FULL COMMUNICATION
Collagen Hydrolysate Intake Increas Skin Collagen Expression
and Suppress Matrix Metalloproteina 2Activity
Vivian Zague,1,2Vanessa de Freitas,1,3
Marina da Costa Rosa,1Geo
´rgia A ´lvares de Castro,2Ruy G.Jaeger,2and Gla
´ucia M.Machado-Santelli 11
Department of Cell and Developmental Biology,Institute of Biomedical Sciences,University of Sa
˜o Paulo,Sa ˜o Paulo,Brazil.2
Rearch and Development Department,GELITA of Brazil Ltd.,Cotia,Brazil.
3Natural Sciences and Humanities Center,Federal University of ABC,Santo Andre
´,Sa ˜o Paulo,Brazil.ABSTRACT The effect of daily ingestion of collagen hydrolysate (CH)on skin extracellular matrix proteins was inves-tigated.Four-week-old male Wistar rats were fed a modified AIN-93diet containing 12%cain as the reference group or CH as the treatment group.A control group was established in which animals were fed a non–protein-modified AIN-93diet.The diets were administered continuously for 4weeks when six fresh skin samples from each group were asmbled and subjected to extraction of protein.Type I and IV collagens were studied by immunoblot,and activities of matrix metalloproteina (MMP)2and 9were assd by zymography.The relative amount of type I and IV collagens was significantly (P <.05)incread after CH intake compared with the reference diet group (cain).Moreover,CH uptake significantly decread both proenzyme and active forms of MMP2compared with cain and control groups (P <.05).In contrast,CH ingestion did not influence on MMP9activity.The results suggest that CH may reduce aging-related changes of the extracellular matrix by stimulating anabolic process in skin tissue.
KEY WORDS: collagen peptides dietary supplements functional foods matrix metalloproteinas skin aging skin health type I collagen western blotting zymography
INTRODUCTION
I
n recent years,functional foods claiming health
benefits have incread enormously,and the food industry has become interested in the field of skin care.
Skin characteristics are known to be affected by endog-enous and environmental factors,including chronological aging,ultraviolet radiation,hormones,and nutrition.The
factors impair skin metabolism,up-regulating synthesis of matrix metalloproteinas (MMPs)that degrade skin col-lagenous extracellular matrix.Collagen atrophy is consid-ered the major characteristic of skin aging.Type I collagen
is the predominant collagen found in dermis,being re-sponsible for skin tensile strength.At the same time,type IV collagen forms a network,highly cross-linked,maintaining the mechanical stability of the bament membrane,and ems to be decread at the bottom of the wrinkles.1MMPs are a family of structurally related molecules,including interstitial collagenas (MMP1in humans and MMP13in rodents)and gelatinas (MMPs 2and 9),that are capable of
degrading all components of the extracellular matrix such as collagen,elastin,fibronectin,proteoglyca
ns,and laminin.2In particular,MMPs 1and 13initiate the degradation of type I collagen,and MMP9further degrades the collagen frag-ments produced.In contrast,MMP2degrades type IV col-lagen,which leads to collagen deficiency and wrinkling.MMPs 2and 9are known to play an important role in the process of skin metabolism and aging.3
Dietary supplements have demonstrated beneficial effects on skin health.Experimental and clinical trials investigating the effects of oral supplementation with vitamins,poly-phenols,micronutrients,and proteins have indicated that
dietary compounds can modulate skin function.4–8More-over,the photoprotective potential of antioxidant intake has been the subject of a considerable number of studies.9,10The classical route of administration of active compounds is by
赞美诗歌1300首
topical application,and manufacturers have substantial ex-perience of formulating ingredients in this field.In recent years,protein hydrolysates have been studied as potential dietary ingredients and in development of functional foods.The newest relationship between food and skin has drawn
considerable attention due to the physiological effects of some dietary compounds on the skin-aging process.11
Collagen hydrolysate (CH)and gelatin are currently ud in diver fields,including food,cosmetic,and biomedical industries.Gelatin is a high-molecular-weight polypeptide
Manuscript received 31March 2010.Revision accepted 13August 2010.
