Treatment of histoplasmosis syndromes and their complications [13, 14].
\r\n\t
",isbn:"978-1-83969-452-3",printIsbn:"978-1-83969-451-6",pdfIsbn:"978-1-83969-453-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"a6e1a11c05ff8853c529750ddfac6c11",bookSignature:"Dr. René Mauricio Barría",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10734.jpg",keywords:"Neonatal Intensive Unit, Neonatal Diagnostic Techniques, Neonatal Nurses, Neonatologists, Newborn Diseases, Premature Diseases, Breast Feeding, Kangaroo-Mother Care Method, Neonatal Survival, Limit of Viability, Minimal Handling, Neonatal Stress",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 5th 2021",dateEndSecondStepPublish:"March 5th 2021",dateEndThirdStepPublish:"May 4th 2021",dateEndFourthStepPublish:"July 23rd 2021",dateEndFifthStepPublish:"September 21st 2021",remainingDaysToSecondStep:"5 days",secondStepPassed:!1,currentStepOfPublishingProcess:2,editedByType:null,kuFlag:!1,biosketch:"The principal investigator and academic expert in epidemiological methods and evidence-based health with an emphasis on children's health. His research interests lie in the areas of Maternal-Child Health, Neonatal Care, and Environmental Health. From 2010 until 2017 he was Director of the Evidence-Based Health Office and currently serves as Director of the Nursing Institute at the Universidad Austral de Chile.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"88861",title:"Dr.",name:"R. Mauricio",middleName:null,surname:"Barría",slug:"r.-mauricio-barria",fullName:"R. Mauricio Barría",profilePictureURL:"https://mts.intechopen.com/storage/users/88861/images/system/88861.jpg",biography:"R. Mauricio Barría, DrPH, is a Principal Investigator and Associate Professor at the Faculty of Medicine at Universidad Austral de Chile. He was trained as an epidemiologist and received his MSc in Clinical Epidemiology from Universidad de la Frontera in Temuco, Chile, and his DrPH from Universidad de Chile in Santiago, Chile. His research interests lie in the areas of Maternal-Child Health, Neonatal Care and Environmental Health. He is skilled in epidemiological studies designs with special interest in cohort studies and clinical trials. Since 2010 until 2017 he was Director of the Evidence-Based Health Office and currently serves as Director of the Nursing Institute at the Universidad Austral de Chile. He has published several articles related to the care and health of the newborn and is a reviewer of several international journals.",institutionString:"Austral University of Chile",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"4",institution:{name:"Austral University of Chile",institutionURL:null,country:{name:"Chile"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"345821",firstName:"Darko",lastName:"Hrvojic",middleName:null,title:"Mr.",imageUrl:"//cdnintech.com/web/frontend/www/assets/author.svg",email:"darko@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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It has the characteristic taste and odor of the plant from which it was derived [1, 2]. This variation in composition could be due to a variety of plants [3], geographical locations [3, 4], harvesting seasons [5–7], drying methods [8] and extraction methods [9, 10].
\nThe quality, freshness and uniqueness of an essential oil are major considerations pertaining to its value [11]. Essential oils are highly sensitive to heat, moisture and oxygen [12]. Formation of oxygenated terpenes, chemical transformations, or polymerization are features of aging processes that led to quality loss. Therefore, quality control of essential oils needs to be monitored by producers, traders, or essential oil manufacturers [13, 14].
\nEssential oils have wide variety of bioactivities and play an important role as ideal natural sources of antimicrobial, antioxidant and chemopreventive agents [15–17]. They have potential therapeutic applications in the prevention of cancer [18].
\nEssential oils (EOs) are volatile constituents obtained from aromatic plant material, including leaves, rhizomes, flowers, roots, bark, seeds, peel, fruits, wood and whole plants [19]. A few of the essential oils are found in animal sources, for example, musk and sperm whale, or are produced by microorganisms [20]. In plants, essential oils occur in oil cells, secretory ducts or cavities, or in glandular hairs. Essential oils can be isolated by steam distillation from an aromatic plant because of their volatility [21]. Different constituents in EOs exhibit varying smell or flavor [9]. The perception of individual volatile compounds depends on their threshold [22].
\nEssential oils are a complex mixture of polar and non‐polar compounds [23]. The essential oil composition depends on the species of the extracted plant, the geographic location of this plant, harvest time and extraction technique [24].
\nEOs can be classified into the four main groups: [25, 26]\n
Terpenes, related to isoprene
Straight‐chain compounds not containing any side chain
Phenylpropanoids (benzene derivatives)
Miscellaneous group having varied structures not included in first three groups (sulfur‐ or nitrogen‐containing compounds)
Essential oils constituents can be divided into two major groups: terpene hydrocarbons and oxygenated compounds [27].
\nTerpenes are the most common class of chemical compounds found in essential oils [28]. Terpenes are synthesized in the cytoplasm of plant cells, through the mevalonic acid pathway [19]. Terpenes have been regarded as polymers of isoprene (C5H8) joined in a repetitive head‐to‐tail manner [29] as shown in Figure 1.
Link of isoprene molecules.
Chemical structures of acyclic and monocyclic monoterpenes.
They could be classified according to the fusion of the isoprene units or to the number of the rings [28]. Terpenes can be classified into hemiterpenes (1 unit), monoterpenes (2 units), sesquiterpenes (3 units), diterpenes (4 units), sesterterpenes (5 units), triterpenes (6 units) and polyterpenes (many units) [30]. The combinations of two isoprene units are called a “terpene unit.” Monoterpenes (C10H16) are formed by the attachment of two isoprene units (at least one double bond). These terpenes have a hydrocarbon skeleton which can be rearranged into acyclic, cyclic, or aromatic (Figure 2). Cyclic monoterpenes can be classified according to their ring size such as monocyclic monoterpenes, bicyclic monoterpenes and tricyclic monoterpenes [31] Figure 3. These compounds oxidize easily because of their rapid reaction to air and heat sources [32].
\nChemical structures of bicyclic and tricyclic monoterpenes.
