Although in smaller quantities than green tea, black tea also contains catechins, although some of them have been oxidized and converted into other compounds during the fermentation process.

Red wine is another source of catechins, especially resveratrol, which is a type of catechin found in the skin of red grapes. Dark chocolate contains catechins, especially when it has a high cocoa content.

These catechins may contribute to some of the health benefits associated with moderate consumption of dark chocolate. Some fruits , such as apples, pears, grapes and cherries, contain catechins in their skins and pulps. Nuts such as walnuts and almonds also contain catechins in small amounts.

When catechins are consumed through food, beverages or supplements, they can be absorbed by the body and have effects throughout the body. This way of acquiring catechins may offer broader benefits, but its effects on the skin are minor.

When applied topically to the skin through skin care products, catechins tend to exert their effects in a more localized manner. This means that they focus on protection against oxidative stress and free radical damage on the skin surface.

Catechins are used in the aesthetic sector to take advantage of their properties and achieve a healthier skin. In general, it is used in the following applications:. Although catechins are mostly safe when consumed in normal amounts through food, beverages or supplements, in some cases they can cause mild side effects such as stomach upset or insomnia, especially when taken in excess.

The appropriate amount of catechins to be consumed may vary depending on the source and the purpose of use.

It is important to follow the dosage recommendations provided on the products or consult a healthcare professional to ensure proper and safe use. This text on catechins has been prepared by professional editors. In addition, we have relied on experts in medicine, engineering and aesthetics as a source of information, as well as specific studies to maintain the quality of what we publish.

At Sisneo Bioscience we are committed to publish truthful and contrasted information. And to update or correct it as soon as new knowledge becomes available. Benefits of catechins Catechins offer a number of general health and aesthetic benefits due to their antioxidant properties and other bioactive effects: They are powerful antioxidants that can neutralize free radicals, harmful substances that can damage cells and accelerate skin aging.

By fighting free radicals, catechins help maintain skin health and prevent premature aging. Catechins, particularly those present in green tea, have been shown to help protect the skin against damage caused by ultraviolet UV radiation.

This can reduce the risk of sunburn and the development of wrinkles and skin spots. Some catechins can promote better blood circulation , which can contribute to healthier, fresher-looking skin. Catechins also have anti-inflammatory properties , which can be beneficial in treating inflammatory skin conditions such as acne or rosacea.

The catechins present in green tea have been associated with weight loss and reduction of body fat , which can have a positive impact on physical appearance.

The consumption of catechins has been found to be associated with reduced risk of cardiovascular disease by helping to maintain healthy cholesterol and blood pressure levels. Finally, catechins can also fight bacteria in the mouth , which can be beneficial for dental health and cavity prevention.

Where are catechins found? Use in aesthetic medicine Catechins are used in the aesthetic sector to take advantage of their properties and achieve a healthier skin. In general, it is used in the following applications: Incorporated in skin care creams, serums and lotions , these products are applied directly to the skin or scalp.

They can help protect the skin against damage caused by free radicals and ultraviolet radiation, as well as act as an anti-inflammatory and lighten the skin, making them useful in the treatment of conditions such as acne and skin blemishes.

As a hair treatment, they can help strengthen hair and improve its appearance. Some aesthetic medicine procedures, such as chemical peels , may include catechins in their formulations to improve skin texture and reduce blemishes.

To prove our hypothesis, the chemistry between the free amine group and EGCG was observed. L-lysine and ethanolamine were reacted with EGCG in a similar condition to SF-EGCG synthesis, and mass spectrometry was measured Fig.

Also, EGCG and amine group compounds were bounded with a ratio of It means the amine compound and EGCG were not bounded by hydrogen bonds. Elevated ROS levels in wounds are known to stimulate dermal fibroblasts to secrete matrix metalloproteinases, predominantly interstitial collagenases, resulting in excessive breakdown of the collagen fibers in extracellular matrix and delayed wound healing [ 5 , 6 ].

The hydroxyl radical scavenging activity of SF-WS, SF-T, and SF-EGCG shown the obvious difference in the low concentration, but the differences became narrow as the increase of concentration. The silk fibroin has hydroxyl radical scavenging activity already. The hydroxyl radical react with the phenol group, and forms a phenoxy radical.