Address correspondence to:Vivian Zague,Department of Cell and Developmental
Biology,Institute of Biomedical Sciences,University of Sa
˜o Paulo,38andar,sala 306=307,Avenue Prof.Lineu Prestes 1524,05508–900Sa
˜o Paulo,SP,Brazil,E-mail:
JOURNAL OF MEDICINAL FOOD J Med Food 14(0)2011,1–7
#Mary Ann Liebert,Inc.and Korean Society of Food Science and Nutrition DOI:10.1089=jmf.2010.0085
1
基质金属蛋白酶/分解酵素
巨大的、庞大的
调节
成胶的细胞外基质萎缩真皮
基膜机械
胶原酶啮齿动物
纤连蛋白蛋白多糖层粘连蛋白调节
光防护的
局部施用
生理学的
derived from collagen,the primary protein component of connective tissues.Industrial preparation of gelatin involves partial hydrolysis of the native structure of collagen to ob-tain the water-soluble form.Gelatin has chemical and physical properties that are interesting from the technolog-ical perspe
ctive.A further enzymatic degradation of gelatin results in a product called CH,which contains peptides with molecular mass ranging from3to6kDa.The most impor-tant sources for gelatin and CH production are bovine hide and bone and pigskin.12,13
There is an agreement that biological effects promoted by food-derived collagen peptides are related to the ingestion of collagen in its hydrolyzed form.14–22CHs have been re-ported to have beneficial biological functions that might justify their u in food supplements and pharmaceutical preparations.Clinical investigations suggest that ingestion of CH reduces pain in patients suffering from osteoarthri-tis.14,15CH is also involved in cartilage matrix synthesis.16 Some collagen peptides exhibit antihypertensive activity,
inhibiting the effect of angiotensin I converting enzyme (ACE)17,18and promoting inhibition of cardiovascular damage to the endothelial cells via its ACE inhibitory ac-tivity and regulation of nitric oxide and intercellular adhe-sion molecule-1.19Bad on in vitro studies,bioactive collagen peptides derived fromfish,bovine,and porcine skin have also shown potent antioxidative activities in dif-ferent oxidative systems.20–22
Before speculating about the mechanism of skin effec-tiveness of CH,it is important to emphasize th
at CH is able to cross the intestinal barrier,reaching blood circulation and becoming available for metabolic process and storage in skin.23–25To date,preclinical and clinical studies have demonstrated that CH may improve skin conditions and protect skin from ultraviolet damage.26–30Elucidating,at least partially,the action mechanism of CH in skin anabolic process after its oral administration may be a major contri-bution to understanding its feasibility for u in dietary products with the purpo of skin improvement.
We investigated the effects of CH intake on type I and IV collagen biosynthesis and MMP activity in order to deter-mine the potential action mechanisms of CH in skin,which could support the value-added utilization of CH and food supplements.
MATERIALS AND METHODS
A bovine hide CH(GELITAÒCPB1000)was kindly supplied by Gelita do Brasil Ltda.(Cotia,SP,Brazil).Ac-cording to the manufacturer,this product was obtained by a quential enzymatic hydrolysis,using a cocktail of en-zymes,including alcala.Commercial cain(CS)as well as the other components of the diet were purchad from MCassab Ltda.(Sa˜o Paulo,SP,Brazil).
贝类海鲜大全Animals and diets
All animal experiments were performed in accordance with the guidelines of the Committee for Animal Rearch of the State University of Campinas,Sa˜o Paulo.
