Chemical shifts of common deuterated solvents (TMS is an internal standard).
\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:{caption:"IntechOpen Maintains",originalUrl:"/media/original/113"}},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
\n'}],latestNews:[{slug:"intechopen-supports-asapbio-s-new-initiative-publish-your-reviews-20220729",title:"IntechOpen Supports ASAPbio’s New Initiative Publish Your Reviews"},{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"}]},book:{item:{type:"book",id:"5181",leadTitle:null,fullTitle:"Powder Metallurgy - Fundamentals and Case Studies",title:"Powder Metallurgy",subtitle:"Fundamentals and Case Studies",reviewType:"peer-reviewed",abstract:"The book presents the fundamentals and the role of powder metallurgy in contemporary technologies and the state of the art of classical powder metallurgy technologies and a general description of new variants and special and hybrid technologies used in powder metallurgy. The next part includes over a dozen case studies provided in the following chapters, comprehensively describing authors' accomplishments of numerous teams from different countries across the world in advanced research areas relating to powder metallurgy and to special and hybrid technologies. The detailed information, largely deriving from own and original research and R&D works pursued by the authors, will be beneficial for the readers to develop their knowledge and harmonise specific information concerning these topics and will convince the manufacturers about the advantages of using the powder metallurgy technology in many branches of industry.",isbn:"978-953-51-3054-3",printIsbn:"978-953-51-3053-6",pdfIsbn:"978-953-51-4892-0",doi:"10.5772/61469",price:139,priceEur:155,priceUsd:179,slug:"powder-metallurgy-fundamentals-and-case-studies",numberOfPages:394,isOpenForSubmission:!1,isInWos:null,isInBkci:!1,hash:"139dae624b1f24ec938f60a45e4ee6e1",bookSignature:"Leszek A. Dobrzanski",publishedDate:"March 29th 2017",coverURL:"https://cdn.intechopen.com/books/images_new/5181.jpg",numberOfDownloads:25785,numberOfWosCitations:31,numberOfCrossrefCitations:32,numberOfCrossrefCitationsByBook:6,numberOfDimensionsCitations:47,numberOfDimensionsCitationsByBook:7,hasAltmetrics:1,numberOfTotalCitations:110,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"October 9th 2015",dateEndSecondStepPublish:"February 2nd 2016",dateEndThirdStepPublish:"April 20th 2016",dateEndFourthStepPublish:"June 8th 2016",dateEndFifthStepPublish:"August 31st 2016",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6,7",editedByType:"Edited by",kuFlag:!1,featuredMarkup:null,editors:[{id:"15880",title:"Prof.",name:"Leszek A.",middleName:null,surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański",profilePictureURL:"https://mts.intechopen.com/storage/users/15880/images/system/15880.png",biography:"Prof. Leszek Adam Dobrzański, DSc, Ph.D., MSc, Eng, Hon Prof, M Dr HC is a full professor and the director of the Science Centre ASKLEPIOS of the Medical and Dental Engineering Centre for Research, Design and Production ASKLEPIOS Ltd., Gliwice, Poland. He is president of World Academy WAMME, vice president of the Academy of Engineers in Poland, and a foreign fellow of engineering academies in Ukraine and Slovakia. He is also editor in chief of JAMME and AMSE journals. He was awarded the title of Professor in 1995 by the President of the Republic of Poland and granted abroad a title of Honorary Professor in Lviv, Ukraine. He was also awarded the title of Doctor Honoris Causa in Ruse, Bulgaria; Khmelnytskyi, Ukraine; and Miskolc, Hungary. He is also a recipient of the Commander\\'s Crosses of Rebirth of Poland by the President of Poland, Merite de L\\'Innvention by King of Belgium, and many other awards.\n\nFrom 1971 to 2017, Dr. Dobrzański was a full professor, director, dean of faculty, and vice-rector at Silesian University of Technology, Gliwice, Poland. He was also vice-rector of the Academy in Bielsko-Biała, Poland, and vice-director of the Institute in Kraków, Poland. He was a visiting professor at more than 100 universities worldwide and an invited lecturer at more than 100 global scientific conferences. \n\nDr. Dobrzański has supervised sixty-two Ph.D. thesis and promoted around 1000 MSc and BSc theses. He is the author or co-author of sixty books and fifty book chapters, sixty patents, and around 3000 publications. He is a scientific editor for many international scientific journals and conferences. He has been cited ca. 15,500 times in scientific journals.",institutionString:"Medical and Dental Engineering Centre for Research, Design and Production ASKLEPIOS in Gliwice",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"12",totalChapterViews:"0",totalEditedBooks:"3",institution:{name:"Silesian University of Technology",institutionURL:null,country:{name:"Poland"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"944",title:"Metallurgy",slug:"metals-and-nonmetals-metallurgy"}],chapters:[{id:"52404",title:"Goals and Contemporary Position of Powder Metallurgy in Products Manufacturing",doi:"10.5772/65378",slug:"goals-and-contemporary-position-of-powder-metallurgy-in-products-manufacturing",totalDownloads:1767,totalCrossrefCites:0,totalDimensionsCites:1,hasAltmetrics:0,abstract:"This chapter is an introduction to the book on powder metallurgy (PM). It presents the basis of the selection of powder metallurgy technologies for manufacturing of products, including such applied in medicine and dentistry, and the state of the art concerning the general characteristic of powder metallurgy. The materials and products manufactured with the classical powder metallurgy methods are generally described. The last section presents the general contents of the book based on the above general information.",signatures:"Leszek A. Dobrzański",downloadPdfUrl:"/chapter/pdf-download/52404",previewPdfUrl:"/chapter/pdf-preview/52404",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"53359",title:"Fabrication Technologies of the Sintered Materials Including Materials for Medical and Dental Application",doi:"10.5772/65376",slug:"fabrication-technologies-of-the-sintered-materials-including-materials-for-medical-and-dental-applic",totalDownloads:1713,totalCrossrefCites:10,totalDimensionsCites:11,hasAltmetrics:0,abstract:"This chapter of the book presents the basis of classical powder metallurgy technologies and discusses powder fabrication, preparation, preliminary moulding, sintering and finish treatment operations. A general description of the materials and products manufactured with the classical powder metallurgy methods is presented. New variants are characterised along with special and hybrid technologies finding their applications in powder metallurgy. Special attention was drawn to microporous titanium and to TiAl6V4 alloy fabricated using hybrid rapid manufacturing technologies with selective laser sintering/selective laser melting (SLS/SLM) used for innovative implant scaffolds in medicine and regenerative dentistry. Laser deposition, thermal spraying and detonation spraying of powders are also discussed as special methods in which powders of metals and other materials are used as raw materials.",signatures:"Leszek A. Dobrzański, Anna D. Dobrzańska-Danikiewicz, Anna\nAchtelik-Franczak, Lech B. Dobrzański, Eugeniusz Hajduczek and\nGrzegorz Matula",downloadPdfUrl:"/chapter/pdf-download/53359",previewPdfUrl:"/chapter/pdf-preview/53359",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"53632",title:"Sintering Prealloyed Powders Fe-Ni-Cu-Mo Modified by Boron Base on Thermodynamic Investigations",doi:"10.5772/66875",slug:"sintering-prealloyed-powders-fe-ni-cu-mo-modified-by-boron-base-on-thermodynamic-investigations",totalDownloads:1704,totalCrossrefCites:0,totalDimensionsCites:1,hasAltmetrics:0,abstract:"One of the methods to reduce porosity and increase mechanical properties of Fe‐Ni‐Cu‐Mo powder type is applying activated sintering with the boron powder. In the experiments, a diffusion bonded prealloyed powder type Distaloy SA (Fe‐1.75%, Ni‐1.5%, Cu‐0.5%, Mo) was used alloyed by 0.2, 0.4, and 0.6 mas.% elemental boron powder with the addition of 0.8 mas.% of zinc stearate lubricant. Powders were 15 min blended, compacted and then sintered. The sintering process was elaborated in detail based on microstructure investigations and thermodynamic analysis, which showed that the liquid phase has to be formed as a result of eutectic reaction between matrix elements (Fe, Mo, Ni) and mixed boride (Fe, Mo, Ni)2B. In alloys with boron excess, the liquid phase may occur already a 1176°C in conformity with the reaction: L ↔ γ‐Fe + Fe2B. Its quantity is increased with liquid solution formed in the eutectic reaction running between boron and copper at 1027°C. If the system tends to be in equilibrium, the chemical composition of the liquid solution should be shifted toward higher Fe levels.",signatures:"Joanna Karwan-Baczewska and Bogusław Onderka",downloadPdfUrl:"/chapter/pdf-download/53632",previewPdfUrl:"/chapter/pdf-preview/53632",authors:[{id:"184968",title:"D.Sc.",name:"Joanna",surname:"Karwan-Baczewska",slug:"joanna-karwan-baczewska",fullName:"Joanna Karwan-Baczewska"},{id:"185097",title:"Dr.",name:"Boguslaw",surname:"Onderka",slug:"boguslaw-onderka",fullName:"Boguslaw Onderka"}],corrections:null},{id:"54398",title:"Application of Direct Current Plasma Sintering Process in Powder Metallurgy",doi:"10.5772/66870",slug:"application-of-direct-current-plasma-sintering-process-in-powder-metallurgy",totalDownloads:1649,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"Direct current (dc) plasma-assisted sintering of metal parts is a promising and relatively new research and development field in powder metallurgy (PM). In the present entry, it is intended to introduce the reader to the main applications of the dc plasma sintering process in PM. To achieve this goal, the present entry is divided in a brief introduction and sections in which the bases of the dc plasma abnormal glow discharge regime and its influence in the sintering process are carefully treated. In this case, a clear language is purposely used to didactically introduce the reader to this “fascinating glow world”, the dc plasma-assisted sintering of metal parts, aiming to put in evidence the main points on physicochemical aspects of the plasma environment, basic knowledge of the plasma heating, and surface-related phenomena during dc plasma sintering of parts. All these aspects are treated considering the main techniques of the dc plasma-assisted sintering process applied to PM. Finally, some results on DC plasma heating, sintering and surface modification are presented.",signatures:"Silvio Francisco Brunatto, Rodrigo Perito Cardoso and Aloísio Nelmo\nKlein",downloadPdfUrl:"/chapter/pdf-download/54398",previewPdfUrl:"/chapter/pdf-preview/54398",authors:[{id:"176640",title:"Dr.",name:"Silvio Francisco",surname:"Brunatto",slug:"silvio-francisco-brunatto",fullName:"Silvio Francisco Brunatto"}],corrections:null},{id:"53448",title:"Composite Materials Infiltrated by Aluminium Alloys Based on Porous Skeletons from Alumina, Mullite and Titanium Produced by Powder Metallurgy Techniques",doi:"10.5772/65377",slug:"composite-materials-infiltrated-by-aluminium-alloys-based-on-porous-skeletons-from-alumina-mullite-a",totalDownloads:1783,totalCrossrefCites:8,totalDimensionsCites:8,hasAltmetrics:0,abstract:"The infiltration technology with reinforcement in the form of porous skeletons fabricated with powder metallurgy methods has been presented in relation to the general characteristics of metal alloy matrix composite materials. The results of our own investigations are presented pertaining to four alternative technologies of fabrication of porous, sintered skeletons, and their structure and their key technological properties are presented. Porous skeletons made of Al2O3 aluminium are sintered reactively using blowing agents or are manufactured by ceramic injection moulding (CIM) from powder. Porous skeletons made of 3Al2O3⋅2SiO2 mullite are achieved by sintering a mixture of halloysite nanotubes together with agents forming an open structure of pores. Titanium porous skeletons are achieved by selective laser sintering (SLS). The structure and properties of composite materials with an aluminium alloy matrix—mainly EN AC-AlSi12 and also EN AC-AlSi7Mg0.3 alloys—reinforced with the so manufactured skeletons are also described. A unique structure of the achieved composite materials, together with good mechanical properties and abrasive wear resistance at low density, ensured by an aluminium alloy matrix, are indicating broad application possibilities of such composites.",signatures:"Leszek A. Dobrzański, Grzegorz Matula, Anna D. Dobrzańska-\nDanikiewicz, Piotr Malara, Marek Kremzer, Błażej Tomiczek,\nMagdalena Kujawa, Eugeniusz Hajduczek, Anna Achtelik-Franczak,\nLech B. Dobrzański and Jagoda Krzysteczko",downloadPdfUrl:"/chapter/pdf-download/53448",previewPdfUrl:"/chapter/pdf-preview/53448",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"53660",title:"Fabrication, Composition, Properties and Application of the AlMg1SiCu Aluminium Alloy Matrix Composite Materials Reinforced with Halloysite or Carbon Nanotubes",doi:"10.5772/65399",slug:"fabrication-composition-properties-and-application-of-the-almg1sicu-aluminium-alloy-matrix-composite",totalDownloads:1353,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"In this chapter, the characterisation of the halloysite nanotubes (HNTs) and multiwalled carbon nanotubes (MWCNTs) as the reinforcement in the composite materials was described. The original and author technology of production of the aluminium AlMg1SiCu matrix composite materials reinforced with halloysite or carbon nanotubes using powder metallurgy techniques, including mechanical alloying and hot extrusion and the range of own research in the case to determine microstructure, as well as mechanical properties of those materials was present. It was investigated that the addition of carbon and halloysite nanotubes causes a significant improvement in mechanical properties of the obtained nanocomposites. The investigation results show that the technology used in manufacturing nanocomposite materials can find the practical application in the production of new light metal matrix nanocomposites.",signatures:"Leszek A. Dobrzański, Błażej Tomiczek and Magdalena Macek",downloadPdfUrl:"/chapter/pdf-download/53660",previewPdfUrl:"/chapter/pdf-preview/53660",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"53425",title:"Porous Selective Laser Melted Ti and Ti6Al4V Materials for Medical Applications",doi:"10.5772/65375",slug:"porous-selective-laser-melted-ti-and-ti6al4v-materials-for-medical-applications",totalDownloads:1690,totalCrossrefCites:8,totalDimensionsCites:11,hasAltmetrics:0,abstract:"This chapter characterises scaffolds manufactured in line with the make-to-order concept according to individual needs of each patient. The clinical data acquired from a patient during computer tomography, nuclear magnetic resonance or using traditional plaster casts is converted by a computer into a virtual solid model of a patient’s loss. The model, through the multiplication of a unit cell, is converted into a porous model on the basis of which an actual object is manufactured with the method of selective laser melting (SLM) from Ti/Ti6Al4V powders. The created scaffold is characterised by good mechanical properties, which is confirmed by the results of the performed tensile and compressive strength tests. The material is additionally subjected to surface treatment consisting of the deposition of atomic layers of titanium dioxide with nanometric thickness.",signatures:"Leszek A. Dobrzański, Anna D. Dobrzańska-Danikiewicz, Anna\nAchtelik-Franczak, Lech B. Dobrzański, Marek Szindler and Tomasz\nG. Gaweł",downloadPdfUrl:"/chapter/pdf-download/53425",previewPdfUrl:"/chapter/pdf-preview/53425",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"53675",title:"Powder Application in Additive Manufacturing of Metallic Parts",doi:"10.5772/66874",slug:"powder-application-in-additive-manufacturing-of-metallic-parts",totalDownloads:2070,totalCrossrefCites:2,totalDimensionsCites:5,hasAltmetrics:0,abstract:"This chapter is going to give up-to-date overview of development in the field of additive manufacturing (AM) of metallic components. There will be briefly mentioned input materials and specific requirement for the input materials (powders and wires). General technology process overview will be presented here, and selective laser melting (SLM) technology and beam melting technologies will be described. Advantages of 3D printing technology will be explained in terms of special designs; special properties and generally multifunctional components of production possibilities will be shown. Postprinting procedures leading to improvement of mechanical properties of printed components like thermal or thermomechanical treatment will also be mentioned here.",signatures:"Jan Džugan and Zbyšek Nový",downloadPdfUrl:"/chapter/pdf-download/53675",previewPdfUrl:"/chapter/pdf-preview/53675",authors:[{id:"184961",title:"Dr.",name:"Jan",surname:"Dzugan",slug:"jan-dzugan",fullName:"Jan Dzugan"}],corrections:null},{id:"52552",title:"Fabrication, Structure, Properties and Application of Gradient Sintered Carbide-Steels with HS6-5-2 Matrix",doi:"10.5772/65379",slug:"fabrication-structure-properties-and-application-of-gradient-sintered-carbide-steels-with-hs6-5-2-ma",totalDownloads:1570,totalCrossrefCites:0,totalDimensionsCites:1,hasAltmetrics:1,abstract:"This chapter presents essential information concerning sintered tool materials containing carbides, i.e. high-speed steels and metal matrix composites, including sintered carbides and carbide steels. Gradient materials, whose properties change gradually according to their volume, are characterised. The results of investigations are presented in the final part into the structure and properties of newly developed sintered graded tool materials fabricated by the conventional metallurgy method from a mixture of high-speed HS6-5-2 steel powder and WC carbides. Investigations are described for four-layer materials, where the successive transition layers with a smaller and smaller volume fraction of tungsten carbide were constituted from the surface layer side, until a substrate layer containing high-speed HS6-5-2 steel only. The outcomes are described of structural examinations in a scanning and transmission electron microscope, an X-ray microanalysis and the results of density, porosity and hardness examinations of sintered gradient materials and the results of structure and hardness examinations of heat-treated materials.",signatures:"Leszek Adam Dobrzański and Anna Kloc-Ptaszna",downloadPdfUrl:"/chapter/pdf-download/52552",previewPdfUrl:"/chapter/pdf-preview/52552",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"54228",title:"Powder Injection Moulding of Tool Materials and Materials Containing One-Dimensional Nanostructural Elements",doi:"10.5772/67353",slug:"powder-injection-moulding-of-tool-materials-and-materials-containing-one-dimensional-nanostructural-",totalDownloads:1479,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"As modern manufacturing methods have been developing, the application methods of powders have changed, and they do not always have to be moulded prior to sintering. The powder injection moulding (PIM) method is suitable for large-lot and mass production; still, powder consumption is not too high. The metal injection moulding (MIM) is an advanced technology and not as developed as classical pressing and sintering but constantly and dynamically developing. The technology is developing towards micro-MIM, that is, production of very small parts for miniaturised devices. The chapter presents the overview of powder injection moulding as specialist powder metallurgy method and its application for fabrication of tool materials. Specially, the fabrication of high-speed steels and carbide-steels on their matrix by powder injection moulding is descripted. In last part of the chapter, the results of own investigations of the structure with nanostructural elements of high-speed steels and carbide-steels on their matrix fabricated by powder injection moulding are presented.",signatures:"Leszek A. Dobrzański and Grzegorz Matula",downloadPdfUrl:"/chapter/pdf-download/54228",previewPdfUrl:"/chapter/pdf-preview/54228",authors:[{id:"15880",title:"Prof.",name:"Leszek A.",surname:"Dobrzański",slug:"leszek-a.