Vlasios Goulas

A Knowledge Base for The Recovery of Natural Phenols with Different Solvents

The current study denotes the prediction of activity coefficients of fifteen natural phenols (tyrosol, hydroxytyrosol, oleuropein, caffeic, cinnamic, p-coumaric, ferulic, gallic, p-hydroxybenzoic, p-hydroxyphenyl acetic, protocatechuic, rosmarinic, sinapic, syringic, and vanillic acid) in seven solvents (water, ethanol, methanol, acetone, dichloromethane, ethyl acetate, and diethyl ether), and three extraction temperatures (298.15, 313.15, and 333.15 K), using the universal functional-group activity coefficient model. Solvents were classified for their ability to dissolve phenols and were compared with experimental data of the literature in order to observe if the solvent extraction of phenols in practice matches with the authors’ theoretical approach. Results indicated the superiority of alcohols and acetone for the recovery of phenols in line with experimental data of previous studies. Furthermore, activity coefficients’ values were found to increase with the increase of temperature. This study provided a knowledge base for the selection of the most appropriate solvents for a given phenolic compound.

Towards an Efficient Protocol for the Determination of Triterpenic Acids in Olive Fruit: A Comparative Study of Drying and Extraction Methods

INTRODUCTION Triterpenic acids, such as maslinic acid and oleanolic acid, are commonly found in olive fruits and have been associated with many health benefits. The drying and extraction methods, as well as the solvents used, are critical factors in the determination of their concentration in plant tissues. Thus, there is an emerging need for standardisation of an efficient extraction protocol that determines triterpenic acid content in olive fruits. OBJECTIVE To evaluate common extraction methods of triterpenic acids from olive fruits and to determine the effect of the drying method on their content in order to propose an optimum protocol for their quantification. METHODOLOGY The efficacy of different drying and extraction methods was evaluated through the quantification of maslinic acid and oleanolic acid contents using the reversed-phase HPLC technique. RESULTS Data showed that ultrasonic assisted extraction with ethanol or a mixture of ethanol:methanol (1:1, v/v) resulted in the recovery of significantly higher amounts of triterpenic acids than other methods used. The drying method also affected the estimated triterpenic acid content; frozen or lyophilised olive fruit material gave higher yields of triterpenic acids compared with air-dried material at both 35°C and 105°C. CONCLUSION This study provides a rapid and low-cost extraction method, i.e. ultrasonic assisted extraction with an eco-friendly solvent such as ethanol, from frozen or lyophilised olive fruit for the accurate determination of the triterpenic acid content in olive fruit.

Drying Technologies: Vehicle to High-Quality Herbs

Herbs are usually marketed as dry due to a consumer demand beyond their seasonality; dehydration leads to a stable, easily moveable product that is available throughout the year. The process of drying, though, leads to modifications in the appearance, composition and quality of the raw material. The extent of these alterations depends on the applied drying methodology and its parameters, rendering the optimization of this process imperative. Numerous studies examining the effect of drying on the main characteristics of herbs have been published in recent years, and this review aims at organizing the available information of the studied herbs, drying methods and measured parameters in a comprehensive manner. Primarily, since aroma is the main characteristic of herbs and the principal aim for the end product is to retain the raw material’s character, this review will focus on the most widely studied effect of drying, which is the essential oil yield and composition. Secondly, results from various studies on the influence of drying on biochemical compounds, organoleptic properties of dried herbs are also presented. The most common approach to the study of drying kinetics is also presented. Finally, novel technologies targeting to minimize the magnitude of changes from the raw material are described.

HPLC-SPE-NMR characterization of major metabolites in Salvia fruticosa Mill. extract with antifungal potential: relevance of carnosic acid, carnosol, and hispidulin.

Plant pathogenic fungi are considered of significant economic importance for adversely affecting both quantitatively and qualitatively fresh and processed produce. Extracts of Salvia fruticosa were initially screened for their antifungal activity, and the ethyl acetate fraction, being the most active, was further analyzed using HPLC-SPE-NMR hyphenation. The methoxylated flavones hispidulin, salvigenin, and cirsimaritin and the diterpenes carnosic acid, carnosol, and 12-methoxycarnosic acid were identified as the major components of the extract. In addition, the concentration levels of all identified components were determined using q-NMR. The antifungal activity of the crude extract and selected phytochemicals was estimated against the fungal species Aspergillus tubingensis, Botrytis cinerea, and Penicillium digitatum. The estimated MIC and MFC values of the ethyl acetate extract of S. fruticosa, as well as three of its major constituents, carnosic acid, carnosol, and hispidulin, support their antifungal activity, especially against B. cinerea and P. digitatum, suggesting their potential use in food and agricultural systems.

