8Non-denatured Soybean Extracts in Skin Care:
Multiple Anti-Aging Effects
Miri Seiberg
The Johnson & Johnson Skin Research Center, CPPW, a division of Johnson & Johnson
Consumer Companies, Inc. Skillman, NJ, 08558,
USA
1. Introduction
Non-denatured soybean extracts contain all the ingredients of the soybean in their naturalconformations, which is different from soy foods that must be denatured, or from soy
isoflavone-based cosmetic products. The multiple benefits of non-denatured soybean
extracts for skin care range from skin lightening, extracellular matrix enhancement anddelayed hair growth, to a protective effect from photodamage and possibly from skincancer.
The discovery of the role of the keratinocyte receptor Protease-Activated Receptor-2 (PAR-2)
in keratinocyte-melanocyte interactions and pigment transfer resulted in the identification ofnovel agents that affect skin color. The inhibition of serine proteases was found to reducePAR-2 activation and therefore to alter skin color. A search for naturally occurring serine
protease inhibitors identified non-denatured soybean extracts. These extracts inhibit PAR-2
activation and induce human skin lightening. Non-denatured soybean extracts also enhancecollagen and elastin synthesis, and protect existing elastin fibers from degradation, therefore
providing anti-aging benefits such as reducing skin sagging and wrinkling. Additionally,
non-denatured soybean extracts delay hair growth, resulting in thinner and smaller hairshafts and reduced visibility of undesired hair growth. Topical pretreatment with nondenatured soybean extracts reduces UV-induced edema and erythema, and additionallyreduces UVB-induced DNA and cellular damage, which could lessen the risk of skin cancer.
Thus non-denatured soybean extracts could serve as inexpensive, safe and efficacious
natural agents, which provide multiple skin care benefits, ranging from protection to
restoration.

2. Non-denatured soybean extracts
Denaturation, a process induced by external agents or stressors, leads to changes in thetertiary structure of proteins, resulting in the loss of their activity. Conventional soybean
extracts used in the cosmetic and food industries are typically denatured. The cosmetic
industry uses mainly organic extracts of soybeans, which are enriched with isoflavones(reviewed in Thornfeldt 2005). The organic solvent extraction process removes orinactivates most of the soybean proteins while concentrating the isoflavones. The foodindustry takes major steps to inactivate the soy proteins, particularly Soybean Trypsin

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Inhibitor (STI), which interferes with the process of food digestion. Industry standards
include heating under pressure and alkaline soaking, which denature most of the soybean
proteins (Liu, 1999). Interestingly, soybeans have been used in Asia for centuries as a
dietary source of protein, and they have always been fermented to inactivate STI, thereby
preventing the inhibition of trypsin activity which is needed for digestion of dietary
proteins.
The non-denatured soybean extracts discussed in this chapter are different from soy foods
and from cosmetic soy preparations in two major aspects. First, the non-denatured soybean
extracts contain all the ingredients at the same proportions as in the native bean. No
soybean constituent is removed or is specifically enriched during the processing of thebeans, and all components of the beans remain in a "soy powder" preparation that serves as
raw material. Second, the soy powder is kept in its natural, non-denatured state, with all thesoybean proteins, and in particular STI, present and active. Non-denatured soybean
extracts, therefore, cannot be used for dietary consumption, as they contain active STI (seee.g. Paine et al, 2001; Seiberg et al, 2001).

3. The Soybean proteins STI and BBI are serine protease inhibitors
The proteins STI, a Kunitz-type trypsin inhibitor, and BBI, the Bowman-Birk protease inhibitor(reviewed in (Birk, 1985; Kennedy, 1998), were first isolated from soybeans in the early 1940's.
STI (reviewed in Birk, 1985) is a protein of 181 amino acid residues and a tertiary structure
which is dependent on two disulfide bridges (reviewed in Song et al., 1998). BBI is an 8 kDa
protein with 81 amino acid residues and seven disulfide bonds that is a weak trypsin inhibitor
and a strong chymotrypsin inhibitor (Billings et al., 1992). STI is heat labile (Song et al., 1998),
while BBI has a stable conformation even after its disulfide bonds are broken by heating or
treatment with acid and pepsin (Birk, 1985). STI and BBI are found only in the bean, and not inany other part of the soy plant (Birk, 1985). BBI is known for its chemo-preventive effects indifferent types of cancer (reviewed in Kennedy, 1998) and a soybean extract enriched in BBI
has been under evaluation for chemo-prevention in clinical studies. STI and BBI have not beenstudied in the past for their topical effect on skin pigmentation, skin aging or skin cancer. This
chapter summarizes the biological activities of STI, BBI, and non-denatured soybean extracts(containing STI and BBI), and describes molecular and cellular mechanisms affected by theseagents, which lead to superior skin care and "anti-aging" benefits.