-dobrzanski",fullName:"Leszek A. Dobrzański"}],corrections:null},{id:"53945",title:"Manufacturing, Composition, Properties and Application of Sintered Hard Metals",doi:"10.5772/66872",slug:"manufacturing-composition-properties-and-application-of-sintered-hard-metals",totalDownloads:2034,totalCrossrefCites:1,totalDimensionsCites:3,hasAltmetrics:0,abstract:"Among other materials, hard metals represent an important family of functional materials. They show properties that are combinations of those of their constituents. The general idea while using hard metals is to exploit their excellent properties in terms of hardness, toughness, wear resistance, and chemical stability. These characteristics made hard metals as promising candidate for use as a cutting tool, which constitutes their main area of application. Depending on the particular use, the most important properties can be achieved: (i) by properly selecting the constituents made up the whole composition, (ii) by varying the relative composition of the phases, or (iii) by applying a suitable hard metal coating layer on the top of the cutting tool. This chapter presents a general overview of the actual scenario concerning different tool materials, including a short history and description of state‐of‐the‐art techniques as regards their composition, their manufacturing routes and their most important properties. Some results of the own research in this field are carried out during the years will integrate this part.",signatures:"Ildiko Peter and Mario Rosso",downloadPdfUrl:"/chapter/pdf-download/53945",previewPdfUrl:"/chapter/pdf-preview/53945",authors:[{id:"32468",title:"Prof.",name:"mario",surname:"rosso",slug:"mario-rosso",fullName:"mario rosso"},{id:"173511",title:"Prof.",name:"Ildiko",surname:"Peter",slug:"ildiko-peter",fullName:"Ildiko Peter"}],corrections:null},{id:"53582",title:"Properties and Testing of Cemented Carbides",doi:"10.5772/66871",slug:"properties-and-testing-of-cemented-carbides",totalDownloads:2246,totalCrossrefCites:1,totalDimensionsCites:1,hasAltmetrics:0,abstract:"This chapter deals with selected properties of cemented carbides and their testing. It consists of three main sub-chapters. The first one covers the degradation processes associated with grinding of cemented carbides. Among those, the focus is on the effects of thermal loads and reactions with the environment—the atmosphere. The second main sub-chapter describes the origin of residual stresses and their impact on cemented carbide properties. Finally, the third main sub-chapter explores the corrosion of cemented carbides in various environments. The choice of these topics was inspired by the impression that many users have of cemented carbides: indestructible materials in which no degradation takes place either during the production of cutting tools or during their use. As evidenced by the authors’ experience described below, which has been acquired over many years in the field, damage in cemented carbides is a very frequent occurrence. Despite that it still receives very little attention, and especially the damage in cemented carbide cutting tools tends to be ascribed to various other factors, such as the applied coatings, the cutting process conditions and the material of the workpiece.",signatures:"Antonín Kříž and David Bricín",downloadPdfUrl:"/chapter/pdf-download/53582",previewPdfUrl:"/chapter/pdf-preview/53582",authors:[{id:"184898",title:"Prof.",name:"Antonín",surname:"Kříž",slug:"antonin-kriz",fullName:"Antonín Kříž"},{id:"189109",title:"Mr.",name:"David",surname:"Bricín",slug:"david-bricin",fullName:"David Bricín"}],corrections:null},{id:"53445",title:"Structure and Properties of the Multicomponent and Nanostructural Coatings on the Sintered Tool Materials",doi:"10.5772/65400",slug:"structure-and-properties-of-the-multicomponent-and-nanostructural-coatings-on-the-sintered-tool-mate",totalDownloads:1463,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,abstract:"This chapter presents a general characteristic of sintered tool materials, in particular sintered sialons, nitride ceramics, injection-moulded ceramic-metallic tool materials and cemented carbides and a general characteristic of their surface treatment technology and especially chemical vapour deposition (CVD) and physical vapour deposition (PVD) techniques. The results of our investigations in technology foresight methods concerning the development prospects of surface engineering of sintered tool materials are presented. In the next subsection, we discuss the outcomes of multifaceted research carried out with advanced materials engineering methods, including high-resolution transmission electron microscopy, into the structure and properties of multicomponent, graded and multilayer coatings on the investigated tool materials, including the newly developed injection moulded ceramic-metallic tool materials. Special attention was drawn to a one-dimensional structure of the studied PVD and CVD coatings and its impact on the properties of coatings.",signatures:"Leszek A. 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The formation of a hardened layer with the structure of eutectic on the metal surface is the creation of a new material with certain mechanical properties. The analysis of different material properties and of alloy addition enabled to work out new eutectic powder alloys based on iron of the Fe-Mn-C-B system. In particular, it enabled to determine eutectic ranges and element contents.",signatures:"Mykhaylo Pashechko, Klaudiusz Lenik, Joanna Szulżyk-Cieplak and\nAneta Duda",downloadPdfUrl:"/chapter/pdf-download/54110",previewPdfUrl:"/chapter/pdf-preview/54110",authors:[{id:"184924",title:"Prof.",name:"Klaudiusz",surname:"Lenik",slug:"klaudiusz-lenik",fullName:"Klaudiusz Lenik"},{id:"185043",title:"Prof.",name:"Mykhaylo",surname:"Pashechko",slug:"mykhaylo-pashechko",fullName:"Mykhaylo Pashechko"},{id:"185044",title:"Dr.",name:"Joanna",surname:"Szulżyk-Cieplak",slug:"joanna-szulzyk-cieplak",fullName:"Joanna Szulżyk-Cieplak"},{id:"185045",title:"Dr.",name:"Aneta",surname:"Duda",slug:"aneta-duda",fullName:"Aneta Duda"}],corrections:null},{id:"54457",title:"Application of Powder Metallurgy Methods for Production of a Novel Cu‐Based Composite Frictional Train Brake Material",doi:"10.5772/67533",slug:"application-of-powder-metallurgy-methods-for-production-of-a-novel-cu-based-composite-frictional-tra",totalDownloads:1965,totalCrossrefCites:1,totalDimensionsCites:4,hasAltmetrics:0,abstract:"A novel Cu-based composite frictional train brake material composed of several elements such as Al, SiO2, Fe, graphite, Sn, Mn and SiO2 re-enforced with other elements was treated under Powder Metallurgy (P/M) route. The materials were sintered at three different temperatures (850°C, 900°C and 950°C) at a constant pressure.",signatures:"Glenn Kwabena Gyimah, Zhongning Guo, Ping Huang and Dong\nChen",downloadPdfUrl:"/chapter/pdf-download/54457",previewPdfUrl:"/chapter/pdf-preview/54457",authors:[{id:"179491",title:"Dr.",name:"Glenn Kwabena",surname:"Gyimah",slug:"glenn-kwabena-gyimah",fullName:"Glenn Kwabena Gyimah"},{id:"193487",title:"Prof.",name:"Ping",surname:"Huang",slug:"ping-huang",fullName:"Ping Huang"},{id:"193488",title:"Prof.",name:"Dong",surname:"Chen",slug:"dong-chen",fullName:"Dong Chen"},{id:"194596",title:"Prof.",name:"Zhongning",surname:"Guo",slug:"zhongning-guo",fullName:"Zhongning Guo"}],corrections:null}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},subseries:null,tags:null},relatedBooks:[{type:"book",id:"5951",title:"Biomaterials in Regenerative Medicine",subtitle:null,isOpenForSubmission:!1,hash:"a4ff8af6190bb48a5857450c9c2612d7",slug:"biomaterials-in-regenerative-medicine",bookSignature:"Leszek A. 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From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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The phytochemicals rich in plants have shown to be beneficial for prevention of diseases as well as long-term health. Plants are generally consumed as sources of essential compounds such as saccharides, coumarins, lignans, flavonoids, terpenoids, and steroids. The health benefits and the composition from plant have been described more and more in the literature. Because of the complexity of plant chemical constituents, pure phytochemicals must to be obtained via extraction and isolation before structure identification, bioactivity screening, and so on. In recent years, new technologies and methods of extraction occurred, which accelerate the extraction and analysis of phytochemicals.
Extraction is the first step of phytochemistry research, which is also the necessary work before the isolation of effective constituents. The purpose of extraction is to get the objective chemical constituents to the utmost extent and avoid or reduce the solution of unwanted constituents.
The separation of phytochemicals is a process of isolating the constituents of plant extracts or effective parts one by one and purifying them into monomer compounds by physical and chemical methods. Classical isolation methods, including solvent extraction, precipitation, crystallization, fractional distillation, salting-out, and dialysis, are still used commonly at present. On the other hand, modern separation technologies such as column chromatography, high performance liquid chromatography, ultrafiltration, and high performance liquid drop countercurrent chromatography also play an important role in the separation of phytochemicals [1, 2, 3].
The chemical structures of plant compounds must be identified, which may provide the necessary basis for further study on the bioactivities, structure-activity relationships, metabolisms
The structural studies are often difficult to carry out with classical chemical methods, such as chemical degradation and derivative synthesis, because of the minute amount of compound isolated from plants. Therefore, spectral analysis is mainly used. That is, consuming sample as little as possible to obtained structural information as much as possible by measuring and analyzing various spectra [4].
Solvent extraction is the commonest method to extract plant material. The main purpose is to select the suitable solvent to extract target plant materials efficiently. During the extraction, the solvent has to diffuse into the cell membrane in the first instance, in the following step it has to dissolve the solutes, then intracellular and extracellular concentration difference is formed, and finally it has to diffuse out of the cells enriched in the extracted solutes [5].
Selecting suitable solvents is the key of the solvent extraction method. Using a solvent of an appropriate polarity according to the principle of “like dissolves like” is the main point to select solvent. Thus, hydrophilic solvents are used to solubilize hydrophilic chemical constituents and vice versa. The hydrophilicity or lipophilicity of solvents and chemical constituents could be predicted by polarity. The plant compounds, such as terpenoids and steroids, possess low polarity, and could be dissolved into lipophilic solvents such as chloroform and ether, while chemical constituents, such as carbohydrates and amino acids, possess rather high polarity and could be dissolved into water and aqueous ethanol.
Solvents commonly used for extracting chemical constituents of plants are in the order of weak to strong polarity as follows: petroleum ether < carbon tetrachloride < benzene < dichloromethane < chloroform < ether < ethyl acetate < n-butanol < acetone < ethanol < methanol < water.
Water is a cheap, easy to get, and nontoxic solvent with strong polarity. It could be used to extract phytochemicals with strong polarity, such as inorganic salts, saccharides, amino acids, tannins, proteins, organic acid salts, alkaloid salts, and glycosides. Acid or alkaline water is applied sometimes to increase the solubility of certain specific components. Acid water could extract alkaline materials, such as alkaloids, via the formation of salts. Similarly, organic acids, anthraquinoids, flavonoids, coumarinoids, phenols, and other acidic materials could be extracted via the formation of salts. The disadvantage to extract chemical constituents with water is that the aqueous extract is easy to go moldy, so difficult to preserve. Additionally, water possesses high boiling point, and the water extract needs to be concentrated for a rather long time. Furthermore, the water extract contains many impurities such as proteins, pectins, tannins, mucilages, and inorganic salts, which make the extraction of target components difficult.
Hydrophilic organic solvents are strong-polarity and water miscible, such as methanol, ethanol, and acetone. Ethanol is the most commonly used hydrophilic organic solvent. Chemical constituents could be extracted by ethanol of different concentrations according to their properties. Furthermore, ethanol is inexpensive, safe, and concentrated easily. Additionally, ethanol extract is not readily moldy and glycosides are hard to be hydrolyzed in ethanol extract. Thus, ethanol is one of the most commonly used solvents in laboratories and industrial production. Methanol possesses similar property to ethanol and lower boiling point. However, methanol has rather strong toxicity, so we have to pay attention to safety when it has to be used. Acetone is a good solvent to extract lipid-soluble chemical constituents. However, acetone is easy to volatilize and flame, and it possesses certain toxicity.
Petroleum ether, benzene, chloroform, ether, ethyl acetate, dichloroform, and so on are lipophilic organic solvents and are not miscible with water. They could be applied to extract lipophilic components, such as volatile oils, fats, chlorophyll, lactones, phytosterols, some alkaloids and some aglycones (aglycones of flavonoids, anthraquinoids, steroids, and so on). These solvents possess low boiling points and are easy to concentrate. However, strong-volatility, large loss, flammability, toxicity, and high price are their disadvantages. Additionally, they are difficult to permeate into plant cell tissues.
Solvent extraction methods could be classified as cold extraction and hot extraction roughly by whether heating or not.
It is a method to dissolve out phytochemicals with appropriate solvents at room or low temperatures (<80°C). It is suitable to extract phytochemicals easily to be destroyed at high temperature. The plants with abundant starches, pectins, gums, or mucilages could also be extracted with this method. Firstly, plant powder or pieces should be loaded in the adequate container, and then the suitable solvents (water, ethanol, aqueous ethanol, and so on) are added into it to immerse the material for the given length of time. Discontinuous stirring or shaking during the process could accelerate dissolution rate. The immersion method is simple but inefficient, and the extraction ratio is also low. Furthermore, aqueous extract is easy to go moldy, so addition of appropriate preservatives is necessary.
The coarse particles of plants should be loaded in percolation apparatus and immersed with suitable solvent for 24–48 h, then collect the percolates at the bottom of percolation apparatus. New solvent should be added at the top of percolation apparatus constantly during the percolation process. It possesses higher efficiency than the immersion method because of the sustained concentration difference during the process. However, this procedure is complex and consumes rather much solvent and long time.
Load short segments, thin pieces, or coarse powder into an appropriate container, add water, and heat it to boiling; the components are then extracted. It is easy to operate; most of the constituents could be extracted in various degrees. Nevertheless, rather much nontargeted components could also be extracted, and it is not suitable to the extraction of volatile compounds and thermal unstable compounds. Furthermore, it is not suitable to extract plants with lots of starches [6].
It is a method to extract plant chemical constituents by organic solvent using heating and refluxing. Refluxing apparatus is necessary so as not to waste solvents, and the toxicity to operators or ruin the environment is deduced. It is applicable to extraction of lipophilic phytochemicals, such as steroids, anthraquinoids, and terpenoids. It is an extraction method of high efficiency but complex, and consumes much more solvent. This method is not applicable to extract thermal unstable chemical constituents because of long time heating.
It is a method developed based on the refluxing method. Soxhlet extractor is the most frequently used constant refluxing apparatus. This method avoids disadvantages of consuming too much solvent and complex operation. However, as a refluxing method, constant refluxing method is not applicable to extract thermally unstable compound either because of long time heating.
In the supercritical state, the supercritical fluid is contacted with the plant tissues. By controlling different temperatures, pressures and different kinds and contents of entrainers, the supercritical fluid can selectively extract the components of different polarities, boiling points, and molecular weights successively. This method is called the supercritical fluid extraction (SFE) method [7].
The critical point of a pure substance is defined as the highest temperature and pressure at which the substance can exist in vapor-liquid equilibrium. At temperatures and pressures above this point, a single homogeneous fluid is formed, which is known as supercritical fluid (SF). SF is heavy like liquid and has low viscosity like gas meanwhile. SF possesses rather large diffusion coefficient and could dissolve many compounds well. A number of materials could be used as SFs, such as ammonia, ethane, difluoro-dichloromethane, heptane, and so on, while the most widely used SF is CO2. The critical temperature of CO2 (
The extraction of nonpolar and medium-polar components by SFE can avoid the sample loss and environmental pollution caused by solvent recovery in traditional extraction methods, especially for the extraction of volatile compounds with thermal instability.
