Phytochem Rev (2015) 14:849–869

DOI 10.1007/s11101-014-9382-0

Bioactive soy isoflavones: extraction and purification

procedures, potential dermal use and nanotechnology-based
delivery systems
Marina Cardoso Nemitz • Renata Cougo Moraes •
Letı́cia Scherer Koester • Valquiria Linck Bassani •
Gilsane Lino von Poser • Helder Ferreira Teixeira

Received: 28 February 2014 / Accepted: 14 October 2014 / Published online: 26 October 2014
Ó Springer Science+Business Media Dordrecht 2014

Abstract Isoflavones are polyphenol compounds Keywords Dermatological application  Extraction

found mainly in legumes such as soybeans (Glycine techniques  Nanostructured delivery systems  Skin
max (L.) Merrill). These compounds can be found in care  Soybean isoflavones
different chemical forms; however, the beneficial
effects for skin care have been mainly credited to their
Abbreviations
free forms. This manuscript claims to review the main
CD Cyclodextrin
effects of isoflavone aglycones on the skin, the
CO2 Carbon dioxide
different techniques for obtaining bioactive forms
DPPH 2,2-Diphenyl-1-picrylhydrazyl
from soybeans, and the interest in incorporating them
ER Estrogen receptor
into topical systems. The benefits of dermatological
HRT Hormone replacement therapies
application of isoflavones, as anti-aging action, estro-
IA Isoflavone aglycones
genic activity, wound healing properties, and anti-
LogP Partition coefficient
photocarcinogenic effects are highlighted. Moreover,
MCT Medium chain triglycerides
the advantages and drawbacks of the extraction
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-
techniques of soybeans, methods for converting glu-
diphenyltetrazolium bromide
cosides into aglycones, and purification procedures are
NMR Nuclear magnetic resonance
described. Different strategies to incorporate these
o/w Oil in water
poorly soluble compounds in conventional or nano-
PAR-2 Protease type 2
structured delivery systems are also discussed. Illus-
ROS Reactive oxygen species
trative examples especially for genistein-loaded
RSM Response surface methodology
liposomes, nanoemulsions, nanocapsules and cyclo-
SLN Solid lipid nanoparticles
dextrin complexation are reported.
TGF-b1 Transforming growth factor b1
UV Ultraviolet
UV–Vis Ultraviolet–visible
w/o Water in oil
M. C. Nemitz  R. C. Moraes  L. S. Koester 
V. L. Bassani  G. L. von Poser  H. F. Teixeira (&) Introduction
Programa de Pós Graduação em Ciências Farmacêuticas,
UFRGS, Av. Ipiranga, 2752, Porto Alegre, RS 90610-000,
Brazil Soy (Glycine max (L.) Merrill) is a legume originating
e-mail: helder.teixeira@ufrgs.br from Asia, widely distributed throughout the world

123
850 Phytochem Rev (2015) 14:849–869

Fig. 1 Chemical structures

of soybean isoflavones
(adapted from Rostagno
et al. 2009)

and of great economic importance (Zuanazzi and 2010). Studies have shown that they play an important
Mayorga 2010; Chen et al. 2012). At first, it role in preventing several chronic diseases, in addition
represented a rich source of protein and vegetable to reducing climacteric symptoms in menopausal and
oil, widely used for preparing food products (Chen postmenopausal women (Barnes 1998; Setchell 1998;
et al. 2012). More recently, however, its pharmaco- Albertazzi and Purdie 2002) and having beneficial
logical importance has been recorded, as its consump- effects on the skin (Wei et al. 1995a; Song et al. 1999;
tion is linked to the reduction of menopausal Miyazaki et al. 2002; Huang et al. 2008).
symptoms and prevention of chronic diseases such Isoflavones are phenolic compounds belonging to the
as certain cancers, cardiovascular disease, osteoporo- flavonoids class and are widely distributed in the plant
sis and diabetes (Barnes 1998; Setchell 1998). kingdom, mainly in the Leguminosae family (Barnes
Among the various compounds present in this plant, 2010). There are twelve main isoflavones in soybeans,
isoflavones have attracted the most attention in the namely genistein, daidzein, glycitein and their respective
pharmaceutical field. Isoflavones are structurally sim- acetyl-glucoside, malonyl-glucoside, and glucoside
ilar to 17-b-estradiol, having proven estrogenic activ- forms. As shown in Fig. 1, the chemical structure of
ity and, therefore, are called phytoestrogens (Barnes these substances is based on the presence of a flavone

123
Phytochem Rev (2015) 14:849–869 851

nucleus, which is composed of two aromatic rings 2009). Its aging is associated with the loss of elasticity,
attached to a heterocyclic ring, with some characteristic an increase of skin roughness and development of
substituents (Rostagno et al. 2009; Shao et al. 2011). wrinkles, whose causes may be of intrinsic or extrinsic
Isoflavones are found in soybeans and their pro- origin. When intrinsic, the phenomenon occurs due to
ducts (Barnes 2010), such as flour, fermented foods aging, leading to a depletion of the extracellular matrix
(tofu, miso, and tempeh), milk, and concentrated soy by a decrease in collagen and elastin synthesis. When
proteins, among others (Chen et al. 2012). To obtain extrinsic, it takes place through exposure to ultraviolet
extracts of these compounds, a number of studies have (UV) radiation, leading to the formation of reactive
proposed increasingly elaborate, fast, and reliable oxygen species (ROS) and causing extensive tissue
extraction and quantification technologies (Rostagno damage (Sudel et al. 2005; Masaki 2010).
et al. 2009; Luthria and Natarajan 2009). As such, the After menopause, women experience a significant
industrial sector has become increasingly interested in reduction in hormone levels, which is one of the
extracts enriched in isoflavones for obtaining herbal intrinsic skin aging factors. Hormone replacement
and phytocosmetic products (Chen et al. 2012). therapies (HRT), both oral and topical, are alternatives
In view of the activities attributed to isoflavones, to maintaining the skin thickness and elasticity. Due to
there are topical products in the market containing soy their similarity to the 17-b-estradiol (Fig. 2), isoflav-
extracts. The Brazilian (AdcosÒ, NaturaÒ, and PayotÒ), ones are strong candidates for this type of therapy
American (AveenoÒ, SkinCeuticalsÒ), and European since they can bind to estrogen receptors (ERs), with
(VichyÒ) brands are examples of companies investing beneficial effects on the skin (Sator et al. 2004).
in products containing soy extracts mainly recom- There are two subtypes of estrogen receptors, ER-a
mended for anti-aging action on the skin. and ER-b, the latter widely found in the skin, present
Nevertheless, the extracts incorporated into cosmetic in the epidermis, fibroblasts, and keratinocytes (Ku-
formulations very often have isoflavones in conjugated iper et al. 1998). Estrogens couple with ERs in the
forms that may, in some cases, limit their biological cell’s nucleus, switching linked genes. This may lead
action due to a lower possibility of penetrating the skin. to proliferative or differentiation responses (Pinnel
To get the desired effects of isoflavones, their free forms 2003). Once bound by estrogen, the ER undergoes a
should preferably be used. However, they are less conformational change allowing the receptor to inter-
soluble than the conjugated forms, which may hinder act with chromatin and modulate transcription of
their incorporation into traditional topical delivery target genes (Kuiper et al. 1998). The presence of ER-
systems (Schmid et al. 2003). b in the skin cells suggests that estrogen may directly
To allow better use of the aglycones in formulations modulate the synthesis of collagen, hyaluronic acid,
and enhance their penetration, some alternatives to the and elastin (Sator et al. 2004).
traditional systems such as liposomes, micro and Among the different varieties of phytoestrogens,
nanosystems, or complexes with dendrimers and genistein and daidzein stimulate the transcriptional
cyclodextrins, are being reported (Schmid et al. activity of both ER subtypes at concentrations of
2003; Silva et al. 2009; Kitagawa et al. 2010; Xavier 1–10 nM, with an affinity about 20 times greater for
et al. 2010; Zhao et al. 2011). ER-b receptors (Kuiper et al. 1998). Even though
In this context, this article aims to report the main genistein and daidzein have showed relative ER
effects of isoflavone aglycones on the skin, as well as affinities 1,000-fold lower than [3H]-17b-estradiol
the different techniques of obtaining bioactive forms (Kuiper et al. 1998), the isoflavones have shown
from soybeans, and the interest in incorporating them beneficial estrogenic effects on the skin. In a clinical
into topical systems, including nanotechnology-based study by Schmid and Zulli (2002), the authors showed
systems. the occurrence of an increase in skin thickness in 20
volunteers who applied a cream containing genistein
at a concentration of 90 mg/g twice a day, during
The action of isoflavones on the skin 3-months’ treatment. Bayerl and Keil (2002) showed
the skin effects in a controlled multi-center study with
The skin is composed of three distinct layers: the 234 post-menopausal volunteers using a cosmetic
epidermis, dermis, and hypodermis (Förster et al. cream preparation including isoflavone (NeovadiolÒ).

123
852 Phytochem Rev (2015) 14:849–869

Fig. 2 Structural similarity

of: a 17-b-estradiol and
b isoflavone aglycones, as
well as the chemical
orientation proposed by
Barnes (2010)

The isoflavone cream was applied twice daily (in the of proteases, specifically elastase (Brincat et al. 2005).
morning at a concentration of 0.0075 % isoflavone Systemic HRT is able to accelerate healing of acute
and in the evening at a concentration of 0.015 % cutaneous wounds in elderly females, linked to its
isoflavone) on the face, neck and one upper arm, potent anti-inflammatory activity. However, the ben-
during 12 weeks. Skin dryness and roughness were eficial effects on skin wound healing are primarily
significantly improved on the treated areas and facial mediated by epidermal ER-b, promoting restoration of
wrinkles were significantly reduced. Moraes et al. the epidermis (Campbell et al. 2010). In this context,
(2009) conducted a randomized, double-blind study of genistein has been considered a good candidate for the
topical administration of estradiol (0.01 %) and 40 % treatment of wounds, since different administration
isoflavones (4 % of which was genistein) on volun- routes have shown wound repairs. Hwang et al. (2001)
teers for 24 weeks. The facial skin was evaluated by reported that topical genistein treatment in rats (1 mg/
biopsy, before and after treatment, to determine 200 g/day and 4 mg/200 g/day) increased the produc-
epidermal thickness parameters and the number of tion of collagen in wounds after 14 days of treatment.
dermal papillae, fibroblasts, and blood vessels. The Emmerson et al. (2010) demonstrated that the subcu-
results showed that women treated with estradiol taneous administration of genistein (50 mg/kg/day) in
experienced more effective results than those treated ovariectomised female mice increased the capacity of
with isoflavones, but the authors reported that addi- wound healing by different pathways. The mecha-
tional studies need to be carried out to confirm the nisms reported were the reduction of inflammation
behavior of collagen, elastic fibers, and glycosamino- along with an increase in fibroblast and macrophage
glycans present in the skin after topical treatment. In migration, which occurred through non-ER depen-
this context, another clinical study was conducted dence, and an increase in keratinocyte migration,
recently by Patriarca et al. (2013) in which 30 post- which occurred through the ER mechanism. Park et al.
menopausal volunteers applied a gel with estradiol (2011) reported the effect of oral genistein treatment
(0.01 %) or genistein (4 %) for 24 weeks. Afterwards, (0.1 %) in mice during 2 weeks in the initial stages of
skin biopsies showed an increased concentration of wound healing. These authors showed the antioxidant
hyaluronic acid in the patients in both treatments. actions of genistein in the tissue repair process,
Moreover, several works have examined the observing a decrease in Cu, Zn-SOD and Mn-SOD
molecular role of estrogen on the cells and metabolic expression and a modulation in ROS production
processes involved in wound repair (Ashcroft et al. during the early stage of wound healing.
1999; Brincat et al. 2005). Age related delays in Conversely, isoflavones have shown potent anti-
wound healing have been partially attributed to low carcinogenic effects that are largely independent of
levels of transforming growth factor-b1 (TGF-b1), their estrogenic activities (Pinnel 2003). Alternative
decreased collagen synthesis, and increased presence mechanisms that promote chemopreventive effects

