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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
2006 Springer Science+Business Media, Inc.
DOI: 10.1007/s11130-006-0006-8
Effect of Different Flavonoids on Collagen Synthesis in Human Fibroblasts
Department of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia;
Department of Molecular Genetics, Rudjer Boskovic Institute,
Zagreb, Croatia;
Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium (
author for correspondence; e-mail:
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
, 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.
,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
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
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
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
gas in 50
C water bath.
The residue was resuspended in 250 µl deionized H
O and
50 µl was mixed with 450 µl of deionized H
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
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
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,
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
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
of flavonoids on protein synthesis
in neonatal human
fibroblasts expressed in µg/dish
Protein (µg/dish)
Compound 80 µmol/l 40 µmol/l 20 µmol/l
-Hydroxyflavanone 915.87 ±58.30 942.47 ±45.89 936.50 ±38.45
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
7-Hydroxyflavone 369.33
±25.04 894.13 ±57.27 963.53 ±43.05
954.40 ±39.92 987.57 ±31.96 913.13 ±71.93
Quercetin (3,3
±24.40 398.47
±21.54 856.13
1055.27 ±71.03 952.80 ±37.87 990.30 ±28.73
Quercetin (3-methyl
±21.18 265.63
±26.92 333.73
Morin (2
938.10 ±35.26 949.53 ±21.54 949.50 ±60.04
915.30 ±57.06 943.00 ±48.93 907.17 ±16.06
Fisetin (3,3
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
Each flavonoid was tested in three concentrations: 80, 40, and 20 µmol/l.
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
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
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
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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... Among the components of the S. oleraceus extract, there are the flavonoids that are common in various types of herbal medicine (LIMA et al., 2009). According Stipcevic et al. (2006), the flavonoids can stimulate the production of some types of collagen but inhibit other and protect them from the action of proteinases. However, the extracts used in this work were not able to alter the expression of collagen type I and III (p>0.05). ...
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The growing of scientific support for the use of medicinal plants has been expanding its use in traditional medicine. Sonchus oleraceus is popularly used for the treatment of various diseases and its healing action was demonstrated in experimental cutaneous wound. The objective of this study was to evaluate the expression of collagen type I and III in wounds treated with the extract of S. oleraceus in the form of ointment or balm, in concentrations of 5%, 10% and 15%. To quantify the collagen types I and III, after 15 days of treatment, biopsies were collected and processed by picrosirius red stain. The type I collagen showed expression of 19.6% in the samples treated with balm and 12.5% on the ointment treated with (p=0.026), regardless of extract concentration. Likewise, the type III collagen showed values of 1.48% for the balm and 0.68% for the ointment (p=0.004). There was no difference between extract concentrations and the control group (p>0.005). The S. oleraceus extract engaged in the repair of skin wounds by a process that does not involve the modulation of the expression of collagen type I and III.
Wound healing is an intricate process consisting of four overlapping phases, namely hemostasis, inflammation, proliferation, and remodelling. Effective treatment of wounds depends upon the interaction of appropriate cell types, cell surface receptors, and the extracellular matrix with the therapeutic agents. Several approaches currently used for treating wounds, such as advanced wound dressing, growth factor therapy, stem cell therapy, and gene therapy, are not very effective and lead to impaired healing. Further, repeated use of antibiotics to treat open wounds leads to multi-drug resistance. Today there is considerable interest in plant-based drugs as they are believed to be safe, inexpensive, and more suitable for chronic wounds. For example, a large number of plant-based extracts and their bioactive compounds have been investigated for wound healing. In recent years the structural and mechanistic diversity of natural products have become central players in the search for newer therapeutic agents. In the present review, a thorough critical survey of the traditionally used plant-based drugs used worldwide for wound healing with special reference to the natural products/bioactive compounds isolated and screened is presented. It is hoped that this review will attract the attention of the research community involved in newer drug design and development for wound healing.
Background: Striae distensae is a skin disorder that causes cosmetic and psychological problems. Purple passion fruit (Passiflora edulis Sims var. Edulis) is widely cultivated, especially in North Sumatra. The seeds are abundant and unused industrial waste. Purple passion fruit seeds contain piceatannol, ascorbic acid, flavonoids, resveratrol, hydroalcohols and sterols which play a role in the repair of striae distensae through anti-inflammatory mechanisms, increase fibroblast proliferation and collagen production, increase crosslinking between collagen fibers and moisturizers. Objective: To determine the effect of 6% purple passion fruit (Passiflora edulis Sims var. Edulis) seed extract cream on striae distensae. Subjects and Methods: This study is a pre-experimental clinical trial with a pretest-posttest research design on 40 subjects with striae distensae. Diagnosis was confirmed by history and clinical evaluation using the Manchester scar scale before and after administration of 6% purple passion fruit extract cream at weeks 0, 2, 4, 6, 8. Adverse effects were recorded during the study and satisfaction levels were assessed at the end. Results: The majority of subjects' ages ranged from 29 to 39 years (72.5%). There was a significant reduction in Manchester scar scale in striae distensae, both after being given a 6% purple passion fruit extract cream or a combination of 1% tretinoin cream for 8 weeks, with a mean reduction of 25% (p = 0.000). Striae distensae after being given 6% purple passion fruit extract cream compared to 6% purple passion fruit extract cream combined with 1% tretinoin cream, there was no significant difference (p = 0.791). From a total of 40 subjects, none experienced side effects (0%). As many as 57.5% of the subjects showed a good level of satisfaction. Conclusion: The use of 6% purple passion fruit seed extract cream can improve the appearance of striae distensae without side effects and the level of satisfaction is good.
