Skin Collagen Research Articles

Preparation and evaluation of stingray skin collagen/oyster osteoinductive composite scaffolds

The regeneration of bone defects is a major challenge for clinical orthopaedics. Herein, we designed and prepared a new type of bioactive material, using stingray skin collagen and oyster shell powder (OSP) as raw materials. A stingray skin collagen/oyster osteoinductive composite scaffold (Col-OSP) was prepared for the first time by genipin cross-linking, pore-forming and freeze-drying methods. These scaffolds were characterized by ATR-FTIR, SEM, compression, swelling, cell proliferation, cell adhesion, alkaline phosphatase activity, alizarin red staining and RT-PCR etc. The Col-OSP scaffold had an interconnected three-dimensional porous structure, and the mechanical properties of the Col-OSP composite scaffold were enhanced compared with Col, combining with the appropriate swelling rate and degradation rate, the scaffold was more in line with the requirements of bone tissue engineering scaffolds. The Col-OSP scaffold was non-toxic, promoted the proliferation, adhesion, and differentiation of MC3T3-E1 cells, and stimulated the osteogenesis-related genes expressions of osteocalcin (OCN), collagen type I (COL-I) and RUNX2 of MC3T3-E1 cells.

Isolation of acid soluble collagen from daggertooth pike conger (Muraenesox cinereus) and evaluation of its antimicrobial activity

The study was conducted to isolate acid soluble collagen (ASC) from daggertooth pike conger eel and to evaluate the antimicrobial activity of the isolated protein. Conger eels were collected from Kochi harbour. The proximate composition of eel skin consisted of 68.73 per cent of moisture on a wet basis with a crude protein, crude fat and total ash of 30.24 per cent, 12.17 per cent and 2.16 per cent respectively on a dry matter basis. Conger eel skin collagen was isolated by treating with 0.5 M acetic acid after pretreatment with 0.1 N NaOH and 10 per cent butanol. Salting out and precipitation of ASC was carried out by adding NaCl to a final concentration of 2.3 M. Precipitated ASC was then dialysed against 0.1 M acetic acid for 24h and then in distilled water until neutral pH was obtained. The yield of ASC was 12.78 per cent on a wet basis and 31.95 per cent on a dry basis. The antimicrobial activity of ASC was determined by the agar diffusion method. ASC showed antimicrobial activity against both E. coli and S. aureus.

Evaluation of the biocompatibility of fish skin collagen with the mesenchymal stem cells in in vitro cultures.

There are scarce published data suggesting, that collagen extracted from fish skin may be an attractive alternative to mammalian-derived collagen for the in vitro cell cultures. In this study, we investigated proliferation potential and differentiation capability into osteogenic and adipogenic lineages of rat adipose-derived mesenchymal stem cells (rASCs) and human adipose-derived mesenchymal stem cells (hASCs) cultured on collagen extracted from silver carp and African sharptooth catfish skins, compared with commercially available mammalian collagen and collagen-free culture dishes. Our results revealed no significant differences between fish collagen and mammalian collagen in supporting cell viability and proliferation capacity. Fish-derived collagen is a cheap material derived from production waste, does not contain transmissible pathogens of mammalian origin, supports human cell cultures at comparable level to conventional collagen sources, and may be considered as the product of choice for the in vitro cell cultures.

Facile construction of electrospun zein nanofiber loaded with Rana chensinensis skin collagen for wound care after caesarean section surgery

