DOI: 10.1002/slct.

201902339 Full Papers

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2 z Materials Science inc. Nanomaterials & Polymers
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Assessment of the Efficiency of a Nanospherical Gallic Acid
6 Dendrimer for Long-Term Preservation of Essential Oils: An
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8 Integrated Chemometric-Assisted FTIR Study
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10 Silvana Alfei,* Paolo Oliveri, and Cristina Malegori[a]
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Dedicated to my parents and Professor Marco Pocci
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Essential oils (EOs) are volatile chemicals very popular in the In this study, a hydrophobic, GA-enriched antioxidant den-
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food, cosmetic and pharmaceutical industry as aromas, fragran- drimer (GAD), is advised as an innovative preservative, after
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ces and alternative therapeutic devices. EOs are susceptible to due evaluations. Besides oxidation chemical indexes, the
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oxidative degradation, loss of quality and alterations of sensory desired information was obtained by FTIR spectroscopy
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and pharmacological properties with production of smelly or assisted by multivariate analysis. For further confirmation, FTIR
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harmful compounds, responsible for allergic reactions. For data were interpreted by considering the area of some selected
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preventing EOs’ spoilage, synthetic preservatives are adopted, bands. Iodometric titration, was performed to determine
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but with poor efficiency and danger of health diseases. Natural peroxide value (PV), selected as chemical index. GAD showed a
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polyphenols as gallic acid (GA) are proposed as safer alter- preservative power extraordinary better than that of GA and no
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natives, but the low compatibility with oils limits their efficacy. pro-oxidant action.
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27 Introduction
a “green” alternative in the nutritional, pharmaceutical ethno-
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Essential oils (EOs) are naturally occurring oily mixtures made pharmaceutical and agricultural fields thus resulting in a broad
29
of a few to more than 100 single substances closely related spectrum of applications. Aromatherapy is an example of EOs
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from plants kingdom.[1] They encompass lipophilic and highly application in alternative medicine with relaxant and tranquil-
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volatile low molecular weight secondary plant metabolites that, lizer purposes.[19]
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according to regulations, can be separated from other plant Obviously, when used for medical purposes, EOs need to
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components or membranous tissue only by physic comply with national or international Pharmacopoeia.[20]
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methodologies.[2–5] Essential oils have been suggested as antioxidants and
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The methods allowed by law are distillation with water or preservatives both in food and into foodstuff packaging
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steam to separate EOs from vegetable raw material, and material to prevent rancidity or spoilage of packed foods
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mechanical processes or dry distillation to extract EOs from the respectively.[21,22] They have been also proposed as plant and
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epicarp of citrus fruits (ISO 9235, 1997). EOs, but often also only crop protectants, due to their nature of secondary plant
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a single EOs component, are extensively adopted as flavoring metabolites whose task is help the plant to survive in its
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or perfuming materials[6] both in food and cosmetic industry environment.[14]
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and they must fulfil specific physical standards. Maximum Early, but promising approaches, have been reported about
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quantities and the specific uses are fixed, even if essential oils EOs or components thereof utilization in medicinal products
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are Generally Regarded As Safe (GRAS) for their use in foods by for human or veterinary use.[17,23] In recent times, consumer’s
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the U.S. Food and Drug Administration (FDA).[7] interest and awareness toward the use of natural ingredients,
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As reported, EOs, in addiction to provide good flavors, especially in food, household and cosmetic products, are
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possess several beneficial properties such as antimicrobial, growing more and more and EOs have gained great
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antiviral, nematocidal, antifungal, insecticidal, and antioxidant popularity.[24,25]
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activity.[8–17] Some of them showed also to work as stimulators Consequently, also research in this field has become
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for the nervous system.[18] Consequently, essential oils represent increasingly pressing. Oils such as eucalyptus or lemon by far
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exceed 1000 metric tons per annum with an estimated value of
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several hundred million euros in 2007.[26] Encompassing an
52 [a] Prof. Dr. S. Alfei, Dr. P. Oliveri, Dr. C. Malegori array of many lipophilic and highly volatile components
53 Department of Pharmacy (DiFAR), University of Genoa, Viale Cembrano, 4
derived from a great range of different chemical classes, EOs
54 - I-16148 - Genova (GE)
E-mail: alfei@difar.unige.it may easily undergo conversion and degradation reactions.