4. Skin aging
Skin aging is a complex phenomenon induced by endogenous (chronological) andexogenous (extrinsic) factors. Chronological aging is a genetically programmed,
unavoidable process, while extrinsic influences (e.g., sunlight, wind, heat, cigarette smoke,or exposure to chemicals) could be at least partially controlled by the individual (reviewed
in Uitto, 1986; Baumann, 2007; Uitto, 2008; Leyden,1990). Among the extrinsic factors,
ultraviolet (UV) irradiation (sunlight, tanning beds) is the single most important cause ofskin aging (reviewed in Uitto, 1986; Baumann, 2007; Uitto, 2008; Leyden, 1990). UV exposure
leads to numerous changes in the skin, including epidermal and dermal thinning, a decline
in collagen production and in the functionality of the dermal elastic network, and an uneven
distribution of pigment deposition, all resulting in an aged-skin phenotype. Additionally,
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induced by the cumulative exposure to UV irradiation, and therefore appear mainly on aged
skins (see e.g. Strom and Yamamura, 1997).

5. Non-denatured soybean extracts reduce pigment production anddeposition
The irregular pigmentation displayed in photo-damaged skin includes numerous types ofhyperpigmented lesions, commonly referred to as "age spots". These UV-induced lesions are
the result of uneven distribution of pigment cells, local loss of melanocytes, hyperactivity ofmelanocytes, and modified keratinocyte-melanocyte interactions (reviewed in Ortonne1990). Therapies for hyperpigmented lesions range from cryotherapy and energy-based
devices (e.g. laser, pulsed light), to chemical peels, and to topical pharmaceutical
depigmenting agents (e.g. hydroquinone, tretinoin, adapalene), (Ortonne 2006). However,many of these treatments are expensive, or are accompanied by undesired effects (reviewed
in Jimbow and Jimbow, 1998). The consumer needs safe, efficacious and cost effective
depigmenting agents, and additionally desires "natural" treatments, with the expectations ofsafety, effectiveness and mildness at reasonable cost.
Early searches for depigmenting agents used cultured melanocytes (pigment cells) andassayed for either inhibition of tyrosinase activity (the rate-limiting enzyme in pigment
production) or for selective melanocyte cytotoxicity. To better represent the complexity of
intact skin, we used keratinocyte-melanocyte co-cultures in our studies. The co-culture
system enabled us to identify keratinocyte-melanocyte interactions that are involved inpigment transfer, and later to modulate these interactions in a way that could alter skincolor (Seiberg et al., 2000a, 2000b). A keratinocyte receptor, PAR-2, was identified as aphagocytic receptor that regulates the keratinocyte ability to ingest melanosomes (Sharow etal, 2000). Since PAR-2 is activated by serine proteases like trypsin (reviewed in (Déry et al.,1998, 1999)), its activation is inhibited by serine protease inhibitors. The modulation of thePAR-2 pathway was indeed shown to affect skin color. Activators of PAR-2 induced avisible skin darkening, and the inhibition of PAR-2 activation by serine protease inhibitors
led to visible skin lightening (Seiberg et al 2000a, 2000b).
A search for botanical agents with a serine protease-inhibitory activity had identified nondenatured soybean extracts, and using the keratinocyte-melanocyte co-culture system
documented that these extracts reduce pigment deposition (Paine et al, 2001). Nondenatured soybean extracts were later shown to reduce pigment deposition in human skinand to decrease the pigmentary levels of human age spots (Hermanns et al, 2000, Wallo et al,2007). Non-denatured soybeans extracts contain the active trypsin inhibitors STI and BBI.
Therefore, these purified proteins were tested, in parallel with the complete non-denatured
soybean extract, for their ability to modulate pigment production and distribution. Nondenatured soybean extracts, or STI or BBI alone, were found to inhibit PAR-2 activation,
resulting in cytoskeletal and cell surface reorganization and reduced keratinocyte
phagocytosis (Paine et al, 2001). The depigmenting activity of these agents, as well as theirability to prevent UV-induced pigmentation, was demonstrated both in vitro and in vivo
(Paine et al, 2001). Importantly, the non-denatured soybean extracts were superior to either
STI or BBI alone in their depigmenting effect, even though the concentrations of STI and BBI
within these extracts were much lower than when tested individually (Paine et al, 2001).
These studies suggest that the non-denatured soybean extracts contain additional active
agents besides STI and BBI, which either contribute directly to the depigmenting effect, or

