Mechanism Of Ethyl Acetate Fraction Research Articles

Anti-inflammatory material basis and mechanism of Artemisia stolonifera based on UPLC-Q-TOF-MS combined with network pharmacology and molecular docking

This study aimed to explore the anti-inflammatory material basis and molecular mechanism of Artemisia stolonifera based on the analysis of the chemical components in different extracted fractions of A. stolonifera and their antioxidant and anti-inflammatory effects in combination with network pharmacology and molecular docking. Thirty-two chemical components were identified from A. stolonifera by ultra-performance liquid chromatography coupled to tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS). Among them, there were 7, 21 and 22 compounds in water, n-butanol and ethyl acetate fractions, respectively. The antio-xidant capacity of different extracted fractions was evaluated by measuring their scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl(DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) free radicals and total antioxidant capacity [ferric reducing antioxidant power(FRAP) assay]. The inflammatory model of RAW264.7 cells was induced by lipopolysaccharide(LPS), and the levels of nitrite oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) in the supernatant and the mRNA expression of related inflammatory factors in cells were used to evaluate the anti-inflammatory effects. The results revealed that ethyl acetate fraction of A. stolonifera was the optimal antioxidant and anti-inflammatory fraction. By network pharmacology, it was found that flavonoids such as rhamnazin, eupatilin, jaceosidin, luteolin and nepetin could act on key targets such as TNF, serine/threonine protein kinase 1(AKT1), tumor protein p53(TP53), caspase-3(CASP3) and epidermal growth factor receptor(EGFR), and regulate the phosphatidylinositol-3-kinase-protein kinase B(PI3K-AKT) and mitogen-activated protein kinase(MAPK) signaling pathways to exert the anti-inflammatory effects. Molecular docking further indicated excellent binding properties between the above core components and core targets. This study preliminarily clarified the anti-inflammatory material basis and mechanism of ethyl acetate fraction of A. stolonifera, providing a basis for the follow-up clinical application of A. stolonifera and drug development.

Cell Cycle Inhibition and Apoptotic Induction of Vernonia amygdalina Del. Leaves Extract on MCF-7 Cell Line.

AIM:The research aimed to determine the cytotoxic activity, cell cycle inhibition, and apoptosis induction of the ethyl acetate extract of the African leaves Vernonia amygdalina Del. on the MCF-7 cancer cells.METHODS:The extraction of Vernonia amygdalina Del. leaves was done using the maceration method whereas the cytotoxic was performed using MTT assay. After that, the cell cycle testing and apoptosis induction were conducted using flow cytometry assay.RESULTS:The IC50 values of n-hexane, ethyl acetate, and ethanol extract of Vernonia amygdalina Del. on the MCF-7 cancer cells were 206.211 ± 0.99, 50,365 ± 0.07, and 967.033 ± 2.68 µg/mL, respectively. The percentage of the cycle cell results in the G0-G1 phase in the cell control with 72.08% decreased in the treatment with ethyl acetate extract 1/2 IC50 with 62.58% and 1/5 IC50 with 44.72%. For the S and G2-M phase, the highest percentage was found in the ethyl acetate extract 1/5 IC50 treatment with 47.27% and 9.50% which were higher than the control cells with 23.26% and 5.90%.CONCLUSION:Based on the results, the Vernonia amygdalina Del. extract provides chemopreventive agent as anti-cancer. Our future study will assess the mechanism of ethyl acetate fraction in inhibiting angiogenesis and metastatic in breast cancer.

CYTOTOXICITY ACTIVITY OF PICRIA FEL-TERRAE LOUR. HERBS AGAINST 4T1 AND MCF-7 BREAST CANCER CELLS

Objective: This study was carried out to investigate the cytotoxic activity toward 4T1 and MCF-7 cell lines of Picria fel-terrae Lour. herb fractions.Methods: P. fel-terrae Lour. herb powder was extracted by maceration method with n-hexane, ethyl acetate, and ethanol solvent. In vitro study was using MTT method toward 4T1 and MCF-7 cell lines.Results: The inhibitory concentration 50% was 234.10 ± 7.85, 50.49 ± 1.07, and 212.53 ± 7.55 μg/mL for 4T1 and 84.62 ± 1.44, 56.79 ± 0.22, and 235.51 ± 4.77 μg/mL for MCF-7 cell lines, respectively.Conclusion: The results reveal that P. fel-terrae Lour. herb fractions provide effective as anticancer. Our further study is to assess the mechanism of ethyl acetate fraction in inhibit angiogenesis and metastatic in breast cancer.

Anti-neuroinflammatory Activity of Elephantopus scaber L. via Activation of Nrf2/HO-1 Signaling and Inhibition of p38 MAPK Pathway in LPS-Induced Microglia BV-2 Cells.

