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Cultivators and livestock farmers are increasingly arranging innovative technical andscientific estimations with the aim to enhance agricultural sustainability,effectiveness, and plant health. Innovative farming technologies incorporatebiology with smart technology (computers and sensor devices) exchanginginformation with one another autonomously in a structured farm managementsystem. This book presents reviews on innovative techniques and methodologiesto complement conventional plant control and breeding attempts toward enhancingcrop yield and production. Reviews covered in this volume include: -Active compounds from pomegranate seeds-Application of Enterococci andtheir bacteriocins for meat biopreservation-Technological advancement in the detection and identification of plantpathogens-Machine learning for precision agriculture-Use of remote sensing technology and geographic information systemsfor agriculture andenvironmental observationThe information presented in this volume will provide helpful updatesfor students, technology experts and professionals in the food security andsustainable agriculture sectors.

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Table of Contents
BENTHAM SCIENCE PUBLISHERS LTD.
End User License Agreement (for non-institutional, personal use)
Usage Rules:
Disclaimer:
Limitation of Liability:
General:
FOREWORD
PREFACE
LIST OF CONTRIBUTORS
Active Compounds from Pomegranate Seed: New Source for Food Applications
Abstract
INTRODUCTION
BOTANICAL ASPECTS OF POMEGRANATE
PS Phytochemical Content
Advanced Analytical Chemistry of Phenolic Compound from PS
Potential Antimicrobial Activity of PS
PS Antioxidant Activity
Biopreservation of Food and Agri-food Products Formulated with PS Extracts
Meat Products
Goat Meat
Pork Meat
Beef and Poultry Meat
Aquatic Meat Product
Pasta
Yoghurts
Animal Feeding
CONCLUSION
FUTURE PERSPECTIVES
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Application of Enterococci and their Bacteriocins for Meat Biopreservation
Abstract
INTRODUCTION
General Characteristics of Enterococci
Bacteriocins Produced by Enterococci
General Characteristics and Inhibitory Spectrum
Classification of Enterocins
Mechanism of Action
Application of Enterococci and their Bacteriocins in Meat
Challenges of their Application in Meat
Advantages of their Application in Meat
Current Applications of Enterococci and their Enterocins in Meat Preservation
Direct Application of Enterocin-producing Enterococci in Meat
Direct Applications of Enterococcal Bacteriocins in Meat
Limits of Enterococci and Enterocins Use in Meat Biopreservation
Limits of Enterococci
Limits of Enterocins
Combinations between Bacteriocinogenic Enterococci and/or Enterococcal Bacteriocins with Other Antimicrobials or Preservation Treatments
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Technological Advancement in the Detection and Identification of Plant Pathogens
Abstract
INTRODUCTION
SEROLOGICAL BASED METHODS
Enzyme Linked Immunosorbent Assay
Immunofluorescence
NUCLEIC ACID-BASED METHODS
DNA-based PCR Methods
RNA-BASED METHODS
NON-INVASIVE DETECTION METHODS
Imaging Methods
Thermography
Fluorescence Imaging
Hyperspectral Imaging
DETECTION OF PLANT PATHOGENS USING BIOSENSORS
conclusions
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Machine Learning for Precision Agriculture: Methods and Applications
Abstract
INTRODUCTION
MACHINE LEARNING AND COMPUTER VISION OVERVIEW
Statistics and Machine Learning
Computer Vision and Deep Learning
APPLICATIONS OF PRECISION AGRICULTURE AND USE-CASES
Crop Yield Prediction and Evaluation
Soil Management and Description
Disease, Weed, Pest Detection and Management
Phenology Recognition
Livestock and Aquaculture Applications
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Use of Remote Sensing Technology and Geographic Information System for Agriculture and Environmental Observation
Abstract
INTRODUCTION
REMOTE SENSING AND GEOGRAPHICAL INFORMATION SYSTEM: GENERALITIES
GIS AND REMOTE SENSING INTEGRATION
SPECTRAL REFLECTANCE OF FOLIAGE
CHARACTERISTICS OF REMOTELY SENSED RECORDS
Spatial Resolution
Spectral Resolution
Radiometric Resolution
Temporal Resolution
IMAGE CLASSIFICATION
Hard Classification
Unsupervised Classification
Supervised Classification
Expert knowledge-Cased Classification
Soft Classification
Object Criented Image Analysis
LAND USE AND LAND COVER
Definitions
Vegetation Observing and Mapping
Ways of Assessing Foliage State
Multi-Temporal Analysis
Macroscopic Visualisation of Vegetation Conversion and Change
APPLICATION OF REMOTE SENSING IN VEGETATION INVENTORY
Field Measuring
Aerial Measuring
Satellites
Synchronized Applications of Modelisation and Remote Sensing in Vegetation Analysis
Empirical Theory
Physical Theory
Dual Mixture: Artificial Neural Networks
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
Emerging Technologies in Agriculture and Food Science
Edited by
Karim Ennouri
University of Sfax,
Sfax,
Tunisia