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enhance the delivery or the activity of STI and/or BBI in the skin. Examples of thedepigmenting activity of non-denatured soybean extracts are shown in figure 1 (Images
courtesy of Dr. C.B. Lin and D. Rossetti).

Control

Non-denatured
soybean extracts

a

b

c

d

Pigmented
epidermal
equivalents

Human skinexplants

Fig. 1. Non-denatured soybean extracts reduce pigment production and deposition. (a-b)
Pigmented epidermal equivalents were topically treated with 2% non-denatured soybean
extracts in PBS, or PBS alone, once daily, 5 days/week. Following nine days in culture thetissues were stained with Fontana-Masson (F&M) staining, to document pigment
deposition. A marked reduction was observed in the treated equivalents as compared tocontrol. (c-d) Human skin explants were obtained from surgical procedures with informed
consent and institutional board approval. Explants were maintained in culture for eightdays, with or without topical daily treatment, 5 days/week, with a formulation containing
2.5% non-denatured soybean extract. F&M staining documented a strong reduction inpigment production and deposition in the human skin following the non-denatured soy
extract treatment. Images are courtesy of Dr. CB Lin and D. Rossetti.

6. Non-denatured soybean extracts enhance the dermal extracellular matrix
and the elastic fiber network
Elastin fiber production is reduced with aging (reviewed in Uitto, 1986, 2008; PasqualiRonchetti and Baccarani-Contri, 1997). The age-induced decline in skin elasticity results fromslower tissue regeneration, from lower elastin synthesis levels, and from the increased
production and secretion of elastases. UV exposure further decreases the functionality of theelastic fiber network, as excessive elastin production and abnormal cross-linking create
elastotic material ("solar elastosis") with reduced elastic capacity (reviewed in Uitto 1986,2008). Preventing or reversing skin ageing includes the use of sunscreens and sun avoidance
behavior, the use of anti-oxidants, and the use of agents like retinoids, which inhibit

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collagenases and promote collagen production (Baily et al, 1990; reviewed in Fisher andVoorhees, 1998). Only a few agents are available to directly enhance the balanced synthesis
and assembly of the elastic fiber network. The inhibition of fibroblast-derived elastases
following chronic UVB irradiation was found to protect the dermal elastic network of the skinfrom fragmentation, and to reduce wrinkle formation in rodents (Tsukahara et al, 2001a, b).
Searching for botanical extracts for anti-aging skin care use, the non-denatured soybean

Control

Non-denatured
soybean extracts

a

b

c

d

e

f

Luna
elastinstaining

Immunohistochemistryof tropoelastin
and fibrilin-1

Herovici
collagenstaining

Fig. 2. Non-denatured soybean extracts enhance elastin and collagen synthesis. Humanskin explants were obtained from surgical procedures with informed consent andinstitutional board approval. Explants were maintained in culture for eight days, with orwithout topical daily treatment, 5 days/week, with a formulation containing 2.5% nondenatured soybean extract. Histological and histochemical stainings were performed to
document elastin and collagen fibers. (a-b) Luna elastin staining. Mature elastin fibers arestained purple-brown. Topical treatment with non-denatured soybean extracts results inenhanced elastic fiber network. (c-d) Immuno-histochemical staining of tropoelastin (theelastin fiber monomer, green) and fibrillin-1 (an elastin-accessory protein, red). Colocalization is viewed as yellow. Non-denatured soybean extracts induce the synthesis of
tropoelastin and fibrillin-1, and their co-localization documents the site of elastin fiber
formation. (e-f) Herovici staining documents mature collagen fibers in magenta-red.
Topical treatment with non-denatured soybean extracts induces the production of collagenfibers. Images are courtesy of Dr. CB Lin and D. Rossetti.

