Effect of selected flavonoids on glycosaminoglycans in human skin fibroblasts.
Purpose: Glycosaminoglycans (GAGs) and proteoglycans (PG) in addition to collagen are the main components of extracellular matrix (ECM). They play an important role in intercellular communication and interactions between cells and ECM. The biological changes in ECM that occur during aging are induced by decrease in GAG biosynthesis. The purpose of this study was to evaluate the effect of selected flavonoids isolated from Cirsium palustre (L.) Scop, on GAG content in human skin fibroblasts.
Materials and methods: Human skin fibroblasts were treated with eriodictyol 7-O-glucoside (C1), 6-hydroxyluteolin 7-O-glucoside (C2), scutellarein 7-O-glucoside (C3) and pedalitin (C4) at 1, 20 and 40 [micro]M for 24 h. Concentration of GAGs in the medium was assayed using method based on their ability to bind the cationic dye 1,9-dimethylmethylene blue (DMMB).
Results: C1, C2 and C4 at concentration of 20 and 40 [micro]M significantly increased content of sulphated GAGs in the medium. In contrast, treatment of cells with compound C3 did not have a statistically significant impact on GAG level. Ascorbic acid used as a positive control at 50 [micro]M showed no effect on GAG concentration and increased their content at 100 [micro]M but to a much lower extent than flavonoids.
Conclusion: Flavonoids C1, C2 and C4 showed greater than ascorbic acid stimulatory impact on GAGs in healthy human skin fibroblasts, demonstrating their therapeutic potential in the aging.
Key words: Fibroblasts, glycosaminoglycans, flavonoids
Glycosaminoglycans (GAGs) are linear polymers of repeated disaccharides, in most cases containing an O-sulphated N-acetylhexosamine and an hexuronic acid, commonly expressed in a variety of tissues. They occur mainly in extracellular matrix (ECM) and associated with the cell membrane [1,2]. GAG family include unsulphated hyaluronic acid (HA), sulphated dermatan sulphate (DS), keratin sulphate (KS), heparin sulphate (HS) and chondroitin sulphates (C4S, C6S). Polysaccharides are very heterogeneous in terms of relative molecular mass, charge density, physicochemical properties as well as biological and pharmacologic activities [3,4]. The specific pattern of their sulphation allow GAGs various electrostatic interactions with many biological molecules and ECM constituents like growth factors and enzymes which facilitate their physiological functions and in some cases prevent their proteolytic degradation [4,5]. Most sulfated GAG chains are components of proteoglycans (PGs) where are they covalently linked to core protein. PGs as major macromolecular components are located at the cell surface and in the extracellular matrix [6,7]. Interactions of these macromolecules with other ECM constituents contribute to the general architecture and permeability properties of the basement membrane. Furthermore, these macromolecules play a role in cellular adhesion, growth, migration and differentiation [6-9].
Flavonoids exhibit multiple biological activities, i.e. antibacterial, antiviral, antioxidant, anti-inflammatory, antiallergic, hepatoprotective, antithrombotic and anti-carcinogenic [10,11].
In the previous study we examined effect of selected flavonoids isolated from Cirsium palustre (L.) Scop., marsh thistle (family Asteraceae) on collagen as the main structural component of the ECM . The aim of current research was to evaluate impact of these compounds on other significant components of ECM which are GAGs.
MATERIALS AND METHODS
The compounds: eriodictyol 7-O-[beta]-glucoside (C1), 6-hydroxyluteolin 7-O-[beta]-glucoside (C2), scutellarein 7-O-[beta]-glucoside (C3) and pedalitin (C4) were isolated from Cirsium palustre (L.) Scop., marsh thistle (family Asteraceae) as described previously .
Estimation of biological action of flavonoids in skin fibroblasts
Human skin fibroblasts purchased from the American Type Culture Collection, (Manassas, Virginia, USA) were cultured in DMEM supplemented with 10 % FBS, 2 mM glutamine, penicillin (50 U/ml), and streptomycin (50 [micro]g/ml). For experiments, cells were plated at a density of 1x[10.sup.6]/well in 6-well culture plates (Costar).
