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Effect of Different Flavonoids on Collagen Synthesis in Human Fibroblasts

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In this study, we discovered that flavonoids belonging to the subclasses: (flavanone, flavone, and flavonol) display differential effects on the synthesis of collagen in human dermal fibroblasts. At 80 microg/ml flavonoids quercetin-3,3',4', 5,7-pentahydroxyflavone, 3-methyl quercetin, and 7-hydroxyflavone significantly decreased the total protein concentration which was a direct consequence of their cytotoxic effect, while naringenin exhibited no effect on total collagen and total protein concentration. Quercetin-3,3'4',7-tetramethyl ether, 4'-hydroxyflavanone, flavanone, and fisetin significantly decreased collagen concentration while morin, rutin, and chrysin increased collagen concentration without changing the overall protein concentration. The initial screening performed in this study enables the identification of compounds that exert significant effects on fibroblast function and show potential as starting material for pharmaceutical preparations targeted against various disorders centered around disturbed collagen metabolism.
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Plant Foods for Human Nutrition
c
2006 Springer Science+Business Media, Inc.
DOI: 10.1007/s11130-006-0006-8
Effect of Different Flavonoids on Collagen Synthesis in Human Fibroblasts
TAMARA STIPCEVIC,
1,
JASENKA PILJAC,
2
& DIRK VANDEN BERGHE
3
1
Department of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia;
2
Department of Molecular Genetics, Rudjer Boskovic Institute,
Zagreb, Croatia;
3
Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium (
author for correspondence; e-mail:
tamara@irb.hr)
Abstract In this study, we discovered that flavonoids belonging to the sub-
classes: (flavanone, flavone, and flavonol) display differential effects on the
synthesis of collagen in human dermal fibroblasts. At 80 µg/ml flavonoids
quercetin-3,3
,4
, 5,7-pentahydroxyflavone, 3-methyl quercetin, and 7-
hydroxyflavone significantly decreased the total protein concentration
which was a direct consequence of their cytotoxic effect, while narin-
genin exhibited no effect on total collagen and total protein concentration.
Quercetin-3,3
4
,7-tetramethyl ether, 4
-hydroxyflavanone, flavanone, and
fisetin significantly decreased collagen concentration while morin, rutin,
and chrysin increased collagen concentration without changing the over-
all protein concentration. The initial screening performed in this study
enables the identification of compounds that exert significant effects on
fibroblast function and show potential as starting material for pharma-
ceutical preparations targeted against various disorders centered around
disturbed collagen metabolism.
Keywords: Collagen, Fibroblasts, Flavonoids, Tissue culture, Woessner
assay
Introduction
Flavonoids are a large group of naturally occurring, low
molecular weight polyphenolic compounds present in all
plants and are, therefore, an integral part of human diet.
Fruits, vegetables, and beverages such as tea and red wine
are especially rich sources of flavonoids. The dietary in-
take of flavonoids has been estimated to vary from 100 to
1000 mg/day [1]. Perhaps the most active area of flavonoid
research at the present time is the possible medicinal con-
tribution that flavonoids make to human health. Flavonoids
have been shown to possess, both in vitro and in vivo, a wide
range of biochemical and pharmacological effects, includ-
ing antiinflammatory and antiallergic effects [2]. While ma-
jor focus of the studies on flavonoids has been their antiox-
idant properties, there is an emerging view that flavonoids
act as modulators of cell signaling [3]. Understanding the
cellular effects of flavonoids is important for predicting
which dietary flavonoids might be most beneficial in vivo.
Fibroblasts are responsible for the synthesis of collagen,
a fibrous protein of extracellular matrix (ECM) and major
constituent of connective tissue (such as skin, tendons, liga-
ments, cartilage, and bones) responsible for scar formation.
Human fibroblasts in culture synthesize both types I and III
collagen, with type I accounting for 70–90% of the total [4].
Studies on the effects of flavonoids on collagen synthesis
have in the past few years progressed to the point of sug-
gesting the potential therapeutic use of these plant derived
compounds in the treatment of various medical conditions
centered on disrupted collagen metabolism, for example,
delayed wound healing, excessive scarring, skin aging, etc.