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FOREWORD

I am delighted to write the foreword of the book titled ‘’Emerging Technologies in Agriculture and Food Science’’ edited by Dr. Karim Ennouri and published by Bentham Science Publishers. I know Dr. Karim Ennouri for more than ten years, and I deeply believe in the research value of interpretive discussion in the biotechnology domain.

Biotechnology is considered as the modern green revolution, offering influential instruments for efficient advanced crop plants, in addition to other organisms through constantly growing technologies aimed at well-organized employment of biological systems to benefit humanity. Applied biotechnology presents an exceptional occasion to propagate scientific perception of a variety of dynamic phenomena and processes related to ecosystems. The exploitation of data sets and the improvement of original data processing algorithms assist in developing aptitudes to process all dimensions of plant observation data and employ these data in making management verdicts and decisions.

I hope and expect that this book will provide an effective learning experience and referenced resource on the topics of agro-biotechnology, bioactive elements, monitoring of vegetation dynamics and modeling, and biotechnological innovations of natural products.

Faiçal Brini Biotechnology and Plant Improvement laboratory Centre of Biotechnology of Sfax-Tunisia

PREFACE

Nowadays, cultivators are increasingly arranging innovative, highly technical and scientific estimations with the aim of enhancing agricultural sustainability, effectiveness, and/or plant health. Innovative farming technologies incorporate biology with smart technology: Computers and devices exchange with one another autonomously in a structured farm management system. Throughout this structure, smart agriculture can be accomplished; cultivators decrease plantation inputs (pesticides and fertilizers) and increase yields via integrated pest management and/or biological control.

Moreover, the intensive use of pesticides creates imbalances in the microbial community, which may be unfavorable for the activity of the beneficial organisms and may also lead to the development of resistant pathogen strains, increasing environmental degradation. Owing to the limitations of chemical control measures, it seems appropriate to seek a more suitable control method. Biological control appears as the most promising strategy, being environmentally safe and cost-effective for controlling several phytopathogens. Therefore, the development of novel agents can be useful in the control of plant diseases. Recently, there has been a growing interest in researching the possible use of functional biomolecules that possess a selective action against these fungi without being toxic to the ecosystem for pest and disease control in agriculture. Natural biomolecules are increasingly becoming an effective and environmentally friendly tool for the control of phytopathogenic agents.

This book resumes present innovative techniques and methodologies to complement usual plant control and breeding attempts toward enhancing crop yield and production and consequently maintaining food security.