Background and Aim: Maytenus ilicifolia has analgesic, healing, antioxidant and anti-inflammatory properties. This study evaluated effect of the hydroalcoholic extract of M. ilicifolia leaves on skin wound repair Experimental procedure Wounds were induced on mice and treated with the extract. The treatment was performed daily, until day 7 after wound induction. Wound closure was measured and the features of the repaired tissue were investigated, including mast cell quantification, neutrophil and macrophage activities, collagen deposition, angiogenesis, and pro-metalloproteases and metalloproteases 2 and 9 activity (pro-MMPs and MMPs) Results and Conclusion The M. ilicifolia extract accelerated the closure of wounds. The extract at a concentration of 4% was found to be effective, presenting anti-inflammatory effects and haemoglobin increased, along with increased soluble, total and type III collagens in the wound. In addition, there was an increase in pro-MMP9 and MMP9 activity after day 7th of treatment. The phenolic compounds and tannins present in this plant could be associated with the anti-inflammatory and healing activities observed in this study. Therefore, the ability to modulate essential parameters for accelerated and adequate healing as shown here suggests that the use of standardised extracts of M. ilicifolia and its fractions enriched in polyphenols may represent a therapeutic strategy for the treatment of wounds.
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Precipitation of copper(II) and zinc(II) by low molecular weight phenols, penta-O-galloyl-β-d-glucose, and commercial tannins is studied at pH 5. The extent of copper precipitation, higher than that of zinc, depends on the initial concentration of both copper and phenolic compounds and on the control of acidification which results from complexation. The copper/phenol ratio in the precipitate, determined by elemental analysis, is independent of the initial concentration of catechol. However, it increases with chestnut tannin when the metal concentration is increased to a value corresponding to complexation with all o-dihydroxyphenyl chelating groups in the molecule. Stability of the precipitates obtained with coppper(II) and various polyphenols in water, acetic acid, and ethylenediaminetetraacetic acid solutions is determined. Models for metal/polyphenol precipitation are proposed. Keywords: Polyphenols; tannins; metal ions; copper; zinc; complexation; precipitation
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Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are important for normal development, wound healing, and a wide variety of pathological processes, including the spread of metastatic cancer cells, arthritic destruction of joints, atherosclerosis, pulmonary fibrosis, emphysema and neuroinflammation. In the central nervous system (CNS), MMPs have been shown to degrade components of the basal lamina, leading to disruption of the blood brain barrier and to contribute to the neuroinflammatory responses in many neurological diseases. Inhibition of MMPs have been shown to prevent progression of these diseases. Currently, certain MMP inhibitors have entered into clinical trials. A goal to the future should be to design selective synthetic inhibitors of MMPs that have minimum side effects. MMP inhibitors are designed in such a way that these can not only bind at the active site of the proteinases but also to have the characteristics to bind to other sites of MMPs which might be a promising route for therapy. To name a few: catechins, a component isolated from green tea; and Novastal, derived from extracts of shark cartilage are currently in clinical trials for the treatment of MMP-mediated diseases.
Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.