Four-week-old male Wistar rats were houd in individ-ual cages at20Æ28C on a12-hour light=12-hour dark cycle, with free(ad libitum)access to water and diets.After the acclimation period,the animals were divided into three groups(six animals per group)such the body weight did not differ significantly among groups.The animals were fed a modified AIN-93diet31(isocaloric and isoproteic)contain-ing12%protein of CS—as the reference group—or a special CH enriched in esntial amino acids,according to Food and Agricultural Organization=World Health Organization guidelines32—as the treatment group.A negative control group was established in which the animals were fed a non–protein-modified AIN-93diet.The diets were administered continuously for4weeks.Body weight change and food intake were recorded three times a week.
The composition of the modified AIN-93diet is shown in Table1,and the centesimal composition of the two protein sources ud in this study is described in Table2. Molecular weight and amino acid compositions
The average molecular weight of CH was determined by gel permeation chromatography on a TSK
gel2000SWXL column(Tosoh,Tokyo,Japan),using a high-performance liquid chromatography system(Waters,Milford,MA,USA). The mobile pha ud was400m M sodium dihydrogen phosphate buffer,pH5.3.The samples were eluted at aflow rate of0.6mL=minute and monitored at214nm at258C.A molecular size calibration curve was prepared from the av-erage retention times of BrCN peptides(human collagen-type I)of38kDa,25kDa,18kDa,13.5kDa,and3.5kDa(Sigma Chemical Co.,St.Louis,MO,USA).Then the amino acid compositions of CH were analyzed by the Waters Pico-Tag system(Waters)described by White et al.35
Skin protein measurement
After the4-week experimental period,animals were sacrificed by cervical dislocation,and six fresh skin samples from each group were asmbled and subjected to extraction Table1.Composition of the Modified AIN-93Diet Ingredient g=kg Cornstarch397.5 Protein source(12%)a200.0 Maldotextrin132.0 Sucro100.0 Soy oil70.0 Fiber50.0 Mineral mix b35.0 Vitamin mix b10.0 l-Cysteine 3.0 Choline bitartarate 2.5 tert-Butylhydroquinone0.014 Source:Reeves et al.31
a Collagen hydrolysate or cain,calculated with the respective nitrogen-to-protein conversion factors(5.55and6.38).
情话英文
b According to the AIN-93diet propod by Reeves et al.31
2ZAGUE ET AL.
软骨碱性蛋白酶
蔗糖
半胱氨酸
胆碱
丁基氢醌
封装在
随意的
等蛋白
of protein.Skin proteins were extracted with a 50m M Tris-HCl buffer (pH 7.4)containing 1%sodium d
eoxycholic acid,150m M NaCl,1%Nonidet P-40,and protea in-hibitor cocktail (catalog number P8340,Sigma)containing 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,pepstatin A,E-64,bestatin,leupeptin,and aprotinin after
being frozen-crushed in liquid nitrogen.36The extract was
parated by centrifugation at 14,000g for 7minutes at 48C.
The supernatant was collected,and the amount of protein was estimated by BCA assay (Pierce Inc.,Rockford,IL,
USA).The water-soluble complex formed exhibits a strong absorbance at 562nm that is nearly linear with increasing protein concentrations.
37
Analysis of type I and IV collagens by immunoblotting
After the protein concentration was measured,samples were loaded in sample buffer containing 0.3M Tris-HCl,5%sodium dodecyl sulfate,50%glycerol,and 100m M dithio-threitol (Pierce).Equivalent
protein samples (40m g)were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10%(wt =vol)polyacrylamide gel.Pro-teins were transferred to a polyvinylidene difluoride mem-brane for 2hours at 100soaked in blocking buffer (Tris-buffered saline [25m M Tris,150m M NaCl,and 2m M KCl,pH 7.4]containing 0.1%Tween-20and 5%skimmed milk)for 1hour.The primary mou
monoclonal antibody against type I collagen (Sigma)and
rabbit monoclonal antibody against type IV collagen (Rockland Immunochemicals,Inc.,Gilbertsville,PA,USA)were ud at a dilution of 1:500in blocking buffer.Sec-ondary anti-mou and anti-rabbit monoclonal antibodies conjugated with horradish peroxida (Amersham Inc.,Arlington Heights,IL,USA)were diluted to 1:1,000in blocking buffer.The resulting bands were detected by the
horradish peroxida reaction with an enhanced chemi-luminescence kit (Amersham).