Numerous types of biomedical applications have found success using electrospun nanofibers. However, electrospinning techniques overlook Zein fibers favoring monolithic and coaxial fibers. Uniaxial electrospinning was used to construct Zein-structured nanofibers for wound healing, with the phase separation of electrospun polycaprolactone (PCL)/polyvinyl alcohol (PVA) in solution, providing the basis for the investigation. Successful loading of silver nanoparticles (AgNPs) and Rana chensinensis skin collagen (RCSCs) into Zein nanofibers (NFs) improved their bioactivity and antibacterial activity as wound dressings. Zein nanofibers were investigated for their heat conductivity, wettability, and mechanical characteristics. The cytotoxic effects of fabricated nanofibers were examined by using L929 fibroblast cells. Ag@RCSCs-NFs also increased cell migration and proliferation, and the wound scratch model was significantly reduced in size using an in vitro scratch assay. The antibacterial studies showed that the Ag@RCSCs-NFs have a potent antibacterial effect against the tested microbial pathogens (Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)). Finally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PCL/PVP, RCSCs, and Ag@RCSCs against two different bacteria were determined. These results suggest that wound care using Zein nanofibers loaded with RCSCs and AgNPs during cesarean section surgery has great promise.

Effects of broiler weight and strain on skin collagen characteristics and their applicability for co-extruded sausage casings

Effects of broiler weight and strain on skin collagen characteristics and their applicability for co-extruded sausage casings

Antioxidaant Activity from Spray Gel Formulation Combination From Ethyl Acetate Fraction Turmeric Rhizome And Collagen From Catfish (Pangasius Hypopthalmus)

Premature aging is the process of aging skin that it faster than time because there is oxidative damage caused by free radicals. This study aims to determine the antioxidant activity of the cosmeceuticals spray gel combination of turmeric rhizome fraction with catfish skin collagen. Curcumin compounds in turmeric are known to have high antioxidant activity to inhibit free radicals and the addition of collagen is intended to be able to provide intake in improving skin structure due to premature aging. The preparations are made into 8 formulas with various concentration. The preparations formed will be evaluated and tested for their antioxidant activity. The result obtained were the extraction of catfish skin collagen the yield of 12.466% collagen. Examination of the ash content, moisture content, fat and protein contain were obtained respectively 0.0105; 0.0525; 5.98; 85.76%. In the evaluation of spray gel preparations, it was found that the preparations F1-F8 met quality standards on the test of spreadability, adhesion resistance, dry time, pH, spray conditions, except for the viscosity value at F4 and F5. The highest % inhibition value was obtained at F8 (0.5% ethyl acetate fractions of turmeric rhizome and 2% collagen of catfish skin) amounted to 48.82%. In statistical t-test showed sig 0.001, indicates there are significant differences in each formula.

Age-related changes in the ratio of Type I/III collagen and fibril diameter in mouse skin.

The content of type I collagen (COL-I) and type III collagen (COL-III) and the ratio between them not only affect the skin elasticity and mechanical strength, but also determine the fibril diameter. In this research, we investigated the age-related changes in COL-I/COL-III ratio with their formed fibril diameter. The experimental result was obtained from high performance liquid chromatography-mass spectrometer, hydroxyproline determination, picrosirius red staining and transmission electron microscopes (TEM), respectively. The result indicated that the COL-I/COL-III ratio in mouse skin increased with aging. From the 0th to 9th week, the COL-I/COLIII ratio increased from 1.3:1 to 4.5:1. From the 9th to the 18th week, it remained between 4.5:1 and 4.9:1. The total content of COL-I and COL-III firstly increased and then decreased with aging. The TEM result showed that the fibril diameter increased with aging. From the 0th to 9th week, the average fibril diameter increased from 40 to 112 nm; From the 9th to 18th weeks, it increased from 112 to 140 nm. After the 9th week. The fibril diameter showed obvious uneven distribution. Thus, the COL-I/COLIII ratio was proportional to the fibril diameter, but inversely proportional to the uniformity of fibril diameter.

Extraction, identification and anti-photoaging activity evaluation of collagen peptides from silver carp (Hypophthalmichthys molitrix) skin

