Dietary flavonoid myricetin 3-O-galactoside suppresses α-melanocyte stimulating hormone-induced melanogenesis in B16F10 melanoma cells by regulating PKA and ERK1/2 activation.

Melanogenesis is the process where skin pigment melanin is produced through tyrosinase activity. Overproduction of melanin causes skin disorders such as freckles, spots, and hyperpigmentation. Myricetin 3-O-galactoside (M3G) is a dietary flavonoid with reported bioactivities. M3G was isolated from Limonium tetragonum and its anti-melanogenic properties were investigated in α-melanocyte stimulating hormone-stimulated B16F10 melanoma cells. The in vitro anti-melanogenic capacity of M3G was confirmed by inhibited tyrosinase and melanin production. M3G-mediated suppression of melanogenic proteins, tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (TRP)-1 and TRP-2, were confirmed by mRNA and protein levels, analyzed by RT-qPCR and Western blot, respectively. Furthermore, M3G suppressed Wnt signaling through the inhibition of PKA phosphorylation. M3G also suppressed the consequent phosphorylation of CREB and nuclear levels of MITF. Analysis of MAPK activation further revealed that M3G increased the activation of ERK1/2 while p38 and JNK activation remained unaffected. Results showed that M3G suppressed melanogenesis in B16F10 cells by decreasing tyrosinase production and therefore inhibiting melanin formation. A possible action mechanism was the suppression of CREB activation and upregulation of ERK phosphorylation which might cause the decreased nuclear levels of MITF. In conclusion, M3G was suggested to be a potential nutraceutical with anti-melanogenic properties.

Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation.

Quercetin 3-O-galactoside (Q3G) is a common dietary flavanol that has been shown to possess several bioactivities, including anti-melanogenesis. However, how Q3G exerts its anti-melanogenic effect has not been studied. The current study, therefore aimed to investigate the anti-melanogenesis potential of Q3G and elucidate the underlying action mechanism in α-melanocyte-stimulating hormone (α-MSH)-induced hyperpigmentation model of B16F10 murine melanoma cells. Results showed that α-MSH stimulation significantly increased tyrosinase (TYR) and melanin production, which were significantly downregulated by Q3G treatment. The treatment with Q3G suppressed the transcriptional and protein expressions of melanogenesis-related enzymes TYR, tyrosinase related protein-1 (TRP-1), and TRP-2, along with the melanogenic transcription factor microphthalmia-associated transcription factor (MITF) in B16F10 cells. It was shown that Q3G downregulated MITF expression and suppressed its transcriptional activity by inhibiting the cAMP-dependent protein kinase A (PKA)-mediated activation of CREB and GSK3ß. In addition, MAPK-regulated MITF activation signaling was also involved in the inhibition of melanin production by Q3G. The results suggest that the anti-melanogenic properties of Q3G rationalize further studies in vivo to confirm its action mechanism and consequent utilization as a cosmetic ingredient against hyperpigmentation.

Salidroside, 8(E)-Nuezhenide, and Ligustroside from Ligustrum japonicum Fructus Inhibit Expressions of MMP-2 and -9 in HT 1080 Fibrosarcoma.

A phenyl ethanoid, salidroside (SAL), and two secoiridoids, 8(E)-nuezhenide (NZD) and ligustroside (LIG), were isolated from fruits of Ligustrumjaponicum, used as traditional folk medicine, and their chemical structures were elucidated by the comparison of spectral data with published literature. Matrix metalloproteinases (MMPs) are major enzymes that play crucial roles in the metastasis and invasive behavior of tumors. In particular, MMP-2 and MMP-9, regulated by the MAPK signaling pathways, including p38, ERK and JNK, are known to play a key role in the degradation of the basement membrane. In the present study, the effects of SAL, NZD and LIG on the expression of MMP-2 and -9 were examined in phorbol 12-myristate 13-acetate (PMA)-induced HT 1080 cells. All the compounds significantly lowered the amount of MMP-2 and MMP-9 released, as determined by gelatin zymography and ELISA. In addition, the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly suppressed, as measured by RT-PCR and Western blotting. According to the Western blotting assay, SAL and LIG effectively reduced the expression of MMP-2 in a dose-dependent manner. NZD lowered the expression of MMP-9 in a similar way. The phosphorylation of p38, ERK and JNK was also significantly suppressed by these compounds. These findings suggest that all the compounds regulate the release and expression of MMP-2 and MMP-9 via MAPK signaling pathways.

