.

Table 1. A summary of extraction techniques used for bioactive components (isoflavones and other polyphenols).

Methods Descriptions Solvents Advantages

Soybeans are left together with the
solvent from hours up to days for 1. very simple;
methanol acetone ethanol
Maceration extraction maceration, and after that, filtration 2.very suitable technique for
water
procedures clean off the extract thermally labile components
from solid suspensions.
It uses solvent heating to the boiling
point and the returning of the
Soxhlet extraction condensed vapors to the flask. This ethanol water 1. simple; 2. low cost
way, it can be run for as many
cycles as desired.
It utilizes ultrasound to develop
bubbles inside the solvent. These
1. shorter extraction time;
bubbles created by cavitation
Ultrasound-assisted 2. a reduced amount of
phenomenon induce wall cells’ ethanol water
extraction (UAE) necessary solvent;
disruption of the targets and speed
3. lower energy consumption
up the penetration of the solvent
into the target material.
It uses microwaves, which can be
1. considerable savings in
absorbed by polar molecules (such
time, solvent amount, energy
Microwave-assisted as water), and facilitates diffusion
Methanol ethanol water consumption;
extraction (MAE) of the solvent into vegetal samples
2. an increase in extraction
and release of the solutes from the
efficiency
target material into the solvent.
The extraction is facilitated by
means of reaching a supercritical
Supercritical fluid state which occurs when the fast, selective and solvent
carbon dioxide co-solvent
extraction (SFE) temperature and pressure of the saving
fluid are raised above its critical
point.
To accelerate extraction, it involves
more suitable for the
Accelerated solvent solvents under high temperature
methanol ethanol acetone extraction of phenolics
extraction (ASE) and pressure but without reaching
compounds than UAE
the critical point.
Int. J. Mol. Sci. 2021, 22, 3212 8 of 23

2.3.1. Maceration Extraction

Maceration is a conventional and very simple extraction method that uses different
solvents for the extraction of components from soybeans or other plant material. Soybeans
or other plant materials are left together with the solvent from a couple of hours up to
several days, and after that, filtration procedures clean the extract from solid suspensions.
It has the advantage of being a very suitable technique for thermally labile components.
The most used solvents for the extraction of the total amount of isoflavones, which are
a kind of polyphenols, are methanol, acetone and water. Maceration extraction has proven
to be an efficient technique for isolation of anthocyanidins, flavan-3-ols, proanthocyanidins,
flavanones, flavones and flavonols subclasses when a mixture of 0.1% HCl in methanol
and water (80:20, v/v) was used as a solvent. Ethanol is preferred to methanol due to
its lower toxicity. Maceration extraction using 80% ethanol kept at room temperature for
7 days and the same solvent at 60–70 ◦ C for 2 days has been applied to the extraction of
other components.

2.3.2. Soxhlet Extraction

Soxhlet extraction is the second conventional method used for bioactive component
extraction, an improvement over maceration, as it uses solvent heating to the boiling point
and the returning of the condensed vapors to the flask. In this way, Soxhlet extraction
can be run over as many cycles as desired. Water and ethanol seem to be the most-used
solvents in this extraction technique due to their availability, efficiency and non-toxicity.
Polyphenolic-polysaccharide conjugates have been extracted using an Soxhlet apparatus.

2.3.3. Ultrasound-Assisted Extraction (UAE)

UAE is an efficient extraction method at the border between classical and modern
techniques and is used for a variety of bioactive compounds. Compared with maceration
and Soxhlet procedures, UAE possesses several strategic advantages: Shorter extraction
times, a reduced amount of necessary solvent and lower energy consumption are the
most important benefits of UAE compared with conventional methods. The operating
principle of UAE is the utilization of ultrasound (waves with a frequency between 20
and 100 MHz) to develop bubbles inside the solvent. These bubbles, once created by
cavitation phenomenon, induce wall cells’ disruption of the targets and then speed up the
penetration of the solvent into the target material. UAE using ethanol–water (1:1, v/v),
with a solvent–sample ratio of 1:8, was applied to extract components in soybeans.

2.3.4. Microwave-Assisted Extraction (MAE)

MAE is a well-established technique and is a promising alternative to conventional
extraction techniques. It uses microwaves (non-ionizing electromagnetic waves with a
frequency between 0.3 and 300 GHz). The absorption of microwaves by polar molecules
(such as water) and, implicitly, the heat generation, facilitates diffusion of the solvent into
vegetal samples and release of the solutes from the target material into the solvent. The
advantages of MAE over the previously presented techniques are considerable savings
in time, solvent amount and energy consumption and therefore an increase in extraction
efficiency. MAE using water, methanol–water (80:20 v/v), ethanol–water (80:20 v/v) and
time/temperature programs were applied for extraction of polyphenols including flavones.

2.3.5. Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction is a valuable and environmentally friendly extraction
technique used for extracting a large variety of bioactive compounds, presenting the
advantages of being fast, selective and solvent saving. A supercritical state occurs when the
temperature and pressure of the fluid are raised above its critical point. Carbon dioxide is
the most used solvent in SFE, being very efficient for extraction of fat, lipids and other non-
polar compounds. For extraction of polar substances, a polar modifier called co-solvent (for
Int. J. Mol. Sci. 2021, 22, 3212 9 of 23

example, methanol, ethanol, acetonitrile, acetone, water, ethyl ether or dichloromethane) is

necessary to be added to the supercritical fluid in order to increase the solubility.

2.3.6. Accelerated Solvent Extraction (ASE)

Accelerated solvent extraction is a modern extraction technique used for the recovery
of bioactive compounds involving solvents under high temperature and pressure but
without reaching the critical point. In this extraction method, different solvents are required;
the most popular are methanol, ethanol or mixtures of other solvents, as well. The 50%
acidified methanol (50MA) followed by 50% acidified acetone (50AA) was the most efficient
solvent for the extraction of phenolic compounds, particularly flavonols, while water (W)
was not beneficial to the extraction of all categories of phenolics. ASE was more suitable
for the extraction of phenolics compounds than UAE.

