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  • 67 Reads
Comparison between different extraction methods in the recovery of bioactive molecules from Melissa officinalis L.under sustainable cultivation: chemical and bioactive characterization

Melissa officinalis L., from the Lamiaceae family, is one of the most important medicinal and aromatic plants with potential in the market. With the passing of time, the use of medicinal plants in the treatment of some illness has gone from the simplest forms of local treatment to the industrial manufacture of phytotherapics. In addition to their medicinal effect, they can also be used in the form of infusions and decoctions and in various food preparations. In this sense, the objective of this work was to compare three different extraction methods: infusion (100% water), maceration (80:20 ethanol: water v:v) and ultrasound assisted extraction (UAE) under previous optimized extraction conditions (33.0 ± 3.2 min, 371.7 ± 19.3 W and 39.9 ± 1.4% ethanol), in plants grown under sustainable cultivation under full irrigation in June. The parameters studied included bioactive evaluation through antimicrobial (microdilution method), antioxidant (thiobarbituric acid reactive substances - TBARS), cytotoxicity (sulforhodamine B) and anti-inflammatory (RAW cells) assays. The composition of phenolic compounds and organic acids was also studied by GC-MS and HPLC-DAD, respectively. According to the obtained results, eight phenolic compounds were identified and quantified, being rosmarinic acid the main one for the three extraction methods (infusion: 107.1±0.9 mg/g extract; maceration: 155.7±0.3 mg/g extract; UAE: 118.7±0.6 mg/g extract). For Lithospermic acid A isomer (25.25 ± 0.01 mg/g) and Hydroxylsalvianolic E (111.70 ± 2.20 mg/g), the UAE revealed the lowest content of individual polyphenols, whereas maceration recorded the highest extractability. On the other hand, the content of six of the eight polyphenols detected for the ultrasound-assisted extraction was similar to the infusion and maceration methods. In terms of antioxidant activity, the infusions showed the highest capacity (3.00 ± 0.14 μg/mL), followed by maceration (5.33 ± 0.30 μg/mL) and UAE (12 ± 0.15 μg/mL). The highest anti-inflammatory activity was verified for the infusion (244 ± 11 μg/mL), followed by UAE (305 ± 9 μg/mL), with no activity recorded for the maceration extract (>400 μg/mL). The antitumor properties were evaluated in 5 cell lines, with the best results being recorded for infusion, except AGS (24 ± 1 μg/mL) where the best results were for the UAE; being the maceration extract more active against NCI-H460 (190 ± 7 μg/mL). It is therefore concluded that the extraction method that contributes to the highest obtainment of phenolic compounds is maceration, followed by infusion and ultrasound-assisted extraction. As for the bioactive and antioxidant compounds, infusion is the most efficient method, followed by maceration and ultrasound. Overall, these natural extracts are interesting ingredients, capable of replacing counterparts of synthetic origin, and can find wide applications in the industrial sector (e.g. food, pharmaceutical and cosmetic companies). Also emphasizing the high contents in rosmarinic acid, and the obtained bioactivity, that turns this samples of great interest to increase their production to obtain extracts enriched with this bioactive molecule.

  • Open access
  • 52 Reads
Sea Fennel (Crithmum maritimum L.): A Promising Biosaline Crop. Extraction, Purification and Chemical Characterization of Polar Extracts

Crithmum maritimum known with several popular names as finocchio marino, critmo, cretamo, spaccasassi, bacicci, basiggia, erba di San Pietro and salissia, is a perennial halophyte, thrives on coastal cliffs and sometimes in sandy beaches along the Mediterranean, Pacific and Atlantic coasts.
This plant, native to southern Europe, survives also under salinity conditions, so it is considered a promising biosaline crop. However, its commercial cultivation potential is not yet fully exploited and there are few studies regarding the agricultural management and best practices to grow this crop in the Mediterranean environment [1].
Sea fennel is an emerging vegetable crop also thanks to its increasing food uses [2]. This is due to its aromatic traits, for instance to make pickles and to flavour and season soups, sauces and salads. The connections between cultivation and nutritional value were investigated. Since the natural environment of the sea fennel has a high concentration of iodine, a biofortification experiment was performed to evaluate the effect on nutritional values [3].
For the reasons mentioned above, interest in this plant is growing and different studies have been performed to understand the total chemical profile of the vegetable.
This work aims to provide a complete quantitative and qualitative overview of the extract obtained from the aerial parts of C. maritimum, provided by a Mediterrean firm. For this purpose aerial parts were dried, ground and extracted by percolation with ethanol 70% at 60°C. The purification was done using an Amberlite® XAD7HP sorbent resin. For the quantification was used an UHPLC-DAD-MS/MS system, equipped with electrospray ionization (ESI) and a Zorbax ODS (250 x 4.6 mm, 5 μm) column.
From the results emerges that the purified extract is richer than the unpurified one, furthermore the extract was mainly composed of phenolic compounds, among which hydroxycinnamic acids and flavonoids were the two main chemical classes. Among the former, chlorogenic acids, including 5-O-caffeoylquinic acid (10.5-22.36 mg g-1), 3,5-di-O-caffeoylquinic acid (5.99-15.82 mg g-1) and 4,5-di-O-caffeoylquinic acid (5.09-15.35 mg g-1), were the most abundant ones. Among flavonoids, rutin (1.60-4.33 mg g-1) and kaempferol-3-O-rhamnoside (0.07-0.33 mg g-1) were the main constituents.
These results support the use of this plant as a functional food or in nutraceuticals, thanks to the abundance of antioxidant compounds. This also suggests the study of the relation between cultivation systems and nutraceutical values.