Elephantopus scaber L. (family: Asteraceae) has been traditionally utilized as a folkloric medicine and scientifically shown to exhibit anti-inflammatory activities in various in vivo inflammatory models. Given the lack of study on the effect of E. scaber in neuroinflammation, this study aimed to investigate the anti-neuroinflammatory effect and the underlying mechanisms of ethyl acetate fraction from the leaves of E. scaber (ESEAF) on the release of pro-inflammatory mediators in lipopolysaccharide (LPS)-induced microglia cells (BV-2). Present findings showed that ESEAF markedly attenuated the translocation of NF-κB to nucleus concomitantly with the significant mitigation on the LPS-induced production of NO, iNOS, COX-2, PGE2, IL-1β, and TNF-α. These inflammatory responses were reduced via the inhibition of p38. Besides, ESEAF was shown to possess antioxidant activities evident by the DPPH and SOD scavenging activities. The intracellular catalase enzyme activity was enhanced by ESEAF in the LPS-stimulated BV-2 cells. Furthermore, the formation of ROS induced by LPS in BV-2 cells was reduced upon the exposure to ESEAF. Intriguingly, the reduction of ROS was found in concerted with the activation of Nrf2 and HO-1. It is conceivable that the activation promotes the scavenging power of antioxidant enzymes as well as to ameliorate the inflammatory response in LPS-stimulated BV-2 cells. Finally, the safety profile analysis through oral administration of ESEAF at 2000 mg/kg did not result in any mortalities, adverse effects nor histopathologic abnormalities of organs in mice. Taken altogether, the cumulative findings suggested that ESEAF holds the potential to develop as nutraceutical for the intervention of neuroinflammatory disorders.

Src/Syk/IRAK1-targeted anti-inflammatory action of Torreya nucifera butanol fraction in lipopolysaccharide-activated RAW264.7 cells

Ethnopharmacological relevanceSeed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. Materials and methodsFor this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. ResultsTn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. ConclusionTn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.

Mechanism(s) of action underlying the gastroprotective effect of ethyl acetate fraction obtained from the crude methanolic leaves extract of Muntingia calabura.

BackgroundMuntingia calabura L. (family Muntingiaceae), commonly known as Jamaican cherry or kerukup siam in Malaysia, is used traditionally to treat various ailments. The aim of this study is to elucidate the possible underlying gastroprotective mechanisms of ethyl acetate fraction (EAF) of Muntingia calabura methanolic leaves extract (MEMC).MethodsMEMC and its fractions were subjected to HPLC analysis to identify and quantify the presence of its phyto-constituents. The mechanism of gastroptotection of EAF was further investigated using pylorus ligation-induced gastric lesion rat model (100, 250, and 500 mg/kg). Macroscopic analysis of the stomach, evaluation of gastric content parameters such as volume, pH, free and total acidity, protein estimation, and quantification of mucus were carried out. The participation of nitric oxide (NO) and sulfhydryl (SH) compounds was evaluated and the superoxide dismutase (SOD), gluthathione (GSH), catalase (CAT), malondialdehyde (MDA), prostaglandin E2 (PGE2) and NO level in the ethanol induced stomach tissue homogenate was determined.ResultsHPLC analysis confirmed the presence of quercetin and gallic acid in EAF. In pylorus-ligation model, EAF significantly (p <0.001) prevent gastric lesion formation. Volume of gastric content and total protein content reduced significantly (p < 0.01 and p < 0.05, respectively), while free and total acidity reduced in the doses of 250 and 500 mg/kg (p <0.001 and p <0.05, respectively). EAF also augmented the mucus content significantly (p < 0.001). Pre-treatment with N-nitro-L-arginine methyl ester (L-NAME) or N-ethylmaleimide (NEM) reversed the gastroprotective activity of EAF. EAF treatment markedly ameliorated the SOD, GSH and CAT activity and PGE2 and NO level while attenuating MDA level, relative to the vehicle group.ConclusionsIn conclusion, the underlying gastroprotective mechanisms of EAF could be associated with the antisecretory, participation of mucus, antiperoxidative, improvement of antioxidant status, modulation of NO and SH compounds, stimulation of PGE2 as well as presence of quercetin and gallic acid.

Swietenia macrophylla King induces mitochondrial-mediated apoptosis through p53 upregulation in HCT116 colorectal carcinoma cells

Ethnopharmacological relevanceSwietenia macrophylla King is a traditional herb used to treat various diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect but the potential mechanisms have not been clearly defined. The current study was to further investigate the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-proliferative effect and apoptosis in HCT116 colorectal carcinoma cell. Materials and methodsCell viability was evaluated in HCT116 cells by trypan blue exclusion assay. Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. The apoptotic gene and protein expression were determined by Real-time quantitative PCR (q-PCR) and immunofluorescence staining using flow cytometry, respectively. ResultsSMEAF significantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive oxygen species (ROS) production. Moreover, the significant increase in p53 was accompanied by a decrease murine double minute 2 (MDM2) expression. SMEAF significantly increased the expression of the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation. ConclusionThese results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-mediated pathway via p53 activation.