The versatility of antioxidant assays in food science and safety—chemistry, applications, strengths, and limitations

Currently, there is a growing interest in screening and quantifying antioxidants from biological samples in the quest for natural and effective antioxidants to combat free radical-related pathological complications. Antioxidant assays play a crucial role in high-throughput and cost-effective assessment of antioxidant capacities of natural products such as medicinal plants and food samples. However, several investigators have expressed concerns about the reliability of existing in vitro assays. Such concerns arise mainly from the poor correlation between in vitro and in vivo results. In addition, in vitro assays have the problem of reproducibility. To date, antioxidant capacities are measured using a panel of assays whereby each assay has its own advantages and limitations. This unparalleled review hotly disputes on in vitro antioxidant assays and elaborates on the chemistry behind each assay with the aim to point out respective principles/concepts. The following critical questions are also addressed: (1) What make antioxidant assays coloured? (2) What is the reason for working at a particular wavelength? (3) What are the advantages and limitations of each assay? and (4) Why is a particular colour observed in antioxidant–oxidant chemical reactions? Furthermore, this review details the chemical mechanism of reactions that occur in each assay together with a colour ribbon to illustrate changes in colour. The review ends with a critical conclusion on existing assays and suggests constructive improvements on how to develop an adequate and universal antioxidant assay

Phenolic compounds analysis of three Euphorbia species by LC-DAD-MSn and their biological properties

Members of the genus Euphorbia have attracted much interest as potential sources of bioactive compounds. In this study, the ethyl acetate, methanolic and aqueous extracts of aerial parts of three Euphorbia species namely Euphorbia hirta L., Euphorbia heterophylla L. and Euphorbia convolvuloides Hochst. ex Benth. were screened for their antioxidant [2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric antioxidant reducing capacity (CUPRAC), metal chelating, total antioxidant capacity (phosphomolybdenum)], and enzymatic properties [alpha-amylase, alpha-glucosidase, tyrosinase, acetyl- (AChE), butyryl-cholinesterase (BChE)]. Total phenolic (TPC), total flavonoid content (TFC) and phytochemical profile were established via LC-DAD-MSn. Results showed that the methanolic and aqueous extracts of the three plants yielded the highest TPC with values ranging from 35.84-141.90 mg gallic acid equivalent/g. E. hirta was the most potent DPPH scavenger and chelator reporting the highest equivalent [DPPH: 195.22 mg Trolox equivalent (TE)/g, chelator: 30.73 mg ethylenediaminetetraacetic acid equivalent (EDTAE)/g], while E. heterophylla demonstrated the most significant Fe3+ and Cu2+ reducing potential (FRAP: 194.28 mg TE/g; CUPRAC: 631.25 mg TE/g) and possessed the highest total antioxidant capacity (3.76 mmol TE/g). The AChE, BChE, alpha-amylase and alpha-glucosidase enzyme inhibitory effects of the three species were relatively the same. However, E. hirta exhibited the highest anti-tyrosinase activity (109.71 mg kojic acid equivalent /g). Euphorbia convolvuloides showed low antioxidant activities and enzymatic inhibitory effects compared to other. LC-DAD-MSn fingerprint of the phenolics of the three species showed the presence of different derivatives. Quantitative data revealed tannins and flavonoids (25 and 20 mg/g respectively) for E. hirta, high amount of tannins 63 and 21 mg/g of flavonoids for E. heterophylla, while high flavonoid contents for E. convolvuloides, 56 mg/g. Euphorbia hirta proved to the most promising among the three tested species and thus can be explored further by isolating and characterizing active bioactive agents that could be used to manage oxidative stress-related diseases and hyperpigmentation problems. (C) 2020 Elsevier B.V. All rights reserved

Chemical characterization, antioxidant and enzyme inhibitory effects of Mitracarpus hirtus extracts

Mitracarpus hirtus (L.) DC. (Family: Rubiaceae) is a tropical annual herb commonly found in America and Mexico. In the present study, the methanol, ethyl acetate, dichloromethane and aqueous extracts of the plant were tested for total phenolic (TPC) and flavonoid content (TFC) and antioxidant activities were evaluated using a battery of assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity, total antioxidant capacity (TAC) (phosphomolybdenum) and metal chelating. Enzyme inhibitory effects were investigated using acetylcholinesterase (AChE), tyrosinase, α-amylase and α-glucosidase as target enzymes. The phytochemical profile was obtained using liquid chromatography-diode array detection-electrospray ionization-mass spectrometry (LC-DAD-ESI-MSn), liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MSn) and nuclear magnetic resonance (NMR) experiments. Results showed that the dichloromethane and ethyl acetate extracts yielded the highest TPC (29.10 ± 0.07 mg gallic acid equivalent/g) and TFC (38.14 ± 0.91 mg rutin equivalent/g), respectively. Aqueous extract showed weak activity against tested enzymes but demonstrated the strongest ABTS scavenging activity (59.39 ± 1.19 mg trolox equivalent/g) and is the strongest Fe3+ reducer (59.42 ± 0.59 mg trolox equivalent/g). Phytochemical analysis revealed the presence of phenolics, pyrrolizidine alkaloids and triterpene acid. This is the first report gathering scientific data on antioxidant, enzyme inhibitory activities and phytochemical composition of M. hirtus and the obtained results can be used as starting point for further investigation on this traditional medicinal herb

Untargeted metabolomic profiling, multivariate analysis and biological evaluation of the true mangrove (Rhizophora mucronata lam.)