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Supporting information for this article is available on the WWW under Since the flavor intensity of the single component does not
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https://doi.org/10.1002/slct.201902339 depend on its concentration, but relies on the specific odor
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threshold that is determined by structure and volatility, even Starting from the assumption that oxidation and radical
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minor components deriving from oxidation or degradation reactive species (ROS, RNS) are the key triggering factors for
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reactions may have a strong impact on the fragrance if their the onset and development of degradation processes in EOs
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aroma value is high enough.[27] once deprived of plant matrix, application to them of synthetic
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Degradation by oxidative and polymerization processes or natural antioxidants is considered as a promising solution.[20]
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may result in a loss of quality, in a reduction or change of flavor Butylated hydroxyl toluene (BHT), butylated hydroxyl
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but also in a loss of pharmacological properties. Temperature, anisole (BHA), tert-butyl hydroquinone (TBHQ), propyl gallate
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light, and oxygen availability are the most responsible for EOs and butyl gallate esters (PGE and BGE) are examples of phenol
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corruption and are crucial for their integrity. or polyphenols synthetic or semisynthetic antioxidant preserva-
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By oxidation, isomerization, cyclisation, or dehydrogenation tives, which can delay or prevent food and EOs oxidation.
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reactions, triggered either enzymatically or chemically, EOs Concerning BHT and BHA, besides a limited efficiency when
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components can easily convert into each other with dramatic compared to natural antioxidants as ellagic acid or gallic
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changes in oil properties. It is well known that EOs original acid,[39] they may work as potential bladder carcinogenic
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chemical composition may begin to vary already in the starting compounds if used at high doses.[40] Therefore, it is desirable to
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plant material, being influenced by plant health, growth stage, explore alternatives to synthetic phenolic antioxidants. In the
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habitat including climate, edaphic factors as well as harvest recent years, many phytochemicals are investigated as valuable
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time.[4,28] compounds possessing scavenging properties towards radical
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Furthermore, EOs constituents depend also on the con- oxygen species and complexing properties towards proteins.
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ditions during processing and storage of the plant material, These abilities make polyphenols interesting for the treatment
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upon distillation, as well as in the course of subsequent of various diseases like inflammation or cancer, but also for
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handling of the oil itself.[29,30] But, once deprived of the anti-ageing purposes in cosmetic formulations or for nutraceut-
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protective compartmentation in the plant matrix, EOs’ constit- ical applications.
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uents are especially prone to oxidative damage, chemical Plant extracts, such as those of olive or tea, have been
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transformations, or polymerization reactions. These aging studied in preservation for extending food shelf-life or for the
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processes generally come along with a more or less pro- preparation of promising preservative materials for packaging
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nounced quality loss,[31] such as development of unpleasant film.[41,42]
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and often pungent flavors, shifting colors, i. e. formation of a As a consequence, several plant polyphenolic antioxidants,
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yellow staining in caraway oil[32] or changes in consistency up that proved to be more efficient and more thermally stable
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to resinification. than top synthetic ones, were considered as safer and more
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These spoilage indicators are visible even if a single capable alternatives and they were introduced into the food
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component degrades.[3,33] Besides sensory alterations and industry. Among natural occurring polyphenols, gallic acid (GA)
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viscosity changes, some aged essential oils, as well as oxidized (Figure 1.a) is able to exert antioxidant activity also in emulsion
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terpenoids have revealed skin-sensitizing capacities[34–36] lead-
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ing to a hypersensitivity reaction synonymous to allergic
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contact dermatitis.[37]
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Oxidative degradation of EOs constituents translates in the
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uncontrolled hyper production and accumulation of the so
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called radical oxygen and nitrogen species, ROS and RNS.[38]
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ROS and RNS in turn trigger an oxidative stress (OS)
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condition that in addition to accelerate the propagation of oil
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oxidative degradation, transform its antioxidant and beneficial
41
properties into pro-oxidant with the risk of damaging DNA and
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establishing of detrimental pathological conditions. Given all
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these considerations on the easy spoilage of EOs, for a safe use Figure 1. Structure of gallic acid (GA) and of GA-enriched dendrimer (GAD).
44 Gallic acid (GA) named 1 in Supporting Information (SI) (a); intuitive
of EOs in the food, cosmetic and pharmaceutical fields,
45 representation of GA-enriched dendrimer (GAD) named 9 in SI (b).
restrictive expiration dates are applied, with a strong impact on
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the production costs of EOs derivatives. Consequently nowa-
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days, a considerable part of research, that aims at a safer and
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more careful use of EOs both for industrial and medicinal or lipid systems[43,44] and in the form of propyl or butyl ester
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applications, urgently needs of being able to handle suitable derivatives, it is used in processed foods, cosmetics and food
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tools for an easy and fast evaluation of the health state of Eos, packing materials to prevent rancidity induced by lipid
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before utilization. In addition, the design and development of peroxidation and spoilage.[45]
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innovative, more efficient and safer preservative additives as an GA is promising not only for industrial application as
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alternative to the existing ones, could represent a better conservative additive but also for the treatment of several
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chance to prevent degradation, spoilage and rancidity of these degenerative diseases.[46] Unfortunately, its clinical application
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precious substances and/or to improve their shelf-life. is limited by its pharmacokinetic drawbacks, poor oral bioavail-
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Figure 2. SEM images of GA-enriched dendrimer nanoparticles. Scale bars represent 300 nm.
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ability, poor gastrointestinal-tract (GIT) absorbability, fast classic chemical indicator of oxidation, was also performed for
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metabolism and rapid excretion in urine.[47–50] confirmation of the results.