123
Phytochem Rev (2015) 14:849–869 853

have been suggested, including induction of cancer growth factor receptor phosphorylation, and suppres-
cell differentiation, inhibition of protein kinases, sion of oncoprotein expression. A clinical study
suppression of angiogenesis, and direct antioxidant indicates that genistein (5 lM genistein/cm2 human
effects (Wei et al. 2003). These alternative pathways skin) effectively protects human skin against UVB-
may occur at isoflavone concentrations much higher induced skin photodamage (Wei et al. 2003). Anal-
([5 lM) than the concentrations at which estrogenic yses of data from six subjects showed that pre-UVB
effects are detected (\100 nM), and show a different application of genistein significantly inhibited both
structure–activity relationship (Kuiper et al. 1998). cutaneous erythema and discomfort whereas post-
Many in vitro and in vivo studies, as well as a UVB application improved the discomfort score with
clinical study with volunteers, show the topical effect a minimal effect on erythema (Wei et al. 2003). A
of genistein on cancer caused by UV-B radiation (Wei phase II clinical trial is underway to determine the
et al. 2003). UV-B from sunlight is closely associated effect of genistein together with interleukin-2 in
with skin diseases such as melanoma and non- patients with metastatic melanoma or renal clear cell
melanoma skin cancer. It produces inflammation and carcinoma. This trial is based on the assumption that
proliferation in skin, both mediated in part by genistein may stop the growth of tumor cells through
activation of protein kinase C and metabolites of its antiangiogenic effect, whereas interleukin-2 may
arachidonic acids (Wei et al. 2002). Evidences from stimulate the immune system to kill tumor cells
studies of the effect of isoflavones, especially geni- (http://www.clinicaltrials.gov).
stein, in cultured tumor cells have demonstrated Table 1 shows a number of pre-clinical studies that
antiproliferative activity due to the inhibition of summarize diverse anti-aging and antiphotocarcino-
certain enzymes, such as tyrosine kinase and topoi- genic actions of isoflavones. It is noteworthy that most
somerase II (Akiyama et al. 1987; Okura et al. 1988). of the studies have reported biological activity for the
The in vitro antiphotocarcinogenic effect of genistein, aglycone forms of isoflavones. Once in the skin,
including the increase of tyrosinase activity and enzymes that hydrolyze the glucosylated molecules
dendrite-like structure formation, was reported by are not found (Schmid and Zulli 2002), contrary to
Kiguchi et al. (1990) in five human melanoma cell what happens when isoflavones are taken orally, in
lines after 6 days of treatment at a concentration of which their transformation takes place while passing
45 lM. Wang et al. (2002) reported that genistein and through the intestinal tract (Sánchez-Calvo et al.
daidzein exerted multiple suppressive effects in 2013). Therefore, when extracting isoflavones
murine and human melanoma cells, including growth intended for topical use, it is important to carry out
inhibition, cell cycle arrest, and induction of cell procedures that allow the increase of aglycone forms,
differentiation effects. The genistein treatment such as soybean extraction followed by hydrolysis and
resulted in arrest of melanoma cells in the G2 phase purification.
of the cell cycle, whereas daidzein, which lacks a
hydroxyl group, induced cell accumulation in the G1
phase. Genistein also induced melanoma cells to Obtaining bioactive isoflavones from soybeans
acquire dendrite-like structures and increased the
production of melanin. In contrast, daidzein only General considerations
retarded the growth of these cells and failed to induce
differentiation, suggesting that these isoflavones can Morphologically, soybeans are generally composed of
inhibit certain malignant phenotypes of melanoma via a seed coat (integument), cotyledon, radicle, hypo-
different mechanisms (Wang et al. 2002). Studies in cotyl, and hilum (Thorne 1981), and isoflavones are
animals have shown that genistein inhibits the UVB- diversely distributed in these parts depending on the
induced skin carcinogenesis and photodamage (Wei degree of the soybeans maturation or variety (Eldridge
et al. 1993, 1995b, 1998, 2002). The possible mech- and Kwolek 1983; Cho et al. 2009; Yuan et al. 2009;
anisms of its action include the inhibition of tyrosine Britz et al. 2011) as shown in Table 2.
protein kinase, the anti-inflammatory pathways, scav- However, when viewed as a whole, the soybean
enging of ROS, blocking of oxidative and photody- typically has isoflavone concentration between 1 and
namic damage to DNA, downregulation of epidermal 2 mg/g, predominantly in the malonyl-glucoside and

123
854 Phytochem Rev (2015) 14:849–869

Table 1 Pre-clinical studies concerning the action of isoflavones on the skin

Samples Evaluation Method Conclusions References

Genistein solution Antioxidant activity In vivo ; H2O2 production Wei et al. (2002)
after acute and Hairless mice ;MDA production
cronic UV-B
exposure ; 8-OHdG production

Isoflavones aglycone Inhibition of retinoid- In vitro ; hyperplasia Varani et al. (2004)

solutions or soy extract induced epidermal Keratinocyte ; keratinocyte
hyperplasia culture cells proliferation
(HDK) : fibroblasts
Fibroblast culture : synthesis of type I
cells (HDF) procollagen
o/w emulsion containing Effects on parameters In vitro : collagen Sudel et al. (2005)
2 % of genistein, of skin aging after Fibroblast culture : hyaluronic acid
daidzein or soy extract exposure to UV-A cells (HDF)
radiation : glycosaminoglycan

Isoflavones aglycone Keratinocytes damage In vitro ; H2O2 production Huang et al. (2008)
solutions after exposure to Keratinocyte Keratinocyte protection
UV-B radiation: culture cells
production of (HaCaT)
intracellular H2O2
and cell viability In vivo
Hairless mice
Isoflavone aglycone Photoprotection after In vivo Decrease of sunburn cell Lin et al. (2008)
solutions UV-B exposure Pig skin formation and/or
erythema
Isoflavones aglycone Photoprotection after In vitro ; oxidative stress Huang et al. (2010)
solutions or Soy extract UV-B exposure Keratinocyte ; inflamation
rich in aglycones and culture cells
acetyl-glucosides Keratinocyte protection
(HaCaT)
Decrease of erythema
In vivo
Hairless mice

H2O2 hydrogen peroxide, MDA malondialdehyde; 8-hydroxy-20 -deoxyguanosine, o/w oil in water, : increase, ; decrease

glucoside forms. The aglycone forms are rarely seen in isoflavones, it is important to note that the glucosy-
the fresh plant; however, some by-products may lated forms are soluble in water, whereas the aglycone
exhibit higher levels of these free forms due to a forms are hydrophobic, although they are highly
chemical change that takes place in the compounds soluble in polar organic solvents, such as ethanol
during production, which involves heat and/or a and methanol (Albulescu and Popovici 2007).
fermentation process (Barnes 2010). The pretreatments of soybeans are very important,
The presence of heat during soybean extractions or because they can actively influence the removal of the
food processing may trigger chemical changes, with chemical compounds of interest. The most frequently
the decarboxylation of malonyl-glucosides to acetyl- used processes for soybeans and their products are
glucosides and breakdown of the ester bond being drying, grinding, sieving, and degreasing (Becker
frequently observed, the latter leading to the formation 1978; Rostagno et al. 2009).
of glucosides. However, sugar hydrolysis for conver-
sion to the aglycone forms only takes place under Techniques for extracting isoflavones
conditions of extreme pH or in the presence of certain from soybeans
enzymes (Albulescu and Popovici 2007; Schwartz and
Sontag 2009; Barnes 2010). Among the most commonly used solid–liquid extrac-
Among the sample physicochemical properties, tion techniques for soybean extraction, the traditional
polarity is a key extraction factor. When working with ones such as reflux, Soxhlet, static and dynamic

123
Phytochem Rev (2015) 14:849–869 855

Table 2 Anatomical Soybean variety Soybean Total isoflavone References

distribution of isoflavones part content (mg/100 g)
in different samples of
soybeans USA—Tiger Hull 20.0 Eldridge and Kwolek (1983)
soybeans (TS-280) Hypocotyl 1,405.2
Cotyledon 319.2
Green soybean (AGA3) Radicle 203.04 Phommalth et al. (2008)
Cotyledon 102.03
Seed coat 61.7
Korea—Sprouts Whole sprout 144.0 Cho et al. (2009)
Hypocotyl 53.0
Cotyledon 218.0
Root 202.0
China—Yellow soybean Seed coat 1.09 Yuan et al. (2009)
Hypocotyl 159.4
Cotyledon 1,239.2