2.4. Analyses of Isoflavones Using HPLC

HPLC can be used for analyses of isoflavones in soybean and related products. HPLC
is a fast, reproducible method requiring small sample sizes and is eligible for both qual-
itative and quantitative analyses as well as for the purpose of sample separation. With
an authentic isoflavone standard available, the appropriate method to analyze isoflavone
extracts from soybeans is usually HPLC coupled with a reversed-phase C18 column (silica-
based packings with covalently bound 18-alkyl chains) and UV spectrophotometer [7].
According to the solubility and polarity of isoflavones in the column between the stationary
and solvent (mobile) phases used, mixed isoflavones in soyfood extracts can be isolated.
Each isoflavone exhibits respective intense absorption in the UV region of the spectrum
ranging from 240 to 280 nm [7]. In addition, using a C18 HPLC column and gradient
solvent phase, a reversed-phase separation can be achieved to isolate isoflavones in the
soyfood extract. Reversed-phase HPLC acts based on lipophilicity and hydrophilicity [7].

3. Utilization of Isoflavones for Menopause-Related Syndromes and Others

3.1. Vasomotor Syndromes (VMS)
Vasomotor syndromes (VMS), such as hot flush and sweating after menopause, are
usually the most troublesome discomforts for affected women, and most of the women
will look for medical help. Nevertheless, because the severity and frequency of hot flush
and sweating are individualized, and such symptoms often subside over time even with-
out any treatment [25], it is difficult to quantify VMS. In 2001, a study conducted by
St. Germain et al. revealed that hot flush declined in all of the patients who respectively
took isoflavone-containing soybeans, isoflavone-free soybeans or whey proteins over
24 weeks, indicating that a placebo effect was inevitable [32]. Likewise, another study re-
ported that the occurrence of hot flush did not differ between the women using isoflavones
and those using placebos over 12 weeks [33].
However, most recent studies favor using isoflavones as a treatment of VMS. A small-
scale prospective study reported a 57% reduction in the severity and frequency of hot flush
for menopausal women who took 60 mg isoflavones daily over 12 weeks [34]. Using the
Kupperman Menopause Index, Cancellieri et al. also reported that 72 mg of isoflavones
retrieved from soybeans and red clover significantly decreased hot flashes over 6 months
of treatment [35].
In contrast to the Western diet, soy nuts were given for 8 weeks as a substitute for
non-soy protein, and this study conducted by Welty et al. noted a >40% reduction in the
symptom of hot flush in menopausal women, regardless of equol-producing status [16].
The results of this study provided an option for Western women to obtain the benefits
of isoflavone by eating an Eastern diet. Furthermore, a systematic review reported that
synthetic isoflavone may have been more effective in reducing the symptom of hot flush
compared with soy isoflavone [36].
Another focus is the optimal dosage of isoflavone for symptomatic women after
menopause. A report conducted in 1999 by Washburn et al. revealed that compared
Int. J. Mol. Sci. 2021, 22, 3212 10 of 23

with administrating the total amount of soy supplement in one dose, dividing the dose
into twice daily better reduced the severity of hot flush, suggestive of a better effect of
phytoestrogens with consistent circulating levels [37]. Another study reported a similar
finding in that both 40 mg and 60 mg of the daidzein-containing isoflavone aglycone given
once daily could reduce the frequency of hot flush by the same degree [38].
Researchers have studied the synergistic action of inulin plus isoflavone. Focusing
on peri-/post-menopausal women treated or untreated with a mixture of soy isoflavone
(40 mg), inulin (3 g), vitamin D3 (300 IU) and calcium (500 mg), a prospective multicenter
study reported the effect of inulin and soy isoflavone on hot flush and life quality [39]. The
mean number of hot flushes was found to decline more for women in the treated group
compared with those in the untreated group. The results of the study revealed the benefits
of using a combination of inulin and soy isoflavone as a dietary supplement for women
with menopausal symptoms [40].
Nonetheless, isoflavone is still less effective when compared with traditional HRT.
Isoflavone 45 mg twice daily, low-dose HRT (E2 1 mg and norethisterone acetate 0.5 mg)
and a placebo have been compared in a RCT. The results showed that isoflavone and
low-dose HRT were more effective than the placebo, yet low-dose HRT was better than
isoflavone [40]. Making indirect comparisons, another report analyzing more than 50 RCTs
disclosed the difference of effects between soy extracts and HRT for relieving hot flush [41].
However, over a research period of 24 months, another study reported that an isoflavone
mixture containing daidzein 22.01 mg, glycitein 13.54 mg and genistein 4.96 mg did not
affect quality of life as measured with Menopause-Specific Quality of Life (MENQOL) [42].
The result of the study mentioned above revealed that isoflavone cannot entirely replace
the role of traditional HRT in relieving symptoms for menopausal women.
Research has revealed that whether or not isoflavone can effectively reduce VMS
depends on the ability to produce S-equol in women. A report analyzing several RCTs
investigated the effects of S-equol and isoflavone to alleviate VMS in women who were
equol-producers or non-producers [43], and the results of the meta-analysis showed a sig-
nificant benefit of equol for lower hot flush scores. Therefore, the meta-analysis concluded
that the supplementation of equol remarkably decreased the occurrence and severity of
VMS in postmenopausal non-producers [43].
Some studies have used skin conductance measurement to avoid subjective evaluation
of the incidence and severity of hot flush. Skin conductance measurement can quantify a
tiny change in individual core temperature as well as later reaction [19]. A study conducted
by Newton showed consistency between the use of a skin conductance monitor and a
diary [28], while Lambert et al. reported inconsistency between hot flush measurement
using all-day ambulatory skin conductance and a subjective report of hot flush with the
Green Climacteric Scale [19]. The latter study revealed that red clover extract significantly
decreased the incidence of hot flush that was measured by skin conductance rather than by
a scale, thus emphasizing the importance of measurement with objective instruments [19].