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Adopting the common practice of making use of nano- All the responses asserted that, the thermally-induced
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particles to ameliorate bioavailability and metabolism profile[51] oxidative degradation of the essayed samples, stops at early
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of chemical bioactive entities (CBEs) affected by poor pharma- stages when they are treated with GAD. Degradation goes on
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cokinetic, a GA-enriched dendrimer (GAD), named 9 in to considerable high degree of spoilage, when samples are not
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Supporting Information (SI) able to carry sixty-four GA units at treated with antioxidants, and goes even further if they are
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once with potentials both for clinical and industrial applications added with GA that, after a very limited initial phase, seems to
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was recently prepared (Figure 1.b).[52] act as a pro-oxidant, improving spoilage rather than limiting it.
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GAD showed an almost perfectly spherical morphology
30
(Figure 2), nanosized particles (Table S4.2.1, Section S4.2 in SI)
31 Results and Discussion
and no cytotoxicity in the 3-(4, 5-dimethylthiazol-2-yl)-2,5-
32
diphenyltetrazolium bromide (MTT) assay, performed on two In order to propose an innovative dendrimer nanomaterial as
33
cell lines (Section S4.2, Table S4.2.1 in SI).[52] powerful antioxidant preservative applicable in lipids substrates
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It proved to be highly compatible with lipids and oily and as an alternative to the existing small molecules, a
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matrices, to own a radical scavenging activity four times higher biodegradable hydrophobic dendrimer decorated peripherally
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than free GA in DPPH test, to be biodegradable through with sixty-four units of GA (GAD)[52] was evaluated as
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esterase hydrolytic actions and to be able to release bioactive conservative additive against the naturally occurring degrada-
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antioxidant GA units.[52] tion of essential oils and was compared with GA.
39
On this background, in this work, GAD was prepared and FTIR spectroscopy was adopted as an evaluation method.
40
characterized again, then it was investigated to evaluate its Iodometric titration was then performed for validation pur-
41
activity as additive to prevent, contrast or postpone the poses. Since atmospheric autoxidation, in turn triggered by
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naturally occurring oxidative degradation of essential oils when ultraviolet (UV) and visible (VIS) light, oxygen in the air and
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subjected to drastic conditions of oxidative stress and its raising in temperature, plays a key role in EOs spoilage, the
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efficiency was compared to that of free GA. designed investigations were developed on model systems of
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Low cost, simple, rapid, conservative and reproducible progressive oxidative degradation.
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Fourier-transform infrared (FTIR) spectroscopy, extensively
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employed in similar investigations,[53–55] was adopted as a
48 Samples Oxidation
leading evaluation method for characterizing samples, identify-
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ing the less degraded ones and tracking down the most Oxidation was artificially induced by treating the selected
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suitable additive, in place of using classic oxidation indices or samples under examination at high temperature (120 °C) in the
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other more expansive and time-consuming analytical techni- presence of light and air for reaching the maximum degree of
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ques. The FTIR spectral data were analyzed both by studying oxidative degradation in a brief time and shortening analysis
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the changes in the areas of significant bands in function of duration. The progression of damage by oxidation was
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oxidation time and by multivariate analysis (MVA). Then, evaluated, in the absence of additives, then in presence of GA
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iodometric titration for obtaining the peroxide value (PV), a and finally of GAD. The samples were heated at 120 °C (for
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3.5 h in the case of β-P, for 8–10 h in the case of EO), then
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20 Figure 3. FTIR spectra of β-pinene and nopinone from Merck (formerly Sigma-Aldrich) database.
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stirred for further time at room temperature, but well exposed Upon temperature increase, dominant oxidative alterations
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to light and air. This additional time was chosen according to in essential oils such as cardamom, clove bud, lavender, pine,
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the physic aspect of samples which changed during the and rosemary were revealed in decreasing amounts of terpene
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experiments: from colorless, samples yellowed and, from fluids, hydrocarbons such as β-caryophyllene, β-myrcene, β-pinene,
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progressively they became more viscous. When viscosity sabinene, or γ-terpinene and in an overall rise of p-cymene.[60,61]
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became excessive and withdrawals to acquire FTIR spectra Obviously, temperatures above 100 °C, as in the experi-
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were not feasible, the experiment was interrupted. In different ments performed in this study, do not necessarily induce only
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cases, when the degradation was terminated, even if viscosity the same changes in terpenoids as real-term storage conditions
31
would have allowed to measure further FTIR spectra, the and lead to alcohols, aldehyde and finally ketones and
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experiment was however stopped. The study was firstly carboxylic acids.[56]
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performed on a single reference compound commonly present β-pinene (β-P) is a bicyclic monoterpene found in pine EOs
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in EOs as a simplified model systems, then on the whole EO. (up to 19% in Pinus mugo,)[62] that may undergo thermal
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The single component model offered the possibility to identify isomerization to α-pinene (up to 23% in Pinus mugo) and vice
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and track much more easily the changes that occurred in the versa.[63]
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FTIR bands during the oxidation process. Therefore, once Among obtainable oxidation products from β-P and its
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obtained the information desired on the compound represen- isoform,[64] nopinone is typically found as the major oxidation
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tative of the most complex system of the whole essential oil, product (SI, Figure S6.1) with reported yields of 30%, 40% and
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additional investigations were performed on the oil itself to even 79% in respect of the initial concentration of β-P.[65]
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achieve the actual goal of the work. All possible compounds possess the ketone or carboxyl
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C=O group and nopinone, the major product, shows a
43
characteristic C=O stretching band around 1700 cm 1 in the
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Selection of samples FTIR spectrum, absent in non-oxidized β-P, which instead
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presents a strong band at 1641 cm 1 not observed in nopinone
46 Choice of the single reference component
spectrum (Figure 3).