Dracunculin Inhibits Adipogenesis in Human Bone Marrow-Derived Mesenchymal Stromal Cells by Activating AMPK and Wnt/ß-Catenin Signaling.

Increased bone marrow adiposity is widely observed in patients with obesity and osteoporosis and reported to have deleterious effects on bone formation. Dracunculin (DCC) is a coumarin isolated from Artemisia spp. but, until now, has not been studied for its bioactive potential except antitrypanosomal activity. In this context, current study has reported the anti-adipogenic effect of DCC in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). DCC dose-dependently inhibited the lipid accumulation and expression of adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) in hBM-MSCs induced to undergo adipogenesis. To elucidate its action mechanism, the effect of DCC on Wnt/ß-catenin and AMPK pathways was examined. Results showed that DCC treatment activated Wnt/ß-catenin signaling pathway via AMPK evidenced by increased levels of AMPK phosphorylation and Wnt10b expression after DCC treatment. In addition, DCC treated adipo-induced hBM-MSCs exhibited significantly increased nuclear levels of ß-catenin compared with diminished nuclear PPARγ levels. In conclusion, DCC was shown to be able to hinder adipogenesis by activating the ß-catenin via AMPK, providing potential utilization of DCC as a nutraceutical against bone marrow adiposity.

Evaluation and Comparison of the Antimelanogenic Properties of Different Solvent Fractionated Cnidium japonicum Extracts in B16F10 Murine Melanoma Cells.

Cnidium japonicum is a biennial halophyte found in the salt marshes and shores of Korea and widely used in traditional Korean medicine as an ingredient. This study investigated and compared the antimelanogenic effect of solventpartitioned fractions of C. japonicum extract (CJEFs) in a B16F10 mouse melanoma cell model, focusing on tyrosinase activity and production. Melanogenesis is the process in which skin pigment melanin is produced through tyrosinase activity. Overproduction of melanin is the primary reason behind several skin disorders such as freckles, spots, and hyperpigmentation. The antimelanogenic capacity of CJEFs was initially screened by their tyrosinase inhibitory effects, prevention of dihydroxyphenylalanine (DOPA) oxidation, and suppression of melanin production. The inhibition of tyrosinase activity and DOPA oxidation by CJEFs was suggested to be related to the downregulation of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2, which was confirmed using mRNA and protein expression levels. Moreover, the glycogen synthase kinase 3 beta- and cyclic adenosine monophosphate response element-binding protein-related signaling pathways were inhibited by treatment with CJEFs, indicating their action mechanism. All the tested CJEFs exerted similar effects on tyrosinase activity and production. However, among those, 85% aq. MeOH was the most active fraction to suppress the signaling pathway that produces tyrosinase. These results suggest that especially the MeOH fraction of C. japonicum extract serves as a potential source of bioactive substances, with effective antimelanogenesis properties.

