In vitro mitochondrial apoptosis of melanoma cells via immature Poncirus trifoliata fruit extract.

OBJECTIVE: Poncirus trifoliata (P. trifoliata) fruits exert phytotherapeutic effects, depending on their maturity level. However, the mechanism by which these phytotherapeutic effects are exerted remains undefined - especially in cancers. Therefore, in this study, we investigated the effects of the immature fruit extract of P. trifoliata on a B16 melanoma cell line. MATERIALS AND METHODS: The effect of immature P. trifoliata extract on B16 cells was evaluated by MTT assay, cell proliferation, FACScan analysis of cell cycles, confocal imaging analysis, nuclear (Hoechst) staining, apoptosis assay (Annexin V-fluorescein isothiocyanate/propidium iodide staining), and Western blot assay. The capacity of immature P. trifoliata extract to inhibit the invasion and migration of B16 cells was assessed using the scratch-wound assay and Matrigel migration assay. The effect of immature P. trifoliata extract on mitochondrial function was determined via the mitochondrial membrane potential assay, activity, and fraction and cytosol proteins. RESULTS: Treating B16 cells with a methanol extract of immature P. trifoliata (MEPT) significantly inhibited cell viability, migration, and invasiveness in a dose- (p<0.01) and time (p<0.01)- dependent manner. MEPT arrested the cells in the G1 phase of the cell cycle and led to the activation of the PI3K/AKT/p21 pathway. Furthermore, MEPT dose-dependently induced apoptosis in B16 cells by increasing the expression of the pro-apoptotic proteins Bax and Apaf-1, while decreasing the expression of the anti-apoptotic protein, Bcl-2. MEPT treatment also decreased mitochondrial membrane potential. CONCLUSIONS: Immature P. trifoliata extract inhibited the growth of melanoma cells by inducing cell apoptosis through mitochondrial pathways. Therefore, further research into immature P. trifoliata extract as a potential therapeutic compound for melanoma treatment is warranted.

Anti-inflammatory character of Phelligridin D modulates periodontal regeneration in lipopolysaccharide-induced human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Phelligridin D is a hispidin analogue from the mushroom Phellinus baumii that is widely used as a food source in East Asia. This study tested phelligridin D for the anti-inflammatory effect and mechanism in lipopolysaccharide (LPS)-induced human periodontal ligament cells (HPDLCs). The objective of this study was to clarify whether the anti-inflammatory function of phelligridin D affects periodontal regeneration for supporting the HPDLCs of teeth. MATERIAL AND METHODS: Primary HPDLCs were isolated from healthy teeth and then cultured. The anti-inflammatory function, mechanism and differentiation molecules were verified with reactive oxygen species generation and western blot analysis in LPS-induced HPDLCs. RESULTS: HPDLCs showed increased inflammatory molecules (intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and decreased osteogenic proteins (bone morphogenetic protein-7, Osterix and runt-related transcription factor 2) by LPS treatment. Phelligridin D decreased inflammatory molecules and increased osteogenic molecules via downregulation of the extracellular signal-regulated kinase and c-jun N-terminal kinases pathway among the mitogen-activated protein kinase, followed by blocking of nuclear factor kappa-B translocation from cytosol to nucleus. In addition, phelligridin D showed antioxidant properties by reducing reactive oxygen species activity. Finally, the anti-inflammatory and antioxidant function of phelligridin D promoted the periodontal differentiation of HPDLCs. CONCLUSION: These results suggest that phelligridin D supports teeth on the alveolar bone against outside stress, and may be used as an anti-inflammatory compound for the prevention of periodontitis or periodontal regenerative related disease.

Impact of opportunistic salpingectomy on anti-Müllerian hormone in patients undergoing laparoscopic hysterectomy: a multicentre randomised controlled trial.