Currently, there is a renewed interest towards the development of plant-based pharmacophores. In this work, 16 extracts prepared from the leaves, twigs, roots and fruits of a hydro-halophyte, Rhizophora mucronata Lam. (Family: Rhizophoraceae), were studied for possible antioxidant activity and the phenolic profiles established. Thereafter, enzymatic inhibitory activities (α-amylase, α-glucosidase, tyrosinase, acetyl-(AChE), butyrylcholinesterase (BChE), lipase, and elastase) were assessed. The total phenolic, flavonoid, phenolic acid, tannin, flavanol and triterpenoid content were estimated using standard assays. An untargeted metabolomics-based approach, based on ultra-high-pressure liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS) followed by multivariate statistics, was then used to comprehensively profile and describe the phenolics present. UHPLC-QTOF-MS allowed for putatively annotating 104 phenolic acids, 103 flavonols, 94 flavones, 71 anthocyanins, 66 tyrosols, 29 lignans, 15 alkylphenols and 10 stilbenes in the extracts. Nine strains (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enteritidis, Sarcina lutea, Proteus mirabilis, Bacillus cereus and Candida albicans) were then used to investigate the antimicrobial properties. The methanolic twig extract exhibited significant reducing potential towards Cu (II)/Cu (I) and Fe (III)/Fe (II) (1336.88 ± 15.70 and 710.18 ± 21.04 mg TE/g, respectively) and was the most potent DPPH radical scavenger (807.07 ± 6.83 mg TE/g). Additionally, the methanolic twig extract showed significant inhibition against most targeted enzymes. Anti-microbial results showed that all extracts were active against MRSA. Multivariate analysis demonstrated that the phenolic profile of ethyl acetate extracts and leaves were the two most discriminative parameters in terms of solvents and organs, respectively. The present findings indicated that R. mucronata may be further explored for the management/prevention of oxidative stress, neurodegenerative complications and hyperpigmentation

UHPLC-QTOF-MS phytochemical profiling and in vitro biological properties of Rhamnus petiolaris (Rhamnaceae)

The genus Rhamnus has a great attention as source of bioactive compounds. So, this work aimed to investigate phytochemical profile and biological activity of water and methanolic extracts of different parts of Rhamnus petiolaris Boiss. & Balansa, namely twigs, leaves, mature and unmature fruits. The in vitro antioxidant activity, enzyme inhibitory properties, along with their polyphenol and anthraquinone profiles were determined by untargeted metabolomics. Results showed that methanolic and aqueous unmature fruit extracts were the most effective 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenger (470.96 mg trolox equivalent (TE)/g and 394.96 mg TE/g, respectively). The aqueous unmature fruit extract displayed the most potent cupric and ferric reducing power and showed the highest total phenolic contents (TPC) (137.17 mg gallic acid equivalent (GAE)/g). The methanolic twig extract showed the highest enzymatic inhibitory property against of α-glucosidase, tyrosinase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). On the basis of the correlation coefficients calculated separately for all experimental parameter pairs, flavonols and anthocyanins highly correlated with DPPH, whereas tyrosols correlated with BChE activity. Multivariate statistics following untargeted metabolomics allowed to describe the differences in polyphenols and anthraquinones, as affected by the extraction solvent used. Mature and unmature fruits were substantially comparable and were affected in a similar way by the extraction conditions, while different profiles were recorded for Rhamnus leaves and twigs. These findings indicate that the recovery efficiencies of specific subclasses of compounds (above all when considering flavonoids, phenolic acids and anthraquinones) as a function of the matrix and extraction chosen, significantly affect the phytochemical profile and biological activity of Rhamnus

Detailed Chemical Characterization and Biological Propensities of Malabaila lasiocarpa Extracts: An Endemic Plant to Turkey

This study focused on the biological evaluation and chemical characterization of Malabaila lasiocarpa Boiss. (M. lasiocarpa) (Family: Apiaceae). The phytochemical profile, antioxidant, enzyme inhibitory of the methanolic, aqueous, dichloromethane, hexane extracts were investigated. Based on UHPLC-HRMS analyses, a total of 101 peaks were annotated or identified for the first time in M. lasiocarpa extracts. They include hydroxybenzoic, hydroxycinnamic, acylquinic acids and their glycosides, C- and O-glycosyl and O-diglycosyl flavonoids. In addition, 10 simple mono- and disubstituted coumarins together with 10 furanocoumarins were tentatively annotated. The methanolic extract possessing the highest phenolic (24.36±0.60 mg gallic acid equivalent/g extract) and flavonoid (69.15±0.37 mg rutin equivalent/g extract) content also exhibited the strongest radical scavenging potential against 2,2-diphenyl-1 picrylhydrazyl (21.73±0.42 mg Trolox equivalent/g extract, respectively), and highest reducing capacity (57.81±0.97 and 28.00±0.40 mg Trolox equivalent/g extract, for cupric reducing antioxidant capacity and ferric reducing antioxidant power, respectively). The dichloromethane extract substantially depressed the tyrosinase (73.92±5.37 mg kojic acid equivalent/g extract), α-amylase (0.63±0.01 mmol acarbose equivalent/g extract) and α-glucosidase (0.69±0.02 mmol acarbose equivalent/g extract) enzymes. This study has produced critical scientific data on M. lasiocarpa which are potential contenders for the development of novel phyto-pharmaceuticals