[58]. It is worth mentioning that flavonoids from plant
extracts are available as over-the counter ‘natural’ ingredi-
ents in a form of oral supplements or skin care cosmetic
products. They are recommended for daily consumption
and can be bought in regular drug stores or easily ordered
via internet. The list of manufacturer’s claimed benefits for
these flavonoids-based products is long and sometimes not
scientifically founded. Therefore, any additional scientific
interest in flavonoids should be welcomed because it will
ultimately drive future research that will distinguish truly
efficacious ingredients from misleading claims. We are re-
porting results of preliminary screening of 11 flavonoids
commonly present in diet, in terms of their influence on
collagen synthesis. Figure 1 shows names of flavonoids
and corresponding general structures.
Materials and Methods
Fibroblast culture was established from a sample of neona-
tal human foreskin under Institutional Review Board Ex-
empted protocol as described [9]. Cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (Invit-
rogen Grand Island, NY) supplemented with 10% fetal
bovine serum FBS (Gemini-Bio-Products, Woodland, CA).
Flavonoids were purchased from Sigma–Aldrich corpora-
tion with the exception of 3-methyl quercetin, a natural
flavonoid, isolated from plant material by Prof. D. Vanden
Berghe. Cells at 50% confluence (2.5 ×10
6
cells/100 mm
dish) were treated with 80 µmol/l (0.05% DMSO/DMEM),
40 µmol/l (0.025% DMSO/DMEM), and 20 µmol/l
(0.0125% DMSO/DMEM) flavonoid concentrations. The
concentration range was selected on the basis of a pre-
vious “trial and error” experiment where it has been
shown that higher concentrations (above 100 µmol/l) of
tested flavonoids were in most cases toxic to cells. 0.05%
DMSO/DMEM, 0.025% DMSO/DMEM, and 0.0125%
DMSO/DMEM solutions were used as controls in order
to rule out the possible effect of DMSO on fibroblasts.
All concentrations were tested in triplicates. Ascorbic acid
Figure 1. Names and general structures of flavonoids.
solution was added every day in concentration of 50 µg/ml
to the medium. After 5 days, cells were collected using
rubber policeman in 1 ml of 10 mM PBS. Cell membranes
were disrupted using sonicator and total protein amount was
quantified using Bio-Rad protein assay kit (Bio-Rad Labo-
ratories, Hercules, CA). The hydroxyproline was quantified
according to Woessner, method suitable for cases when the
proportion of hydroxyproline to other amino acids in the
sample is very small [10]. 0.5 ml of sonicated samples
wasmixedwith0.5mlof12NHClin2mlglassam-
pules (Wheaton Science Products, Millville, NJ). The vials
were flame-sealed and placed in 115
C oven for 18 hrs.
The samples were dried under N
2
gas in 50
C water bath.
The residue was resuspended in 250 µl deionized H
2
O and
50 µl was mixed with 450 µl of deionized H
2
O to per-
form assay. The standard was prepared in glass test tubes
using 10 µg/ml hydroxyproline standard (Sigma–Aldrich,
St. Louis, MO). Following, were the incubations of sam-
ples: 20 min incubation with 250 µl of 0.05 M chloramine
T solution [chloramine T solution = 1.41 g sodium p-
toluenesulfonylchloramide (chloramine T, Sigma–Aldrich,
St. Louis, MO)] dissolved in 20 ml dH
2
O, 30 ml
methyl cellosolve (2-methoxyethanol, Fisher Scientific,
Pittsburgh, PA) and 50 ml citric acid buffer]; 5 min
incubation with 250 µl of 3.15 M perchloric acid
(Fisher Scientific, Pittsburgh, PA); 20 min incubation with
250 µl of 20% p-dimethylbenzaldehyde (Fisher Scientific,
Pittsburgh, PA) in 60
C water bath dissolved in methyl
cellosolve to a final volume of 50 ml and heated to 60
Cto
dissolve the reagent. After cooling to room temperature, the
optical density of samples was read at 557 nm in a Milton-
Roy spectrophotometer Model 601 (Milton-Roy, Ivyland,
PA).
The data was analyzed using one-way analysis of vari-
ance (ANOVA) accepting
p < 0.05 as level of significance.