The biggest advantage of SFE is that it can be performed at near-room temperature, and almost all the active ingredients in the product can be retained. There is no residual organic solvent in the process. The product has high purity and high yield. Additionally, the operation is simple and energy saving.
Compared with other conventional separation methods, SFE possesses the following advantages: (1) No residual organic solvents, fast extraction speed, simple process, high yield, and easy operation; (2) no flammable and explosive dangers, no environmental pollution. Low extraction temperature, suitable for the extraction of thermal unstable components; (3) the dissolution properties of SF are easy to improve, only the pressure needs to be changed at a certain temperature; (4) entrainers can be added to change the polarity of the extraction medium to extract polar substances; extraction medium can be recycled with low cost; (5) it could be applied combined with other chromatographic techniques, such as GC, IR, GC–MS, and HPLC, to extract, separate, and determine phytochemicals efficiently and quickly, so as to achieve the integration of extraction and quality analysis. However, supercritical extraction has some limitations: strong solubility of fat-soluble components, weak solubility of water-soluble components, high cost of equipment, resulting in higher product costs, and cleaning equipment is difficult.
Supercritical fluid extraction (SFE) technology has achieved gratifying results in the fields of medicine, chemical, food, light industry, and environmental protection. Especially, it has been widely used in phytochemical extraction field, such as the extraction of alkaloids, volatile oils, phenylpropanoids, flavonoids, organic acids, glycosides, terpenoids, and so on.
It is a method of solvent extraction assisted by ultrasound. Ultrasonic wave is a kind of elastic mechanical vibration wave. The vibration frequency is as high as 20 KHz in elastic medium. The ultrasonic wave could vibrate the liquid medium. When the vibration is sparse, many small holes are formed in the medium. The instantaneous closure of these small holes can cause a pressure of up to thousands of atmospheric pressures. At the same time, the local temperature can rise to 1000°C. It can cause instantaneous rupture of the cell wall of plants and the whole organism, and make the solvent permeate into the cells of plants. This accelerates the dissolution of active ingredients in plants into solvents. Ultrasonic wave extraction could shorten the extraction time and improve the extraction efficiency, but could not change the structures of chemical constituents meanwhile.
Ultrasonic extraction technology has been widely used in the extraction of natural products in recent years, for example, extraction of soy isoflavones; see [8].
Microwave refers to the electromagnetic wave whose wavelength is in the range of 0.1–100 cm (the corresponding frequency is 300–300,000 MHz), which is between infrared and radio waves. Polar molecules can absorb microwave energy, then release energy in the form of thermal energy, which makes the temperature inside the medium rise rapidly, causes the rather high pressure inside, and then the components flow out and dissolve in the solvent. On the other hand, the electromagnetic field produced by microwave can make some components diffuse to the interface of the extraction solvent, accelerating their thermal movement, which not only improves the extraction efficiency but also reduces the extraction temperature [9].
Microwave-assisted extraction has the advantages of less decomposition of chemical constituents, shorter time, lower energy consumption and less environmental pollution. Microwave-assisted extraction has been widely used in a series of fields of perfume, condiments, natural pigments, herbal medicine, cosmetics, soil and environmental analysis, and so on. In China, microwave-assisted extraction technology has been used in hundreds of Chinese herbal medicine extraction, such as
Steam distillation is suitable for the extraction of volatile components which can be distilled with steam without being destroyed and are insoluble in water. These compounds’ boiling points of are mostly higher than 100°C, and they possess certain vapor pressures at about 100°C. The principle of steam distillation is that the vapor pressure of each component is equal to that of their pure state, while the existence of another liquid does not affect their vapor pressure. The total vapor pressure of the mixing system is equal to the sum of the vapor pressures of the two components. Because the total vapor pressure of the system is higher than that of any single component, so the boiling point of the mixture is lower than that of any component. It is mainly used to extract volatile oils, some alkaloids, and phenolic substances of small molecules from plants.
The process that solid material converts into steam directly without melting after heating is called sublimation. The phenomenon that steam condenses into solid after cooling is called deposition. Some natural chemicals have sublimation properties, which can be extracted directly with the sublimation method, for example, the extraction of camphor from camphor wood and caffeine from tea. In addition, some small molecular alkaloids, coumarins, organic acids, and other components also have sublimation properties, such as aesculetin and benzoic acid. However, it is easy to carbonize natural products because of long heating time. The volatile tar-like substances often adhere to sublimates, which are difficult to remove and often accompanied with thermal decomposition. The yield of this method is often low, and it is not suitable for large-scale production.
When the content of active ingredients is relatively high and exists in the juice of plants, the juice can be extracted directly from fresh raw materials. Volatile oils can also be extracted from plant tissues by mechanical pressing, such as orange peel oil and lemon oil. It is performed at room temperature, so its components will not be decomposed by heat. However, the products obtained are impure and often contain impurities such as water, mucoid substances, and cell tissues, so they are often turbid, and it is not easy to press the volatile oil in plants entirely. Therefore, the crushed residue is often distilled by steam to extract volatile oils completely. For example, the black soybean oil from black soybean is often extracted with the low-temperature pressing method.
The separation of phytochemicals is a process of isolating the constituents of plant extracts or effective parts one by one and purifying them into monomer compounds by physical and chemical methods. Classical isolation methods, including solvent extraction, precipitation, crystallization, fractional distillation, salting-out, and dialysis, are still used commonly at present. On the other hand, modern separation technologies such as column chromatography, high performance liquid chromatography, ultrafiltration, and high performance liquid drop countercurrent chromatography also play an important role in the separation of phytochemicals. This section describes the common methods and their specific applications in isolation of phytochemicals.
It is carried out according to the different acidity and alkalinity of each component in the mixture. Water-insoluble alkaline organic components, such as alkaloids, could react with inorganic acids and form salts, which can be separated from nonalkaline and water-insoluble components. Acid components with carboxyl or phenolic hydroxyl groups can be salted by bases and dissolved in water. Components with lactone or lactam substructures can be saponified and dissolved in water and then isolated from other water-insoluble components. The total extract can be dissolved in lipophilic organic solvents (ethyl acetate is commonly used) and extracted respectively with acid water and alkali water, and then the total extract would be divided into acidic, alkaline, and neutral parts. Of course, the total extract can also be dissolved in water and extracted with organic solvents after adjusting the pH value. The alkalinity or acidity of the fractions are different and can be separated further by pH gradient extraction.
When using the acid and basic solvent method, attention should be paid to the strength of acidity or alkalinity, the contact time with the separated components, heating temperature, and time, so as to avoid the structural changes of some compounds under severe conditions or the chemical structures cannot be restored to the original states.
This method is to achieve the separation aim based on the different polarity of each component in plant extracts and the different partition coefficients in two-phase solvents. Generally, different two-phase solvent systems are selected according to the polarity of components in plant extracts. For example, the components with strong polarity can be separated by n-butanol-water system, the components with medium polarity can be separated by ethyl acetate-water system, and the components with weak polarity can be separated by chloroform (or ether)-water system. During the operation, the plant extract should be dissolved by water firstly, and then the solution or suspension is extracted in a separating funnel with different organic solvent which is not miscible with water based on the polarity difference. Usually, the extract was extracted with petroleum ether (or cyclohexane) firstly, then ethyl acetate (or chloroform), and finally with water saturated n-butanol, as shown in Figure 1. Petroleum ether layer contains lipid-soluble compounds with low polarity. Ethyl acetate layer contains medium polar compounds such as monoglycosides, flavonoids, and compounds with more polar functional groups. N-butanol layer contains compounds with strong polarity, such as oligoglycosides and other water-soluble components. Compounds in water layer possess strongest polarity, such as glycosides with more glycosyl groups, carbohydrates, amino acids, proteins, and other water-soluble compounds.
Flow charts of common polarity gradient extraction method.
It is a method based on the formation of precipitation of some phytochemicals by reaction with specific reagents, or the precipitation of some components from the solution by adding specific reagents, which can reduce the solubility of some components in the solution. The precipitation reaction must be reversible if the target components are required to form precipitation. While if the components are nontarget, the precipitation generated will be removed, so the precipitation reaction can be irreversible. According the addition of reagents or solvents, this method could be classified as follows [11].
The solubility of some components in the mixed component solution can be changed by adding a specific solvent that can be mutually soluble with the solution, so it can be precipitated from the solution. The gradual precipitation by changing the polarity or amount of solvent added is called fractional precipitation. For example, using water as an extracting solvent to extract phytochemicals, ethanol is added to the water extracting concentrate to make its alcohol content more than 80%, and then polysaccharides, proteins, starch, gum, and so on will be precipitated and removed after filtration. The preceding procedure is called water extraction and ethanol precipitation. Crude polysaccharides from plants are often separated with this method. For example, see [12].
Some reagents could react selectively with certain chemical constituents to produce reversible precipitation, and the separation aims are achieved, which is called the exclusive reagent precipitation method. For example, alkaloid precipitation reagents such as Reynolds ammonium salt can precipitate after reacting with alkaloids, which can be used to separate alkaloids and nonalkaloids, or water-soluble alkaloids and other alkaloids. As another example, reactions of cholesterol and sterol saponins could form precipitation, which can separate them from triterpene saponins. Additionally, gelatin can precipitate tannins, which can be used to separate or remove tannins. In practical application, appropriate precipitation reagents should be selected according to the properties of target constituents and impurities in plants.
Adding inorganic salts to a certain concentration or saturated state in the water extract of plants can reduce the solubility of some components in water, thus they could be separated from water-soluble compounds. The inorganic salts commonly used for salting out are sodium chloride, sodium sulfate, magnesium sulfate, ferric sulfate, etc. For example, extractions of tetrandrine from
It is a method to let substances selectively penetrate through natural or synthetic semi-permeable membranes (or dialysis bags) under the action of concentration difference, pressure difference, or potential difference, so as to achieve the purpose of separation, classification, purification, or concentration. For example, when saponins, proteins, polypeptides, polysaccharides, and other substances in plants are separated and purified, dialysis can be used to remove inorganic salts, monosaccharides, and other impurities. On the contrary, large molecular impurities can also be left in the semi-permeable membrane, while small molecular substances can be separated and purified through the semi-permeable membrane into the solution outside the membrane [14].
Fractional distillation is a method of separating components in liquid mixtures based on their different boiling points. It is usually categorized into atmospheric, vacuum, molecular distillation, and so on. It is mainly used for the separation of volatile oils and some liquid alkaloids in plants. For example, the boiling points of the two alkaloids in total alkaloids of
Crystallization is the process of solute precipitation from mother liquor with complex components, and it is an effective method to prepare pure substances. The initial crystallization is often impure and needs to crystallize again, which is called recrystallization. It is a method to separate compounds from the mixture by using the difference of solubility of each component in the solvent. Crystallization is one of the important technologies for plant chemists to prepare pure compounds.
When the content of a phytochemical is very high in one plant, crystals can be obtained by cooling or slightly concentrating the extract after extraction with appropriate solvent. For example, see [16].
Selecting suitable crystallization solvent is the key of the crystallization method. The ideal solvents for crystallization should possess the following characteristics: high solubility for the components to be purified at high temperature, low solubility at low temperature, insoluble for the impurities at high and low temperature, or soluble for the impurities at high and low temperature, moderate boiling point, no chemical reaction with the components to be crystallized, safe, low price, easy to obtain, and so on. Solvents commonly used for crystallization are methanol, ethanol, acetone, ethyl acetate, acetic acid, pyridine, etc. When crystals cannot be obtained with a single solvent, the crystallization operation can be carried out with a mixture of two or more solvents. Mixed solvents generally consist of two miscible solvents, one of which has high solubility for the component to be crystallized, and the other has low solubility. Firstly, the sample to be crystallized is heated and dissolved in as few solvents as possible with high solubility. Then the second solvent with low solubility is added to the hot solution to make it turbid. Then the first solvent is added to dissolve the sample. The solution reaches saturation at this point and crystallizes when it is cooled. The purity of crystallization can be preliminarily identified by the crystal form, color, melting point, melting range, thin layer chromatography, paper chromatography, etc.
Chromatography is the most commonly used method for the separation of chemical constituents of natural products. It possesses advantages of high separation efficiency, rapidity, and simplicity. By choosing different separation principles, different operation modes, different chromatographic packings, or applying various chromatographic methods jointly, the separation and purification of various types of phytochemicals could be achieved. It can also be used for the identification of compounds.
It is a kind of chromatography based on the difference of adsorptive capacity of adsorbents to different compounds. The commonly used adsorbents include silica gel, alumina, activated carbon, polyamide, and so on. Silica gel adsorption chromatography is widely used, and it is suitable to the separation of most of the plant chemical constituents. Alumina adsorption chromatography is mainly used for the separation of alkaline or neutral lipophilic components, such as alkaloids, steroids, and terpenoids. Activated carbon is mainly used for the separation of water-soluble substances, such as amino acids, carbohydrates and some kinds of glycosides. Polyamide, which allows the separation to take place based on the formation of kinds of hydrogen bonds, is mainly used for the separation of phenols, quinones, flavonoids, anthraquinones, tannins, etc. [17].
Molecular sieve is the main principle of gel chromatography, which can separate mixture compounds according to the pore size of the gel and the molecular size of the compounds. Gel is a kind of solid material with a porous network structure. The molecules of the separated substances are different in size, so their ability to enter the gel is different. When the mixture solution passes through the gel column, the molecules smaller than the gel pores can enter the gel interior freely, while the molecules with larger size than the gel pores cannot enter the gel, and only pass through the gel particle gaps. Therefore, different movement rates are emerged. The molecules with large sizes are not excluded, and the retention time is shorter. The molecules with small sizes are detained because of its diffusion into the pores, thus the retention time is longer. There are many kinds of commercial gels, dextran gel and hydroxypropyl dextran gel are used most commonly [18].
It is to separate chemical constituents according to the difference of dissociation degrees. In this method, ion exchange resin is applied as stationary phase and water or solvent mixed with water as mobile phase. The ionic components existing in the mobile phase are absorbed by ion exchange resin after ion exchange reaction. Ion exchange chromatography is suitable for the separation of ionic compounds, such as alkaloids, amino acids, organic acids, peptides, and flavonoids. The ability of ion exchange reaction between compounds and ion exchange resins mainly depends on the compounds’ dissociation degree and the amount of electric charges. If the dissociation degree of a compound is high (acidic or alkaline), it is easily exchanged on resins and difficult to elute. Therefore, when the compounds with different degree of dissociation are exchanged on the resin, the compounds with lower degree of dissociation are eluted before those with higher degree of dissociation [19].
It is a chromatographic method which combines the principle of adsorption and molecular sieve. Its chromatographic behavior possesses reversed-phase properties. Macroporous resin is a kind of solid macromolecule material with no dissociable group and porous structure and is insoluble in water. It is widely used in the separation and enrichment of natural compounds because of its stable physical and chemical properties (insoluble in acids, bases, and organic solvents).
In practical work, the water solution of the mixture to be separated is usually washed by water, water-containing alcohol solution with low to high concentration. The mixture can be separated into several components. The regeneration of macroporous adsorbent resin is convenient. It is often washed by 1 mol/L hydrochloric acid and 1 mol/L sodium hydroxide solution, respectively, first, then washed by distilled water to neutral, and stored in methanol or ethanol. The alcohol should be washed out with distilled water before using.
It is a kind of chromatography method to separate components by using different partition coefficients between stationary phase and mobile phase, which are immiscible liquids. Partition chromatography could be divided into normal phase chromatography and reverse phase chromatography. The polarity of stationary phase is stronger than that of mobile phase in normal phase partition chromatography, which is mainly used to separate polar and moderately polar molecular compounds. Carriers commonly used in normal phase distribution chromatography include silica gel, diatomite, cellulose powder, etc. Silica gel with water content of more than 17% can be used as a carrier for partition chromatography because of its loss of adsorption. It is the most widely used carrier for partition chromatography. In reverse phase partition chromatography, the polarity of mobile phase is stronger than that of stationary phase. The commonly used stationary phase is octadecyl-silylated silica (ODS). The mobile phase is usually methanol-water or acetonitrile-water system, which is mainly used for the separation of nonpolar and moderately polar molecular compounds.
High performance liquid chromatography (HPLC) is a rapid separation and analysis technology developed on the basis of conventional column chromatography. Its separation principle is the same as regular column chromatography, including adsorption chromatography, gel chromatography, partition chromatography, ion exchange chromatography, and other methods. HPLC columns are produced with particle fillers (particle diameter 5–20 μm) and high pressure homogenate column loading technology. The eluents are pressed into the column by a high pressure infusion pump and equipped with high sensitive detectors and automatic recording and collection devices. As a result, it is far superior to conventional column chromatography in separation speed and efficiency. It has the characteristics of high efficiency, high speed, and automation. Preparative HPLC can be used to prepare a large amount of samples of high purity. HPLC has played an increasingly important role in the separation, qualitative identification, and quantitative analysis of plant chemical constituents. During the separation of many plant chemical constituents, it is necessary to separate trace constituents from a large amount of crude extracts. Usually, in the final stage of separation, samples with high purity are prepared by high or medium pressure liquid chromatography. Constant concentration eluents are mostly used in preparative HPLC. However, gradient elution is sometimes applied for samples that are difficult to be separated. Moreover, HPLC retains the advantages of liquid chromatography, such as a wide range of application and flexibility of mobile phase change. It can be applied to chemical constituents of difficult gasification, high molecular weight, or thermal instability.