[1] Zenobi S, Fiorentini M, Zitti S, Aquilanti L, Foligni R, Mannozzi C, Mozzon M and Orsini R. Crithmum maritimum L.: First Results on Phenological Development and Biomass Production in Mediterranean Areas. Agronomy. 2021 April.
[2] Pereira AG, Fraga-Corral M, García-Oliveira P, Jimenez-Lopez C, Lourenço-Lopes C, Carpena M, Otero P, Gullón P, Prieto MA, Simal-Gandara J. Culinary and nutritional value of edible wild plants from the northern Spain rich in phenolic compounds with potential health benefits. Food & Function. 2020 Oct 21; 11(10):8493-8515.
[3] Sarrou E, S.Siomos A,Riccadona S, Aktsoglou D, Tsouvaltzis P, Angeli A, Franceschi P, Chatzopoulou P, Vrhovsek U, Martens S. Improvement of sea fennel (Crithmum maritimum L.) nutritional value through iodine biofortification in a hydroponic floating system. Food Chemistry, 296, 150-159. October 2019.

  • Open access
  • 31 Reads
Effects of Chitosan Oligosaccharide Lactate on Growth and Overwintering of Evergreen Fern Cyrtomium fortunei var. clivicola

Chitosan derivatives with low molecular weight can enhance plant growth and improve tolerance to various stress. Hardy ferns form a group of attractive garden perennials with unknown response to plant biostimulants. Cold winter temperature limits the growth and reduces the decorative value of evergreen fern species. This study was carried out to explore the effects of chitosan oligosaccharide lactate (COL) with Mn = 5,000 on growth of evergreen fern Cyrtomium fortunei var. clivicola. COL sprayed at 50 and 100 mg/L significantly increased height of plant and fresh weight of the above-ground and the underground parts of C. fortunei var. clivicola compared to control plants. Moreover, ferns sprayed with 50 and 100 mg/L COL also had higher leaf total chlorophyll contents and value score. These results indicate that COL improved C. fortunei var. clivicola growth and overwintering and may be used for high quality hardy ferns production.

  • Open access
  • 21 Reads
Different Strategies to Tolerate Salinity Involving Water Relations

Salinity is one of the main limiting factors in agriculture, which can affect plants growth and development, as a result of a disruption of homeostasis. Because of that, to understand the plants resistance mechanisms in response to salinity stress is essential in order to develop new techniques that may improve tolerance and, in this way, optimize crops yield. In this paper we compare the response of Cucumber (Cucumis sativus L.) and tomato (Solanum lycopersicum L.), grown by hydroponic culture, to a moderate salinity of NaCl 60 mM. For that, root hydraulic conductivity, leaves relative water content (RWC), stomatal conductance, fresh weight and dry weight relation and Na+ concentration in aerial part and root were measured. The results showed a significant decrease of root hydraulic conductivity in both species treated with NaCl, revealing a higher resistance to water passage from root to aerial part, probably influenced by the increase of Na content after the treatment. In addition, stomatal conductance in cucumber was reduced, accompanied with a decrease of fresh/dry weight relation in root. Conversely, neither of those parameters changed in tomato. These experiments confirm the evidence that cucumber and tomato follow different strategies in the adaptation to salinity, being tomato more resistant probably due to the role of membrane water transporters. Despite that, more specific studies would be needed in order to support this conclusion.