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extracts were found to have elastase-inhibitory activities (Zhao et al, 2009). In addition, nondenatured soybean extracts were found to induce the synthesis of collagen and elastin, andto promote the correct assembly of new elastin fibers, providing a complete protection andrestoration to the dermal extracellular matrix (see Figure 2, images courtesy of Dr. C.B. Lin
and D. Rossetti).
In vitro studies using both purified elastases and cultured fibroblasts demonstrated that
non-denatured soybean extracts could affect the extracellular matrix. The enzymatic activityof several elastases was inhibited by these extracts, and, to a lesser extent, by STI or BBI,
while soy isoflavones did not show any elastase-inhibitory activity (Zhao et al, 2009).. The
non-denatured soybean extracts also protected elastic fibers produced by cultured
fibroblasts from degradation by exogenously-added elastases (Zhao et al, 2009).
Additionally, these extracts exhibited elastin-enhancement activities. A dose-dependent
induction of expression of the elastin gene was documented using an elastin promoterluciferase reporter gene, and was confirmed by mRNA analysis of treated fibroblasts. The
synthesis of new tropoelastin monomers, as well as of elastin-accessory proteins, and theassembly of new elastin fibers, was documented by histological staining (Zhao et al, 2009).
The elastin-enhancing activity of the non-denatured soybean extracts was confirmed in vivo.
Histological analysis of mice and swine skins topically treated with non-denatured soybean
extracts showed a significant increase in the elastic fibers network, with an accompanied
increase in elastin mRNA and in desmosine content (Zhao et al, 2009). When human skins,transplanted onto immuno-deficient mice, were topically treated with these extracts, anincrease was documented in the expression of elastin, elastin-accessory proteins, andcollagen (Zhao et al, 2009). Human skin explants treated ex-vivo with non-denatured
soybean extracts and analyzed by histological staining and immunohistochemistry showed
an increase in elastin, fibrilin-1 and collagen production (see Figure 2; Images courtesy of
Dr. C.B. Lin and D. Rossetti). These data suggest that non-denatured soybean extracts not
only protect extracellular matrix from degradation, they also induce collagen and elastin
synthesis, and increase the total amount of stable, cross-linked elastin fibers.

7. Non-denatured soybean extracts reduce hair growth
Unlike other mammals, humans no longer use their hair for environmental protection, but
keep or remove their hair for social and cosmetic purposes. Many procedures are used toremove unwanted hair, from simple home treatments like shaving, to laser and lighttherapies. These methods differ in the duration of hair elimination after removal, their pricerange, their pain and discomfort levels, and their possible undesired effects (reviewed in
Olsen, 1999). Shaving, the most popular hair-removal method, requires daily treatments,
may result in nicks and cuts, may increase the risk of infection, may leave a perception of anincreased rate of hair growth, and may leave undesirable stubble. An alternative to at-homehair removal is hair dying or bleaching, used to reduce hair visibility in particular bodyareas. However, such methods are less preferred as the emerging portions of the hair shafts
are always darker than the already-treated parts. An alternative to these methods is desired;
particularly a method that would reduce undesired hair growth, with a safe and simple athome procedure. As the biological activities of the non-denatured soybean extracts were
further explored, they were found to delay hair growth, resulting in smaller and thinner hairshafts (Seiberg et al, 2001). The use of a skin care product containing these extracts,
therefore, would reduce the visibility of undesired hair growth.

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Topical daily treatments with STI or BBI visually delayed hair growth and reduced thelength of the hair shafts of treated mice, with reduced hair follicles size observed
histologically. Non-denatured soybean extracts (containing STI, BBI and isoflavones) weresuperior to either STI or BBI alone in this inhibitory activity. Non-denatured soybean
extracts led to delayed and reduced hair growth, and hairs were visibly thinner, more“directionally organized”, and smoother to touch, relative to untreated controls (Seiberg etal, 2001). Histological analysis confirmed these observations, and documented smaller sizeand less developed hair follicles (see Figure 3). A statistically significant effect on hairfollicle dimensions was observed, with hair shaft diameter reduced by an average of 42%,and hair bulb diameter reduced by an average of 23.8% (Seiberg et al, 2001). Additionally,
the depigmenting effect of STI and BBI led to lighter colored hair shafts, which could
potentially contribute to the reduced visibility of undesired hair growth. Heat-denatured
soybean extract, or commercially available pasteurized soymilk, had no effect on hair
growth or hair appearance (Seiberg et al, 2001), further supporting the involvement of intact
STI and BBI in the hair growth inhibitory effect.