Karim Ennouri University of Sfax Sfax Tunisia

LIST OF CONTRIBUTORS

Ahlem Chakchouk-MtibaaLaboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, TunisiaEnnio OttavianiOnAIR Ltd, Genoa, Italy Department of Mathematics, University of Genoa, Genoa, ItalyEmna BouaziziLaboratoire Les Ressources Génétiques de l’Olivier: Caractérisation, Valorisation et Protection Phytosanitaire, Institut de l’Olivier, Université de Sfax, TunisieEnrico BarelliOnAIR Ltd, Genoa, ItalyHajer Ben HlimaUnité de Biotechnologie des Algues, Biological Engineering Department, National School of Engineers of Sfax, University of Sfax, Sfax, TunisiaImen SellemLaboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, TunisiaKarim EnnouriTechnopark of Sfax, Sfax, Tunisia Olive Tree Institute, Sfax, TunisiaKhaoula ElhadefLaboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, TunisiaLotfi MellouliLaboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, TunisiaManel CheffiLaboratoire Les Ressources Génétiques de l’Olivier: Caractérisation, Valorisation et Protection Phytosanitaire, Institut de l’Olivier, Université de Sfax, TunisieMariam FouratiLaboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, TunisiaMohamed Ali TrikiOlive Tree Institute, Sfax, Tunisie Laboratoire Les Ressources Génétiques de l’Olivier: Caractérisation, Valorisation et Protection Phytosanitaire, Institut de l’Olivier, Université de Sfax, Sfax, TunisiaOlfa Ben BraïekLaboratory of Transmissible Diseases and Biologically Active Substances (LR99ES27), Faculty of Pharmacy, University of Monastir, TunisiaPaola CremonesiInstitute of Agricultural Biology and Biotechnology, Italian National Research Council (CNR IBBA), Lodi, ItalyStefano MorandiInstitute of Sciences of Food Production, Italian National Research Council (CNR ISPA), Milan, ItalySlim SmaouiLaboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, TunisiaYaakoub GharbiLaboratoire Les Ressources Génétiques de l’Olivier: Caractérisation, Valorisation et Protection Phytosanitaire, Institut de l’Olivier, Université de Sfax, Tunisie

Active Compounds from Pomegranate Seed: New Source for Food Applications

Slim Smaoui*,1,Mariam Fourati1,Hajer Ben Hlima2,Khaoula Elhadef1,Olfa Ben Braïek3,Ahlem Chakchouk-Mtibaa1,Imen Sellem1,Lotfi Mellouli1
1 Laboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, Tunisia
2 Unité de Biotechnologie des Algues, Biological Engineering Department , National School of Engineers of Sfax, University of Sfax , Sfax, Tunisia
3 Laboratory of Transmissible Diseases and Biologically Active Substances (LR99ES27) , Faculty of Pharmacy, University of Monastir, Tunisia

Abstract

Pomegranate is an essential fruit bearing tree well cultivated in the world. Biological potential and nutritional value were very reputed in both of pomegranate fruit and its by-products, such as seeds. According to the presented information in literature, the use of pomegranate seed as a natural food preservative can be explained by its phytochemicals richness. Based on this phenolics content of pomegranate seed (PS) extracts, the current chapter will talk about its successful use as natural preservative agent in the development of healthier and shelf stable food products. This document speaking of the antioxidant and antimicrobial activities evaluation of PS and its principal active phenolic compounds identified and quantified by advances in the separation sciences and spectrometry, will perform a comprehensive review of the scientific literature. Furthermore, the impact of using PS on the food quality and agri-food products was also evaluated.

Keywords: Advanced analytical chemistry, Antioxidant and Antimicrobial activities, Biopreservation, Food and agri-food products, Pomegranate seed, Phenolic compounds.
*Corresponding author Dr. Slim Smaoui: Laboratoire de Microorganismes et de Biomolécules du Centre de Biotechnology de Sfax, Sfax, Tunisia; E-mails: [email protected]; [email protected]

INTRODUCTION

Because food and agri-food products preservation has become an international problem, the used chemicals are severely regulated [1, 2]. For that reason, in the previous decade, the exploitation of natural products with biological properties has been revived attention given its remarkable phytochemical and reliable

approaches for the preservation of food products. In this line, bioactive compounds of fruits and vegetables, including sources of flavonoids, phenolic acids and pigments, have been examined for enhancing human health and ensuring food security due to their biological potential [3-5].

Given its remarkable phytochemical content, pomegranate (Punica granatum) has captured increased interest [6-9]. Considered usually as waste, pomegranate seeds (PS) have been described as being abundant in polyphenols such as flavonoids (anthocyanins, catechins and other complex flavanoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) [10, 11]. Equally, PP’s Phenolic compounds contain anthocyanins, gallotannins, ellagitannins, gallagylesters, hydroxyl benzoic acids, hydroxyl cinnamic acids and dihydro flavonol [12, 13]. Fig. (1) demonstrates the polyphenols structure found in pomegranate [13]. Phenolics of PS have been reported to display realistically elevated free radical scavenging activities and also powerful antimicrobial activity [14, 15]. Furthermore, their nutraceutical application, PS exposes important properties and techno-functional food applications, (e.g. antioxidant, antimicrobial, colorant and flavoring) [16-18]. PS, evenly, can proceed as notable natural additives for food and agri-food products and their quality development thus affording a well-founded alternative to synthetic antioxidants [18-20].

