Biological activities, identification, method development, and validation for analysis of polyphenolic compounds in Nymphaea rubra flowers and leaves by UHPLC-Q-cIM-TOF-MS and UHPLC-TQ-MS.

INTRODUCTION: Nymphaea rubra belongs to the Nymphaea family and is regarded as a vegetable used in traditional medicine to cure several ailments. These species are rich in phenolic acid, flavonoids, and hydrolysable tannin. OBJECTIVE: This study aimed to assess the biological activities of Nymphaea rubra flowers (NRF) and leaves (NRL) by identifying and quantifying their polyphenolic compounds using ultra-performance liquid chromatography coupled to quadrupole cyclic ion mobility time-of-flight mass spectrometry (UHPLC-Q-cIM-TOF-MS) and triple quadrupole mass spectrometry (UHPLC-TQ-MS). METHODOLOGY: NRF and NRL powder was extracted with methanol and fractionated using hexane, ethylacetate, and water. Antioxidant and α-glucosidase, and tyrosinase enzyme inhibitory activities were evaluated. The polyphenolic components of NRF and NRL were identified and quantified using UHPLC-Q-cIM-TOF-MS and UHPLC-TQ-MS. The method was validated using linearity, precision, accuracy, limit of detection (LOD), and lower limit of quantification (LLOQ). RESULTS: Bioactive substances and antioxidants were highest in the ethylacetate fraction of flowers and leaves. Principal component analysis showed how solvent and plant components affect N. rubra's bioactivity and bioactive compound extraction. A total of 67 compounds were identified, and among them 21 significant polyphenols were quantified. Each calibration curve had R2 > 0.998. The LOD and LLOQ varied from 0.007 to 0.09 µg/mL and from 0.01 to 0.1 µg/mL, respectively. NRF contained a significant amount of gallic acid (10.1 mg/g), while NRL contained abundant pentagalloylglucose (2.8 mg/g). CONCLUSION: The developed method is simple, rapid, and selective for the identification and quantification of bioactive molecules. These findings provide a scientific basis for N. rubra's well-documented biological effects.

Tyrosinase inhibitory activity of Sargassum fusiforme and characterisation of bioactive compounds.

INTRODUCTION: Sargassum fusiforme (Harvey) Setchell, also known as Tot (in Korean) and Hijiki (in Japanese), is widely consumed in Korea, Japan, and China due to its health promoting properties. However, the bioactive component behind the biological activity is still unknown. OBJECTIVES: We aimed to optimise the extraction conditions for achieving maximum tyrosinase inhibition activity by using two sophisticated statistical tools, that is, response surface methodology (RSM) and artificial neural network (ANN). Moreover, high-resolution mass spectrometry (HRMS) was used to tentatively identify the components, which are then further studied for molecular docking study using 2Y9X protein. METHODOLOGY: RSM central composite design was used to conduct extraction using microwave equipment, which was then compared to ANN. Electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was used to tentatively identify bioactive components, which were then docked to the 2Y9X protein using AutoDock Vina and MolDock software. RESULTS: Maximum tyrosinase inhibition activity of 79.530% was achieved under optimised conditions of time: 3.27 min, temperature: 128.885°C, ethanol concentration: 42.13%, and microwave intensity: 577.84 W. Furthermore, 48 bioactive compounds were tentatively identified in optimised Sargassum fusiforme (OSF) extract, and among them, seven phenolics, five flavonoids, five lignans, six terpenes, and five sulfolipids and phospholipids were putatively reported for the first time in Sargassum fusiforme. Among 48 bioactive components, trifuhalol-A, diphlorethohydroxycarmalol, glycyrrhizin, and arctigenin exhibited higher binding energies for 2Y9X. CONCLUSION: Taken together, these findings suggest that OSF extract can be used as an effective skin-whitening source on a commercial level and could be used in topical formulations by replacing conventional drugs.

Antioxidant, Tyrosinase, α-Glucosidase, and Elastase Enzyme Inhibition Activities of Optimized Unripe Ajwa Date Pulp (Phoenix dactylifera) Extracts by Response Surface Methodology.

The Ajwa date (Phoenix dactylifera L., Arecaceae family) is a popular edible fruit consumed all over the world. The profiling of the polyphenolic compounds of optimized unripe Ajwa date pulp (URADP) extracts is scarce. The aim of this study was to extract polyphenols from URADP as effectively as possible by using response surface methodology (RSM). A central composite design (CCD) was used to optimize the extraction conditions with respect to ethanol concentration, extraction time, and temperature and to achieve the maximum amount of polyphenolic compounds. High-resolution mass spectrometry was used to identify the URADP's polyphenolic compounds. The DPPH-, ABTS-radical scavenging, α-glucosidase, elastase and tyrosinase enzyme inhibition of optimized extracts of URADP was also evaluated. According to RSM, the highest amounts of TPC (24.25 ± 1.02 mgGAE/g) and TFC (23.98 ± 0.65 mgCAE/g) were obtained at 52% ethanol, 81 min time, and 63 °C. Seventy (70) secondary metabolites, including phenolic, flavonoids, fatty acids, and sugar, were discovered using high-resolution mass spectrometry. In addition, twelve (12) new phytoconstituents were identified for the first time in this plant. Optimized URADP extract showed inhibition of DPPH-radical (IC50 = 87.56 mg/mL), ABTS-radical (IC50 = 172.36 mg/mL), α-glucosidase (IC50 = 221.59 mg/mL), elastase (IC50 = 372.25 mg/mL) and tyrosinase (IC50 = 59.53 mg/mL) enzymes. The results revealed a significant amount of phytoconstituents, making it an excellent contender for the pharmaceutical and food industries.

Activation of Nrf2/HO-1 by Peptide YD1 Attenuates Inflammatory Symptoms through Suppression of TLR4/MYyD88/NF-κB Signaling Cascade.

In this study, we investigate the immunomodulatory effects of a novel antimicrobial peptide, YD1, isolated from Kimchi, in both in vitro and in vivo models. We establish that YD1 exerts its anti-inflammatory effects via up-regulation of the Nrf2 pathway, resulting in the production of HO-1, which suppresses activation of the NF-κB pathway, including the subsequent proinflammatory cytokines IL-1ß, IL-6, and TNF-α. We also found that YD1 robustly suppresses nitric oxide (NO) and prostaglandin E2 (PGE2) production by down-regulating the expression of the upstream genes, iNOS and COX-2, acting as a strong antioxidant. Collectively, YD1 exhibits vigorous anti-inflammatory and antioxidant activity, presenting it as an interesting potential therapeutic agent.

Lablab Purpureus Protects HaCaT Cells from Oxidative Stress-Induced Cell Death through Nrf2-Mediated Heme Oxygenase-1 Expression via the Activation of p38 and ERK1/2.

Ultraviolet B (UV-B) radiation induces the extreme production of either reactive oxygen species (ROS) or inflammatory mediators. The aim of this study was to evaluate the antioxidant activities of 70% ethanolic extract of Lablab purpureus (LPE) and the underlying mechanisms using HaCaT cells exposed to UV-B. High-performance liquid chromatography (HPLC) confirmed the presence of gallic acid, catechin, and epicatechin in LPE. LPE was shown to have a very potent capacity to scavenge free radicals. The results showed that LPE prevented DNA damage and inhibited the generation of ROS in HaCaT cells without causing any toxicity. LPE increased the expression of endogenous antioxidant enzymes such as superoxide dismutase-1 and catalase. Furthermore, LPE treatment facilitates the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), boosting the phase II detoxifying enzyme heme oxygenase-1 (HO-1) leading to the combatting of oxidative stress. However, pretreatment of LPE also caused the phosphorylation of mitogen-activated protein kinases (MAPK kinase) (p38 kinase) and extracellular signal-regulated kinase (ERK), whereas treatment with p38 and ERK inhibitors substantially suppressed LPE-induced Nrf2 and heme oxygenase (HO)-1 expression. These findings suggest that LPE exhibits antioxidant activity via Nrf-2-mediated HO-1 signaling through the activation of p38 and ERK, indicating that LPE can potentially be used as a remedy to combat oxidative stress-induced disorder.

Metabolite Profiling of Manilkara zapota L. Leaves by High-Resolution Mass Spectrometry Coupled with ESI and APCI and In Vitro Antioxidant Activity, α-Glucosidase, and Elastase Inhibition Assays.

High-resolution mass spectrometry equipped with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources was used to enhance the characterization of phytochemicals of ethanol extracts of Manilkara zapota L. leaves (ZLE). Sugar compounds, dicarboxylic acids, compounds of phenolic acids and flavonoids groups, and other phytochemicals were detected from the leaves. Antioxidant activity and inhibition potentiality of ZLE against α-glucosidase enzyme, and elastase enzyme activities were evaluated in in vitro analysis. ZLE significantly inhibited activities of α-glucosidase enzyme at a lower concentration (IC50 2.51 ± 0.15 µg/mL). Glucose uptake in C2C12 cells was significantly enhanced by 42.13 ± 0.15% following the treatment with ZLE at 30 µg/mL. It also exhibited potential antioxidant activities and elastase enzyme inhibition activity (IC50 27.51 ± 1.70 µg/mL). Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) detected more m/z peaks than electrospray ionization mass spectrometry (ESI-MS), and both ionization techniques illustrated the biological activities of the detected compounds more thoroughly compared to single-mode analysis. Our findings suggest that APCI along with ESI is a potential ionization technique for metabolite profiling, and ZLE has the potential in managing diabetes by inhibiting α-glucosidase activity and enhancing glucose uptake.

Inhibition of Migration and Invasion in Melanoma Cells by ß-Escin via the ERK/NF-κB Signaling Pathway.

ß-Escin, a natural triterpene saponin was extracted from Aesculus hippocastanum seeds, which have been widely used to treat inflammation in traditional medicine. In an effort to study the possible anti-tumor effects of ß-escin, we performed wound healing, invasion, and adhesion assays to examine the effects of ß-escin on cell migration, invasion, and angiogenesis. Our results revealed that ß-escin inhibits cell migration as well as motility in B16F10 and SK-MEL5 cells in a dose-dependent manner. RT-PCR and Western blot analysis showed that ß-escin increased TIMP-1, -2 while significantly downregulated phosphorylated extracellular signal-regulated kinase (p-ERK) expression, and suppressing nuclear factor-kappa B (NF-κB) and inhibitor of nuclear factor-kappa B (IκB) expression. Overall, the data from the current study suggest that ß-escin has the potential for inhibiting both metastatic and angiogenic activities, and are the earliest evidence for the involvement of the NF-κB/IκB signaling in ß-escin-induced anti-tumor effects.

Cytotoxic properties of the anthraquinone derivatives isolated from the roots of Rubia philippinensis.

BACKGROUND: Cancer is one of the most frequently occurring diseases and is the second leading cause of death worldwide. In this study, anthraquinone derivatives (Compounds 1-5) were evaluated for their anti-cancer potential against various skin and breast cancer cell lines to assess whether these anthraquinone derivatives may serve as a lead for the augmentation of anti-cancer drug. METHODS: Anthraquinone derivatives, 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone-3-O-(6'-O-acetyl)-α-rhamnosyl(1 → 2)-ß-glucoside (Comp 1), 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone (Comp 2), and alizarin (Comp 3) were isolated from the dichloromethane fraction of the roots of Rubia philippinensis., whereas ethyl acetate fraction yielded xanthopurpurin (Comp 4) and lucidin-ω-methyl ether (Comp 5). Structures of all the isolated compounds were determined by spectral data analysis. All isolated compounds (Comp 1-5) were assessed for cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against four different cancer cell lines, i.e. human melanoma (SK-MEL-5), murine melanoma (B16F10), and human breast adenocarcinoma (MCF7 and MDA-MB-231). RESULTS: Significant activity of the compounds 4 and 5 was observed against the breast cancer cell line MDA-MB-231 with IC50 values of 14.65 ± 1.45 and 13.03 ± 0.33 µM, respectively. Encouragingly, IC50 values of 67.89 ± 1.02 and 79.01 ± 0.03 µM against normal kidney epithelial cells (MDCK) were also obtained for compounds 4 and 5, respectively, which indicated very low toxicity and favorable selectivity indices for compounds 4 and 5 in the range of 1.85 to 3.95 and 2.11 to 6.06 against skin cancer cell lines (SK-MEL-5, and B16F10), and breast cancer cell lines (MCF7 and MDA-MB-231), respectively. CONCLUSION: Our results suggested that the compounds 4 (xanthopurpurin) and 5 (lucidin-ω-methyl ether) showed high selective toxicity towards breast cancer cells at lower concentrations without showing toxicity towards normal cells, thus could be of potential as new lead molecules in cancer treatment.

Antimelanogenic Effect of an Oroxylum indicum Seed Extract by Suppression of MITF Expression through Activation of MAPK Signaling Protein.

In this study, the antimelanogenic effect of an ethyl acetate fraction of Oroxylum indicum Vent. seeds (OISEA) and its underlying mechanisms in melan-a cells were investigated. Antimelanogenesis activity was confirmed by assessing inhibition of tyrosinase activity and melanin content in the cells. Both transcriptional and translational expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein-1 and 2 (TYRP-1 and TYRP-2), were also examined. The results depicted that pretreatment of OISEA significantly inhibits not only tyrosinase activity, but melanin production and intracellular tyrosinase activity. By repressing the expression of tyrosinase, TYRP-1, TYRP-2, and MITF, OISEA interrupted melanin production. Additionally, OISEA interfered with the phosphorylation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase (JNK), with the reversal of OISEA-induced melanogenesis inhibition after treatment with the specific inhibitors SB239063, U0126, and SP600125. Overall, these results suggest that OISEA can stimulate p38, ERK1/2, JNK phosphorylation, and subsequent suppression of melanin, leading to the inhibition of melanogenic enzymes and melanin production, possibly owing to the presence of polyphenolic compounds.

DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases.

This study was performed to investigate the antioxidant activities of Nymphaea nouchali flower (NNF) extract and the underlying mechanism using RAW 264.7 cells. The presence of gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate, caffeic acid, quercetin, and apigenin in the NNF was confirmed by high-performance liquid chromatography (HPLC). The extract had a very potent capacity to scavenge numerous free radicals. NNF extract was also able to prevent DNA damage and quench cellular reactive oxygen species (ROS) generation induced by tert-Butyl hydroperoxide (t-BHP) with no signs of toxicity. The NNF extract was able to augment the expression of both primary and phase II detoxifying enzyme, resulting in combat the oxidative stress. This is accomplished by phosphorylation of mitogen-activated protein kinase (MAP kinase) (p38 kinase and extracellular signal-regulated kinase (ERK)) followed by enhancing the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2). This attenuates cellular ROS generation and confers protection from cell death. Altogether, the results of current study revealed that Nymphaea nouchali flower could be a source of natural phytochemicals that could lead to the development of new therapeutic agents for preventing oxidative stress associated diseases and attenuating disease progression.

Lannea coromandelica (Houtt.) Merr. Induces Heme Oxygenase 1 (HO-1) Expression and Reduces Oxidative Stress via the p38/c-Jun N-Terminal Kinase-Nuclear Factor Erythroid 2-Related Factor 2 (p38/JNK-NRF2)-Mediated Antioxidant Pathway.

The leaves of Lannea coromandelica (Houtt.) Merr. are used in the Garo, Pahan, and Teli tribal communities of Bangladesh as a traditional medicinal plant to treat hepatitis, diabetes, ulcers, heart disease, and dysentery. However, there have been limited phytochemical and biological studies on the bark of L. coromandelica. This study aimed to investigate the antioxidant activities of L. coromandelica bark extract (LCBE) and the underlying mechanism using RAW 264.7 cells. The LCBE was analysed by high-pressure liquid chromatography (HPLC) to detect its key polyphenolic compounds. Various in vitro antioxidant assays were performed using RAW 264.7 cells to assess the antioxidant effects of the LCBE and to understand the underlying molecular mechanism. HPLC revealed the presence of gallic acid, (-)-epigallocatechin-3-gallate, catechin, chlorogenic acid, and caffeic acid in the LCBE. The extract showed a very potent capacity to scavenge numerous free radicals through hydrogen atom transfer and/or electron donation and also quenched cellular reactive oxygen species (ROS) generation without showing any toxicity. The LCBE was found to combat the oxidative stress by enhancing the expression, at both transcriptional and translational levels, of primary antioxidant enzymes as well as phase II detoxifying enzymes, especially heme oxygenase 1, through the upregulation of the nuclear factor erythroid 2-related factor 2 (NRF2)-mediated pathway in RAW 264.7 cells via the phosphorylation of p38 kinase and c-Jun N-terminal kinase (JNK). The LCBE exhibited strong antioxidant activities and mitigated the cellular ROS production. These results provide scientific evidence of its potential as an ideal applicant for a cost-effective, readily available, and natural phytochemical, as well as a strategy for preventing diseases associated with oxidative stress and attenuating disease progress.

Anti-Melanogenic Activities of Heracleum moellendorffii via ERK1/2-Mediated MITF Downregulation.

In this study, the anti-melanogenic effects of Heracleum moellendorffii Hance extract (HmHe) and the mechanisms through which it inhibits melanogenesis in melan-a cells were investigated. Mushroom tyrosinase (TYR) activity and melanin content as well as cellular tyrosinase activity were measured in the cells. mRNA and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), TYR-related protein-1 (TYRP-1) and -2 were also examined. The results demonstrate that treatment with HmHe significantly inhibits mushroom tyrosinase activity. Furthermore, HmHe also markedly inhibits melanin production and intracellular tyrosinase activity. By suppressing the expression of TYR, TYRP-1, TYRP-2, and MITF, HmHe treatment antagonized melanin production in melan-a cells. Additionally, HmHe interfered with the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, with reversal of HmHe-induced melanogenesis inhibition after treatment with specific inhibitor U0126. In summary, HmHe can be said to stimulate ERK1/2 phosphorylation and subsequent degradation of MITF, resulting in suppression of melanogenic enzymes and melanin production, possibly due to the presence of polyphenolic compounds.

Effect of Novel Compound LX519290, a Derivative of l-allo Threonine, on Antioxidant Potential in Vitro and in Vivo.

We investigated the antioxidative activity of LX519290, a derivative of l-allo threonine, in vitro and in vivo. To evaluate the antioxidative activity of LX519290, we performed several in vitro assays (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical-scavenging assays, a ferric reducing antioxidant power assay, cupric-reducing antioxidant capacity, and oxygen radical absorbance capacity assay) and evaluated inhibition against the generation of nitric oxide (NO) and reactive oxygen species (ROS) in murine macrophage (RAW264.7) cells. The results showed that LX519290 possessed very strong radical scavenging activity and reducing power, and inhibited NO and ROS generation in a dose-dependent manner without showing any cytotoxicity. LX519290 treatment also increased the total thiol content and glutathione S-transferases (GST) activities in RAW264.7 cells. Finally, we also determined whether LX519290 affects the mRNA levels of antioxidant enzymes in vitro and in vivo. The expression of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were markedly higher in the sample-treated group than in the oxidative stress group. LX519290 treatment also increased the transcriptional and translational activities of NF-E2-related factor-2 (Nrf-2) with corresponding increases in the transcriptional and translational activities of haeme oxygenase-1 (HO-1). Collectively, the data demonstrated that LX519290 has potent antioxidative activity, decreases NO and ROS generation, increases total thiol content and GST activities in RAW264.7 cells, and increases the transcriptional and translational levels of antioxidant enzymes in vitro and in vivo.

Optimization, Metabolomic Analysis, Antioxidant Potential andDepigmenting Activity of Polyphenolic Compounds fromUnmature Ajwa Date Seeds (Phoenix dactylifera L.) Using Ultrasonic-Assisted Extraction.