OBJECTIVE: The aim of the study was to investigate whether opportunistic salpingectomy has any deleterious effects on ovarian reserve and increases surgical risk in patients undergoing laparoscopic hysterectomy. DESIGN: A multicentre, randomised controlled trial. SETTING: Three university hospitals in Korea. POPULATION: Sixty-eight patients undergoing laparoscopic hysterectomy for the treatment of symptomatic benign uterine diseases. METHODS: Patients were randomised to undergo either opportunistic salpingectomy (n = 34) or no salpingectomy (n = 34) during laparoscopic hysterectomy. MAIN OUTCOME MEASUREMENTS: The primary and secondary outcome measures were the change of ovarian reserve, determined by the rate of decline in anti-Müllerian hormone (AMH) level from before surgery to 3 months post-surgery and surgical outcomes, respectively. RESULTS: Baseline demographic and clinical characteristics were similar between the two groups. There was also no difference in operative outcomes such as operative time, operative bleeding, or complications between the two groups. In both groups, postoperative AMH levels were significantly lower than preoperative AMH levels (both, P < 0.01). The decline rate in AMH was 12.5% (interquartile range 0.8-60.9%) in the opportunistic salpingectomy group and 10.8% (interquartile range 6.9-27.4%) in the no salpingectomy group, with no significant difference between both groups (P = 0.898). CONCLUSIONS: Opportunistic salpingectomy at the time of laparoscopic hysterectomy did not have any negative effects on ovarian reserve or increased surgical risk. TWEETABLE ABSTRACT: Opportunistic salpingectomy did not have any negative effects on ovarian reserve or increased surgical risk.

Use of vasopressin vs epinephrine to reduce haemorrhage during myomectomy: a randomized controlled trial.

OBJECTIVE: To compare the effectiveness and safety of vasopressin with epinephrine for reducing blood loss during laparoscopic myomectomy. STUDY DESIGN: Sixty patients undergoing laparoscopic myomectomy were allocated at random to receive either dilute vasopressin or epinephrine into the serosal and/or overlying myometrium, and just around the myoma. The surgeon was blinded to the group allocation. Blood loss, duration of surgery, degree of surgical difficulty, postoperative pain scores and complications were compared. RESULTS: Patient characteristics (e.g. age, body mass index, demographic data), number of myomas, and location and size of the largest myoma were similar between the two study groups. There were no differences in operative blood loss, operative time, subjective surgical difficulty or postoperative pain between the two groups. Transient and non-serious increases in systolic and diastolic blood pressure and heart rate following intra-operative intramyometrial and/or perimyometrial injection of the vasoconstrictive agent only occurred in the epinephrine group, but the difference between the groups was not significant (13% vs 0%, p=0.112). No significant postoperative complications were observed in either group. CONCLUSIONS: Injection of dilute epinephrine before laparoscopic myomectomy was comparable to injection of dilute vasopressin in terms of operative blood loss, operative time, subjective surgical difficulty, postoperative pain and complications.

Eckols reduce dental pulp inflammation through the ERK1/2 pathway independent of COX-2 inhibition.

OBJECTIVES: The aim of this study was to elucidate the role of 6-6 bieckol (EB1) and pholorofucofuroeckol-A (EB5) from brown seaweed marine algae (Eisenia bicyclis) on lipopolysaccharide (LPS)-induced inflammation in human dental pulp cells (HDPCs). METHODS: The cytotoxicity of EB1 and EB5 was examined by MTT assay on LPS-induced human dental pulp cells. Their role on expression of inflammatory, odontogenic, and osteogenic molecules was determined by Western blot analysis. The dentin mineralization was checked by alkaline phosphatase activity. RESULTS: The five compounds from E. bicyclis have different structure with non-cytotoxic in HDPCs. EB1 and EB5 showed anti-inflammatory properties and inhibited phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) and phosphorylated-c-jun N-terminal kinases (p-JNK) without any cytotoxicity. In particular, EB1 inhibited cyclooxygenase-2 (COX-2) and p-ERK1/2 signaling, and EB5 inhibited only p-ERK1/2 signaling but not COX-2. Both compounds inhibited nuclear factor kappa-B (NF-κB) translocation. Furthermore, EB1 and EB5 increased dentinogenic and osteogenic molecules, and dentin mineralized via alkaline phosphatase activity (ALP) in LPS-induced HDPCs. CONCLUSIONS: This study elucidates that EB1 and EB5 have different types of anti-inflammatory property and help in dentin formation. Therefore, these compounds derived from marine algae of E. bicyclis may be used as selective therapeutic strategies for pulpitis and oral diseases.