Biologically active peptides are particularly important for anti-photoaging studies. In this study, silver carp (Hypophthalmichthys molitrix) skin collagen polypeptide (SCSCP) with collagenase inhibition activity was obtained by optimizing the enzymatic hydrolysis process. Fourier-transform infrared spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and liquid-liquid mass spectrometry were used to analyze the secondary structure, molecular weight distribution and characteristics of peptides, respectively. The effects of SCSCP on the proliferation of mouse fibroblasts (L929) and secretion of matrix metalloproteinase-1 (MMP-1) after UVB irradiation were also detected. Process-optimized SCSCP inhibited collagenase activity up to 54.47%. In SCSCP, α-helical and β-folded structures accounted for 17.70% and 22.65%, respectively. The relative molecular weights of SCSCP were more widely distributed below 1600 Da, with higher response values at 665.93 and 980.95 Da. The molecular weights of the characteristic peptides were 358.68–863.89 Da, and the peptide sequences with anti-photoaging activity were GPPGPPGTPGPQ, SGLPGPIGPPGPR, and GLPGPIGPPGPR. SCSCP at 10 mg/mL concentration had high DPPH and hydroxyl radical scavenging activity, while 0.1 mg/mL SCSCP significantly restored UVB-induced L929 cell viability and inhibited MMP-1 secretion in L929 cells after UVB irradiation. These results indicate that SCSCP achieves anti-photoaging effects by scavenging DPPH and hydroxyl radicals and inhibiting MMP-1 secretion.

Physicochemical and Fibril Formation Properties of Pufferfish (Takifugu obscurus) Skin Collagen from Solvent Extraction in Different Conditions.

Acid-solubilized (ASC) and pepsin-solubilized collagen (PSC) extracted at 4 °C (ASC-4 and PSC-4), 12 °C (ASC-12 and PSC-12), and 20 °C (ASC-20 and PSC-20) from the skin of farmed pufferfish (Takifugu obscurus) was characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy (FTIR), and fibril-forming tests. The results indicate that extraction at 12 °C can effectively improve the extraction efficiency of natural collagen compared with extraction at 4 °C. However, extraction at 20 °C results in a decrease in molecular integrity, thus, inducing the resultant collagen to degrade or even lose fibril-forming ability. Transmission electron microscope (TEM) images revealed that ASC-4, PSC-4, ASC-12, and PSC-12 can assemble into fibrils with D-periodicities, and ASC-20 associated into molecular aggregates alongside partial D-banded fibrils, while no well-defined fibrils were observed in PSC-20. Scanning electron microscope (SEM) analysis confirmed the well-defined fibril morphologies of ASC-4, PSC-4, ASC-12, and PSC-12 with imino acid contents between 190.0 and 197.8 residues/1000 residues. The denaturation temperature of ASC-4, PSC-4, ASC-12 and PSC-12 was 30.0, 27.6, 25.9 and 22.7 °C, respectively. This study indicates that ASC and PSC extracted at 4 °C and 12 °C could be alternatives to terrestrial collagens for industrial applications.

A new iron supplement: The chelate of pig skin collagen peptide and Fe2+ can treat iron-deficiency anemia by modulating intestinal flora.

Iron deficiency anemia (IDA) is one of the most common nutritional diseases encountered all over the world. Nowadays, oral iron supplementation is still the mainstay of IDA treatment. In this study, a new iron nutritional supplement named pig skin collagen peptides ferrous chelates (PSCP-Fe) was prepared, and its structure was characterized by the scanning electron microscopy, sykam amino acid analyzer and Fourier transform infrared spectroscopy (FTIR). The anti-IDA activity of PSCP-Fe was evaluated in low-Fe2+ diet-induced IDA in rats. 16S amplicon sequencing technology was then used to reveal the mechanism of PSCP-Fe against IDA. The results of amino acid analysis and FTIR showed that aspartic acid (Asp), arginine (Arg), histidine (His), glutamic acid (Glu), cystine (Cys), and lysine (Lys) residued in PSCP chelated readily with Fe2+ through their functional groups. PSCP-Fe treated reversed the hematology-related indexes, such as red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentrate (MCHC), serum ferritin (SF), serum hepcidin (HEPC) and serum transferrin receptor (TFR). And its regulatory action was better than that of FeSO4. Moreover, PSCP-Fe alleviated the hepatocyte apoptosis and necrosis, Fe2+ loss, and injury in IDA rats. In addition, PSCP-Fe could significantly retrace the disturbed profile of gut microbiota in IDA rats (p < 0.05) and significantly up-regulated the relative abundances of nine bacterial genus, including Lactobacillus, Alloprevotella, unclassified_of_Oscillospiraceae, and NK4A214_group (p < 0.05). It could also downgrade the relative abundances of Subdoligranulum and Coriobacteriaceae_UCG-002 (p < 0.05). The results of Spearman's correlation analysis and distance-based redundancy analysis (db-RDA) revealed that Subdoligranulum and Christensenellaceae_R-7_group may be potential microbial markers for effective PSCP-Fe action in the treatment of IDA. Overall, our results elucidate the interactions between gut bacteria and related cytokines and reveal the mechanisms underlying the anti-IDA effect of PSCP-Fe. They will thus provide a theoretical foundation for PSCP-Fe as a new iron nutritional supplement.