The phenoxy radical react with the other phenoxy radical, hydroxyl radical and superoxide radical, and forms diphenol, catechol, and tyrosine hydroperoxide, respectively. In the low concentration, it seems that the hydroxyl radical scavenging activity is more dominantly affected by the tyramine or EGCG modified to silk fibroin than the tyrosine residue in the silk fibroin, and as the increase of the concentration, the effect of tyrosine residue become higher.

EGCG has been reported to exhibit about fold higher affinity towards human serum albumin than — -epicatechin or — -epigallocatechin, which lacks a galloyl moiety, suggesting that the galloyl moiety plays a crucial role in EGCG-protein interactions via hydrogen-bonding and hydrophobic forces [ 44 ].

Also, the collagenase inhibitory activity of EGCG was improved after the conjugation with SF. Although the silk fibroin backbone can make steric hindrance, other hydrogen bond sites in SF can bind collagenase.

For instance, due to the scavenging effect of EGCG moieties against phenoxy free radicals, the concentration of H 2 O 2 6. The SF-EGCG exhibited greater ROS-scavenging activity than SF-T although DS of SF-EGCG 0.

After the conjugation, the hydroxyl radical scavenging activity was increased, but the superoxide radical scavenging activity was decreased. It is considered that the conjugation attenuates the activity of EGCG, but inherent hydroxyl radical scavenging activity of silk fibroin supplemented attenuation of activity in the hydroxyl radical scavenging assay Fig.

In addition, the specific collagenase activity was calculated. After the conjugation, the collagenase inhibitory activity was increased. The silk fibroin backbone can make steric hindrance after the conjugation. However, in this work, it is considered that additional hydrogen bond sites in the silk fibroin backbone supplemented the steric hindrance.

Also, after the conjugation, once an EGCG is bound to the collagenase, other conjugated EGCG get a chance to bind to the collagenase more frequently.

It is a similar mechanism that the polymer-antibody conjugation improves the binding affinity of the antibody. The replacement of the gauze dressing not only causes pain and bleeding, but also delays the wound healing process by removing regenerating skin cells.

Neither bleeding nor skin detachment was observed from the wounds treated with all SF hydrogels and DuoDERM® gel. Wound closure profile of all experimental groups except the control group was almost completed after 14 days.

Because the efficacy of hydrogel wound dressings is excellent than other type of wound dressings. Therefore, the day 14 observation is not significant different. But, inner part of regenerated tissue showed matured collagen deposition with reduced inflammatory cells.

It suggests that these hydrogels effectively maintained a moist environment within the wounds. In the present study, SF-EGCG conjugate was synthesized for the first time using the nucleophilic addition reaction of lysine residues in silk proteins with EGCG quinone under a mild basic condition.

This conjugate exhibited superior ROS-scavenging and collagenase-inhibitory activities than SF-T and native SF. With these attractive properties, SF-EGCG-based composite hydrogels hold great promise as functional biomaterials for wound healing applications. The supplementary data are available online at www.

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Biomed Mater. Bae JW, Choi JH, Lee Y, Park KD. TAKE THE QUIZ. shop refills. shop sets. GET STARTED QUIZ WHY IT WORKS CLINICAL RESULTS THE LEGIT LIST.

Time Rewind Retinol Pro System Activated Serum. Adding Add to cart. Firming Filter CoQ10 Pro System Add On Powder with Precision Dispenser. Firming Filter CoQ10 Power System Activated Serum.

SHOP ALL. Starter Systems POWER SERUM. Refills PRO SYSTEM REFILLS POWER SYSTEM REFILLS. GET STARTED QUIZ. WHY IT WORKS. CLINICAL RESULTS. THE LEGIT LIST. ABOUT US. OUR STORY. THE STANDARD. THE DOSE. Green Tea: Skincare Ingredient Spotlight Green Tea. Share on: Twitter Facebook Linkedin.

What is Green Tea? Does Green Tea Lose Potency Over Time? Words By: Ana Vasilescu. Footnotes Ohishi T, Goto S, Monira P, Isemura M, Nakamura Y.

Anti-inflammatory Action of Green Tea. Antiinflamm Antiallergy Agents Med Chem. Source Saric S, Notay M, Sivamani RK. Green Tea and Other Tea Polyphenols: Effects on Sebum Production and Acne Vulgaris.

Antioxidants Basel. Source Pazyar N, Feily A, Kazerouni A.