Fig. (1)) Structure of polyphenols present in pomegranate.

For that reason, the existing chapter presents a cumulative in-depth knowledge on the analytical techniques exploited in categorization of PS phenolic compounds, anti oxidant and antibacterial potentials and successful exploitation of PS in food and agri-food products.

BOTANICAL ASPECTS OF POMEGRANATE

Pomegranate probably originated from Saxifragales belonging to the order Myrtales [21]. It was in 1753 that the genus Punica, having tropical ancestors close to Lythraceae and Sonneratiaceae, was described for the first time by Linnaeus [22]. The evolution of Punica along the xero- and cryophilic lines of development, caused its Arogenesis. Punicaceae is a monogeneric family that includes a single genus Punica of two species, Punica granatum L. and P. protopunica Balf. f., (syn. Socotria protopunica) with the latter endemic to Socotra Island (Yemen) whereas Punica nana, another form of P. granatum is frequently considered as third species of Punica [23].

More than 1000 cultivars of Punica granatum are present [24], native from the Middle East, prolonged throughout the Mediterranean, eastward to China and India, and on to the American Southwest, California and Mexico in the New World. The fruit itself donates rise to three parts: the seeds, about 3% of the fruit weight, and themselves possessing around 20% oil, the juice, near 30% of the fruit mass, and the peels (pericarp) who also contain the inner network of membranes. Other functional parts of the plant comprise the roots, bark, leaves, and flowers [25].

PS Phytochemical Content

From Tunisian pomegranate fruits, PS total phenolic content (TPC) was 326.7 ± 1.4 mg gallic acid equivalent/100 g fresh matters (FM) [26]. These authors confirmed that this value was in accord with preceding finding in Indian PS, which ranged from 230 to 510 mg gallic acid equivalent (GAE)/100 g FM [27].

According to Elfalleh et al. [28], Tunisian PS extracted with methanol had higher TPC values (11.84 GAE mg/g dry weight), flavonoids (6.79 mg rutin equivalents per dry weight (mg RE/g DW/g DW), anthocyanins (40.84 mg of cyanidin-3-glucoside equivalents per g DW (mg CGE/g DW) and hydrolysable tannins (29.57 mg tannic acid equivalent per g of DW (mg TAE/g DW). Gozlekci et al. [29] investigated the total phenolic of four Turkish PS.

PS contained 3.3% of the overall fruit phenolic content and TPC was ranged from 117.0 to 177.4 mg GAE/L. In fact, TPC of PS from cultivars “Asinar,” “Lefan,” “Katirbasi,” and “Cekirdeksiz-IV” was 177.4 mg/L, 125.3 mg/L, 121.2 mg/L, and 117.0 mg/L, respectively [24]. Kalaycıoğlu and Erim [30] calculated the levels of bioactive compounds and antioxidant activities in juice, peel and seed of 3 genotypes of pomegranate cultured in Turkey. Results discovered that the peel extract had about 12.4-fold higher total flavonoid than that of juice extract, and seed extract had 13.4-fold more total flavonoid than that of juice extract. Pande and Akoh [31] reported that TPC in Georgian PS are 365 mg GAE/g FW. In the study of Derakhshan et al. [32], TPC of Iranian PS extracted from three varieties of pomegranate was ranged between 72.4 and 73 mg GAE/g. The TPC of Iranian PS extracts assorted with solvent at a ratio of 1:10 (w/v) were calculated [33]. In this study, solvents were water, methanol, acetone, ethyl acetate and hexane. The authors indicated that the effectiveness of the solvents for extraction of the phenolic compounds was in the following order: methanol > water > acetone > butanol > ethyl acetate > hexane, which was confirmed by Singh et al. [34] results. These authors also described that the water extract of PS (3%, w/w) had the highest TPC, succeed by methanol extract (2.6%) and ethyl acetate extract (2.1%). However, methanol extract (27.93 mg/L seed extract) of PS presented highest phenolics and hexane extract (0.29 mg/L seed extract) showed the minimum of phenolics.