The detectors commonly used in HPLC are ultraviolet detectors and differential refractive index detectors, but both have limitations. Differential refractive index detectors are sensitive to temperature change, the detection of a small amount of substances is often not ideal, and gradient elution cannot be used. As for ultraviolet detectors, they cannot detect samples without ultraviolet absorption. In recent years, a kind of mass detector, called evaporative light scattering detector (ELSD), has been applied in HPLC. It can not only detect samples without ultraviolet absorption, but also use gradient elution. It is suitable for most nonvolatile components [20].
DCCC is an improved liquid-liquid partition chromatography based on the counter-current partition method. The formation of droplets is required when the mobile phase passes through a liquid stationary phase column. Droplets of mobile phase contact with stationary phase effectively, and form new surfaces in thin partition extraction tubes constantly, which promote the partition of solutes in two-phase solvents, and the chemical components of mixtures are isolated in immiscible two-phase droplets due to different partition coefficients. This method is suitable for the separation of phytochemicals with strong polarity. The separation effect is usually better than counter-current partition chromatography, and there is no emulsification phenomenon. Furthermore, nitrogen is used to drive the mobile phase, so the separated substance will not be oxidized by oxygen in the atmosphere. However, the solvent system which can generate droplets must be selected in this method, the amount of sample treated is small, and special equipment is needed.
DCCC possesses good reproducibility, and can handle crude extract samples of milligram to gram grade. It can be used in either acidic or basic conditions. Because no solid separation carriers are used, the phenomenon of irreversible adsorption and band broadening of chromatographic peaks can be avoided. Compared with preparative HPLC, DCCC consumes less solvent, but the separation time is longer and the resolution is lower. For example, see [21].
HSCCC is also a liquid-liquid partition chromatography. It is another mild form of chromatography with no solid support and hence no chance of loss of substrate by binding to the column. The only media encountered by the sample are solvent and Teflon tubing. The former is common to all forms of chromatography and the latter to most. The chemical constituents with higher partition coefficient in mobile phase are eluted first, whereas those with higher partition coefficient in stationary phase are eluted later.
HSCCC chromatography could avoid the shortcomings of irreversible adsorption and abnormal tailing of chromatographic peaks caused by solid carriers in liquid chromatography because it does not need solid carriers. The sample recovery is near 100% from a chromatography. It also has advantages of good reproducibility, high purity of separated compounds, and fast speed. It is suitable for the isolation and purification of wide kinds of phytochemicals, such as saponins, alkaloids, flavonoids, anthraquinoids, lignans, triterpenes, proteins, and carbohydrates. For example, see [22].
It is an instrumental analysis method developed in the late 1980s combining classical electrophoresis with modern microcolumn separation technologies. In pharmaceutical analysis, the most commonly used separation modes are capillary zone electrophoresis, micellar electrokinetic capillary chromatography, and capillary gel electrophoresis. It is an efficient separation technology of large and small molecules in a hollow and thin inner diameter capillary (10–200 μm). The two ends of the capillary are immersed in a buffer solution and electrodes connected with a high voltage power supply are inserted separately. The voltage makes samples migrate along the capillary. According to the charge and volume of the separated substances, various molecules are separated under high voltage. In zone capillary electrophoresis, separation could be achieved by the movement of electrophoresis and electroosmotic flow. The strength of electroosmotic flow depends on the strength of electric field, PH value of electrolyte, composition of buffer solution, ionic strength, internal friction, and so on. Sample injection could be accomplished by pressing the sample into a capillary tube by atmospheric pressure or voltage.
HPCE has the advantages of high efficiency, microamount, economy, high automation, and wide application. However, it has the disadvantages of poor preparation ability, low sensitivity, and poor separation reproducibility. For example, see [23].
Affinity chromatography is a unique chromatographic separation method based on the principle of reversible combination of high affinity and specificity between molecules. By simulating the reversible and specific interaction between biological molecules, affinity chromatography uses the adsorption medium coupled with affinity ligands as the stationary phase to adsorb target compounds. It is a development of adsorption chromatography. This method can selectively separate and analyze specific chemical constituents from complex samples. Firstly, ligands that can specifically bind to the target compounds are fixed on the filler carrier to make the chromatographic column. Then the mixture containing the target compounds is passed through the column. Only the target compounds which show affinity with the ligands can bind to the ligands and remain in the column. Finally, the adsorbed target compounds are eluted by changing the composition of the mobile phase and are separated from other chemical constituents. AC is mainly used for the separation and purification of proteins, especially enzymes, antigens, and antibodies. Its application range has been expanding along with the continuous development of technology in recent years. For example, see [24].
The chemical structures of plant compounds must be identified or elucidated, which may provide the necessary basis for further study on the bioactivities, structure-activity relationships, metabolisms in vivo, structural modification, and synthesis of the active phytochemicals.
The quality of physiological active substances isolated from plants is often small, sometimes only a few milligrams, and the structural studies are often difficult to carry out with classical chemical methods, such as chemical degradation, derivative synthesis, etc. Therefore, spectral analysis is mainly used, that is, consuming sample as little as possible to obtain structural information as much as possible by measuring various spectra. Then comprehensive analysis is carried out with the assistance of literature data. If necessary, chemical means would be integrated into the former methods to determine the planar- and even the stereo-structures of the compounds.
Before the structural investigation of an active compound, the purity must be determined, which is a prerequisite for the structural identification.
The crystals of each compound have certain shape, color, and melting point, which can be used as the basis for the preliminary determination of the purity. Generally, the crystal shape of a specific compound under the same solvent is consistent, the color is pure, and has a short melting range (generally at 1~2°C). But for compounds with double melting points or amorphous substances, the purity cannot be determined by this method.
TLC, such as silica gel and paper chromatography, is the most commonly used method to determine the purity of compounds. Generally, a specific sample, showing an only spot (Rf value at 0.2~0.8) in three different developing agents, could be considered as a pure compound. In some cases, both normal and reverse phase chromatographic methods are needed.
GC and HPLC are important methods in the purity determination of phytochemicals. GC is widely used in the analysis of volatile compounds. Both volatile and nonvolatile substances could be analyzed with HPLC, which possesses various advantages of high speed, high efficiency, sensitivity, and accuracy.
The general procedures of structural determination of phytochemicals are shown roughly in Figure 2.
The main procedures for studying the structures of phytochemicals.
The structural identification of phytochemicals can be greatly simplified according to the researchers’ habits, experiences, and skill levels of different technologies. However, the literature search almost runs through the whole process of structural research, no matter for known or new compounds. A large number of facts have been proved that taxonomically related plants, that is to say, plants of same or similar genus often contain chemical constituents of similar or even same chemical structures. Therefore, it is necessary to investigate literatures of chemical studies of the study object and the plants of its same and similar genera. It is necessary to understand not only the components from different plants of similar genera, but also their extraction methods, physicochemical properties, spectral data, and biosynthesis pathways before the extraction and separation of one specific plant. The SciFinder Scholar database is used most widely to quickly determine whether the compound was “known” or “unknown”.
At present, spectrum analyses have become the main means to determine the chemical structures of plant chemicals. Particularly, with the developing of the superconducting nuclear magnetic resonance (NMR) and mass spectroscopic (MS) technologies, the speed of structural determination is greatly accelerated and the accuracy is improved. Here, the applications of infrared (IR), ultraviolet (UV), nuclear magnetic resonance (NMR), and mass (MS) spectra in the structural identification of phytochemicals are introduced briefly.
UV-vis spectrum is a kind of electron transition spectrum, which is generated after the molecules absorbing the electromagnetic waves with wavelength at the range of 200–800 nm. The valence electrons in the molecules absorb light of certain wavelengths and jump to the excited state from the ground state, and then UV spectra are recorded.
Compounds containing conjugated double bonds, α,β-unsaturated carbonyl groups (aldehydes, ketones, acids, and esters), and aromatic compounds could show strong absorption in UV spectra because of n → π* or π → π* transitions. Therefore, UV spectrum is mainly used to identify the presence of conjugated systems in the structures.
UV spectra could provide the following information: (1) the compounds show no UV absorption at 220–800 nm, indicating the compounds were aliphatic hydrocarbons, aliphatic cyclic hydrocarbons, or their simple derivatives. (2) The compounds show strong absorption at 220–250 nm, indicating that the compounds possess conjugated diene, α,β-unsaturated aldehyde, or ketone substructures. (3) The absorption at 250–290 nm is moderately strong, indicating that the compounds possess benzene rings or aromatic heterocycles. (4) Weak absorption at 250–350 nm indicates the presence of carbonyl or conjugated carbonyl groups. (5) Strong absorptions at above 300 nm indicate that the structures possess long conjugated chains.
Generally, UV spectrum can only provide part of the structural information, rather than the whole structural information of a compound, so it can only be used as an auxiliary method to identify the structures. It possesses practical value to determine the structures of phytochemicals with conjugated substructures.
IR is caused by the vibration-rotational energy level transition of the molecule, ranging from 4000 to 625 cm−1. The region above 1250 cm−1 is functional group region, and the absorption of characteristic functional groups such as hydroxyl, amino, carbonyl, and aromatic rings occurs in this region. The region of 1250 to 625 cm−1 is fingerprint region, and the peaks appear mainly due to the stretching vibrations of C-X (X = C, O, N) single bonds, and various bending vibrations. IR is mainly used for the determination of functional groups and the types of aromatic ring substitution. In some cases, IR can also be used to determine the configuration of plant chemical constituents. For example, there is a significant difference between 960 and 900 cm−1 for 25R and 25S spirostanol saponins.
In a mass spectrometer, mass and strength information of molecular and fragment ions is recorded after the molecules are ionized and enter into the collector under the action of electric and magnetic fields. The abscissa represents the mass-to-charge ratio (m/z) and the ordinate represents the relative intensity in a MS spectrum. Unlike IR, UV, and NMR spectra, MS is mass spectrum, which characterizes fragment ions, not an absorption spectrum. Its role is to determine weights, formulas, and fragment structures of molecules.
With the rapid development of modern techniques, new ion sources have emerged in recent years, which make MS play more important role in determining the molecular weights, elemental composition, detecting functional groups by cleavage fragments, identifying compound types, and determining carbon skeletons [25]. In the structural analysis, the information of molecular weights could be obtained on the basis of molecular ion peaks, and the molecular formula could be obtained by high-resolution mass spectrometry (HR-MS). Fragment ion peaks, combined with molecular ion peak, could be applied to conjecture chemical structures. Tandem mass spectrometry even can isolate and analyze the mixed ions again. According to the types of ion sources, common mass spectrometry could classified as electron impact mass spectrometry (EI-MS), chemical ionization mass spectrometry (CI-MS), field desorption mass spectrometry (FD-MS), fast atom bombardment mass spectrometry (FAB-MS), matrix-assisted laser desorption mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS–MS), and so on.
With the birth of Fourier transform spectrometer, the great progress of radionuclide research such as 1H, 13C, 15N, 19F, 31P, and the advancement of two-dimensional and three-dimensional nuclear magnetic technology, NMR has become the most important spectroscopic method to determine chemical structures. Particularly, hydrogen spectrum and carbon spectrum are most widely used. During the operation of nuclear magnetic resonance spectrometer, compound molecules are irradiated by electromagnetic waves in a magnetic field, energy level transitions occur after the atomic nuclei with magnetic distance absorb a certain amount of energy, and then NMR spectrum is obtained by mapping the absorption strength with the frequencies of the absorption peaks. It can provide structural information about the type and number of hydrogen and carbon atoms in the molecule, the modes they are connected, the surrounding chemical environment, configuration, and conformation [26].
Samples used to measure NMR spectra include solids, liquids, and gases. Liquid high-resolution NMR is most widely used. The solvent used in the measurement of NMR must be deuterated. The commonly used deuterated reagents to dissolve samples and their chemical shifts of their residual proton and carbon signals are shown in Table 1.
Solvent | ||
---|---|---|
CDCl3 | 77.0 | 7.24 |
CD2Cl2 | 53.8 | 5.32 |
CD3OD | 49.0 | 3.3 |
Acetone- | 29.8, 206.0 | 2.04 |
D2O | — | 4.7 |
DMSO- | 39.5 | 2.49 |
C6D6 | 128.0 | 7.16 |
C5D5N | 123.6135.6149.9 | 7.2, 7.6, 8.7 |
Chemical shifts of common deuterated solvents (TMS is an internal standard).
Resonance absorption peaks are generated after hydrogen protons absorb electromagnetic waves of different frequencies in an external magnetic field. 1H-NMR possesses high sensitivity, easy measurement, and wide application. 1H-NMR spectrum can provide structural information of chemical shifts (
Because of the different surrounding chemical environment, the 1H nuclei possess different magnetic cloud densities and magnetic shielding effects caused by the rotation around the nucleus, and then different types of 1H nuclear resonance signals appear in different regions. Tetramethylsilane (TMS) is usually used as a reference compound. Compared with the general compounds, the shielding effect of protons and carbons on the methyl groups is stronger in TMS. Therefore, regardless of the hydrogen spectrum or the carbon spectrum, the absorption peaks generated by the general compounds appear in the lower field than TMS, that is to say,
1H-NMR chemical shift range of common hydrogen protons.
In addition to the normal 1H-NMR spectrum technique, there are some auxiliary techniques that assist in structural analysis, such as selective decoupling, heavy hydrogen exchange, addition of reaction reagents, and dual irradiations.
13C-NMR spectra can provide structural information of organic compounds, including the number, types, and chemical environment of carbon atoms [27]. It is one of the important means for the structural identification of organic compounds. Especially, where there are serious signal peak overlaps in the 1H-NMR spectrum, or the molecules contain several quaternary carbon atoms, 13C-NMR spectra will provide crucial information for the structure identification. The chemical shifts of common carbon signals are shown in Figure 4 [4].
13C-NMR chemical shifts of common carbon signals.
Common 13C-NMR techniques include proton broadband decoupling, off resonance decoupling (OFR), insensitive nuclei enhanced by polarization transfer (INEPT), and distortionless enhancement by polarization transfer (DEPT). Proton broadband decoupling and DEPT spectra are most commonly used at present.
Proton broadband decoupling spectrum is measured after 1H nuclei are saturated with broadband electromagnetic radiation. At this point, the couplings between 1H and 13C are completely eliminated, and all 13C signals are shown as singlets, so it is very convenient to determine the chemical shift of 13C signals. In addition, because of the NOE effect of 1H after irradiation, the signal of 13C signal connected with 1H will be increased, while the quarterly carbon signal will show weak absorption peaks.
It is an improved method of INEPT, in which a
The DEPT spectrum of Arctiin (CD3OD).
Two-dimensional correlation spectroscopy (2D-COSY) is the most important and widely used in 2D-NMR spectroscopy. 2D-COSY spectra can be divided into homonuclear and heteronuclear correlation spectra. Both abscissa and ordinate represent chemical shifts in 2D-COSY. Common correlation spectrum types are show as follows.
It is a kind of chemical shift correlation spectrum between 1H and 1H. It is the coupling correlation spectrum between protons in the same coupling system. The adjacent hydrogen groups could be determined by their coupling relationships (3
In addition, for compounds of aromatic systems, double bond systems, and some particular configuration systems, 1H-1H COSY spectra can show 4
1H detected heteronuclear single quantum coherence (HSQC) and 1H detected heteronuclear multiple quantum coherence (HMQC) can display the correlations between 1H and 13C. HSQC possesses higher sensitivity and wider application than HMQC. In the HMQC or HSQC spectrum, the signals occurred at the crosses of chemical shifts generated by corresponding carbons and protons (Figure 6).
Schematic diagram of correlations between 1H and 13C in the HSQC or HMQC spectrum.
HMBC spectrum is short for 1H detected heteronuclear multiple bond correlation, which associates the 1H nucleus with 13C nucleus of long-range coupling. HMBC could detect the long-range coupling of 1H-13C sensitively (n
Schematic diagram of correlations between 1H and 13C in the HMBC spectrum.
When two groups of protons are located at rather close spatial distances, irradiation of one group will enhance the signal strength of another, which is known as nuclear Overhauser enhancement (NOE). The NOE spectrum can determine the spatial relative position, stereoscopic configuration, and dominant conformation of some groups in the molecule, which is very important for the study of the stereostructures of organic compounds.
2D-NOE (NOESY) spectra could show the NOE correlations of protons. The greatest advantage of NOESY is that all the NOE information between protons of a compound could be shown in one spectrum. However, not all the cross peaks are NOE correlation signals, the residual correlation signals of COSY are often shown in NOESY spectrum as well, which should be paid attention during spectroscopic analysis.
The TOCSY spectrum shows the correlation of the entire spin system, which is different from the ordinary 1H-1H COSY. The relationships between the nuclei that generated the correlation peaks are shown in Figure 8. Not only the correlation signals of a proton with protons connected to the adjacent carbons, but also its correlation signals with other protons in a whole spin system could be shown in the TOCSY spectrum, which provides important basis for the connection of structural fragments.