Libanoridin Isolated from Corydalis heterocarpa Inhibits Adipogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow adiposity is a complication in osteoporotic patients. It is a result of the imbalance between adipogenic and osteogenic differentiation of bone marrow cells. Phytochemicals can alleviate osteoporotic complications by hindering bone loss and decreasing bone marrow adiposity. Corydalis heterocarpa is a biennial halophyte with reported bioactivities, and it is a source of different coumarin derivatives. Libanoridin is a coumarin isolated from C. heterocarpa, and the effect of libanoridin on adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) was evaluated in the present study. Cells were induced to undergo adipogenesis, and their intracellular lipid accumulation and expression of adipogenic markers were observed under libanoridin treatment. Results showed that 10 µM libanoridin-treated adipocytes accumulated 44.94% less lipid compared to untreated adipocytes. In addition, mRNA levels of PPARγ, C/EBPα, and SREBP1c were dose-dependently suppressed with libanoridin treatment, whereas only protein levels of PPARγ were decreased in the presence of libanoridin. Fluorescence staining of adipocytes also revealed that cells treated with 10 µM libanoridin expressed less PPARγ compared to untreated adipocytes. Protein levels of perilipin and leptin, markers of mature adipocytes, were also suppressed in adipocytes treated with 10 µM libanoridin. Analysis of MAPK phosphorylation levels showed that treatment with libanoridin inhibited the activation of p38 and JNK MAPKs observed by decreased levels of phosphorylated p38 and JNK protein. It was suggested that libanoridin inhibited adipogenic differentiation of hBM-MSCs via suppressing MAPK-mediated PPARγ signaling. Future studies revealing the anti-adipogenic effects of libanoridin in vivo and elucidating its action mechanism will pave the way for libanoridin to be utilized as a nutraceutical with anti-osteoporotic properties.

Myricetin 3-O-ß-D-Galactopyranoside Exhibits Potential Anti-Osteoporotic Properties in Human Bone Marrow-Derived Mesenchymal Stromal Cells via Stimulation of Osteoblastogenesis and Suppression of Adipogenesis.

Natural bioactive substances are promising lead compounds with beneficial effects on various health problems including osteoporosis. In this context, the goal of this study was to investigate the effect of myricetin 3-O-ß-D-galactopyranoside (M3G), a glycoside of a known bioactive phytochemical myricetin, on bone formation via osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). The hBM-MSCs were induced to differentiate into osteoblasts and adipocytes in the presence or absence of M3G and the differentiation markers were analyzed. Osteoblastogenesis-induced cells treated with M3G exhibited stimulated differentiation markers: cell proliferation, alkaline phosphatase (ALP) activity, and extracellular mineralization. In terms of intracellular signaling behind the stimulatory effect of M3G, the expression of RUNX2 and osteopontin transcription factors were upregulated. It has been shown that M3G treatment increased the activation of Wnt and BMP as a suggested mechanism of action for its effect. On the other hand, M3G treatment during adipogenesis-inducement of hBM-MSCs hindered the adipogenic differentiation shown as decreased lipid accumulation and expression of PPARγ, SREBP1c, and C/EBPα, adipogenic transcription factors. In conclusion, M3G treatment stimulated osteoblast differentiation and inhibited adipocyte differentiation in induced hBM-MSCs. Osteoblast formation was stimulated via Wnt/BMP and adipogenesis was inhibited via the PPARγ pathway. This study provided necessary data for further studies to utilize the therapeutic potential of M3G against osteoporosis via regulation of bone marrow stromal cell differentiation.

Ligustrum japonicum Thunb. Fruits Exert Antiosteoporotic Properties in Bone Marrow-Derived Mesenchymal Stromal Cells via Regulation of Adipocyte and Osteoblast Differentiation.

Ligustrum japonicum fruits have been used as a part of traditional medicinal practices and supplements in Korea and Japan. It has been reported to possess various bioactivities, but its antiosteoporotic potential and active substances have not been reported yet. The present study followed an ALP activity and lipid accumulation-guided screening of L. japonicum fruits for antiosteoporotic compounds and isolated salidroside as an active compound. Antiosteoporotic effects of L. japonicum fruits and salidroside were examined in mesenchymal stromal cells by their ability to enhance osteoblast formation by increased ALP activity and osteogenic marker gene expression while suppressing adipogenesis by inhibition of lipid accumulation and adipocyte marker gene expressions. Results showed that salidroside was able to enhance osteoblast differentiation via Wnt/BMP signaling pathway overactivation and suppress the PPARγ-mediated adipocyte differentiation, both through the MAPK pathway. In conclusion, L. japonicum fruits were suggested to possess antiosteoporotic activities and to be a source of antiosteoporotic substances such as salidroside.

Effect and Comparison of Luteolin and Its Derivative Sodium Luteolin-4'-sulfonate on Adipogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells through AMPK-Mediated PPARγ Signaling.