Production and antioxidant activity of peptides from sturgeon head

The head is a significant, but typically unused, fish by-product. Here, we developed an industrially applicable new method to make head tissue peptides (HTPs) by autoclaving, blending and enzymatic hydrolysis of sturgeon head tissue. Four conditions were tested: head tissue autoclaved in distilled water (DW) and hydrolyzed with papain (DW-P) or papain and bromelain (DW-P + B), or head tissue autoclaved without DW and hydrolyzed with papain (WDW-P) or papain and bromelain (WDW-P + B). Among these conditions, DW-P + B resulted in the highest peptide yield, more peptides of <500 Da, and peptides with the highest antioxidant activity. Next, the same method produced sturgeon skin tissue peptide (STP) and skin collagen peptide (SCP) and the bioactivities were compared. The gross yield of HTP per fish was higher than that of STP and SCP because the sturgeon's head is ∼3 times heavier than its skin. The HTP contained more reactive oxygen species (ROS)-scavenging amino acids (Glx, His, Leu, Lys, Met, Phe, Tyr, Val) and showed significantly higher ABTS• scavenging activity than STP and SCP. The extracellular cell-protection and intracellular anti-ROS activities of HTP on the application of oxidative stress were similar to those of STP and SCP. Moreover, HTP showed high transparency and high safety (a low endotoxin level). All these features suggest that HTP is suitable for percutaneous applications. Extracellular oxidative stress promotes human skin aging; thus, HTP may be a novel, low-cost ingredient for anti-aging cosmetics. The present study will help to exploit zero industrial discards, resulting in blue transformation of by-products.

Gelatin Microspheres Loaded with Wharton's Jelly Mesenchymal Stem Cells Promote Acute Full-Thickness Skin Wound Healing and Regeneration in Mice.

Objective: At present, there is an urgent need to develop a novel and practical therapeutic approach to accelerate the healing of acute wounds. Mesenchymal stem cell (MSC)-based therapy is emerging as a promising therapeutic approach for acute skin wounds. However, there are still challenges in clinical application of this strategy, such as low survivability, low retention time, and less engraftment in skin wounds. Approach: Wharton's jelly mesenchymal stem cells (WJMSCs) were seeded into three-dimensional (3D) gelatin microspheres (GMs) to identify the biocompatibility of GMs. WJMSCs were embedded in GMs and then encapsulated with Pluronic F-127 (PF-127) and sodium ascorbyl phosphate (SAP) combination to transplant onto acute full-thickness skin wound in mice. Histology, immunohistochemistry, and immunofluorescence assay were used to investigate the skin wound healing, dermis regeneration, collagen deposition, cell proliferation, and neovascularization. Results: Three-dimensional GM had strong biocompatibility, compared with two-dimensional adherent culturing, GM loading increased the cell viability and proliferation ability of WJMSCs. WJMSCs+GM+PF-127+SAP transplantation increased skin wound healing rate, dermis regeneration, and type III collagen deposition through improving macrophage polarization, cell proliferation, neovascularization, cell retention, and engraftment at skin wound site. Innovation: The effective 3D encapsulation technology for WJMSCs solved the main problems of cell activity and residence time during MSC transplantation. WJMSCs+GM+PF-127+SAP transplantation will be a new and effective MSC biomaterials-based therapeutic strategy for acute skin traumatic wounds. Conclusion: WJMSCs+GM+PF-127+SAP transplantation facilitated acute full-thickness skin wound healing and regeneration and might be a new and effective therapy for acute skin traumatic wounds.