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Diabetes Metab Res Rev. J Nutr. Download references. This work was supported by the Mishima Kaiun Memorial Foundation. The authors are very grateful to Tetsuya Tanigawa for his advice. Department of Plastic Surgery, Osaka University Graduate School of Medicine, Suita-shi, Osaka, Japan.

Department of Child Development and Molecular Brain Science, United Graduate School of Child Development, Osaka University, Suita-shi, Osaka, Japan. Division of Molecular Brain Science, Research Institute of Traditional Asian Medicine, Kinki University, Osakasayama, Osaka, Japan.

Division of Plastic and Reconstructive Surgery, Niigata University Graduate School of Medicine, Niigata-shi, Niigata, Japan. You can also search for this author in PubMed Google Scholar. Correspondence to Shigeyuki Kanazawa.

TT designed and performed the study and wrote the manuscript. SK designed and performed the study and revised the manuscript. RY, TF, TK, KY, and KH helped to perform the study. TM, YH, KT, KM, KS, SM, and MT provided technical support.

All authors read and approved the final manuscript. Open Access This article is published under license to BioMed Central Ltd.

Reprints and permissions. Tanigawa, T. et al. BMC Complement Altern Med 14 , Download citation. Received : 24 September Accepted : 24 March Published : 08 April Anyone you share the following link with will be able to read this content:.

Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Abstract Background Oxidative stress has been suggested as a mechanism underlying skin aging, as it triggers apoptosis in various cell types, including fibroblasts, which play important roles in the preservation of healthy, youthful skin.

Methods Fibroblasts NIH3T3 under oxidative stress induced by hydrogen peroxide 0. Results Hydrogen peroxide induced apoptotic cell death in fibroblasts, accompanied by induction of phosphorylation of JNK and p38 and activation of caspase Background Skin wrinkles and sagging are important factors defining skin youthfulness.

Figure 1. Full size image. Methods Cell culture NIH 3T3 fibroblasts were used for all experiments. Cell viability assay Cell survival was determined using the 3- 4,5-dimethylthiazoleyl -2,5-diphenyltetrazolium bromide MTT assay CellTiter 96 ® AQueous One Solution Cell Proliferation Assay; Promega, WI, USA.

Statistical analysis All data shown are expressed as the mean ± SE of three independent experiments. Results Catechin increases the viability of fibroblasts Oxidative stress is known to promote fibroblast cell death[ 14 ]. Figure 2. Figure 3. Figure 4.

Figure 5. Figure 6. Figure 7. In addition to antioxidant benefits, green tea also has anti-inflammatory and anti-microbial properties. For these reasons, green tea has become a popular ingredient for use in skin creams and beauty masks to soothe and protect skin, reduce sebum, and minimize signs of aging.

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Anti-infective properties of epigallocatechingallate EGCG , a component of green tea. Br J Pharmacol. doi: PMID: ; PMCID: PMC Create your account Lost password?

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Therefore, maintaining the population of dermal fibroblasts is important for both preventing and treating age-related skin changes. Oxidative stress has been indicated in a variety of pathological processes, such as atherosclerosis, diabetes, neurodegenerative diseases, and aging.

Reactive oxygen species induce DNA damage, intracellular lipid peroxidation, and abnormal protein oxidation reactions, all of which result in cell damage. Oxidative stress also promotes skin aging[ 3 ]; it reduces the number of skin fibroblasts by inducing apoptosis and decreasing their regenerative capacity, which in turn leads to increased skin sagging.

Therefore, suppression of oxidative stress-induced apoptosis in skin fibroblasts is a potential treatment and prevention strategy for maintaining healthy youthful skin.

Green tea, which is routinely consumed in Japan and China, is widely known as a healthy drink containing various antioxidants, vitamins, and minerals.

Catechins are thought to not only possess antioxidant effects to control active oxygen[ 4 — 7 ] but also exert various actions, such as anti-inflammatory[ 8 ], antibacterial[ 9 , 10 ], and anti-cancer[ 11 — 13 ] actions. Chemical Structures of catechins.

NIH 3T3 fibroblasts were used for all experiments. All experiments were performed in triplicate. Cell survival was determined using the 3- 4,5-dimethylthiazoleyl -2,5-diphenyltetrazolium bromide MTT assay CellTiter 96 ® AQueous One Solution Cell Proliferation Assay; Promega, WI, USA.

