scholarly journals The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent

Antioxidants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 177 ◽  
Author(s):  
Guillermo Cásedas ◽  
Francisco Les ◽  
Carmen Choya-Foces ◽  
Martín Hugo ◽  
Víctor López
Keyword(s):  
Oxidative Stress ◽  
Enzyme Inhibitor ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Monoamine Oxidase A ◽  
Mitochondrial Activity ◽  
Radical Scavenger ◽  
Dependent Manner ◽  
Urolithin A ◽  
Ic50 Values

Urolithin A is a metabolite generated from ellagic acid and ellagitannins by the intestinal microbiota after consumption of fruits such as pomegranates or strawberries. The objective of this study was to determine the cytoprotective capacity of this polyphenol in Neuro-2a cells subjected to oxidative stress, as well as its direct radical scavenging activity and properties as an inhibitor of oxidases. Cells treated with this compound and H2O2 showed a greater response to oxidative stress than cells only treated with H2O2, as mitochondrial activity (MTT assay), redox state (ROS formation, lipid peroxidation), and the activity of antioxidant enzymes (CAT: catalase, SOD: superoxide dismutase, GR: glutathione reductase, GPx: glutathione peroxidase) were significantly ameliorated; additionally, urolithin A enhanced the expression of cytoprotective peroxiredoxins 1 and 3. Urolithin A also acted as a direct radical scavenger, showing values of 13.2 μM Trolox Equivalents for Oxygen Radical Absorbance Capacity (ORAC) and 5.01 µM and 152.66 µM IC50 values for superoxide and 2,2-diphenyss1-picrylhydrazyl (DPPH) radicals, respectively. Finally, inhibition of oxidizing enzymes, such as monoamine oxidase A and tyrosinase, was also detected in a dose-dependent manner. The cytoprotective effects of urolithin A could be attributed to the improvement of the cellular antioxidant battery, but also to its role as a direct radical scavenger and enzyme inhibitor of oxidases.

scholarly journals Persicaria lapathifolia Essential Oil: Chemical Constituents, Antioxidant Activity, and Allelopathic Effect on the Weed Echinochloa colona

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1798
Author(s):  
Ahmed M. Abd-ElGawad ◽  
Giuliano Bonanomi ◽  
Sarah A. Al-Rashed ◽  
Abdelsamed I. Elshamy
Keyword(s):  
Antioxidant Activity ◽  
Ascorbic Acid ◽  
Essential Oil ◽  
Bioactive Compounds ◽  
Chemical Constituents ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Allelopathic Effect ◽  
Dependent Manner ◽  
Ic50 Values

The exploration of new green, ecofriendly bioactive compounds has attracted the attention of researchers and scientists worldwide to avoid the harmful effects of chemically synthesized compounds. Persicaria lapathifolia has been reported to have various bioactive compounds, while its essential oil (EO) has not been determined yet. The current work dealt with the first description of the chemical composition of the EO from the aerial parts of P. lapathifolia, along with studying its free radical scavenging activity and herbicidal effect on the weed Echinochloa colona. Twenty-one volatile compounds were identified via GC–MS analysis. Nonterpenoids were the main components, with a relative concentration of 58.69%, in addition to terpenoids (37.86%) and carotenoid-derived compounds (1.75%). n-dodecanal (22.61%), α-humulene (11.29%), 2,4-dimethylicosane (8.97%), 2E-hexenoic acid (8.04%), γ-nonalactone (3.51%), and limonene (3.09%) were characterized as main compounds. The extracted EO exhibited substantial allelopathic activity against the germination, seedling root, and shoot growth of the weed E. colona in a dose-dependent manner, showing IC50 values of 77.27, 60.84, and 33.80 mg L−1, respectively. In addition, the P. lapathifolia EO showed substantial antioxidant activity compared to ascorbic acid as a standard antioxidant. The EO attained IC50 values of 159.69 and 230.43 mg L−1, for DPPH and ABTS, respectively, while ascorbic acid exhibited IC50 values 47.49 and 56.68 mg L−1, respectively. The present results showed that the emergent leafy stems of aquatic plants such as P. lapathifolia have considerably low content of the EO, which exhibited substantial activities such as antioxidant and allelopathic activities. Further study is recommended to evaluate the effects of various environmental and climatic conditions on the production and composition of the EOs of P. lapathifolia.

