Collagen Peptides Derived from Sipunculus nudus Accelerate Wound Healing

Marine collagen peptides have high potential in promoting skin wound healing. This study aimed to investigate wound healing activity of collagen peptides derived from Sipunculus nudus (SNCP). The effects of SNCP on promoting healing were studied through a whole cortex wound model in mice. Results showed that SNCP consisted of peptides with a molecular weight less than 5 kDa accounted for 81.95%, rich in Gly and Arg. SNCP possessed outstanding capacity to induce human umbilical vein endothelial cells (HUVEC), human immortalized keratinocytes (HaCaT) and human skin fibroblasts (HSF) cells proliferation and migration in vitro. In vivo, SNCP could markedly improve the healing rate and shorten the scab removal time, possessing a scar-free healing effect. Compared with the negative control group, the expression level of tumor necrosis factor-α, interleukin-1β and transforming growth factor-β1 (TGF-β1) in the SNCP group was significantly down-regulated at 7 days post-wounding (p < 0.01). Moreover, the mRNA level of mothers against decapentaplegic homolog 7 (Smad7) in SNCP group was up-regulated (p < 0.01); in contrast, type II TGF-β receptors, collagen I and α-smooth muscle actin were significantly down-regulated at 28 days (p < 0.01). These results indicate that SNCP possessed excellent activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to reducing inflammation, improving collagen deposition and recombination and blockade of the TGF-β/Smads signal pathway. Therefore, SNCP may have promising clinical applications in skin wound repair and scar inhibition.     

Artocarpus plants as a potential source of skin whitening agents

Artocarpus plants have been a focus of constant attention due to the potential for skin whitening agents. In the in vitro experiment, compounds from the Artocarpus plants, such as artocarpanone, norartocarpetin, artocarpesin, artogomezianol, andalasin, artocarbene, and chlorophorin showed tyrosinase inhibitory activity. Structure-activity investigations revealed that the 4-substituted resorcinol moiety in these compounds was responsible for their potent inhibitory activities on tyrosinase. In the in vitro assay, using B16 melanoma cells, the prenylated polyphenols isolated from Artocarpus plants, such as artocarpin, cudraflavone C, 6-prenylapigenin, kuwanon C, norartocarpin, albanin A, cudraflavone B, and brosimone I showed potent inhibitory activity on melanin formation. Structure-activity investigations revealed that the introduction of an isoprenoid moiety to a non-isoprenoid-substituted polyphenol enhanced the inhibitory activity of melanin production in B16 melanoma cells. In the in vivo investigation, the extract of the wood of Artocarpus incisus and a representative isolated compound from it, artocarpin had a lightening effect on the skin of guinea pigs' backs. Other in vivo experiments using human volunteers have shown that water extract of Artocarpus lakoocha reduced the melanin formation in the skin of volunteers. These results indicate that the extracts of Artocarpus plants are potential sources for skin whitening agents.     

Tuliposides H–J and Bioactive Components from the Bulb of Amana edulis

Three new tuliposides H–J (1–3) and 11 known compounds were obtained from the methanolic extracts of the bulbs of Amana edulis for the first time. Their structures were elucidated by NMR, MS, and IR spectroscopic data, optical rotation, and Mosher’s method. The melanogenesis properties of all the isolates were evaluated in B16 melanoma cells. Consequently, tributyl citrate (9) had anti-melanogenesis activity but was cytotoxic toward B16. (+)-Pyroglutamic acid (4), (+)-butyl 5-oxopyrrolidine-2-carboxylate (6), (–)-3-hydroxy-2-methylbutyrolactone (10), and 5-(hydroxymethyl)furfural (12) had increased melanin productions and tyrosinase activities. Those active components could be further studied as the candidates against melanoma and vitiligo for skin diseases or whitening/hypopigmentation for hair.     