3.2. Hormone-Related Osteoporosis

After menopause, another noteworthy change is a gradual loss of bone mineral density
(BMD), resulting in osteoporosis. Because expression of ERβ is higher in bone tissues [24],
theoretically, isoflavone can alleviate loss of bones. A prior study investigated the effect of
ipriflavone, one of the synthetic isoflavones; however, the results were been inconclusive [25].
There is a wider distribution and stronger expression of ERβ in trabecular bone [44];
hence, the spine is deemed the site most sensitive to isoflavone due to a higher content of
trabecular bone than cortical bone. In contrast, the hip consists of more cortical bones, and
thus undergoes slower remodeling compared with the spinal bones [45]. In a report of a
meta-analysis, spinal bone loss was significantly alleviated after a daily 90 mg isoflavone
supplement for 6 months [45]. A study conducted by Amato et al. revealed that although
daily 120 mg isoflavone failed to prevent loss of local bones, systemic BMD loss was
attenuated [42]. A meta-analysis reported in 2017 mentioned that isoflavone alleviated
Int. J. Mol. Sci. 2021, 22, 3212 11 of 23

BMD loss, but more prominently at the lumbar spine than the femoral neck, and the form
of isoflavone in aglycone was even more efficacious [44].
Because bone remodeling needs from 4 to 8 months, research with a longer duration
could assist in elucidating a more conclusive result [44]. Meanwhile, for postmenopausal
women without hormone replacement therapy, even a slight improvement in BMD can
be beneficial [25].

3.3. Female Urogenital System

Research has identified the variable distribution of both ERα and ERβ in female
urogenital organs such as the vagina, urethra and bladder [46]. In addition, the aforemen-
tioned organs contain GPERs, which possess a greater affinity for daidzein and genistein.
In the past, topical estrogen was applied to vaginal and urethral surfaces for treating
vaginal dryness and urinary incontinence; however, the detailed mechanisms of improving
incontinence remain unknown. Currently, isoflavone is being tried for the treatment of
urogenital symptoms after menopause. However, a longitudinal study over a 10-year
duration revealed that neither higher nor lower intake of dietary isoflavone could prevent
urge or stress incontinence [46], though equol-producer status was not considered. On
the other hand, Burton and Wells have not found any substantial evidence to confirm
that phytoestrogen affects the female genital system, but this can be attributed to a lack
of research rather than a paucity of correlation and urges further investigation into this
issue [47]. A study reported no difference in the cytology of the vagina for women who
used dietary soy or black cohosh for one year [48], although the percentage of isoflavone in
the supplement was not clearly calculated.
A prospective, randomized study identified the effect of nutraceuticals on the sexual
function of postmenopausal women. This trial evaluated a combined use of isoflavone
(40 mg), inulin (3 g), vitamin D (300 IU) and calcium (500 mg) in postmenopausal status [49].
The researchers found that patients in the treatment group had better scores in the physical
and vasomotor domains of life quality evaluation and better scores in all domains of sexual
function evaluation after 12 months, suggestive of a favorable effect of the combination
of isoflavone, inulin, vitamin D and calcium on life quality, menopausal symptoms and
sexual function [49].
Recently, a randomized clinical trial was conducted on 60 postmenopausal women,
who were randomly assigned to receive oral isoflavone (150 mg dry extract of glycine
max) alone or isoflavone plus probiotic or hormone therapy (1 mg estradiol and 0.5 mg
norethisterone acetate) to evaluate genitourinary symptoms and vaginal atrophy [50]. The
result revealed that after 16 weeks of treatment, the urogenital symptoms including vaginal
dryness and complaints of sexual problem as well as the maturation value, vaginal pH and
vaginal flora improved significantly in the hormone therapy group. There was a significant
increase in daidzein, glycitein, equol intermediate, and O-dimethylangolensin contents
after 16 weeks in the isoflavone plus probiotic group. The vaginal health score increased in
the isoflavone and hormone therapy groups. Therefore, it was concluded that probiotics
improved the metabolism of isoflavones after 16 weeks of treatment. However, the increase
in the contents of isoflavones and their metabolites failed to yield an estrogenic effect on
the urogenital tract and relieve vulvovaginal symptoms [50].

3.4. Metabolic Syndrome

After menopause, relative changes of metabolism can lead to obesity, which is a
major risk factor for cardiovascular events [51]. Current evidence obtained from one study
supported that the use of isoflavone can ameliorate glycemic control and facilitate weight
loss [52]. However, there are limited studies researching the relationship between soy
isoflavone supplementation and body weight loss in obese menopausal women. Although
another study has shown that isoflavone and exercise can result in a reduction of fat mass,
its conclusion is somewhat questionable due to a high dropout rate and poor patient
compliance to treatment [51].
Int. J. Mol. Sci. 2021, 22, 3212 12 of 23

An in vitro study showed that daidzein, genistein and equol all had good binding affinity
to activate the peroxisome-proliferator-activated-receptor (PPAR)γ, which played a key role in
many metabolic syndromes and diabetes [52]. In comparison with rosiglitazone, which was a
well-known drug for diabetes, the maximal isoflavone-induced PPARγ activity was between
23% and 32% [52]. Because soybeans rarely induced gains in body weight as rosiglitazone
did [52], isoflavone might be considered for the treatment of metabolic syndromes.