Schematic diagram of correlations between 1H and 13C in the TOCSY spectrum.
HSQC-TOCSY is a kind of combined 2D-NMR spectrum. Comprehensive results of HSQC and HMBC are obtained by using a long pulse sequence. The correlation is shown in Figure 9. It is very useful for the assignment of carbon and proton signals in complex chemical structures. For example, for saponins with a series of glycosyl groups, the signals generated by glycosyl groups are often overlapped seriously in common NMR spectra, which causes difficulty to assign signals of glycosyls. HSQC-TOCSY spectrum will play an important role in this case. The spectrum includes the information of HSQC, HMBC, and 1H-1H COSY.
Schematic diagram of correlations between 1H and 13C in the HSQC-TOCSY spectrum.
Polarimetry is an optical method used widely in the studies of asymmetric structures, which appeared very early. The progress of the sensitive method such as ORD and CD made it possible to study stereostructures of chiral compounds more deeply. Both of them are spectra related to the optical activity of compounds, and could provide information of absolute configurations, dominant conformations, and reaction mechanisms of chiral compounds, that cannot be replaced by any other spectroscopic methods [28].
The specific rotation [α] of a chiral compound depends upon the wavelength of the monochromatic light wave. The measurement of specific rotation as a function of wavelength is called optical rotator dispersion (ORD). The common types of ORD curves are as follows.
The ORD spectrum of an optically active compound with no chromophores is plain without peaks and troughs. An ORD curve of specific rotation increases with decrease of wavelength which is called positive plain curve, while in the case of negative plain curve, negative rotation increases with decrease of wavelength (see Figure 10).
ORD plain curves (A: Positive plain curve; B: Negative plain curve).
If there is a simple chromophore in the molecule, the ORD curve is very different from plain curve. Near the absorption wavelength region of chromophore, a peak and a trough are exhibited, which is called the Cotton effect, and the spectrum drawn is called the Cotton effect curve. The spectrum with only one peak and one trough is called pure Cotton effect curve, while the spectrum with several peaks and troughs is called complex Cotton effect curve. The Cotton effect is called positive when the trough is observed at a shorter wavelength then peak. Conversely, the Cotton effect is called negative if the trough is observed at a longer wavelength than the peak. Cotton curves of △5-cholestenone are shown in Figure 11, which shows A and B possess the same structural formula, while different opposite configurations.
The Cotton effect curves of △5-cholestenone (A) natural cholesterone (+) cotton; (B) Cholesterone in the opposite absolute configuration (−) cotton.
For compound with two or more different chromophores, its ORD curve may possess multiple peaks and troughs, which is called complex Cotton effect curve. Each ORD curve is the average effect of each chromophore in the molecule, and the contribution of each orientation and conformation of the molecule. Hence the Cotton effect curve is often complex.
Optically active compounds have different molar absorption coefficients for left-circularly and right-circularly polarized light that make up plane polarized light, which is called circular dichroism (CD). The difference value between the two molar absorption coefficients (Δє = єL−єR) changes with the wavelength of the incident polarized light. With Δє as the ordinate, the wavelength as the abscissa, the spectrum obtained is called circular dichroism spectrum. Because the absolute value of Δє is very small, it is often replaced by molar ellipticity [
Because Δє could be positive or negative, the circular dichroism curve also could be classified as positive and negative. In the CD spectrum showing positive Cotton effect, only a peak appears near the λmax of the chromophore in the molecule. Conversely, a trough appears in the CD spectrum showing negative Cotton effect. Therefore, CD spectra are simpler and easier to analyze than ORD spectra. For example, the ORD and CD spectra of (+)-camphor are shown in Figure 12. CD is more widely used than ORD in the study of chiral compounds.
The ORD and CD spectrum of (+)-camphor.
Single crystal X-ray diffraction could be applied independently to analyze the structures, components, contents, configurations, conformations, solvents, and crystal forms of samples. It is widely used in the stereostructural study of natural compounds, synthetic compounds, peptides, proteins, etc. Therefore, X-ray diffraction analysis is a necessary physical method in the field of structure and function research of modern natural drugs.
Single crystal X-ray diffraction is a kind of quantitative analysis technology, which can provide three-dimensional structural information of molecules, including atomic coordinates, bond length, bond angles, dihedral angles, hydrogen bonds, salt bonds, coordinate bonds, and so on. In addition, it is also a reliable method to determine the absolute configuration of chiral drug molecules and the epimers in the stereochemical structures. For example, see [29].
In recent years, study on phytochemicals from plants becomes more and more popular due to their demonstrated health benefits. A number of plants having high contents of phytochemicals (particularly phenolic acids and flavonoids) with associated antioxidant activities have been increasingly utilized. Complementary research is also needed to enhance the potential functionalities of the phytochemicals in future, where such plants have shown to contain numerous phytochemicals that may be beneficial to human health. The compiled results indicated that many of their bioactive compounds remain to be fully isolated, identified, and characterized (alkaloids, diterpenoids, and so on).
Therefore, phytochemicals can be considered as the source of natural medicines. The compounds of plants are bioaccessible and bioavailable in humans with some demonstrated health benefits, including antioxidant, anti-inflammatory, anti-cancer, anti-microbial, hypoglycemic action, etc. Additional well-designed human intervention studies and clinical trials are needed to validate the health benefits of phytochemicals.
In women, breast cancer is a prevalent cause of cancer worldwide [1]. It affected 2.3 million women globally in 2020, with 685,000 deaths. It has been diagnosed in 7.8 million women in the past 5 years, making it the most common type of cancer in the world [1]. Although breast cancer diagnostic methods and therapeutic procedures have improved in the past decade, the long-term survival of these patients remains low due to a high rate of postsurgical relapse. The efficacy of breast cancer treatment is limited by drug toxicity, multidrug resistance, and a lack of definitive prognostic biomarkers [2]. Thus, there is an urgent need to develop novel biomarkers and therapeutics to cure the disease.
In recent years, many studies have suggested that intercellular communication plays a key role in driving various cellular functions and homeostasis in physiological as well as pathological conditions such as cancer, cardiovascular diseases, and neurological disorders. Cancer development is mainly dependent on interactions between cancerous cells and their microenvironment components. Some of these interactions are mediated by extracellular vesicles, which alter the phenotype of recipient cells [3, 4, 5].
Extracellular vesicles (EVs) are spherical nanoparticles shed by all types of cells, including archaea, prokaryotes, eukaryotes, and fungi in the extracellular milieu [6]. These typically range from 30 nm to 5 μm in diameter based on their type and vary widely in composition [7]. In addition to being released during disease pathology, EVs allow various cells to send and receive messages to crosstalk with other cells, thus carrying out various biological functions [7]. These are mainly composed of different proteins, lipids, nucleic acids, and enzymes [8]. EVs circulate through many body fluids, such as blood, serum, and urine. Owing to their structural similarity to the parental source, they are considered potential biomarkers for diseases such as cancer [9]. To study the characteristics and functions of EVs, they are isolated using different techniques such as differential ultracentrifugation, size-exclusion, and ultrafiltration [10].
EVs are generally categorized into exosomes, microvesicles, and apoptotic bodies according to their release mechanism, size, and composition [3]. Exosomes are 30–150 nm in diameter and are formed by inward budding of the plasma membrane of the cell [9]. Microvesicles are formed by direct outward budding of the cell’s plasma membrane and range in size from 100 to 1000 nm in diameter. Consequently, they are reported to contain mainly cytosolic and plasma membrane proteins, such as tetraspanins. Apoptotic bodies are shed during cell death into the extracellular space, ranging from 50 to 5000 nm in diameter. These generally contain intact organelles, glycosylated proteins, and chromatin, unlike the other two types of EVs. Among these, exosomes have been widely studied since their role in intercellular communication has been reported. This chapter will focus on exosomes and their potential applications as therapeutics for breast cancer.
Exosomes are generated by the endocytic pathway from late endosomes (LE) [11, 12]. LEs are formed by inward budding of the multivesicular body (MVB) membrane. LE membranes invaginate to form intraluminal vesicles (ILVs) within MVBs. During this process, some proteins are engulfed and packaged within the ILVs. ILVs then fuse with the cell’s plasma membrane and release the vesicles into the extracellular space.
As reported previously, the formation of ILVs can occur either dependent or independent of the ESCRT complex. The ESCRT complex is a set of proteins that function together to facilitate the formation of MVBs, vesicle release, and protein cargo sorting [13, 14, 15]. ESCRT 0 has two subunits, HRS and STAM ½, which bind together and recognize specific ubiquitinated proteins in early endosomes. This leads to the recruitment of ESCRT 1 containing Tsg 101, Vps28, Vps37, and Mvb 12, which further recruits ESCRT II. ESCRT II is composed of four subunits, Vps22-EAP30, Vps36—EAP45 and Vps25—EAP20 which starts the invagination of endosomal membranes encapsulating different molecules/cargo such as proteins and nucleic acids. The ESCRT II subunit Vsp25 then binds with Vsp20 to activate and recruit ESCRT III. It deubiquitinates proteins and allows complete membrane invagination, generating ILVs. Other adaptor proteins such as Vps4 interact with ESCRT III to finally start budding of the membrane, ECSRT subunit removal, and cargo delivery. Hence, the ESCRT complex regulates the whole process of vesicle budding and cargo sorting into exosomes [16, 17]. In cancer, an increased amount of exosomes is often observed in the bodily fluids of cancer patients as a result of deregulation of exosomal formation and secretion [18]. Specifically, in breast cancer, the amount of exosomes released by the human tumor cell line B42 clone 16 was much larger than that released by the parental normal mammary epithelial cells (HMEC B42), as shown by Azmi et al. [19].
Exosomes are composed of a heterogeneous set of cytosolic, nuclear, mitochondrial, ribosomal, and membrane-bound proteins derived from donor cells [20]. Some of these proteins are conserved irrespective of their origin; therefore, they are considered exosomal markers such as tetraspanins, ESCRT proteins, and major histocompatibility complex (MHC) molecules [21]. In addition, some proteins are related to the phenotype of producing cells, such as cancer-derived exosomes, which in turn determines their biological mechanisms. The lipid bilayer membrane of exosomes contains transmembrane proteins, transporter proteins, adhesion molecules, and lipid raft-associated proteins. Exosomes contain nucleic acids such as DNA (ssDNA, mtDNA, dsDNA, and RNA (mRNA, miRNA, and lncRNA) [22]. Exosomal miRNAs and mRNAs are transferred from donor cells to recipient cells, thus modulating the latter’s phenotype. Although there are numerous reports indicating the presence of DNA within exosomes, the mechanisms leading to this phenomenon remain unclear. Exosomes also exhibit an exclusive set of lipids distributed in their bilayers, such as sphingolipids, arachidonic acid, cholesterol, phosphatidylserine, and ganglioside [23, 24]. Lipids such as lysobisphosphatidic acid are abundant in the inner membranes of multivesicular bodies and play a crucial role in exosome formation [25, 26]. ExoCarta is a database containing all the data on exosomal content, with over 47,000 protein, mRNA, and lipid entries. Furthermore, ExoCarta is an excellent source of information for exosome characterization (Figure 1) [27].
Schematic representation of exosome biogenesis and composition. Exosomes originate from multivesicular bodies and shed into extracellular space packaging motley of proteins such as ESCRT associated protein, chaperones, along with ssDNA, RNA and dsDNA, miRNA and lncRNA.
Metastasis is the process by which primary tumor cells/tumor cells/cancer cells invade the surrounding tissues and colonize the blood vessels to proliferate and give rise to the tumor [28]. Controlling metastasis, which is mainly responsible for high patient mortality, is the main challenge in breast cancer therapy. Hence, several investigations are ongoing to understand the molecular mechanisms underlying metastasis in breast cancer. Recently, exosomes have attracted great attention as key players in regulating complex intracellular pathways from initiation to progression to metastasis in the development of breast cancer [29, 30, 31]. These mainly interact with the recipient cells in three ways: direct fusion with the cell membrane, interaction with the surface receptors, or internalization via endocytosis. Upon cellular uptake, exosomes deliver their cargo and initiate a cascade of events leading to various biological functions. Many breast cancer cell lines have been shown to release exosomes containing several proteins with signaling molecules, miRNAs, and long non-coding RNAs involved in migration, invasion, angiogenesis, and metastasis [32, 33, 34]. Proteomic profiling of exosomes secreted from breast cancer cell lines was shown to contain matrix metalloproteinases, which might be linked to the enhanced metastatic properties of breast cancer cells [32]. These findings suggest that exosomes act as key mediators in the tumor microenvironment by communicating various signaling molecules essential for breast cancer development [31].
Exosome-mediated transfer of genetic material from breast cancer cells has been shown to mediate resistance to chemotherapy and enhance tumor growth [35, 36]. Accumulating evidence suggests that exosomes may also play a role in the resistance of breast cancer radiotherapy and cancer immunotherapy [37, 38]. In breast cancer, drug-resistant cancer cells transmit resistance in drug-sensitive cells via the intercellular horizontal transfer of exosomal miRNAs [38]. Exosomes also transfer the drug efflux pump from docetaxel-resistant to sensitive ones in MCF-7 breast cancer cells [39]. Lv MM et al. showed that exosomes from drug-resistant cancer cells contain miRNAs that alter the phenotype of recipient breast cancer cells by altering their transcriptome [40]. Exosomes from stromal fibroblasts transmit non-coding RNA to breast cancer cells, thus contributing to treatment resistance by expanding therapy-resistant cells [41]. Thus, exosomes contribute to drug resistance in breast cancer.
The role of exosomes in carcinogenesis has been extensively investigated in recent years. Cancer cells have been shown to use exosomes as a novel mechanism to transfer the malignant phenotype to normal healthy cells and establish a niche for tumor growth. Cancer cell-derived exosomes are reported to contain miRNAs, proteins, or long non-coding RNAs that mediate cancer development, growth, and progression [42, 43]. Exosomes derived from breast cancer cells contain a variety of proteins and RNAs that are transmitted among these cells as well as normal cells, thus altering the phenotype of healthy mammary epithelial cells. Wang J et al. showed that cancer exosomes were able to transform normal mammary epithelial cells into cancerous cells via transfer of microRNAs packaged within exosomes [44]. Similarly, Melo et al. showed enhanced expression of exosomal miR-10b in metastatic breast cells compared to non-metastatic or non-malignant breast cells [45]. Thus, it can be used as a therapeutic target for breast cancer therapy.
These characteristics make exosomes ideal biomarkers, and exosomal profiling in the absence of tissue holds great promise for early diagnosis. Owing to their crucial functional role in breast cancer, exosomes have been investigated for their potential development as breast cancer biomarkers and therapeutic targets. Singh R et al. have shown that psoralen reduces the formation and secretion of exosomes, thus reversing multidrug resistance in breast cancer cells [46]. The presence of diverse content within and on the surface of exosomes has led to their application as biomarkers, diagnostics, and drug delivery. A large number of exosomes circulate within bodily fluids of not only healthy individuals but also cancer patients, according to some studies. Since exosomes play various significant roles in breast cancer, exosomes can be developed as potential therapeutic agents in biomarkers, diagnostics, and drug delivery. Kumar et al. investigated the release of exosomes from breast cancer stem cells to characterize their constituent exosomal markers. They detected tetraspanin proteins, Alix, and tumor susceptibility gene-101 (TSG101) in breast cancer stem cell-derived exosomes. This study indicates that secreted exosomes can be utilized as biomarkers for breast cancer to understand their development, progression, and metastasis [47]. Kumar et al. showed that miRNAs 155 and 205 are expressed in serum exosomes derived from breast cancer cells and modulate the epithelial-to-mesenchymal transition (EMT), growth, and metastasis of cancer, suggesting their employability as breast cancer biomarkers [48]. Zhang et al. studied the role of long non-coding RNA MALAT 1 which is highly expressed in exosomes derived from breast cancer cells in tumor progression, representing a potential treatment strategy for breast cancer [49]. Dong et al. investigated the role of exosomal long non-coding RNA in the chemoresistance of HER2+ breast cancer cells. They found that exosomal lncRNA-SNHG14 was not only upregulated in trastuzumab-resistant cells but also transmitted the lncRNA into drug-sensitive cells, thus disseminating trastuzumab resistance. Furthermore, when compared to patients who responded to trastuzumab, the expression level of serum exosomal lncRNA-SNHG14 was higher in patients who were resistant. This suggests that lncRNA-SNHG14 is a promising therapeutic target for HER2+ breast cancer patients [50].
Exosomes are emerging as promising therapeutic agents because of their role in tumor-related processes and their ability to deliver their cargo, such as proteins, lipids, and nucleic acids, into the tumor sites. However, their full clinical applicability has not yet been realized. This is because of many factors, including low yield and relatively low percentage loading to the therapeutic moiety. As such, new approaches for mass production and enhancement of the percent loading need to be explored. In general, these approaches are divided into two categories: passive and active loading, which are discussed in detail in the following sections.