Sesquiterpenes (C15H24) are the second to the dominant monoterpenes. They are formed from the combination of three isoprene units [29]. Sesquiterpenes are unsaturated compounds. There are linear, branched, or cyclic sesquiterpenes (Figure 4). Sesquiterpenes are unsaturated compounds. Cyclic sesquiterpenes can be classified into monocyclic, bicyclic, or tricyclic [31] (Figure 5). Diterpenes are formed by the head‐to‐tail combinations of four isoprene units followed by rearrangement and/or substitutions (Figure 6).
\nChemical structures of acyclic and monocyclic sesquiterpenes.
Chemical structures of bicyclic and tricyclic sesquiterpenes.
Chemical structures of acyclic, monocyclic and bicyclic diterpenes.
They are important components of plant resins [33]. Diterpenes, triterpenes and tetraterpenes are present at a very low concentration in essential oils [27, 34]. Their recovery increases with increasing steam distillation times [35] and influenced by the extraction method.
\nThe oxygenated compounds are highly odoriferous [36]. Terpenoids are volatile secondary metabolites which give plants their fragrance [37]. Terpenoids can be subdivided into aldehydes, ethers, alcohols, esters, ketones, phenols and epoxides [19]. Examples: (+)‐Borneol occurs in camphor, rosemary and lavender oils. It has a camphor‐like odor, with a slightly peppery note [38]. Carvacrol is found in oregano and thyme [39] oils. It has a herbaceous, phenolic odor [32]. (-)‐Carvone has a herbal scent and is found in caraway seed oil; (+)‐carvone with a spicy‐minty odor can be found in spearmint oil [40]. 1,8‐Cineole is obtained primarily by isolation from eucalyptus oil. It has a camphor odor [41].
\nThis group contains only straight chain non‐terpenoid hydrocarbons and their oxygen derivatives: alcohols, aldehydes, ketones, acids, ethers and esters. These hydrocarbons range from n‐heptane, to compounds with 35 carbon atoms. Heptane content represented 3.8 and 36.8% of the wood volatile oils of Pinus jeffreyi and Pinus sabiniana, respectively [42]. The leaf alcohol (3(Z)‐hexen‐1‐ol, Figure 7) and its esters are responsible for the intense grassy‐green odor of freshly cut green grass and leaves via the octadecenoic pathway [43–45].
Chemical structure of leaf alcohol.
Chemical structures of phenylpropenes.
These aromatic compounds are an important group to flavor and fragrance industry though it constitutes a relatively small part of the essential oils. This non‐terpenoid group comprises of constituents derived from n‐propyl benzene. The aromatic ring may carry hydroxy, methoxy and methylene dioxy groups; the propyl side chain may contain hydroxyl or carboxyl group [25].
\nPhenylpropenes constitute a subfamily of phenylpropanoids that are synthesized from the amino acid phenylalanine and
The representatives of this group are the compounds, which are not included in the above mentioned three groups [25]. They are different degradation products originating from unsaturated fatty acids, lactones, terpenes, glycosides and sulfur‐ and nitrogen‐containing compounds [48].
\n\nChemical structures of sulfur‐ and nitrogen‐containing compounds.
Sulfur‐ and nitrogen‐containing compounds occur mainly as aglycones or glucosinolates, or their breakdown products, which include isothiocyanates [49]. The Brassicaceae is an important source of glucosinolates and isothiocyanates [50]. Garlic, onion, leek and shallots contain volatile sulfur compounds, namely allyl sulfide, dimethyl sulfide, diallyl disulfide and dimethylthiophene [51]. The sulfur compounds are responsible for the characteristic aroma and taste [52]. Cysteine sulfoxides including alliin predominate in mature, intact Allium spp., along with γ‐glutamylcysteines [53]. Upon rupture, such as when chopped or pressed, the action of a class of enzymes known as alliinases catalyzes the conversion of cysteine sulfoxides into the volatile thiosulfinates [54] including allicin. Allicin makes up 70–80% of the thiosulfinates. Allicin and other thiosulfinates decompose diallyl sulfide, diallyl disulfide, diallyl trisulfide, dithiins and ajoene, while the γ‐glutamylcysteines are converted to S‐allyl cysteine through a non‐alliin/allicin pathway [53]. Nitrogen‐containing compounds are found in only a few essential oils. Examples include methyl anthranilate, indole, pyridine and pyrazine. Methyl anthranilate is present in orange, lemon and bergamot oils [35] and jasmine oil [55]. Indole occurs in neroli and some citrus fruit oils [32]. Pyridines and pyrazines occur in black pepper, sweet orange and vetiver oils [35] (Figure 9).
\nThe studies have shown that a differentiation in oil quality and volatile components is associated with climatic conditions, geographical location of collection sites and other ecological and genetic factors. The influence of those factors on the accumulation of distinct volatile compounds defines its chemotypes [56–58].
\nBlack pepper oil is obtained from the dried unripe fruits of Piper nigrum [59]. The monoterpenes hydrocarbons (i.e., limonene, sabinene, β‐pinene, 3‐carene and phellandrene) to sesquiterpenes hydrocarbons (β‐caryophyllene) ratio represents the quality of the oil and indicates the aroma value; the monoterpenes provide the body of the flavor, but the sesquiterpenes provide the spicy note. A lower value represents a higher quality of the essential oil [60, 61]. A high total monoterpenes content results in a strong “peppery” top‐note and a predominantly pinene content of the terpene fraction gives a turpentine‐like note, while a high caryophyllene content results in a sweet, flowery note, which is more desirable in the flavor industry [62]. In addition to the ratio of monoterpenes to sesquiterpenes, the oxygenated compounds are supposed to provide the heart of the aroma of pepper oil [63]. The pepper oil extracted with supercritical fluid carbon dioxide was found to have a higher content of caryophyllene and oxygenated sesquiterpene compounds. The volatile oil obtained under these conditions was considered superior to the steam‐distilled oil, using aroma, taste and monoterpene to sesquiterpene hydrocarbons ratio as the criteria [64, 65]. Tipsrisukond et al. [66] reported that this ratio reached 3.02 and 0.95 in the steam distilled oil and supercritical fluid extract, respectively. On the other hand, this ratio was found to be 2.21 in the hydro‐distilled oil while it was 1.66 in the supercritical fluid extract [67].