3.5. Cardiovascular Disease

The etiology and pathophysiology of cardiovascular diseases are complex and multi-
factorial, and cardiovascular events increase in postmenopausal women because of aging
and estrogen deficiency [53]. A paucity of estrogen brings about elevation in cholesterol
and low density lipoprotein and thus endothelial dysfunction [54]. Acting as an estrogen
substitute, isoflavone may be capable of reducing the risk of cardiovascular events.
Soy products are generally deemed healthy foods due to nearly no cholesterol content,
and therefore the FDA has stated that soybean proteins may lower the risks of cardiovas-
cular diseases [25]. Nevertheless, there is habitually a low intake of soybeans in Western
countries. Interestingly, another study reported no connection between a habitual phytoe-
strogen intake (including isoflavone and lignan) in the Western-style diet and the risk of
cardiovascular diseases, possibly due to an overall low intake of isoflavone [53].
The detailed mechanisms of isoflavone to prevent cardiovascular diseases remain
unclear. Previously, a study showed the benefits of isoflavone in ameliorating systemic
arterial compliance of perimenopausal and postmenopausal women, even if it did not
influence plasma lipids [54]. Similarly, a study conducted in 2007 revealed no difference in
lipoprotein levels after using 60 mg isoflavone per day for 12 weeks [34]. Hence, isoflavone
may exert its protective effect on cardiovascular diseases in other ways [55].
Daidzein and genistein have been thought to cause arterial relaxation by means
of releasing nitric oxide [55]. Nonetheless, a study conducted by Wong et al. showed
no significant difference in blood pressure after using daily 80 mg soy isoflavone for
6 weeks [56]. In contrast, animal research has indicated a deleterious effect of soybeans or
HRT in vessels with a preceding change of atherosclerosis. Neither soybeans nor HRT could
reduce the incidence of myocardial ischemia or reperfusion injuries in ovariectomized or
diet-induced atherosclerotic monkeys. Furthermore, a combination of soybeans and HRT
practically increased myocardial changes after local ischemia [57]. However, the inferences
from animal research must be made with caution. Before suggesting that women avoid
consumption of soybean products once they have cardiovascular events, more studies are
required in the future to draw a reliable conclusion [55].
Recently, an RCT indicated that isoflavone in soybeans could reduce systolic blood
pressure in the early stages of menopause. Sathyapalan et al. noted that supplementation
with soy isoflavone for 6 months could reduce systolic blood pressure in women who
were menopausal for less than 2 years, although both diastolic blood pressure and lipid
profiles (triglycerides, HDL, LDL and total cholesterol) remained unchanged [58]. Using
the Framingham equation, a reduction of risk in cardiovascular events can be calculated
using personal blood pressure [58]. Based on the findings mentioned above, it can be
inferred that the best and safest treatment window for cardiovascular diseases will be early
menopausal stages and before critical atherosclerotic changes occur.
Isoflavone probably only plays a minor role in the prevention of cardiovascular
diseases, and the major benefits disclosed are from soybeans themselves. Compared
with animal proteins, soybeans contain minimal saturated fats and cholesterol [25] and
subsequently make it healthier to meet daily nutritional requirements. Hence, the American
Heart Association and FDA recommend soybean foods for their benefits to overall and
cardiovascular health [25,51] whether the rationale is based on soybeans containing copious
amounts of fibers, vitamins and polyunsaturated fats [51] or simply on the fact that they
replace a dietary intake of animal proteins [26].
Int. J. Mol. Sci. 2021, 22, 3212 13 of 23

Recently, a study investigated the health benefits of soy products for the reduction
of coronary heart disease (CHD). A total of 74,241 women from the Nurses’ Health Study
(1984–2012), 94,233 women from the Nurses’ Health Study II (1991–2013) and 42,226 men
from the Health Professionals Follow-Up Study (1986–2012), who were free of cardio-
vascular disease and cancer at baseline, were included [59]. In multivariable-adjusted
analyses, isoflavone intake was inversely associated with CHD (pooled hazard ratio (HR)
comparing the extreme quintiles: 0.87 (95% CI 0.81–0.94); p < 0.01). Consumption of
tofu, but not soy milk, was inversely associated with the risk of CHD, with pooled HRs
0.82 (95% CI 0.70–0.95; p < 0.01) and 0.87 (95% CI 0.69–1.10; p = 0.41), respectively. The study
concluded that higher intake of isoflavones and tofu was associated with a moderately
lower risk of developing CHD [59].

3.6. Cognitive Function and Neuromuscular Systems

Although the natural aging process is inevitably a confounding factor, research has
still explored the effect of isoflavone on the cognitive function of postmenopausal women.
Research enrolled menopausal populations with various intakes of isoflavones over 6 years
and found that perimenopausal and postmenopausal women in Asia with a higher intake
of isoflavone had better function in processing speed but worse function in verbal mem-
ory [60]. However, the overall effect of isoflavone on cognition was small, thus inducing
doubt about the importance of the above findings. A meta-analysis reviewing 12 RCTs
reported that isoflavone and soybeans might improve the cognitive function of menopausal
populations; however, several obtained RCTS had problematic methods and designs to
some extent [61]. In 2011, the North American Menopause Society (NAMS) reported the
benefits of soybeans in the function of cognition for postmenopausal women aged younger
than 65 years old, yet not for those older [26].
A cross-sectional study evaluated the associations between intake of soy products and
isoflavones with depressive symptoms in 1745 pregnant Japanese women [62]. The result
revealed that higher intake of total soy products, tofu products, fermented soybeans, boiled
soybeans, miso soup and isoflavones was independently related to a lower prevalence of
depressive symptoms during pregnancy: The adjusted prevalence ratios (95% CI) between
extreme quartiles were 0.63 (0.47–0.85), 0.74 (0.56–0.98), 0.57 (0.42–0.76), 0.73 (0.55–0.98),
0.65 (0.49–0.87) and 0.63 (0.46–0.86), respectively. A significant positive exposure–response
relationship was found between miso intake and depressive symptoms during pregnancy.
No material relationship was observed between soymilk intake and depressive symptoms
during pregnancy. The study showed independent inverse relationships between intake of
soy products and isoflavones and depressive symptoms during pregnancy [62].
A study explored whether the dietary isoflavone aglycone (AglyMax) at 0.6% could
prevent denervation-mediated muscle atrophy based on the modulation of atrogin-1- or
apoptosis-dependent signaling [63]. Mice were fed either a normal diet or 0.6% AglyMax
diet, and then the right sciatic nerve was cut for examination. The 0.6% AglyMax diet signif-
icantly attenuated denervation-induced decreases in fiber atrophy but not the muscle wet
weight. In addition, dietary isoflavone suppressed denervation-induced apoptosis. The au-
thors concluded that the isoflavone aglycone significantly modulated muscle atrophy after
denervation in mice, probably due to the decrease in apoptosis-dependent signaling [63].