Luteolin is a common phytochemical from the flavonoid family with a flavone structure. Studies reported several bioactivities for luteolin and similar flavones. Attenuating the increased adipogenesis of bone marrow cells (hBM-MSCs) has been regarded as a therapeutic target against osteoporotic bone disorders. In the present study, the potential roles of luteolin and its sulfonic acid derivative luteolin-OSO3Na in regulating adipogenic differentiation of hBM-MSCs were investigated. Adipo-induced cells were treated with or without compounds, and their effect on adipogenesis was evaluated by adipogenic marker levels such as lipid accumulation and PPARγ pathway activation. Luteolin hindered the adipogenic lipid accumulation in adipo-induced hBM-MSCs. Immunoblotting and reverse transcription-polymerase chain reaction analysis results indicated that luteolin downregulated PPARγ and downstream factors of C/EBPα and SREBP1c expression which resulted in inhibition of adipogenesis. Luteolin-OSO3Na showed similar effects; however, it was significantly less effective compared to luteolin. Investigating p38, JNK, and ERK MAPKs and AMPK activation indicated that luteolin suppressed the MAPK phosphorylation while stimulating AMPK phosphorylation. On the other hand, luteolin-OSO3Na was not able to notably affect the MAPK and AMPK activation. In conclusion, this study suggested that luteolin inhibited adipogenic differentiation of hBM-MSCs via upregulating AMPK activation. Replacing its 4'-hydroxyl group with sulfonic acid sodium salt diminished its antiadipogenic effect indicating its role in regulating AMPK activation. The general significance is that luteolin is a common phytochemical with various health-beneficial effects. The current study suggested that luteolin may serve as a lead compound for developing antiosteoporotic substances with antiadipogenic properties.

Effect of Quercetin 3-O-ß-D-Galactopyranoside on the Adipogenic and Osteoblastogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Natural products, especially phenols, are promising therapeutic agents with beneficial effects against aging-related complications such as osteoporosis. This study aimed to investigate the effect of quercetin 3-O-ß-D-galactopyranoside (Q3G), a glycoside of a common bioactive phytochemical quercetin, on osteogenic and adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). hBM-MSCs were induced to differentiate into osteoblasts and adipocytes in the presence or absence of Q3G and the differentiation markers were analyzed to observe the effect. Q3G treatment stimulated the osteoblastogenesis markers: cell proliferation, alkaline phosphatase (ALP) activity and extracellular mineralization. In addition, it upregulated the expression of RUNX2 and osteocalcin protein as osteoblastogenesis regulating transcription factors. Moreover, Q3G treatment increased the activation of osteoblastogenesis-related Wnt and bone morphogenetic protein (BMP) signaling displayed as elevated levels of phosphorylated ß-catenin and Smad1/5 in nuclear fractions of osteo-induced hBM-MSCs. The presence of quercetin in adipo-induced hBM-MSC culture inhibited the adipogenic differentiation depicted as suppressed lipid accumulation and expression of adipogenesis markers such as PPARγ, SREBP1c and C/EBPα. In conclusion, Q3G supplementation stimulated osteoblast differentiation and inhibited adipocyte differentiation in hBM-MSCs via Wnt/BMP and PPARγ pathways, respectively. This study provided useful information of the therapeutic potential of Q3G against osteoporosis mediated via regulation of MSC differentiation.

Compression injury of the circular stapler for gastrointestinal end-to-end anastomosis: preliminary in-vitro study.

PURPOSE: This preliminary in-vitro study was designed to evaluate the risk factors of compression injury from use of a circular stapler for end-to-end anastomosis. METHODS: Transparent collagen plates were prepared in dry and wet conditions. Physical properties of collagen plates and porcine colon tissue were examined using a rheometer. Adjustable and fixed-type circular staplers were applied on the collagen plates and the gap distance and compressive pressure were measured during anvil approximation. Tissue injury was evaluated using a compression injury scale. Compression properties were accessed to optimal or overcompression based on gap distance. RESULTS: Unacceptable injuries were rarely observed on the dry collagens, regardless of compression device. In the adjustable compression, the compressibility ratio was similar between dry and wet collagen. Overcompression and unacceptable injury increased on the wet collagens. In the fixed compression, the compressibility ratio increased significantly and unacceptable injuries were observed in more than 50% of wet collagens. Peak pressure was significantly higher in the fixed-compression types than those of adjustable type. On bivariate correlation analysis, fixed-compression type and wet collagens were respectively associated with overcompression. On multivariate analysis, edematous collagen condition was the most important risk factor and proximal anvil side, fixed compression type, and overcompression were also independent risk factors for unacceptable compression injury. CONCLUSION: In the edematous tissue condition, unintentional overcompression could be increased and result in tissue injury on the compression line of the circular stapler.