maceration, and such unconventional ones as ultra- regardless of the solvent used. According with Carrão-
sound, microwave, supercritical fluid, and extraction Panizzi et al. (2002), soybean flour extraction with or
with pressurized liquid, are the most noteworthy without constant agitation at room temperature, both
(Vacek et al. 2008; Rostagno et al. 2009; Luthria and with ethanol 70 % (v/v), had similar results for
Natarajan 2009). isoflavone concentration after 1 or 24 h of extraction;
The earliest studies of extraction of soy isoflavones however, comparing the average total isoflavone
reported extensive use of Soxhlet and reflux (Walter recovery, the authors reported the highest isoflavone
1941; Naim et al. 1974). Reflux is a widely described concentration when extraction was performed with
technique for laboratory use, facilitating routine agitation. A technique involving shaking and heating
analysis because it is an easy and quick process that is often described for soybeans (Rostagno et al. 2009)
allows high yields during extraction. Despite having and, more recently, was optimized by Zhang et al.
some similar features to reflux, Soxhlet has the (2007). The use of high temperatures during dynamic
advantage to exhaust the drug extraction when maceration usually modifies the prevalent isoflavone
subjected to a prolonged period of time with a suitable forms extracted, but it is not normally used to increase
solvent (Seidel 2006). However, both techniques have their total yield of isoflavones at the end of process
the disadvantage of using heat with a refrigeration (Coward et al. 1998).
system and, therefore, rarely used in obtaining extracts Another conventional method widely described for
on a large scale. soybeans extraction is the ultrasound process, which
Publications began appearing in the 1980s report- allows high yields of isoflavones (Rostagno et al.
ing conditions for optimizing the extraction of 2009). Ultrasound extraction, in general, is made
isoflavones aimed at ensuring better performance, through the use of sound wave energy generated at
reliability, and making it easier to enhance the specific frequencies (20–100 kHz), which causes
production scale. Thus, the use of static and dynamic cavitation in the sample, generating ruptures that
maceration began to be explored, because it enables allow the chemical constituents to be extracted from
greater large-scale use (Murphy 1981; Carrara et al. the matrix. Its advantages over other techniques are
2009). Murphy (1981) published one of the earliest the speed of extraction and low solvent consumption,
studies of soy extraction by dynamic maceration at which makes this technique specifically applicable to
room temperature and showed that the 2-h-long analyzing the raw material in laboratories, but the
extraction process was similar for isoflavone recovery drawback is the difficulties concerning scaling up
when compared with the Soxhlet extraction process, (Seidel 2006). Rostagno et al. (2003) optimized the

123
856 Phytochem Rev (2015) 14:849–869

ultrasound extraction (200 W and 24 kHz) of soy- best results were obtained using 100 atm, 100 °C, three
beans and evaluated variables such as the type of static cycles at 7 min each, and ethanol 70 % in water.
solvent, with ethanol, methanol, or acetonitrile at When extraction is done using microwaves, sub-
concentrations of 30–70 % in water, temperature of 10 stances are extracted from the matrix through changes
or 60 °C, a drug ratio of 0.5 g per 25 mL of solvent, in their cellular structures due to the action of
and time of 10, 20, or 30 min. For quantification electromagnetic waves (0.3–300 GHz), with heat
purposes, the best conditions for extracting all pro- generation. This allows the solvent molecules to be
posed isoflavones were a temperature of 60 °C and exposed, resulting in high yields at the end of the
50 % ethanol for 20 min. process (Rostagno et al. 2007; Wijngaard et al. 2012).
Other techniques aimed at increasing extraction Rostagno et al. (2007) evaluated the extraction of
yields, process speed, and duplication of results have isoflavones on a laboratory scale using microwaves at
been evaluated for the extraction of soy isoflavones. 500 W. Different situations were assessed in that
Extractions with supercritical fluid, microwaves, or study, such as the use of methanol or ethanol at
pressurized solvents are examples of these techniques, different concentrations (30–70 %), temperature
and for this reason, they were recently optimized by between 50 and 150 °C, amount of sample and
Rostagno et al. (2002, 2004, 2007). solvent, and extraction time (5–30 min) in samples
Supercritical fluid extraction employs a fluid in a of ground, lyophilized soybeans. The best extraction
supercritical state that has intermediate properties of all forms of isoflavones was: 0.5 g of the drug and
between gas and liquid, with carbon dioxide (CO2) 25 mL of 50 % ethanol at 50 °C for 20 min.
being the most common solvent used. Despite the high In summary, there is a vigorous motivation to find
cost of purchasing the equipment, this technique is different conditions for extracting isoflavones from
being increasingly used as it offers benefits such as soy. Conventional techniques are generally slower,
high precision and sensitivity, good selectivity, low whereas modern ones are considerably faster. How-
solvent consumption, speed, and safety (Rostagno ever, isoflavone yield needs to be taken into account to
et al. 2002; Sovová and Stateva 2011). Rostagno et al. assess the real advantage between the different
(2002) evaluated this procedure for extracting soy techniques, along with the possibility of scale up.
isoflavones on a laboratory scale, using soybean seeds, Table 3 shows several comparative studies on differ-
static extraction for 10 min, followed by dynamic ent methods of isoflavone extraction from soybean
extraction for 20 min at different temperatures seeds. Thus, it may be suggested that extraction by
(40–70 °C), pressures (200–360 bar), and percentage microwave, ultrasound, and pressurized solvent are
of the modifier (0.5 and 10 % ethanol 70 %). The best the most predisposed candidates for extracting iso-
condition for all compounds was 1 g of the drug and flavones from soybeans.
55.2 g of CO2, with 10 % of ethanol: water modifier
(7:3, v/v). Hydrolysis of isoflavones
A highly efficient extraction may be carried out
using the pressurized solvent extraction technique, Hydrolysis of isoflavones is a procedure that aims at
which employs high temperatures and pressures rang- transforming conjugated compounds into their free
ing from 50–200 °C and 40–200 bar, respectively. forms (Lee et al. 2008) and is interesting for two
These conditions allow a high extraction of compounds, reasons: obtaining their active forms in the extract
given that this technique facilitates the diffusion of the when the goal is the incorporation into products
solvent from the pores of the matrix, increasing mass (medicines and cosmetics) or reducing the time of
transfer to the liquid extractant (Rostagno et al. 2004; quantitative analysis in routine laboratory quality
Wijngaard et al. 2012). Rostagno et al. (2004) con- control by simplifying the number of substances to
ducted a study to optimize isoflavone extraction using be analyzed (Lee et al. 2008; Schwartz and Sontag
the pressurized solvent technique on a laboratory scale. 2009).
Parameters evaluated were the type of solvent (ethanol For analytical purposes or the industrial production
or methanol at concentrations of 30–80 %), tempera- of isoflavones from soybean, the literature describes
ture (60–200 °C), pressures (100–200 atm), amount of three types of hydrolysis: acidic, basic, and enzymatic
sample (0.5–0.05 g), and cycle time (5–10 min). The (Schwartz and Sontag 2009). Acidic and enzymatic

123
Phytochem Rev (2015) 14:849–869 857

Table 3 Comparative studies on different methods of isoflavone extraction from soybean seeds
Techniques Experimental conditions Best conditions based on isoflavone References
yield extraction

SE SE: 80 % MeOH, 9 h US-B Rostagno et al.

US-B US-B: 22 kHz, 80 % MeOH (3 cycles, 1 h each one) (2002)
SFE SFE: 40–70 °C; 200–360 bar CO2, with 10 % modifier
agent
US-B Different combination of solvents, time and temperature US-B Rostagno et al.
US-P 50 % EtOH, 60 °C, 20 min (2003)
HSE
PLE PLE: hexane followed of MeOH during 2 cycles of PLE ? US Kledjus et al.
5 min, 145 °C, 140 bar (2005)
US US-P: 38 kHz, 90 % MeOH, 5 min
SE SE: 90 % MeOH, 70 min
US-P US-P: kHz, 50 % EtOH, 60 °C, 20 min Comparable yields Rostagno et al.
MW MW: 500 W, 50 % EtOH, 50 °C, 20 min (2007)
Different solvents by:
VE VE: 1 min PLE Luthria et al.
SHE SHE: 60 min DMSO:ethanol: water (5:75:25, v/v/v) (2007)
US-B US-B: 15 min
SE SE: 3 h
PLE PLE 1,000 psi, 100 °C (3 static cycles, 7 min each one)
Different solvents by:
SHE SHE: 15 h US: 50 % acetone Chung et al.
SE SE: 4 h, 80 °C (2010)
US US: 40 W, 3 min
DMSO dimethylsulfoxide, EtOH ethanol, HSE hot and shaking extraction, MeOH methanol, MW microwave extraction, PLE
pressurized liquid extraction, SHE shaker extraction, SE Soxhlet extraction, SFE supercritical fluid extraction, US ultrasound
extraction, US-B ultrasonic bath extraction, US-P ultrasonic probe extraction, VE vortex extraction

hydrolysis is responsible for the breakdown of the the procedure are optimized; and enzymatic hydro-
isoflavone between the glucoside and aglycone, lysis, in which the choice of the enzyme varied
resulting in a higher yield of the latter. Conversely, considerably in an attempt to find the best yields and
in basic hydrolysis, breakdown takes place in the ester percentage of isoflavone recovery (Rostagno et al.
parts of the compound, resulting in greater conversion 2009).
for the glucosylated forms (Schwartz and Sontag The best known and most frequently used acid for
2009; Lee et al. 2008). the hydrolysis of isoflavones is hydrochloric acid
Some studies attempting to compare the best (HCl); while for enzymatic hydrolysis, the most
conditions to obtain the isoflavone aglycones (IA) frequently used enzymes are b-glucosidase (from
after acidic or enzymatic hydrolysis can be found in almonds or Escherichia coli) and b-glucuronidase
the literature as shown in Table 4. However, the (from Helix pomatia). However, in recent years, there
choice of technique is always in accordance with the has been an increase in the number of studies for the
intended purpose (Schwartz and Sontag 2009). For discovery of new sources of those enzymes involved in
analytical purposes or quality control in laboratories, the process of transforming isoflavones into their active
studies can be found with acidic hydrolysis, in which forms. Some examples are b-glucosidase from Asper-
the concentration of acid, time, and temperature of gillus oryzae, Paecilomyces thermophila, Thermotoga

123
858 Phytochem Rev (2015) 14:849–869

Table 4 Comparative studies for different methods of isoflavone hydrolysis

Samples Type of hydrolysis Best conditions to obtain References
isoflavone aglycones