\nStorage of black pepper hammer milled powder for 6 months at 4°C, markedly increased monoterpene to sesquiterpene hydrocarbons ratio from 1.31 to 4.47. They attributed this increase to the degradation of some sesquiterpenes, as a result of oxidative decomposition during storage [68].
\nCardamom oil is obtained from the dried ripe fruits of Elettaria cardamomum [59]. Salzer [60] reported that 1,8‐cineole and α‐terpinyl acetate together with the terpene alcohols are important for the evaluation of the aroma quality of cardamom. 1,8‐Cineole is reported to contribute to pungency while terpinyl acetate is known for its desirable, pleasant flavor of cardamom [69, 70]. The higher level of α‐terpinyl acetate compared to that of 1,8‐cineole could be used as an indicator of the superior quality of cardamom essential oil [71, 72].
\nMonoterpenes hydrocarbons are less odoriferous than oxygenated monoterpenes [73]. Amma et al. [74] recorded superior flavor quality for the Malabar variety with the highest α‐terpinyl acetate/1,8‐cineole ratio (1.55) when compared with the Mysore variety (1.34). Morsy [75] found that this ratio exceeded 1.6 when ultrasound‐assisted extraction (30 W, for 30 min) was used as a pretreatment for hydrodistillation (30 min) compared to 1.13 when hydrodistillation was conducted for 6 h. The presence of a low level of hydrocarbons in cardamom oil could be used as an indicator of its high quality [74].
\nCaraway oil is obtained from the dried ripe fruits of Carum carvi [59]. The main components of caraway essential oil are carvone and limonene, whose mixture constituted from 97.69 to 98.62% of total oil composition [76]. The overall quality of fruits is considered to correlate with the content of essential oil and its carvone/limonene (C/L) ratio: the higher the ratio, the better the quality. C/L ratio varies from 0.90 to 2.74 [77, 78]. This ratio is variable during ripening. Limonene is of high level in green immature seeds [79]. Aćimović et al. [76] considered the quality of essential oil poor when C/L ratio recorded 0.47. The essential oil of biennial caraway varieties usually has a higher C/L ratio compared to that of the annual varieties [80, 81]. The C/L ratio decreased with the time of storage (70 days, at room temperature) of caraway seed samples [82].
\nPeppermint (Mentha piperita) has bright green leaves, with a fresh, slightly sweet, tangy, peppery and strong menthol notes [83]. The principal constituents of peppermint oil are menthol (30–55%), menthone, menthyl acetate and other esters [84]. The ratio of menthol/menthone is the major determinant of the flavoring quality of distilled essential oil [85]. For commercial purposes, a high oil yield with a ratio of menthol to menthone of 2:1 is desired [86]. In general, high‐quality peppermint oil (i.e., high menthol/menthone ratio) develops during full bloom [87]. The English oil contains 60–70% of menthol, the Japanese oil contains 85% and the American only about 50%. The odor and taste afford a good indication of the quality of the oil.
\nLavender flowers (Lavendula angustifolia) have a strong perfumery odor with crusty woody undertones a camphor‐like note. The leaves have herbaceous and more pronounced bitter notes [83]. Linalool and its ester form, linalyl acetate, are the most abundant monoterpenes in lavender varieties and are the most desired components of the lavender oil, while trace amounts of camphor generally contribute an undesirable odor, diminishing the quality of the oil [88–90]. The linalyl acetate to linalool ratio may change in different distillation times and may affect the final odor of the oil [91]. The oil samples that were obtained after 1 and 2 h of steam distillation had linalyl acetate to linalool ratio of 0.57 and 0.44, respectively. The ratio of linalyl acetate to linalool should be higher than one in high‐quality lavender oil [92].
\nRosa damascena is an ornamental plant. This plant is used for food flavoring as dried flowers, dried bud and dried petals [93]. The flavor compounds that contribute to the distinctive scent of rose oil are β‐damascenone, β‐damascone, β‐ionone and rose oxide. Even though these compounds exist in <1% of rose oil, they make up for more than 90% of the odor content due to their low odor thresholds. The concentration of β‐damascenone is considered as the marker for the quality of the rose oil [94, 95].
\nCitronellol/geraniol (C/G) ratio could be used for evaluating the quality of rose oil [96]. The finest quality rose oil has C/G ratio between 1.25 and 1.30 [97]. In the rose oil trade, the citronellol content should be higher than 35%. The oils from non‐fermented petals generally contain citronellol lower than this level. Therefore, a short‐term fermentation is conducted to increase the citronellol content. The C/G ratios were higher in the oils distilled from long‐term fermented petals (e.g., 10.3 in 36 h fermentation) than non‐fermented petals (0.56). Based on these results, rose oils distilled from long‐term fermented petals are of poor quality [98].
\nGinger oil is obtained from the rhizomes of Zingiber officinale [59]. α‐Zingiberene is the major sesquiterpene hydrocarbon of ginger oil [99]. Salzer [60] reported that citral, zingiberene, β‐sesquiphellandrene and ar‐curcumene could be used for evaluating the quality of the ginger oil. Govindarajan [100] reported that citral and citronellyl acetate are co‐determinants of the odor of ginger oil, while zingiberene and β‐sesquiphellandrene are the main components of the freshly prepared oil. ar‐Curcumene increased with storage. A good quality oil has a ratio of zingiberene + β‐sesquiphellandrene to ar‐curcumene = 2:3.
\nThe lemony note is attributed to citral together with α‐terpineol, while nerolidol is responsible for the woody note. β‐Sesquiphellandrene and ar‐curcumene contribute to the characteristic ginger flavor [101].
\nJuniper (Juniperus communis) has green and sharp leaves (needles) [102]. Juniper\'s odor is woody and astringent with sweet, lemon and pinelike overtones. It has a ginlike aroma. Its flavor is released when it is lightly crushed [83].