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EOs components are mainly lipophilic terpenoids, phenyl- The progressive advancement of the oxidative degradation
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propanoids, or short-chain aliphatic hydrocarbon derivatives[56] of β-P could therefore be easily monitored through FTIR
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of low molecular weight. Terpenoids are the most affected by analysis by observing the appearance and then the increase in
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oxidative degradation and deteriorate through a free radical intensity of the band around 1700 cm 1 and in parallel the
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chain mechanism with aerial oxygen that leads to a range of decrease in intensity of the band at 1641 cm 1. So, β-P was
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primary and secondary oxidation products. Among terpenoids, selected as single component system representative of the
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heat-labile terpenes, at elevated temperatures, are susceptible more complex system of the essential oil.
54
to rearrangement processes and terpene conversion reactions
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that have been reported both for isolated compounds[57,58] as
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well as for essential oils.[59]
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Choice of the EO
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After collecting information about the oxidative degradation
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profile of β-P, the investigation was extended to an essential
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oil. To be consistent with the preliminary investigations, an
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essential oil that contains a considerable concentration of the
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compound used as a simplified model was chosen. Then, the
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pine essential oil was selected and among the various species
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of pine, Pinus mugo Turra was chosen because in addition to
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having various uses as a fragrance, it is also used in
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pharmaceutical preparations for its recognized healthy proper-
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ties. Table 1 reports the main constituent of Pinus mugo Turra
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essential oil (PEO).
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14 Figure 4. FTIR spectra of β-pinene (β-P).
15 Table 1. Chemical composition of the essential oil from the needles and
16 twigs of Pinus mugo Turra.[62]
17 Chemical Compound Pinus mugo essential oil (PEO) During the progression of the β-P’s oxidative degradation,
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δ-3-carene 7.6–33.2 FTIR spectra were acquired at fixed time points under the three
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α-pinene 7.9–22.8 conditions examined, i. e. without any additive, with the
20 β-pinene 1.5–19.0 addition of GA in the concentration approved by regulations
21 β-phellandrene 2.4–18.0
myrcene 1.7–21.5
(200 ppm; 200 mg kg 1; 0.2 mg g 1; 1.2 μmol g 1) and, finally,
22
bornyl acetate 1.5–8.0 with the equivalent mole amount of the GA-enriched den-
23
drimer (GAD) (1.2 μmol g 1; 20 mg g 1).
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For every time point, the FTIR spectra were acquired in
25
triplicate.
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For samples without additive and with GA, an excessive
27
viscosity was reached after 3.5 h at 120 °C and 16 hours at
28 FTIR spectra
room temperature; therefore, the experiments were stopped
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FTIR spectroscopy is a versatile method of analysis as it can after a total of 19.5 h.
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measure samples directly without preparation; it does not Differently, the sample treated with GAD did not become
31
involve harmful chemicals, provides fast results and gives too viscous, but the experiment was interrupted after 24 h
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highly reproducible results.[53–55] because the degradation did not progress further.
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In SI (Figure S6.2) an image of all the FTIR spectra acquired
34
in three diverse colors for the three conditions is available.
35 FTIR spectra of β-pinene (β -P) oxidative degradation
In all the three conditions examined, the small bands
36
FTIR spectrum of a sample of β-P acquired by Merck showed indicating trace of oxidation increased as a function of time,
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the existence of bands related to specific functional groups of reaching an intensity plateau after a certain time.
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the compound (Table 2, Figure 4) and in particular an intense As far as the bands indicating degradation are concerned,
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characteristic band at 1641 cm 1 interesting for our purposes. the highest intensity was reached, both by the sample without
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additive and by that one added with GA. In this cases the
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bands of the oxidations products completely triumphed on the
42 Table 2. Major bands observed from the FTIR spectra of β-pinene (Fig- β-P band at 1641 cm 1 which was practically no longer visible
43 ure 4).
(Figure 5.a and 5.b).