scholarly journals Protective Effect of Dealcoholized Persimmonwine on H2O2 - Induced Oxidative Injury in H9c2 Cardiomyocytes

2013 ◽  
Vol 2 (4) ◽  
pp. 61
Author(s):  
Jin Taek Hwang ◽  
Chan Kyu Han ◽  
Sang Yoon Choi ◽  
Sung Soo Kim
Keyword(s):  
Oxidative Stress ◽  
Gallic Acid ◽  
High Performance ◽  
Cell Injury ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Dependent Manner ◽  
Protective Effects ◽  
H9c2 Cardiomyocytes ◽  
Dose Dependent

<p>In this study, we investigated the antioxidant capacity of persimmon wine (PW) and dealcoholized persimmon wine (DPW). Both PW and DPW showed radical scavenging activity in the DPPH (1-diphenyl-2-picrylhydrazyl) assay. We next analyzed the phenolic content and major compounds present in PW using high-performance liquid chromatography (HPLC). Phenolic compounds, including gallic acid, catechin, and epicatechin, were found in PW. Gallic acid was the most abundant phenolic compound (157.5 µg/ml) in PW. In addition, the protective effects of DPW and gallic acid against H<sub>2</sub>O<sub>2</sub>-induced cell injury in H9c2 cardiomyocytes were investigated. Pretreatment with DPW or gallic acid strongly inhibited H<sub>2</sub>O<sub>2</sub>-induced cell death in a dose-dependent manner. These results suggested that PW and its major phenolic component, gallic acid, were effective inhibitors of oxidative stress and oxidative stress-induced cardiomyocyte injury.</p>

scholarly journals Antioxidant and Enzyme Inhibitory Properties of the Polyphenolic-Rich Extract from an Ancient Apple Variety of Central Italy (Mela Rosa dei Monti Sibillini)

Plants ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 9 ◽  
Author(s):  
Joice Guileine Nkuimi Wandjou ◽  
Serena Mevi ◽  
Gianni Sagratini ◽  
Sauro Vittori ◽  
Stefano Dall’Acqua ◽  
...  
Keyword(s):  
High Performance ◽  
Radical Scavenging Activity ◽  
Central Italy ◽  
Radical Scavenging ◽  
Monoamine Oxidase A ◽  
Diode Array ◽  
Chemical Profile ◽  
Apple Variety ◽  
First Choice ◽  
Ic50 Values

This study was undertaken to evaluate the nutraceutical potential of the Mela Rosa dei Monti Sibillini (MR), an ancient apple variety of the Sibillini Mountains, central Italy. The chemical profile of the apple’s polyphenolic-rich extract (MRE) obtained from first- and second-choice samples using the Amberlite® XAD7HP resin was analyzed by High Performance Liquid Chromatography with Diode-Array and Mass spectrometry (HPLC-DAD-MS) and 21 phytochemicals were quali–quantitatively determined. For comparative purposes, the polyphenol-rich extract of Annurca (ANE), a southern Italian variety, was analyzed. The antioxidant capacity of MREs was evaluated by Folin–Ciocalteu, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), and 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. The inhibitory capacity of MREs for the enzymes α-glucosidase, lipase, monoamine oxidase A, tyrosinase, and acetylcholinesterase was also determined. The MREs showed higher polyphenolic and triterpene profiles than the ANE. Their radical scavenging activity was higher than that of ANE and comparable to the reference trolox. The MRE from the second-choice apples displayed higher contents of the 21 phytochemicals investigated. Either MRE from second-choice or first-choice samples showed enzymatic inhibition with IC50 values higher than those of reference inhibitors but worthy of nutraceutical consideration. Taken together, these results show the potential of MRE as a source of bioactive compounds to be used for pharmaceutical, nutraceutical, and cosmeceutical applications has been confirmed.

scholarly journals IN VITRO ANTIOXIDANT POTENTIAL AND HEPATOPROTECTIVE ACTIVITY OF TAXUS WALLICHIANA

2018 ◽  
Vol 11 (8) ◽  
pp. 237
Author(s):  
Muzafar Ahmad Bhat ◽  
Showkat Ahmad Ganie ◽  
Khalid Bashir Dar ◽  
Rohaya Ali ◽  
Rabia Hamid
Keyword(s):  
Ethyl Acetate ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Reducing Power ◽  
Hepatoprotective Activity ◽  
Antioxidant Potential ◽  
Dependent Manner ◽  
Scavenging Activity ◽  
Ethnic Population ◽  
Ic50 Values