Soybeans seeds and tofu Acid Acid hydrolysis, during 1 h for Franke et al.
Reflux of 1.0 g using 50 mL of 2 M HCl in genistein and 2 h for daidzein (1994)
77 % ethanol during 1–4 h
Enzimatic
Reflux of 1.0 g using 50 mL of 77 % ethanol
during 3 h, solvent evaporation and addition of
pH 5.0 acetate buffer ? 2 mg
b-glucosidase from almonds ? 40 lL
b-glucoronidase from Helix pomatia during
24 h at 37 °C
Soybeans flour Acid Enzimatic with cellulase from Liggins et al.
2.5 g in 5 mL of 80 % methanol, sonication A. niger (1998)
during 10 min followed by addition of 3 M
HCl and reaction during 2 h at 80 °C
Enzimatic
5,000 U of b-glucuronidase from H. pomatia in
5 mL of pH 5.0 acetate buffer
Enzimatic
100 U of cellulase from Aspergllus niger in
5 mL of pH 5.0 acetate buffer
Enzimatic
100 U of b-glucosidase from almonds in 5 mL
of pH 6.8 acetate buffer
Note for the enzimatic procedures, the reaction
was conducted overnight in a concentration of
0.5 mg/mL
Standards of isoflavones Enzimatic 30 U of b-glycosidase from Ismail and
3, 6 or 30 U of b-glycosidase from E. coli in pH E. coli during 4 h Hayes
7.0 phosphate buffer at 37 °C (2005)
Enzimatic
3, 6 or 30 U of b-glycosidase from almonds in
pH 7.0 phosphate buffer at 37 °C
Note Reactions using standards solutions at
500 ppm
Soybeans seeds, tofu, soybean Alkaline All treatments showed an Shao et al.
milk or soybeans cereals 400 lL of extract ? 30 lL of 2 M NaOH equivalent quantification for (2011)
during 10 min at room temperature the isoflavones evaluated,
however only the acid and
Acid
enzimatic hydrolysis
100 lL of extract ? 900 lL 1.3 M HCl at converted all them until the
80 °C during 2 h free forms
Enzimatic
1 mL of dry extract followed by addition of
1 mL of enzimatic solution (1 mg/mL de b-
glucuronidase, type H-5 from H. pomatia in pH
5.0 phosphate buffer) during 16 h at 37 °C
Note extracts were obtained by shaking 1.0 of
sample with 10 mL of 80 % methanol during
12 h

123
Phytochem Rev (2015) 14:849–869 859

maritime, Pyrococcus furiosus, and Sulfolobus solfa- latter process. After purification, the antioxidant
taricus (Aguiar and Park 2004; Yang et al. 2009; Sun activity was studied within the range of 0.1–10 mM,
et al. 2010; Song et al. 2011; Yeom et al. 2012; Kim demonstrating that at no less than 1 mM concentra-
et al. 2012; Kuo et al. 2012). tions, the rich IA fraction inhibits phospholipid
Some studies have reported the optimization of the peroxidation registered through the peroxide forma-
hydrolysis process to successfully convert the isoflavone tion and malonic dialdehyde accumulation. Zhang
glucosides into their free forms. Chiang et al. (2001) et al. (2007) optimized the extraction of isoflavones
optimized the acid hydrolysis conditions of soybean from soybean flour, followed by the optimization of
hypocotyls by response surface methodology (RSM). acid hydrolysis. The best condition to obtain the IA
Optimum hydrolysis conditions were obtained using was 0.13 M HCl in ethanolic media at 80 °C for 6 h.
3.42 M HCl at 44.6 °C for 205.5 min in a water bath, After purification, the authors not only obtained a rich
resulting in an isoflavone recovery close to 100 %. César fraction with high level of IA but also identified its
et al. (2006) optimized the acid hydrolysis efficiency of estrogenic activity at 0.4 lg of total isoflavones/mL
soybean flour at five different concentrations of HCl (1.0, (0.84 lM of genistein and 0.64 lM of daidzein) by the
1.5, 2.0; 2.5, and 3.0 M) at different hydrolysis times. MTT assay using MCF-7 cells. Deshmukh and Amin
After sonication for 5 min, the highest IA content was (2013) obtained a rich IA fraction from a commercial
achieved after 40 min of steam bath (temperature not soy extract by refluxing the extract with 4 M HCl in an
indicated), using a 3.0 M HCl in ethanolic solution. ethanolic media for 2 h followed by purification. The
Using the Plackett–Burman design and RSM, Tipkanon antioxidant activity was studied within the range of
et al. (2010) optimized the enzymatic hydrolysis condi- 50–500 lg/mL, demonstrating that at a concentration
tions of soybean flour using the b-glucosidase enzyme. of 125 lg/mL the fraction was able to inhibit 50 % of
The optimized process was obtained with a proportion of DPPH reaction.
soy: deionized water (1:5, w/v), b-glucosidase at 1 U/g of
soy flour, pH 5, and incubation temperature/time of Extract purification
45 °C/5 h. Lee and Choung (2011) studied the optimal
acid hydrolysis condition using drying oven and micro- To obtain isoflavone-rich extracts, subsequent extrac-
wave assisted methods. All isoflavone glucosides were tion steps may be carried out to purify the sample by
completely converted into their aglycones at 120 min removing impurities and making it highly concen-
with drying oven and 50 min with microwave using 1 M trated in isoflavones. Such steps include centrifuga-
HCl at 100 °C. Shao et al. (2011) studied the acid and tion, precipitation, ultrafiltration, passage through
enzymatic hydrolysis processes to compare the conver- columns containing specific resins, or purification by
sion of isoflavone glucosides into aglycones. The opti- polarity selectivity (Murphy 1981; Xu et al. 2004; Cho
mized acid method (1.2 M HCl in ethanolic media, et al. 2009).
80 °C, 2 h) converted only 92 % of isoflavones, while Among these processes, the solid phase extraction
the enzymatic method with 1 mg/mL of b-glucuronidase technique has been well-described for the purification
(from H. pomatia) effectively converted 100 % of of soy extracts on a laboratory scale. This is based on
isoflavone into their aglycone forms at 37 °C, pH 5.0 separating compounds by the interaction between a
for 16 h. stationary and a mobile phase through different
Moreover, a few studies have reported the hydro- physical–chemical phenomena. To purify soy isoflav-
lysis processes of soybeans as a way to obtain a rich IA ones, the use of different types of adsorbents, such as
fraction to be used in the pharmaceutical field. Utkina silica gel or C18 polymeric materials, such as Diaion
et al. (2004) optimized the hydrolysis conditions to HP-20 and Amberlite XAD16, has been reported in the
obtain a rich IA fraction from a soybean supplement literature (Thorne 1981; Hirota et al. 2004; Li-Hsun
(NovaSoyÒ). The authors studied the enzymatic et al. 2004).
hydrolysis using Aspergillus heteromorphous 3010, The liquid–liquid partition in conjunction with
or the acidic hydrolysis of the glycosides in ethanol solid phase extraction is a complementary alternative
solution using 1 M HCl for 2 h at 100 °C, and the acid for purifying soybean extract on a laboratory scale, as
hydrolysis of glycoside powder using 6 M HCl for 5 h demonstrated in the study by Cho et al. (2009). The
at 100 °C. The best IA yield was achieved with the authors used three purification steps: passing the

123
860 Phytochem Rev (2015) 14:849–869

hydroethanolic extract through a column of Diaion procedure consisting of ethanol precipitation, ethyl
HP-20, followed by acid hydrolysis, and liquid–liquid acetate liquid–liquid extraction, and water rinse. The
partition with an appropriate solvent polarity. A good final amount of aglycone isoflavones obtained was
recovery of genistein and daidzein was obtained by 1.279 mg/g of defatted soybean.
enriching the extract by about 20 times when using
solvent partition with ethyl ether. The final amount of Obtaining isolated isoflavones
IA obtained was 229 mg/g of fraction.
Purification of aglycone isoflavones may also occur As previously mentioned, the aglycone forms of
with other separation techniques, including those that isoflavones are of great industrial interest (Setchell
would allow scale-up production, as reported by 1998). Isoflavones may be isolated by purifying the
Zhang et al. (2007). The authors used a high polar soybean extract or they may also be purchased
solvent, such as water, to turn the aglycone forms commercially, since their synthesis routes have been
insoluble in the medium, thereby causing their reported for these compounds (Baker and Robinson
precipitation, which, with subsequent filtration and 1928; Baker et al. 1933; Antus et al. 1975).
drying of the residue, resulted in obtaining an enriched Genistein was first isolated from a plant called
fraction of these substances. This study was carried out Dyer’s Broom (Genista tinctoria) by Perkin and
to optimize the amount of antisolvent, and it was Newbury (1899); three decades later, it was synthe-
concluded that, by using a 1:4 ratio (hydrolyzed sized by Baker and Robinson (1928). In soybeans,
extract:water), it is possible to get a high recovery of genistein was first isolated by Walz (1931), and its
genistein and daidzein, thereby showing the process to physical and chemical characteristics were reported by
be efficient in obtaining a fraction with isoflavone Walter (1941), who studied and further described this
content twice that of the extract prior to the process. compound. Daidzein was also isolated from soybeans
The final amount of IA obtained was 0.87 mg of total by Walz (1931), and its synthesis was successfully
isoflavones (aglycone equivalency)/g of soybeans. accomplished by Baker et al. (1933). Glycitein was
Ultrafiltration is an easily scale-up purification isolated years later by Naim et al. (1973). This
process, in which the choice of filters may play a role compound is a minor isoflavone in soybean; therefore,
in selecting compounds with regard to the separation it is the most difficult to obtain. Glycitein was
of interesting substances. The study performed by Xu successfully synthesized few years later (Antus et al.
et al. (2004) reveals the efficacy of this process 1975).
concentrating isoflavones from soybean milk. The Even though soy isoflavone aglycones are individu-
process consisted of centrifuging the aqueous extract ally available for purchase, studies have described their
of soybean seeds, passing it through a 30,000 Da isolation from soybeans. Farmakalidis and Murphy
cellulose membrane, diafiltratering, followed by pass- (1984) isolated genistein and daidzein from soybean by
ing it through a 1,000 Da cellulose membrane, and a semi-preparative high-performance liquid chromato-
finally passing it through a reverse osmosis membrane; graphic method. First, the flakes were extracted with
in the latter step, only substances that were not of acetone 0.1 M HCl, passed through silica gel column
commercial interest were able to permeate, with only eluted with chloroform–methanol with increasing polar-
isoflavones being retained in the filter. This process ity. Further, isoflavones have been isolated by the semi-
allowed for increased isoflavone concentration, result- preparative chromatography method using a reversed-
ing in a final IA amount of 10.9 mg/g of fraction. phase 250 9 9.4 mm Partisil ODS-3 column, a non-
Recently, Wang et al. (2013b) developed a green linear methanol–water gradient, and flow-rate at 5 mL/
strategy to obtain IA from soybean. First an ethanol- min. Yang et al. (2001) reported the isolation of daidzin,
alkaline extraction method was designed and opti- glycitin, genistin, acetyldaidzin, glycitein, acetylgeni-
mized. The high extraction yield of isoflavones was stin, and daidzein from soybeans using high-speed
achieved by the optimal extraction conditions of pH counter-current chromatography. Three solvent systems
9.0 at 70 °C for 60 min, using ethanol 65 % (v/v). were used: chloroform–methanol–water (4:3:2, v/v);
Next, enzymatic hydrolysis with cellulase (GC-220) chloroform–methanol-n-butanol-water (4:3:0.5:2, v/v);
gave an excellent conversion of 95 %. Lastly, the and methyl tert-butyl ether-tetrahydrofuran-0.5 %
crude isoflavone aglycones were purified by a aqueous trifluoroacetic acid (2:2:0.15:4, v/v). The

123
Phytochem Rev (2015) 14:849–869 861

isolated compounds presented 98–99 % purity. More

recently, Wang et al. (2013a) reported a chromato-
graphic method for isolating and purifying isoflavones
from soybean extracts by using 12 % cross-linked
agarose gel as the separation media, and methanol at
concentrations of 30 % or 65 % as gradient elution.
Soybean extracts were separated by the proposed
method and six soy isoflavones, including glycitin,
daidzin, genistin, glycitein, daidzein, and genistein,
were obtained with purity higher than 97 %.