Anti-inflammatory effect of pachymic acid promotes odontoblastic differentiation via HO-1 in dental pulp cells.

OBJECTIVES: Heme oxygenase-1 (HO-1) is contributed to odontoblast differentiation in human dental pulp cells (HDPCs). In this study, pachymic acid from mushroom Formitopsis niagra is examined to determine whether it affects pulpal inflammation and promotes odontogenesis via HO-1 gene expression. MATERIALS AND METHODS: The HDPCs were given H2O2 for inflammation. The anti-inflammatory character and odontoblast differentiation by pachymic acid were analyzed by Western blotting, alkaline phosphatase activity, and alizarin red S staining. To understand the mechanism of pachymic acid via HO-1 induction, the cells were treated with zinc protoporphyrin IX (ZnPP: HO-1 inhibitor). RESULTS: H2O2 induced pulp inflammation and disturbed odontoblast differentiation. However, the HDPCs treated with pachymic acid affected anti-inflammatory effect and induction of odontoblast differentiation through increasing HO-1 expression. In addition, pachymic acid has potent cytoprotection and mineralization under H2O2 treatment. Furthermore, pachymic acid significantly suppressed nuclear factor-kappa B (NF-κB) translocation into nucleus and induced NE-E2-related factor-2 (Nrf2) translocation into nucleus. Overall, NF-κB and Nrf2 translocation were regulated by the HO-1 pathway. CONCLUSIONS: The pachymic acid showed anti-inflammatory function and odontoblast differentiation via HO-1 pathway. These results suggested that pachymic acid may be applicable for prevention of oral inflammation or to improve dentin mineralization against several stresses.

Phellinus baumii extract influences pathogenesis of Brucella abortus in phagocyte by disrupting the phagocytic and intracellular trafficking pathway.

AIMS: To clarify the effects of Phellinus baumii ethanol extract (PBE) on Brucella abortus pathogenesis in phagocytes focusing on the phagocytic and intracellular trafficking pathway. METHODS AND RESULTS: The effects of PBE on Br. abortus infection in macrophages were evaluated through an adherence and infection assays and an analysis of LAMP-1 staining. The phosphorylation of ERK1/2 and the F-actin polymerization associated with PBE during Br. abortus uptake were detected by immunoblotting and FACS, respectively. The survival of Br. abortus in pure culture was remarkably reduced by PBE in a dose-dependent manner. PBE-treated cells showed significantly decreased uptake, intracellular replication and adherence of Br. abortus. The declines of ERK1/2 phosphorylation and F-actin polymerization following Br. abortus entry were apparent in PBE-treated cells compared with the control. Moreover, the co-localization of Br. abortus-containing phagosomes with LAMP-1 was elevated in PBE-treated cells compared with the control during intracellular trafficking. CONCLUSION: Phellinus baumii ethanol extract may possess the modulatory effect on pathogenesis of Br. abortus through disrupting the phagocytic and intracellular trafficking pathway in phagocyte. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential modulation of PBE to Br. abortus pathogenesis could provide an alternative approach to control of brucellosis, contributing to attenuate Br. abortus manifestation in hosts.

Src kinase-targeted anti-inflammatory activity of davallialactone from Inonotus xeranticus in lipopolysaccharide-activated RAW264.7 cells.