ControlaNon-denatured
soybean extractsbFig. 3. Non-denatured soybean extracts delay hair growth. C57Bl/6 mice were wax
depilated to induce a new hair cycle, and were treated daily with non-denatured soymilk (b)or remain untreated (a) as in (Seiberg et. al., 2001). F&M staining of skin biopsies taken at
day 7 of the hair cycle demonstrate delayed follicular development, and reduced hair follicle
dimensions and pigment deposition in the soy-treated mice.
The non-denatured soybean extracts contain about 0.001% isoflavones. Phytoestrogens wereshown to reduce hair growth in vitro (Hoffman et al, 1997), but to promote hair growth inmice and in volunteers with alopecia (Okajima and Harada, 2008). Therefore, the effect of
the soybean-derived phytoestrogens on hair growth was studied, alone (0.001%), and incombination with the whole non-denatured soybean extract (total 0.002% isoflavones).
Isoflavones alone reduced mouse hair growth, but not as effectively as non-denatured
soybean extracts alone. The combination of non-denatured soybean extracts, supplemented
with 0.001% soybean-derived isoflavones, reduced hair growth to a higher degree than each
preparation alone (Seiberg et al, 2001), suggesting complementary roles for STI, BBI andisoflavones in the hair growth inhibitory effect. These data suggest that non-denatured
soybean extracts may serve as a natural alternative treatment for reducing the visibility ofundesired grown hair..

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8. Non-denatured soybean extracts inhibit the COX-2 pathway, contributing
to anti-aging and chemo-preventive effects
Chronic inflammation has been linked to numerous skin diseases and conditions, including
skin aging (Reviewed in Thornfeldt, 2008), and many skin care products, therefore, containanti-inflammatory agents. Additionally, inflammation has been linked to epithelial skintumors, and anti-inflammatory drugs are being studied for the prevention and treatment of
non-melanoma skin cancers (Reviewed in Mueller, 2006). Cyclooxygenase-2 (COX-2), themain UV-responsive COX isoform in human skin, is involved in UV-induced skininflammation and carcinogenesis (reviewed in Zhan and Zheng (2007); Rundhaug et al,2007). UV-induced COX-2 expression plays a major role in UV-induced inflammation,
edema, keratinocyte proliferation and epidermal hyperplasia, as well as in the generation ofoxidative DNA damage. Repeated exposure to UV leads to chronic upregulation of COX-2
expression and chronic inflammation, contributing both to accelerated skin aging and to anincreased risk of skin cancer. The induction of COX-2 expression by UV is higher in aged
human skin as compared to young human skin, and aged human skin produces higher
amounts of prostaglandin E2 (PGE2, the product of the COX-2 pathway ) relative to young
skin (Seo et al, 2003). These data suggest that the aging of the skin may increase thesusceptibility for developing both photoaging and carcinogenic processes. Oral and topical
COX-2 inhibitors have chemopreventive activity against chemically- and UV-induced skincancer in numerous animal models (reviewed in Zhan and Zheng (2007); Rundhaug et al,2007). The topical applications of soy isoflavones to mouse skins before UVB exposure
reduced the expression of COX-2 (Chiu et al, 2009).
Non-denatured soybean extracts were found to inhibit the COX-2 pathway both in vitro andin vivo (Chen et al, 2008). Cultured keratinocytes or epidermal equivalents were pretreatedwith non-denatured soybean extracts and then thoroughly washed prior to UV exposure. In
the absence of treatment, UV irradiation induced COX-2 expression and PGE2 secretion, andinduced the activation of p38 MAP kinase. Non-denatured soybean extracts reduced UVBinduced COX-2 expression and PGE2 secretion, and inhibited p38 MAP kinase activation invitro (Chen et al, 2008). Mice pre-treated topically with non-denatured soybean extracts,
then extensively washed prior to UV-exposure, had reduced levels of COX-2 expression in
their skins at 24 hr post UVB exposure, as compared to mice exposed to UVB alone,
confirming the COX-2 inhibitory activity of the non-denatured soybean extracts in vivo
(Chen et al, 2008). Examples of the COX-2 inhibitory activity of the non-denatured soybean
extracts, and of the reduction in UV-induced PGE2 secretion are shown in Figure 4. The
COX-2 inhibitory activity of the non-denatured soybean extracts suggests the possible delayof photoaging and skin cancer processes in human skin.