Syringaresinol Inhibits UVA-Induced MMP-1 Expression by Suppression of MAPK/AP-1 Signaling in HaCaT Keratinocytes and Human Dermal Fibroblasts.

Ultraviolet (UV) irradiation induces detrimental changes in human skin which result in photoaging. UV-induced intracellular changes cause degradation of extracellular matrix (ECM). UV-stimulated cleavage of collagen in ECM occurs via matrix metalloproteinases (MMPs). (±)-syringaresinol (SYR), a phytochemical which belongs to the lignan group of polyphenols, was investigated for its ability to reverse the UVA-induced changes in human HaCaT keratinocytes and dermal fibroblasts (HDFs) in vitro. Effect of SYR on UVA-induced changes was investigated by production and activation of MMPs and its transcriptional upstream effectors; mitogen-activated protein kinases (MAPKs) and pro-inflammatory mediators. Levels of expression were determined using ELISA, RT-PCR and immunoblotting. UVA irradiation stimulated the production of MMP-1 and inhibited collagen production. SYR treatment suppressed MMP-1 and enhanced collagen production in UVA-irradiated HaCaT keratinocytes and HDFs. SYR repressed the UV-induced phosphorylation of p38, ERK and JNK MAPKs in HaCaT keratinocytes while only suppressing JNK phosphorylation in HDFs. In addition, SYR was able to inhibit UVA-induced production of inflammatory cytokines; TNF-α, COX-2, IL-1ß and IL-6. Moreover, SYR suppressed the activator protein-1 (AP-1), a heterodimer of phosphorylated transcription factors c-Jun and c-Fos. SYR-treatment decreased nuclear levels of activated c-Fos and c-Jun as a mechanism to inhibit UVA-induced transcriptional activities leading to MMP-1 production. In conclusion, current results demonstrated that SYR could inhibit UVA-induced upregulation of MMP-1 by suppressing MAPK/AP-1 signaling in HaCaT keratinocytes and HDFs. Therefore, SYR was suggested as a potential compound with antiphotoaging properties against UVA-induced skin aging.

3,5-dicaffeoyl­epi-quinic acid from Atriplex gmelinii enhances the osteoblast differentiation of bone marrow-derived human mesenchymal stromal cells via WnT/BMP signaling and suppresses adipocyte differentiation via AMPK activation.

BACKGROUND: Impaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment. PURPOSE: In this context, 3,5-dicaffeoyl­epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro. METHODS: Anti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways. RESULTS: At 10 µM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of ß-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes. CONCLUSION: DCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.

6-Acetyl-2,2-Dimethylchroman-4-One Isolated from Artemisia princeps Suppresses Adipogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells via Activation of AMPK.

Obesity is a world-wide health concern with increasing mortality and morbidity rates. Development of novel therapeutic agents for obesity from phytochemicals may lead to the effective prevention and control of obesity and obesity-related complications. 6-acetyl-2,2-dimethylchroman-4-one (1) was isolated from a dietary plant, Artemisia princeps. The antiobesity effect of compound 1 was determined in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) induced to differentiate into adipocytes. Treatment with compound 1 resulted in decreased lipid accumulation and expression of key adipogenic markers, proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, and sterol regulatory element-binding transcription factor 1. It was also shown that compound 1 downregulated the adipogenesis-induced p38 and JNK MAPK activation, while upregulating adipogenesis inhibitory ß-catenin-dependent Wnt10b pathway. Compound 1 was also able to stimulate adenosine monophosphate-activated protein kinase phosphorylation, which was suggested to be the underlying mechanism that resulted in inhibition of adipogenesis in hBM-MSCs. In conclusion, 6-acetyl-2,2-dimethylchroman-4-one was identified as a bioactive constituent of A. princeps that exerts antiobesity properties via suppressing adipocyte formation.