BACKGROUND AND PURPOSE: Mushrooms are popular both as food and as a source of natural compounds of biopharmaceutical interest. Some mushroom-derived compounds such as beta-glucan have been shown to be immunostimulatory; this study explores the anti-inflammatory properties of hispidin analogues derived from the mushroom, Inonotus xeranticus. We sought to identify the molecular mechanism of action of these hispidin analogues by determining their effects on lipopolysaccharide (LPS)-mediated inflammatory responses in a macrophage cell line. EXPERIMENTAL APPROACH: The production of inflammatory mediators was determined by Griess assay, reverse transcription-PCR and ELISA. The inhibitory effect of davalliactone on LPS-induced activation of signalling cascades was assessed by western blotting, immunoprecipitation and direct kinase assay. KEY RESULTS: In activated RAW264.7 cells, davallialactone strongly downregulated LPS-mediated inflammatory responses, including NO production, prostaglandin E2 release, expression of proinflammatory cytokine genes and cell surface expression of co-stimulatory molecules. Davallialactone treatment did not alter cell viability or morphology. Davallialactone was found to exert its anti-inflammatory effects by inhibiting a signalling cascade that activates nuclear factor kappa B via PI3K, Akt and IKK, but not mitogen-activated protein kinases. Treatment with davallialactone affected the phosphorylation of these signalling proteins, but not their level of expression. These inhibitory effects were not due to the interruption of toll-like receptor 4 binding to CD14. In particular, davallialactone strongly inhibited the LPS-induced phosphorylation and kinase activity of Src, implying that Src may be a potential pharmacological target of davallialactone. CONCLUSIONS AND IMPLICATIONS: Our data suggest that davallialactone, a small molecule found in edible mushrooms, has anti-inflammatory activity. Davallialactone can be developed as a pharmaceutically valuable anti-Src kinase agent.

Antioxidant polyphenols from the mycelial culture of the medicinal fungi Inonotus xeranticus and Phellinus linteus.

AIMS: The medicinal fungi Inonotus xeranticus and Phellinus linteus in the family Hymenochaetaceae have been used as traditional medicines for the treatment of various diseases. However, the compound responsible for the antioxidant activity is still unknown. Therefore, this study was conducted to characterize the antioxidant substances present in cultured broths made from these fungi. METHODS AND RESULTS: Antioxidant fractions of the cultured broths obtained from I. xeranticus and P. linteus were analysed using reversed-phase HPLC, which revealed several peaks that exhibited a potent free radical scavenging activity. To identify these antioxidant peaks, an I. xeranticus strain was mass-cultured, and the cultured broth was separated using antioxidant activity-guided fractionation. Four major active substances were purified and identified as hispidin and its dimers, 3,14'-bihispidinyl, hypholomine B, and 1,1-distyrylpyrylethan based on spectroscopic analyses. All compounds exhibited a significant scavenging activity against these radical species in a concentration-dependent manner. CONCLUSIONS: Antioxidant substances found in the cultured broths of the medicinal fungi I. xeranticus and P. linteus were identified as hispidin and its dimers, 3,14'-bihispidinyl, hypholomine B, and 1,1-distyrylpyrylethan. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyphenol antioxidants were isolated from the cultured broth of the medicinal fungi I. xeranticus and P. linteus and identified based on extensive spectroscopic analyses. These compounds exhibited a strong antioxidant activity.

Suillusin, a unique benzofuran from the mushroom Suillus granulatus.

A unique benzofuran named suillusin was isolated from the methanolic extract of the fruiting body of the mushroom Suillus granulatus. Its structure was assigned on the basis of various spectroscopic analyses as a highly substituted novel 1H-cyclopenta[b]benzofuran (1). Suillusin is suggested to be biogenerated from polyporic acid.

Coumarins with monoamine oxidase inhibitory activity and antioxidative coumarino-lignans from Hibiscus syriacus.

A previously undescribed coumarin and a new coumarino-lignan, together with the known compounds scopoletin and cleomiscosins A, C, and D, have been isolated from the root bark of Hibiscus syriacus, and their structures were assigned on the basis of various spectral studies. The coumarin analogue and scopoletin inhibited monoamine oxidase with moderate IC(50) values. The new coumarino-lignan and cleomiscosin C showed lipid peroxidation inhibitory activity comparable to vitamin E.

Complestatin is a noncompetitive peptide antagonist of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors: secure blockade of ischemic neuronal death.