This study sought to optimize the ultrasonic-assisted extraction of polyphenolic compounds from unmature Ajwa date seeds (UMS), conduct untargeted metabolite identification and assess antioxidant and depigmenting activities. Response surface methodology (RSM) utilizing the Box-Behnken design (BBD) and artificial neural network (ANN) modeling was applied to optimize extraction conditions, including the ethanol concentration, extraction temperature and time. The determined optimal conditions comprised the ethanol concentration (62.00%), extraction time (29.00 min), and extraction temperature (50 °C). Under these conditions, UMS exhibited total phenolic content (TPC) and total flavonoid content (TFC) values of 77.52 ± 1.55 mgGAE/g and 58.85 ± 1.12 mgCE/g, respectively, with low relative standard deviation (RSD%) and relative standard error (RSE%). High-resolution mass spectrometry analysis unveiled the presence of 104 secondary metabolites in UMS, encompassing phenols, flavonoids, sesquiterpenoids, lignans and fatty acids. Furthermore, UMS demonstrated robust antioxidant activities in various cell-free antioxidant assays, implicating engagement in both hydrogen atom transfer and single electron transfer mechanisms. Additionally, UMS effectively mitigated tert-butyl hydroperoxide (t-BHP)-induced cellular reactive oxygen species (ROS) generation in a concentration-dependent manner. Crucially, UMS showcased the ability to activate mitogen-activated protein kinases (MAPKs) and suppress key proteins including tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (Trp-1 and -2) and microphthalmia-associated transcription factor (MITF), which associated melanin production in MNT-1 cell. In summary, this study not only optimized the extraction process for polyphenolic compounds from UMS but also elucidated its diverse secondary metabolite profile. The observed antioxidant and depigmenting activities underscore the promising applications of UMS in skincare formulations and pharmaceutical developments.

Glycoprotein from Sargassum fusiforme exhibiting anti-inflammatory responses in vitro and in vivo via modulation of TLR4/MyD88 and NF-κB signaling.

This study focuses on the identification and characterization of a glycoprotein from Sargassum fusiforme (Harvey) Setchell (SFGP), as well as investigating its potential anti-inflammatory properties both in vitro and in vivo, along with the underlying mechanism. SDS-PAGE analysis revealed a prominent band with a molecular weight of <10 kDa, consisting of 58.39 % protein and 41.61 % carbohydrates, which was confirmed through glycoprotein staining and Coomassie blue staining. Various analytical techniques, including high-resolution mass spectrometry (HRMS), FTIR, amino acid analysis, and UV-visible spectrometry, provided evidence for the presence of monosaccharides (such as d-glucose and mannose) and 17 amino acids linked by an O-glycopeptide bond. In vitro and in vivo studies were conducted to assess the anti-inflammatory activities of SFGP. The results demonstrated that SFGP effectively attenuated nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in LPS-treated RAW264.7 cells. Moreover, SFGP administration significantly and dose-dependently suppressed TLR4/MyD88 signaling as well as the phosphorylation of MAPKs, IκB, and NF-κB, leading to a reduction in the production of TNF-α, IL-1ß, and IL-6 in LPS-stimulated RAW264.7 cells. Furthermore, the anti-inflammatory efficacy of SFGP was validated in a carrageenan-induced inflammatory mouse model. These findings indicate that SFGP exhibits anti-inflammatory characteristics and has the potential to be utilized as a novel anti-inflammatory agent.

RSM- and ANN-Based Multifrequency Ultrasonic Extraction of Polyphenol-Rich Sargassum horneri Extracts Exerting Antioxidative Activity via the Regulation of MAPK/Nrf2/HO-1 Machinery.

