A high throughput method for detection of cyclooxygenase-2 enzyme inhibitors by effect-directed analysis applying high performance thin layer chromatography-bioassay-mass spectrometry.

A high throughput method was developed to detect bioactive molecules with inhibitory activity over cyclooxygenase (COX-2) enzyme applying effect-directed analysis and planar chromatography hyphenated with bioassay and mass spectrometry. The assay was based on the indirect measurement of arachidonic acid transformation into prostaglandin with the colorimetric co-substrate N,N,N',N'-tetramethyl-p-phenylenediamine. Inhibitory zones were observed as colorless bands over a blue background. Using a central composite design the critical factors like substrate concentration, enzyme: substrate ratio, reaction time, and co-substrate concentration were optimized. Optimal conditions were achieved with 0.03 mg/mL of arachidonic acid, 0.15 U/mL of COX-2, and 8.21 mg/mL of chromogenic reagent. Method usefulness was challenged analyzing fresh Chiloe's giant garlic (Allium ampeloprasum L) ethanol: water (8:2 v/v) extract, finding COX-2 inhibitors that were preliminarily identified as the isomers γ-glutamyl-S-allyl-l-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L- cysteine.

Bioactive Compounds and Antioxidant Activity in the Fruit of Rosehip (Rosa canina L. and Rosa rubiginosa L.).

Rosehips (Rosa spp., Rosaceae) are wild rose bushes with more than 100 species. Its fruits vary in colour and size, depending on the species, and are recognised for their nutritional characteristics. Ten samples of Rosa canina L. and Rosa rubiginosa L. fruits were collected at different geographical points from Southern Chile. Nutrients such as crude protein and minerals and functional properties such as phenolic compounds, ascorbic acid, and also antioxidant activities were evaluated by HPLC-DAD-ESI-MS/MS. The results revealed a high content of bioactive compounds, primarily ascorbic acid (6.0 to 8.2 mg g-1 fresh weight (FW)), flavonols (427.9 ± 0.4 µg g-1 FW) and antioxidant activity. We established a relationship between the antioxidant activity using Trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC) and 2,2-diphenyl radical (DPPH) methods and the concentration of uncoloured compounds, such as flavonols and catechin. This antioxidant activity was primarily associated with the samples from Gorbea, Lonquimay, Loncoche, and Villarrica localities, and all of them were of the species Rosa rubiginosa L. The results here obtained represent novel information of rosehip fruits. In this sense, the reported information about compounds and antioxidant activities in rosehip fruits allowed us to continue new lines of research in relation to the potential formulation of new functional foods and also in the treatment and/or prevention of some diseases.

Effect of Inoculation with Arbuscular Mycorrhizal Fungi and Fungicide Application on the Secondary Metabolism of Solanum tuberosum Leaves.

In potato (Solanum tuberosum) crops, the use of fungicides to control some diseases is widespread; however, it has been reported that this practice can modify the potato polyphenolic content, and new strategies oriented to the potato defense system are necessary. One alternative is the use of arbuscular mycorrhizal fungi (AMF) to improve the defense mechanisms of plants. In this study, phenolic profiles and antioxidant activities in leaves of three potato genotypes (CB2011-509, CB2011-104, and VR808) were evaluated in crops inoculated with three AMF strains (Claroideoglomus claroideum, Claroideoglomus lamellosum, and Fumneliformis mosseae) and with AMF in combination with the use of two commercial fungicides (MONCUT [M] and ReflectXtra [R]). Eight phenolic compounds were detected, mainly hydroxycinnamic acids (HCAD) and flavonols, in samples where the highest concentrations of HCAD were obtained, 5-caffeoylquinic acid was the most abundant phenolic. The antioxidant activity was higher using the cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) methods. The association of AMF with plants had benefits on the secondary metabolism; however, the response differed according to genotype. The different combinations of potato genotypes, AMF strain, and fungicide modified the content of phenolic compounds in leaves in different ways; the treatment using C. lamellosum and ReflectXtra was the ideal combination for the genotypes analyzed here, with the higher antioxidant response, which supports the further technological evaluation of efficient AMF strains and fungicides in potato crops.

Metabolic and antioxidant effects of inoculation with arbuscular mycorrhizal fungi in crops of flesh-coloured Solanum tuberosum treated with fungicides.