\nButkienë et al. [103] identified 143 components in the juniper leaves essential oil. They found that monoterpenes (M)/sesquiterpenes (S) ratio ranged from 2:1 to 5:1 according to localities in Vilnius district, Lithuania. Sela et al. [104] found that M/S ratio ranged from 1:1 to 3:1 for leaves essential oil of Macedonian juniper, from different localities. Orav et al. [105] reported that M/S ratio of juniper berries oils was higher (4:1) than that obtained from leaves samples (2.5:1). Pourmortazavi et al. [106] found that supercritical fluid extraction products from J. communis L. leaves using carbon dioxide were markedly different from the hydrodistilled oil. The hydrodistilled oil was characterized by a high concentration of β‐phellanderene. The ratio of α‐pinene to 3‐carene in hydrodistilled oil was high (0.62) compared with that of the supercritical carbon dioxide extracts (0.04–0.065). Looman and Svendsen [107] reported that the average ratio of α‐pinene:sabinene:limonene was generally 21:45:5 in the leaf essential oil of Norwegian mountain juniper (Juniperus communis L. var. saxatilis Pall).
\nOregano, wild marjoram, Origanum vulgare, has dark green fresh leaves. Fresh oregano is available whole, chopped, or minced. The dried light green leaves are available whole, flaked, or ground [83]. The thyme plant, Thymus vulgaris L., is an evergreen herb that is used for its flavor [108]. Summer savory, Satureja hortensis, is often used as a culinary herb either as a fresh or dried herb [109].
\nIn oregano essential oil, the total content of the thymol and carvacrol was the highest and amounted to 67.51%; in thyme essential oil, 47.47%; and in savory essential oil, 49.71%. The thymol and carvacrol types have sharp, warm and penetrating herbal (thyme type) odors, with woody, spicy and tobacco‐like notes; thymol itself has a powerful, medicated and herbaceous odor while carvacrol itself has a tar‐like odor [110]. The ratio of carvacrol to thymol in oregano, thyme and savory essential oils was 15:1, 1:19 and 1.8:1, respectively [111]. Oils containing predominantly thymol are generally considered of superior quality [112, 113].
\nLemongrass (Cymbopogon spp.) is a tall perennial C4 grass belonging to the family Poaceae (Gramineae), commonly known as the “sweet grass family” [114]. Lemongrass gives a refreshing lemon‐lime‐like taste with a tinge of mint and ginger. It has a citral odor with floral‐like (rose) and a fresh, grassy aroma. Its flavor is found in the lower tender part of the stem. The root‐end stalk gives the most flavor. The whole fresh stalk becomes aromatic when it is crushed or cut. The dried form has a very little aroma [83]. The quality of the lemongrass essential oil is measured by its citral content [115, 116]. Citral is a natural mixture of two monoterpene aldehydes, geranial (trans isomer) and neral (cis isomer). Supercritical fluid extraction was found to be a superior process than steam distillation, producing better quality lemongrass oil containing 90% citral [117]. The leaves yield aromatic oil, containing 70–90% citral. C. flexuosus has higher citral content than C. citratus [118, 119].
\nEssential oils from different plant parts exhibit different biological activities [120]. Biological activities of essential oils include antioxidant, antimicrobial, antiviral, anti‐mutagenic and anticancer [34]. Essential oils are complex mixtures of terpenoids and phenylpropanoids compounds extracted by distillation or solvent extraction [121]. Overall activity cannot be attributed to only one of the major constituents [122]. The inactive compounds might influence resorption, the rate of reactions and biological activity of the active compounds. The combination of the major and minor constituents modifies the activity to exert significant synergistic or antagonistic effect [123].
\nAntioxidants are substances that protect cells from being oxidized by free radicals. Reactive oxygen species (ROS) are highly reactive toxic molecules. ROS induced oxidative diseases such as ageing, arteriosclerosis, cancer, Alzheimer\'s disease and Parkinson\'s disease [124]. Living cells possess scavenging activity to diminish excess ROS that induced cellular injury. These mechanisms become inefficient, with ageing and under external stress. Therefore, dietary supplementation of natural and synthetic antioxidants is required [125]. Some synthetic antioxidants cause liver damage and have carcinogenic effects. Natural antioxidants are preferred to synthetic ones [126].
\nThe essential oil of lemon balm, containing neral/citral, citronellal, menthone and isomenthone, showed strong antioxidant activity [127].
\nThe essential oils with good radical‐scavenging activity could be arranged in the following order, clove > cinnamon > nutmeg > basil > oregano > thyme [128].
\nMisharina and Samusenko [129] stored a mixture of lemon, coriander and clove buds essential oils (1:1:1) at room temperature in the light for 145 days. They found that the mixture of the three essential oils increased the stability of limonene and γ‐terpinene significantly higher than in the individual essential oils and inhibited oxidation of hexanal efficiently. It was shown that terpene hydrocarbons break free‐radical chain reactions, which is accompanied by their irreversible oxidation into inert compounds, such as p‐cymene. Therefore, terpene hydrocarbons do not exhibit the properties of prooxidants.
\nWei and Shibamoto [130] proposed that terpenes and terpenoids that contribute to the antioxidant activity of essential oils include α‐terpinene, β‐terpinene and β‐terpinolene in tea tree; 1,8‐cineole in water mint, menthone and isomenthone in peppermint; thymol, eugenol and linalool in black cumin, cinnamon bark and ginger; and thymol and eugenol in thyme and clove leaf.
\nEugenol, the major constituent of clove essential oil, was found to have an inhibitory activity against lipid peroxidation by interfering with chain reactions of free radicals. The inhibitory activity of eugenol was about fivefold higher than that observed for alpha‐tocopherol [131]. Thymol and carvacrol the main constituents of thyme oil are shown to act as strong antioxidants [132]. The antioxidant activity of caraway oil may be due to the presence of linalool, carvacrol, anethole and estragole [133]. Marjoram and clove essential oils exerted a powerful antioxidant activity in beef burger prepared with sunflower oil during storage at -18°C for 3 months. The addition of marjoram oil at 250 mg/kg kept thiobarbituric acid value of the beef burger samples after 3 months of storage at a level not significantly different from that of control samples stored frozen for 1 month [134]. Flavoring refined corn oil with thyme increased its oxidative stability (induction time) from 4.36 to 6.48 h [135]. Blending cold‐pressed oregano (Origanum vulgare) oil with sunflower oil at 10 and 20% levels increased its induction time from 3 to 6 h and 8 h, respectively. Assiri et al. [136] attributed the superior antioxidant activity of sunflower oil blends to the high levels of phenolic compounds in oregano oil.