Identification and mycotoxigenic capacity of fungi associated with pre- and postharvest fruit rots of pomegranates in Greece and Cyprus

Pre- and postharvest fruit rots of fungal origin are an important burden for the pomegranate industry worldwide, affecting the produce both quantitatively and qualitatively. During 2013, local orchards were surveyed and 280 fungal isolates from Greece (GR) and Cyprus (CY) were collected from pomegranates exhibiting preharvest rot symptoms, and additional 153 isolates were collected postharvest from cold-stored fruit in GR. Molecular identification revealed that preharvest pomegranate fruit rots were caused predominately by species of the genera Aspergillus (Aspergillus niger and Aspergillus tubingensis) and Alternaria (Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens). By contrast, postharvest fruit rots were caused mainly by Botrytis spp. and to a lesser extent by isolates of Pilidiella granati and Alternaria spp. Considering that a significant quota of the fungal species found in association with pomegranate fruit rots are known for their mycotoxigenic capacity in other crop systems, their mycotoxin potential was examined. Alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) production was estimated among Alternaria isolates, whereas ochratoxin A (OTA) and fumonisin B2 (FB2) production was assessed within the black aspergilli identified. Overall in both countries, 89% of the Alternaria isolates produced AOH and AME in vitro, while TEN was produced only by 43.9%. In vivo production of AOH and AME was restricted to 54.2% and 31.6% of the GR and CY isolates, respectively, while none of the isolates produced TEN in vivo. Among black aspergilli 21.7% of the GR and 17.8% of the CY isolates produced OTA in vitro, while in vivo OTA was detected in 8.8% of the isolates from both countries. FB2 was present in vitro in 42.0% of the GR and 22.2% of the CY isolates, while in vivo the production was limited to 27.5% and 4.5% of the GR and the CY isolates, respectively. Our data imply that mycotoxigenic Alternaria and Aspergillus species not only constitute a significant subset of the fungal population associated with pomegranate fruit rots responsible for fruit deterioration, but also pose a potential health risk factor for consumers of pomegranate-based products.

Classification, Biotransformation and Antioxidant Activity of Olive Fruit Biophenols: A Review

Olive biophenols affect the organoleptic and nutraceutical properties of fruits; therefore, it is an emerging re- search field. In addition, a plethora of health-promoting properties have been attributed to the phenolic content of olive fruits. The main biophenols can be divided into different sub-groups such as phenolic acids and alcohols, flavonoids, lig- nans and secoiridoids. The latter sub-group of biophenols is typical in olive fruits, since oleuropein and the products of its biotransformation characterize olive fruit. An array of genetic, agronomic and technological factors that affect the rate of synthesis and biotransformation of secoiridoids in olive fruits is discussed. Furthermore, the relationship between antioxi- dant activity and structures of olive biophenols is explored.

1H NMR Metabolic Fingerprinting to Probe Temporal Postharvest Changes on Qualitative Attributes and Phytochemical Profile of Sweet Cherry Fruit

Sweet cherry fruits (Prunus avium cvs. ‘Canada Giant’, ‘Ferrovia’) were harvested at commercial maturity stage and analyzed at harvest and after maintenance at room temperature (storage at ∼20°C, shelf life) for 1, 2, 4, 6, and 8 days, respectively. Fruit were initially analyzed for respiration rate, qualitative attributes and textural properties: ‘Canada Giant’ fruit were characterized by higher weight losses and stem browning index, being more intense over the late stages of shelf life period; meanwhile ‘Ferrovia’ possessed appreciably better performance even after extended shelf life period. A gradual decrease of respiration rate was monitored in both cultivars, culminated after 8 days at 20°C. The sweet cherry fruit nutraceutical profile was monitored using an array of instrumental techniques (spectrophotometric assays, HPLC, 1H-NMR). Fruit antioxidant capacity was enhanced with the progress of shelf life period, concomitant with the increased levels of total anthocyanin and of phenolic compounds. ‘Ferrovia’ fruit presented higher contents of neochlorogenic acid and p-coumaroylquinic acid throughout the shelf life period. We further developed an 1H-NMR method that allows the study of primary and secondary metabolites in a single running, without previous separation and isolation procedures. Diagnostic peaks were located in the aliphatic region for sugars and organic acids, in the aromatic region for phenolic compounds and at 8.2–8.6 ppm for anthocyanins. This NMR-based methodology provides a unifying tool for quantitative and qualitative characterization of metabolite changes of sweet cherry fruits; it is also expected to be further exploited for monitoring temporal changes in other fleshy fruits.