TPC of 50% aqueous acetone like extraction solvent assorted from 1.29 to 2.17 mg GAE/g in four pomegranate China cultivars [35]. These authors demonstrated that the TFC attained 80% aqueous methanol between 0.37 and 0.58 mg catechin equivalents (CAE) in PS. Extracted by 80% methanol, China PS restricted the highest TFC value (0.58 mg CAE/g) among these four cultivars. Total proan-thocyanidins considerably wide-ranging from 68 to182 μg cyanidin equivalents/g seeds in the four seed samples [35].

Thailand PS extracted with ethanol showed a higher yield (11.9%) than that extracted with acetone (10.0%) extract [36].

For TPC, acetone extracts (0.175 mg GAE/g extract) of PS showed higher TPC than ethanol (0.136 mg GAE/g extract) and water (0.084 mg GAE/g extract) extracts. Among three extraction solvents extract, acetone extracts of PS had higher (P < 0.05) TFC [36].

TPC of the fraction involving free and esterified phenolics (soluble) from PS extracted from pomegranate fruits grown in California-USA were resolved by Folin−Ciocalteu’s method.

The TPC value of PS was 3.39 mg GAE/g of sample. According to He et al. [37], the TPC of PS extracted with 70% acetone was 24.28 mg catechin equivalents per gram (dw).

Malaysian ethanol (70%) PS extract was analyzed for its TPC. In fact, TPC in PS was 165 ± 49 mg GAE/L [38].

These variations in TPC might be due to the divergence between the extraction methods; in fact, phenolic contents of PS extracts are projected to robustly depend on extraction circumstances as well as the sort of solvent used [38, 39].

The phenolic compound solubilities are depending on the polarization of solvents. They are extracted greatly in polar solvents (e.g. water and methanol), however, and these compounds are not extracted with nonpolar solvents.

Table 1 summarizes some results found in relation to this topic in recent years.

Advanced Analytical Chemistry of Phenolic Compound from PS

By using high-performance liquid chromatography-diode array detection-electrospray ionization/mass spectrometry (HPLC-DAD-ESI-MS/MS), a total of 47 phenolic compounds were identified in the extracts of PS [40].

Thirteen phenolic acids were recognized by UV spectra, the MS and literature data. According to Ambigaipalan et al. [40], protocatechuic acid, vanillic acid, gallic acid, brevifolin carboxylic acid, p-hydroxybenzoic acid hexoside, cis- and trans-caffeic acid hexoside, derivative of caffeic acid hexoside, vanillic acid hexoside, ferulic acid hexoside, catechin, quercetin hexoside, cis- and trans-dihydrokaempferol-hexoside, ellagic acid, ellagic acid pentoside, ellagic acid deoxyhexose and ellagic acid hexoside, valoneic acid bilactone, digalloyl hexoside, and galloyl-HHDP hexoside were known for the first time in PS. On the other hand, Fischer et al. [48] confirmed that cyanidin– pentoside–hexoside, valoneic acid bilactone, brevifolin carboxylic acid, vanillic acid 4-glucoside and dihydrokaempferol-hexoside are identified for the first time in pomegranate fruits.