\nEssential oils display broad‐spectrum inhibitory activities against various bacterial pathogens [137]. Essential oil is easily permeable through the cell wall and cell membrane due to its lipophilic characteristic. Interaction of essential oil components with polysaccharides, fatty acids and phospholipids causes loss of membrane integrity, leakage of the cellular contents, interference in proton pump activity and leads to cell death [124, 138–140]. Other important mechanisms of action include denaturation of cellular proteins [9, 34, 141]. Carvone partitioned in the lipid membrane [142], while terpinen‐4‐ol inhibits cellular respiration and both damage cell membrane function as a permeable barrier [143]. Carvacrol and p‐cymene accumulate in the lipid phase of the membrane by causing expansion of the phospholipids bilayer and increasing spaces through which ion leakage might occur [144].
\nSage essential oil is rich in antimicrobial agents [145]. The single layer wall of S. aureus is highly sensitive to essential oils with a high content of p‐cymene [34]. Cold‐pressed oregano (Origanum vulgare) oil exhibited antimicrobial activity against Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Listeria monocytogenes with minimum lethal concentrations ranging between 160 and 320 μg/mL. The antimicrobial activity of the oregano oil could be due to the presence of phenolic constituents and the differences in the permeability of cell wall of those bacteria [136]. Ghabraie et al. [146] found that essential oils of Red bergamot (Flower top) and Chinese cinnamon (bark) inhibited Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella Typhimurium. The inhibition zone ranged from 20 to more than 70 mm depending on the target bacteria. Chinese cinnamon inhibited S. aureus and E. coli at minimum inhibition concentration of 470 ppm in microbroth dilution assay. Antibacterial activity of Red bergamot oil could be due to carvacrol and p‐cymene, while trans‐cinnamaldehyde is responsible for this effect in Chinese cinnamon oil. Radaelli et al. [147] studied the antimicrobial activities of basil, rosemary, marjoram, peppermint, thyme and anise essential oils against Clostridium perfringens strain A. They found that all oils showed bactericidal activity at their minimum inhibitory concentration, except anise oil, which displayed bacteriostatic effect.
\nFungi are important causes of human infections [148]. Several crops are susceptible to fungal attack either in the field or during storage [149]. Fungicide residues are problems for the food industry [150]. Prevention of fungal growth is an effective way of impeding mycotoxin accumulation [151]. Essential oils have the ability to attack the life cycle of molds [152].
\nThe high cost of essential oils production and the low concentration of active constituents limit their direct use in the control of fungal diseases of plants and animals. Therefore, investigation of antifungal compounds of the essential oils is considered important because of the possibility of synthesizing these compounds for the use in the control of fungal diseases [153].
\nBouchra et al. [154] reported that the major essential oils constituents of Moroccan Labiatae Origanum compactum and Thymus glandulosus consisted of carvacrol, linalyl acetate and thymol. Both oils inhibited completely the growth of the mycelium of Botrytis cinerea at 100 ppm.
\nSerrano et al. [155] reduced the growth of yeasts and molds for stored cherries by developing active packaging materials containing eugenol, menthol, thymol and eucalyptol.
\nThe essential oil of cinnamon had a high antifungal effect (very low minimum inhibitory concentration) against Aspergillus flavus [156, 157]. The antimycotic activity of Cinnamomum zeylanicum bark essential oil is due to the presence of cinnamaldehyde [158].
\nCarvacrol, the major active ingredient, of oregano oil was found to cause complete inhibition of Saccharomyces cerevisiae growth at 0.01%. Its potency was 1500 times that of the oregano oil. In contrast, the γ‐terpinene, which is the biosynthetic precursor of carvacrol, was ineffective as a fungicide. Carvacrol interfered in the target of rapamycin signaling pathway, resulting in loss of viability. Eugenol, thymol and carvacrol affect Ca2+ and H+ homeostasis leading to loss of ions and inhibition of Saccharomyces cerevisiae [159]. Citral, citronellol, geraniol and geranyl acetate that are the major components of eucalyptus oil, tea tree oil and geranium oil possess cell cycle inhibitory activities against Candida albicans [160].
\nSourmaghi et al. [161] found that hydrodistilled coriander essential oil had a potent antifungal activity against Candida albicans. Linalool was the major component in coriander oil. The essential oil of coriander had synergistic antimicrobial effect with amphotericin B [162].
\nRahman et al. [153] reported that the hydrodistilled essential oil of the leaves of Piper chaba Hunter displayed potent antifungal activity against Fusarium oxysporum, Phytophthora capsici, Colletotrichum capsici, Fusarium solani, and Rhizoctonia solani. They attributed this activity to α‐humulene, caryophyllene oxide, viridiflorol, globulol, β‐selinene, spathulenol, (E)‐nerolidol, linalool, 3‐pentanol and p‐cymene that present in the oil. At a concentration of 125 μg/mL, the fungicidal action of oil showed complete spore germination inhibition of Phytophthora capsici.
\nde Oliveira et al. [163] found that the essential oil of Piper ilheusense was active in combatting activity of Candida albicans, Candida krusei, and Candida parapsilosis. (E)‐Caryophyllene and patchouli alcohol were the major components of the oil. γ‐Cadinene, germacrene B and gleenol were found in significant quantities. Marino et al. [164] reported that the minor components might be involved in some type of synergism with the other active compounds.
\nCancer is a multifactorial disease contributing towards the uncontrolled growth of the abnormal cells, leading to the formation of a tumor [165]. Carcinogenesis is a multistep process and oxidative damage that is linked to the formation of tumors. Secondary metabolites from different plants are capable of halting development of cancer [166]. Essential oil constituents have cytotoxic and antitumor activities. They play an important role in cancer prevention and treatment [18, 167]. Essential oils can be used in combination with cancer therapy to decrease the side effects of the drugs [168]. Cytotoxicity of essential oils is due to its action upon cellular integrity, leading to necrosis and apoptosis, cell cycle arrest and loss of key organelles function [169, 170]. Therefore, the evaluation of the anticancer activity of essential oils and their safety on normal cell lines are of great importance [171].
\nThe essential oil of Ricinus communis leaves exhibited a moderate antiproliferative activity against cervical cancer line. The composition of the oil was predominantly by α‐thujone and 1,8‐cineole [172]. The Eugenia caryophyllata essential oil showed significant cytotoxic effects against HT29, A549 and Hep2 cancer cell lines. The cytotoxicity is likely due to the high concentrations of phenolic compounds, particularly eugenol [173].
\nEugenol displayed cytotoxic action in a dose‐dependent manner against human hepatoma cells HepG2 and colon cells Caco‐2 [174], HL‐60 leukemia cells [175] and osteoblastic cell line U2OS [176].
\nTerpenoids of essential oils prevent tumor cell proliferation [34]. Linalool and linalyl acetate represented the major constituents in lavender essential oil. They were more cytotoxic than the whole essential oil against 153BR, HNDF and HMEC‐1 cancer cell lines [177]. Geraniol suppressed the growth of MCF‐7 breast cancer cells [178] and PC‐3 prostate cancer cells [179]. It was reported to interfere with membrane functions, ion homeostasis; inhibit DNA synthesis; and reduce the size of colon tumors [180]. β‐Caryophyllene did not inhibit cell growth of MCF‐7 breast cancer cell lines, but α‐humulene was cytotoxic. However, β‐caryophyllene potentiated the cytotoxicity of α‐humulene [181]. Limonene and linalyl acetate had no effect on neuroblastoma cells. However, their combination induced apoptosis [182]. This synergic effect was consistent in several studies for antitumor properties [183].
\nCarvacrol is a major component of oregano and thyme essential oils. It inhibits tumor cell proliferation and induces apoptosis in human colon cancer cell lines, HCT116 and LoVo [184] and in human oral squamous cell carcinoma [185]. Perillyl alcohol decreases the growth of HCT116 cells [186].
\nThe essential oils are multi‐component systems, while their key components represent single component systems. Quality assurance of essential oils is imperative to ensure authenticity and product quality. The ratio of one special component or group of components to another is one of the quality indices of an essential oil as it affects its aroma.
\nSome of the essential oil constituents contribute to the essential oil antioxidant, antimicrobial and anticancer activities, regardless their concentration. They contribute by their synergistic effect to the property of the essential oil.
\nHistoplasmosis is an uncommon endemic mycosis caused by the fungus Histoplasma capsulatum that usually causes an asymptomatic infection but occasionally results in severe multisystem disease [1, 2]. Two main varieties of the saprophytic, thermally dimorphic fungus of the genus Histoplasma affect humans; Histoplasma capsulatum var. capsulatum and Histoplasma capsulatum var. duboisii [3, 4, 5]. Histoplasmosis affects over 10,000 people globally; it is neglected, worryingly under-diagnosed, and often misdiagnosed as cancer or tuberculosis with fatal consequences [6, 7, 8]. The spectrum of the clinical manifestation of histoplasmosis is very broad, ranging from an asymptomatic or minimally symptomatic acute pulmonary disease following inhalation of a large inoculum of Histoplasma microconidia to chronic pulmonary disease in patients with underlying structural lung disease, to acute progressive disseminated disease in patients with severe immunodeficiency [9, 10, 11, 12, 13]. In immunocompetent patients, acute histoplasmosis is typically a self-limiting disease with no need for antifungal therapy [14]. Pneumonia remains the most common disease presentation but extrapulmonary dissemination can occur, especially in immunocompromised patients [12]. A definite diagnosis of histoplasmosis is based on the isolation of the organisms in fungal culture [15]. Rapid detection of the H. capsulatum polysaccharide antigen using enzyme immunoassay (EIA) in urine, blood or bronchoalveolar lavage fluid is also available and very useful especially among immunocompromised patients with disseminated or acute pulmonary disease [13, 16]. Serologic testing for antibodies can be achieved by EIA, immunodiffusion, and complement fixation [13, 16, 17]. These antibody tests may be falsely negative in immunosuppressed patients and are most valuable when combined with a compatible clinical presentation and epidemiologic risk factors.
This chapter provides an overview of the currently available treatment options for the different manifestations of histoplasmosis.
Histoplasmosis is primarily treated with 2 distinct classes of antifungal agents: The triazoles and the polyenes. Data is lacking on the use of the echinocandins for the treatment of histoplasmosis.
The triazole class of antifungals examples of which includes drug like fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole [18, 19]. Of these, itraconazole is the drug of choice for the treatment of the various forms of histoplasmosis as a sole therapy or as a step-down therapy following amphotericin B infusion [13, 14, 20].
Triazole antifungals inhibit the cytochrome P450-dependent enzyme 14-alpha-lanosterol demethylase (CYP51) encoded by the ERG11 gene that converts lanosterol to ergosterol in the cell membrane, inhibiting fungal growth and replication (fungistatic) [18, 19]. Drug-drug and drug-food interactions, as well as side effects are major issues associated with itraconazole therapy. Common side effects of itraconazole include drug-induced hepatitis, gastrointestinal discomfort, heart failure, ankle edema, alopecia, erectile dysfunction, gynaecomastia, peripheral neuropathy, visual disturbance and headaches [21]. Itraconazole interacts with agents used in the treatment of HIV (especially, nevirapine) and tuberculosis (especially, rifampicin) leading to decreased or increased exposures to itraconazole and therefore increased risk of hepatotoxicity. With the exception of fluconazole, voriconazole and posaconazole are used as alternative agents for the treatment of histoplasmosis if there is a contraindication to the use of itraconazole [13, 14, 20]. However, to the best of our knowledge, there is no data to support the use of isavuconazole for the treatment of histoplasmosis.
The polyene antibiotic, amphotericin B derived from the fermentation product of the filamentous bacteria, Streptomyces nodosus is a broad-spectrum antifungal agent administered as deoxycholate (conventional amphotericin B) or liposomal formulation [18, 19].
In its mechanism of action, amphotericin B preferentially binds ergosterol in the fungal cell membrane (and to a lesser extent, cholesterol in cell membrane of humans) to form channels through which small molecules leak from the inside of the fungal cell to the outside, resulting in death of the fungal cell (fungicidal) [18, 19].