44 Peak no Wavelength (cm 1) Functional group Differently, for the sample with GAD, the oxidative degrada-
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1 3070 H-C=C stretching tion was considerably more limited with improvement in β-P
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2 2980, 2939 C–H stretching[a] shelf life and the bands between 1706 and 1653 cm 1 were far
47 3 2869, 2918 C–H stretching[a] less intense, leaving the band at 1641 cm 1 still clearly visible
48 4 1641 C=C stretching
5 1458 C–H bending[a]
(Figure 5.a and 5.b).
49
50
51 FTIR spectra of essential oil of Pinus mugo (PEO) oxidative
52 degradation
As asserted by very small bands at 1674, 1740 and
53
1706 cm 1 (Figure 4 and Figure 5) and consistent with func- To follow the progression of oxidative degradation of PEO, a
54
tional groups of oxidation products, commercial β-P already procedure similar to that described for β-P was performed.
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showed traces of degradation before the thermal treatment. Firstly, the FTIR spectrum of the fresh EO was acquired.
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Then, other FTIR spectra were measured at fixed time points
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19 Figure 5. FTIR spectra of β-P samples. FTIR spectrum of not heated β-P compared to the FTIR spectra of the three samples evaluated at their own maximum
level of degradation reached (a); expansion of the significant region of the spectra in the range 1800–1500 cm 1 (b).
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during the oxidation progression and until the degradation Areas ratios were chosen in order to minimize the error
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reached its final point and did not progress further. related to the difference in optical path of the sample aliquots
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For every time point, the FTIR spectra were acquired in deposited between the KBr dishes.
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triplicate. The values obtained as an average of three ratios �
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Images of the initial FTIR spectrum of PEO and of the all Standard Dediation (S.D.) obtained by the three FTIR spectra
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other spectra acquired are available in SI (Section S7, Fig- acquired for each time point, were reported in graph as a
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ure S7.1-S7.3). function of time, finding curves in Figure 6.
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Qualitatively, at an early and hasty observation, the most
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visible changes were similar to those seen during the experi-
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ments on β-P.
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34
35 FTIR spectra analysis
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FTIR spectra acquired during progression of thermally-induced
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oxidative degradation were exploited to get information about
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the efficiency of GAD as a conservative additive in comparison
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with free GA, both in the case of a single component model
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system and in the case of a real EO.
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The desired information was achieved analyzing FTIR
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spectra by two methods. The evaluation of the areas ratio
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between bands (subsequently abbreviated BAR) individuated
44 Figure 6. Oxidative degradation profiles reported as area ratios of bands
as indicators of oxidation and the characteristic β-P band
45 (BAR) indicating oxidation and β-P characteristic band (C=C stretching)
(1641 cm 1) was the first method, while the multivariate expressed in A % as a function of time (min).
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analysis (MVA) by Principal Components Analysis (PCA) was the
47
second one.
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49
According to the obtained curves, the addition of GA
50 FTIR spectra of β -P oxidative degradation analysis by BAR
resulted in reducing only slightly the maximum degree of
51 and MVA methods
oxidative degradation of β-P (’ 10%), while the addition of
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BAR method. The percentage ratios expressed as absorbance GAD decreased it by ’ 75%.
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(A/A %) between the increasing area of the bands in the range Considering the time necessary to reach a spoilage degree
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1706–1653 cm 1 (C=O stretching) and the decreasing area of of a medium entity [namely D50 (A/A%) ’ 2.9% ], both the
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the band of β-P at 1641 cm 1 (C=C stretching) measured in the sample without additive and that one added with GA reached
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range 1653–1506 cm 1 was considered for each experiment. the D50 in a time included in the range 150–210 minutes.
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Differently, in the presence of GAD, such a level of oxidation PC1 information precisely matches the response previously
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was never achieved. β-P degradation was initially remarkably obtained. At the extreme right of PC1 β-P and β-P + GA are
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slowed down then, after 210 minutes, it was totally blocked at both present with a trivial distance between them, indicating
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only 0.86% and no progression was observed also after 3.5 h at that the addition of GA, as a preservative additive, only slightly
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120 °C and 4.5 h at room temperature. decreases the maximum degree of degradation achievable.
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PCA method. All the FTIR spectra in the spectral region Looking, instead, at the respective positions of the samples
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range from 1800 cm 1 and 1500 cm 1 acquired at fixed time with or without GA along PC1, from left to right, the GA
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points during the progression of the oxidative degradation of samples are always found behind the samples without GA
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β-P and PEO were subjected to PCA, to identify important indicating a slight degradation delaying effect provided by GA.
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spectral regions that differentiated the samples and clustering No sample of β-P added with GAD reached the extreme
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patterns among different experiment times. As a result, the right of PC1 scores, stopping about in the middle of the
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desired information about the degree of oxidation achievable distance elapsing between not oxidized β-P and degraded
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by β-P and PEO without any additive, with GA and with GAD, ones. Every GAD sample remained always behind in the trend
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and consequently the efficiency of GA and GAD as conservative towards the positive scores on PC1, causing a delay effect on
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additives against oxidative degradation, was obtained from the progress of oxidation and confirming GAD as a strong
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PC1 and PC2. As far as β-P is concerned, on PC1, which conservative additive to prevent β-P spoilage. Loading profiles
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accounts for 86.1% of explained variance (Figure 7.a), the of PC1 (blue line in Figure 7.b), considered jointly with the
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score trend described above, confirm that the degradation
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phenomena can be followed thanks to a decrease of the FTIR
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band at 1641 cm 1 and a simultaneous increase of the spectral
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features between 1700 and 1750 cm 1.
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Analysis of FTIR spectra of Pinus mugo essential oil
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oxidative degradation by PCA and by BAR methods
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Similarly to β-P investigations, when Pinus mugo’s essential
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oil (PEO) degradation was studied, information about the
25
efficiency of GA and GAD as conservative additives against its
26
oxidative degradation was obtained processing FTIR spectra,
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firstly by PCA, whose results are showed in Figure 8, and then
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by measuring the area ratios of the bands (BAR) in the ranges
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1724–1650 cm 1 and 1650–1506 cm 1, respectively.
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The area ratios were expressed as absorbance % (A/A %)
31
and reported in graph as a function of time. The obtained
32
curves are shown in Figure S8.1 available in Section S8, SI.
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PCA results. In accordance with the respective positions of
34
PEO sample not yet heated and of the completely degraded
35
one at the end of experiment in the score plot (Figure 8.a), PC2,
36
which accounts for 26.5% of the explained variance, shows the
37
progression of oxidative degradation proceeding from bottom
38
to top.
39
Otherwise, with the help of results obtained from peroxide
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value (PV) determinations, that give a measure of primary
41
oxidation products (Figure 9), it was understood that, on PC1
42
(58.7%) can be observed the presence and separation on the
43
right side of apparently “anomalous” and unexpected data,
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corresponding to FTIR analysis made at 320, 490, 587 and 1787
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minutes. By observing the graph PV/time (Figure 9) at these
46
time points, plateaus or downhills, i. e. temporary or definitive
47
arrests or even a decrease of the level of oxidative degradation,
48
are noticeable. All the determinations, in which there was a
49
Figure 7. PCA score plot and loading plot concerning β-P. PCA score plot: ”normal” and continuative progression of the degradation,
50
PC1 vs. PC2. β-P samples in red, GA-added samples in green, GAD-added from a minimum to a maximum level, are grouped at negative
51 samples in blue (a); PCA loading plot: PC1 versus PC2 (b). scores on PC1, where evolution in spoilage can be read in
52
vertical, along PC2, from negative to positive score values.
53
Loading profiles on PC2 (red line in Figure 8.b) consistently
54
progressive degree of oxidative degradation was observed show that the FTIR bands involved in explaining the degrada-
55
proceeding from left to right, as required by the original tion process are those at around 1641 cm 1 (decreasing) and
56
position of β-P not subjected to oxidative stress. around 1700–1750 cm 1 (increasing). Stop or reduction in the
57

ChemistrySelect 2019, 4, 8891 – 8901 8897 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
exceeds that of initiation and propagation (also thanks to the
1
presence of additives), the amount of oxidation products and
2
PV does not change or even decrease. From these consider-
3
ations, GAD is confirmed to be the most efficient preservative
4
additive. In fact, it was capable of blocking the degradation of
5
PEO to levels significantly lower than those achieved both by
6
PEO without preservatives and GA-added. By examining the
7
scores of samples on PC2, it may be estimated that oxidative
8
degradation of GAD-added samples was lowered by around
9
25–30%. Furthermore, the GAD-added PEO sample at the end
10
of the oxidation experiment (1787 min), appears at the highest
11
positive PC1 score value and at the highest negative score on
12
PC2, with a value even lower than that of not oxidized PEO.
13
This apparently anomalous outcome was also verified in the
14
chemical determinations. In the PV/time graph, it gave place to
15
a descent of the GAD-treated PEO curve (Figure 9). Therefore,
16
in addition to block PEO degradation, GAD showed a high
17
radical scavenging power that, for prolonged time (1787 min)
18
of heating and exposition to air and light, translates in a
19
degradation products elimination faster than their formation.
20
BAR results. As in the case of β-P, the results obtained were
21
strictly in accordance with PCA responses. GAD was confirmed
22
as the most efficient preservative, while GA, although endowed
23
with appreciable preservative activity in the initial phase of
24
oxidative degradation, for longer times, lost efficiency until it
25
promoted rather than limited the oil spoilage. In particular,
26
GAD was able, both to delay the process of oxidative
27
degradation – increasing the duration of the health state of
28
PEO – and to block the degradation in much earlier stages with
29
respect to the PEO untreated or even treated with GA. By
30 Figure 8. PCA score plot and loading plot concerning PEO. PCA score plot: observing the pathway of the blue curve related to PEO with
31 PC1 vs. PC2. PEO samples in red, GA-added samples in green, GAD-added
samples in blue (a); PCA loading plot: PC1 versus PC2 (b).