Incorporation of isoflavones into topical systems

As shown in Fig. 3, both commercially obtained pure

substances and the extract obtained without the
hydrolysis step (preferably containing glucosylated
forms), or hydrolyzed and purified extracts (primarily
containing aglycone forms), can be incorporated into
different types of topical formulations.
When developing products (medicines and cosmet- Fig. 3 Steps described in this review for the development of
topical products containing isoflavones from soybeans or
ics) for topical use, parameters such as skin character- isolated commercial compounds
istics, type of formulation, and solubility of substances,
are of great importance to maintaining product stability values (log P) ranging between 1 and 3 (Förster et al.
and allowing the active ingredients to penetrate into the 2009).
skin (Förster et al. 2009; Weiss 2011). Different vehicles used in topical formulations may
The epidermis is the first layer of the skin, whose also influence the amount and extent to which a
function is to protect the skin from the external substance is able to permeate the skin. In the case of
environment. The stratum corneum is found on the highly permeable substances, the rate of penetration
surface and consists of dead cells that function as a can be controlled by using a suitable carrier. In these
protective lipophilic barrier. When a topical formula- conditions, the diffusion of the active ingredient in the
tion is administered, it is precisely this barrier that formulation is the limiting step. Therefore, the partition
active substances must penetrate to effect action on the coefficient of a substance of low solubility in water
skin (Förster et al. 2009). between a hydrophilic matrix and the stratum corneum
In this context, it is important to point out that skin promotes its migration to the latter, characterizing a
penetration depends on the physicochemical proper- good ‘‘transfer’’ to the skin (Förster et al. 2009).
ties of each substance, their behavior when added to a It has been postulated that the highly hydrophilic
formulation, and, last but not least, the conditions of isoflavone glucosides will have difficulty penetrating
the user’s skin (Förster et al. 2009). the stratum corneum, whereas genistein, daidzein, and
The physical and chemical characteristics of the glycitein have a tendency to more easily penetrate this
active substances are relevant for predicting behavior layer because they have values near the partition
after cutaneous application. Those with a high coefficients of 2.98, 2 78 and 2.57, respectively (these
hydrophilicity will have difficulty penetrating the values are determined through the ACD/I-Lab online
skin. Conversely, if they have high lipophilicity, they service) (http://www.acdlabs.com). However, even
will tend to be retained in the formulation when it is with a suitable log P, the type of formulation will
also lipophilic in nature. For this reason, it is influence their penetration, a parameter that should
important that the substance has characteristics that always be taken into account when developing a
allow its partition, with favorable partition coefficient product.

123
862 Phytochem Rev (2015) 14:849–869

Conventional topical systems It is worth mentioning that the glucosylated forms

may have unfavorable penetration, due to their high
In traditional cosmetics systems, cream type formu- water solubility, in addition to not being metabolized
lations are widely used, and the choice of system is into active forms due to the lack of specific enzymes in
determined by the solubility of the active substance to the skin. This situation was reported by Schmid et al.
be incorporated. Thus, when the compound is hydro- (2003) through a clinical study with ten volunteers,
philic, it will be more readily incorporated into oil-in- showing no transformation of genistin into genistein in
water (o/w) emulsions and, when hydrophobic, in the skin’s outer layers after topical application of the
water-in-oil (w/o) emulsions. However, the latter is glucosylated form.
characterized as a more oily formulation and, there- Kitagawa et al. (2010) developed two types of
fore, less accepted by users (Weiss 2011). microemulsions with the intent of incorporating
In the case of incorporating isoflavones into these genistein, daidzein, and biochanin A. The formula-
types of emulsions, two situations can be observed: (1) tions were composed of the following components:
when the purpose is to incorporate glucosylated forms, isopropyl myristate, aqueous solution containing
o/w emulsions may be used, although penetration sodium chloride (NaCl) 150 mM, Tween 80 and
could be somewhat difficult, and (2) when the goal is ethanol, which, when in a ratio of 8:25:20:47,
to use aglycone forms, the use of w/o emulsions is constituted an o/w emulsion, and when in a ratio of
more described, despite lower user acceptance (Sch- 33:7:30:30, formed a w/o emulsion. Based on these
mid et al. 2003; Georgetti et al. 2006; Kitagawa et al. results, the authors concluded that the best formulation
2010). for isoflavones was the one that used the second ratio,
In a study by Georgetti et al. (2006), four o/w given that the substance solubility was higher, pro-
emulsion formulations were prepared with the aim of viding better system stability and greater cutaneous
incorporating 2 % commercial extract, containing retention of isoflavone aglycone in porcine skin.
both glucosylated and aglycone isoflavones (Isoflavin
BetaÒ). These formulations were evaluated using the Topical systems involving different technologies
physicochemical parameters of the products and
in vitro antioxidant activity. The systems comprised Due to the poor water solubility of the active forms of
distilled water, methylparaben, propylparaben, mac- isoflavones, several alternative technologies have
adamia oil, propylene glycol, imidazolidinyl urea, and been studied to improve this feature and make it
a self-emulsifying base (PolawaxÒ or CrodabaseÒ) possible to incorporate them into hydrophilic phar-
with or without carboxypolymethylene as a stabilizer. maceutical forms. Some of them are listed in Table 5,
Among these formulations, all showed a similar which describes the technologies and type of sample
antioxidant potential when in the presence of isoflav- used.
ones, but the formulation consisting of CrodabaseÒ Delivery systems containing active substances can
without the added stabilizer was found to be unstable be formed by (1) incorporation in micelles and
(Georgetti et al. 2006). vesicles formed by polymers, surfactants, or phospho-
In a subsequent study, the same authors evaluated lipids, and (2) complexation techniques to host
the stability of formulations containing the seemingly macromolecules. In both cases, it is possible to
more promising PolawaxÒ for 6 months, and also increase the compounds’ water solubility, making
studied the in vitro dermal absorption and in vivo them very attractive in terms of the technological
antioxidant activity of formulations containing the development of topical formulations (Förster et al.
isoflavones. The formulations were stable in the 2009). Among the main types of such systems, the
evaluated conditions (4 and 40 °C and 70 % RH, literature reports the use of liposomes, nanoemulsions,
30 °C and 70 % RH) for 6 months. The evaluation of nanocapsules, nanospheres, or cyclodextrin and den-
cutaneous antioxidant activity in mice was shown by drimer complexes. These structures offer a number of
the inhibition of lipid peroxidation and the cutaneous advantages when compared with the hydrophobic
retention in porcine skin was found to be low, as active substance, for, in addition to improving solu-
quantification was not possible, neither on the skin nor bility, they also increase stability and control delivery
in the Franz cell receptor (Georgetti et al. 2008). (Förster et al. 2009).

123
Phytochem Rev (2015) 14:849–869 863

Table 5 Technological strategies to enhance the release characteristics of isoflavone aglycones

Sample System Other constituents References

Daidzein Liposome Phosphatidilcoline Dwiecki et al. (2009)

Genistein Liposome Phospholipids, polysorbate 80 Schmid et al. (2003)
Genistein Nanoemulsion MCT, egg lecithin Silva et al. (2009)
Genistein Nanocapsule PLA Zampieri et al. (2013)
Soybean extract Microcapsule NaCMC Sansone et al. (2013)
Rich fraction of IA Solid Lipid Nanoparticle Softsan 601, polysorbate 20 Deshmukh and Amin (2013)
Genistein CD complex b-CD; hydroxypropyl-b-CD or methyl-b-CD Crupi et al. (2007)
b-CD or c-CD Daruhazi et al. (2008)
b-CD Xavier et al. (2010)
Amphiphilic CD Cannavà et al. (2010)
Genistein ? daidzein CD complex Hydroxypropyl-b-CD Stancanelli et al. (2007)
Rich fraction of IA CD complex b-CD or hydroxypropyl-b-CD Yatsu et al. (2013)
Daidzein CD complex b-CD; methyl-b-CD or hydroxypropyl-b-CD Borghetti et al. (2011)
Daidzein Dendrimers PAMAM or PPI Zhao et al. (2011)
CD cyclodextrin, IA isoflavone aglycones, MCT medium chain triglycerides, NaCMC sodium-carboxymethylcellulose, PAMAM
poly(amidoamine), PLA poly (acid lactic), PPI poly(propylene imine); Softsan 601 glyceryl cocoate (and) hydrogenated coconut Oil
(and) ceteareth—25

Liposomes are formed by a lipid bilayer primarily Like liposomes, nanoemulsions have been consid-
consisting of phospholipids, which form a colloidal ered as a potential colloidal system for topical
vesicular system. These structures have the ability to administration of hydrophobic molecules. These sys-
incorporate hydrophilic substances in their core, or tems are defined as a nano-dispersion of oily droplets
even hydrophobic ones in the phospholipid bilayer. in an external aqueous phase, stabilized by a suitable
The main benefit of this system is its biocompatibility surfactant system (Förster et al. 2009; Vargas et al.
with the skin’s outer layer, which increases the 2012).
likelihood that the active compounds will be able to A study describing the incorporation of genistein in
penetrate it (Förster et al. 2009). nanoemulsions was reported by Silva et al. (2009),
The use of liposomes to encapsulate daidzein was who developed a topical nanoemulsion containing
evaluated by Dwiecki et al. (2009), who proposed a 1 mg/mL of this substance by using the spontaneous
weak interaction of the molecule with a lipid bilayer emulsification technique. The best formulation was
composed of egg phosphatidylcholine and a greater composed of water, medium chain triglycerides
interaction with the polar portion of this membrane. The (MCT), stabilized with egg lecithin, and presented
in vitro antioxidant activity of this system was evalu- an average particle size ranging between 200 and
ated and it was found that there was an increase in such 300 nm, with association efficiency very close to
activity when compared to the free substance. Schmid 100 %. In the same study, the cutaneous permeation in
et al. (2003) conducted a study of the ability of genistein porcine skin was also studied, and the results indicated
to penetrate skin under different formulations in ten that the use of such nanoemulsions led to a delay in the
volunteers. The objective was to compare the accumu- cutaneous flow of genistein and an increase in its
lation of genistein on the skin, whether or not the retention in the skin layers. In a continuation of the
compound had been incorporated into liposomes. The study, Vargas et al. (2012) incorporated genistein
results showed that the best penetration of this isoflav- nanoemulsions in Carbopol 940Ò hydrogels dispersed
one into the stratum corneum took place when the in water at a 5 % concentration. An evaluation of
volunteers applied the formulation containing genistein genistein permeation in porcine skin once again
associated with the liposomes, thereby demonstrating showed better retention when using MCT as an oily
one of the strong points of this alternative technology. core, and the main benefit of using hydrogel is based