Long term and repeated exposure of UV light on the skin often induces chronic skin diseases such as skin cancer as well as photoaging, and the mechanisms of these skin damages are closely associated with up-regulation of matrix metallopro-teinase's (MMPs) activities. We investigated the effect of 2,4,7-trihydroxyisoflavone purified from the whole plants of Viola hondoensis W. BECKER et H BOISSIEU (Violaceae) on the expres-sion of MMPs in UV-B irradiated old aged human skin fibrob-lasts. 2,4,7-trihydroxyisoflavone markedly reduced UV-induced MMP-1 expression, but not MMP-2, at the both mRNA and protein levels in a dose-dependent manner. Our report is the first description for the ability of 2,4,7-trihydroxyisoflavone to regulate MMP-1 expression from ultraviolet-B irradiated primary cultured old aged human skin fibroblasts. Key words Viola hondoensis; 2,4,7-trihydroxyisoflavone; matrix met-alloproteinase (MMP)-1; MMP-2; aged human skin fibroblast The skin aging process can be divided into intrinsic aging and photoaging. Damage to human skin due to repeated exposure to ultraviolet (UV) radiation (photoaging) and dam-age occurring as a result of the passage of time (intrinsic aging) are considered to be distinct entities rather than simi-lar skin aging processes. Photoaging of skin is caused by genetically determined or intrinsic processes superimposed upon the degenerative changes due to solar radiation. 1) Alter-ations and deficiencies of collagen, the major structural com-ponent of skin, have been suggested to be a cause of the skin wrinkling observed in photoaged and naturally aged skin. 2,3) The dermis contains predominantly type I and type III colla-gen, elastin, proteoglycans, and fibronectin. Since collagen fibrils and elastin are responsible for the strength and re-siliency of skin, their disarrangement during photoaging causes the skin to appear aged. Recently, it was suggested that excessive matrix degradation by UV-induced MMP-1 se-creted by various cells, including, keratinocytes, fibroblasts, and inflammatory cells, contributes substantially to the con-nective tissue damage that occurs during photoaging. 4—6) The expression of MMPs proteins in the dermis of sun-exposed and sun-protected skin of elderly subjects is known to differ. 5) Photodamaged skin of the elderly shows higher MMP-1 protein expression, than the intrinsically aged skin in the same individual. 5) Thus, this resulting higher expression of MMP-1 in photoaged skin may cause severe collagen defi-ciency and wrinkling. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases that play pivotal roles in the dynamic remodeling of extracellular matrix. Based on substrate preference and structural homology, MMPs are sub-classified into functional groups: collage-nases, gelatinases, stromelysins, matrilysins, membrane type-MMPs (MT-MMPs) and other non-classified MMPs. 7) MMPs are frequently overexpressed by the various extracellular stimuli including growth factors, cytokines, tumor promot-ers, and UV, and this increase in MMP-related activities may be involved in the pathogenesis of diseases such as cancer and inflammation as well as in physiologic processes. 7,8) It has been also reported that up-regulation of some MMPs is responsible for the enhanced degradation of dermal collagen during chronological and UV-induced skin aging. 4) With in-creasing age, collagen synthesis becomes lower and MMP-1 levels become higher in sun-protected human skin in vivo. 3) Therefore, the regulation of MMP activity might be a poten-tial strategy for prevention and/or treatment of UV-induced skin damage. During our screening program for MMP-1 in-hibitors from Viola species. Very recently, we reported the isolation of the MMP-1 inhibitor from the whole plants of V. hondoensis. 9) In this study, we investigated the rare occur-rence effect of 2,4,7-trihydroxyisoflavone (2,4,7-THIF), isoflavonoid from V. hondoensis W. BECKER et H BOISSIEU, on the expression of UV-B induced MMP-1 and MMP-2 mRNA and protein in aged human skin fibroblasts.
Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase. Complete resistance to the action of the mammalian enzyme may be achieved by incubating 0.6 mg of collagen (dry weight) with 0.1 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid. Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation.
A method is presented for the quantitative determination of hydroxyproline in biological materials containing as little as one part of hydroxyproline in 4000 parts of amino acids. Thus method has been applied to a study of hydroxyproline distribution in cell particulates, tissue fluids, and purified plant and animal proteins. Significant amounts of hydroxyproline were found in crystalline preparations of pepsin, elastase. and ficin.
No doubt can remain that the flavonoids have profound effects on the function of immune and inflammatory cells as determined by a large number and variety of in vitro and some in vivo observations. That these ubiquitous dietary chemicals may have significant in vivo effects on homeostasis within the immune system and on the behavior of secondary cell systems comprising the inflammatory response seems highly likely but more work is required to strengthen this hypothesis. Ample evidence indicates that selected flavonoids, depending on structure, can affect (usually inhibit) secretory processes, mitogenesis, and cell-cell interactions including possible effects on adhesion molecule expression and function. The possible action of flavonoids on the function of cytoskeletal elements is suggested by their effects on secretory processes. Moreover, evidence indicates that certain flavonoids may affect gene expression and the elaboration and effects of cytokines and cytokine receptors. How all of these effects are mediated is not yet clear but one important mechanism may be the capacity of flavonoids to stimulate or inhibit protein phosphorylation and thereby regulate cell function. Perhaps the counterbalancing effect of cellular protein tyrosine phosphatases will also be found to be affected by flavonoids. Some flavonoid effects can certainly be attributed to their recognized antioxidant and radical scavenging properties. A potential mechanism of action that requires scrutiny, particularly in relation to enzyme inhibition, is the redox activity of appropriately configured flavonoids. Finally, in a number of cell systems it seems that resting cells are not affected significantly by flavonoids but once a cell becomes activated by a physiological stimulus a flavonoid-sensitive substance is generated and interaction of flavonoids with that substance dramatically alters the outcome of the activation process.
Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase. Complete resistance to the action of the mammalian enzyme may be achieved by incubating 0.6 mg of collagen (dry weight) with 0.1 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid. Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation.