Methyl jasmonate induces oxidative/nitrosative stress and the accumulation of antioxidant metabolites in Phoenix dactylifera L.

OBJECTIVES: The present study aimed to explore the eliciting effects of increasing concentrations (50, 100, and 200 µM) of methyl jasmonate (MeJA). We cultivated actively proliferating buds of Phoenix dactylifera L. cv. Barhee in a temporary immersion system and we monitored the bioactive compound accumulation after 7 days of culture. METHODS: Total phenolic (TPC) and flavonoid (TFC) contents were determined by high-performance liquid chromatography (HPLC), Fourier-transform infrared (FTIR), and radical scavenging activity using DPPH and ABTS assays. We also explored the activity of phenylpropanoid pathway enzymes, namely phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL) and polyphenol oxidase (PPO). RESULTS: Our results revealed that MeJA treatment induced oxidative stress, and at the same time increased the activity of related defense enzymes in a dose-dependent manner. Exogenous application of MeJA at 200 µM increased ROS (two fold), hydrogen peroxide (3.7 fold), nitric oxide (14 fold), MDA (6.3 fold), superoxide dismutase (5.9 fold), catalase (4.4 fold) and guaiacol peroxidase (3.87 fold). Furthermore, the results demonstrated that 200 µM MeJA treatment enhanced the activities of PAL (3.65 fold), TAL (4.35 fold), PPO (threefold) and increased TPC (twofold) and TFC (1.75 fold) contents in buds cultures higher than the control. HPLC analysis showed that buds cultures exposed to 200 µM MeJA accumulated maximum amount of catechin (11 fold), 4-hydroxybenzoic acid (1.48 fold), caffeic acid (2.5 fold) and p-coumaric acid (1.76 fold) and demonstrate antioxidant capacity with the lowest DPPH (114.5 µg ml-1) and ABTS (90.2 µg ml-1) IC50 values on day 7 of culture as compared to the control. The MeJA in the culture medium directly reduced cell viability in a dose dependent manner up to 35% with the highest concentration. CONCLUSION: The results of this study has revealed, for the first time, that MeJA offers a promising potential for the production of phenolic compound in Phoenix dactylifera L. buds.

Influence of the sulfate content of the exopolysaccharides from Porphyridium sordidum on their elicitor activities on date palm vitroplants.

Given the increasing interest that is being paid to polysaccharides derived from algae as plant natural defense stimulators, the degree of sulfation of exopolysaccharides produced by P. sordidum for inducing defense responses in date palm vitroplants was investigated. Firstly, the culture parameters of P. sordidum were optimized to maximize the amount of sulfate in EPS using a Box-Behnken experimental design and the elicitor effects of two EPS which differ in the sulfation degrees were compared. Results demonstrated that the concentrations of NaCl, NaNO3 and MgSO4 set at 28, 0.54 and 16.31 g/L, respectively yielded the best sulfate contents. To elucidate defense-inducing activities in date palm vitroplants, EPS with the highest sulfate content (EPS1) were prepared for comparison with those obtained under standard conditions (EPS0). A fucoidan extracted from Cystoseira compressa was used as positive control and MgSO4 as negative control. Both EPS and the fucoidan displayed H2O2 accumulation and expression of PR1, SOD, PAL and WRKY genes. Interestingly, EPS1 was significantly more bioactive than EPS0 and the fucoidan suggesting that the elicitor activity is positively correlated with the sulfate groups content of this polysaccharide.

Sakuranetin Induces Melanogenesis in B16BL6 Melanoma Cells through Inhibition of ERK and PI3K/AKT Signaling Pathways.

Sakuranetin (Sak) is considered one of the most important flavanone phytoalexins in regard to antimicrobial activity, and accumulation, in the rice plant. The current study determined that Sak strongly stimulates melanogenesis in B16BL6 melanoma cells in a dose-dependent manner. This flavonoid upregulates the expression of microphthalmia transcription factor (MITF) and reaches its maximum after 24 h. In addition, Sak was found to increase in vitro tyrosinase (Tyr) activity, along with time-dependent upregulation of Tyr, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2). Sakuranetin also decreased the proliferation rate in these cells without directly affecting their viability, as revealed by MTT and trypan blue assays. Further, Sak was shown to inhibit phosphorylation and activation of ERK1/2 from 12 h, without significantly affecting p38 and JNK phosphorylation. Sakuranetin was also found to inhibit the phosphorylation of AKT at threonine 308 and serine 473 and leads to activation of GSK3ß via decreased phosphorylation at serine 9. Taken together, these results demonstrate that Sak stimulates melanogenesis in B16 melanoma cells via inhibition of ERK1/2 and PI3K/AKT signaling pathways, which lead to upregulation of Tyr, TRP1, and TRP2. Copyright © 2016 John Wiley & Sons, Ltd.

