LEGEND
Dermal Restructuring effect of Trifolium pratense extract demonstrated by in vitro comparative studies
Authors: Laura Olariu, Brandusa Dumitriu, Manuela Diana Ene, Lenuta Zglimbea, Mariana Constantinovici
S.C. Biotehnos S.A., 3-5 Gorunului Street, 075100-Otopeni, Ilfov, Romania, Phone: +40317102402, Fax:
+40317102400,
e-mail: lolariu@biotehnos.com
AIM of EXPERIMENTAL STUDY
FIBROBLAST CELLS PROLIFERATION
The results were estimated as proliferation index, i.e. the sum of the percentages of cells in S and
G2 phases of multiplication/ M calculated with a specific analysis software (FACS Express V3 DNA
cell cycle and proliferation module).
Many dermatocosmetics have in their composition complex biological extracts, the most traced
of them being the anti-aging ones. Given the development of this kind of preparations in
Biotehnos laboratories, it was desired to establish the complementary action of the active
principles components (isoflavones: genistein, daidzein, biochanin, formononetin and flavonoid
quercetin) of the clover extract isolated from Herba Trifolium pratense in delaying the aging
process (acceleration of cellular and protein turn-over, balancing synthesis and degradation of
extracellular matrix and enhancing the links cell - matrix structural proteins by overexpression of
integrins). Thus, restoring dermo-epidermal structures (the first step in blocking the mechanism
of aging) under the action of clover extract was the objective of this work conducted with
complementary in vitro investigative techniques on fibroblasts cultures from standardized cell
line Hs27. The applied in vitro techniques were: evaluation of collagen synthesis, inhibition of
MMP 2 and 9, cell proliferation of fibroblasts and highlighting the overexpression of integrins
responsible for cell adhesion and therefore the inter firmness of skin tissue. The clover extract is
physico-chemically characterized in the below chromatograms (being called Dermo ET), in
respect to the combination of isoflavones standards - daidzein, genistein, formononetin and
biochanin (Fig. 1 and 2).
10
0
CONTROL
formononetin
5
Fig. 1. HPLC chromatogram for isoflavones standard combination.
10
15
20
25
30
Fig. 2. HPLC chromatogram for Dermo-ET extract.
MATERIALS and METHODS
Cell culture: Hs27 - Human Skin Fibroblasts, originating from ECACC, catalog no. 94041901
Kits: - Cell Trace CFSE Cell Proliferation Kit for flow cytometry, Invitrogen C34554
- Cycle Test Plus DNA Reagent Kit, BD 340242
- Monoclonal antibodies for α1β1 and α2β1 integrins, BD cat. 559596, 555498, 559883
Other reageants: Sigma origin
Collagen synthesis: spectrophotometrical measurement of hydroxyproline (OH-Pro)
concentration in culture media allows the indirect estimation of total collagen content
synthesized by cells from a specific treated sample. The hydroxiproline level is correlated with
that of biosynthesized collagen (1 mg of collagen correspond to 0.0122 mg OH-Pro) and it is
quantified from a standard curve generated using synthetic OH-Pro (Sigma Chemical Co.).
MMP 2 and 9 inhibition: application of a gelatin-zymography protocol which allows their
identification by the degradation of MMPs preferential substrate (gelatin) and by their molecular
weight. The zymograms are scanned to densitometer the area of the gel where these
gelatinases have acted.
Cellular proliferation: evaluated by fluorescence quantification of CFSE labeled probes using
a flow-cytometer BD FACSCanto II and data interpretation with a specific soft, FCS Express –
proliferation module, which enables calculation of relevant statistical parameters: proliferation
index (Ip), division index, number of generations, percent of cells in 0 generation (which are not
dividing).
Sequentiation of cell cycle enables identification of DNA aberrations and estimation of mitotic
index by applying nuclei fluorescence labeling with propidium iodide, followed by flowcytometric quantification of aneuploidies.
α1β1 and α2β1 integrins overexpression: using monoclonal antibodies to α and β chains
(CD49a, PE fluorescent labeled, corresponding α2 subunit, CD49b, FITC fluorescent labeled,
corresponding α1 subunit, and CD 29, APC fluorescent labeled, corresponding β1 subunit).
RESULTS and DISSCUSSION
STIMULATION of COLLAGEN SYNTHESIS
Degradation of collagen in the extracellular matrix is largely due to the proteolytic activity of
metalloproteinases (MMPs) that are overexpressed by dermal fibroblasts cytokines or growth
factors’ action in physiological and pathological processes. Adjusting the extracellular matrix
involves a balance between synthesis and degradation of structural components under catalytic
action of MMP – sites.