Effect of electrospun non-woven mats of dibutyryl chitin/poly(lactic acid) blends on wound healing in hairless mice

The aim of this study was to examine the proliferative ability of dibutyryl chitin (DBC) on scratch wounds in HaCaT keratinocytes and to evaluate the effect of nanoporous non-woven mat (DBCNFM) on skin wound healing in hairless mice using the advantages of DBCNFM, such as high porosity and high surface area to volume. The cell spreading activity of DBC was verified through a cell spreading assay in scratched human HaCaT keratinocytes. Scratch wound experiments showed that DBC notably accelerates the spreading rate of HaCaT keratinocytes in a dose dependent manner. The molecular aspects of the healing process were also investigated by hematoxylin & eosin staining of the healed skin, displaying the degrees of reepithelialization and immunostaining on extracellular matrix synthesis and remodeling of the skin. Topical application of DBCNFM significantly reduced skin wound rank scores and increased the skin remodeling of the wounded hairless mice in a dose dependent way. Furthermore, DBCNFM notably increased the expression of the type 1 collagen and filaggrin. These results demonstrate that DBC efficiently accelerates the proliferation of HaCaT keratinocytes and DBCNFM notably increases extracellular matrix synthesis on remodeling of the skin, and these materials are a good candidate for further evaluation as an effective wound healing agent.     

Inhibitory effects of a novel ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate on melanogenesis

We investigated the inhibitory effects of a novel amphiphilic ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), synthesized from a hydrophilic ascorbic derivative, sodium-2-O-L-ascorbyl phosphate (VCP-Na), on melanogenesis in cultured human melanoma cells, normal human melanocytes, and three-dimensional cultured human skin models. Melanin synthesis in melanoma cells treated with VCP-IS-2Na at 300 muM and melanocytes treated with VCP-IS-2Na at 100 muM decreased to 23% and 52% of that in non-treated cells, respectively, and the cell viability was not affected. VCP-IS-2Na also significantly suppressed the cellular tyrosinase activity of melanoma cells and melanocytes. Melanin synthesis in human skin models was evaluated by macro- and microscopic observations of its pigmentation and quantitative measurements of melanin. Treatment of the human skin models with 1.0% VCP-IS-2Na did not inhibit cell viability, while melanin synthesis was decreased to 21% of that in the control. In contrast, L-ascorbic acid (VC) and VCP-Na did not seem to inhibit melanin synthesis and cellular tyrosinase activity. These results indicate that VCP-IS-2Na may be an effective whitening agent for the skin, and we expect the application of VCP-IS-2Na in the cosmetic industry.     

Mealworm Oil (MWO) Enhances Wound Healing Potential through the Activation of Fibroblast and Endothelial Cells

Mealworm and mealworm oil (MWO) have been reported to affect antioxidant, anti-coagulation, anti-adipogenic and anti-inflammatory activities. However, the function of MWO in wound healing is still unclear. In this study, we found that MWO induced the migration of fibroblast cells and mRNA expressions of wound healing factors such as alpha-smooth muscle actin (α-SMA), collagen-1 (COL-1) and vascular endothelial growth factor (VEGF) in fibroblast cells. The tube formation and migration of endothelial cells were promoted through the activation of VEGF/VEGF receptor-2 (VEGFR-2)-mediated downstream signals including AKT, extracellular signal-regulated kinase (ERK) and p38 by MWO-stimulated fibroblasts for angiogenesis. Moreover, we confirmed that MWO promoted skin wound repair by collagen synthesis, re-epithelialization and angiogenesis in an in vivo excisional wound model. These results demonstrate that MWO might have potential as a therapeutic agent for the treatment of skin wounds.     