3,5-Dicaffeoyl-epi-quinic acid inhibits the PMA-stimulated activation and expression of MMP-9 but not MMP-2 via downregulation of MAPK pathway.

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, are very important gelatinases that are overexpressed during tumor metastasis. Up to date, several MMP inhibitors have been developed from natural sources as well as organic synthesis. In the present study, the MMP-2 and MMP-9 inhibitory effects of 3,5-dicaffeoyl-epi-quinic acid (DCEQA), a caffeoylquinic acid derivative isolated from Atriplex gmelinii, were investigated in phorbol 12-myristate 13-acetate (PMA)-treated human HT1080 fibrosarcoma cells. Gelatin zymography and immunoblotting showed that DCEQA significantly inhibited the PMA-induced activation and expression of MMP-9 but was not able to show any effect against MMP-2. DCEQA treatment was also shown to upregulate the protein expression of tissue inhibitor of MMP-1 along with decreased MMP-9 protein levels. Moreover, the effect of DCEQA on phosphorylation of mitogen activated protein kinases (MAPKs), analyzed by immunoblotting, indicated the DCEQA inhibited the MMP-9 by downregulation of MAPK pathway. Collectively, current results suggested that DCEQA is a potent MMP-9 inhibitor and can be utilized as lead compound for treatment of pathological complications involving enhanced MMP activity such as cancer metastasis.

Phlorofucofuroeckol A from Edible Brown Alga Ecklonia Cava Enhances Osteoblastogenesis in Bone Marrow-Derived Human Mesenchymal Stem Cells.

The deterioration of bone formation is a leading cause of age-related bone disorders. Lack of bone formation is induced by decreased osteoblastogenesis. In this study, osteoblastogenesis promoting effects of algal phlorotannin, phlorofucofuroeckol A (PFF-A), were evaluated. PFF-A was isolated from brown alga Ecklonia cava. The ability of PFF-A to enhance osteoblast differentiation was observed in murine pre-osteoblast cell line MC3T3-E1 and human bone marrow-derived mesenchymal stem cells (huBM-MSCs). Proliferation and alkaline phosphatase (ALP) activity of osteoblasts during differentiation was assayed following PFF-A treatment along extracellular mineralization. In addition, effect of PFF-A on osteoblast maturation pathways such as Runx2 and Smads was analyzed. Treatment of PFF-A was able to enhance the proliferation of differentiating osteoblasts. Also, ALP activity was observed to be increased. Osteoblasts showed increased extracellular mineralization, observed by Alizarin Red staining, following PFF-A treatment. In addition, expression levels of critical proteins in osteoblastogenesis such as ALP, bone morphogenetic protein-2 (BMP-2), osteocalcin and ß-catenin were stimulated after the introduction of PFF-A. In conclusion, PFF-A was suggested to be a potential natural product with osteoblastogenesis enhancing effects which can be utilized against bone-remodeling imbalances and osteoporosis-related complications.

Protective effect of 3,5­dicaffeoyl­epi­quinic acid against UVB­induced photoaging in human HaCaT keratinocytes.

Derivatives of caffeoylquinic acid (CQA) have been studied and reported as potent bioactive molecules possessing various health benefits including antioxidant and anti­inflammatory activities. In the present study, the protective effect of 3,5­dicaffeoyl­epi­quinic acid (DCEQA) isolated from Atriplex gmelinii on UVB­induced damages was investigated in human HaCaT keratinocytes. The effect of DCEQA against UVB­induced oxidative stress­mediated damages was determined measuring its ability to alleviate UVB­induced elevation of oxidative stress, proinflammatory response and antioxidant enzyme suppression through nuclear factor­like 2 (Nrf2). Treatment with DCEQA hindered the generation of intracellular reactive oxygen species. Increased levels of proinflammatory cytokines TNF­α, COX­2, IL­6 and IL­1ß following UVB exposure were suppressed by the introduction of DCEQA. Additionally, DCEQA upregulated the mRNA and protein expression of antioxidant enzymes superoxide dismutase­1 and heme oxygenase­1 which were inhibited under UVB irradiation. Antioxidant enzyme regulation transcription factor Nrf2 was also upregulated in the presence of DCEQA. These results suggest that DCEQA prevents photoaging via protection of keratinocytes from UVB irradiation by ameliorating the oxidative stress and pro­inflammatory response.