  • Open access
  • 51 Reads
Genome-Wide Association Analysis of Yield-Related Traits of Soybean Using Haplotype-Based Framework

Haplotype-based breeding involving multi-marker association analysis is a promising approach to developing custom-designed high-yielding crop varieties. Here, we reported multi-marker association analysis for the number of pods per plant (PNP), the number of seeds per plant (SNP), 100-seed weight (HSW), and seed yield per plant (SYP) using 211 cultivated soybean accessions. The field experiment was conducted across six environments following a randomized complete block design with three replications. Genome-wide association study (GWAS) using 12,617 single-nucleotide polymorphism (SNP) markers from NJAU 355K SoySNP Array identified 57 significant marker associations with the studied traits across the six environments. Out of these, six markers that were consistently associated with the yield traits in two or more environments were considered stable and selected as the reference markers for building haplotype block/loci. The multi-marker association analysis within the haplotype-based framework revealed various allelic combinations regulating the phenotypic variations for the studied yield-related traits in soybean. These haplotype alleles may serve as genomic resources in breeding programmes aimed at improving the yield potential of soybean.

  • Open access
  • 36 Reads
Image Based Phenotyping of Shell Thickness Revealed Strong Association with Kernel Recovery in Macadamia

Phenotyping in macadamia breeding programs is usually laborious, time-coming, and costly. The development of rapid and cost-effective phenotyping technologies can reduce costs and increase breeding efficiency. As macadamia kernel is the only edible part of the nut, a key indicator of farm profitability is kernel recovery (KR), which is the amount of total kernel in a nut in shell (NIS). There was a general consensus, without scientific records, that a thicker nutshell results in a smaller kernel, thus lower kernel recovery. This project aims to assess the relationships between nutshell thickness and KR by using a rapid and cost-effective method. Nut samples were collected from second-generation macadamia breeding progenies grown in Bundaberg station of the Department of Agriculture and Fisheries, Queensland, Australia. A digital slide caliper and an analytical balance were used to measure manually phenotypic traits of each nut, including nutshell thickness. Pictures of samples were captured with a digital camera and processed with Image J to extract phenotypic information. Negative correlations between shell thicknesses with kernel recovery were recorded in both manual and image-based approaches. Particularly, manual measurements of nutshell thickness had correlation coefficients ranged from -0.54 to -0.59 with KR while image-based measurements of average shell thickness had a correlation coefficient of -0.87 with KR. The outcomes indicated that shell thicknesses can be used as a predictor for KR in macadamia breeding programs and more importantly, image-based measurements offered higher prediction accuracy of KR than manual measurements.

  • Open access
  • 11 Reads
Determination of CBD in Ethanol Extracts Prepared from Hemp (Cannabis sativa L.) Cultivar Beniko Using Dynamic Maceration and Ultrasound Assisted Extraction

Hemp (Cannabis sativa L.) has been known as a source of fibres, oil and medicines since ancient times (4000 B.C.). The most known cannabinoids responsible for pharmacological properties are psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabinoids such as cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC) or cannabinol (CBN). Fast and increasing interest in using low-THC (<0.2%) hemp requires the development of efficient methods for extracting bioactive substances. The right choice of extraction method is important for obtaining valuable cannabinoids. The aim of the work was comparison of the conventional solid-liquid extraction method using dynamic maceration (DM) and ultrasound assisted extraction (UAE). For extraction with ethanol (40%, 60% and 80%) were selected fresh panicles of monoecious variety Beniko (2.55% CBD and 0.06% THC) allowed to cultivation in Europe. After harvesting, the panicles were frozen in liquid nitrogen and stored in a freezer (-20°C). For 40%, 60% and 80% ethanol extraction, dynamic maceration was used during which the plant material was shaken in the dark (100 rpm, 72h) and ultrasound assisted extraction (45 Hz, 0.5 h). The conducted research showed the highest content of CBD (361.3 µg/ml) during extraction with 80% ethanol combined with ultrasound. On the other hand, dynamic extraction with 60% ethanol allowed to obtain 352.7 µg/ml of CBD. The obtained results indicate that the most efficient method of extraction is ultrasound assisted extraction, however, dynamic maceration with shaking may be an alternative method.

The research was funded by Polish Ministry of Agriculture and Rural Development, resolution of the Council of Ministers no: 171/2017.