Ultraviolet B Inhibits Skin Wound Healing by Affecting Focal Adhesion Dynamics

As the most important interface between human body and external environment, skin acts as an essential barrier preventing various environmental damages, among which DNA‐damaging UV radiation from the sun remains the major environmental risk factor causing various skin diseases. It has been well documented that wavelengths in the ultraviolet B (UVB) radiation range (290–320 nm) of the solar spectrum can be absorbed by skin and lead to cutaneous injury and various other deleterious effects. During process such as wound healing, the orchestrated movement of cells in a particular direction is essential and highly regulated, integrating signals controlling adhesion, polarity and the cytoskeleton. Cell adhesion and migration are modulated through both of actin and microtubule cytoskeletons. However, little was known about how UVB affects skin wound healing and migration of epidermal keratinocytes. Here, we demonstrate that UVB can delay the wound healing progress in vivo with a murine model of full‐thickness skin wound. In addition, UVB significantly inhibited keratinocyte motility by altering focal adhesion turnover and cytoskeletal dynamics. Our results provide new insights into the etiology of UVB exposure‐induced skin damages.     

Bee Venom in Wound Healing

Bee venom (BV), also known as api-toxin, is widely used in the treatment of different inflammatory diseases such as rheumatoid arthritis or multiple sclerosis. It is also known that BV can improve the wound healing process. BV plays a crucial role in the modulation of the different phases of wound repair. It possesses anti-inflammatory, antioxidant, antifungal, antiviral, antimicrobial and analgesic properties, all of which have a positive impact on the wound healing process. The mentioned process consists of four phases, i.e., hemostasis, inflammation, proliferation and remodeling. The impaired wound healing process constitutes a significant problem especially in diabetic patients, due to hypoxia state. It had been found that BV accelerated the wound healing in diabetic patients as well as in laboratory animals by impairing the caspase-3, caspase-8 and caspase-9 activity. Moreover, the activity of BV in wound healing is associated with regulating the expression of transforming growth factor (TGF-β1), vascular endothelial growth factor and increased collagen type I. BV stimulates the proliferation and migration of human epidermal keratinocytes and fibroblasts. In combination with polyvinyl alcohol and chitosan, BV significantly accelerates the wound healing process, increasing the hydroxyproline and glutathione and lowering the IL-6 level in wound tissues. The effect of BV on the wounds has been proved by numerous studies, which revealed that BV in the wound healing process brings about a curative effect and could be applied as a new potential treatment for wound repair. However, therapy with bee venom may induce allergic reactions, so it is necessary to assess the existence of the patient’s hypersensitivity to apitoxin before treatment.     

Inhibitory Effect of Elaeagnus umbellata Fractions on Melanogenesis in α-MSH-Stimulated B16-F10 Melanoma Cells

When skin is exposed to UV radiation, melanocytes produce melanin. Excessive melanin production leads to skin pigmentation, which causes various cosmetic and health problems. Therefore, the development of safe, natural therapeutics that inhibit the production of melanin is necessary. Elaeagnus umbellata (EU) has long been widely used as a folk medicinal plant because of pharmacological properties that include anti-ulcer, antioxidant, and anti-inflammatory properties. In this study, we investigated the antioxidant activity and melanogenesis inhibitory effects of EU fractions in B16-F10 melanoma cells. EU fractions showed a dose-dependent increase in antioxidant activity in radical scavenging activity. In addition, we evaluated the effect of EU fractions on tyrosinase activity and melanogenesis in α-melanocyte-stimulating hormone-induced B16-F10 melanoma cells. EU was noncytotoxic at 12.5–50 μg/mL. EU fractions effectively inhibited tyrosinase activity and melanogenesis, suppressed the phosphorylation of CREB and ERK involved in the melanogenesis pathway, and down-regulated expression of melanogenesis-related proteins. Interestingly, the anti-melanogenesis effect was most effective at a concentration of 50 μg/mL EU, and the effects of the fractions were superior to those of the extract. Therefore, our study suggests that EU has potential as a safe treatment for excessive pigmentation or as a natural ingredient in cosmetics.     

Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9(th) and 10(th) type III domains of fibronectin, and 4(th) FAS1 domain of betaig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9(th) and 10(th) type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4(th) FAS1 domain of betaig-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4(th) FAS1 domain of betaig-h3 or 9(th) and 10(th) type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.     