Common side effects of amphotericin B include, acute infusion-related reactions characterised by nausea, vomiting, rigors, fever, hyper or hypotension, and hypoxia mainly driven by the effects of amphotericin B on pro-inflammatory cytokine production. Nephrotoxicity is another very important side effect of amphotericin B occurring in about 34–60% of patients. Blood disorders, especially anaemia and electrolyte disturbances, especially hypomagnesaemia and hypokalemia are common but can be part of the rare Fanconi syndrome. Other side effects include infusion site phlebitis, and acute kidney injury [21].
It is important to understand the indications and the choices of antifungal agents in the treatment of histoplasmosis. Manifestations of histoplasmosis that are a typically treated include moderate to severe acute pulmonary histoplasmosis, symptomatic chronic cavitary pulmonary histoplasmosis, acute progressive disseminated disease, and histoplasmosis in immunocompromised individuals [12, 13], (Table 1). Treatment is also indicated for complications of histoplasmosis such as mediastinal granulomas and adenitis [14], (Table 1). Itraconazole can be used for the treatment of symptomatic immunocompetent patients with indolent non-meningeal infection, including chronic cavitary pulmonary histoplasmosis [12, 13]. Itraconazole is also used as a step-down oral agent following initial treatment of severe disease with amphotericin B [12, 13]. Itraconazole is administered at a dose 200 mg 3 times daily for 3 days, as loading dose and then 200 mg once or twice daily, for maintenance therapy [12, 13]. Duration of itraconazole therapy depends on the histoplasmosis syndrome being managed (Table 1).
Histoplasmosis syndrome or complication | Risk factor | Clinical time-course | Indication for treatment | Treatment | Antifungal therapy duration | |
---|---|---|---|---|---|---|
Acute progressive disseminated histoplasmosis | Immunosuppression such as advanced HIV disease | 1–2 weeks | Always | Mild disease | Itraconazole | 12 months followed by maintenance antifungal suppression until immune recovery |
Moderate to severe disease | Amphotericin B for 1–2 weeks in non-meningeal and 4–6 weeks in meningeal disease followed by itraconazole as step-down therapy | |||||
Acute pulmonary histoplasmosis | High inoculum exposure to Histoplasma conidia | 1–2 weeks | Moderate or severe disease or immunosuppressed patient | Mild disease | Itraconazole | 12 weeks |
Severe disease | Amphotericin B for 1–2 weeks followed by itraconazole as step-down therapy | |||||
Subacute pulmonary histoplasmosis | Low inoculum exposure to Histoplasma conidia | Weeks to months | If symptoms last >1 months or immunosuppressed patient | Itraconazole | 6–12 weeks | |
Chronic cavitary pulmonary histoplasmosis | Chronic obstructive pulmonary disease and other lung diseases Smoking | Months to years | Always | Itraconazole | 1–2 years and until radiologic resolution or stabilisation | |
Mediastinal adenitis | Reactive and enlarged mediastinal lymph nodes | Early complication | If compressive symptoms present or adenitis last >1 months | Itraconazole and steroids | 6–12 weeks | |
Mediastinal granuloma | Coalesced necrotic mediastinal lymph nodes | Early or late complication | If compressive symptoms present | Surgery and itraconazole | 6–12 weeks | |
Mediastinal fibrosis | Fibrosis of mediastinal structures | Late complication | If compressive symptoms present | Stenting, arterial embolization or surgery | Not applicable |
Amphotericin by intravenous infusion is the drug of choice for the initial treatment of fulminant or moderate to severe infections, followed by a course of oral. Following successful treatment, itraconazole can be used for secondary prophylaxis against relapse until immune reconstitution is realised [12, 13]. The deoxycholate formulation of amphotericin B is administered at a dose of 0.7–1.0 mg/kg daily by intravenous infusion, meanwhile the lipid formulation of amphotericin B can be administered at a higher dose of 3.0–5.0 mg/kg daily. Liposomal formulation is preferred to the deoxycholate due to its superior side effect profile, better response rate and survival benefit [14, 20].
There are no known prospective studies that have evaluated treatment of central nervous system (CNS) histoplasmosis. Treatment recommendations are guided by several case reports, retrospective case series and expert opinion [12, 13].
Liposomal amphotericin B achieves higher CNS concentrations than deoxycholate formulations and together with triazoles, is the recommended therapy for CNS histoplasmosis [22]. While the choice of the optimal triazole is in doubt, there is evidence from an animal model to suggest that fluconazole may be antagonistic when combined with amphotericin B [23]. Voriconazole could have a role among patients with good performance status as monotherapy or in combination with amphotericin B, but the evidence is limited and the occurrence of hepatotoxicity and hypersensitivity may limit its use [24, 25, 26]. One case series of 11 cases reported a morbidity free survival of 54.5% when patients were treated with intravenous amphotericin B deoxycholate for 8 weeks followed by maintenance therapy with fluconazole or itraconazole for 12–18 months [27].
The Infectious Diseases Society of America (IDSA) recommends treating CNS disease with liposomal amphotericin B (3.0–5.0 mg/kg daily for a total of 175 mg/kg given over 4–6 weeks), followed by a maintenance phase with itraconazole for at least a year at a dose of 200 mg, given 2–3 times a day [14]. Resolution of CSF abnormalities, including a negative antigen test is the recommended treatment target. Specific recommendations for CSF monitoring are: (a) worsening disease in the initial 2 weeks of therapy or lack of improvement by 1 month of therapy, in order to re-evaluate the diagnosis, (b) when amphotericin B is being replaced by a triazole, (c) if relapse is suspected and (d) after 1 year of therapy, to make the decision of treatment continuation or stoppage. Therapeutic drug monitoring for itraconazole is recommended during treatment to ensure adequate drug exposure. Treatment recommendations for histoplasmosis meningitis or CNS masses are the same. Also, for patients with concurrent pulmonary disease, chronic suppressive therapy with 200 mg of itraconazole, given once a day, is indicated until the patient’s immune system is reconstituted [13, 14, 28].
The role of steroids is not well described, although case reports indicate they can be successfully used [14] . Similarly, routine brain or spinal cord surgery is not recommended by IDSA but is chronicled in case reports [14, 28]. Hydrocephalus complicating CNS histoplasmosis may be managed with shunt placement when the patient has received at least 2 weeks of amphotericin B therapy [22]. The management of increased intracranial pressure and the long-term sequelae that require rehabilitation is similar to what is done for stroke and brain tumours [14, 28].