The production of formazan by viable cells was detected by measuring the absorbance at nm using a well plate reader. Oxidative stress was induced by addition of 0. Next, permeabilization was performed by incubation with 0. Fluorescence images were taken using a microscope IX; Olympus equipped with a charge-coupled device camera CoolSNAP HQ; Nippon Roper, Chiba, Japan.

For each experiment, cells were randomly selected, and the percentage of TUNEL-positive cells was measured. After H 2 O 2 challenge for 1 h, cells were harvested and lysed in radioimmunoprecipitation assay buffer containing 1 mM Na 3 VO 4 , 1 mM NaF, and Protease Inhibitor Cocktail Roche Diagnostics, Basel, Switzerland for 20 min at 4°C.

After centrifugation at 15, × g for 15 min at 4°C, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon-P Transfer Membranes Millipore Japan, Tokyo, Japan.

All antibodies were purchased from Cell Signaling Technology, MA, USA. Next, membranes were incubated for 1 h with an anti-mouse or anti-rabbit HRP-linked secondary antibody ,; Cell Signaling Technology. Reaction products were visualized by chemiluminescence detection using the ECL Western Blotting Detection System GE Healthcare, Piscataway, NJ, USA.

Quantification of relative band densities was performed by densitometry using Image J software National Institutes of Health, Bethesda, MD, USA. All data shown are expressed as the mean ± SE of three independent experiments. Data from each experiment were normalized to the respective control sample.

Oxidative stress is known to promote fibroblast cell death[ 14 ]. The cell numbers were analyzed after 24 h. After 24 h, cell viability was evaluated. Cell viability was assessed by the MTT assay c.

a The image of non-loading control cells. And then subjected to oxidative stress induction with 0. After 24 h, microscopic morphological changes were evaluated.

After 24 h, apoptosis was evaluated by TUNEL staining. a Microscopic findings of TUNEL staining for detection of apoptotic cells. b For evaluation of apoptosis, cells were randomly selected and the percentage of TUNEL-positive cells was measured. After 1 h, activation of caspase-3 was determined by SDS-PAGE and western blotting analysis using an anti-cleaved caspase-3 antibody.

a Representative images of western blot analysis for cleaved and total caspase b Expression levels of cleaved caspase-3 were normalized to those of total caspase After 1 h, cells were collected, and phosphorylation of p38 and JNK was determined by SDS-PAGE and western blotting analysis using anti-phospho p38 and anti-phospho JNK antibodies.

a Results of western blotting for phosphorylation of a p38 and b JNK. Phosphorylation levels of p38 and JNK were normalized to those of total p38 and JNK, respectively. In contrast, EGCG at and μM significantly decreased cell viability Figure 7.

Cell viability was assessed by the MTT assay. We focused on fibroblasts because they participate in skin maintenance and renewal. In the skin, fibroblasts play a key role in the production of extracellular matrix components, including collagen, elastin, and hyaluronic acid.

In clinical aesthetic medicine, epidermal or intradermal injection of hyaluronic acid is performed to obtain glossy and healthy skin microinjections of hyaluronic acid, vitamins, minerals, and amino acids into the superficial layer of the skin [ 15 ].

Other techniques, such as implanting activated fibroblasts in the skin, are also known to revive the skin to be glossy and healthy intradermal injection of cultivated skin fibroblasts into wrinkles [ 16 — 18 ].

However, these therapies are associated with a high cost and may provoke adverse events, including misplacement, allergy, nodules, necrosis, abscesses, and rejection.

In contrast, the use of health supplements, such as green tea and food-derived active substances, is a safer and beneficial anti-aging method. The integrity and functions of the skin barrier may be impaired by excessive exposure to allergens, chemicals, ultraviolet light, and dehydration.

Failure of the skin barrier would subsequently lead to infections with pathogens and result in inflammatory responses. Locally produced reactive oxygen species are also known to inhibit the growth of epithelial cells and fibroblasts by inducing apoptosis and inhibiting collagen and hyaluronic acid production, all of which have been implicated in aging processes leading to skin wrinkles and sagging.

JNK and p38 belong to the family of stress kinases and have been shown to be required for biological stress responses, such as apoptosis induced by UV, radiation, oxidative stress, heat shock, and tumor necrosis factor TNF -α stimulation.

It has been reported that H 2 O 2 signaling through TNF receptor 1 selectively activates JNK and p38[ 20 , 21 ]. JNK plays an important role in controlling cell death and is known to affect the function of Bcl-2 family molecules, which suppress apoptosis.

