DOI:10.1111/j.1600-0625.2009.00862.x
www.blackwellpublishing.com/EXD

Letter to the Editor

Extracts from Glycine max (soybean) induce elastin
synthesis and inhibit elastase activity
Renbin Zhao1, Elizabeth Bruning1, Dianne Rossetti1, Barry Starcher2, Miri Seiberg1 andVioletta Iotsova-Stone1
1

The Johnson and Johnson Skin Research Center, CPPW, A division of Johnson and Johnson Consumer Product Companies, Skillman, NJ, USA;
Health Center at Tyler, University of Texas, TX, USA
Correspondence: Miri Seiberg, The Johnson and Johnson Skin Research Center, CPPW, A division of Johnson and Johnson Consumer ProductCompanies, Skillman, NJ, USA, Tel.: 1-908-874-2325, Fax: 1-908-874-1254, e-mail: mseiber@its.jnj.com
2

Abstract: Elastic fibres are essential extracellular matrix

components of the skin, contributing to its resilience andelasticity. In the course of skin ageing, elastin synthesis is reduced,
and elastase activity is accelerated, resulting in skin sagging andreduced skin elasticity. Our studies show that non-denatured
Glycine max (soybean) extracts induced elastin promoter activity,
inhibited elastase activity and protected elastic fibres fromdegradation by exogenous elastases in vitro. Mouse and swine
skins topically treated with soybean extracts showed enhanced

elastic fibre network and increased desmosine content. Elastin
expression was also augmented in human skin transplanted onto
SCID mice in response to soy treatment. These data suggest that
non-denatured soybean extracts may be used as skin care agentsto reduce the signs of skin ageing.
Keywords: elastase – elastin – Glycine max – non-denatured –skin ageing – soy – soybean

Accepted for publication 15 December 2008. Please cite this paper as: Extracts from Glycine max (soybean) induce elastin synthesis and inhibit
elastase activity. Experimental Dermatology 2009; 18: 883–886.

The resilience of the skin depends on the functionality of
its elastic fibres. Elastic fibre formation is a multi-step process engaging a variety of factors. It involves tropoelastin
synthesis and secretion, and its deposition on microfibrils
followed by cross-linking into mature fibres (1–4). In addition, growth factors, such as TGF-b, IGF-1 and IL-1b
induce elastin expression (5–9), while TNF-a and EGF
inhibit elastin synthesis (10,11). Loss of elastic fibres is amajor contributing factor of skin ageing (12,13). Chronological ageing, UV exposure and inflammation, all increase
the release of elastases, and in particular of human neutrophil and human macrophage elastases (HLE and HME)
(14–16). These elastases degrade elastic fibres, resulting inskin sagging and wrinkling (14,16–21). Indeed, the inhibition of fibroblast-derived elastase protects skin’s elastic
fibres and reduces wrinkle formation in rodents (19,22,23).
Our previous studies showed that treatments with nondenatured soybean extracts and with the soybean derived
protease inhibitors (STI and BBI) result in skin lightening(24–26) and in delayed hair growth (26). During the clinical studies, we noticed an improvement in skin elasticity ofAbbreviations: HLE, human leukocyte elastase; HME, humanmacrophage elastase; STI, soybean trypsin inhibitor; BBI,
Bowman-Birk inhibitor; SCID, severe-combined immunodeficient; IHC, immuno-histochemistry; ECM, Extracelluar matrix;
DE, dermal–epidermal.

ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 883–892

soy-treated subjects (27). Here we report that soybean
extracts enhance the elastic fibres in skin by inducing
elastin promoter activity and by inhibiting elastases.
To understand the mechanism of action of thesoybean extracts, we generated two elastin promoterreporter constructs, and their activities were examined intransiently transfected H9c2 cells (rat cardiac myoblasts).
A 2.2 kb promoter-reporter construct (Elp2.2, Fig. S1a)showed lower basal promoter activity than a shorter,
0.5 kb construct (Elp0.5, Fig. S1a,b), because of negative
cis-acting elements between )2260 and )495 (28). Treatment of Elp2.2 transfected cells with soybean extracts
resulted in a dose-dependent increase in elastin promoter
activity (Fig. 1a). No induction was observed with the
Elp0.5 promoter (not shown), suggesting a possible
de-repression, via regulatory elements located between)2260 and )495 bp.
HME and HLE activities were measured in vitro using
fluorescent-labelled peptide substrates (Appendix. S1). Soybean extracts inhibited both HME (Fig. 1b) and HLE(Fig. 1c) in a dose-dependent manner, yet with different
degrees of inhibition. HLE was inhibited by 70% with 0.1%
soy extracts, while HME was only inhibited by 40%(Fig. 1b,c). The soy proteins STI and BBI showed only
30% and 40% inhibition of HLE respectively, at concentrations ten times higher than those present in the tested soybean extracts, while soy isoflavones and BSA did not883