Calculation of the total collagen was based on the analy-
sis of total hydroxyproline which is for the most purposes,
a reasonably specific marker for collagen, as the amount
present in elastin and in the Clq component of complement
(the only two potential sources) is negligible by comparison
[11]. The conversion factor of hydroxyproline to collagen
was taken as 7.8.
Results and Discussion
The results obtained clearly show that tested flavonoids
exert differential effects on the total protein concentra-
tion and total collagen concentration in fibroblast cultures
(Table 1, Figure 2). Quercetin (3,3
,4
,5,7-pentahy-
droxyflavone) showed cytotoxic effect and significantly de-
creased total protein and total collagen concentration in a
dose-dependent manner at all tested concentrations (more
Table 1. Effects of three different concentrations
a
of flavonoids on protein synthesis
b
in neonatal human
fibroblasts expressed in µg/dish
Protein (µg/dish)
Compound 80 µmol/l 40 µmol/l 20 µmol/l
Flavanone
4
-Hydroxyflavanone 915.87 ±58.30 942.47 ±45.89 936.50 ±38.45
Naringenin
(4
,5,7-trihidroxyflavanone)
963.07 ±81.50 948.93 ±31.59 946.80 ±25.28
Flavanone 914.20 ±9.25 1060.07 ±90.95 950.60 ±32.34
Flavone
7-Hydroxyflavone 369.33
∗∗
±25.04 894.13 ±57.27 963.53 ±43.05
Chrysin
(5,7-dihidroxyflavone)
954.40 ±39.92 987.57 ±31.96 913.13 ±71.93
Flavonol
Quercetin (3,3
,4
,5,7-
pentahydroxyflavone)
176.23
∗∗
±24.40 398.47
∗∗
±21.54 856.13
±32.82
Quercetin
(3,3
,4
,7-tetramethylether)
1055.27 ±71.03 952.80 ±37.87 990.30 ±28.73
Quercetin (3-methyl
quercetin)
232.20
∗∗
±21.18 265.63
∗∗
±26.92 333.73
∗∗
±5.25
Morin (2
,3,4
,5,7-
pentahydroxyflavone)
938.10 ±35.26 949.53 ±21.54 949.50 ±60.04
Rutin
(quercetin-3-rutinoside)
915.30 ±57.06 943.00 ±48.93 907.17 ±16.06
Fisetin (3,3
,4
,7-
tetrahydroxyflavone)
919.13 ±10.61 938.10 ±62.48 943.00 ±33.43
Control 957.10 ±39.38 950.57 ±25.03 947.33 ±23.72
a
Each flavonoid was tested in three concentrations: 80, 40, and 20 µmol/l.
b
Values were calculated using means of three measurements.
Significant difference from control values was determined by ANOVA (single factor analysis) where
p < 0.05;
∗∗
p < 0.01.
than 50% inhibition) which can be attributed to its ability of
inducing DNA damage [12]. Overall, 80 µmol/l was deter-
mined to be concentration at which flavonoids exert maxi-
mum effect on the concentration of collagen in fibroblasts.
At 80 µmol/l, fisetin, quercetin-tetramethylether, flavanone,
and 4
-hydroxyflavanone decreased collagen concentra-
tion with no effect on the overall protein concentration
(Table 1, Figure 2). Such cases, where the overall protein
amount remains unaffected while the collagen has either
been decreased or increased, are of particular interest be-
cause at these concentrations flavonoids specifically act on
collagen production and at the same time are not harmful
to the cells.
One of the possible explanations for collagen inhibit-
ing activity of various flavonoids could lie in their ability
to interfere with the biosynthesis of the precursor colla-
gen molecule known as procollagen. In the study involving
genistein and quercetin, it has been shown that flavonoids
have the ability to directly influence the synthesis of colla-
gen by inhibiting type I procollagen mRNA in concentra-
tions ranging from 6.25 to 70 µmol/l, indicating effect at a
pretranslational level [13]. Similarly, quercetin was shown
to have growth-inhibitory effect on keloid derived fibrob-
last and significantly inhibited collagens I and III expres-
sion [7,14]. The inhibition of collagen by some flavonoids
is a property relating positively to cases of deranged heal-
ing resulting in excessive scarring. Compounds capable of
reducing collagen could modulate the deposition of extra-
cellular matrix thus favoring the repair process with very
little fibrosis.