Specifically, phosphorylation of Bcl-2 by JNK results in the inhibition of Bcl-2 function and therefore induces the activation of apoptosis[ 20 , 21 ].

In contrast, p38 MAPK is known to be involved in the activation of apoptosis-modulating proteins, such as Fas and Bax[ 21 ]. In particular, EGCG, a molecule in the same catechin group, was suggested to play a role in growth inhibition and apoptosis induction in a variety of cancer cells[ 22 ].

In contrast, EGCG was reported to have an anti-apoptotic effect in renal mesangial cells[ 23 ] and endothelial cells[ 24 ], similar to our results in the present study.

Therefore, we speculate that the effect of catechins on apoptosis may vary according to cell type and the nature of pathogenesis. Given the different cell-specific responses of catechins, it is important to establish an appropriate strategy for using catechins for treatment and prevention of various diseases.

It would be ideal for catechins have suppressive actions against cancers and protective effects for organs such as the kidneys and cardiovascular system. Accumulating evidence on the preventive effect of catechins and green tea against various systemic diseases, including cancers, diabetes, and hypertension, suggests little potential harm to human health from high consumption of catechins and green tea for maintenance of skin beauty.

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All animal experiments were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the Kyungpook National University. All rats were divided into 5 groups randomly and acclimatized for 5 days before surgery. A full thickness skin defect 2 cm in diameter was created in dorsal region.

The wounds treated with a cotton gauze Daehan Medical, Korea served as a negative control. For comparison, a commercial hydrogel dressing DuoDERM ® hydroactive ® gel, ConvaTec, USA was tested.

The wound size was recorded and the wound dressings were replaced on days 1, 3, 5, 7, 10, and The wound size was calculated using an image analyzer I-solution lite, IMT i-solution, Korea. The fixed tissue was embedded in paraffin and then cross-sectioned to 5 μm thickness. Each slide was mounted and observed using an optical microscope ECLIPSE TS, Nikon, Japan equipped with a digital camera DS-Fi-2, Nikon, Japan.

The quantitative area of inflammatory cells and collagen deposition was calculated using an image analyzer ImageJ, 1. All data were analyzed by Shapiro-wilk test to confirm the normal distribution. Statistical significance was considered with p values less than 0.

Natural SF fibers contain crystalline regions mainly composed of β-sheet structure and thus require a regeneration process to obtain an amorphous random-coil conformation with high water solubility for biomedical use [ 36 ].

However, regenerated SF tends to reform β-sheet structure and aggregate into water-insoluble fibrils upon lyophilization. To circumvent this limitation, we carried out thermal hydrolysis of SF. It was confirmed through GPC that the molecular weight of the SF prepared through heat treatment was reduced.

Water-soluble SF derivative SF-WS was prepared by thermal hydrolysis of SF over 7 h at ºC to produce short chain [ 31 ]. Gel permeation chromatography Table S1 revealed the weight-average molecular weight M w of SF-WS The regenerated SF-WS powder was fully soluble in water without β-sheet formation, and thus used for all subsequent experiments.

Figure 1 a depicts the synthesis scheme for SF-EGCG conjugate having multiple EGCG moieties along the SF-WS backbone. Under mild alkaline condition pH 7. The nucleophilic addition of a lysine residue of SF-WS to the ortho -quinone moiety leads to the formation of amine-quinone adduct at the B ring of EGCG.

Of note, amine-quinone adduct is considered the dominant final product as the formation of EGCG-quinone imine is unfavorable due to rapid hydrolysis of the imine bond in aqueous solution Fig. The resulting SF-EGCG conjugate was purified by extensive dialysis using nitrogen-purged water and then lyophilized to obtain a dry product.

a Scheme of the SF-EGCG conjugate formation through autoxidation of EGCG at basic pH 7. c Optimization of EGCG feeding amount and reaction time. UV-visible spectrum of SF-EGCG conjugate showed much larger absorption peak at nm than SF-WS, demonstrating the conjugation of EGCG moieties on SF-WS Fig.

UV-visible spectroscopy of the products revealed that absorbance at nm gradually increased over 4 h, indicating time-dependence of EGCG conjugation reaction Fig.

Since a further incubation up to 5 h did not increase the absorbance significantly, the optimal reaction time was determined as 4 h. The absorbance at nm was the largest when the feeding amount of EGCG was 40 µmol.