Letters to the Editor

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Figure 1. Soybean extracts induce elastin promoter and inhibit
elastases. (a) Soybean extracts induce elastin promoter activity. Data
were presented as fold of induction. Results from 5 experiments are
summarized. Each bar represents mean ± SD * significant withP £ 0.001. (b, c) HME (b) and HLE (c) activities were measured in thepresence of soybean extracts, STI, BBI, soy isoflavones and BSA, atdifferent concentrations as indicated. The extract contains
approximately 0.001% STI and BBI and 0.0001% isoflavones. Results
are presented as percent activity of control. Each bar representsmean ± SD * significant with P < 0.01 (N = 5 for soy and N = 3 for STI,
BBI). (d–g) Elastic fibres produced by primary fibroblast cultures werevisualized by IHC with polyclonal anti-elastin antibody. (d) Untreatedcontrol culture; (e) Fibroblasts treated with 0.1 U ⁄ ml HLE for 1 h; (f, g)
Fibroblasts treated with 0.1 U ⁄ ml HLE in the presence of 0.02% (f) and
0.05% (g) soybean extracts. Bar = 50 lM.

inhibit HLE (Fig. 1c). Heat-denatured soybean extracts
showed similar elastase inhibitory activity to that of thenon-denatured soy, (Fig. S1c), suggesting that STI, BBI andisoflavones are not the main elastase inhibitors in soy.
Further studies were carried out in vitro, to evaluate theprotective effect of soybean extracts against elastases. Soybean extracts protected elastic fibres, produced by primary884

dermal fibroblasts (Fig. 1d), from degradation by exogenous HLE. Elastic fibres appeared fragmented within one
hour of exposure to 0.1 U ⁄ ml HLE (Fig. 1e). When incubated with soybean extracts during the HLE treatment, theelastic fibres were protected from degradation, partially at
0.02% (Fig. 1f) and completely at 0.05% (Fig. 1g). Ellagic
and tannic acid can protect newly synthesized elastic fibres
from elastase degradation, and therefore enhance elastogenesis in fibroblast cultures (29). The protective capacities ofsoybean extracts against elastases suggest that they may also
contribute to elastogenesis.
To test the in vivo activity of the soybean extracts,
SKH-1 hairless mice (Fig. 2a,b) and Yucatan swine(Fig. 2c,d) were treated for 8 and 12 weeks, respectively,
with either soybean extracts or vehicle (Appendix S1).
Luna elastin staining of the soy-treated mouse and swine
skins demonstrated an increase in elastic fibre staining in
the upper dermis, adjacent to the dermal–epidermal (DE)junction (Fig. 2b,d). Soy-treated swine skins revealed anincrease in branched, fine elastic fibre that were perpendicular to the DE junction (Fig. 2d). Skins treated withsoybean extracts showed no abnormal elastin accumulationor any other histological abnormalities. Next, we showed
the induction of tropoelastin expression in mouse skins. A
2.4-fold increase in tropoelastin mRNA was demonstrated
in soy-treated mouse skins relative to vehicle (QPCR,
Fig. 2e). To measure elastin content in skin, we quantified
desmosine crosslinks, which are unique to elastic fibres(30). Desmosine analysis of swine skins showed anincrease from 104 ± 15 pmole ⁄ mg protein (vehicle-treated)
to 163 ± 17 pmole ⁄ mg protein in soy-treated skins, withan average increase of about 57% (Fig. 2f), suggesting thatsoy not only induces elastin synthesis, but also increases
the amount of mature elastic fibres.
To relate the findings to human skin, two facial skins(facelifts, obtained with informed consent), transplanted
onto SCID mice, were treated for 7 weeks with soy extracts
or vehicle. Treatment with soy extracts significantly
induced tropoelastin expression, compared with respective
controls (3.4- and 1.8-fold, QPCR) (Fig. 2g). Different
basal levels of tropoelastin expression were documented
with the two individuals; however, soy treatment induced
tropoelastin expression in both donors, relative to theirbaselines (Fig. 2g). The levels of tropoelastin induction inthese human skin grafts were in the range of thoseobserved in the soy-treated mouse skins. Confirmation ofelastin induction by histology was not feasible due tomassive solar elastosis in these skins both pre and posttransplantation. Treatment of these human skins with soy
extracts not only induced tropoelastin expression, but also
increased the expression of fibulin-5, a major microfibril
component (Fig. 2g), suggesting the concerted regulationof elastic fibre assembly. Soy extracts also induced theª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 883–892

Letters to the Editor

(a)

(b)

We also show the inhibition of elastases by soybean extracts,
which prevents elastic fibre degradation. These two activitiesof the soybean extracts suggest their usefulness in skin care,particularly in the prevention and reduction of the loss ofelastic fibres associated with skin ageing.
Acknowledgements

(c)

(d)

The authors would like to thank Frank Liebel, David Appel and Jeff
Pote for technical help, Wen-Hwa Li for human ⁄ SCID support, andDrs Magdalena Eisinger and Stanley Shapiro for fruitful discussions.
All in vivo studies were completed by December 2007.