  • Open access
  • 50 Reads
Effect of Salicylic Acid and Methyl Jasmonate on Stress Indices in Papaver bracteatum Lindl

Persian poppy (Papaver bracteatum Lindl.) is a perennial medicinal plant belonging to the Papaveraceae family that is endemic to the mountainous areas in Northern Iran. It is known for high amounts of the valuable benzylisoquinoline alkaloid thebaine. In this study, the effect of salicylic acid and methyl jasmonate elicitors on stress indices was investigated. For this purpose, an experiment was conducted in the form of a factorial experiment with a randomized complete block design on three different populations of Persian poppies. The interactions of the population×salicylic acid×methyl jasmonate were significant (level of 1%) among the chlorophyll fluorescence, ion leakage, malondialdehyde, and proline indices. The highest Fv/Fm (0.838) was observed in the German population and 100 μM salicylic acid treatment. The lowest ion leakage (20.51%) was observed in the polour population and 100 μM methyl jasmonate treatment. The lowest amount of malondialdehyde (19.36 mM/g fresh weight) was observed in Fil zamin population and 100 μM salicylic acid treatment. The highest amount of proline (6.29 μmol/g fresh weight) was also observed in the polour population and 100 μM salicylic acid treatment. In general, salicylic acid and methyl jasmonate treatments were shown to improve stress-related indices well. It seems that the best treatments to increase plant capacity to deal with environmental stresses are 100 μM salicylic acid and 100 μM methyl jasmonate in Persian poppy.

  • Open access
  • 44 Reads
Evaluating Seven Macadamia Seedling and Cutting Rootstocks for Their Effect on Scion Growth

Plant vigour is one of the key obstacles of efficient orchard system in macadamia (Macadamia integrifolia, M. tetraphylla, and hybrids). Vigorous trees restrict low-density planting in commercial orchards. In absence of low vigour scion and dwarfing rootstocks this problem is addressed by pruning and hedging in mature macadamias, which appear to be the major expenses for nut production. However, the ability of rootstocks to reduce tree vigour has been explained as an important advantage for many fruit-crop growers. To identify vigour managing rootstocks, this study investigates 21 diverse genotypes of macadamia including 12 elite selections from the national breeding program, an accession of the wild species Macadamia jansenii, and eight cultivars. Scions of HAES 741 were whip-grafted onto 245 seedlings and 188 cuttings. Both rootstocks and scions were phenotyped for growth parameters (tree height, rootstock and scion height and trunk circumference, canopy width and depth) from 2017 to 2021. The variability in plant vigour traits due to the effect of rootstock genotypes and propagation types (grafted and ungrafted / seedlings and cuttings) are being evaluated. The growth parameters of the rootstocks in nursery and in the field will be compared. Results from this study will provide a basis of selecting superior rootstocks for high performance in future high density orchard systems.

  • Open access
  • 50 Reads
Biostimulant Based on Algae Extract and Fulvic Acids Is Able to Improve Photosynthetic Performance and Mitigate the Effects of Salinity in Soybean

The expansion of salinization of agricultural areas limits the production of crops of high economic importance such as soybeans. To attenuate the effects of salts on the plant and improve photosynthetic performance under salinity conditions, the application of a biostimulant based on seaweed extract (Ascophyllum nodosum (L)) and fulvic acids as a physiological enhancer was adopted. In the present study, we evaluated whether applications of the biostimulant at different phenological stages can reduce damage to the photosynthetic apparatus in soybean plants under salt stress. The experiment was conducted in a greenhouse and the design adopted was completely randomized in a double factorial, consisting of three applications (V3, V3/R1 and V3/R1/R4), two levels of salinity of the irrigation water (S0 - absence of salt and S1 - saline solution at 5.0 dS m-1 prepared with the salts NaCl, CaCl2.2H2O and MgCl2.6H2O in the ratio 7:2:1) with two additional controls without application of the biostimulant (with and without stress) and five repetitions. Soybean plants were irrigated daily with the solutions and a weekly depth of 25% higher than the demand of the culture was applied. The evaluations were carried out 49 days after sowing, evaluating the potential quantum efficiency of photosystem II (Fv/Fm), the effective quantum yield of PSII (ɸFSII), the photochemical (qP ) and non-photochemical (qN) quenching and the rate of electron transport (ETR). Plants subjected to irrigation with saline water showed reductions in the evaluated parameters, suggesting that the salts caused damage to the photosynthetic apparatus in the photochemical stage. The application of the biostimulant was effective in reducing damage to the photosynthetic apparatus, providing greater efficiency in dissipating excess energy and less reduction in ETR. The application that provided the best results was in V3/R1.