Raising the feeding amount of EGCG up to 80 µmol did not help to increase the absorbance, suggesting that the reaction reached a saturation state above 40 µmol of EGCG. Thus, SF-EGCG conjugate produced with 40 µmol of EGCG was used for subsequent studies.

The extent of EGCG conjugation was measured by comparing the difference in absorbance between SF-EGCG and SF-WS with a series of EGCG standards Fig. The degree of substitution DS of SF-EGCG conjugate was determined as 0. This DS value is considered reasonable because native SF contains only 0.

Next, we performed fluorescamine assay to examine the extent of lysine side chain modifications [ 40 ]. Fluorescamine assay Fig. These results demonstrated that EGCG conjugation reactions occurred primarily at lysine residues in SF-WS. For comparison, SF-T conjugate with DS of 1. S3 according to the previous report [ 27 ].

It is worth noting that SF-EGCG exhibited greater ROS-scavenging activity than SF-T although DS of SF-EGCG 0. The superior ROS scavenging effect of SF-EGCG conjugates was likely ascribed to the existence of aromatic ring structures in EGCG moiety, which are capable of capturing and neutralizing free radicals [ 28 , 33 ].

The SF-EGCG conjugate has scavenging efficacy. Considerable attention has been paid to EGCG because of its ability to inactivate collagenase in a competitive manner by binding to its catalytic domain [ 42 , 43 ]. As shown in Fig. Even though DS of SF-EGCG 0. Hence, it is conceivable that SF-EGCG could bind to collagenase more strongly than SF-T, leading to more effective inhibition of its enzymatic activity.

It has been reported that a catalytic cycle of HRP converts phenolic moieties of tyramine and EGCG to phenoxy free radicals while consuming H 2 O 2 and releasing water molecules as a byproduct [ 32 , 45 ]. The phenoxy free radicals can react with each other to produce cross-linkages between SF-EGCG and SF-T conjugates.

b Storage modulus and gelation time of SF-T hydrogels as a function of H 2 O 2 concentration. The concentration of HRP was fixed to 0. c Storage modulus and gelation time of SF-T hydrogels as a function of HRP concentration. The concentration of H 2 O 2 was fixed to 5 mM.

The concentration of H 2 O 2 and HRP was fixed to 5 mM and 0. First, we attempted to fabricate the SF-T hydrogel without SF-EGCG Fig. As reported in the previous report, high concentrations of H 2 O 2 increased the storage modulus of hydrogel but denatured HRP over excessive concentration [ 46 ].

Also, the increase of HRP concentration highly shortened gelation time without affecting the mechanical strength [ 47 ]. Generally, the mechanical properties of hydrogels should be matched with those of native skin tissue to promote the restoration of defected wound area.

Hence, the concentration of H 2 O 2 and HRP was set to 5 mM and 0. This finding can be explained by the scavenging of phenoxy free radicals by greater amounts of EGCG moieties, leading to an inhibition in the crosslinking reaction [ 39 ].

As presented in Fig. SEM images revealed the presence of interconnected microscopic pores within the optimized hydrogels Fig. ATR-FTIR spectra of the hydrogels on e day 1 and f day 9. In vitro stability of SF hydrogels was assessed by measuring their weight loss in PBS solution pH 7.

To understand the reason for the stabilization, ATR-FTIR was conducted to monitor changes in the β-sheet content in the hydrogels. The wounds treated with the cotton gauze failed to heal completely even after 14 days Fig.

In addition, bleeding was consistently observed from the gauze group on day 3 and 7. Since the cotton gauze is a dry dressing and unable to keep the wounds moisture, strong adhesion between the gauze and wound bed usually occurs.

The gauze group exhibited incomplete re-epithelialization and significant appearance of inflammatory cells, such as macrophages, lymphocytes and neutrophils Fig.

It means that typical hydrogel dressings with a moisture maintaining ability were not sufficient to promote effective wound healing. Both groups contained a far smaller number of inflammatory cells compared to SF-T group.

Cytocompatibility experiments revealed that both SF and SF-EGCG were totally non-toxic to NIH3T3 fibroblasts, indicating that the hydrogel components are not likely to cause skin cell death even if they are released as a result of potential degradation Fig.

The insets represent inflammatory cells macrophages, lymphocytes, neutrophils, eosinophils at the interface between matured and healed wound area.