References

(e)

(f)

(g)

Figure 2. Soybean extracts enhance the elastic fibre network in vivo.
(a, b) SKH-1 mice were treated with soy extracts (b) and vehicle control(a), for 8 weeks, and elastic fibres were visualized histologically by Lunaelastin staining. Elastic fibres stained purple in a yellow background. (c,d) Swine skins were topically treated with soybean extract (d) or vehicle(c), for12 weeks and stained for elastin fibres (stained purple in ablue ⁄ green background). Bar = 50 lM (e) RT-PCR analysis of RNAs
isolated from SKH-1 mouse skins treated with soy extracts and control
for 1 week. Tropoelastin expression was normalized to GAPDH andresults were averaged from three mice per group. Fold of induction was
calculated as ratio of treatment to control. (f) Desmosine analysis ofswine skins topically treated with soybean extracts or vehicle for
12 weeks. Desmosine results were averaged from both left and rightside of the treated swine. (g) Soy extracts induced elastin, collagen-1
alpha-1 and fibulin-5 expression in human skins transplanted onto SCID
mice. Fold of induction was calculated as the level of expression in soy
treated skin, relative to control from the same donor.

expression of collagen1-a1, a major component of thedermal extracellular matrix, suggesting that soy may haveeffects on additional ECM genes (Fig. 2g).
We present in vitro and in vivo data for soybean extracts
to induce the expression of tropoelastin and fibulin-5, which
are both essential for the de novo formation of elastic fibres.

ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 883–892

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Supporting information

Figure S1. (a) Two promoter fragments of 2.2 kb (Elp2.2) and 0.5 kb(Elp0.5) in length upstream of the elastin coding region, were cloned into
luciferase reporter constructs. A promoterless vector was used as negative
control. An internal control carrying Thymidine kinase promoter with the
Elastin promoter
Renila luciferase (pRL-TK) was also included.
sequence.
Elastin coding sequence;
Firefly luciferase;
Thymidinekinase promoter; Renila luciferase. (b) Relative promoter activities of thetwo elastin- promoter fragments were measured by transient transfection
into rat cardiac myoblast H9c2 cells. The results are expressed as relative
promoter activities of the ratio between Firefly and Renila luciferase measurements. Each bar represents mean ± SD. (c) HLE activity was measured
in the presence of non-denatured and heat-inactivated soybean extracts, atdifferent concentrations as indicated. Results are presented as percent activity of control and averaged from triplicate of readings. Each bar representsmean ± S.D.
Appendix S1. Materials and methods.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries(other than missing material) should be directed to the corresponding
author for the article.

Additional Supporting Information may be found in the online version ofthis article:

DOI:10.1111/j.1600-0625.2009.00848.x
www.blackwellpublishing.com/EXD

Letter to the Editor

A method for the analysis of milk and egg allergens
for the atopy patch test
Cinzia Ballabio1, Alessandro Fiocchi2, Alberto Martelli2, Silvia Pecora3, Samuele E. Burastero4 andPatrizia Restani1
1

Department of Pharmacological Sciences, University of Milan, Milan, Italy;
Melloni Paediatria, University of Milan Medical School at the Melloni Hospital, Milan, Italy;
3
ALK Abellò Italy, Lainate, Italy;
4
San Raffaele Scientific Institute, Milan, Italy
Correspondence: Samuele Burastero, MD, San Raffaele Scientific Institute, 58, via Olgettina, 20132 Milan, Italy, Tel.: +39 2 2643 4730,
Fax: +39 2 2643 4723, e-mail: s.burastero@hsr.it
2

Abstract: The patch test with food antigens (atopy patch test, APT)
has been reported as a more specific method than prick or RAST forthe early detection of cow’s milk and ⁄ or egg sensitizations inchildren. Standardization of APT extracts is a major issue on theroad towards full clinical exploitation of this assay. Here, we usedsodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) to characterize sensitivity and specificity of commercial
preparations of APT for milk and egg allergies, which are expected
to improve the reliability of this test, when compared with fresh

food allergen sources. We found that: (i) SDS-PAGE is anappropriate technique for quality control of APT and (ii)commercial milk and egg APT are equivalent to fresh foodpreparations in terms of allergen content. Clinical trials aimed atcharacterizing sensitivity and specificity of APT in the diagnosis offood allergy in children will benefit from this technique.
Key words: allergy – atopy patch test – egg allergens – milkallergens – SDS-PAGE

Accepted for publication 22 December 2008. Please cite this paper as: A method for the analysis of milk and egg allergens for the atopy patch test.
Experimental Dermatology 2009; 18: 886–888.

Abbreviation: APT, atopy patch test.

886ª 2009 The Authors
Journal compilation ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 883–892