Complestatin, a peptide derived from Streptomyces, was found to protect cultured cortical neurons from excitotoxicity induced by N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate. This neuroprotective behavior of complestatin was attributed to a blockade of Ca2+ ion entry and accumulation, after the activation of NMDA and AMPA/kainate receptors. Complestatin reversibly interfered with NMDA- and AMPA-mediated excitatory synaptic transmission. Complestatin also protected cortical neurons from prolonged deprivation of oxygen and glucose, more effectively than combined antagonists of NMDA and AMPA/kainate receptors. Neurotoxicity, evolving within 1 to 2 days after continuous exposure to combined NMDA and AMPA/kainate antagonists, was not observed in cortical cell cultures that were exposed to complestatin. Finally, complestatin dose dependently prevented neuronal death evolving within the inner nuclear and ganglion cell layers, after transient retinal ischemia. We conclude that complestatin possesses novel pharmacological properties that effectively prevent excitotoxicity under certain pathological conditions.

Ellagic acid rhamnosides from the stem bark of Eucalyptus globulus.

Four ellagic acid rhamnosides were isolated from the stem bark of Eucalyptus globulus. Their structures have been established on the basis of the analysis of their 1H NMR, 13C NMR, HMBC, IR and MS spectral data. The HMBC data of these compounds were most useful for their structure determinations, with these bring determined to be 3-O-methylellagic acid 3'-O-alpha-rhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-3''-O-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-2''-O-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-4''-O-acetylrhamnopyranoside, respectively. Their antioxidant activities were evaluated by measuring the inhibition of lipid peroxidation using rat liver microsomes, with IC50 values of 10.0-14.0 microg/ml.

Neuroprotectins A and B, bicyclohexapeptides protecting chick telencephalic neuronal cells from excitotoxicity. I. Fermentation, isolation, physico-chemical properties and biological activity.

Glutamate, an excitatory amino acid, is known to induce neurotoxicity in central nervous system under abnormal conditions such as ischemia, hypoglycemia, epilepsy, Huntington's chorea, Parkinson's disease and Alzheimer's disease. In our search for neuroprotective agents of microbial origin against excitatory neurotoxins, we have isolated two new bicyclohexapeptides, neuroprotectins A and B, together with a known compound complestatin, from the fermentation broth of Streptomyces sp. Q27107. Neuroprotectins protected primary cultured chick telencephalic neurons from glutamate- and kainate-induced excitotoxicities in a dose-dependant fashion.

Neuroprotectins A and B, bicyclohexapeptides protecting chick telencephalic neurons from excitotoxicity. II. Structure determination.

In the course of our search for neuroprotective agents of microbial origin against kainate-induced neurotoxicity, we have succeeded in the isolation of two new bicyclohexapeptides, neuroprotectins A and B, together with a known compound, complestatin, from the fermentation broth of Streptomyces sp. Q27107. They are closely related in structure to complestatin and possess an oxindolylalanine moiety in place of the tryptophan residue present in complestatin.

Phellinsin A, a novel chitin synthases inhibitor produced by Phellinus sp. PL3.

Phellinsin A, a novel chitin synthases inhibitor was isolated from the cultured broth of fungus PL3, which was identified as Phellinus sp. PL3. Phellinsin A was purified by solvent partition, silica gel, ODS column chromatographies, and preparative HPLC, consecutively. The structure of phellinsin A was assigned as a phenolic compound on the basis of various spectroscopic analyses including UV, IR, Mass, and NMR. Its molecular weight and formula were found to be 358 and C18H14O8, respectively. Phellinsin A selectively inhibited chitin synthase I and II of Saccharomyces cerevisiae with an IC50 value of 76 and 28 microg/ml, respectively, in our cell free assay system. This compound showed antifungal activity against Colletotrichum lagenarium, Pyricularia oryzae, Rhizoctonia solani, Aspergillus fumigatus, and Trichophyton mentagrophytes.

Cyclo(dehydroala-L-Leu), an alpha-glucosidase inhibitor from Penicillium sp. F70614.

A diketopiperazine (1) has been isolated from the culture broth of Penicillium sp. F70614 and its structure has been determined to be cyclo(dehydroala-L-Leu) by various spectroscopic analyses. This compound selectively inhibited yeast alpha-glucosidase and porcine intestinal alpha-glucosidase with IC50 values of 35 and 50 microg/ml, respectively. However, it did not show significant inhibitory effects against almond beta3-glucosidase, Aspergillus alpha-galactosidase, Escherichia coli beta-galactosidase and jack bean alpha-mannosidase.