GAD, after a certain time, the entity of oxidation damage tends
32
to diminish, leading to samples with decreasing amounts of
33
oxidation products, as observed in PCA (PEO + GAD 1787 min).
34
It should also be noted that the curves related to the samples
35
treated with preservatives (GA or GAD), for treatments shorter
36
than 500 minutes, are very similar but different from that of the
37
untreated sample, thus showing a similar significant influence
38
of the additives on the system. In all three cases, the curves
39
present plateaus or even a downhill that indicate the presence
40
of anomalous samples for which the degradation temporarily
41
or permanently stops or decreases, confirming what was seen
42
in PCA (PC1). According to data obtained by FTIR, the
43
maximum level of degradation reached by PEO without
44
additives after 1787 min matches the area ratio of 1.6 (A/A %).
45
After the same total time, the oxidative damage affecting the
46
oil added with GA, always expressed as area ratio A/A%, was
47
Figure 9. Peroxide value (PV) determined by iodometric titration of PEO
reduced by 3% while when GAD was addicted by 42%.
48
along thermally-induced oxidative degradation.[a]of CH2 and CH3[66] Consequently, GAD reduced the maximum oxidative damage
49
obtained with the addition of GA by 60%.
50
Considering a damage of about 50% of the maximum
51
degradation level (D50 = 1.24%), the PEO sample without
52
oxidative degradation level can be explained by the instability additive reached it after 317 minutes, while the sample GA-
53
both of the free radicals and of the primary oxidation products added just before the 320 minutes, thus showing that the
54
themselves, which may undergo a termination phase after degradation was only slightly delayed by GA. Otherwise, the
55
propagation or transformation in secondary oxidation products. sample added with GAD never reached such a level of
56
If the termination and/or transformation speed match or oxidation. PEO degradation was initially remarkably slowed
57

ChemistrySelect 2019, 4, 8891 – 8901 8898 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
down, touched a maximum after 320 minutes and then (Figure 9), then, linear regressions were built by correlating PV
1
decreased to 0.9% and no longer progression was observed. to FTIR analysis as made in the β-P study and discussion is
2
available in Section S9, SI. PV were reported as an average of
3
three determinations � S.D.
4 Investigation of GA and GAD preservative efficiency by
As far as oil without additive is concerned, the PV initially
5 chemical method
rapidly increased, then stopped to increase and maintained a
6
As previously mentioned, the feasibility of MVA-assisted FTIR plateau for a considerable time. Then PV increased rapidly
7
spectroscopy for the determination of the conservative again and, at around 23 hours (eight hours at 120 °C and
8
efficiency of oils, fats or foods has been repeatedly confirmed further exposition to air and light), reached a definitive plateau.
9
and reported.[53–55] In the present work, as a further confirma- It has to be remarked, that a plateau does not mean
10
tory method, peroxide value (PV) determinations by iodometric necessarily a stop in the degradation progress, but means
11
titration were performed. instability of peroxides and primary oxidation products (POP)
12
which depends on their highly susceptibility to further changes
13
that result in the formation of secondary oxidation products.[53]
14 Peroxide value (PV)
The final definitive plateau means that, after a fixed
15
PV is commonly used to determine the spoilage of a samples degradation time, corruption of instable radicals matches their
16
containing fat or oils[67,68] even if commented on by several production. PV evaluations of GAD-added oil, confirmed the
17
authors.[69–71] PV shows the degree of oxidation in the substance high efficiency of GAD as a preservative additive, firstly
18
and measures the amount of total peroxides as a product of observed for β-P.
19
primary oil oxidation. This value is a commonly used indicator The maximum value was reached with a slower increase
20
of the shelf life of a product, since an elevated PV accompanies after 490 minutes and it did not increase further. This value was
21
a high degree of degradation. As far as fresh edibles vegetable reduced by ’ 37% if compared to PEO without additives at the
22
oils are concerned, they usually have PV well below 10 meq O2 same time, and by ’ 43% if the final time is considered. For
23
kg-oil 1,[72] though this also depends on the type of oil and on further experiment time, PV related to GAD-treated PEO fell
24
the refining process. down to value close to the initial ones. Previous studies already
25
reported a spontaneous PV decrease during oxidative degrada-
26
tion of oils or fats non treated with additive, after an initial
27 β -pinene (β -P)
increase,[55,73] due to the easy degradability of radicals and POP.