3.7. Adverse Effects

Generally, isoflavone was well-tolerated by human bodies in all of the studies available
for this review. Adverse effects were usually mild and mainly gastrointestinal such as
bloating, nausea, constipation and diarrhea [8].
Previous results of animal studies have raised concerns about S-equol because in-
fertility and reproductive anomalies have been noted among animals fed with soybean-
containing diets or red clover [29]. In mice undergoing oophorectomies, feeding with a
high dose of equol for 3 months induced obvious mammotropic effects [64]. Nevertheless,
the use of S-equol seems safe for the uteri of humans among all of the studies available.
Int. J. Mol. Sci. 2021, 22, 3212 14 of 23

Since estrogen is likely to contribute to the differentiation of male external genitalia,

dietary intake of isoflavone, which has a similar structure to human estrogen, may be
associated with the occurrence of hypospadias. A nationwide birth cohort study recruited
women in Japan between 2011 and 2014 [65]. Among 41,578 mothers who delivered
singleton, live male births, the median genistein intake was 15.3 mg/day, and 51 cases of
hypospadias were identified. Compared with mothers in the reference group (genistein
intake 11th–89th percentiles), those in the low-intake group (≤10th percentile) had an
elevated risk of hypospadias in their sons (multivariable-adjusted OR = 2.8, 95% CI 4–5.8).
Adverse or beneficial effects of genistein on hypospadias were not observed in the high
intake group (≥90th percentile) (OR = 0.9, 95% CI 0.4–2.4). It is concluded that low
maternal intake of isoflavone in early pregnancy was associated with an elevated risk
of hypospadias [65].

3.8. The Risks of Cancers

Isoflavone can bind to estrogen receptors, thus raising concerns about its potential
to induce estrogen-dependent malignancies, especially in the population of survivors of
breast malignancy [51]. Nonetheless, research has indicated proliferation of breast cancer
depends on the activity of ERα, while ERβ activation appears to suppress proliferation
of ERα-induced cancer cells [66]. Hence, isoflavone is postulated to protect breasts from
cancers by binding to ERβ. In addition, phytoestrogen has been noted to prolong the
menstrual cycle, which is a possible reason for its protective effects against hormone-
sensitive malignancies [25]. However, a detailed mechanism has not been fully clarified.
Focusing on healthy women, research has shown a decreased incidence of estrogen-
related cancer in women using isoflavone. In Japan, a case–control study revealed that
consumption of soybeans was inversely associated with the risk of breast cancer in women
before menopause but not in those post menopause [67]. In a 12-week study, although soy
isoflavone elicited some benign ultrasonographic changes of the breast, all of these changes
were subclinical and did not need further intervention or follow-up after the study [68].
Moreover, soy isoflavone did not appear to stimulate proliferation of endometrial cells if
given over a short term [26]. On the contrary, Shike et al. conducted an RCT to explore
the effects of soybean intake on the gene expression of breast cancer. In their study, it was
noted that supplementation with soybeans was related to enhanced gene expression, and
there was a connection between high plasma levels of genistein and over-expression of
FGFR2 genes to activate the proliferative pathway. Hence, they concluded that soybeans
could unfavorably affect gene expression of breast cancer cells in a subset of women [69].
A meta-analysis aimed to systematically evaluate and summarize available evidence
on the effect of dietary isoflavone intake on the risk of developing breast cancer [70].
PubMed, Embase, and the Cochrane Library were searched for prospective cohort studies
that evaluated the effect of dietary isoflavone intake on the development of breast cancer.
Among 16 studies, 11,169 breast cancer cases and 648,913 participants were identified
and included in the data analysis. The pooled relative risk (RR) of breast cancer was
0.99 for high versus low intake of isoflavones (95% CI 0.91–1.09; p = 0.876) and 0.99 for
moderate versus low intake of isoflavones (95% CI 0.92–1.05; p = 0.653). While a moderate
consumption of soy-based foods did not significantly affect breast cancer risk, a high intake
of soy-based foods was associated with a lower risk of developing breast cancer. The
present meta-analysis indicated that women with a high dietary intake of soy foods might
experience a statistically significant reduction in breast cancer risk [70].
Limited research has explored the role of soy isoflavone in postmenopausal patients
who had breast cancer and underwent surgeries. The study conducted by Kang et al.
revealed that a higher intake of soy isoflavone was related to a lower risk of breast cancer
recurrence in the women who had positive ERs and progesterone receptors (PRs) and used
anastrozole after surgeries [71].
A study attempted to evaluate the association of soy food or isoflavone intake with
endometrial cancer risk in Japanese women [72]. This prospective cohort study enrolled
Int. J. Mol. Sci. 2021, 22, 3212 15 of 23