Collagen
µg OH-Pro/2*105cells/ mL
Activ principle/ Control
% of variation
Measured
value
Designation
Dose
Measured
value
Cellular Control
-
0,0712
-
Solvent Control
-
0,0617
1/1000
Dermo ET
Daidzein
Genistein
Biochanin
Formononetin
(Daidzein+Genistein+Biochanin+Formo
nonetin) equivalent to Dermo ET
MMP
MMP9 (pixels)
MMP2 (pixels)
% of
variation
Measured
value
% of
variation
90,84
-
94,26
-
-
89,11
-
93,3
-
0,1012
39,03
73,18
-21,8
94,61
1,4
1/2000
0,0774
20,28
78,55
-13,4
94,32
1,1
2.6 µM
0,0758
18,60
74,08
-20,3
90,61
-3,0
1.3 µM
0,0761
18,92
73,83
-20,7
89,69
-4,0
3.4 µM
0,0325
-89,85
76,21
-16,9
91,95
-1,5
1.7 µM
0,0287
-114,98
79,08
-12,7
92,98
-0,3
6.4 µM
0,0370
-66,76
81,52
-9,3
91,63
-1,8
3.2 µM
0,0436
-41,51
72,71
-22,6
89,96
-3,7
32.7 µM
0,0583
-5,83
71,96
-23,8
95,95
2,8
16.4 µM
0,0396
-55,81
77,11
-15,6
94,32
1,1
1/1000
0,0786
21,50
92,95
4,1
96,71
3,5
1/2000
0,0455
-35,60
91,04
2,1
95,86
2,7
Table 1. Comparative evaluation of collagen biosynthesis towards MMPs 9 and 2 inhibition in
fibroblasts treated with each of the active principles from Dermo ET.
• only daidzein induces, both biosynthesis of collagen type I and III,
and inactivation of proteolytic enzyme MMP 9;
•other phytohormones individual analysis showed an inhibitory
effect on proteolytic enzymes, but matrix collagen biosynthesis
could not be stimulated in their presence;
•phytohormones in various combinations boosted
by at least 13.5% daidzein effects, aspect
observed also for Dermo ET (see herewith zymogram).
Dermo ET
1/2000
Dermo ET
1/2000
min
INDUCED OVEREXPRESSION OF INTEGRINS α1β1 and α1β2
Integrins are functional glycoproteins composed of two subunits (α and β) that extend across the
membrane, being capable of binding multiple ligands, including extracellular matrix molecules,
and having a role in cell adhesion, cell movement and migration. α1β1 integrin mediates
feedback control for the synthesis of collagen, making links cell - collagen or cell - laminin 1 in the
extracellular matrix. α2β1 integrin mediates the stimulation of the type I collagenase (MMP1)
involved in fibrillogenesis, binding type I collagen. The balance between α1β1 and α2β1 integrins
is important for maintaining the equilibrium between collagen degradation and synthesis.
Specimen_002_H1_H01.fcs
5
10
0.00%
Specimen_002_H1_H01.fcs
0.00%
4
10
3
10
2
10
1
10
5
10
APC-A
biochanin A
genistein
daidzein
min
Dermo ET
1/1000
100.00%
3
10
2
10
1.23%
0.07%
4
10
3
10
5
98.07%
3
Tested
compound
APC-A
Mean
( CD 49b % of
(CD 49a % de
Integrin variation Integrin variation
alfa2)
alfa1)
(CD 29 Integrin
beta1)
5
10
4
10
% of
variation
1.56%
5
3
2 0 2
86.38%
2 0 2
-
5837
-
4167
-
Solvent control
TGF beta 4ng/ml
(negative
control)
Dermo ET
1/1000
Dermo ET
1/2000
15122
4,67
6330
8,45
4984
19,61
44217
206,04
7005
20,01
8123
94,94
29921
107,09
4851
-16,89
3719
-10,75
25796
78,54
4092
-29,9
4314
3,53
1.74%
3
10
4
5
10
10
5
10
0.27%
3.73%
Dermo- Sks 1/60000
4
10
3
10
2
-10 10 10
14448
97.59%
-10 10 10
2.10%
2
Control
Cellular control
Specimen_001_D12_D12.fcs
10
2
0.04%
FITC-A
4
-10
0.63%
3
10
10
10
5
10
10
10
2
-10
Specimen_001_D12_D12.fcs
PE-A
Mean
4
10
4
PE-A
FITC-A
Mean
3
10
2
0.63%
10
2
10
10
2
2 2
1
10
Specimen_001_C1_C01.fcs
10
-1010
0.00%
FITC-A
Specimen_001_C1_C01.fcs
10
2
-10
100.00%
10
0
10
PE-A
5
Isotipic control
0
10
0
1
2
3
4
5
10 10 10 10 10 10
10
0.00%
4
10
0
0.00%
10
1
0.00%
APC-A
30
% of variation
SOLVENT
CONTROL
APC-A
25
Dermo ET
1/1000
APC-A
biochanin A
0
20
SOLVENT
CONTROL
APC-A
formononetin
genistein
0
15
CONTROL
5
200
10
0
The results demonstrate the accelerating rate of cell multiplication induced by clover extract,
Dermo ET, with over 30% above control. Accelerating S phase and entry into mitosis is strongly
influenced especially by formononetin and biochanin, weakly by daidzein and at all by genistein.