Sargassum horneri (SH) is widely consumed as a healthy seaweed food in the Asia-Pacific region. However, the bioactive components contributing to its biological activity remain unknown. Herein, we optimized multifrequency ultrasonic-assisted extraction conditions to achieve higher antioxidant activity using a response surface methodology and an artificial neural network. High-resolution mass spectrometry (HRMS; negative mode) was used to tentatively identify the secondary metabolites in the optimized SH extract, which were further tested against oxidative stress in RAW264.7 cells. Additionally, the identified compounds were analyzed in silico to determine their binding energies with the Keap1 protein (4L7B). We identified 89 compounds using HRMS, among which 19 metabolites (8 polyphenolics, 2 flavonoids, 2 lignans, 2 terpenes, 2 tannins, 2 sulfolipids, and 1 phospholipid) were putatively reported for the first time in SH. The in vitro results revealed that optimized SH extract inhibited oxidative stress via the Nrf2/MAPKs/HO-1 pathway in a dose-dependent manner. This result was validated by performing in silico simulation, indicating that sargaquinoic acid and glycitein-7-O-glucuronide had the highest binding energies (-9.20 and -9.52 Kcal/mol, respectively) toward Keap1 (4L7B). This study offers a unique approach for the scientific community to identify potential bioactive compounds by optimizing the multivariant extraction processing conditions, which could be used to develop functional and nutraceutical foods.

Development of a rapid screening method utilizing 2D LC for effect-directed analysis in the identification of environmental toxicants.

Effect-directed analysis (EDA) is a crucial tool in environmental toxicology, effectively integrating toxicity testing with chemical analysis. The conventional EDA approach, however, presents challenges such as significant solvent consumption, extended analysis time, labor intensity, and potential contamination risks. In response, we introduce an innovative alternative to the conventional EDA. This method utilizes the MTT bioassay and online two-dimensional liquid chromatography (2D LC) coupled with high-resolution mass spectrometry (HR-MS), significantly reducing the fractionation steps and leveraging the enhanced sensitivity of the bioassay and automated chemical analysis. In the chemical analysis phase, a switching valve interface is employed for comprehensive analysis. We tested the performance of both the conventional and our online 2D LC-based methods using a household product. Both methods identified the same number of toxicants in the sample. Our alternative EDA is 22.5 times faster than the conventional method, fully automated, and substantially reduces solvent consumption. This novel approach offers ease, cost-effectiveness, and represents a paradigm shift in EDA methodologies. By integrating a sensitive bioassay with online 2D LC, it not only enhances efficiency but also addresses the challenges associated with traditional methods, marking a significant advancement in environmental toxicology research.

γ-Mangosteen, an autophagy enhancer, prevents skin-aging via activating KEAP1/NRF2 signaling and downregulating MAPKs/AP-1/NF-κB-mediated MMPs.

BACKGROUND: Mangosteens, a naturally occurring xanthones, found abundantly in mangosteen fruits. The anti-skin aging potential of γ-mangosteen (GM) remains unexplored; therefore, we investigated the UVB-induced anti-skin aging of GM via activation of autophagy. HYPOTHESIS: We hypothesized that GM exerts antioxidant and anti-aging capabilities both in vitro and in vivo through activation of autophagy as well as control of KEAP1/NRF2 signaling and MAPKs/AP-1/NF-κB-mediated MMPs pathways. METHODS: The anti-skin aging effects of GM were studied using HDF cells and a mice model. Various assays, such as DPPH, ABTS, CUPRAC, FRAP, and ROS generation, assessed antioxidant activities. Kits measured antioxidant enzymes, SA-ß-gal staining, collagen, MDA content, si-RNA experiments, and promoter assays. Western blotting evaluated protein levels of c-Jun, c-Fos, p-IκBα/ß, p-NF-κB, MAPK, MMPs, collagenase, elastin, KEAP1, NRF2, HO-1, and autophagy-related proteins. RESULTS: GM exhibited strong antioxidant, collagenase and elastase enzyme inhibition activity surpassing α- and ß-mangosteen. GM competitively inhibited elastase with a Ki value of 29.04 µM. GM orchestrated the KEAP1-NRF2 pathway, enhancing HO-1 expression, and suppressed UVB-induced ROS in HDF cells. NRF2 knockdown compromised GM's antioxidant efficacy, leading to uncontrolled ROS post-UVB. GM bolstered endogenous antioxidants, curbing lipid peroxidation in UVB-exposed HDF cells and BALB/c mice. GM effectively halted UVB-induced cell senescence, and reduced MMP-1/-9, while elevated TIMP-1 levels, augmented COL1A1, ELN, and HAS-2 expression in vitro and in vivo. Additionally, it suppressed UVB-induced MAPKs, AP-1, NF-κB phosphorylation. Pharmacological inhibitors synergistically enhanced GM's anti-skin aging potential. Moreover, GM inhibited UVB-induced mTOR activation, upregulated LC3-II, Atg5, Beclin 1, and reduced p62 in both UVB induced HDF cells and BALB/c mice, while blocking of autophagy successfully halt the GM effects against the UVB-induced increase of cell senescence, degradation of collagen through upregulation of MMP-1, underscoring GM's substantial anti-skin aging impact via autophagy induction in vitro and in vivo. CONCLUSION: Together, GM has potent antioxidant and anti-skin aging ingredients that can be used to formulate skin care products for both the nutraceutical and cosmeceutical industries.