On the other hand, at 80 µmol/l concentration morin,
rutin, and chrysin increased collagen concentration 42, 61,
and 42% respectively as compared to control (p = 0.01
and p < 0.01) without affecting the total protein concentra-
tion (Table 1, Figure 2). Collagenolytic enzymes, precisely
matrix metalloproteinases (MMPs) are neutral proteinases
that are inhibited by decrease in free zinc concentrations
caused by chelating agents. With regards to this, flavonoid
catechin, isolated from green tea, is currently in clinical
trials for the treatment of MMP-medicated diseases [15].
Because flavonoids have already been proven to be very
effective in chelating metal ions [1618] it is possible that
they potentially act as collagenase inhibitors which would
result in the overall increase in the measurable collagen con-
centration. Novel results suggest that certain flavonols have
strong inhibitory activity against collagenase [19]. Simi-
larly, catechins, in particular ( )epigallocatechin gallate
(EGCG) has been shown to inhibit membrane type I matrix
Figure 2. Effect of 80 µmol/l (A), 40 µmol/l (B), and 20 µmol/l (C) flavonoid concentrations on collagen
production, expressed as µg of total collagen per dish. t-bars represent standard deviation. Significant
difference from control values determined by ANOVA (single factor analysis) where
p < 0.05;
∗∗
p < 0.01.
metalloproteinase which hydrolyzes type I collagen [20].
Additionally, certain flavonoids have the ability to bind di-
rectly to collagen fibers, thus making them resistant to the
action of mammalian collagenase [21]. Flavonoids of sub-
class proanthocyanidins display high affinity for collagen
and elastin fiber in addition to preventing their enzymatic
hydrolysis by matrix metalloproteinases [22, 23]. This im-
plies that the treatment of fibroblasts with these flavonoids
would result in increase of measurable extracellular
collagen.
Cutaneous aging resulting in wrinkled, rough, and pig-
mented skin is considered to be as a consequence of the
interplay of extrinsic damage to skin by UV radiation, in-
trinsic increases in collagen-degrading matrix metallopro-
teinases, and decreased collagen synthesis [24]. As men-
tioned previously, some flavonoids are capable of increasing
collagen and inhibiting the degradation of matrix (reduction
in dermal thickness). Flavonoids can also have photopro-
tective properties against UVB radiation [25].
Collagen stimulating activity observed for some flavo-
noids might be beneficial in the cases of delayed healing of
cutaneous wounds. By inhibiting MMP, flavonoids could
increase the rate and the amount of collagen synthesized
by fibroblasts necessary for the formation of new wound
matrix, thereby speeding up the healing process.
Overall, it is reasonable to consider the use of flavonoids,
having the ability to accelerate wound healing, con-
trol scar formation and prevent or slow-down the pro-
cess of skin aging for dermatological and cosmetic
purposes.
Knowing the important function of collagen in main-
taining structure, storing energy, altering its diameter and
modality of packing and its role in physiological processes
such as aging and wound healing, the ability of flavonoids
to influence the collagen synthesis should be further ex-
plored. The initial screening performed in this study, by
testing the activity of flavonoids on collagen synthesis, en-
ables the identification of compounds that exert significant
effects on fibroblast function and show potential as start-
ing material for various antiwrinkling cosmetic prepara-
tions, wound healing or even antifibrotic drug development
programs.
Acknowledgments
This study was performed within the Masters degree pro-
gram at the Faculty of Medicine and Pharmaceutical Sci-
ences, University of Antwerp, Belgium. The authors would
like to thank Prof. Karen Reiser, Department of Internal
Medicine, University of California at Davis, for helping in
analysing collagen data.
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... The degradation of the ECM has directly been linked to skin aging, with an increase in the activity of certain enzymes such as collagenases or matrix metalloproteinase. causing the deterioration of connective tissue proteins such as collagen in the primary human skin dermal fibroblasts, leading to a loss of strength and flexibility in the skin [21,22]. Studies have evidenced that probiotics accelerate the fibrosis process, causing the deposition of collagen [8]. ...
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