123
864 Phytochem Rev (2015) 14:849–869

on its better viscosity and product appearance, while and evaluating the permeation profile in nitrocellulose
maintaining the nanostructures’ physicochemical membranes and the compounds’ dissolution profile.
characteristics. The authors suggested that microparticles of isoflav-
Solid lipid nanoparticles (SLNs) are composed of ones could be potentially used for topical and oral use.
physiological and biodegradable solid lipids. SLNs Cyclodextrins (CD) are structures formed by cyclic
have the advantage of protection against chemical oligosaccharides composed of 6, 7, or 8 toroidal-
degradation when compared with liposomes and shaped dextrose molecules whose interior is charac-
nanoemulsions (which are also lipid carriers but terized as a hydrophobic cavity whereas the exterior is
without a solid structure). However, the main draw- hydrophilic. These characteristics make complexation
back of SLNs is that during storage the bioactive possible with apolar substances, thus increasing their
component can be expulsed due to a change in lipid water solubility (Förster et al. 2009; Xavier et al.
conformation to a lower energy crystal state (Förster 2010). Several studies have demonstrated the effi-
et al. 2009). Deshmukh and Amin (2013) reported the ciency of complexation of genistein with cyclodex-
production of an isoflavone aglycone-rich fraction trins, thus increasing the solubility of that particular
from commercial soy extract intended to be incorpo- isoflavone in water (Crupi et al. 2007; Stancanelli et al.
rated into a SLN-based gel at a final concentration of 2007; Daruhazi et al. 2008; Cannavà et al. 2010;
1 mg/mL. To prepare the SLN, the fraction was added Xavier et al. 2010). Among them, the efficiency of
in a warm microemulsion followed by dilution in cold complexation and characterization of complexes are
water (2–3 °C) under mechanical stirring. The well described; nevertheless, their incorporation into
selected compounds for microemulsion formation final topical formulation still needs to be evaluated as
were glyceryl cocoate (and) hydrogenated coconut well as their release behavior and activities.
Oil (and) ceteareth—25 (Softsan 601) as solid lipid. Crupi et al. (2007) obtained genistein complexes by
The microemulsions were stabilized by the surfactant using the co-precipitation technique with b-cyclodex-
tween 20. Carbopol (971 P) was selected as the gelling trin (b-CD), hydroxypropyl-b-CD, and methyl-b-CD,
agent based on compatibility with the nanoparticulate characterizing the associations by infrared and UV–Vis
dispersion, ease of preparation, and aesthetic appeal. spectrophotometry. Daruhazi et al. (2008) achieved
The SLN-based gel was characterized by pH, spread- complexation with b-CD and c-CD, with the technique
ability, rheology, bioactive content, and in vitro having enabled an association with 9.9 % genistein,
permeation using porcine ear skin model; its safety using a molar ratio of 2:1 CD-genistein, and the
was assessed using primary skin irritation studies. The complexes were characterized by nuclear magnetic
developed SLN-based gel showed about 60 % depo- resonance (1H NMR). Inclusion complexes were
sition of soy isoflavones in dermal matrix and showed prepared by Xavier et al. (2010) in an aqueous solution
no skin irritation on intact rabbit skin. with b-CD in 1:1 molar ratio, dried by lyophilization to
Nanocapsules, or nanospheres, are another type of obtain a powder with 19.22 % genistein concentration.
nanometric system that can be formed from suitable Molecular modeling studies carried out in the latter
techniques when in the presence of polymers. Zampi- work suggest that it is the isoflavone’s A ring that is
eri et al. (2013) encapsulated genistein in nanostruc- included in the cyclodextrin’s hydrophobic cavity.
tures formed with the poly (lactic acid) biodegradable Cannavà et al. (2010) also evaluated the association
polymer by the nanoprecipitation technique and of genistein to amphiphilic CD by the emulsification-
evaluated their physical and chemical characteristics, diffusion technique, with evaluation by UV–Vis,
stability, and in vitro cutaneous permeation of porcine circular dichroism, and infrared analysis. The authors
skin. Permeation studies showed that genistein found that the complexes organized themselves to
reached the deepest layers, characterizing a penetra- form nano-aggregates due to the presence of polyeth-
tion enhancement when incorporated into gel-type ylene structures, thus increasing the solubility of
formulations. isoflavones by about ten times. Stancanelli et al.
Sansone et al. (2013) described a sodium carboxy- (2007) complexed genistein and daidzein into
methylcellulose matrix obtained by spray-drying to hydroxypropyl-b-CD in an aqueous phase, and the
microencapsulate soy extract rich in isoflavones, associations were later evaluated by UV–Vis and
thereby increasing the solubility of these substances, circular dichroism. Both substances successfully

123
Phytochem Rev (2015) 14:849–869 865

complexed with CD, thereby, increasing the water present in this plant with benefits to the skin are mainly
solubility of isoflavones. genistein and daidzein, but they are more easily
Multiple complexation of a fraction enriched in extracted in conjugated forms with sugars. In this
isoflavone aglycones (genistein, daidzein, and glyci- sense, hydrolysis of glucosides present in the extracts,
tein) with b-CD or hydroxypropyl-b-CD was recently with subsequent purification to enrich the aglycone
proposed by Yatsu et al. (2013). Different techniques forms or the use of these synthetic compounds is
for obtaining complexes were studied, which were essential to developing skin permeable delivery
characterized by infrared spectroscopy, x-ray diffrac- systems.
tion, scanning electron microscopy, differential scan- However, the active forms of isoflavones are poorly
ning calorimetry, and NMR. The results showed that water soluble, often requiring the use of alternative
the complexes obtained with hydroxypropyl-b-CD technologies, such as nanotechnology, to facilitate
allowed a greater increase in solubility for isoflavones, their ability to incorporate into topical formulations of
and complexation may occur by inserting the isoflav- hydrophilic character and promote their retention
one’s A or B ring into the hydrophobic cavity of CD. within the skin.
Among the studies found in the literature concern-
ing cyclodextrin complexation with isoflavones, only Acknowledgments This work was supported by the Brazilian
Federal Agency for Support and Evaluation of Graduate
the study by Borghetti et al. (2011) reported complex-
Education (CAPES)—Rede Nanobiotec-Brasil (grant agreement
ation of daidzein aimed at its incorporation into n8 902/2009) and State Foundation for Research Support
hydrogels for topical application. The study was (FAPERGS)—PRONEM (Grant Agreement No. 11/2206-7).
conducted by using different gels, with hydroxypro- M.C.N. wishes to thank CAPES for her scholarship.
pylmethylcellulose or polyvinylpyrrolidone present-
ing results of being viable for use. In this case,
molecular modeling assumes that the B and C rings References
enter into the hydrophobic cavity of CD, contrary to
what was found for genistein in earlier studies. Aguiar CL, Park YK (2004) Conversão de daidzina e genistina
Another organized system known as dendrimers is de soja por b-glicosidase de Aspergillus oryzae. B CEPPA
22:183–195
a class of macromolecules of nanometer size with a Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe SI,
certain degree of repetition of a known grouping, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific
forming branches around a core substance. Dendri- inhibitor of tyrosine-specific protein kinases. J Biol Chem
mers have a part that allows complexation with apolar 262:5592–5595
Albertazzi P, Purdie DW (2002) The nature and utility of the
substances, while the remainder is considered polar phytoestrogens: a review of the evidence. Maturitas
and, therefore, a macromolecule with hydrophilic 42:173–185
characteristics (Zhao et al. 2011). Albulescu M, Popovici M (2007) Isoflavones—biochemistry,
Zhao et al. (2011) evaluated daidzein complexation pharmacology and therapeutic use. Rev Roum Chim
52:537–550
with dendrimers formed by poly-amidoamine (PA- Antus S, Farkas L, Kardos-Balogh Z, Nogradi M (1975) Syn-
MAM) or poly-propyleneimine (PPI). The results thesis des dalpatins, fujikinins, glyciteins und anderer
showed that both complexes effectively enhanced the natürlicher Isoflavone. Chem Ber 108:3883–3893
solubility of isoflavone, improved the pharmaceuti- Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM, Fer-
guson MW (1999) Topical estrogen accelerates cutaneous
cals’ delivery profile, prolonged their action, and were wound healing in aged humans associated with an altered
stable for 30 days. However, the complex formed with inflammatory response. Am J Pathol 155:1137–1146
PAMAM dendrimer was the best candidate to be used Baker W, Robinson R (1928) Synthetical experiments in the
for antioxidant action, as it presented less cytotoxicity isoflavone group. Part III. Synthesis of genistein. J Chem
Soc 131:3115–3118
against two cellular lines. Baker W, Robinson R, Simpson NM (1933) Synthetical
experiments in the isoflavone group. Part VII. Synthesis of
daidzein. J Chem Soc 1933:274–275
Conclusions Barnes S (1998) Evolution of the health benefits of soy iso-
flavones. Proc Soc Exp Biol Med 217:386–396
Barnes S (2010) The biochemistry, chemistry and physiology of
Soy is a legume that is widely exploited economically the isoflavones in soybeans and their food products.
and pharmacologically. The chemical constituents Lymphat Res Biol 8:89–98