Table 1Phenolic contents reported in PS from different regions.SourceSolvent ExtractionAnalyteMethodQuantificationDetectionReferenceThailand- Water (W) - Ethanol (95%) (E) - Acetone (70%) (A)TPC (mg GAE/g)Folin–Ciocalteu0.084 (W) 0.136 (E) 0.175 (A)765 nm[36]Total flavonoid content (TFC) (mg quercetin equivalent per g (mg QE/g)Aluminium chloride colorimetric technique0.027 (W) 0.042 (E) 0.069 (A)415 nmIranEthanol (80%)Total TFC (mg GAE/g)Folin–Ciocalteu72.4-73.13765 nm[32]TFC (mg rutin/g)Aluminium chloride colorimetric technique7.55-38415 nmTFC (mg rutin/g)Aluminium chloride colorimetric technique3.4-22440 nmCanada+BrazilDefatted with HexaneTPC (mg GAE/g)Folin-Ciocalteu0.62-1.39765 nm[40]Monomeric anthocyanin contents (mg/100g)pH-differential method3.7-7.04520 nm and 700 nmChinaAcetone (70%)TPC (mg/g d.w)Folin–Ciocalteu6.17-12.44765 nm[41]EgyptWaterTotal phenolic (GAE mg/g FW)Folin–Ciocalteu8.67-12.37765 nm[42]TFC (RE mg/g FW)Colourimetric method19.84-26.45440 nmMalaysiaEthanol (70%)TPC (mg GAE/L)Folin–Ciocalteu165760 nm[38]IranWater (W) Methanol(M) Acetone (A) Butanol (B) Ethyl acetate(EA) Hexane (H)TPC (mg/L seed extract)Folin–Ciocalteu22.61 (W) 27.93 (M) 3.41 (A) 0.57 (B) 0.37 (EA) 0.29(H)760 nm[33]Tunisia- Untreated seeds (US) - Osmotic Dehydration Treatment (ODT) - Air-Drying Experiment 40-50-60°C (ADE)TPC (mg/100 g)Folin–Ciocalteu326.68 (US) 184.39 (ODT) 134.58-151.56 (ADE)410 nm[26]Total anthocyanin (mg/100 g)pH differential method82.3 (US) 68.43 (ODT) 20.1-40.11 (ADE)510 nm and 700 nmIran- Ethanol - Chloroform - WaterTFC Tannins- Ferric chloride test - Alkaline reagent Test - Lead acetate solution Test - Gelatin Test(- in all tests) (+ in Ethanol)760 nm[43]Tunisia- Water (W) - Methanol (M)Total polyphenol (GAE mg/g dry weight)Folin–Ciocalteu7.94 (W) 11.84 (M)765 nm[28]TFC (RE mg/g dry weight)colourimetric method3.35 (W) 6.79 (M)430 nmTotal anthocyanins (CGE mg/g dry weight)pH differential method19.62 (W) 40.84 (M)510 nm and 700 nmHydrolysable tannin (TAE mg/g dry weight)32.86 (W) 29.57 (M)550nmChina-Acetone (50%) (A) - Methanol (80%) (M) Acetone/ Methanol/WaterTPC (mg GAE/g)Folin–Ciocalteu1.29-2.17 (A) 1.29-1.79 (M)765 nm[35]TFC (mg CAE/g)colourimetric method0.37-0.58 (A) 0.42-0.62 (M)510 nmChinaWater (W) Methanol (M) Ethanol (E) Acetone (A)TPC (mg/100 g DW)Leaching extraction (all solvents)1116-2194.3765 nm[37]ultrasound-assisted extraction (all solvents)312.9-566.4Soxhlet extraction (M) (A)10.92-1965.7Subcritical water extraction (120 – 160 - 180 °C) (W)992.3-2752Thailand- Ethanol (70%) (E) - Water (W)TPC (mg GAE/mg)Folin–Ciocalteu77.93 (E) 51.58 (W)760 nm[44]TFC (mg GAE/mg)Colorimetric method16.66 (E) 10.55 (W)510 nmTurkeyWater (W) Methanol (M) Ethanol (E)TPC (μg gallic acid equivalents/mg extract)Folin–Ciocalteu49.67-56.89 (W) 54.64-63.77 (M) 54.48-56.89 (E)760 nm[45]TFC (μg QE /mg)colourimetric method18.73-24.94 (W) 5.92-15.59 (M) 9.35-16.92 (E)430 nmTotal tannin content (μg tannic acid eqivalent/mg extract)AOAC16.32-29.07 (W) 32.43-56.01 (M) 10.45-25.14 (E)760 nmTurkeyEthanol/ Water 50% (v/v)TPC (mg GAE/L)Folin-Ciocalteu117-177.4765 nm[29]USAWaterTPC (%)Folin–CiocalteuDry seeds 6% Wet seeds 5%760 nm[46]USAHexaneTPC (mg GAE/ 100 g FW)Folin–Ciocalteu84.9-91.1765 nm[31]