Ice Plant (Mesembryanthemum crystallinum) Extract Promotes Lipolysis in Mouse 3T3-L1 Adipocytes Through Extracellular Signal-Regulated Kinase Activation.

The antiobesity effect of ice plant (IP) (Mesembryanthemum crystallinum), a salt-resistant African plant, has recently attracted increased attention. IP is rich in pinitol, which lowers blood sugar, and myo-inositol, which prevents fatty liver disease. Furthermore, IP can potentially prevent or reduce the symptoms of metabolic syndrome. However, the details of the physiological mechanisms and mechanisms of action of IP are unclear. A previous study by our group demonstrated the capability of IP extract to prevent adipogenesis in 3T3-L1 preadipocytes. In this study, we analyzed the physiological function of IP extract on lipolysis in 3T3-L1 cells and the underlying mechanisms of this process. We found that the release of glycerol from cells treated with IP extract increased in an IP dose-dependent manner. IP extract exhibited cytotoxic activity at concentrations above 4 mg/mL. Real-time polymerase chain reaction and western blotting showed that IP extract downregulated peroxisome proliferator-activated receptor (PPAR-)γ, hormone-sensitive lipase (HSL), and adipose triglyceride lipase (ATGL) in a concentration-dependent manner, but did not affect HSL-Ser563, HSL-Ser660, or perilipin phosphorylation. Although the cAMP-dependent protein kinase A (PKA)-specific inhibitor H89 did not affect IP extract-induced lipolysis, the extracellular signal-regulated kinase (ERK1/2) inhibitor U0126 significantly abrogated IP extract-activated glycerol release. Furthermore, IP extract strongly enhanced ERK1/2 phosphorylation at the concentrations used in the study. These results suggest that IP extract augments lipolysis by enhancing ERK phosphorylation.

Isosakuranetin, a 4'-O-methylated flavonoid, stimulates melanogenesis in B16BL6 murine melanoma cells.

AIMS: The beneficial effects of 4'-O-methylated flavonoids on induction of melanogenesis are well established. Here, we report the effect of isosakuranetin (Iso) on melanogenesis in B16BL6 melanoma cells and an analysis of the signaling pathways involved in this activity. METHODS: B16BL6 melanoma cells were treated with several concentrations of Iso and melanin content was measured. Activation and expression of factors involved in melanogenesis were assessed via western blotting. KEY FINDINGS: Iso (15 and 30µmol/L) strongly stimulated melanogenesis in a dose-dependent manner. Iso increased tyrosinase activity and up-regulated tyrosinase (Tyr), tyrosinase related protein 1 (TRP1), and tyrosinase related protein 2 (TRP2) in a time-dependent manner. Iso decreased B16 cell proliferation at a concentration above 45µmol/L, and had no effect on cell viability as revealed by MTT and trypan blue assays. Iso up-regulated expression of microphthalmia transcription factor (MITF), with a maximum effect after 12h. H89, a specific inhibitor of PKA, showed that MITF up-regulation is mediated through PKA/CREB activation. Furthermore, Iso decreased phosphorylation of MITF at Ser73 after 24h and 48h of exposure, activating MITF and leading to up-regulation of Tyr, TRP1, and TRP2. Iso inhibited phosphorylation and activation of ERK1/2 after 12h, while no significant effects on p38 and JNK phosphorylation were observed. Iso inhibited AKT phosphorylation and led to activation of GSK3ß. SIGNIFICANCE: Iso stimulates melanogenesis in B16 melanoma cells via up-regulation of MITF. Furthermore, Iso-induced inhibition of ERK1/2 and PI3K/AKT signaling pathways activate MITF and subsequent expression of Tyr, TRP1, and TRP2.

Hydroxytyrosol stimulates lipolysis via A-kinase and extracellular signal-regulated kinase activation in 3T3-L1 adipocytes.