Objectives: Taxus wallichiana, the member of family Taxaceae is used for the cure of various ailments by a large ethnic population native to the forests in Kashmir valley. The aim of this study is to evaluate antioxidant potential and hepatoprotective action of T. wallichiana as claimed by the ethnic population segment. Methods: Antioxidant potential of different T. wallichiana extracts was evaluated by estimating the total phenolics, 1,1-diphenyl -2-picrylhydrazyl (DPPH), reducing power, microsomal lipid peroxidation (LPO), and hydroxyl radical scavenging activity through standard protocols. Hepatoprotective activity of T. wallichiana was determined by evaluation of liver marker enzyme (aspartate transaminase, alanine transaminase, and alkaline phosphatase) levels using standard protocols and by histological analysis of rat liver tissues of different animal groups against CCl4 induced liver damage. Results: Phenolic content expressed as gallic acid equivalents (GAE/g) was found maximum in case of ethanolic extract (153.34 mg GAE/g), followed by extracts of methanol (149.24 mg GAE/g), hexane (134 mg GAE/g), ethyl acetate (128.9 mg GAE/g), chloroform (121.37 mg GAE/g), and aqueous (113.00 mg GAE/g). At concentration of 700 μg/ml, DPPH radical scavenging activity of methanol extract was 88.29% with IC50 (212.00 μg/ml), aqueous (82.47%) with IC50 (258.29 μg/ml), and ethyl acetate (79.57%) with IC50 (301.80%μg/ml). The reducing power of the extracts increased in a concentration dependent manner. At concentration of 700 μg/ml, 79%, 68%, and 56% inhibition was observed with extracts of methanol, aqueous, and ethyl acetate on microsomal LPO with IC50 values of 126.09, 168.83, and151.96 μg/ml, respectively. Superoxide radical scavenging activity of T. wallichiana extracts increased in a dose-dependent manner with IC50 values 170.30 μg/ml (methanol), 257.00 μg/ml (aqueous), and 297.55 μg/ml ethyl acetate extract. T. wallichiana extracts exhibited antioxidant effects on calf thymus DNA damage. The results further depict the hepatoprotective action of T. wallichiana extract at doses of 100 and 300 mg/kg and were comparable with that of standard treatment comprising 50 mg/kg-day of Vitamin C, a known hepatoprotective agent. The data were substantiated with histopathological studies of rat liver sections. Conclusion: These results comprehensively depict that T. wallichiana extracts are endowed with the free radical sequestering potential and be employed as potential antioxidant and hepatoprotective arsenal against many oxidative stress linked diseases. 

Curcumin-based antioxidant and glycohydrolase inhibitor compounds: Synthesis and in vitro appraisal of the dual activity against diabetes

2020 ◽  
Vol 16 ◽  
Author(s):  
Sajjad Esmaeili ◽  
Nazanin Ghobadi ◽  
Donya Nazari ◽  
Alireza Pourhossein ◽  
Hassan Rasouli ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Enzyme Inhibitor ◽  
Radical Scavenging Activity ◽  
Curcuma Longa ◽  
Radical Scavenging ◽  
Reducing Power ◽  
Inhibitory Effect ◽  
Curcumin Derivatives ◽  
Reducing Power Assay

Background: Curcumin, as the substantial constituent of the turmeric plant (Curcuma longa), plays a significant role in the prevention of various diseases, including diabetes. It possesses ideal structure features as enzyme inhibitor, including a flexible backbone, hydrophobic nature, and several available hydrogen bond (H-bond) donors and acceptors. Objective: The present study aimed at synthesizing several novel curcumin derivatives and further evaluation of these compounds for possible antioxidant and anti-diabetic properties along with inhibitory effect against two carbohydrate-hydrolyzing enzymes, α-amylase and α-glucosidase, as these enzymes are therapeutic targets for attenuation of postprandial hyperglycemia. Methods: Therefore, curcumin-based pyrido[2,3-d]pyrimidine derivatives were synthesized and identified using an instrumental technique like NMR spectroscopy and then screened for antioxidant and enzyme inhibitory potential. Total antioxidant activity, reducing power assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH• ) radical scavenging activity were done to appraisal the antioxidant potential of these compounds in vitro. Results: Compounds L6-L9 showed higher antioxidant activity while L4, L9, L12 and especially L8 exhibited the best selectivity index (lowest α-amylase/α-glucosidase inhibition ratio). Conclusion: These antioxidant inhibitors may be potential anti-diabetic drugs, not only to reduce glycemic index but also to limit the activity of the major reactive oxygen species (ROS) producing pathways.