Antioxidant Potential-Rich Betel Leaves (Piper betle L.) Exert Depigmenting Action by Triggering Autophagy and Downregulating MITF/Tyrosinase In Vitro and In Vivo.

Each individual has a unique skin tone based on the types and quantities of melanin pigment, and oxidative stress is a key element in melanogenesis regulation. This research sought to understand the in vitro and in vivo antioxidant and depigmenting properties of betel leaves (Piper betle L.) extract (PBL) and the underlying mechanism. Ethyl acetate fractions of PBL (PBLA) demonstrated excellent phenolic content (342 ± 4.02 mgGAE/g) and strong DPPH, ABTS radicals, and nitric oxide (NO) scavenging activity with an IC50 value of 41.52 ± 1.02 µg/mL, 45.60 ± 0.56 µg/mL, and 51.42 ± 1.25 µg/mL, respectively. Contrarily, ethanolic extract of PBL (PBLE) showed potent mushroom, mice, and human tyrosinase inhibition activity (IC50 = 7.72 ± 0.98 µg/mL, 20.59 ± 0.83 µg/mL and 24.78 ± 0.56 µg/mL, respectively). According to gas chromatography-mass spectrometry, PBL is abundant in caryophyllene, eugenol, O-eugenol, 3-Allyl-6-methoxyphenyl acetate, and chavicol. An in vitro and in vivo investigation showed that PBLE suppressed tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (Trp-1 and Trp-2), and microphthalmia-associated transcription factors (MITF), decreasing the formation of melanin in contrast to the untreated control. PBLE reduced the cyclic adenosine monophosphate (cAMP) response to an element-binding protein (CREB) phosphorylation by preventing the synthesis of cAMP. Additionally, it activates c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38), destroying Tyr and MITF and avoiding melanin production. Higher levels of microtubule-associated protein-light chain 3 (LC3-II), autophagy-related protein 5 (Atg5), Beclin 1, and lower levels of p62 demonstrate that PBLE exhibits significant anti-melanogenic effects via an autophagy-induction mechanism, both in vitro and in vivo. Additionally, PBLE significantly reduced the amount of lipid peroxidation while increasing the activity of several antioxidant enzymes in vivo, such as catalase, glutathione, superoxide dismutase, and thioredoxin. PBLE can therefore be employed in topical formulations as a potent skin-whitening agent.

Identification and quantification of photodegradation products of disposed expanded polystyrene buoy used in aquaculture.

This study investigated the chemicals extracted from an EPS buoy used in aquaculture, which were subsequently collected from a recycling center. It was observed that the chemicals generated upon photodegradation make disposed buoys more toxic. Analysis of the extracted chemicals revealed the presence of 37 compounds, with four compounds quantitatively determined. Further analysis showed that the quantity of compounds dissolved in seawater was significantly higher than the amount remaining on the buoy surface. Based on the assumption that the buoy was exposed to sunlight for a year, it was estimated that 14.44 mg of the four compounds dissolved into the ocean. Given that South Korea used over 7 million EPS buoys, photodegraded EPS buoys are expected to represent a significant source of potentially hazardous chemicals.