PURPOSE: The principal function of the adipose tissue is the storage of energy in the form of triglyceride through the process of adipogenesis, as well as the provision of the stored energy through lipolysis. In the present study, we investigated the effect of hydroxytyrosol on lipolysis in 3T3-L1 adipocytes. METHODS: 3T3-L1 adipocytes, used as in vitro model in this study, were treated with several concentration of hydroxytyrosol. Glycerol release was measured to identify the lipolytic rate activation. All factors activation and expression were carried out via Western blotting and qRT-PCR. RESULTS: Our results showed that hydroxytyrosol, over a range of concentrations, attenuated triglyceride accumulation and stimulated glycerol release in fully differentiated adipocytes in a dose- and time-dependent manner. Moreover, hydroxytyrosol had no effect on adipocyte viability. To understand the mechanism underlying hydroxytyrosol-stimulated lipolysis, we used inhibitors of PKA, PKC, PKG, ERK1/2, and nitric oxide production. Pretreatment with a PKA inhibitor (Rp-cAMPs) and an ERK1/2 inhibitor (U0126) significantly attenuated hydroxytyrosol-stimulated lipolysis. In contrast, a PKC inhibitor (Calphostin C), 2 PKG inhibitors (KT 5823 and Rp-cGMPs), and a nitric oxide inhibitor (S-ethyl ITU) had no effect on hydroxytyrosol-stimulated lipolysis. Over the same range of concentrations, hydroxytyrosol downregulated the expression of adipose triglyceride lipase, hormone sensitive lipase (HSL), and adipogenesis-related transcription factors PPARγ and C/EBPα. In addition, hydroxytyrosol increased the phosphorylation rate of HSL at Ser563 and Ser660, as well as perilipin and ERK phosphorylation. CONCLUSION: Hydroxytyrosol induced lipolysis in 3T3-L1 adipocytes via the activation of PKA and ERK1/2 pathway.

Modulation of adipogenesis, lipolysis and glucose consumption in 3T3-L1 adipocytes and C2C12 myotubes by hydroxytyrosol acetate: a comparative study.

Hydroxytyrosol acetate (Hd-Ac) is a polyphenol that is present in the olive fruit and oil at a concentration similar to that of hydroxytyrosol (Hd). The effects of Hd-Ac on adipogenesis, lipolysis, and glucose consumption in 3T3-L1 cells were investigated. Treatment with Hd-Ac at concentrations of 0-75 µmol/L inhibited 3T3-L1 differentiation and lipid accumulation in a dose-dependent manner. At the same concentration range, no effect on cell viability was observed in the MTT assay. Inhibition of adipogenesis was associated with the downregulation of PPARγ, C/EBPα, SREBP-1c, and their downstream target genes (GLUT4, CD36, and FAS) as revealed by qRT-PCR. On the other hand, Hd-Ac dose dependently activated glycerol release in fully differentiated 3T3-L1 adipocytes, indicating lipolysis. This stimulation of lipolysis was mediated via the activation of hormone-sensitive lipase (HSL) by phosphorylation at Ser563 and Ser660, and the phosphorylation of perilipin. Further investigation of the in vitro activities of this polyphenol showed that Hd-Ac has the capability to increase glucose consumption in 3T3-L1 adipocytes and C2C12 myotubes.

Oleuropein and hydroxytyrosol inhibit adipocyte differentiation in 3 T3-L1 cells.

AIMS: Oleuropein and hydroxytyrosol, which are antioxidant molecules found in olive leaves and oil, have been reported to exert several biochemical and pharmacological effects. These polyphenols are able to prevent low-density lipoprotein oxidation and protect cells against several diseases. Here, we studied the effect of these compounds on adipocyte differentiation in 3 T3-L1. MAIN METHODS: To perform this study, 3 T3-L1 preadipocytes viability was analysed via Trypan blue and MTT assays, and triglycerides were stained with Oil Red O. Adipogenesis related genes expression were checked by RT-PCR and qRT-PCR. Also, cells counting and flow cytometry were used to analyse the mitotic cell cycle during the adipogenesis clonal expansion phase. RESULTS: Oleuropein and hydroxytyrosol dose-dependently suppressed intracellular triglyceride accumulation during adipocyte differentiation without effect on cell viability. PPARγ, C/EBPα and SREBP-1c transcription factors and their downstream targets genes (GLUT4, CD36 and FASN) were down-regulated after treatment by oleuropein and hydroxytyrosol. At 200 and 300 µmol/L oleuropein or 100 and 150 µmol/L hydroxytyrosol, the greatest effect on the adipogenesis process was observed during the early stages of differentiation. Flow cytometry revealed both polyphenols to inhibit the division of 3T3-L1 preadipocytes during mitotic clonal expansion and cause cell cycle delay. Furthermore, oleuropein and its derivate hydroxytyrosol decreased the transcriptional activity of SREBP-1c in a stable transfected 3T3-L1 cell line. SIGNIFICANCE: These findings indicate that both compounds are able to prevent 3T3-L1 differentiation by inhibition of the mitotic clonal expansion and downregulation of the adipogenesis related genes.