49,121 women aged 45–74 years, among whom 112 newly diagnosed endometrial cancer
cases were identified. Cox proportional hazards regression models revealed that energy-
adjusted intakes of soy food and isoflavone were not associated with a risk of endometrial
cancer. Therefore, there was no evidence of a harmful association between soy food or
isoflavone intake and endometrial cancer risk [72].
Another study aimed to determine whether long-term isoflavone soy protein (ISP)
supplementation affected endometrial thickness and rates of endometrial hyperplasia
and cancer in postmenopausal women [73]. In this randomized, double-blind, placebo-
controlled trial, women aged 45 to 92 years were randomized to a total daily dose of 154 mg
in the ISP group or a milk protein-matched placebo. During a 3-year period, a total of
224 participants were evaluated. The treatment group did not significantly differ from the
mean baseline in endometrial thickness. Although the rate of hyperplasia/malignancy was
higher in the placebo group (14.3% vs. 0%), the difference was not statistically significant.
The study concluded that 3-year ISP supplementation had no effect on endometrial thick-
ness or on the rates of endometrial hyperplasia and cancer in postmenopausal women [73].
Furthermore, a prospective study analyzed 46,027 non-hysterectomized postmenopausal
women who were recruited into the Multiethnic Cohort (MEC) Study [74]. Among these
women, 489 women diagnosed with endometrial cancer were identified during a median
follow-up period of 13.6 years. Cox proportional hazards models revealed a reduced
risk of endometrial cancer was associated with total isoflavone intake (highest vs. lowest
quintile, relative risk (RR) = 0.66, 95% CI = 0.47–0.91), daidzein intake (highest vs. lowest
quintile, RR = 0.64, 95% CI = 0.46–0.90) and genistein intake (highest vs. lowest quintile,
RR = 0.66, 95% CI = 0.47–0.91). This study suggests that greater consumption of isoflavone-
containing foods is associated with a reduced risk of endometrial cancer in this population
of non-hysterectomized postmenopausal women [74].
A population-based prospective study evaluated the associations of soy and isoflavone
intakes with bladder cancer incidence in Japan and enrolled 14,233 men and 16,584 women
aged 35 years or older [75]. During a mean follow-up of 13.6 years, 120 men and 41 women
had developed bladder cancer. After adjustments for multiple confounders, compared
with the lowest quartile of soy food intake, the estimated hazard ratios for the second,
third and highest quartiles of soy food intake in men were 0.74, 0.52 and 0.55, respectively.
The corresponding values in women were 0.60, 0.75 and 0.64, respectively. Similar inverse
associations were observed between isoflavone intake and bladder cancer risk. A significant
decreased risk of bladder cancer was observed among individuals who had higher intakes
of total soy and isoflavones. The study explored the potential benefit of consuming soy
foods and isoflavones against bladder cancer [75].
In addition, isoflavone has also played a role in protecting against colorectal cancer.
Isoflavone treats diabetes, hyperlipidemia and obesity, all of which are potential risk
factors for colorectal cancer. Hence, it would be a reasonable hypothesis to state isoflavone
prevents colon cancer. In Korea, a study reported that a high intake of isoflavone or soy
foods was related to an overall reduced risk of colon cancer, though an over-ingestion of
fermented soyfoods appeared to elevate the risks of male colon cancer, perhaps owing
to their salty contents [76]. Future investigations are still needed for confirmation of this
hypothesis. Other than isoflavone, soy fibers may also have a role in the prevention of
colorectal cancer and warrant a comparison between soybeans and isoflavone rich extracts
in the future.
Table 2 is a brief summary showing current evidence of studies regarding the preven-
tive and therapeutic effects of isoflavones and soy foods on menopausal syndromes and
other diseases.
Int. J. Mol. Sci. 2021, 22, 3212 16 of 23

Table 2. A brief summary of current evidence of studies regarding the preventive and therapeutic effects of isoflavones and soyfoods.

Studies (Ref. No.) Study Design Contents Main Preventive (P) and Therapeutic (T) Effects
Hot flushes
[32] St Germain randomized controlled trial (RCT) soy T no difference
[33] Tice RCT isoflavone tablets T no difference
[35] Cancellieri RCT isoflavone from herbal supplement T isoflavones more effective than placebo
[34] Cheng prospective study isoflavones extracted from soya bean T isoflavones more effective than placebo
[16] Welty RCT, crossover soy nut T soy more effective than placebo
[36] Thomas systematic review natural vs. synthetic isoflavones T synthetic or combination isoflavones more effective than natural soy
[37] Washburn randomized crossover trial soy protein T soy protein more effective than placebo
[38] Khaodhiar RCT daidzein-rich isoflavone aglycones T daidzein-rich isoflavone aglycones more effective than placebo
[39] Cianci observational prospective study calcium, vitamin D3, inulin, soy isoflavones T soy supplement + inulin effective
[40] Carmignani RCT soy vs. hormone replacement therapy (HRT) T HRT more effective than soy; both are superior to placebo
[41] Bolanos-Dıaz meta-analysis soy extracts vs. HRT T HRT more effective than soy extracts; both are superior to placebo
[42] Amato multicenter RCT aglycone hypocotyl soy isoflavone T no difference
[43] Daily systematic review, meta-analysis soy isoflavone and equol T equol or isoflavone in equol-producers more effective than placebo
[28] Newton observational study equol-producer status T soy in equol-producers more effective than non-producers
[19] Lambert RCT red clover extracts T red clover extracts more effective than placebo
Hormone-related osteoporosis
[45] Ma meta-analysis isoflavone T increase spinal bone mineral density (BMD)
[42] Amato multicenter RCT aglycone hypocotyl soy isoflavone T slow BMD loss
[44] Lambert systematic review and meta-analysis isoflavone aglycone T preserve BMD
Urogenital tract
[48] Reed RCT black cohosh or dietary soy T no effect on vaginal cytology
[46] Waetjen prospective cohort study dietary intake of isoflavones T no effect on stress or urge incontinence
prospective, randomized,
[49] Vitale isoflavones, calcium, vitamin D, inulin T May improve sexual function
placebo-controlled study
failed to yield an estrogenic effect on the urogenital tract and to
[50] Ribeiro RCT isoflavone T
relieve the vulvovaginal symptoms
Metabolic syndrome
[51] Stuenkel randomized clinical trial isoflavone supplements T loss of weight and fat mass, but interpretation difficult
[52] Mueller in vitro study PPARγ binding and transactivational activity T red clover extracts may be used to treat metabolic syndrome
Int. J. Mol. Sci. 2021, 22, 3212 17 of 23