Immunoblot band intensities were quantified by Image J software (National Institute of Mental Health,Rockville,MD,USA).To confirm equal loading conditions mem-branes were stripped and reprobed with b -actin antibody (Sigma).
Activities of MMPs 2and 9by zymography
The same skin extracts as described above were ud for
the zymographic study.After the protein concentration was
measured,samples were resuspended in sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer (without b -mercaptoethanol)(Pierce).A total of 10m g of each sample was loaded in polyacrylamide gels containing 0.2%gelatin type A from porcine skin (Sigma).After electrophoresis,the gels were washed in 2.5%Triton X-100
for 30minutes,equilibrated in 10m M Tris (pH 8.0),and incubated at 378C in a developer buffer containing 50m M Tris (pH 8.0),5m M CaCl 2,and 0.02%NaN 3for 16hours.The gels were stained with 0.2%Coomassie Brilliant Blue and destained with a solution of acetic acid and methanol.Gelatinolytic bands were obrved as clear zones against the
blue background,and the intensity of bands was estimated
石锅饭的做法using Image J software.Statistical analysis
Data were analyzed using Minitab software,version 15.1.1.0(Minitab Inc.,State College,PA,USA).Differences among groups were assd by analysis of variance,fol-lowed by the Tukey–Kramer multiple comparison post-test.
Values of P <.05were considered statistically significant.
RESULTS
Molecular weight and amino acid compositions
The gel permeation chromatography technique was applied to analyze the molecular size distribution of CH,as shown in Figure 1.The results revealed an average molecular mass of
3,551Da and a main fraction of peptides with molecular sizes ranging from 1,000to 3,000Da (66.06%of total protein).The
molecular mass of the main peak of CH was 1,547Da,and its relative proportion was more than 90%.Table 2.Percentage Composition of Cain and Collagen
Hydrolysate Ud as Protein Sources in the Diets
Basic chemical characterization (%)CS CH Protein (N ÂF)a,*
84.8090.00Protein (dry basis)93.5098.60
小学生玩的游戏Fat { 2.000.05
Ash* 2.30 1.00
Moisture*12.008.70Carbohydrate { 1.200.25a Nitrogen factor for commercial cain (CS)and collage hydrolysate (CH)
of 6.38and 5.55,respectively.
*Determined by the Association of Official Analytical Chemists’method.33
{Determined by the methodology propod by Bligh and Dyer.34{Calculated by difference:carbohydrate ¼100–(protein þfat þash þmoisture).FIG.1.Molecular weight of CH analyzed by the gel permeation
chromatography technique.ABS,absorbance.COLLAGEN HYDROLYSATE INCREASES SKIN COLLAGEN
谁污染谁治理
3
去氧胆酸诺乃清洁剂
氨乙基
苯磺酰氟化物盐酸盐胃酶抑制剂抑肽酶十二烷基硫酸盐
二硫苏糖醇聚乙烯
二氟化物吸收
封闭代盐的
脱脂
单克隆抗体辣根过氧化物酶
化学发光明胶分解液偏二氟乙烯膜
In order to know whether the effect of CH ingestion is collagen-specific or is due to the ingestion of p
rotein itlf, animals were fed a diet containing the same amount of protein,12%of CS or CH,changing the protein source.CS is considered by the Food and Agriculture Organization= World Health Organization32as a reference standard be-cau of its esntial amino acid profile.In this study,CS contained all amino acids,whereas CH showed deficiency in all esntial amino acids and the complete abnce of tryptophan(Table3).CH had high contents of glycine (24.5%),glutamic acid(10.1%),arginine(8.1%),proline (13.8%),and hydroxyproline(7.4%)residues and small amounts of tyrosine,cysteine,histidine,and methionine residues.