Silkworm-pheophorbide alpha mediated photodynamic therapy against B16F10 pigmented melanoma

In order to apply photodynamic therapy (PDT) to pigmented melanoma, the efficacy of PDT mediated by pheophorbide alpha from silkworm excreta (SPbalpha) and commercial Photofrin against B16F10 melanoma was comparatively studied from the in vivo assay using C57BL/6J mice. From in vitro PDT assay, the proliferation of B16F10 cells treated with SPbalpha (more than 0.5 microg/ml) and light illumination (1.2 J/cm2) were significantly inhibited with the necrotic response. This indicated that the photocytotoxicity of SPbalpha (665 nm) was not influenced by melanin from melanoma. From the assessment of the in vivo photosensitizing activity, the tumor growth was further delayed in groups treated with SPbalpha/PDT compared to that treated with Photofrin /PDT. The survival rate of tumor bearing mice treated with SPbalpha/PDT was closely associated with its photosensitizing effect. In addition, the photosensitizing effect of SPbalpha/PDT showed a dose dependent tendency in light illumination. These results demonstrated that B16F10 melanoma cells were significantly photosensitized by SPbalpha/PDT, regardless of the influence of melanin from melanoma, and SPbalpha/PDT at very low drug dose (1 mg/kg) and light dose (1.2 J/cm2) showed the photosensitizing efficacy surpassing Photofrin against B16F10 melanoma in mice system.     

Anti-oxidative, anti-inflammatory and whitening effects of phenolic compounds from Bambusae Caulis in Liquamen

Bambusae Caulis in Liquamen (BCL) is the distilled product of the condensation from the burning of Phyllostachys nigra var. henosis (Gramineae). The activity-guided isolation of BCL yielded four phenolic compounds: 2,6-dimethoxyphenol (1), 1,2-dihydroxybenzene (2), 1,3-dihydroxybenzene (3) and 1,2-dibenzenecarboxylic acid (4). We evaluated the anti-oxidative, anti-inflammatory and whitening effects of these compounds, via assays, of 1,1-diphenyl-2-picrylhydazyl (DPPH) radical scavenging activity and inhibition of nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells as well as inhibition of tyrosinase activity and melanin production in B16F10 melanoma cells. The results showed that 2 and 3 exhibited significant DPPH radical scavenging activity as well as inhibitory effects on NO production, tyrosinase activity and melanin production. These results suggested that BCL and compounds 2 and 3 can be developed as anti-oxidative, anti-inflammatory and whitening ingredients.     

Anti-Melanogenic Properties of Velutin and Its Analogs

Velutin, one of the flavones contained in natural plants, has various beneficial activities, such as skin whitening, as well as anti-inflammatory, anti-allergic, antioxidant, and antimicrobial activities. However, the relationship between the structure of velutin and its anti-melanogenesis activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7, C3′, and C4′ of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chemical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that the substitution of functional groups at C5, C7, C3′, and C4′ of the flavone backbone affects biological activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7 position is essential for inhibiting tyrosinase activity. However, 1,2-diol compounds substituted at C3′ and C4′ of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C3′ and C4′ with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would be helpful for the development of natural-derived functional materials to regulate melanin synthesis.     

In vitro wound healing evaluation,antioxidant and chemical profiling of Baeckea frutescens leaves ethanolic extract