Carboxymethyl Chitosan/Tannic Acid Hydrogel with Antibacterial, Hemostasis, and Antioxidant Properties Promoting Skin Wound Repair.

Local causes of slow wound healing include infection and wound hemorrhage. Using sodium bicarbonate as a neutralizer, a variety of carboxymethyl chitosan-tannic acid (CMC-TA) composite hydrogels solidify through hydrogen bonding in this study. The best-performing hydrogel was synthesized by altering the concentration of TA and exhibited remarkable mechanical properties and biocompatibility. Following in vitro characterization tests, the CMC-TA hydrogel exhibited remarkable antibacterial and antioxidant properties, as well as quick hemostasis capabilities. In the in vivo wound healing study, the results showed that the CMC-TA hydrogel could relieve inflammation and promote the recovery of skin incision, re-epithelialization, and collagen deposition. Overall, this multifunctional hydrogel could be an ideal wound dressing for the clinical therapy of full-thickness wounds.

Delivery of bone morphogenetic protein-2 by crosslinking heparin to nile tilapia skin collagen for promotion of rat calvaria bone defect repair.

Collagen has been widely used as a biomaterial for tissue regeneration. At the present, aqua-collagen derived from fish is poorly explored for biomedical material applications due to its insufficient thermal stability. To improve the bone repair ability and thermal stability of fish collagen, the tilapia skin collagen was crosslinked by EDC/NHS with heparin to bind specifically to BMP-2. The thermal stability of tilapia skin collagen crosslinked with heparin (HC-COL) was detected by differential scanning calorimetry (DSC). Cytotoxicity of HC-COL was assessed by detecting MC3T3-E1 cell proliferation using CCK-8 assay. The specific binding of BMP-2 to HC-COL was tested and the bioactivity of BMP-2-loaded HC-COL (HC-COL-BMP-2) was evaluated in vitro by inducing MC3T3-E1 cell differentiation. In vivo, the bone repair ability of HC-COL-2 was evaluated using micro-CT and histological observation. After crosslinking by EDC/NHS, the heparin-linked and the thermostability of the collagen of Nile Tilapia were improved simultaneously. HC-COL has no cytotoxicity. In addition, the binding of BMP-2 to HC-COL was significantly increased. Furthermore, the in vitro study revealed the effective bioactivity of BMP-2 binding on HC-COL by inducing MC3T3-E1 cells with higher ALP activity and the formation of mineralized nodules. In vivo studies showed that more mineralized and mature bone formation was achieved in HC-COL-BMP-2 group. The prepared HC-COL was an effective BMP-2 binding carrier with enough thermal stability and could be a useful biomaterial for bone repair.

Biomimetic multifunctional hybrid sponge via enzymatic cross-linking to accelerate infected burn wound healing

Preparing sponge dressings with stable wet adhesion remains difficult in wound repair, especially in burn wounds with bleeding and large amounts of exudate. In this work, a multifunctional hybrid sponge dressing (DHGT+PHMB+TiO2NPs) with good wet adhesion was developed by combining biomimetic and enzymatic cross-linking reactions. The sponge dressing matrix (DHGT) was prepared by tyrosinase-catalyzed cross-linking of dopamine-modified hyaluronic acid (DOPA-HA) and gelatin. The multifunctional hybrid sponge dressing was obtained by loading polyhexamethylene biguanide (PHMB) and titanium dioxide nanoparticles (TiO2NPs) onto the DHGT matrix. The newly developed sponge dressing exhibited high mechanical properties, good wet adhesion, antibacterial activity, reactive oxygen species (ROS) scavenging, biocompatibility, and excellent hemostasis ability. In vivo studies showed that the multifunctional hybrid sponge dressing could significantly accelerate the healing of infected full-thickness burn wounds by inhibiting bacterial growth, accelerating skin tissue reepithelialization, collagen deposition, and angiogenesis, as well as regulating the expression of inflammatory factors and cytokines.