The first agenda of this study was fabricating a silk fibrin based hydrogel with secured quality. To achieve this goal, we have taken the water-soluble SF SF-WS by thermal treatment [ 31 ].

The thermal treated SF-WS powder was fully soluble. Because the Mw of SF-WS was significantly reduced from kDa to As mentioned before, the EGCG has beneficial function for wound dressing due to its strong ROS scavenging and anti-inflammatory activities. The synthesis of SF-EGCG conjugate was successfully achieved.

To prove our hypothesis, the chemistry between the free amine group and EGCG was observed. L-lysine and ethanolamine were reacted with EGCG in a similar condition to SF-EGCG synthesis, and mass spectrometry was measured Fig.

Also, EGCG and amine group compounds were bounded with a ratio of It means the amine compound and EGCG were not bounded by hydrogen bonds. Elevated ROS levels in wounds are known to stimulate dermal fibroblasts to secrete matrix metalloproteinases, predominantly interstitial collagenases, resulting in excessive breakdown of the collagen fibers in extracellular matrix and delayed wound healing [ 5 , 6 ].

The hydroxyl radical scavenging activity of SF-WS, SF-T, and SF-EGCG shown the obvious difference in the low concentration, but the differences became narrow as the increase of concentration.

The silk fibroin has hydroxyl radical scavenging activity already. The hydroxyl radical react with the phenol group, and forms a phenoxy radical. The phenoxy radical react with the other phenoxy radical, hydroxyl radical and superoxide radical, and forms diphenol, catechol, and tyrosine hydroperoxide, respectively.

In the low concentration, it seems that the hydroxyl radical scavenging activity is more dominantly affected by the tyramine or EGCG modified to silk fibroin than the tyrosine residue in the silk fibroin, and as the increase of the concentration, the effect of tyrosine residue become higher.

EGCG has been reported to exhibit about fold higher affinity towards human serum albumin than — -epicatechin or — -epigallocatechin, which lacks a galloyl moiety, suggesting that the galloyl moiety plays a crucial role in EGCG-protein interactions via hydrogen-bonding and hydrophobic forces [ 44 ].

Also, the collagenase inhibitory activity of EGCG was improved after the conjugation with SF. Although the silk fibroin backbone can make steric hindrance, other hydrogen bond sites in SF can bind collagenase.

For instance, due to the scavenging effect of EGCG moieties against phenoxy free radicals, the concentration of H 2 O 2 6. The SF-EGCG exhibited greater ROS-scavenging activity than SF-T although DS of SF-EGCG 0. After the conjugation, the hydroxyl radical scavenging activity was increased, but the superoxide radical scavenging activity was decreased.

It is considered that the conjugation attenuates the activity of EGCG, but inherent hydroxyl radical scavenging activity of silk fibroin supplemented attenuation of activity in the hydroxyl radical scavenging assay Fig. In addition, the specific collagenase activity was calculated.

After the conjugation, the collagenase inhibitory activity was increased. The silk fibroin backbone can make steric hindrance after the conjugation.

However, in this work, it is considered that additional hydrogen bond sites in the silk fibroin backbone supplemented the steric hindrance. Also, after the conjugation, once an EGCG is bound to the collagenase, other conjugated EGCG get a chance to bind to the collagenase more frequently.

It is a similar mechanism that the polymer-antibody conjugation improves the binding affinity of the antibody. The replacement of the gauze dressing not only causes pain and bleeding, but also delays the wound healing process by removing regenerating skin cells. Neither bleeding nor skin detachment was observed from the wounds treated with all SF hydrogels and DuoDERM® gel.

Wound closure profile of all experimental groups except the control group was almost completed after 14 days. Because the efficacy of hydrogel wound dressings is excellent than other type of wound dressings.

Therefore, the day 14 observation is not significant different. But, inner part of regenerated tissue showed matured collagen deposition with reduced inflammatory cells. It suggests that these hydrogels effectively maintained a moist environment within the wounds. In the present study, SF-EGCG conjugate was synthesized for the first time using the nucleophilic addition reaction of lysine residues in silk proteins with EGCG quinone under a mild basic condition.

This conjugate exhibited superior ROS-scavenging and collagenase-inhibitory activities than SF-T and native SF. With these attractive properties, SF-EGCG-based composite hydrogels hold great promise as functional biomaterials for wound healing applications. The supplementary data are available online at www.

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