Occurrence of the wound or chronic wound, which results to disability, amputation and diminish quality of life, are leading to increased healthcare expenditure around the globe. Despite effective conventional wound healing medicines, the exploration of alternative medicines are continuing process as researchers seek for the approach to reduce the cost of wound healing management. In present study, wound healing properties of ethanolic extract of Baeckea frutescens leaves were determined by evaluating their cytotoxicity, proliferation and migration rate on two types of cells, keratinocytes (HaCaT) and fibroblasts (BJ). Furthermore, the antioxidant properties of this plant were determined by DPPH scavenging, ferric reducing antioxidant power (FRAP) and total phenolics content (TPC) assays. The phytochemistry of the extract was evaluated by phytochemicals screening and liquid chromatography mass spectrometry (LCMS) analysis. Results of this study indicated Baeckea frutescens extract increased the rate of proliferation and migration on both HaCat and BJ cells within their nontoxic doses. The extract also possessed a very good antioxidant property as demonstrated with high DPPH radical scavenging, FRAP and TPC values, comparable to that of Green tea extract, a widely known antioxidant. The phytochemistry analyses and LCMS exhibited the presence saponins, flavonoids, tannins and steroids in Baeckea frutescens extract possibly responsible to their antioxidant and wound healing properties.     

Phytochemical Analysis,Antioxidant, and Wound Healing Activity of Pluchea indica L. (Less) Branch Extract Nanoparticles

Proliferation and migration of keratinocytes and fibroblasts play an important role in cutaneous wound healing, while oral mucosal squamous cell proliferation and migration are crucial for oral wound healing. In this study, the phytochemical profile of Pluchea indica branch ethanolic extract was characterized. The bioactive compound of Pluchea indica branch ethanolic extract was identified and analyzed by the validated HPLC method. The nanoparticles of P. indica branch extract were formulated by solvent displacement method to increase the solubility and the colloidal stability of the extract. The stability of the nanoparticles was investigated by using the dynamic light scattering technique. Effects of P. indica crude extract and nanoparticles on cell viability, proliferation and migration of primary epidermal keratinocytes, human dermal fibroblasts, and oral mucosal keratinocyte cells were investigated by MTT assay and scratch assay, respectively. The results showed that P. indica branch extract contained a high content of total phenolic and total flavonoids. The HPLC analysis revealed that the main compound in the extract was 4,5-O-dicaffeoylquinic acid. The cell viability of the extract and nanoparticles decreased when cells were exposed to a high concentration of extract and nanoparticles. These results demonstrate that P. indica branch extract and extract nanoparticles at specific concentrations possess in vitro wound healing activity and they may be possibly used to treat different types of wounds including dermal and oral mucosal wounds.     

In vitro and In vivo Antiproliferative Effect of a Combination of Ultraviolet‐A and Alkoxy Furocoumarins Isolated from Umbelliferae Medicinal Plants,in Melanoma Cells

We examined the effects of six furocoumarins with alkoxy groups at the C‐5 or C‐8 position isolated from Umbelliferae medicinal plants on cell proliferation, and their mechanisms of action against B16F10 melanoma cells or in melanin‐possessing hairless mice implanted with B16F10 cells, under UVA irradiation. Three furocoumarins with an alkoxy group at C‐5, isoimperatorin (1), oxypeucedanin (2) and oxypeucedanin hydrate (3), showed antiproliferative activity and caused G₂/M arrest at concentrations of 0.1–10.0 μm . Furthermore, three furocoumarins with an alkoxy group at C‐8, imperatorin (4), heraclenin (5) and heraclenol (6), inhibited the proliferation of melanoma cells and cell cycle at G₂/M at concentrations of 0.1–1.0 μm . UVA plus 1, 2, 3, 4 and 6 reduced tumor growth and final tumor weight in B16F10‐bearing mice at a dose of 0.3, 0.5 or 1.0 mg kg^?1 (intraperitoneal injection). UVA plus 1, 3 and 6 increased Chk1 phosphorylation and reduced cdc2 (Thr 161) phosphorylation in melanoma cells. We suggest that the antitumor actions of UVA plus furocoumarins with an alkoxy group at C‐5 or C‐8 were due to G₂/M arrest of the cell cycle by an increase in phosphor‐Chk1 and decrease in phospho‐cdc2.     

Selection of malignant melanoma variant cell lines for ovary colonization

Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16-010 cells grew in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melanin pigment (B16-010 less than B16-05 less than B16-01 approximately equal to B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.