Edible coating composed of chitosan and Salvia fruticosa Mill. extract for the control of grey mould of table grapes

BACKGROUND Consumer concerns regarding high-quality produce, free of pesticide residues, direct research towards disease management strategies that minimise or even exclude the use of synthetic chemistries in crop production. The efficacy of a chitosan-based edible coating combined with the acetonic extract of Salvia fruticosa Mill. (ASF) was assessed against the grey mould of table grapes. RESULTS HPLC-SPE-NMR and q-NMR analyses defined major constituents of ASF to be the flavonoids hispidulin, salvigenin and cirsimaritin and the diterpenes carnosic acid, carnosol and the 12-methoxycarnosic acid. The extract was found to be efficacious in reducing spore germination and mycelial growth of Botrytis cinerea in vitro at 10 and 25 °C. However, the combination of the ASF with chitosan 1% (w/v; CHIT) significantly improved fungal inhibition. Similarly, in fruit inoculation trials at 10 °C, the efficacy of the combined application of the ASF at 500 mg L-1 with CHIT against grey mould was statistically equal to the synthetic fungicide thiabendazole, ranging from 98.4% to 92.7% at 12 and 21 days post-inoculation, respectively. Furthermore, chitosan coating alone and in combination with ASF decreased the rate of fruit weight loss during cold storage, while preserved soluble solids content and titratable acidity. Chitosan-based coatings did not affect quality attributes and the bioactive compounds in table grapes. CONCLUSION The combined application of the ASF in the form of an edible coating with chitosan could effectively control B. cinerea without deteriorating quality and physico-chemical properties of grapes.

Regulation of On-Tree Vitamin E Biosynthesis in Olive Fruit during Successive Growing Years: The Impact of Fruit Development and Environmental Cues

The term vitamin E refers to a group of eight lipophilic compounds known as tocochromanols. The tocochromanols are divided into two groups, that is, tocopherols and tocotrienols, with four forms each, namely α-, β-, γ-, and δ-. In order to explore the temporal biosynthesis of tocochromanols in olive (Olea europaea cv. ‘Koroneiki’) fruit during on-tree development and ripening over successive growing years, a combined array of analytical, molecular, bioinformatic, immunoblotting, and antioxidant techniques were employed. Fruits were harvested at eight successive developmental stages [10–30 weeks after flowering (WAF)], over three consecutive years. Intriguingly, climatic conditions affected relative transcription levels of vitamin E biosynthetic enzymes; a general suppression to induction pattern (excluding VTE5) was monitored moving from the 1st to the 3rd growing year, probably correlated to decreasing rainfall levels and higher temperature, particularly at the fruit ripening stage. A gradual diminution of VTE5 protein content was detected during the fruit development of each year, with a marked decrease occurring after 16 WAF. Alpha-tocopherol was the most abundant metabolite with an average percentage of 96.82 ± 0.23%, 91.13 ± 0.95%, and 88.53 ± 0.96% (during the 1st, 2nd, and 3rd year, respectively) of total vitamin E content in 10–30 WAF. The concentrations of α-tocopherol revealed a generally declining pattern, both during the on-tree ripening of the olive fruit and across the 3 years, accompanied by a parallel decline of the total antioxidant capacity of the drupe. Contrarily, all other tocochromanols demonstrated an inverse pattern with lowest levels being recorded during the 1st year. It is likely that, in a defense attempt against water deficit conditions and increased air temperature, transcription of genes involved in vitamin E biosynthesis (excluding VTE5) is up-regulated in olive fruit, probably leading to the blocking/deactivating of the pathway through a negative feedback regulatory mechanism.

Temporal analysis reveals a key role for VTE5 in vitamin E biosynthesis in olive fruit during on-tree development

The aim of this work was to generate a high resolution temporal mapping of the biosynthetic pathway of vitamin E in olive fruit (Olea europaea cv. “Koroneiki”) during 17 successive on-tree developmental stages. Fruit material was collected from the middle of June until the end of January, corresponding to 6–38 weeks after flowering (WAF). Results revealed a variable gene regulation pattern among 6–38 WAF studied and more pronounced levels of differential regulation of gene expression for the first and intermediate genes in the biosynthetic pathway (VTE5, geranylgeranyl reductase, HPPD, VTE2, HGGT and VTE3) compared with the downstream components of the pathway (VTE1 and VTE4). Notably, expression of HGGT and VTE2 genes were significantly suppressed throughout the developmental stages examined. Metabolite analysis indicated that the first and intermediate stages of development (6–22 WAF) have higher concentrations of tocochromanols compared with the last on-tree stages (starting from 24 WAF onwards). The concentration of α-tocopherol (16.15 ± 0.60−32.45 ± 0.54 mg/100 g F.W.) were substantially greater (up to 100-fold) than those of β-, γ-, and δ-tocopherols (0.13 ± 0.01−0.25 ± 0.03 mg/100 g F.W., 0.13 ± 0.01−0.33 ± 0.04 mg/100 g F.W., 0.14 ± 0.01−0.28 ± 0.01 mg/100 g F.W., respectively). In regard with tocotrienol content, only γ-tocotrienol was detected. Overall, olive fruits (cv. “Koroneiki”) exhibited higher concentrations of vitamin E until 22 WAF as compared with later WAF, concomitant with the expression profile of phytol kinase (VTE5), which could be used as a marker gene due to its importance in the biosynthesis of vitamin E. To the best of our knowledge, this is the first study that explores the complete biosynthetic pathway of vitamin E in a fruit tree crop of great horticultural importance such as olive, linking molecular gene expression analysis with tocochromanol content.