scholarly journals Comparison of Phenolic Contents and Scavenging Activities of Miang Extracts Derived from Filamentous and Non-Filamentous Fungi-Based Fermentation Processes

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1144
Author(s):  
Aliyu Dantani Abdullahi ◽  
Pratthana Kodchasee ◽  
Kridsada Unban ◽  
Thanawat Pattananandecha ◽  
Chalermpong Saenjum ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Protective Effects ◽  
Tea Leaves ◽  
Antioxidant Power ◽  
Phenolic Contents ◽  
Ic50 Values ◽  
Ferric Reducing Antioxidant Power ◽  
The Impact

The study investigated the impact of the fermentation process on the phenolic contents and antioxidant and anti-inflammatory activities in extracts of Miang, an ethnic fermented tea product of northern Thailand. The acetone (80%) extraction of Miang samples fermented by a non-filamentous fungi-based process (NFP) and filamentous fungi-based process (FFP) had elevated levels of total polyphenols, total tannins, and condensed tannins compared to young and mature tea leaves. The antioxidant studies also showed better the half-maximal inhibitory concentration (IC50) values for fermented leaves in both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity assays as well as improved ferric reducing antioxidant power (FRAP) compared to young and mature tea leaves. Extracts of NFP and FFP samples at concentrations of 50 and 100 ppm showed better protective effects against hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species (ROS) production in HT-29 colorectal cells without exerting cytotoxicity. Additionally, lipopolysaccharide (LPS)-induced production of nitric oxide (a proinflammatory mediator as well as a reactive nitrogen species) was also inhibited by these fermented Miang extracts with an IC50 values of 17.15 μg/mL (NFP), 20.17 μg/mL (FFP), 33.96 μg/mL (young tea leaves), and 31.33 μg/mL (mature tea leaves). Therefore, both NFP-Miang and FFP-Miang showed the potential to be targeted as natural bioactive functional ingredients with preventive properties against free radical and inflammatory-mediated diseases.

scholarly journals Antioxidant Activity in Rheum emodi Wall (Himalayan Rhubarb)

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2555
Author(s):  
Sang Koo Park ◽  
Yoon Kyung Lee
Keyword(s):  
Antioxidant Activity ◽  
Ethyl Acetate ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Ethyl Acetate Fraction ◽  
Total Phenolic ◽  
Scavenging Activity ◽  
Polyphenol Content ◽  
Abts Assay ◽  
Ic50 Values

Using natural products as antioxidant agents has been beneficial to replace synthetic products. Efforts have been made to profile the antioxidant capacities of natural resources, such as medicinal plants. The polyphenol content of Himalayan rhubarb, Rheum emodi wall, was measured and the antioxidant activity was determined using DPPH and ABTS+ assay, and the oxidative stress was assessed using SOD enzymatic assay. Five different solvent fractions, n-hexane, n-butanol, ethyl acetate, dichloromethane, and water, were used for screening the antioxidant capacity in effort to determine the optimum extraction solvent. The total phenolic contents for R. emodi fractions ranged from 27.76 to 209.21 mg of gallic acid equivalents (GAE)/g of dry weight. DPPH and ABTS+ assay results are presented into IC50 values, ranged from 21.52 to 2448.79 μg/mL and 90.25 to 1718.05 μg/mL, respectively. The ethyl acetate fraction had the highest antioxidant activity among other fractions. Also, n-butanol and water fractions showed significantly lower IC50 values than the positive control in DPPH radical scavenging activity. The IC50 values of SOD assay of fractions ranged from 2.31 to 64.78 μg/mL. A similar result was observed with ethyl acetate fraction showing the highest SOD radical scavenging activity. The study suggests that the ethyl acetate fraction of R. emodi possess the strongest antioxidant activity, thus the most efficient in extracting antioxidant contents. Moreover, a highly significant correlation was shown between total polyphenol content and antioxidant activity screening assays. The compounds related to the antioxidant activity of R. emodi were identified to myricitrin, myricetin 3-galloyl rhamnoside, and myricetin, which have not been reported in studies about R. emodi before.

scholarly journals Inhibitory Effect of Antidesma bunius Fruit Extract on Carbohydrate Digestive Enzymes Activity and Protein Glycation In Vitro