Instead, the four components combination is optimal for stimulating cell proliferation, empowering
themselves.
600
5
Proliferation index
5
% of variation
15
800
400
10
20
1000
100
15
% phase S+G2/M
25
1200
200
20
Fig. 3. Cell cycle sequentation and proliferation index for fibroblasts treated 48 h with Dermo ET.
1600
daidzein
300
25
30
1400
400
45
35
mAU
500
30
40
DAD1 A, Sig=260,16 Ref=off (ISOFLAVONE\ISOFLAVONE2 2011-03-28 15-05-38\DERMO-ET-EX6D.D)
mAU
50
APC-A
DAD1 A, Sig=260,16 Ref=off (ISOFLAVONE\STD-DGFB000056.D)
35
9.96%
3
10
4
10
PE-A
5
10
10
2
-10
37.09%
2
0 2
-10 10 10
58.91%
3
10
4
10
5
10
FITC-A
Example of flow cytometry analysis (dot
plot and fluorescence histograms)
estimating the integrins expression
based on multicolor antibodies staining.
Table 2. Comparative evaluation of the three glico-proteins chains
expression as corresponding median fluorescence channel.
The tests revealed Trifolium pratense Herba extract action, Dermo ET, in a dose-effect manner, only
on the induction of glycoprotein α2 chain overexpression, indicating an increase in fibroblast - collagen
type I ties and stimulating the collagenase activity with a role in fibrillogenesis. Evidence indicates that
this action is mainly due to the components extract biochanin and genistein, and in a lesser extent to
daidzein, with formononetin not acting at this level. The determined effect is similar to the positive
control (TGF beta 4ng/ mL).
CONCLUSIONS
The obtained results for testing clover extract with the three mechanisms of dermal tissue
reconstruction demonstrates the complementary role of daidzein, genistein, biochanin and
formononetin and the importance of their combination in a specific ratio to maximize the effect, as they
are naturally mixed in Dermo ET.
REFERENCES
1. Kähäri V-M, Saarialho-Kere U. “Matrix metalloproteinases in skin”, Exp Dermatol 6:199-213, 1997.
2. GT Wondrak, “Mechanisms and potential for therapeutics in skin photodamage”, Curr Opin in Invest
Drugs vol 8, 390 – 400 2007.
3. Zhang Z, Bothe I, Hirche F, Zweers MC, Gullberg D, Pfitzer G et al.; „Interactions of primary
fibroblasts and keratinocytes with extracellular matrix proteins: contribution of alpha-2 beta-1 integrin”,
J Cell Sci, 119:1886–1895, 2006.
4. Fujumura T., Moriwaski S., Imokawa G., Takema Y., „Crucial role of fibroblast integrins alpha2 and
beta1 in maintaining the structural and mechanical properties of the skin”, J Dermatol Sci,
Jan;45(1):45-53, 2007.
ACKNOWLEDGEMENT
CELLULAR CONTROL
MOLECULAR MARKER
SOVENT CONTROL
DERMO ET 1µL
DERMO ET 0.5 µL
1
2
3
4
5
The input in Biotehnos’ biotechnology research infrastructure was made ​on the basis of the project POS 275 /
CNRS 6009 CTR 74/2009
Experimental developement and inovation activities in dermatocosmetics are done with financial help from POS
CCE ID 383 SMIS CSNR 6009 CTR 107/2010
Project title: POS 275 / CNRS 6009 CTR 74/2009 –BTHCD “Infrastructure development activity of biotech
R&D in BIOTEHNOS as part of SMEs competitive in Europe”
Project title: POS CCE ID 383 SMIS CSNR 6009 CTR 107/2010 - DERMOLAB “International standards
implementation for the organisation of a dermato-cosmetic research and testing core”