In a sizable cohort of patients, the one-year survival of patients with CNS histoplasmosis was reported to be 75% among patients who were initially treated with liposomal or deoxycholate formulations of amphotericin [28].
All presenting clinical syndromes of histoplasmosis in pregnancy require antifungal therapy due to the increased risk of trans-placental transmission of the infection to the developing fetus [29]. Preferably, pregnant women can be treated with amphotericin B preparations for 4–6 weeks. Lipid formulation of amphotericin B is given at a dose of 3–5 mg/kg/day. For pregnant women with low risk for nephrotoxicity, amphotericin B deoxycholate (0.7–1.0 mg/kg/day) may be offered as a substitute [14, 30]. Coupled with this, post-partum monitoring of the child for any evidence of the infection is vital. In any case of the newborn showing signs of histoplasmosis infection, it is recommended that treatment of amphotericin B deoxycholate (1 mg/kg/day) is given for 4 weeks [14, 30].
Itraconazole is generally considered teratogenic to the growing fetus and is therefore best avoided in pregnancy. However, itraconazole may be considered for treatment in pregnant women with systemic histoplasmosis, but only after the first trimester [31, 32]. Amphotericin B therefore remains the drug of choice in managing histoplasmosis in pregnancy, as azole antifungals should generally be avoided.
Immune reconstitution inflammatory syndrome (IRIS) is a spectrum of inflammatory disorders linked with paradoxical worsening of pre-existing infectious diseases (previously diagnosed or subclinical) in HIV-infected patients. It typically follows, the initiation of antiretroviral therapy (ART): whereby the ART improves the patient’s immune system enough to mount an inflammatory response tends to unmask the underlying infectious processes, such as histoplasmosis [33, 34, 35, 36].
In studies done among HIV patients, histoplasmosis-associated - IRIS was associated with histoplasmosis; IRIS was reported to be uncommon with incidence rates of 0.74 cases per 1000 HIV-infected person-years 0.5% [37, 38, 39]. However according to another study among 271 patients, the emergence of IRIS tended to be quite common in people with HIV and disseminated histoplasmosis; whether the IRIS is triggered by the Histoplasma or other co-infections, is still unclear [40].
For HIV patients with histoplasmosis to be considered to have histoplasmosis -associated IRIS, they should fulfil most or all of the following criteria: AIDS with low pretreatment CD4 count (≤100 cells/μL), a positive immunological and virological response to ART, clinical manifestations of an inflammatory condition, association between ART initiation and appearance of clinical features of the inflammatory condition and; absence of evidence of ART resistance, patient non-compliance, drug allergy or adverse reactions, a concomitant non- fungal infection, or decreased drug levels due to malabsorption or drug-drug interactions [36, 38, 41, 42, 43].
The clinical manifestations vary from case to case, and they commonly include fever, lymphadenopathy, mucocutaneous lesions, and disseminated disease. The timeline of occurrence from initiation of ART is also varied from a few days to months, and symptoms can set in when the patient is already on the ART course or when the ART is just introduced [39, 44, 45]. Histoplasmosis-associated IRIS in HIV patients may have a predilection for females over males, with one study showing that it was four times more frequent in females than males [39].
If the symptoms of IRIS are mild, the patient is managed symptomatically. The role of steroids in treating histoplasmosis-IRIS is yet unclear [39].
However if the presentation is severe, corticosteroids may be used as they have been seen to be of benefit in TB-associated IRIS, though they should be used with caution [46].
Once a patient exhibits features of IRIS while already on effective ART, the ART should be continued and the patient should be initiated on histoplasmosis treatment immediately. However if the patient is not receiving ART, a two-week delay is encouraged before starting ART while the patient is on antifungal therapy (amphotericin B or itraconazole) as per the guidelines [14].
Itraconazole alone is used for the treatment of mild forms of histoplasmosis and as a step-down therapy in severe disease and for secondary prophylaxis to prevent relapse in the immunocompromised after induction therapy with amphotericin B. Moderate-to-severe acute pulmonary histoplasmosis as well as acute progressive disseminated histoplasmosis require intravenous amphotericin B therapy for at least 2 weeks (4–6 weeks if meningeal involvement) or until a patient can tolerate oral therapy, then oral itraconazole (or an alternative triazole) for at least 12 weeks (for acute pulmonary) or 12 months (for acute progressive disseminated histoplasmosis). Chronic cavitary pulmonary histoplasmosis is treated with itraconazole for 12–24 months. There is insufficient evidence to support the use of isavuconazole and the echinocandins for the treatment of histoplasmosis.
IntechOpen celebrates Open Access academic research of women scientists: Call Opens on February 11, 2018 and closes on March 8th, 2018.
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\\n\\nAPPLYING FOR THE “INTECHOPEN WOMEN IN SCIENCE 2018” OPEN ACCESS BOOK COLLECTION
\\n\\nWomen scientists can apply for one book topic, either as an editor or with co-editors, for a publication of an OA book in any of the scientific categories that will be evaluated by The Women in Science Book Collection Committee, led by IntechOpen’s Editorial Board. Submitted proposals will be sent to designated members of the IntechOpen Editorial Advisory Board who will evaluate proposals based on the following parameters: the proposal’s originality, the topic’s relation to recent trends in the corresponding scientific field, and significance to the scientific community.
\\n\\nThe submissions are now closed. All applicants will be notified on the results in due time. Thank you for participating!
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\n\nAPPLYING FOR THE “INTECHOPEN WOMEN IN SCIENCE 2018” OPEN ACCESS BOOK COLLECTION
\n\nWomen scientists can apply for one book topic, either as an editor or with co-editors, for a publication of an OA book in any of the scientific categories that will be evaluated by The Women in Science Book Collection Committee, led by IntechOpen’s Editorial Board. Submitted proposals will be sent to designated members of the IntechOpen Editorial Advisory Board who will evaluate proposals based on the following parameters: the proposal’s originality, the topic’s relation to recent trends in the corresponding scientific field, and significance to the scientific community.
\n\nThe submissions are now closed. All applicants will be notified on the results in due time. Thank you for participating!
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