The amount of protein intake throughout the study dif-fered between the CS and CH groups(57.2Æ6.2g and 46.5Æ6.0g,respectively)(data not shown).Conquently, animals grew quite differently,and thefinal body weight in the CS group was significantly higher(268.9Æ31.6g) compared with the CH group(197.4Æ22.2g).Although CH is deficient in esntial amino acids,it has an excellent di-gestibility38and is often ud to supplement other proteins to give a higher protein value.Moreover,it is interesting to note that from the nutritional perspective CH is character-ized by a considerably high safety profile.12
CH ingestion incread skin type I and IV collagens
In immunoblots,antibodies recognized type I collagen protein in both the CH and CS groups.However,CH re-sulted in a statistically significant(P<.05)increa of type I collagen synthesis,after4weeks of daily ingestion(Fig.2).The relative amount of type I collagen incread up to fourfold after CH intake(3.4Æ1.7)compared with the reference diet CS group(0.8Æ0.5).Similar results were found relating to type IV collagen expression:the collagen IV amount incread up to threefold after CH intake (6.7Æ1.1)compared with the reference diet CS group (2.3Æ1.2)(P<.05)as obrved in Figure2.
CH decread skin MMP2activity
Zymography showed gelatinolytic bands at molecular weights of tho corresponding to MMPs2andÀ9(Fig.3). Gelatin is a general substrate for proteolytic activity,suit-able for the detection of different proteas.However,gel-atin zymography is particularly suitable for MMPs2and9. The oral administration of CH was associated with a sta-tistically significant decrea(P<.05)of MMP2activity shown by zymography(Fig.4)in relation to the reference group(CS).In contrast,MMP9activity was not influenced by administration of CH in the diet as shown in Figure4.
DISCUSSION
毕业后
CHs have long been ud in pharmaceuticals and food supplements for improving skin and cartilage tissues.It is digested and absorbed in the digestive tract,appears in the human blood partly in a peptide form,23,24and is accumu-lated in skin for up to96hours as shown by Oesr et al.25 Becau of its biochemical similarity to genuine collagen in connective tissue and especially to type I collagen in dermis, it has been suggested that oral ingestion of CH might have beneficial effects and can promote both collagen type I biosynthesis and the repair process in a dermal wound.Thus CH ingestion would have a potential positive impact,pro-tecting skin from the aging process.
Studies have reported that food-derived collagen peptides in human blood exert a chemotactic role on skinfibro-blasts39and increa the migration and growth of mou skinfibroblasts.40However,the effects of CH on expression of skin collagen protein and activity of MMPs have not yet been examined to date.Our prent study provides addi-tional evidence for an effect of CH on skin.皇甫轸
Despite of its reduced nutritional profile,CH promoted stimulatory effects on skin tissue,increasing expression lev-els of the most abundant extracellular matrix protein in skin, type I collagen(Fig.2).The CH effect appears collagen-specific becau CS(reference diet group)failed to stimulate collagen expression.Our results agree with tho of Matsuda et al.41They investigated effects of CH ingestion onfibro-blast and collagen densities of pig skin;CH was administered orally to
pigs at0.2g=kg of body weight=day for62days,and its effects were compared with tho of lactalbumin and water controls.Fibroblast density and diameter and density of col-lagenfibrils were significantly larger in the collagen peptide group than in the control groups.This implies that the effect of CH was protein-specific and did not depend merely on an increa of amino acid intake.