9. Non-denatured soybean extracts reduce skin cancer progression in highrisk mice
The photoprotective effects of polyphenols, including green tea polyphenols, resveratrol, andsoy isoflavones like genistein, on UV-induced skin inflammation, oxidative stress and DNA
damage, have been extensively documented (Reviewed in Nichols and Katiyar, 2010).
Additionally, the chemopreventive effects of the soy protein BBI have been demonstrated, andan extract of soybeans enriched in BBI has been under evaluation in numerous human clinicalstudies for more than ten years (Kennedy, 1998). Oral feeding of mice with a non-denatured

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a

b

127

c

PGE2(pg/ml)

d
160
140
120
100
80
60
40
20
0
Control

UVB

soy+UVB

Fig. 4. Non-denatured soybean extracts inhibit the COX-2 pathway. Epidermal equivalents
were treated with 2% non-denatured soybean extracts in PBS or with PBS alone for 24 hours,
then washed extensively, followed by UVB exposure (100 mJ/cm2) as in (Chen et al., 2008).
Samples were collected at 24 hours post UVB exposure for COX-2 immuno-histochemical
staining. (a) PBS. (b) PBS followed by UVB. (c) 2% non-denatured soybean extracts
followed by UVB. COX-2 staining of the treated equivalents demonstrates the inhibitory
effect of non-denatured soybean extracts on UVB-induced COX-2 protein levels. (d)Culture media were analyzed for PGE2 secretion. UV induced, and non-denatured soybean
extracts inhibited the secretion of the inflammatory mediator PGE2.
soymilk protein supplement led to reductions in skin tumor numbers and volume whentumors were chemically induced (Limtrakul et al, 1993), however undesired gastrointestinal effects were observed during this study. Since non-denatured soybean extracts
contain isoflavones and BBI, and since they possess COX-2 inhibitory activity, the use of
these extracts as topical chemo-preventive agents was evaluated.
Hairless mice exposed for 20 weeks to chronic low levels of UV light become "high riskmice", which are tumor-free, but with a high risk of developing skin tumors during the next
several months in the absence of additional UV exposure. The high-risk, UV-irradiated mice(with no visible skin lesions) were topically treated with non-denatured soybean extract,heat-denatured soybean extract, STI, or BBI, or with water or BSA as controls (Huang et al,2004). Topical applications of non-denatured soybean extract but not of heat-denatured
soybean extract to these high risk mice reduced the incidence and slowed the growth andprogression of skin tumors (see Fig. 5a). Similar topical applications of STI or BBI also
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but to a lesser extent than the whole non-denatured soybean extract. The number of tumorbearing mice, the number of tumors per mouse, and the volume of the visible part of thetumors, were all reduced the most upon the topical treatment of non-denatured soybean
extracts (Huang et al, 2004). Histopathological examination of skin sections from theuntreated mice and from mice treated with heat-denatured soybean extracts all documented
numerous squamous cell carcinoma (SCC) lesions. The non-denatured soybean extractstreated skins, in contrast, had only very small lesions, with no dysplasia or carcinoma(Figure 5b-d, images courtesy of C. Paine). These data might suggest that non-denatured
soybean extracts could be topically applied to sun-exposed human skin to prevent sunlightinduced skin damage and to reduce the risk of skin tumor formation and progression.

SCC

d
a

b

ControlcSoy, lesion
Largest size
Untreated, tumor
Average size
Fig. 5. Non-denatured soybean extracts delay the progression of skin cancer in high-riskmice. SKH-1 mice were irradiated with UVB (30 mJ/cm2) twice weekly for 20 weeks andUVB irradiation was then stopped, as in (Huang et al., 2004). Three weeks later, the mice
were topically treated with non-denatured soybean extract ("soymilk"), heat-denatured
soybean extract ("heated soymilk"), or water control, once a day, 5 days/week, for 21 weeks.
(a) The number of visible tumors per mouse was determined every 3 weeks. Topicaltreatment with non-denatured soymilk, but not with heat-denatured soymilk, led to amarked reduction in the number of tumors per mouse. (b-d) H&E staining of visible skinlesions and visually unaffected skins was performed at the end of the study. (b) Control skin(not exposed to UV and untreated). (c) Largest visible lesion of a non-denatured soymilktreated skin. (d) An average lesion in the high-risk, untreated group shows a welldifferentiated SCC. All images are of same magnification. Topical treatment with nondenatured soymilk significantly reduced the severity of the developing skin lesions. Images
are courtesy of C. Paine.

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10. Non-denatured soybean extracts reduce skin cancer risk by multiple
mechanisms
Non-denatured soybean extracts, as all botanical extracts, contain multiple agents, which are
only partially characterized. It is expected, therefore, that such extracts would act bymultiple mechanisms. Studies of the mechanisms of action of the chemopreventive activityof non-denatured soybean extracts identified effects both at the initiation and theprogression of skin tumors, with multiple mechanisms affecting the tumor and themicroenvironment (Chen et al, 2008). Topical pretreatment of hairless mice or Yucatanswine with non-denatured soybean extracts for several days prior to UVB exposure,prevented or reduced UVB-induced DNA damage (T-T dimers) and apoptosis. This
reduction in the cumulative DNA damage in skin is, at least partially, mediated via theactivation of the cell-cycle regulatory protein Chk1 (Chen et al, 2008). Non-denatured
soybean extracts inhibit metallo-proteinases (MMPs) expression in vivo, suggesting theinhibition of dermal ECM remodeling, which is required for tumor progression. Vascularendothelial growth factor (VEGF)-induced endothelial tube formation in Matrigel was
inhibited in vitro by non-denatured soybean extracts, suggesting a possible inhibitory effecton angiogenesis and tumor progression. Taken together with their ability to inhibit multiple
proteases and to enhance ECM production (Zhao et al, 2009), their ability to inhibit the
COX-2 pathway (Chen et al, 2008), and their content of intact STI and BBI, non-denatured
soybean extracts could affect the tumor and the microenvironment at multiple stages of skincancer initiation and development. Topical treatment with non-denatured soybean extracts,
therefore, could reduce the risk of skin cancer development, by reducing UVB-induced
DNA and cellular damage, and reducing microenvironment processes.

11. Clinical studies with non-denatured soybean extracts
An initial human clinical study using non-denatured soybean extracts for skin care was
performed on hyperpigmentary lesions (Hermanns et al, 2000). Non-invasive image analysis
was performed with high magnification pictures obtained with a UV-emitting unit and asensitive fluorescence video recording, which could detect subclinical mottled skinpigmentation. Non-denatured soybean extracts led to significant skin lightening after a 3week treatment (Hermanns et al, 2000), suggesting, for the first time, that the inhibition of
the PAR-2 pathway might present a novel approach for the treatment of pigmentary skinlesions.
The effect of non-denatured soybean extracts on hair growth was first evaluated in three
Caucasian males with dark facial hair. Panelists treated one side of their face with theextracts, daily, immediately after shaving. By the forth week, the hair of the treated side
was visually lighter and shorter, and felt smoother to touch. Image analysis of the treatedareas four hours after shaving documented reduced length and thickness of the hair stubble
at the treated side, with a statistical significant difference in all measured parameters(Seiberg et al, 2001). Examples of images from this study are shown in figure 6a-b. For the
next proof of concept study, hair was wax-depilated of two symmetrical areas of the legs,
below the knee, in two females, and one leg was treated daily, for four weeks, with nondenatured soybean extracts. Hair growth on the treated site was visually reduced, with thehair shafts visibly shorter and thinner than those of the control sites (Seiberg et al, 2001).

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Examples of images from this study are shown in Figure 6c-d. Later studies documented
similar effects on women's facial hair. These observations confirmed the effect of nondenatured soybean extracts on delaying human hair growth, and suggested that this effect is
not related to the method of hair removal or to the body part being treated.

Control

Non-denatured
soybean extracts

a

b

c

d

Fig. 6. Non-denatured soybean extracts reduce human hair length and thickness. (a-b)
Caucasian males treated the right side of their face with non-denatured soybean extract,
immediately after shaving. Digital pictures taken after four weeks of treatment, four hours
after shaving, demonstrate reduced growth rate and dimensions of the hair stubble at thetreated side (b), relative to the untreated control (a). (c-d) Hair was wax-depilated of twosymmetrical areas of female legs, below the knee, and one leg was treated daily, for fourweeks, with non-denatured soybean extract . Images taken at four weeks of treatment, sixdays after shaving, document reduced density and dimensions of the hair shafts at thetreated side (d), relative to the untreated control (c).
The non-denatured soybean extracts were next evaluated in a placebo-controlled clinicalstudy for their effect on UV-induced erythema, hyperpigmentation and peeling (Wu et al,2001). Subjects were exposed to UV irradiation of about 1.5-2 MED, following by daily

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applications of the non-denatured soybean extract. Diffused reflectance spectroscopy
measurements and independent dermatologist assessment concluded that UV-induced
erythema was reduced at 24 hrs after the application of non-denatured soybean extracts. At
7 days post irradiation, peeling was observed on some placebo or untreated sites, but not on
the non-denatured soy-treated sites (Wu et al, 2001). This clinical, placebo-controlled study
clearly demonstrates that non-denatured soybean preparations are beneficial for daily
defense against solar irradiation. An example of the effect of pretreatment with nondenatured soybeans extracts on UV-induced erythema and peeling is shown in figure 7.

ControlaNon-denatured
soybean extractsbFig. 7. Non-denatured soybean extracts reduce UV-induced erythema and peeling of
human skin. A Caucasian male treated the right side of his face with non-denatured
soybean extract, immediately after shaving, for six weeks, and was then exposed to solar
irradiation during recreational activity. Digital Hi-Scope pictures taken 24 hrs post UV
exposure, demonstrate reduced redness and peeling at the treated side (b), relative to theuntreated control (a).
A double blind, placebo-controlled study of the efficacy of a moisturizer containing nondenatured soybean extracts was performed to evaluate the effects of this moistuizer on skin
tone, skin pigmentation, and other photoaging-induced effects, in 63 women with moderate
photo-damage (Wallo et al, 2007). Efficacy was monitored by clinical observations and selfassessments on weeks 0, 2, 4, 8, and 12, and by non-invasive measurements (such ascolorimetric evaluations and digital photography) on weeks 0, 4, 8, and 12. The nondenatured soybean extract-containing moisturizer was well tolerated, and was more
effective than the placebo in improving numerous photoaging parameters of skin color andtexture, including overall skin tone, mottled pigmentation, blotchiness, dullness, fine lines,
overall texture, and overall appearance (Wallo et al, 2007). Examples of the effect of the nondenatured soy extract moisturizer on skin pigmentation and on facial wrinkles are shown in

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ControlaNon-denatured
soybean extracts
b

c

d

e

f

g

h

Fig. 8. Non-denatured soybean extracts reduce signs of photoaging. Women's faces weretreated for 12 weeks, twice daily, with a moisturizer containing 2% non-denatured soybean
extracts. A cross-polarized (a-b, e-f) and a black-and-white (blue-channel) of cross-polarized(c-d, g-h, enable visual enhancement of pigmentation changes) photographs were taken atbaseline and at the end of the treatment period. (a-d) The images demonstrate improvementin skin tone, texture and radiance parameters following the treatment with non-denatured
soybean extracts. (e-h) Images demonstrate improvement in wrinkling ("crow's feet") and in
pigmentation parameters. Images are courtesy of W. Wallo.

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figure 8 (Images courtesy of W. Wallo). The differences in photoaging parameters weresignificant from week 2 to week 12 for all above parameters (except for dullness, whichstarted at week 4), suggesting that a moisturizer containing non-denatured soybean extracts
is very effective in ameliorating photoaging-induced skin effects.

12. Conclusion
Preclinical and clinical studies, as well as mechanistic understanding, confirm that nondenatured soybean extracts, their formulations and their products, are safe and effective in
improving numerous skin care parameters. Thus, the use of non-denatured soybean extracts
provides multiple skin care benefits, ranging from protection and restoration, to aesthetic
benefits.

13. Acknowledgement
The author would like to acknowledge the many individuals who had contributed to thestudies of non-denatured soybean extracts over the years. Special thanks to Dr. C.B. Lin, D.
Rossetti, C. Paine and W. Wallo for providing unpublished images for this publication.
Many thanks to Drs. C.B. Lin and D. Cavender for critical reading of this chapter.

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Soybean - Biochemistry, Chemistry and Physiology
Edited by Prof. Tzi-Bun Ng

ISBN 978-953-307-219-7
Hard cover, 642 pages
Publisher InTech

Published online 26, April, 2011

Published in print edition April, 2011
Soybean is an agricultural crop of tremendous economic importance. Soybean and food items derived from it
form dietary components of numerous people, especially those living in the Orient. The health benefits ofsoybean have attracted the attention of nutritionists as well as common people.

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