Evaluation of MMP Inhibitors Isolated from Ligustrum japonicum Fructus.

The current study investigated the ability of two secoiridoids, GL-3 (1) and oleonuezhenide (2), isolated from the fruits of Ligustrum japonicum to inhibit MMP-2 and -9 activity in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 human fibrosarcoma cells. Both compounds 1 and 2 were able to exert lowered gelatin digestion activity for MMP-2 and -9 tested by gelatin zymography via suppressing the release of MMPs to culture medium according to ELISA results. Treatment with compounds was also able to suppress the expression of both mRNA and protein levels of MMP-2 and -9. Action mechanism behind the MMP inhibitory effect of the compounds was suggested to be via MAPK pathway indicated by decreased levels of phosphorylated p38, ERK and JNK proteins evaluated employing immunoblotting. Compound 1 was shown to be slightly more active to inhibit MMP-2 and -9, however, compound 2 showed more regular dose-dependency during inhibition. In conclusion, this study suggested that GL-3 and oleonuezhenide were notable natural origin potent MMP inhibitors and could serve as lead compounds for development of anti-invasive MMP inhibitors against tumor metastasis.

3,5-Dicaffeoyl-Epi-Quinic Acid Isolated from Edible Halophyte Atriplex gmelinii Inhibits Adipogenesis via AMPK/MAPK Pathway in 3T3-L1 Adipocytes.

Atriplex gmelinii is an edible halophyte that has been suggested to possess various health benefits. In the present study, 3,5-dicaffeoyl-epi-quinic acid (DEQA) isolated from A. gmelinii was tested for its ability to prevent adipogenesis in 3T3-L1 cells. Also, the molecular mechanisms by which DEQA affects differentiation of 3T3-L1 cells were investigated. The introduction of DEQA to differentiating 3T3-L1 preadipocytes resulted in suppressed adipogenesis and lowered expression of adipogenesis-related factors, PPARγ, C/EBPα, and SREBP-1c. Treatment of 3T3-L1 adipocytes with DEQA notably decreased the levels of phosphorylated p38, ERK, and JNK. In addition, presence of DEQA upregulated the levels of both inactive and phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its substrate, acetyl-CoA carboxylase (ACC). Taken together, current results indicated that DEQA exhibited a significant antiadipogenesis activity by activation of AMPK and downregulation of MAPK signal pathways in 3T3-L1 preadipocytes.

MMP-Inhibitory Effects of Flavonoid Glycosides from Edible Medicinal Halophyte Limonium tetragonum.

Limonium tetragonum has been well-known for its antioxidative properties as a halophyte. This study investigated the antimetastasis effect of solvent-partitioned L. tetragonum extracts (LTEs) and isolated compounds on HT1080 mouse melanoma cell model with a focus on matrix metalloproteinase (MMP) activity and TIMP and MAPK pathways. Upregulation and stimulation of MMPs result in elevated degradation of extracellular matrix which is part of several complications such as metastasis, cirrhosis, and arthritis. The anti-MMP capacity of LTEs was confirmed by their MMP-inhibitory effects, regulation of MMP and TIMP expression, and suppression of MAPK pathway. Among all tested LTEs, 85% aq. MeOH and n-BuOH were found to be most active fractions which later yielded two known flavonoid glycosides, myricetin 3-galactoside and quercetin 3-o-beta-galactopyranoside. Anti-MMP potential of the compounds was confirmed by their ability to regulate MMP expression through inhibited MAPK pathway activation. These results suggested that L. tetragonum might serve as a potential source of bioactive substances with effective anti-MMP properties.