28
Once all the experiments performed on β-P were completed In the present case, GAD, i. e. an additional chemical entity, has
29
(16-20 h), the three oxidized samples were subjected both to been annexed to the chemical system of PEO, consequently PV
30
FTIR analysis and to iodometric titration, to obtain the PV at decrease could be provoked, 1) by the radical scavenging
31
their maximum degree of degradation. Since the main aim of power of GAD; 2) by instability of POP. So, the question is: who
32
the work was not the evaluation of the goodness of samples is the main responsible? The answer is obvious and again
33
but the efficiency of GAD and GA, the easily degradable β-P confirms the high efficiency of GAD. Since in absence of GAD
34
was subjected to drastic conditions for long time and, only a stable plateau was reached and no PV decrease was
35
consequently, the PV determined at the end of the three observed, the first assumption is rationally the most correct.
36
experiments, were exceptionally high (Figure S6.3, SI). There is Radicals and POP instability alone, would fix PV at the value
37
no official maximum limit in PV for essential oils, because not achieved after 490 min (PV = 78, ’ 37% fewer than in non-
38
edible as vegetable oils, but it has been noted that sensitive addicted PEO), while at the experiment end it was inferior by
39
essential oils stored in small vials regularly opened and not about 80%, that means a decrease much more robust than the
40
protected from light and warm temperatures can easily reach one previously reported.[55]
41
200–300 meq O2 kg 1, as obtained here. Even just by looking at Realistically, GAD scavenging activity is more rapid in
42
the bar plot (Figure S6.2, SI), the lowest PV was determined on removing radical species than oxidation in producing then and
43
sample added with GAD. Increasing PV was obtained for GA- this action translates in PV remarkable decrease. PV evaluation
44
added sample followed by the sample without additive with a of the degradation profile of GA-addicted PEO sample, showed
45
PV > 400 meq O2 kg 1, in accordance with the results from FTIR some differences if compared to β-P study. Within short times
46
spectra MVA and BAR processed. PV versus the area ratios of heating (< 2.5 h), GA showed a significant anti-degradative
47
obtained from FTIR spectra were used to build a linear action causing a slower increase in PV if compared to PEO
48
regression and to evaluate correlation index. The relative without additive, but for further times, PV continued to grow
49
discussion is available in Section S9 in SI. linearly exceeding its values. A short plateau followed and
50
then, a steady increase began to reach at the experiment end,
51
a PV higher by 45%. The differences with β-P may be explained
52 Pinus mugo essential oil (PEO)
hypothesizing additional side reactions of GA with other
53
As far as PEO is concerned, PV by iodometric titrations were chemical compounds naturally present in PEO or formed
54
determined along all the period of oxidation at fixed time during primary oxidation, that do not occur with β-P alone,
55
points, every time that a FTIR spectrum was acquired. First, PV such as esterification and/or transesterification reactions. Such
56
determinations were reported in graph as a function of time reactions may cause GA rapid consumption or inactivation.
57

ChemistrySelect 2019, 4, 8891 – 8901 8899 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
When instead GAD is added in the oil system, the phenomenon Supporting Information Summary
1
was not observed. In this case, side reactions are rationally
2
hindered and prevented by the particular architecture of the Experimental Section of the current article, details concerning
3
dendrimer matrix that transports GA units. Thanks to dendrimer the synthesis, complete characterization, cytotoxicity and
4
scaffold, the GA reactive functions, i. e. OH and COOH groups, Radical Scavenging Activity (RSA %) of GAD dendrimer (9), as
5
are either less accessible (OH groups) or inactivate by well as significant images and other details concerning
6
esterification (COOH group). Another appealing explanation of oxidative degradations of β-pinene and Pinus mugo essential
7
the PV constant increasing in presence of GA may be that only oil reported in this study, are available in a single file (PDF).
8
at early stages of the thermally-induced oxidation, GA acts as
9
antioxidant, while it works as pro-oxidant for prolonged times
10 Acknowledgements
and particular conditions.[74,75]
11
The authors are very thankful to Mr Gagliardo Osvaldo for
12
Elemental Analysis and to University of Genoa. Furthermore a
13 Conclusions
special thanks to Mrs Deirdre Kantz for language help.
14
By merging FTIR analysis processed by two different methods
15
(named along the text BAR and MVA) and chemical inves-
16 Conflict of Interest
tigations, reliable information about the efficiency of a
17
spherical dendrimer nanomaterial peripherally esterified with The authors declare no conflict of interest.
18
sixty-four gallic acid moieties (GAD) in preventing essential oils
19
spoilage, was achieved. As every analytical technique has limits, Keywords: antioxidant additives · dendrimer · essential oils ·
20
more rather than one have been performed to achieve a cross FTIR spectroscopy · gallic acid · multivariate analysis
21
validation. Since auto oxidation is considered the key triggering
22
factor for fats spoilage, the analysis were executed on model
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systems of oxidative degradation thermally-induced. The study Band 1: Grundlagen des Arznei- und Gewurzpflanzenbaus I, (Eds.: B.
24
was performed firstly on a single component as simplified Hoppe, K. Hoppe, W. Junghanns, L. Kabelitz, F. Pank, E. Schliephake),
25 Grafisches Centrum Cuno GmbH & Co. KG, Calbe (Saale), 2009, pp. 383–
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