123
866 Phytochem Rev (2015) 14:849–869

Bayerl C, Keil D (2002) Isoflavonoide in der Behandlung der complexes with native cyclodextrins. J Pharm Biomed
Hautalterung postmenopausaler Frauen. Aktuelle Derma- Anal 48:636–640
tol 28:14–18 Deshmukh K, Amin P (2013) Formulation and evaluation of
Becker W (1978) Solvent extraction of soybeans. J Am Oil solid–lipid nanoparticle based 0.1% Soy isoflavone dermal
Chem Soc 5:754–761 gels. J Pharm BioSci 1:7–18
Borghetti GS, Pinto AP, Lula IS, Sinisterra RD, Teixeira HF, Dwiecki K, Neunert G, Polewski P, Polewski K (2009) Anti-
Bassani VL (2011) Daidzein/cyclodextrin/hydrophilic oxidant activity of daidzein, a natural antioxidant, and its
polymer ternary systems. Drug Dev Ind Pharm 37:886–893 spectroscopic properties in organic solvents and phospha-
Brincat MP, Baron YM, Galea R (2005) Estrogens and the skin. tidylcholine liposomes. J Photochem Photobiol B Biol
Climacteric 8:110–123 96:242–248
Britz SJ, Schomburg CJ, Kenworthy WJ (2011) Isoflavones in Eldridge AC, Kwolek WF (1983) Soybean isoflavones: effect of
seeds of field-grown soybean: variation among genetic environment and variety on composition. J Agric Food
lines and environmental effects. J Am Oil Chem Soc Chem 31:394–396
88:827–832 Emmerson E, Campbell L, Ashcroft GS, Hardman MJ (2010)
Campbell L, Emmerson E, Davies F, Gilliver SC, Krust A, The phytoestrogen genistein promotes wound healing by
Chambon P, Ashcroft GS, Hardman MJ (2010) Estrogen multiple independent mechanisms. Mol Cell Endocrinol
promotes cutaneous wound healing via estrogen receptor b 321:184–193
independent of its antiinflammatory activities. J Exp Med Farmakalidis E, Murphy PA (1984) Semi-preparative high-
207:1825–1833 performance liquid chromatographic isolation of soybean
Cannavà C, Crupi V, Ficarra P, Guardo M, Majolino D, Maz- isoflavones. J Chromatogr A 295:510–514
zaglia A, Stancanelli R, Venuti V (2010) Physicochemical Förster M, Bolzinger MA, Fessi H, Briançon S (2009) Topical
characterization of an amphiphilic cyclodextrin/genistein delivery of cosmetics and drugs. Molecular aspects of
complex. J Pharm Biomed Anal 51:1064–1068 percutaneous absorption and delivery. Eur J Dermatol
Carrão-Panizzi MC, Favoni SPG, Kikuchi A (2002) Extraction 19:309–323
time for soybean isoflavone determination. Braz Arch Biol Franke AA, Custer LJ, Cerna CM, Narala KK (1994) Quanti-
Technol 45:515–518 tation of phytoestrogens in legumes by HPLC. J Agric
Carrara VS, Amato AA, Neves FAR, Bazotte RB, Mandarino Food Chem 42:1905–1913
JMG, Nakamura CV, Filho BPD, Cortez DAG (2009) Georgetti SR, Casagrande R, Vicentini FTMC, Verri WA Jr,
Effects of a methanolic fraction of soybean seeds on the Fonseca MJV (2006) Evaluation of the antioxidant activity
transcriptional activity of peroxisome proliferator-acti- of soybean extract by different in vitro techniques and
vated receptors (PPAR). Braz J Med Biol Res 42:545–550 investigation of this activity after its incorporation in top-
César IC, Braga FC, Soares CD, Nunan EA, Pianetti GA, ical formulations. Eur J Pharm Biopharm 64:99–106
Condessa FA, Barbosa TA, Campos LM (2006) Develop- Georgetti SR, Casagrande R, Verri WA Jr, Lopez RFV, Fonseca
ment and validation of a RP-HPLC method for quantifi- MJV (2008) Evaluation of in vivo efficacy of topical for-
cation of isoflavone aglycones in hydrolyzed soy dry mulations containing soybean extract. Int J Pharm
extracts. J Chromatogr B 836:74–78 352:189–196
Chen KI, Erh MH, Su NW, Liu WH, Chou CC, Cheng KC Hirota A, Inaba M, Chen YC, Abe N, Taki S, Yano M, Kawaii S
(2012) Soyfoods and soybean products: from traditional (2004) Isolation of 8-hydroxyglycitein and 6-hydroxyd-
use to modern applications. Appl Microbiol Biotechnol aidzein from soybean miso. Biosci Biotechnol Biochem
96:9–22 68:1372–1374
Chiang W-D, Shih C-J, Chu Y-H (2001) Optimization of acid Huang ZR, Hung CF, Lin Y, Fang JY (2008) In vitro and in vivo
hydrolysis conditions for total isoflavones analysis in evaluation of topical delivery and potential dermal use of
soybean hypocotyls by using RSM. Food Chem soy isoflavones genistein and daidzein. Int J Pharm
72:499–503 364:36–44
Cho SY, Lee YN, Park HJ (2009) Optimization of ethanol Huang CC, Hsu BY, Wu NL, Tsui WH, Lin TJ, Su CC, Hung CF
extraction and further purification of isoflavones from (2010) Anti-photoaging effects of soy isoflavone extract
soybean sprout cotyledon. Food Chem 117:312–317 (aglycone and acetylglucoside form) from soybean cake.
Chung H, Ji X, Canning C, Sun S, Zhou K (2010) Comparison of Int J Mol Sci 12:4782–4795
different strategies for soybean antioxidant extraction. Hwang K, Chung RS, Schmitt JM, Buck D, Winn SR, Hollinger
J Agric Food Chem 58:4508–4512 JO (2001) The effect of topical genistein on soft tissue
Coward L, Smith M, Kirk M, Barnes S (1998) Chemical mod- wound healing in rats. J Histotechnol 24:95–99
ification of isoflavones in soyfoods during cooking and Ismail B, Hayes K (2005) b-Glycosidase activity toward dif-
processing. Am J Clin Nutr 68:1486S–1491S ferent glycosidic forms of isoflavones. J Agric Food Chem
Crupi V, Ficarra R, Guardo M, Majolino D, Stancanelli R, 53:4918–4924
Venuti V (2007) UV–vis and FTIR–ATR spectroscopic Kiguchi K, Constantinou A, Huberman E (1990) Genistein
techniques to study the inclusion complexes of genistein induced cell differentiation and protein-linked DNA strand
with b-cyclodextrins. J Pharm Biomed Anal 44:110–117 breakage in human melanoma cells. Cancer Commun
Daruhazi AE, Szente L, Balogh B, Matyus P, Beni S, Takacs M, 2:271–278
Gergely A, Horvath P, Szoke E, Lemberkovics E (2008) Kim BN, Yeom SJ, Kin YS, Oh DK (2012) Characterization of a
Utility of cyclodextrins in the formulation of genistein Part b-glucosidase from Sulfolobus solfataricus for isoflavone
1. Preparation and physicochemical properties of genistein glycosides. Biotechnol Lett 34:125–129

123
Phytochem Rev (2015) 14:849–869 867

Kitagawa S, Inoue K, Teraoka R, Morita SY (2010) Enhanced Okura A, Arakawa H, Oka H, Yoshinari T, Monden Y (1988)
skin delivery of genistein and other two isoflavones by Effect of genistein on topoisomerase activity and on the
microemulsion and prevention against UV irradiation- growth of [Val 12]Ha-ras-transformed NIH 3T3 cells.
induced erythema formation. Chem Pharm Bull 58: Biochem Biophys Res Commun 157:183–189
398–401 Park E, Lee SM, Jung IK, Lim Y, Kim JH (2011) Effects of
Kledjus B, Mikelová R, Petrlová J, Potesil D, Adam V, Stibo- genistein on early-stage cutaneous wound healing. Bio-
rová M, Hodek P, Vacek J, Kizek R, Kuban V (2005) chem Biophys Res Commun 410:514–519
Determination of isoflavones in soy bits by fast column Patriarca MT, Moraes ARB, Nader HB, Petri V, Martins JRM,
high-performance liquid chromatography coupled with Gomes RCT, Junior JMS (2013) Hyaluronic acid concen-
UV–visible diode-array detection. J Chromatogr A 1084: tration in postmenopausal facial skin after topical estradiol
71–79 and genistein treatment: a double-blind, randomized clin-
Kuiper GGJM, Lemmen JG, Carlsson B, Corton JC, Safe SH, ical trial of efficacy. Menopause 20:336–341
Saag PTV, Burg BV, Gustafsson JÅ (1998) Interaction of Perkin AG, Newbury FG (1899) The colouring matters con-
estrogenic chemicals and phytoestrogens with estrogen tained in dyer’s broomn (Genista tinctoria) and heather
receptor b. Endocrinology 139:4252–4263 (Calluna vulgaris). J Chem Soc 75:830–839
Kuo LC, Wu RY, Lee KT (2012) A process for high-efficiency Phommalth S, Jeong Y-S, Kim Y-H, Hwang Y-H (2008) Iso-
isoflavone deglycosylation using Bacillus subtilis natto flavone composition within each structural part of soybean
NTU-18. Appl Microbiol Biotechnol 94:1181–1188 seeds and sprouts. J Crop Sci Biotechnol 11:57–62
Lee JH, Choung M-G (2011) Determination of optimal acid Pinnel SR (2003) Cutaneous photodamage, oxidative stress, and
hydrolysis time of soybean isoflavones using drying oven topical antioxidant protection. J Am Acad Dermatol
and microwave assisted methods. Food Chem 129: 2003(48):1–19
577–582 Rostagno MA, Araújo JMA, Sandi D (2002) Supercritical fluid
Lee J, Doo EH, Kwon DY, Park JB (2008) Functionalization of extraction of isoflavones from soybean flour. Food Chem
isoflavones with enzymes. Food Sci Biotechnol 17: 78:111–117
228–233 Rostagno MA, Palma M, Barroso CG (2003) Ultrasound-assited
Liggins J, Bluck LJC, Coward WA, Bingham A (1998) extraction of soy isoflavones. J Chromatogr A 1012:
Extraction and quantification of daidzein and genistein in 119–128
food. Anal Biochem 264:1–7 Rostagno MA, Palma M, Barroso CG (2004) Pressurized liquid
Li-Hsun C, Ya-Chuan C, Chieh-Ming C (2004) Extracting and extraction of soy isoflavones. Anal Chim Acta 522:
purifying isoflavones from defatted soybean flakes using 169–177
superheated water at elevated pressures. Food Chem Rostagno MA, Palma M, Barroso CG (2007) Microwave
84:279–285 assisted extraction of soy isoflavones. Anal Chim Acta
Lin JY, Tournas JA, Burch JA, Monteiro-Riviere NA, Zielinski 588:274–282
J (2008) Topical isoflavones provide effective photopro- Rostagno MA, Villares A, Guillamón E, Gárcia-Lafuente A,
tection to skin. Photodermatol Photoimmunol Photomed Martinéz JA (2009) Sample preparation for the analysis of
24:61–66 isoflavones from soybeans and soy foods. J Chromatogr A
Luthria DL, Natarajan SS (2009) Influence of sample prepara- 1216:2–29
tion on the assay of isoflavones. Planta Med 75:704–710 Sánchez-Calvo JM, Rodrı́guez-Iglesias MA, Molinillo JMG,
Luthria DL, Biswas R, Natarajan S (2007) Comparison of Macı́as FA (2013) Soy isoflavones and their relationship
extraction solvents and techniques used for the assay of with microflora: beneficial effects on human health in
isoflavones from soybean. Food Chem 105:325–333 equol producers. Phytochem Rev 12:979–1000
Masaki H (2010) Role of antioxidants in the skin: anti-aging Sansone F, Picerno P, Mencherini T, Russo P, Gasparri F,
effects. J Dermatol Sci 58:85–90 Giannini V, Lauro MR, Puglisi G, Aquino RP (2013)
Miyazaki K, Hanamizu T, Lizuka R, Chiba K (2002) Genistein Enhanced technological and permeation properties of a
and daidzein stimulate hyaluronic acid production in microencapsulated soy isoflavones extract. J Food Eng
transformed human keratinocyte culture and hairless 115:298–305
mouse skin. Skin Pharmacol Appl Skin Physiol 15: Sator PG, Schmidt JB, Rabe T, Zouboulis CC (2004) Skin aging
175–183 and sex hormones in women—clinical perspectives for
Moraes AB, Haidar MA, Junior JMS, Simões MJ, Baracat EC, intervention by hormone replacement therapy. Exp Der-
Patriarca MT (2009) The effects of topical isoflavones on matol 13:36–40
postmenopausal skin: double-blind and randomized clini- Schmid D, Zulli F (2002) Topically applied soy isoflavones
cal trial of efficacy. Eur J Obstet Gynecol Reprod Biol increase skin thickness. Cosmet Toilet 117:45–50
146:188–192 Schmid D, Zulli F, Nissen HP, Prieur H (2003) Penetration and
Murphy PA (1981) Separation of genistin, daidzin and their metabolism of isoflavones in human skin. Cosmet Toilet
aglucones, and coumesterol by gradient high-performance 118:71–74
liquid chromatography. J Chromatogr A 211:166–169 Schwartz H, Sontag G (2009) Comparison of sample prepara-
Naim BM, Kirson GI, Bondi BA (1973) A new isoflavone from tion techniques for analysis of isoflavones in foodstuffs.
soya beans. Phytochemistry 12:169–170 Anal Chim Acta 633:204–215
Naim M, Gestetner B, Zilkah S, Birk Y, Bondi A (1974) Soy- Seidel V (2006) Initial and bulk extraction. In: Sarker SD, Latif
bean isoflavones: characterization, determination, and Z, Gray AI (eds) Natural products isolation, vol 20. Hu-
antifungal activity. J Agric Food Chem 22:806–810 mana Press Inc, Totowa, pp 27–46