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 32
Author(s):  
Pattamaporn Aksornchu ◽  
Netima Chamnansilpa ◽  
Sirichai Adisakwattana ◽  
Thavaree Thilavech ◽  
Charoonsri Choosak ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Radical Scavenging Activity ◽  
Digestive Enzyme ◽  
Radical Scavenging ◽  
Protein Glycation ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Fruit Extract ◽  
Antioxidant Power ◽  
Ic50 Values

Antidesma bunius (L.) spreng (Mamao) is widely distributed in Northeastern Thailand. Antidesma bunius has been reported to contain anthocyanins, which possess antioxidant and antihypertensive actions. However, the antidiabetic and antiglycation activity of Antidesma bunius fruit extract has not yet been reported. In this study, we investigated the inhibitory activity of anthocyanin-enriched fraction of Antidesma bunius fruit extract (ABE) against pancreatic α-amylase, intestinal α-glucosidase (maltase and sucrase), protein glycation, as well as antioxidant activity. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) chromatogram revealed that ABE contained phytochemical compounds such as cyanidin-3-glucoside, delphinidin-3-glucoside, ellagic acid, and myricetin-3-galactoside. ABE inhibited intestinal maltase and sucrase activity with the IC50 values of 0.76 ± 0.02 mg/mL and 1.33 ± 0.03 mg/mL, respectively. Furthermore, ABE (0.25 mg/mL) reduced the formation of fluorescent AGEs and the level of Nε-carboxymethyllysine (Nε-CML) in fructose and glucose-induced protein glycation during four weeks of incubation. During the glycation process, the protein carbonyl and β-amyloid cross structure were decreased by ABE (0.25 mg/mL). In addition, ABE exhibited antioxidant activity through DPPH radical scavenging activity and Trolox equivalent antioxidant capacity (TEAC) with the IC50 values 15.84 ± 0.06 µg/mL and 166.1 ± 2.40 µg/mL, respectively. Meanwhile, ferric reducing antioxidant power (FRAP) showed an EC50 value of 182.22 ± 0.64 µg/mL. The findings suggest that ABE may be a promising agent for inhibiting carbohydrate digestive enzyme activity, reducing monosaccharide-induced protein glycation, and antioxidant activity.

scholarly journals A Comparative Study of the Antioxidative Effects of Helichrysum italicum and Helichrysum arenarium Infusions

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 380
Author(s):  
Katja Kramberger ◽  
Zala Jenko Pražnikar ◽  
Alenka Baruca Arbeiter ◽  
Ana Petelin ◽  
Dunja Bandelj ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
High Performance ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Morphological Type ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Spectrometry Analysis ◽  
Stress Related Genes

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

scholarly journals Hydroxycinnamic Acid Antioxidants: An Electrochemical Overview

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
José Teixeira ◽  
Alexandra Gaspar ◽  
E. Manuela Garrido ◽  
Jorge Garrido ◽  
Fernanda Borges
Keyword(s):  
Rational Design ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Phenoxyl Radical ◽  
Free Radical Scavenger ◽  
Hydroxycinnamic Acid ◽  
Hydroxycinnamic Acids ◽  
Radical Scavenger ◽  
Redox Potentials ◽  
Structure Property

Hydroxycinnamic acids (such as ferulic, caffeic, sinapic, andp-coumaric acids) are a group of compounds highly abundant in food that may account for about one-third of the phenolic compounds in our diet. Hydroxycinnamic acids have gained an increasing interest in health because they are known to be potent antioxidants. These compounds have been described as chain-breaking antioxidants acting through radical scavenging activity, that is related to their hydrogen or electron donating capacity and to the ability to delocalize/stabilize the resulting phenoxyl radical within their structure. The free radical scavenger ability of antioxidants can be predicted from standard one-electron potentials. Thus, voltammetric methods have often been applied to characterize a diversity of natural and synthetic antioxidants essentially to get an insight into their mechanism and also as an important tool for the rational design of new and potent antioxidants. The structure-property-activity relationships (SPARs) correlations already established for this type of compounds suggest that redox potentials could be considered a good measure of antioxidant activity and an accurate guideline on the drug discovery and development process. Due to its magnitude in the antioxidant field, the electrochemistry of hydroxycinnamic acid-based antioxidants is reviewed highlighting the structure-property-activity relationships (SPARs) obtained so far.

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