Circ004463 promotes fibroblast proliferation and collagen I synthesis by sponging miR-23b and regulating CADM3/MAP4K4 via activation of AKT/ERK pathways

Skin thickness is closely related to the appearance of human skin, such as sagging and wrinkling, which primarily depends on the level of collagen I synthesized by fibroblasts in the dermal layer. To explore the underlying genetics of the development of skin thickness, we used the indigenous Chinese Chenghua pigs, considered to have superior skin thickness, as model animals. We first performed whole transcriptome sequencing analysis to identify significant skin morphological differences between Chenghua pigs and Large White pigs and obtained some differentially expressed coding RNAs (454 mRNAs) and noncoding RNAs (612 circRNAs, 188 miRNAs, and 19 lncRNAs); moreover, some competing endogenous RNA (ceRNA) networks were constructed. Interestingly, we then identified a circRNA, namely circ0044633, which plays an important role in promoting fibroblast proliferation along with myofibroblast transition and collagen I synthesis by sponging miR-23b and regulating CADM3 and MAP4K4 expression via activation of the downstream AKT and ERK pathways in vitro. Furthermore, overexpression of circ004463 increased the mouse skin thickness and collagen I content in vivo. These results revealed a whole transcript profile of skin tissue and identified an important circ0044633-miR-23b-CADM3/MAP4K4 axis related to fibroblast proliferation and collagen I synthesis during the development of skin thickness.

A novel peptide isolated from Catla skin collagen acts as a self-assembling scaffold promoting nucleation of calcium-deficient hydroxyapatite nanocrystals.

Catla collagen hydrolysate (CH) was fractionated by chromatography and each fraction was subjected to HA nucleation, with the resultant HA-fraction composites being scored based on the structural and functional group of the HA formed. The process was repeated till a single peptide with augmented HA nucleation capacity was obtained. The peptide (4.6kDa), exhibited high solubility, existed in polyproline-II conformation and displayed a dynamic yet stable hierarchical self-assembling property. The 3D modelling of the peptide revealed multiple calcium and phosphate binding sites and a high propensity to self-assemble. Structural analysis of the peptide-HA crystals revealed characteristic diffraction planes of HA with mineralization following the (002) plane, retention of the self-assembled hierarchy of the peptide and intense ionic interactions between carboxyl groups and calcium. The peptide-HA composite crystals were mostly of 25-40nm dimensions and displayed 79% mineralization, 92% crystallinity, 39.25% porosity, 12GPa Young's modulus and enhanced stability in physiological pH. Cells grown on peptide-HA depicted faster proliferation rates and higher levels of osteogenic markers. It was concluded that the prerequisite for HA nucleation by a peptide included: a conserved sequence with a unique charge topology allowing calcium chelation and its ability to form a dynamic self-assembled hierarchy for crystal propagation.

Preparation of Nile tilapia skin collagen powder by low-temperature and comprehensive evaluation of hemostasis and wound healing.

Nile tilapia (hereinafter referred to as tilapia) is a species with high economic value and extensive cultivation. In this study, the low-temperature Nile tilapia skin collagen powder (TSCP) was prepared by liquid nitrogen freeze pulverization. After physical and chemical analysis of its properties, it was found that its characteristics were similar to those of type I collagen. The three-dimensional helix structure of protein peptide is good and non denatured. It shows that cryogenic temperature guarantees the activity of TSCP. In addition, TSCP has good biocompatibility. Specifically, it has good blood compatibility, lacks cytotoxicity, will not cause intradermal stimulation and acute systemic toxicity, and has no obvious rejection after implantation. In the rat liver hemorrhage model and wound repair model, compared with the commercially available bovine collagen powder (BSCP), TSCP has better blood coagulation ability: the shortest hemostatic time (135 s) and wound healing efficiency: the wound healing is obvious on the 14th day. The results of this study indicate that the TSCP is an ideal candidate for hemostatic agents and wound healing dressings.