Table 2. Cont.

Studies (Ref. No.) Study Design Contents Main Preventive (P) and Therapeutic (T) Effects
Cardiovascular disease
[53] van der Schouw prospective study food phytoestrogens P low dose phytoestrogen not protective
[54] Nestel randomized crossover trial purified soybean extract T may improve systemic arterial compliance
[34] Cheng prospective study isoflavones extracted from soya bean T no difference in lipoprotein lipids
[56] Wong RCT soy hypocotyl isoflavones T no effect on nitric oxide metabolism or blood pressure
[57] Suparto animal study soy protein T HRT + soy harmful, soy or HRT not beneficial
soy protein with isoflavones improved cardiovascular markers
[58] Sathyapalan double-blind randomized study soy protein +/− soy isoflavone T
compared to soy protein alone
higher intake of isoflavones and tofu was associated with a
[59] Ma 3 prospective cohort studies isoflavones, tofu and soymilk P
moderately lower risk of developing coronary heart disease
Cognitive function and neuromuscular systems
[61] Clement systematic review isoflavones and soy T may improve cognition
[60] Greendale cohort study dietary phytoestrogens T better processing speed but worse verbal memory
independent inverse relationships between intake of soy products
[62] Miyake cross-sectional study soy products and isoflavones T
and isoflavones and depressive symptoms during pregnancy
significantly modulated muscle atrophy after denervation in mice,
[63] Tabata animal study isoflavone aglycone T
probably due to the decrease in apoptosis-dependent signaling
Cancer risk
[67] Hirose case-control study soy products as part of daily intake P lower risk of breast cancer in premenopausal women
[68] Alipour case-control study soy extracts soy extracts may cause benign changes in breast
lower recurrence of estrogen- and progesterone-receptor positive
[71] Kang cohort study dietary intake of soy isoflavones P
breast cancers receiving anastrazole therapy after surgery
[76] Shin case-control study dietary soyfood and isoflavone intake P reduced risk for overall colorectal cancer
[70] Zhao meta-analysis soy foods P a high dietary intake of soy foods may reduce breast cancer risk
[72] Budhathoki prospective study soy food and isoflavone were not associated with the risk of endometrial cancer.
[73] Quaas double-blind RCT isoflavone soy protein no effect on the rates of endometrial hyperplasia and cancer
[74] Ollberding prospective study isoflavone, daidzein and genistein intake P are associated with a reduced risk of endometrial cancer
[75] Wada population-based prospective study total soy and isoflavones P are associated with a decreased risk of bladder cancer
Int. J. Mol. Sci. 2021, 22, 3212 18 of 23

4. Discussion
Regarding the preventive effects on diseases or cancers, a higher intake of isoflavones
and tofu is associated with a moderately lower risk of developing coronary heart disease.
Moreover, a high dietary intake of isoflavones or soyfoods may reduce the risks of breast
and colorectal cancer as well as the incidence of breast cancer recurrence. Consumption of
isoflavones and soy products is associated with a reduced risk of endometrial and bladder
cancer. Regarding the therapeutic effects on menopausal syndrome or other diseases,
isoflavones and soy extracts are superior to placebo in relieving vasomotor syndrome, but
appear less effective than HRT. Use of isoflavones increases spinal BMD and ameliorates
hypertension and in vitro glycemic control. Soy products and isoflavones may alleviate
depressive symptoms during pregnancy, yet they fail to relieve vulvovaginal symptoms
and stress or urge incontinence.
Because the concentrations of isoflavones are low in soybeans and related products,
effective extraction prior to further analyses is quite important. Before extraction, the
originals should be well prepared during the process, which includes cleaning, removing
water from the samples and grinding. Different processing conditions produce soybean
products with a variety of isoflavone composition and content. The chemical structures
and abundance of isoflavones in soybean foods may have an important impact on their
biological effects and bioavailability.
In general, the findings of previous studies are highly heterogenic, and it is quite
difficult to draw conclusions regarding the effects of isoflavones [77–79]. Since 2011,
the NAMS has recommended in a considerable way the prescription of isoflavone for
menopausal syndrome. The recommendation is to initiate the use of isoflavone at a higher
dose (more than 50 mg/day) for twelve weeks and monitor potential side effects. However,
the treatment should be stopped in case of no response after a 12-week treatment [26]. In
some women with menopausal symptoms, isoflavone treatment was considered relatively
safe and beneficial. Another report also deemed isoflavone as an acceptable alternative for
the menopausal population who suffered VMS and did not use HRT [80].
The physiological pathways of soybeans and isoflavones as well as their derivatives are
not fully understood, as well as the interactions between these compounds and the human
body. A previous in vitro study has shown that isoflavone can function as an agonist of
estrogens after menopause due to a low-estrogen environment. In contrast, isoflavone can
play the role of an antagonist of estrogens before menopause because of a higher circulatory
level of endogenous estrogen [10]. Nevertheless, many questions still remain unanswered.
Currently, it is not known which component of isoflavone produces the estrogen-like effect.
Previous research has reflected this condition by examining a variety of isoflavone extracts
and different parts of soybeans [81]. It is probable that substances in soybeans other than
isoflavones can exert a favorable effect as well. It is thought that a wide range of health
benefits from soy phytochemicals (lipids, fatty acids, soy sterols, vitamins, minerals and
soyasaponins) contributes to the overall nutritional well-being of humans. The result was
similar in another study where isoflavone has been removed from soybean products and
compared with soybean extracts containing isoflavone [82]. Furthermore, a systematic
review showed that synthetic isoflavone was superior to soybean isoflavone in alleviating
hot flush [83]. In summary, the current evidence suggests that soybeans, isoflavones
and their derivatives may function as an alternative via both estrogen-dependent and
independent pathways [26].
There are some controversies regarding the trials to standardize the level of isoflavone
for relieving menopausal symptoms in Asian women. A study reported hot flush occurred
less in women of Eastern countries irrespective of soybean ingestion yet admitted that their
equol-producing status was not fully considered [17]. Burton and Wells found uterotrophic
effects in rats with neonatal exposure to phytoestrogen [47]. On the other hand, another
human study recognized early-life exposure to dietary isoflavones as a precondition of
the effects, suggestive of a possible early transformation of genes [26]. Because of the
unpredictability in the action and response of isoflavone, an optimal dose of isoflavone
Int. J. Mol. Sci. 2021, 22, 3212 19 of 23

remains to be determined. A study disclosed the response to S-equol exposure was

approximately linear with dose, and a twice-daily dose might be better [27]. A previously
study indicated that the total amount of isoflavone, estimated in aglycone-equivalent
weight, was possibly 40–50 mg/day for symptom relief in women [83]. However, there
was no study suggesting the upper limit for isoflavone use. Utian et al. reported twice
per day instead of quotidian use of S-equol had a better effect [84]. Likewise, Crawford
and his colleagues reported that isoflavone given twice per day could provide greater
relief of hot flush than daily use, but the benefit of doubling the dose may have been for
equol producers only [85]. Compared with these studies, a report did not show a linear
but instead a biphasic effect of glabridin, a different isoflavone on the Ishikawa cell [86].
Therefore, daidzin, genistin, S-equol and other isoflavones appear idiosyncratic in their
respective actions on humans and should be investigated individually. The limitations in
previous studies warrant further investigations of isoflavone use for women’s health.

5. Conclusions
The previous studies that elucidated the structures and functions of bioactive molecules
in soybeans have provided an insight into correlation between the reported health benefits
associated with soy intake and soy phytochemicals. The processes of preparation (cleaning,
drying, crushing and dehulling) and extraction of soybeans are implemented to produce
refined soy oil, soy lecithin, free fatty acids, glycerol and soybean meal. Because the concen-
trations of isoflavones are low in soybeans and related products, effective extraction prior
to further analyses is very important. Different processing conditions produce soybean
products with a variety of isoflavone composition and content. Isoflavones including
daidzin and genistin and their derivatives daidzein, genistein and S-equol (structurally
similar to estrogen) can act to affect human endocrine functions. The chemical structures
and abundance of isoflavones in soybean foods may have an important impact on their
biological effects and bioavailability.
To summarize the therapeutic effects on menopausal syndrome or other diseases,
isoflavones appear to reduce hot flush and other VMS even after accounting for a placebo
effect. They may alleviate lumbar spine loss, improve BMD and have a positive effect on
the control of blood pressure and sugar during early menopause. They may also ameliorate
depressive symptoms during pregnancy. Currently, research has not drawn consistent
conclusions regarding the benefit of isoflavone on the cognitive and urogenital systems.
Regarding the preventive effects on diseases or cancers, a higher intake of isoflavones is
associated with a moderately lower risk of developing coronary heart disease. It may also
reduce the risks of breast and colorectal cancers as well as the incidence of breast cancer
recurrence. Consumption of isoflavones or soy foods is associated with reduced risks of
endometrial and bladder cancer.
Even though isoflavones are not as effective as traditional HRT in alleviating menopausal
syndrome, a previous survey reported that 70% of women were satisfied with a non-
hormonal intervention that decreased VMS by more than half [79]. The relative safeness
of isoflavone as well as its benefits to overall health make it a considerable option for the
treatment of postmenopausal women unable or unwilling to use HRT.
To minimize the heterogeneity of future studies, standardization of some influential
factors involved in isoflavone research should be considered. One of the influential factors
is “time to menopause”, which has an important impact on the individual response to
isoflavones. The aglycone ratio in isoflavones needs to maintain consistency among future
studies for a superior comparison of the therapeutical effects and to ensure reliability.
Moreover, study duration and outcome measures also need standardization together with
key endocrinal profiles of affected women. Furthermore, a larger sample sizes are required
to draw reliable conclusions and improve the efficacy and replicability of study results.

Author Contributions: K.-H.C. and L.-R.C. conceived the review and designed the search methods
for the literature. K.-H.C. and L.-R.C. collected the data in the literature. K.-H.C. and L.-R.C.
Int. J. Mol. Sci. 2021, 22, 3212 20 of 23

performed the data analysis. K.-H.C. and L.-R.C. wrote the review. All authors have read and agreed
to the published version of the manuscript.
Funding: This review and APC were funded by a grant of Taipei Tzu-Chi Hospital, Taiwan (TCRD-
TPE-110-06) for K.-H.C.; The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in writing the manuscript, or in deciding to publish the results.
Conflicts of Interest: The authors declare no conflict of interest.

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