Although our results are related to expression levels of type I collagen in normal rat skin,they are also in accor-
Table3.Amino Acid Composition of Cain
and Collagen Hydrolysate
Level(g=100g)
Amino acid CS CH
Aspartic acid7.91Æ0.2 6.02Æ0.2
Glutamic acid21.17Æ0.210.12Æ0.2
Serine 6.05Æ0.2 3.43Æ0.2
Glycine 1.96Æ0.224.47Æ0.2
Histidine 2.82Æ0.20.70Æ0.2
Arginine 3.53Æ0.28.05Æ0.2 Threonine 4.93Æ0.2 1.79Æ0.2
Alanine 3.21Æ0.29.74Æ0.2
Proline9.97Æ0.213.81Æ0.2
Tyrosine 4.46Æ0.20.58Æ0.2
Valine 5.97Æ0.2 2.36Æ0.2 Methionine 2.46Æ0.20.98Æ0.2
Cysteine 1.07Æ0.20.24Æ0.2 Isoleucine 4.83Æ0.2 1.60Æ0.2
Leucine9.71Æ0.2 3.08Æ0.2 Phenylalanine 4.57Æ0.2 1.94Æ0.2
Lysine7.91Æ0.2 3.68Æ0.2 Tryptophan 1.12Æ0.2ND Hydroxyproline ND7.41Æ0.2
ND,not determined.
4ZAGUE ET AL.
dance with tho of Tanaka et al.42The authors demon-strated that CH ingestion inhibited ultraviolet B-induced decrea of type I collagen,thus improving skin conditions in mice.
Taking into account that a gradual decrea in type I collagen level is known to occur during the cour of a lifetime and induces skin aging,3an increa of type I col-lagen after consumption of CH is desired for anti-aging benefits.
Our results complement tho reported by Nishimoto et al.,43investigating the effects of oral administration of CH on the hydroxyproline content of mou skin.CH was administered at 50and 100mg =kg for 21days and incread the hydroxyproline content in unshaved and shaved skin,which may be related to skin collagen synthesis.
Bad on in vitro studies,Rodrigues 44showed that CH at concentrations ranging from 0.1mg =mL up to 6mg =mL stimulated fibroblast to crete 1.8-fold more collagen than the untreated cells.Furthermore,fibroblast cell cultures treated with CH showed a higher percentage of cells in S pha (25.8Æ4.0%)than control cells (18.9Æ2.0%)and decread percentage of cells in apoptosis (8.2Æ4.0%)compared with controls (22.4Æ3.0%).
In our study,it was also found that daily CH ingestion for 4weeks significantly suppresd MMP2activity compared with the control and reference groups.In contrast,MMP9
activity was not influenced by CH ingestion (Fig.4).MMPs can degrade skin collagen and contribute to the connective tissue breakdown,resulting in collagen deficiency.This deficiency may be followed by skin wrinkling.3MMPs 2and 9are known to play an important roles in the process of skin aging and metabolism.3Inhibition of MMPs can take place at different levels,from their gene expression to en-zyme activity.Becau MMPs 2and 9belong to the
group
FIG.2.(Left panel)Type I and (right panel)type IV collagen expression in the skin of rats determined by immu-noblotting.Control animals were fed a non–protein-modified AIN-93diet.CS animals were fed a modified AIN-93diet containing 12%CS,and CH ani-mals were fed one with 12%CH.(A )Reprentative immunoblot from three independent experiments.(B )Relative collagen protein expression levels,normalized using b -actin as a marker for equal protein loading.Each column reprents the mean ÆSEM value from three determinations.ab Different letters indicate a significant difference among groups (P <
.05).
FIG.3.Zymograms for the determination of activities of matrix metalloproteinas (MMPs)2and 9in the skin of rats fed diets containing non-protein (control),CS,and CH.Gelatinolytic activities of MMPs
2and 9in skin lysates were detected by electrophoresis of soluble protein on a gelatin-containing 10%polyacrylamide gel.Reprentative zymograms were obtained from three independent
experiments.
FIG.4.Densitometry of both latent and active forms of MMPs (A )2and (B )9for the non-protein (control),CS,and CH groups.Each column reprents the mean ÆSD value from three determinations.abcdef Different letters indicate a significant difference among groups (P <.05).AU,arbitrary units.
COLLAGEN HYDROLYSATE INCREASES SKIN COLLAGEN 5