The flavonoids were dissolved in DMSO and added to the medium to a final concentration of 1, 20 and 40 [micro]M. The same concentration of DMSO solution was used as control in order to rule out the possible effect of DMSO on fibroblasts. After 24 h incubation, the exposure medium was used for analysis of GAG content.
GAGs content assay
The assay for GAGs quantification is based on their ability to bind the cationic dye 1,9-dimethylmethylene blue (DMMB) [13,14]. After 24h incubation of fibroblasts with different concentrations of flavonoids the concentrated medium was digested with 50 [micro]g/ml proteinase K in 100 mM solution of [K.sub.2]HP[O.sub.1] pH 8.0 at 56[degrees]C overnight. Proteinase K was then inactivated by heating for 10 min at 90[degrees]C. Next, 1 ml of DMMB was added to the samples and vigorously vortexed for 30 min to promote complexation of GAGs with DMMB. The GAG/DMMB complex was then separated from solution by centrifugation (12 000 g, 10 min). The pellet was dissolved by adding 1 ml of decomplexation solution and shaking the mixture during 30 min. Absorbance was measured at 656 nm. Sulfated GAGs quantities were determined by comparison with a calibration curve of CS solutions used as standard and normalized to total protein. Protein concentration was measured using BCA Protein Assay Kit (Pierce).
The results were subjected to statistical analysis using the one-way analysis of variance (ANOVA) followed by the Duncan's multiple range post hock test. Differences were recognized as statistically significant at p<0.05.
Human dermal fibroblasts were exposed to the compounds (C1-C4), which were isolated from methanolic extract of C. palustre leaves as described previously , at the concentration of 1, 20 and 40 [micro]M each for 24 h. Fig. 1 presents their names and structures. All compounds showed no cytotoxicity to the cells at concentration up to 100 [micro]M (results not shown). GAG quantification is based on their ability to bind the cationic dye DMMB [13,14]. In this assay, we used modified method of Barbosa et al.  which has improved specificity, reproducibility and sensitivity as compared to the method of Farndale .
As shown in Fig. 2 flavonoids C1, C2 and C4 exerted stimulatory effect on GAGs in a dose-dependent pattern. The differences between amount of GAGs in control (DMSO-treated) and compound-treated cells were significant at higher doses of the compounds (20 and 40 [micro]M). Compound C1 increased GAG content 2.8- and 3.7times at 20 and 40 [micro]M, respectively. Compound C2 showed similar stimulatory impact at 20 and 40 [micro]M increasing GAG content about 4-times. In the case of compound C4 1.7- and 4-times increase of GAG content at 20 and 40 [micro]M, respectively was noted. In contrast, treatment of cells with compound C3 did not have a statistically significant effect on GAG level as compared to control. Ascorbic acid as known antioxidant was simultaneously used in experiments as a positive control. At 50 [micro]M it showed no effect on GAG concentration, but incubation of cells in the presence of 100 [micro]M increased their content by 76%.
The extracellular matrix is composed mainly of proteoglycans, hyaluronic acid, collagens and other glycoproteins, which all interact with each other. Proteoglycans and their attached glycosaminoglycans are closely associated with collagen fibers and these interactions play a dominant role in the maintenance of tissue structure and cell adhesion [15,16]. GAGs with negatively charged residues can interact with basic amino acids of proteins and change their conformation. Because of their potential reactivity with other components, they play important biological role in cell--cell and cell--matrix interactions during normal and pathological conditions. Furthermore, they have been shown to regulate protein secretion, enzyme activity and gene expression in certain tissues by mechanisms involving both membrane and nuclear events, including the binding of GAGs to transcription factors [3,4,8,9].