Direct modification, also known as non-cell-based loading or exogenous loading, refers to the direct loading of therapeutic moieties such as siRNA, miRNA, drugs, and proteins after the isolation and purification of exosomes from the cells. This may encompass a series of procedures such as incubation, freeze-thaw cycles, sonication, and electroporation, and thus can further be categorized into passive and active loading. Passive loading includes loading of therapeutic moiety into exosomes by diffusion; on the other hand, active loading includes disrupting the exosomal membrane by electroporation, sonication, or freeze thawing, thus allowing the therapeutic moiety to enter into these vesicles. In passive drug loading, exosomes are incubated with drugs and allowed to diffuse into vesicles along a concentration gradient. Because exosomes consist of a lipid bilayer, the drug loading efficiency depends largely on the hydrophobicity of the drugs. Dong et al. loaded curcumin into milk exosomes by incubating at 4°C overnight and reported 70.46% drug loading using an incubation method [51]. Similarly, Sun et al. incorporated curcumin into exosomes derived from a mouse lymphoma cell line by incubating in PBS at room temperature (22°C) for 5 min and showed a binding capacity of 2.9 g curcumin to 1 g of exosomes [52]. Sun et al. packaged Cho-miR159 (cholesterol-modified miRNA 159) along with doxorubicin into exosomes derived from the human monocytic cell line THP-1 by incubating in PBS at 37°C to deliver to triple-negative breast cancer cells [53]. Linezolid was incorporated into exosomes derived from the mouse macrophage cell line RAW 264.7, by mixing both and incubating at 37°C for 1 h, resulting in ~5% drug loading. The exosomal formulation of linezolid was more effective against MRSA infections than the free drug [54]. Although several studies have reported the use of incubation with exosomes for drug or any therapeutic agent loading, it often suffers from issues of low percent drug loading, urging a requirement for improved methods for higher drug loading percent. Another method (less common) of passive loading includes incubating the exosome donor cells with the drugs/therapeutic agents. First, the donor cells are exposed to drugs or therapeutic agents, followed by isolation of released exosomes (supposedly) containing the loaded drugs or therapeutic agents. This method was used in a study by Pascucci et al., wherein they exposed bone marrow-derived mesenchymal stromal cells (MSCs) with a very high concentration of paclitaxel followed by incubation at 37°C for 24 h. After incubation, the cells were washed twice with PBS, trypsinized, and seeded in a fresh flask for 48 h. After 48 h, cell-conditioned medium was collected to isolate exosomes containing paclitaxel. They found that MSC-PTX-derived exosomes had a greater inhibitory effect on tumor cell proliferation (Figure 2) [55].
Illustration representing different methods of cargo loading in exosomes. (A) Passive cargo loading is achieved by incubating the therapeutic moiety directly with isolated exosomes or by exposing to the exosome secreting donor cells followed by isolation of loaded exosomes. (B) Active cargo loading methods include use of physical treatments to disrupt the membrane integrity thus allowing entry of cargo in the interiors of exosomes. These treatments include sonication, electroporation, freeze thawing cycles and extrusion method.
For active cargo loading, the exosomal membrane is temporarily disrupted using different methods and then restored once the drug/therapeutic agent was loaded. These methods may include sonication, extrusion, freeze-thawing, electroporation, use of membrane permeabilizers, conjugation using click chemistry, and antibodies against exosomal surface proteins. Electroporation uses an electric field to generate small pores in the exosomal membrane to disturb the phospholipid bilayer of exosomes. Drug/therapeutic agents can enter these vesicles via the generated pores. Once they entered, the pores were closed to recover the exosomal membrane integrity. This method has mostly been used to encapsulate siRNA or miRNA into exosomes and has been reported to enhance the percent loading compared to the simple diffusion method. Jia et al. loaded exosomes derived from RAW 264.7 cells with curcumin and superparamagnetic iron oxide nanoparticles (SPIONs) synchronously using optimal electroporation conditions of 400 V, 150 μF, and 1 ms discharge time. They observed that electroporation had no effect on the membrane integrity of exosomes and efficiently encapsulated curcumin and SPIONs [56]. Similarly, Jia et al. incorporated doxorubicin into exosomes isolated from MDA-MB-231 and HCT-116 cell lines using an electroporation method, which resulted in ~1.5% drug loading [57]. According to published studies, although electroporation enhanced the percentage of drug loading in exosomes compared to the incubation method, it was still low. Therefore, scientists have employed sonication methods to load cargo more efficiently. The mechanical shear force of a sonicator/homogenizer probe is applied to disrupt the membrane integrity of exosomes, thus allowing the mixed drug/therapeutic agent to enter into the exosomes. In 2017, Kim et al. compared the incubation, electroporation, and sonication method of cargo loading in RAW264.7 cell derived exosomes to develop an exosomal formulation of paclitaxel (PTX). For the incubation method, the authors mixed and incubated PTX with exosomes at 37°C for 1 h. Using electroporation, exosomes and PTX were added to a pre-chilled electroporation cuvette and applied at 1000 kV for 5 ms followed by incubation at 37°C for half an hour to fully recover the exosome membrane. For sonication, the PTX-exosome mixture was sonicated at 20% amplitude, given 6 cycles of 30 s on/off for 3 min and a 2 min cooling period between each cycle. After sonication, the solution was incubated at 37°C for 1 h to fully recover the membrane of the exosomes. They showed the highest percent drug loading of ~28% using sonication followed by ~5% using electroporation and the lowest at ~1.4% with the incubation method [58].
In the extrusion method, exosomes mixed with the drug are passed through a syringe-based lipid extruder with a membrane ranging from to 10 to 400 nm pore size. In this process, the membrane of exosomes is disrupted by the extensive mechanical force of the extruder. In a study by Kim et al. when breast cancer cell-derived exosomes loaded with porphyrin were extruded, it altered the surface charge of blank exosomes, leading to cytotoxic effects [59]. On the other hand, in another study by Fuhrmann et al., loading cargo in exosomes using the extrusion method did not render them cytotoxic [60]. In the freeze-thaw method, the drug was first incubated with exosomes at ambient temperature and then frozen at −80°C. The mixture was then repeatedly thawed at room temperature to ensure drug loading into these vesicles. The main disadvantage is that this method often leads to particle aggregation, resulting in a wide size distribution. This method has also been reported to result in a lower percent drug loading than other methods, such as sonication.
Although exosomes have been shown to mediate cancer development, they are an emerging platform for drug delivery to cancerous sites because of their excellent biocompatibility, low immunogenicity (since they are derived from the patient’s own cells), good tolerance, and remarkable biodistribution. Owing to their small size, they can readily pass through different bodily barriers such as the blood-brain barrier [61]. Compared to synthetic nanoparticles, exosomes are relatively easy to manipulate through surface modification in order to enhance their targeting efficiency to cancer cells. Recently, the use of exosomes for drug delivery in breast cancer cells has been proven to be efficient. Alvarez-Erviti et al. delivered the chemotherapeutic drug doxorubicin to breast cancer tissues in a mouse model [62]. First, they engineered these cells by expressing Lamp2b, a lysosome-associated membrane glycoprotein 2b, on their surface and fused with a targeting peptide for integrins. They then isolated exosomes from immature dendritic cells (with low immunogenicity because of the absence of immunostimulatory markers on their surface) and used an electroporation technique to load doxorubicin within. They have shown that the exosomal formulation of doxorubicin has greater efficiency in targeting mouse tumors and hence, exhibits a novel propitious approach in breast cancer treatment in the clinical context. Li et al. loaded milk exosomes with doxorubicin to target CD44 overexpressed human breast cancer cell lines and found an exosomal formulation capable of delivering the drug into cancerous sites in a target-specific manner [63]. Vakshiteh et al. used dental pulp-derived mesenchymal stem cells to isolate exosomes and loaded them with miRNA, which was then targeted to breast cancer cells. They found that exosomes significantly decreased the proliferation of cancer cells and reduced the migratory and invasive properties of breast cancer cells
Currently, there are some hurdles in realizing the clinical potential of exosomes as drug delivery nanovehicles. These include low yield, long-term stability, and lack of understanding of their therapeutic effects. Hence, more research is required to develop techniques that can be used universally to enhance the yield in a time-efficient manner and increase the stability of exosomes.
The authors declare that they have no competing interests.
IntechOpen - where academia and industry create content with global impact
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\\n\\nAdrian Assad De Marco
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\n\nSara Uhac, COO
\n\nSara Uhac was appointed Managing Director of IntechOpen at the beginning of 2014. She directs and controls the company’s operations. Sara joined IntechOpen in 2010 as Head of Journal Publishing, a new strategically underdeveloped department at that time. After obtaining a Master's degree in Media Management, she completed her Ph.D. at the University of Lugano, Switzerland. She holds a BA in Financial Market Management from the Bocconi University in Milan, Italy, where she started her career in the American publishing house Condé Nast and further collaborated with the UK-based publishing company Time Out. Sara was awarded a professional degree in Publishing from Yale University (2012). She is a member of the professional branch association of "Publishers, Designers and Graphic Artists" at the Croatian Chamber of Commerce.
\n\nAdrian Assad De Marco
\n\nAdrian Assad De Marco joined the company as a Director in 2017. With his extensive experience in management, acquired while working for regional and global leaders, he took over direction and control of all the company's publishing processes. Adrian holds a degree in Economy and Management from the University of Zagreb, School of Economics, Croatia. A former sportsman, he continually strives to develop his skills through professional courses and specializations such as NLP (Neuro-linguistic programming).
\n\nDr Alex Lazinica
\n\nAlex Lazinica is co-founder and Board member of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his Ph.D. in Robotics at the Vienna University of Technology. There, he worked as a robotics researcher with the university's Intelligent Manufacturing Systems Group, as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and, most importantly, co-founded and built the International Journal of Advanced Robotic Systems, the world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career since it proved to be the pathway to the foundation of IntechOpen with its focus on addressing academic researchers’ needs. Alex personifies many of IntechOpen´s key values, including the commitment to developing mutual trust, openness, and a spirit of entrepreneurialism. Today, his focus is on defining the growth and development strategy for the company.
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Catarina Guedes and F. Xavier Malcata",authors:[{id:"83136",title:"Prof.",name:"F. Xavier",middleName:null,surname:"Malcata",slug:"f.-xavier-malcata",fullName:"F. 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Labella, C. Berbel, M. Manchado, D. Castro and J.J. Borrego",authors:[{id:"67855",title:"Prof.",name:"Juan J.",middleName:null,surname:"Borrego",slug:"juan-j.-borrego",fullName:"Juan J. Borrego"},{id:"71146",title:"Dr.",name:"Alejandro",middleName:null,surname:"Labella",slug:"alejandro-labella",fullName:"Alejandro Labella"},{id:"71148",title:"Dr.",name:"Concepcion",middleName:null,surname:"Berbel",slug:"concepcion-berbel",fullName:"Concepcion Berbel"},{id:"71149",title:"Dr.",name:"Manuel",middleName:null,surname:"Manchado",slug:"manuel-manchado",fullName:"Manuel Manchado"},{id:"71151",title:"Dr.",name:"Dolores",middleName:null,surname:"Castro",slug:"dolores-castro",fullName:"Dolores Castro"}]}],mostDownloadedChaptersLast30Days:[{id:"35141",title:"Antibiotics in Aquaculture – Use, Abuse and Alternatives",slug:"antibiotics-in-aquaculture-use-abuse-and-alternatives",totalDownloads:19347,totalCrossrefCites:137,totalDimensionsCites:293,abstract:null,book:{id:"2052",slug:"health-and-environment-in-aquaculture",title:"Health and Environment in Aquaculture",fullTitle:"Health and Environment in Aquaculture"},signatures:"Jaime Romero, Carmen Gloria Feijoo and Paola Navarrete",authors:[{id:"72898",title:"Dr.",name:"Jaime",middleName:null,surname:"Romero",slug:"jaime-romero",fullName:"Jaime Romero"},{id:"79684",title:"Dr.",name:"Paola",middleName:null,surname:"Navarrete",slug:"paola-navarrete",fullName:"Paola Navarrete"},{id:"83411",title:"Dr.",name:"Carmen",middleName:null,surname:"Feijoo",slug:"carmen-feijoo",fullName:"Carmen Feijoo"}]},{id:"69948",title:"Floating Cage: A New Innovation of Seaweed Culture",slug:"floating-cage-a-new-innovation-of-seaweed-culture",totalDownloads:970,totalCrossrefCites:2,totalDimensionsCites:2,abstract:"Eucheumatoid cultivation continues to expand with a variety of methods that can increase production. This chapter will discuss an innovation in seaweed cultivation of the genus Eucheuma, which is the prime marine commodity in the tropical regions of the world. Research conducted during 2015-2017 and 2019 in Southeast Sulawesi Province, Indonesia, provided an overview of the use of floating cage that showed very significant growth results. The research result showed that the growth rates of Eucheuma denticulatum and Kappaphycus alvarezii in floating cage seemed faster and resulted in better thallus morphology. Daily production of E. denticulatum and K. alvarezii that were cultivated in floating cage was higher than daily production of E. denticulatum and K. alvarezii cultivated on longline. Specific growth rate (SGR) of E. denticulatum and K. alvarezii cultivated by using floating cage method was also higher than E. denticulatum and K. alvarezii cultivated by using longline method. Moreover, the cultivation by using floating cages produces good growth rates with no effect of herbivore attacks.",book:{id:"8928",slug:"emerging-technologies-environment-and-research-for-sustainable-aquaculture",title:"Emerging Technologies, Environment and Research for Sustainable Aquaculture",fullTitle:"Emerging Technologies, Environment and Research for Sustainable Aquaculture"},signatures:"Ma’ruf Kasim, Abdul Muis Balubi, Ahmad Mustafa, Rahman Nurdin, Rahmad Sofyan Patadjai and Wardha Jalil",authors:[{id:"309893",title:"Prof.",name:"Maruf",middleName:null,surname:"Kasim",slug:"maruf-kasim",fullName:"Maruf Kasim"},{id:"313040",title:"MSc.",name:"Abdul Muis",middleName:null,surname:"Balubi",slug:"abdul-muis-balubi",fullName:"Abdul Muis Balubi"},{id:"313041",title:"MSc.",name:"Wardha",middleName:null,surname:"Jalil",slug:"wardha-jalil",fullName:"Wardha Jalil"},{id:"313042",title:"MSc.",name:"Ahmad",middleName:null,surname:"Mustafa",slug:"ahmad-mustafa",fullName:"Ahmad Mustafa"},{id:"313043",title:"MSc.",name:"Rahman",middleName:null,surname:"Nurdin",slug:"rahman-nurdin",fullName:"Rahman Nurdin"},{id:"313044",title:"MSc.",name:"Rahmat Sofyan",middleName:null,surname:"Patadjai",slug:"rahmat-sofyan-patadjai",fullName:"Rahmat Sofyan Patadjai"}]},{id:"62842",title:"Integrated Rice and Aquaculture Farming",slug:"integrated-rice-and-aquaculture-farming",totalDownloads:1915,totalCrossrefCites:2,totalDimensionsCites:3,abstract:"The burning problems like scarcity of food for ever-growing human population in the present world are addressed by adapting various methods for production of protein, carbohydrate, oils and other food materials. One of the methods to produce high amount of food is integrated farming including rice-aquaculture farming, which produces protein and carbohydrate as major components besides others. Rice-aquaculture farming produces grain (carbohydrate) and animal protein without affecting the quality and quantity of rice yield on the same piece of land and renders additional financial gain besides main crop (rice) like conventional monoculture. The aquatic species grown in the integrated culture are mainly distinct types of fishes, selected crustaceans and other selected species. Profitable rice-aquaculture integrated farming is popular in Asian countries than in Western countries. However, the integrated rice-aquaculture farming has its own limitations. The type of methods, culture species, influencing factors, and pros and cons of rice-aquaculture integrated farming are discussed in the present chapter.",book:{id:"7229",slug:"aquaculture-plants-and-invertebrates",title:"Aquaculture",fullTitle:"Aquaculture - Plants and Invertebrates"},signatures:"Pamuru Ramachandra Reddy and Battina Kishori",authors:[{id:"242524",title:"Dr.",name:"Ramachandra Reddy",middleName:null,surname:"Pamuru",slug:"ramachandra-reddy-pamuru",fullName:"Ramachandra Reddy Pamuru"},{id:"255022",title:"Dr.",name:"Kishori",middleName:null,surname:"Battina",slug:"kishori-battina",fullName:"Kishori Battina"}]},{id:"24074",title:"Embryonic and Larval Development of Freshwater Fish",slug:"embryonic-and-larval-development-of-freshwater-fish",totalDownloads:7462,totalCrossrefCites:1,totalDimensionsCites:2,abstract:null,book:{id:"612",slug:"recent-advances-in-fish-farms",title:"Recent Advances in Fish Farms",fullTitle:"Recent Advances in Fish Farms"},signatures:"Faruk Aral, Erdinç Şahınöz and Zafer Doğu",authors:[{id:"25600",title:"Prof.",name:"Faruk",middleName:null,surname:"Aral",slug:"faruk-aral",fullName:"Faruk Aral"},{id:"29132",title:"Dr.",name:"Zafer",middleName:null,surname:"Dogu",slug:"zafer-dogu",fullName:"Zafer Dogu"},{id:"39952",title:"Dr.",name:"Erdinc",middleName:null,surname:"Sahinoz",slug:"erdinc-sahinoz",fullName:"Erdinc Sahinoz"}]},{id:"68966",title:"Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ",slug:"novel-biofloc-technology-bft-for-ammonia-assimilation-and-reuse-in-aquaculture-in-situ",totalDownloads:1948,totalCrossrefCites:2,totalDimensionsCites:8,abstract:"Ammonia is one of the most harmful risks for success of fish and shrimp culture. There is no effective solution for harmlessness of ammonia in traditional aquaculture operations except exchanging water, which would bring negative effects on environment, or fixing expensive equipment. Biofloc technology (BFT) that appeared in recent years supplies a novel solution for this issue without exchanging huge water and fixing equipment. This technology could assimilate ammonia almost in real time with many other supplemental benefits. Because of the very high nutritional value for fish and shrimp, bioflocs, the by-product of BFT, could also be reused as a complemented food in situ or a gradient for feedstuff to replace expensive fishmeal or be processed to pellet diet to feed fish and shrimp directly. 