Cranberry Juice Polyphenols Inhibited the Formation of Advanced Glycation End Products in Collagens, Inhibited Advanced Glycation End Product-Induced Collagen Crosslinking, and Cleaved the Formed Crosslinks.

Collagens in the human skin are susceptible to glycation due to their long half-life of about 15 years, accumulating advanced glycation end products (AGEs). The formation of AGEs and the subsequent AGE-induced collagen crosslinking are major factors for skin aging. The objective of this study was to determine the capacity of cranberry juice polyphenols (CJPs) and their fractions to inhibit collagen glycation and to break AGE-induced crosslinks in collagens. Concentrated cranberry juice was extracted to obtain the CJP, which was further fractionated into an ethyl acetate fraction, water fraction, 30% methanol (MeOH) fraction, 60% MeOH fraction, MeOH fraction, and acetone fraction. CJPs and their fractions contained different ratios of anthocyanins, procyanidins, and flavonols. All the fractions significantly inhibited collagen glycation assessed with the collagen-methylglyoxal (MGO) or collagen-dehydroascorbic acid (DHAA) assays. The ethyl acetate fraction and 60% MeOH had the lowest IC50 values in the collagen-MGO and collagen-DHAA assays. The methanol fraction (IC50 = 0.52 μg/mL) and acetone fraction (IC50 = 0.019 mg/mL) had the lowest IC50 values in the inhibition and breakage of AGE-induced collagen crosslinking, respectively. The ethyl acetate fraction significantly scavenged the highest amount of MGO and DHAA after incubation compared to the other fractions. Results suggested that procyanidins were the most effective antiglycation agent in both collagen glycation assays, followed by flavonols and anthocyanins. High-performance liquid chromatography-electrospray ionization─tandem mass spectrometry showed that the reactions of DHAA with quercetin or epicatechin formed several adducts with unreported proposed structures. This study suggested that CJPs may be used as active ingredients in cosmetics to prevent skin collagen glycation and crosslinking and to break the formed crosslinks.

Protective Effects of Withagenin A Diglucoside from Indian Ginseng (Withania somnifera) against Human Dermal Fibroblast Damaged by TNF-α Stimulation.

Human skin is constructed with many proteins such as collagen and elastin. Collagen and elastin play a key role in providing strength and elasticity to the human skin and body. However, damage to collagen causes various symptoms such as wrinkles and freckles, which suggests that they are important to maintain skin condition. Extrinsic or intrinsic skin aging produces an excess of skin destructive factors such as tumor necrosis factor (TNF)-α, which is a major mediator of the aging process. In aged skin, TNF-α provokes the generation of intracellular ROS (reactive oxygen species). It triggers the excessive secretion of MMP-1, which is a collagen-degrading enzyme that causes the collapse of skin collagen. Therefore, we aimed to search for a natural-product-derived candidate that inhibits the skin damage caused by TNF-α in human dermal fibroblasts. In this study, the protective effect of withagenin A diglucoside (WAD) identified from Withania somnifera against TNF-α-stimulated human dermal fibroblasts is investigated. W. somnifera (Solanaceae), well-known as 'ashwagandha', is an Ayurvedic medicinal plant useful for promoting health and longevity. Our experimental results reveal that WAD from W. somnifera suppresses the generation of intercellular ROS. Suppressing intracellular ROS generation inhibits MMP-1 secretion and the collapse of type 1 collagen. The effect of WAD is shown to depend on the inhibition of MAPK phosphorylation, Akt phosphorylation, c-Jun phosphorylation, COX-2 expression, and NF-κB phosphorylation. Further, WAD-depressed expression of the pro-inflammatory cytokines IL-6 and IL-8 triggers various inflammatory reactions in human skin. These findings suggest that WAD has protective effects against skin damage. Accordingly, our study provides experimental evidence that WAD can be a potential agent that can be applied in various industrial fields, such as cosmetics and pharmaceuticals related to skin aging.