GAGs posses the ability to bind water and therefore are responsible for skin hydration, elasticity and firmness. Synthesis and degradation of the main ECM constituents are influenced by a number of internal factors (vitamins, mineral compounds, hormones, metalloproteinase inhibitors - TIMPs) as well as external ones (UV radiation, environment, stress) [17,18]. Skin exposure to UV radiation contributes to cellular imbalance, oxidative stress, stimulation of pro-inflammatory cytokines, increase MMP activity resulted in increased fragmentation of collagen fibres and impaired function of fibroblasts . In these conditions as well as with age the content of glucosaminoglycans and collagen considerably decreases [17-20]. Breen et al.  reported that there was an decrease in total glycosaminoglycan (HA and DS) content in adult skin relative to fetal skin. Takeda et al.  studied an age-related changes in PG and collagen synthesis in skin fibroblasts of donors in different age. They found decrease in collagen expression at both protein and mRNA level as well as impaired proteoglycan synthesis, whereas overall protein synthesis was slightly affected.
The mostly studied group of natural compounds with great potential antioxidant properties and relatively low toxicity are flavonoids. They are widely distributed in plants. Cirsium palustre (L.) Scop., marsh thistle (family Asteraceae, subtribe Cardueae) is a herbaceous biennial plant widely distributed in Europe and found on marshes, hedgerows and moorland pastures. Nazaruk and Jakoniuk  demonstrated antimicrobial activity of the polyphenolic constituents extracted from this plant.
In our previous study, we have examined the effect of the compounds C1-C4 obtained from the methanolic extract of C. palustre leaves, during multistep chromatographic separations, on collagen expression in human skin fibroblasts . Among them 6-hydroxyluteolin 7-O-glucoside (C2) at concentration of 40 [micro]M and pedalitin (C4) at all used concentrations significantly enhanced amount of collagen in the medium. In current study we showed that the same compounds exerted significant stimulatory impact on GAG content at concentration of 20 and 40 [micro]M. In contrast, eriodictyol 7-O-glucoside (C1) affected these macromolecules differently increasing significantly GAGs (at 20 and 40 [micro]M) without influence on collagen level. Treatment of cells with scutellarein 7-O-glucoside (C3) showed opposite effects with tendency to increase GAG and decrease collagen concentration but the differences in treated and non-treated cells were not significant. The analysis of relationship between the structure of the compounds and their activity suggests that the most important factor influencing the activity seems to be the presence of two hydroxyl groups in the ring B (Fig. 1) since C3 with one--OH group not revealed its activity or to GAGs or to collagen.
Ascorbic acid is a natural anti-oxidant and protects the skin against the harmful effects of free radicals. As a cofactor of prolyl hydroxylase is essential in biosynthesis of collagen and elastin . Except of up-regulation of collagen and collagen-degrading inhibitors (TIMPs), its stimulatory action on GAGs synthesis in fibroblasts was demonstrated . In our study, we used as a positive control. However, flavonoids showed significantly higher stimulatory activity and at lower concentration than ascorbic acid.
Summarizing, in light of these data, flavonoids C1, C2 and C4 may be considered in the future as a therapeutic option in the prevention of biological alterations in the skin aging.
This study was conducted with the use of equipment purchased by the Medical University of Bialystok as part of the OP DEP 2007-2013, Priority Axis 1.3, contract No POPW.01.03.00-20-008/09.
Conflicts of interest
The authors declare that they have no conflicts interests.
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Wosek J. (1,A,B,C), Kuzmicz I. (2,B,C), Wisniewska R. (3,C,E,F), Nazaruk J. (4,C,G), Galicka A, (1.A-F*)
(1.) Department of Medical Chemistry, Medical University of Bialystok, Poland
(2.) Laboratory of Cosmetology, Medical University of Bialystok, Poland
(3.) Department of Pharmacology, Medical University of Bialystok, Poland
(4.) Department of Pharmacognosy, Medical University of Bialystok, Poland
(A) - Conception and study design, (B) - Data collection, (C)--Data analysis, (D) - Writing the paper, (E)--Review article, (F) - Approval of the final version of the article
(*) Corresponding author:
Department of Medical Chemistry, Medical University ul. Mickiewicza 2A, 15-222, Bialystok, Poland
Tel.: 857485675; e-mail: email@example.com
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|Author:||Wosek, J.; Kuzmicz, I.; Wisniewska, R.; Nazaruk, J.; Galicka, A.|
|Publication:||Progress in Health Sciences|
|Date:||Dec 1, 2016|
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