The HPLC-DAD-ESI-MS/MS quantification of phenolic acids from PS demonstrated that gallic acid (~1037 μg/100 g dry weight) was the major phenolic acid present in PS, which followed the order of insoluble-bound > esterified > free. A total phenolic acid obtained from PS was 1164 μg/100 g DW. Eight flavonoids, namely (+)-catechin, dihydroxygallocatechin, naringenin hexoside, quercitrin-3-O-rhamnoside, quercetin hexoside, kaempferol-3-O-glucoside, as well as cis- and trans-dihydrokaempferol-hexoside were absolutely recognized in PS. Catechin was identified only in the free phenolic fraction of PS with its characteristic molecular ion [M-H]- at m/z 289. Naringenin hexoside was detected in the free phenolic fraction of PS as its deprotonated molecular ion at m/z 433 [40].

Thirteen hydrolysable tannins were identified in PS [40]. Ellagic acid (m/z 301) and MS2 fragments m/z 185, 229, 257 and 283 were identified in all fractions of PS based on the retention time, UV spectra and MS data of the authentic standard. Ellagic acid-derivative II (m/z 425) and ellagic acid pentoside (m/z 433) were detected as isomers of ellagic acid derivative in PS, respectively. Isomers of hexahydroxydiphenoyl (HHDP) hexoside have already been known in PS [49-51].

Corilagin or galloyl-HHDP hexoside was also recognized for the first time in PS by Ambigaipalan et al. [40].

Ellagic acid was the most important hydrolysable tannin present in PS (~ 220 μg/100 g dry weight), which followed the order of free > insoluble-bound > esterified. Twelve anthocyanins were detected in PS based on UV spectra (520 nm), MS2data in positive mode, as well as data from the literature. Four anthocyanins, namely cyanidin-3-O-pentoside, pelargonidin-3-O-glucoside, cyan-idin-3-O-glucoside and delphinidin-3-O-glucoside were identified in PS [47].

In the study of He et al. [37], phenolic compounds were extracted and isolated from PS residue (PSR). TPC and proanthocyanidin (PC) contents of the extracts were concluded as 2427.90 and 505.63 mg catechin equivalent of 100 g/DW, respectively. Seventeen compounds in PSR extracts were discovered with antioxidant capacity and HPLC–ESI–MS was used to identify them. The main identified phenolics in PSR were flavol-3-ols, phenolic acids, flavonoid glycosides and hydrolysable tannin. Phenolic acid derivatives of PSR are caffeic acid glycoside dimmer and fabric acid derivate. Equally, procyanidin trimer type C, procyanidin dimer type B, procyanidin dimer and (E) catechin are the four flavan-3-ols identified from PSR. These four molecules exhibited a very strong antioxidant activity with the same λmax of 280 nm. Procyanidin trimer type C showed [M-H]− ion of m/z 865 and fragments of m/z 849, 739, 713, 577, 407 and 425, and it was identified as proanthocyanidin trimer. Procyanidin dimer type B had a molecular ion of m/z 577 with fragments of m/z at 451, 425, 407, 289 and 245. The main fragments of procyanidin dimer were detected: m/z 289 and m/z 273 corresponding to catechin and afzelechin units, respectively. (+)-catechin presented at 23.9 min in HPLC profile with MS [M-H]−of m/z 289 and fragments of m/z 245, 205, 125 [37].

Quercetrin 3-O-rhamnoside, kaempferol 3-O-glucoside and kaempferol 3-O- rutinoside were characterized as flavonoid glycosides. These composites offered two maximum absorbances between 190 and 400 nm and could be related to flavonoid. Flavonoid has two maximum absorbances in ultraviolet region: 240–285 nm (band II) and 300–400 nm (band I) [51].

The MS/MS spectra of the reference substances quercetin, kaempferol 3-O-rutinoside-7-rhamnoside, and kaempferol 3-Oglucoside-7-rhamnoside showed a different ion [M - H - 146]- that visibly signify the removal of rhamnosyl moiety from the hydroxyl group of carbon 7 and confirmed approximately the same intensity as the aglycone [51].