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He is an academic staff member of the Department of Reproduction and Artificial Insemination, Selçuk University, Turkey. He manages several studies on sperms and embryos and is an editorial board member for several international journals. His studies include sperm cryobiology, in vitro fertilization, and embryo production in animals.",institutionString:"Selçuk University, Faculty of Veterinary Medicine",institution:null},{id:"90846",title:"Prof.",name:"Yusuf",middleName:null,surname:"Bozkurt",slug:"yusuf-bozkurt",fullName:"Yusuf Bozkurt",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/90846/images/system/90846.jpg",biography:"Yusuf Bozkurt has a BSc, MSc, and Ph.D. from Ankara University, Turkey. He is currently a Professor of Biotechnology of Reproduction in the field of Aquaculture, İskenderun Technical University, Turkey. His research interests include reproductive biology and biotechnology with an emphasis on cryo-conservation. He is on the editorial board of several international peer-reviewed journals and has published many papers. Additionally, he has participated in many international and national congresses, seminars, and workshops with oral and poster presentations. He is an active member of many local and international organizations.",institutionString:"İskenderun Technical University",institution:{name:"İskenderun Technical University",country:{name:"Turkey"}}},{id:"61139",title:"Dr.",name:"Sergey",middleName:null,surname:"Tkachev",slug:"sergey-tkachev",fullName:"Sergey Tkachev",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/61139/images/system/61139.png",biography:"Dr. Sergey Tkachev is a senior research scientist at the Institute of Fundamental Medicine and Biology, Kazan Federal University, Russia, and at the Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia. He received his Ph.D. in Molecular Biology with his thesis “Genetic variability of the tick-borne encephalitis virus in natural foci of Novosibirsk city and its suburbs.” His primary field is molecular virology with research emphasis on vector-borne viruses, especially tick-borne encephalitis virus, Kemerovo virus and Omsk hemorrhagic fever virus, rabies virus, molecular genetics, biology, and epidemiology of virus pathogens.",institutionString:"Russian Academy of Sciences",institution:{name:"Russian Academy of Sciences",country:{name:"Russia"}}},{id:"310962",title:"Dr.",name:"Amlan",middleName:"Kumar",surname:"Patra",slug:"amlan-patra",fullName:"Amlan Patra",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/310962/images/system/310962.jpg",biography:"Amlan K. Patra, FRSB, obtained a Ph.D. in Animal Nutrition from Indian Veterinary Research Institute, India, in 2002. He is currently an associate professor at West Bengal University of Animal and Fishery Sciences. He has more than twenty years of research and teaching experience. He held previous positions at the American Institute for Goat Research, The Ohio State University, Columbus, USA, and Free University of Berlin, Germany. His research focuses on animal nutrition, particularly ruminants and poultry nutrition, gastrointestinal electrophysiology, meta-analysis and modeling in nutrition, and livestock–environment interaction. He has authored around 175 articles in journals, book chapters, and proceedings. Dr. Patra serves on the editorial boards of several reputed journals.",institutionString:null,institution:{name:"West Bengal University of Animal and Fishery Sciences",country:{name:"India"}}},{id:"53998",title:"Prof.",name:"László",middleName:null,surname:"Babinszky",slug:"laszlo-babinszky",fullName:"László Babinszky",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/53998/images/system/53998.png",biography:"László Babinszky is Professor Emeritus, Department of Animal Nutrition Physiology, University of Debrecen, Hungary. He has also worked in the Department of Animal Nutrition, University of Wageningen, Netherlands; the Institute for Livestock Feeding and Nutrition (IVVO), Lelystad, Netherlands; the Agricultural University of Vienna (BOKU); the Institute for Animal Breeding and Nutrition, Austria; and the Oscar Kellner Research Institute for Animal Nutrition, Rostock, Germany. In 1992, Dr. Babinszky obtained a Ph.D. in Animal Nutrition from the University of Wageningen. His main research areas are swine and poultry nutrition. He has authored more than 300 publications (papers, book chapters) and edited four books and fourteen international conference proceedings.",institutionString:"University of Debrecen",institution:{name:"University of Debrecen",country:{name:"Hungary"}}},{id:"201830",title:"Dr.",name:"Fernando",middleName:"Sanchez",surname:"Davila",slug:"fernando-davila",fullName:"Fernando Davila",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/201830/images/5017_n.jpg",biography:"I am a professor at UANL since 1988. My research lines are the development of reproductive techniques in small ruminants. We also conducted research on sexual and social behavior in males.\nI am Mexican and study my professional career as an engineer in agriculture and animal science at UANL. Then take a masters degree in science in Germany (Animal breeding). Take a doctorate in animal science at the UANL.",institutionString:null,institution:{name:"Universidad Autónoma de Nuevo León",country:{name:"Mexico"}}},{id:"309250",title:"Dr.",name:"Miguel",middleName:null,surname:"Quaresma",slug:"miguel-quaresma",fullName:"Miguel Quaresma",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/309250/images/9059_n.jpg",biography:"Miguel Nuno Pinheiro Quaresma was born on May 26, 1974 in Dili, Timor Island. He is married with two children: a boy and a girl, and he is a resident in Vila Real, Portugal. He graduated in Veterinary Medicine in August 1998 and obtained his Ph.D. degree in Veterinary Sciences -Clinical Area in February 2015, both from the University of Trás-os-Montes e Alto Douro. He is currently enrolled in the Alternative Residency of the European College of Animal Reproduction. He works as a Senior Clinician at the Veterinary Teaching Hospital of UTAD (HVUTAD) with a role in clinical activity in the area of livestock and equine species as well as to support teaching and research in related areas. He teaches as an Invited Professor in Reproduction Medicine I and II of the Master\\'s in Veterinary Medicine degree at UTAD. Currently, he holds the position of Chairman of the Portuguese Buiatrics Association. He is a member of the Consultive Group on Production Animals of the OMV. He has 19 publications in indexed international journals (ISIS), as well as over 60 publications and oral presentations in both Portuguese and international journals and congresses.",institutionString:"University of Trás-os-Montes and Alto Douro",institution:{name:"University of Trás-os-Montes and Alto Douro",country:{name:"Portugal"}}},{id:"38652",title:"Prof.",name:"Rita",middleName:null,surname:"Payan-Carreira",slug:"rita-payan-carreira",fullName:"Rita Payan-Carreira",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRiFPQA0/Profile_Picture_1614601496313",biography:"Rita Payan Carreira earned her Veterinary Degree from the Faculty of Veterinary Medicine in Lisbon, Portugal, in 1985. She obtained her Ph.D. in Veterinary Sciences from the University of Trás-os-Montes e Alto Douro, Portugal. After almost 32 years of teaching at the University of Trás-os-Montes and Alto Douro, she recently moved to the University of Évora, Department of Veterinary Medicine, where she teaches in the field of Animal Reproduction and Clinics. Her primary research areas include the molecular markers of the endometrial cycle and the embryo–maternal interaction, including oxidative stress and the reproductive physiology and disorders of sexual development, besides the molecular determinants of male and female fertility. She often supervises students preparing their master's or doctoral theses. She is also a frequent referee for various journals.",institutionString:null,institution:{name:"University of Évora",country:{name:"Portugal"}}},{id:"283019",title:"Dr.",name:"Oudessa",middleName:null,surname:"Kerro Dego",slug:"oudessa-kerro-dego",fullName:"Oudessa Kerro Dego",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/283019/images/system/283019.png",biography:"Dr. Kerro Dego is a veterinary microbiologist with training in veterinary medicine, microbiology, and anatomic pathology. Dr. Kerro Dego is an assistant professor of dairy health in the department of animal science, the University of Tennessee, Institute of Agriculture, Knoxville, Tennessee. He received his D.V.M. (1997), M.S. (2002), and Ph.D. (2008) degrees in Veterinary Medicine, Animal Pathology and Veterinary Microbiology from College of Veterinary Medicine, Addis Ababa University, Ethiopia; College of Veterinary Medicine, Utrecht University, the Netherlands and Western College of Veterinary Medicine, University of Saskatchewan, Canada respectively. He did his Postdoctoral training in microbial pathogenesis (2009 - 2015) in the Department of Animal Science, the University of Tennessee, Institute of Agriculture, Knoxville, Tennessee. Dr. Kerro Dego’s research focuses on the prevention and control of infectious diseases of farm animals, particularly mastitis, improving dairy food safety, and mitigation of antimicrobial resistance. Dr. Kerro Dego has extensive experience in studying the pathogenesis of bacterial infections, identification of virulence factors, and vaccine development and efficacy testing against major bacterial mastitis pathogens. Dr. Kerro Dego conducted numerous controlled experimental and field vaccine efficacy studies, vaccination, and evaluation of immunological responses in several species of animals, including rodents (mice) and large animals (bovine and ovine).",institutionString:"University of Tennessee at Knoxville",institution:{name:"University of Tennessee at Knoxville",country:{name:"United States of America"}}},{id:"251314",title:"Dr.",name:"Juan Carlos",middleName:null,surname:"Gardón Poggi",slug:"juan-carlos-gardon-poggi",fullName:"Juan Carlos Gardón Poggi",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/251314/images/system/251314.jpeg",biography:"Juan Carlos Gardón Poggi received University degree from the Faculty of Agrarian Science in Argentina, in 1983. Also he received Masters Degree and PhD from Córdoba University, Spain. He is currently a Professor at the Catholic University of Valencia San Vicente Mártir, at the Department of Medicine and Animal Surgery. He teaches diverse courses in the field of Animal Reproduction and he is the Director of the Veterinary Farm. He also participates in academic postgraduate activities at the Veterinary Faculty of Murcia University, Spain. His research areas include animal physiology, physiology and biotechnology of reproduction either in males or females, the study of gametes under in vitro conditions and the use of ultrasound as a complement to physiological studies and development of applied biotechnologies. Routinely, he supervises students preparing their doctoral, master thesis or final degree projects.",institutionString:null,institution:{name:"Valencia Catholic University Saint Vincent Martyr",country:{name:"Spain"}}},{id:"309529",title:"Dr.",name:"Albert",middleName:null,surname:"Rizvanov",slug:"albert-rizvanov",fullName:"Albert Rizvanov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/309529/images/9189_n.jpg",biography:'Albert A. Rizvanov is a Professor and Director of the Center for Precision and Regenerative Medicine at the Institute of Fundamental Medicine and Biology, Kazan Federal University (KFU), Russia. He is the Head of the Center of Excellence “Regenerative Medicine” and Vice-Director of Strategic Academic Unit \\"Translational 7P Medicine\\". Albert completed his Ph.D. at the University of Nevada, Reno, USA and Dr.Sci. at KFU. He is a corresponding member of the Tatarstan Academy of Sciences, Russian Federation. Albert is an author of more than 300 peer-reviewed journal articles and 22 patents. He has supervised 11 Ph.D. and 2 Dr.Sci. dissertations. Albert is the Head of the Dissertation Committee on Biochemistry, Microbiology, and Genetics at KFU.\nORCID https://orcid.org/0000-0002-9427-5739\nWebsite https://kpfu.ru/Albert.Rizvanov?p_lang=2',institutionString:"Kazan Federal University",institution:{name:"Kazan Federal University",country:{name:"Russia"}}},{id:"210551",title:"Dr.",name:"Arbab",middleName:null,surname:"Sikandar",slug:"arbab-sikandar",fullName:"Arbab Sikandar",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/210551/images/system/210551.jpg",biography:"Dr. Arbab Sikandar, PhD, M. Phil, DVM was born on April 05, 1981. He is currently working at the College of Veterinary & Animal Sciences as an Assistant Professor. He previously worked as a lecturer at the same University. \nHe is a Member/Secretory of Ethics committee (No. CVAS-9377 dated 18-04-18), Member of the QEC committee CVAS, Jhang (Regr/Gen/69/873, dated 26-10-2017), Member, Board of studies of Department of Basic Sciences (No. CVAS. 2851 Dated. 12-04-13, and No. CVAS, 9024 dated 20/11/17), Member of Academic Committee, CVAS, Jhang (No. CVAS/2004, Dated, 25-08-12), Member of the technical committee (No. CVAS/ 4085, dated 20,03, 2010 till 2016).\n\nDr. Arbab Sikandar contributed in five days hands-on-training on Histopathology at the Department of Pathology, UVAS from 12-16 June 2017. He received a Certificate of appreciation for contributions for Popularization of Science and Technology in the Society on 17-11-15. He was the resource person in the lecture series- ‘scientific writing’ at the Department of Anatomy and Histology, UVAS, Lahore on 29th October 2015. He won a full fellowship as a principal candidate for the year 2015 in the field of Agriculture, EICA, Egypt with ref. to the Notification No. 12(11) ACS/Egypt/2014 from 10 July 2015 to 25th September 2015.; he received a grant of Rs. 55000/- as research incentives from Director, Advanced Studies and Research, UVAS, Lahore upon publications of research papers in IF Journals (DR/215, dated 19-5-2014.. He obtained his PhD by winning a HEC Pakistan indigenous Scholarship, ‘Ph.D. fellowship for 5000 scholars – Phase II’ (2av1-147), 17-6/HEC/HRD/IS-II/12, November 15, 2012. \n\nDr. Sikandar is a member of numerous societies: Registered Veterinary Medical Practitioner (life member) and Registered Veterinary Medical Faculty of Pakistan Veterinary Medical Council. The Registration code of PVMC is RVMP/4298 and RVMF/ 0102.; Life member of the University of Veterinary and Animal Sciences, Lahore, Alumni Association with S# 664, dated: 6-4-12. ; Member 'Vets Care Organization Pakistan” with Reference No. VCO-605-149, dated 05-04-06. :Member 'Vet Crescent” (Society of Animal Health and Production), UVAS, Lahore.",institutionString:"University of Veterinary & Animal Science",institution:{name:"University of Veterinary and Animal Sciences",country:{name:"Pakistan"}}},{id:"311663",title:"Dr.",name:"Prasanna",middleName:null,surname:"Pal",slug:"prasanna-pal",fullName:"Prasanna Pal",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/311663/images/13261_n.jpg",biography:null,institutionString:null,institution:{name:"National Dairy Research Institute",country:{name:"India"}}},{id:"202192",title:"Dr.",name:"Catrin",middleName:null,surname:"Rutland",slug:"catrin-rutland",fullName:"Catrin Rutland",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/202192/images/system/202192.png",biography:"Catrin Rutland is an Associate Professor of Anatomy and Developmental Genetics at the University of Nottingham, UK. She obtained a BSc from the University of Derby, England, a master’s degree from Technische Universität München, Germany, and a Ph.D. from the University of Nottingham. She undertook a post-doctoral research fellowship in the School of Medicine before accepting tenure in Veterinary Medicine and Science. Dr. Rutland also obtained an MMedSci (Medical Education) and a Postgraduate Certificate in Higher Education (PGCHE). She is the author of more than sixty peer-reviewed journal articles, twelve books/book chapters, and more than 100 research abstracts in cardiovascular biology and oncology. She is a board member of the European Association of Veterinary Anatomists, Fellow of the Anatomical Society, and Senior Fellow of the Higher Education Academy. Dr. Rutland has also written popular science books for the public. https://orcid.org/0000-0002-2009-4898. www.nottingham.ac.uk/vet/people/catrin.rutland",institutionString:null,institution:{name:"University of Nottingham",country:{name:"United Kingdom"}}},{id:"283315",title:"Prof.",name:"Samir",middleName:null,surname:"El-Gendy",slug:"samir-el-gendy",fullName:"Samir El-Gendy",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRduYQAS/Profile_Picture_1606215849748",biography:"Samir El-Gendy is a Professor of anatomy and embryology at the faculty of veterinary medicine, Alexandria University, Egypt. Samir obtained his PhD in veterinary science in 2007 from the faculty of veterinary medicine, Alexandria University and has been a professor since 2017. Samir is an author on 24 articles at Scopus and 12 articles within local journals and 2 books/book chapters. His research focuses on applied anatomy, imaging techniques and computed tomography. Samir worked as a member of different local projects on E-learning and he is a board member of the African Association of Veterinary Anatomists and of anatomy societies and as an associated author at local and international journals. Orcid: https://orcid.org/0000-0002-6180-389X",institutionString:null,institution:{name:"Alexandria