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

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).

Fig. 1. HPLC chromatogram for isoflavones standard combination. 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 flow-cytometric 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.

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, aspectobserved also for Dermo ET (see herewith zymogram).

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DAD1 A, Sig=260,16 Ref=off (ISOFLAVONE\STD-DGFB000056.D)

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DAD1 A, Sig=260,16 Ref=off (ISOFLAVONE\ISOFLAVONE2 2011-03-28 15-05-38\DERMO-ET-EX6D.D)

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Activ principle/ ControlCollagen

µg OH-Pro/2*105cells/ mL

MMP

MMP9 (pixels) MMP2 (pixels)

Designation DoseMeasured

value% of variation

Measured value

% of variation

Measured value

% of variation

Cellular Control - 0,0712 - 90,84 - 94,26 -

Solvent Control - 0,0617 - 89,11 - 93,3 -

Dermo ET1/1000 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

Daidzein2.6 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

Genistein3.4 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

Biochanin6.4 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

Formononetin32.7 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

(Daidzein+Genistein+Biochanin+Formononetin) equivalent to Dermo ET

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

FIBROBLAST CELLS PROLIFERATIONThe 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).

Fig. 3. Cell cycle sequentation and proliferation index for fibroblasts treated 48 h with Dermo ET .

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.

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.

Tested compound

FITC-A Mean

  PE-A Mean

 APC-A Mean

% of variation

( CD 49b - Integrin alfa2)

% of variation

(CD 49a -Integrin alfa1)

% de variation

(CD 29 -Integrin beta1)

Control 14448 -  5837  - 4167  -

Solvent control 15122 4,67 6330 8,45 4984 19,61TGF beta 4ng/ml

(negative control) 44217 206,04 7005 20,01 8123 94,94

Dermo ET 1/1000 29921 107,09 4851 -16,89 3719 -10,75

Dermo ET 1/2000 25796 78,54 4092 -29,9 4314 3,53

Specimen_001_D12_D12.fcs

PE-A

APC-

A

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-102

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105 1.56% 2.10%

86.38% 9.96%

Specimen_001_C1_C01.fcs

PE-A

APC-

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98.07% 0.63%

Specimen_001_D12_D12.fcs

FITC-A

APC-

A

-10210

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-102

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105 0.27% 3.73%

37.09% 58.91%

Specimen_002_H1_H01.fcs

FITC-A

APC-

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0.00%0.00%

Specimen_001_C1_C01.fcs

FITC-A

APC-

A

-10210

010

210

310

410

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-102

102

103

104

105 0.63% 0.04%

97.59% 1.74%

Specimen_002_H1_H01.fcs

PE-A

APC-

A

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105

100

101

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103

104

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0.00%100.00%

0.00%0.00%Isotipic control

Cellular control

Dermo- Sks 1/60000

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.

ACKNOWLEDGEMENT

The input in Biotehnos’ biotechnology research infrastructure was made on the basis of the project POS 275 / CNRS 6009 CTR 74/2009Experimental 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”

CELLULAR CONTROL 1MOLECULAR MARKER 2SOVENT CONTROL 3DERMO ET 1µL 4DERMO ET 0.5 µL 5

LEGEND