Determination of the existence of biologically active O-prenylated umbelliferone derivatives, such as auraptene and umbelliprenin in ethanolic PS extracts, ultra high performance liquid chromatography (UHPLC) methodology with spectrophotometric (UV/Vis) detection, coupled with different extraction procedures, has been perfected by Fiorito et al. [10]. The highest concentration values recorded under short ultrasound-assisted conditions were 1.99 μg/g of dry extract and 6.53 μg/g for auraptene and umbelliprenin, respectively. The parent metabolite umbelliferone was also detected (0.67 μg/g).

In Table 2, some of the most commonly used methods to identify and quantify phenolic compounds (chromatographic conditions; mobile phase and gradient, quantification and detection) from PS extracts are shown.

Table 2Phenolic compounds characterization by analytical techniques reported in PS from different regions.SourceSolvent ExtractionAnalyteMethodQuantificationDetectionReferenceCanada+ BrazilDefatted with HexanePhenolic compounds (Free, esterified and insoluble-bound phenolic compounds)HPLC-DAD-ESI-MS47 phenolic compounds were foundPhenolic acids and Flavonoids (280nm) Anthocyanins (520nm)[40]MalaysiaWaterPhenolic acidsLiquid chromatography–mass spectrometry LC-MS7 compounds were found220 nm[52]ChinaWaterPhenolic acidsLC-MS/MS HPLC–DADGallic acid 0.02-0.03 Ellagic acid 0.02-0.05 Punicalagin A et B (nd)377 nm[41]EgyptWaterPhenolic composition (mg/g)HPLC- Gallic acid 0.42-2.29 - Chlorogenic acid 0.04-0.73 -Caffeic acid 0.05-0.51 - Catechol 0.07-0.62 - Vanillic acid 0.03-0.21 - P-coumaric acid 0.01-0.05220 nm[42]China- Acetone (50%) (A) - Methanol (80%) (M) Acetone/ Methanol/WaterPhenolic acid composition Total soluble conjugated and insoluble bound phenolic acids (µmol/g)HPLC1.22-1.85220 nm[35]

Potential Antimicrobial Activity of PS

The study of Gaber et al. [53] was planned to evaluate the antibacterial activity of the DMSO, ethanol and methanol extracts of Egyptian and Taif (The Kingdom of Saudi Arabia) cultivars of PS extracts against five opportunistic pathogens namely Staphylococcus aureus, Pseudomonas sp. and Bacillus sp. (Gram positive bacteria), Escherichia coli and Aeromonas hydrophila (Gram negative bacteria). The inhibitory activity was found to be dose dependent.

In fact, the maximum antimicrobial activity for the PS extracts was evaluated at 60 mg/mL. Escherichia coli was reported to have considerable vulnerability against most of the extract, followed by Staphylococcus aureus and Pseudomonas sp.

The ethanolic extracts of Iranian PS were arranged and the antibacterial activity of extracts was determined by agar diffusion and micro-broth dilution methods against clinical isolates of P. aeruginosa and S. aureus strains [54]. PS showed inhibitory effects and the minimum inhibitory concentrations (MICs) of PS extracts were 25.0 mg/mL. In addition, the minimum bactericidal concentration ns (MBCs) of PS extracts were found to be 50 mg/mL.

According to Nozohour et al. [54], for all of the studied bacterial isolates, the MICs and MBCs values for PS extract were higher than those of pomegranate peel extract (P < 0.05). From aqueous PS extracts, Tanveer et al. [55] evaluated the antibacterial activity in opposition to chosen pathogenic microorganisms: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus feacalis using disc inhibition zone technique and the results were evaluated with a commercial antibiotic (Amoxicillin). Lowest inhibition zone was distinguished for the seed aqueous extract of pomegranate. In fact, PS extract was three times inferior (21.12mm/7.13m) than amoxicillin [55].

PS Antioxidant Activity

The study of Ambigaipalan et al. [40] shows that phenolics from all PS fractions (free, ester, insoluble bound) are capable of scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. In this line, the total antioxidant aptitude of PS was resoluted by evaluating potential scavenging of the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS•+) and reported as micromoles of Trolox equivalents (TE). PS displayed ABTS radical cation scavenging activity with values of 11-18 μmol of Trolox equivalents/g sample. Under other conditions, the DPPH and hydroxyl radicals scavenging activities of PS were studied via an electron paramagnetic resonance (EPR) spectrometry.