University",country:{name:"Egypt"}}},{id:"246149",title:"Dr.",name:"Valentina",middleName:null,surname:"Kubale",slug:"valentina-kubale",fullName:"Valentina Kubale",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/246149/images/system/246149.jpg",biography:"Valentina Kubale is Associate Professor of Veterinary Medicine at the Veterinary Faculty, University of Ljubljana, Slovenia. Since graduating from the Veterinary faculty she obtained her PhD in 2007, performed collaboration with the Department of Pharmacology, University of Copenhagen, Denmark. She continued as a post-doctoral fellow at the University of Copenhagen with a Lundbeck foundation fellowship. She is the editor of three books and author/coauthor of 23 articles in peer-reviewed scientific journals, 16 book chapters, and 68 communications at scientific congresses. Since 2008 she has been the Editor Assistant for the Slovenian Veterinary Research journal. She is a member of Slovenian Biochemical Society, The Endocrine Society, European Association of Veterinary Anatomists and Society for Laboratory Animals, where she is board member.",institutionString:"University of Ljubljana",institution:{name:"University of Ljubljana",country:{name:"Slovenia"}}},{id:"258334",title:"Dr.",name:"Carlos Eduardo",middleName:null,surname:"Fonseca-Alves",slug:"carlos-eduardo-fonseca-alves",fullName:"Carlos Eduardo Fonseca-Alves",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/258334/images/system/258334.jpg",biography:"Dr. Fonseca-Alves earned his DVM from Federal University of Goias – UFG in 2008. He completed an internship in small animal internal medicine at UPIS university in 2011, earned his MSc in 2013 and PhD in 2015 both in Veterinary Medicine at Sao Paulo State University – UNESP. Dr. Fonseca-Alves currently serves as an Assistant Professor at Paulista University – UNIP teaching small animal internal medicine.",institutionString:null,institution:{name:"Universidade Paulista",country:{name:"Brazil"}}},{id:"245306",title:"Dr.",name:"María Luz",middleName:null,surname:"Garcia Pardo",slug:"maria-luz-garcia-pardo",fullName:"María Luz Garcia Pardo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/245306/images/system/245306.png",biography:"María de la Luz García Pardo is an agricultural engineer from Universitat Politècnica de València, Spain. She has a Ph.D. in Animal Genetics. Currently, she is a lecturer at the Agrofood Technology Department of Miguel Hernández University, Spain. Her research is focused on genetics and reproduction in rabbits. The major goal of her research is the genetics of litter size through novel methods such as selection by the environmental sensibility of litter size, with forays into the field of animal welfare by analysing the impact on the susceptibility to diseases and stress of the does. Details of her publications can be found at https://orcid.org/0000-0001-9504-8290.",institutionString:null,institution:{name:"Miguel Hernandez University",country:{name:"Spain"}}},{id:"350704",title:"M.Sc.",name:"Camila",middleName:"Silva Costa",surname:"Ferreira",slug:"camila-ferreira",fullName:"Camila Ferreira",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/350704/images/17280_n.jpg",biography:"Graduated in Veterinary Medicine at the Fluminense Federal University, specialist in Equine Reproduction at the Brazilian Veterinary Institute (IBVET) and Master in Clinical Veterinary Medicine and Animal Reproduction at the Fluminense Federal University. She has experience in analyzing zootechnical indices in dairy cattle and organizing events related to Veterinary Medicine through extension grants. I have experience in the field of diagnostic imaging and animal reproduction in veterinary medicine through monitoring and scientific initiation scholarships. I worked at the Equus Central Reproduction Equine located in Santo Antônio de Jesus – BA in the 2016/2017 breeding season. I am currently a doctoral student with a scholarship from CAPES of the Postgraduate Program in Veterinary Medicine (Pathology and Clinical Sciences) at the Federal Rural University of Rio de Janeiro (UFRRJ) with a research project with an emphasis on equine endometritis.",institutionString:null,institution:null},{id:"41319",title:"Prof.",name:"Lung-Kwang",middleName:null,surname:"Pan",slug:"lung-kwang-pan",fullName:"Lung-Kwang Pan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/41319/images/84_n.jpg",biography:null,institutionString:null,institution:null},{id:"125292",title:"Dr.",name:"Katy",middleName:null,surname:"Satué Ambrojo",slug:"katy-satue-ambrojo",fullName:"Katy Satué Ambrojo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/125292/images/system/125292.jpeg",biography:"Katy Satué Ambrojo received her Veterinary Medicine degree, Master degree in Equine Technology and doctorate in Veterinary Medicine from the Faculty of Veterinary, CEU-Cardenal Herrera University in Valencia, Spain.Dr. Satué is accredited as a Private University Doctor Professor, Doctor Assistant, and Contracted Doctor by AVAP (Agència Valenciana d'Avaluació i Prospectiva) and currently, as a full professor by ANECA (since January 2022). To date, Katy has taught 22 years in the Department of Animal Medicine and Surgery at the CEU-Cardenal Herrera University in undergraduate courses in Veterinary Medicine (General Pathology, integrated into the Applied Basis of Veterinary Medicine module of the 2nd year, Clinical Equine I of 3rd year, and Equine Clinic II of 4th year). Dr. Satué research activity is in the field of Endocrinology, Hematology, Biochemistry, and Immunology in the Spanish Purebred mare. She has directed 5 Doctoral Theses and 5 Diplomas of Advanced Studies, and participated in 11 research projects as a collaborating researcher. She has written 2 books and 14 book chapters in international publishers related to the area, and 68 scientific publications in international journals. Dr. Satué has attended 63 congresses, participating with 132 communications in international congresses and 19 in national congresses related to the area. Dr. Satué is a scientific reviewer for various prestigious international journals such as Animals, American Journal of Obstetrics and Gynecology, Veterinary Clinical Pathology, Journal of Equine Veterinary Science, Reproduction in Domestic Animals, Research Veterinary Science, Brazilian Journal of Medical and Biological Research, Livestock Production Science and Theriogenology, among others. Since 2014 she has been responsible for the Clinical Analysis Laboratory of the CEU-Cardenal Herrera University Veterinary Clinical Hospital.",institutionString:null,institution:null},{id:"201721",title:"Dr.",name:"Beatrice",middleName:null,surname:"Funiciello",slug:"beatrice-funiciello",fullName:"Beatrice Funiciello",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/201721/images/11089_n.jpg",biography:"Graduated from the University of Milan in 2011, my post-graduate education included CertAVP modules mainly on equines (dermatology and internal medicine) and a few on small animal (dermatology and anaesthesia) at the University of Liverpool. After a general CertAVP (2015) I gained the designated Certificate in Veterinary Dermatology (2017) after taking the synoptic examination and then applied for the RCVS ADvanced Practitioner status. After that, I completed the Postgraduate Diploma in Veterinary Professional Studies at the University of Liverpool (2018). My main area of work is cross-species veterinary dermatology.",institutionString:null,institution:null},{id:"291226",title:"Dr.",name:"Monica",middleName:null,surname:"Cassel",slug:"monica-cassel",fullName:"Monica Cassel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/291226/images/8232_n.jpg",biography:'Degree in Biological Sciences at the Federal University of Mato Grosso with scholarship for Scientific Initiation by FAPEMAT (2008/1) and CNPq (2008/2-2009/2): Project \\"Histological evidence of reproductive activity in lizards of the Manso region, Chapada dos Guimarães, Mato Grosso, Brazil\\". Master\\\'s degree in Ecology and Biodiversity Conservation at Federal University of Mato Grosso with a scholarship by CAPES/REUNI program: Project \\"Reproductive biology of Melanorivulus punctatus\\". PhD\\\'s degree in Science (Cell and Tissue Biology Area) \n at University of Sao Paulo with scholarship granted by FAPESP; Project \\"Development of morphofunctional changes in ovary of Astyanax altiparanae Garutti & Britski, 2000 (Teleostei, Characidae)\\". She has experience in Reproduction of vertebrates and Morphology, with emphasis in Cellular Biology and Histology. She is currently a teacher in the medium / technical level courses at IFMT-Alta Floresta, as well as in the Bachelor\\\'s degree in Animal Science and in the Bachelor\\\'s degree in Business.',institutionString:null,institution:null},{id:"442807",title:"Dr.",name:"Busani",middleName:null,surname:"Moyo",slug:"busani-moyo",fullName:"Busani Moyo",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Gwanda State University",country:{name:"Zimbabwe"}}},{id:"439435",title:"Dr.",name:"Feda S.",middleName:null,surname:"Aljaser",slug:"feda-s.-aljaser",fullName:"Feda S. Aljaser",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"King Saud University",country:{name:"Saudi Arabia"}}},{id:"423023",title:"Dr.",name:"Yosra",middleName:null,surname:"Soltan",slug:"yosra-soltan",fullName:"Yosra Soltan",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Alexandria University",country:{name:"Egypt"}}},{id:"349788",title:"Dr.",name:"Florencia Nery",middleName:null,surname:"Sompie",slug:"florencia-nery-sompie",fullName:"Florencia Nery Sompie",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Sam Ratulangi University",country:{name:"Indonesia"}}},{id:"428600",title:"MSc.",name:"Adriana",middleName:null,surname:"García-Alarcón",slug:"adriana-garcia-alarcon",fullName:"Adriana García-Alarcón",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"428599",title:"MSc.",name:"Gabino",middleName:null,surname:"De La Rosa-Cruz",slug:"gabino-de-la-rosa-cruz",fullName:"Gabino De La Rosa-Cruz",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"428601",title:"MSc.",name:"Juan Carlos",middleName:null,surname:"Campuzano-Caballero",slug:"juan-carlos-campuzano-caballero",fullName:"Juan Carlos Campuzano-Caballero",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}}]}},subseries:{item:{id:"18",type:"subseries",title:"Proteomics",keywords:"Mono- and Two-Dimensional Gel Electrophoresis (1-and 2-DE), Liquid Chromatography (LC), Mass Spectrometry/Tandem Mass Spectrometry (MS; MS/MS), Proteins",scope:"With the recognition that the human genome cannot provide answers to the etiology of a disorder, changes in the proteins expressed by a genome became a focus in research. Thus proteomics, an area of research that detects all protein forms expressed in an organism, including splice isoforms and post-translational modifications, is more suitable than genomics for a comprehensive understanding of the biochemical processes that govern life. The most common proteomics applications are currently in the clinical field for the identification, in a variety of biological matrices, of biomarkers for diagnosis and therapeutic intervention of disorders. From the comparison of proteomic profiles of control and disease or different physiological states, which may emerge, changes in protein expression can provide new insights into the roles played by some proteins in human pathologies. Understanding how proteins function and interact with each other is another goal of proteomics that makes this approach even more intriguing. Specialized technology and expertise are required to assess the proteome of any biological sample. Currently, proteomics relies mainly on mass spectrometry (MS) combined with electrophoretic (1 or 2-DE-MS) and/or chromatographic techniques (LC-MS/MS). MS is an excellent tool that has gained popularity in proteomics because of its ability to gather a complex body of information such as cataloging protein expression, identifying protein modification sites, and defining protein interactions. The Proteomics topic aims to attract contributions on all aspects of MS-based proteomics that, by pushing the boundaries of MS capabilities, may address biological problems that have not been resolved yet.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/18.jpg",hasOnlineFirst:!0,hasPublishedBooks:!0,annualVolume:11414,editor:{id:"200689",title:"Prof.",name:"Paolo",middleName:null,surname:"Iadarola",slug:"paolo-iadarola",fullName:"Paolo Iadarola",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bSCl8QAG/Profile_Picture_1623568118342",biography:"Paolo Iadarola graduated with a degree in Chemistry from the University of Pavia (Italy) in July 1972. He then worked as an Assistant Professor at the Faculty of Science of the same University until 1984. In 1985, Prof. Iadarola became Associate Professor at the Department of Biology and Biotechnologies of the University of Pavia and retired in October 2017. Since then, he has been working as an Adjunct Professor in the same Department at the University of Pavia. His research activity during the first years was primarily focused on the purification and structural characterization of enzymes from animal and plant sources. During this period, Prof. Iadarola familiarized himself with the conventional techniques used in column chromatography, spectrophotometry, manual Edman degradation, and electrophoresis). Since 1995, he has been working on: i) the determination in biological fluids (serum, urine, bronchoalveolar lavage, sputum) of proteolytic activities involved in the degradation processes of connective tissue matrix, and ii) on the identification of biological markers of lung diseases. In this context, he has developed and validated new methodologies (e.g., Capillary Electrophoresis coupled to Laser-Induced Fluorescence, CE-LIF) whose application enabled him to determine both the amounts of biochemical markers (Desmosines) in urine/serum of patients affected by Chronic Obstructive Pulmonary Disease (COPD) and the activity of proteolytic enzymes (Human Neutrophil Elastase, Cathepsin G, Pseudomonas aeruginosa elastase) in sputa of these patients. More recently, Prof. Iadarola was involved in developing techniques such as two-dimensional electrophoresis coupled to liquid chromatography/mass spectrometry (2DE-LC/MS) for the proteomic analysis of biological fluids aimed at the identification of potential biomarkers of different lung diseases. He is the author of about 150 publications (According to Scopus: H-Index: 23; Total citations: 1568- According to WOS: H-Index: 20; Total Citations: 1296) of peer-reviewed international journals. He is a Consultant Reviewer for several journals, including the Journal of Chromatography A, Journal of Chromatography B, Plos ONE, Proteomes, International Journal of Molecular Science, Biotech, Electrophoresis, and others. He is also Associate Editor of Biotech.",institutionString:null,institution:{name:"University of Pavia",institutionURL:null,country:{name:"Italy"}}},editorTwo:{id:"201414",title:"Dr.",name:"Simona",middleName:null,surname:"Viglio",slug:"simona-viglio",fullName:"Simona Viglio",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRKDHQA4/Profile_Picture_1630402531487",biography:"Simona Viglio is an Associate Professor of Biochemistry at the Department of Molecular Medicine at the University of Pavia. She has been working since 1995 on the determination of proteolytic enzymes involved in the degradation process of connective tissue matrix and on the identification of biological markers of lung diseases. She gained considerable experience in developing and validating new methodologies whose applications allowed her to determine both the amount of biomarkers (Desmosine and Isodesmosine) in the urine of patients affected by COPD, and the activity of proteolytic enzymes (HNE, Cathepsin G, Pseudomonas aeruginosa elastase) in the sputa of these patients. Simona Viglio was also involved in research dealing with the supplementation of amino acids in patients with brain injury and chronic heart failure. She is presently engaged in the development of 2-DE and LC-MS techniques for the study of proteomics in biological fluids. The aim of this research is the identification of potential biomarkers of lung diseases. She is an author of about 90 publications (According to Scopus: H-Index: 23; According to WOS: H-Index: 20) on peer-reviewed journals, a member of the “Società Italiana di Biochimica e Biologia Molecolare,“ and a Consultant Reviewer for International Journal of Molecular Science, Journal of Chromatography A, COPD, Plos ONE and Nutritional Neuroscience.",institutionString:null,institution:{name:"University of Pavia",institutionURL:null,country:{name:"Italy"}}},editorThree:null,series:{id:"11",title:"Biochemistry",doi:"10.5772/intechopen.72877",issn:"2632-0983"},editorialBoard:[{id:"72288",title:"Dr.",name:"Arli Aditya",middleName:null,surname:"Parikesit",slug:"arli-aditya-parikesit",fullName:"Arli Aditya Parikesit",profilePictureURL:"https://mts.intechopen.com/storage/users/72288/images/system/72288.jpg",institutionString:null,institution:{name:"Indonesia International Institute for Life Sciences",institutionURL:null,country:{name:"Indonesia"}}},{id:"40928",title:"Dr.",name:"Cesar",middleName:null,surname:"Lopez-Camarillo",slug:"cesar-lopez-camarillo",fullName:"Cesar Lopez-Camarillo",profilePictureURL:"https://mts.intechopen.com/storage/users/40928/images/3884_n.png",institutionString:null,institution:{name:"Universidad Autónoma de la Ciudad de México",institutionURL:null,country:{name:"Mexico"}}},{id:"81926",title:"Dr.",name:"Shymaa",middleName:null,surname:"Enany",slug:"shymaa-enany",fullName:"Shymaa Enany",profilePictureURL:"https://mts.intechopen.com/storage/users/81926/images/system/81926.png",institutionString:"Suez Canal University",institution:{name:"Suez Canal University",institutionURL:null,country:{name:"Egypt"}}}]},onlineFirstChapters:{paginationCount:3,paginationItems:[{id:"82956",title:"Potential Substitutes of Antibiotics for Swine and Poultry Production",doi:"10.5772/intechopen.106081",signatures:"Ho Trung Thong, Le Nu Anh Thu and Ho Viet Duc",slug:"potential-substitutes-of-antibiotics-for-swine-and-poultry-production",totalDownloads:0,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Antibiotics and Probiotics in Animal Food - Impact and Regulation",coverURL:"https://cdn.intechopen.com/books/images_new/11578.jpg",subseries:{id:"20",title:"Animal Nutrition"}}},{id:"82905",title:"A Review of Application Strategies and Efficacy of Probiotics in Pet Food",doi:"10.5772/intechopen.105829",signatures:"Heather Acuff and Charles G. 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