123
868 Phytochem Rev (2015) 14:849–869

Setchell KDR (1998) Phytoestrogens: the biochemistry, physi- Walz E (1931) Isoflavon and saponin-glucoside in soja hispida.
ology, and implications for human health of soy isoflav- Justus Liebigs Ann Chem 489:118–155
ones. Am J Clin Nutr 68:1333S–1343S Wang HZ, Zhang Y, Xie LP, Yu XY, Zhang RQ (2002) Effects
Shao S, Duncan AM, Yang R, Marcone MF, Rajcan I, Tsao R of genistein and daidzein on the cell growth, cell cycle, and
(2011) Systematic evaluation of pre-HPLC sample pro- differentiation of human and murine melanoma cells.
cessing techniques on total and individual isoflavones in J Nutr Biochem 13:421–426
soybeans and soy products. Food Res Int 44:2425–2434 Wang J, Sun AL, Liu RM, Zhang YQ (2013a) Isolation and
Silva APC, Nunes BR, De Oliveira MC, Koester LS, Mayorga P, purification of soy isoflavones from soybean extracts by
Bassani VL, Teixeira HF (2009) Development of topical adsorption chromatography on 12% cross-linked agarose
nanoemulsions containing the isoflavone genistein. Phar- gel media. J Liq Chromatogr Relat Technol 36:2307–2316
mazie 64:32–35 Wang M, Guo J, Qi W, Su R, He Z (2013b) An effective and green
Song TT, Hendrich S, Murphy PA (1999) Estrogenic activity of method for the extraction and purification of aglycone iso-
glycitein, a soy isoflavone. J Agric Food Chem 47:1607–1610 flavones from soybean. Food Sci Biotechnol 22:705–712
Song X, Xue Y, Wang Q, Wu X (2011) Comparison of three Wei H, Wei L, Frenkel K, Bowen R, Barnes S (1993) Inhibition
thermostable b-Glucosidases for application in the hydro- of tumor promoter-induced hydrogen peroxide production
lysis of soybean isoflavone glycosides. J Agric Food Chem in vitro and in vivo by genistein. Nutr Cancer 20:1–12
59:1954–1961 Wei H, Bowen R, Cai Q, Barnes S, Wang Y (1995a) Antioxidant
Sovová H, Stateva PR (2011) Supercritical fluid extraction from and antipromotional effects of the soybean isoflavone
vegetable materials. Rev Chem Eng 27:79–156 genistein. Proc Soc Exp Biol Med 208:124–130
Stancanelli R, Mazzaglia A, Tommasini S, Calabrò ML, Villari Wei H, Barnes S, Wang Y (1995b) The inhibition of tumor
V, Guardo M, Ficarra P, Ficarra R (2007) The enhancement promoter-induced proto-oncogene expression mouse skin
of isoflavones water solubility by complexation with by soybean isoflavone genistein. Oncol Rep 3:125–128
modified cyclodextrins: a spectroscopic investigation with Wei H, Bowen R, Zhang X, Lebwohl M (1998) Isoflavone
implications in the pharmaceutical analysis. J Pharm Bio- genistein inhibits the initiation and promotion of two-stage
med Anal 44:980–984 skin carcinogenesis. Carcinogenesis 19:1509–1514
Sudel KM, Venzke K, Mielke H, Breitenbach U, Mundt C, Wei H, Zhang X, Wang Y, Lebw M (2002) Inhibition of ultra-
Jaspers S, Koop U, Sauermann K, KnuBmann-Hartig E, violet light-induced oxidative events in the skin and
Moll I, Gercken G, Young AR, Stab F, Wenck H, Gallinat S internal organs of hairless mice by isoflavone genistein.
(2005) Novel aspects of intrinsic and extrinsic aging of Cancer Lett 185:21–29
human skin: beneficial effects of soy extract. Photochem Wei H, Saladari R, Lu Y, Wang Y, Palep SR, Moore J, Phelps R,
Photobiol 81:581–587 Shyong E, Lebwohl MG (2003) Isoflavone genistein:
Sun Y, Zhang Y, Liu N, Li D, Li M (2010) Hydrolysis of gen- photoprotection and clinical implications in dermatology.
istin and daidzin by a b-glucosidase purified from Lentin- J Nutr 133:3811S–3819S
ula edodes. J Med Plants Res 4:2684–2690 Weiss SC (2011) Conventional topical delivery systems. Der-
Thorne JH (1981) Morphology and ultrastructure of maternal matol Ther 24:471–476
seed tissues of soybean in relation to the import of photo- Wijngaard H, Hossain MB, Rai DK, Brunton N (2012) Tech-
synthate. Plant Physiol 67:1016–1025 niques to extract bioactive compounds from food by-pro-
Tipkanon S, Chompreeda P, Haruthaithanasan V, Suwonsichon ducts of plant origin. Food Res Int 46:505–513
T, Prinyawiwatkul W, Xu Z (2010) Optimizing time and Xavier CR, Silva APC, Schwingel LC, Borghetti GS, Koester
temperature of enzymatic conversion of isoflavone gluco- LS, Mayorga P, Teixeira HF, Bassani VL (2010)
sides to aglycones in soy germ flour. J Agric Food Chem Improvement of genistein content in solid genistein/b-
58:11340–11345 cyclodextrin complexes. Quim Nova 33:587–590
Utkina EA, Antoshina SV, Selishcheva A, Sorokoumova GM, Xu L, Lamb K, Layton L, Kumar A (2004) A membrane-based
Rogozhkina EA, Shvets VI (2004) Isoflavones daidzein process for recovering isoflavones from a waste stream of
and genistein: preparation by acid hydrolysis of their gly- soy processing. Food Res Int 37:867–874
cosides and the effect on phospholipid peroxidation. Bio- Yang F, Ma Y, Ito Y (2001) Separation and purification of
org Khim 30:429–435 isoflavones from a crude soybean extract by high-speed
Vacek J, Klejdus B, Lojkova L, Kuban V (2008) Current trends counter-current chromatography. J Chromatogr A 928:
in isolation, separation, determination and identification of 163–170
isoflavones: a review. J Sep Sci 31:2054–2067 Yang S, Wang L, Yan Q, Jiang Z, Li L (2009) Hydrolysis of
Varani J, Kelley EA, Perone P, Lateef H (2004) Retinoid- soybean isoflavone glycosides by a thermostable b-gluco-
induced epidermal hyperplasia in human skin organ cul- sidase from Paecilomyces thermophila. Food Chem
ture: inhibition with soy extract and soy isoflavones. Exp 115:1247–1252
Mol Pathol 77:176–183 Yatsu FKJ, Koester LS, Lula I, Passos JJ, Sinisterra R, Bassani
Vargas BA, Bidone J, Oliveira LK, Koester LS, Bassani VL, VL (2013) Multiple complexation of cyclodextrin with soy
Teixeira HF (2012) Development of topical hydrogels isoflavones present in an enriched fraction. Carbohydr
containing genistein-loaded nanoemulsions. J Biomed Polym 98:726–735
Nanotechnol 8:1–7 Yeom SJ, Kim BN, Kim YS, Oh DK (2012) Hydrolysis of
Walter ED (1941) Genistin (an Isoflavone glucoside) and its isoflavone glycosides by a thermostable b-glucosidase
aglucone, genistein, from soybeans. J Am Chem Soc 63: from Pyrococcus furiosus. J Agric Food Chem 60:
3273–3276 1535–1541

123
Phytochem Rev (2015) 14:849–869 869

Yuan JP, Liu YB, Peng J, Wang JH, Liu X (2009) Changes of Zhao C, Wang Y, Su Y, Zhang H, Ding L, Yan X, Zhao D, Shao
isoflavone profile in the hypocotyls and cotyledons of N, Ye X, Cheng Y (2011) Inclusion complexes of isoflav-
soybeans during dry heating and germination. J Agric Food ones with two commercially available dendrimers: solu-
Chem 57:9002–9010 bility, stability, structures, delivery behaviors, cytotoxicity,
Zampieri ALTC, Ferreira FS, Resende ÉC, Gaeti MPN, Diniz and antioxidant activities. Int J Pharm 421:301–309
DGA, Taveira SF, Lima EM (2013) Biodegradable poly- Zuanazzi JAS, Mayorga P (2010) Fitoprodutos e desenvolvi-
meric nanocapsules based on poly(DL-lactide) for geni- mento econômico. Quim Nova 33:1421–1428
stein topical delivery: obtention, characterization and skin
permeation studies. J Biomed Nanotechnol 9:527–534
Zhang EJ, Ng KM, Luo KQ (2007) Extraction and purification
of isoflavones from soybeans and characterization of their
estrogenic activities. J Agric Food Chem 55:6940–6950

123