BACKGROUND: Solanum tuberosum tubers have higher content of phenolic compounds such as hydroxycinnamic acid derivatives (HCAD) and anthocyanins in coloured genotypes. The use of fungicides for crops is common, but there are few studies regarding the interaction of fungicides and arbuscular mycorrhizal fungi (AMF). Here, the AMF-plant interactions and the metabolic responses of three potato genotypes with different tuber colorations (VR808, CB2011-509 and CB2011-104) inoculated with Claroideoglomus claroideum (CC), Claroideoglomus lamellosum (HMC26) or Funneliformis mosseae (HMC7) were studied together with the use of the fungicides MONCUT (M) and ReflectXtra (R). Mycorrhizal traits, phenolic compound profiles and antioxidant activity (AA) were evaluated. RESULTS: Despite only two HCADs being identified, with 5-caffeolquinic acid the most abundant, four anthocyanins were detected only in purple potato genotypes. The anthocyanin and HCAD profiles, as well as AA, showed that the CB2011-104 genotype had better characteristics than the other genotypes, while VR808 and CB509 showed similar responses. The responses were dependent on the specific combinations of genotype, fungicide and the AMF strain, and generally showed better responses when colonized by AMFs. CONCLUSION: The three potato genotypes had differential responses depending on the inoculated AMFs and the fungicide applied before sowing, where the optimal combinations for antioxidant response, mycorrhization degree and performance were HMC26/R for VR808, HMC7/M for CB2011-509 and HMC26/M for CB2011-104. Our results suggest the existence of functional compatibility that can be registered as beneficial effects even at the genotypic level of the host regarding a specific AMF strain. © 2021 Society of Chemical Industry.

Shifts in biochemical and physiological responses by the inoculation of arbuscular mycorrhizal fungi in Triticum aestivum growing under drought conditions.

BACKGROUND: A decrease in rainfall is one of the main constraints on wheat production, although the association of wheat with arbuscular mycorrhizal fungi (AMF) may be an alternative for crop production under drought conditions. In the present study, we used three wheat cultivars (Purple, Ilustre and Maxi Baer) inoculated with two AMF strains, one obtained from the hyperarid Atacama Desert (northern Chile; Fm) and the other obtained from southern Chile (Cc). Plants were maintained under two irrigation conditions (normal irrigation and drought) and the physiological behaviour and enzymatic and nonenzymatic antioxidant activities in the shoots were determined. In addition, the phenolic compounds were identified by high-performance liquid chromatography-dioide array detection-electrospray ionization-tandem mass spectrometry and quantified. RESULTS: AMF colonization produced higher levels of efficiency of photosystem II and photosynthetic pigments. High values of catalase in Purple-Cc, ascorbate peroxidase in Purple-Cc, glutathione reductase in Maxi-Cc and superoxide dismutase in Purple-Cc, all under stress, were registered. Of the inoculated cultivars, Purple-Cc showed the highest flavonoid levels, whereas hydroxycinnamic acids were higher in Maxi-Fm without drought, with apigenin and luteolin being the most abundant. High levels of phenols were present in the Ilustre-Fm plants without drought. Under normal irrigation, high levels of antioxidant activity were registered in the AMF treatments, whereas, under stress conditions, in general, high values were observed under the Fm inoculation. CONCLUSION: Our results showed that the greatest antioxidant activity and phenolic content occurred in wheat plants inoculated with AMF, indicating their influence on coping with water stress, which is of importance in vast areas where global climate change is resulting in diminished rainfall. © 2021 Society of Chemical Industry.

Effect of fertilization and arbuscular mycorrhizal fungal inoculation on antioxidant profiles and activities in Fragaria ananassa fruit.

BACKGROUND: The main methods used to increase the yield and quality of strawberry fruit produced in acidic soils with low P availability include root colonization by arbuscular mycorrhizal (AM) fungi and adjusting the type of P supplementation by means of traditional (TF) or organic (OF) fertilization, and adjusting nutrient doses. In this study, the antioxidant properties of strawberry fruit were evaluated under different treatments of fertilization (TF or OF) and different doses of P supplied at planting (0, 50 or 100% of the agronomic recommendation) and in the presence or absence of AM fungus as a bioinoculant. RESULTS: Fruits without fertilization treatments and with TF presented with higher anthocyanin concentrations. In general, higher values were obtained without AM colonization. However, spectrophotometric tests showed the highest activity and concentration in the AM-inoculated treatments. It is likely that phenolic compounds other than anthocyanins are present in the extracts. These other compounds could not be identified by the method used but could be detected by the Folin-Ciocalteu method. CONCLUSION: This study provides information that allows for improvements in strawberry fruit quality by agronomic management, with a potential beneficial effect on the health of consumers. © 2018 Society of Chemical Industry.

Changes in the content of anthocyanins, flavonols, and antioxidant activity in Fragaria ananassa var. Camarosa fruits under traditional and organic fertilization.

BACKGROUND: Strawberries are consumed worldwide. In Chile, strawberry production has been established in Andisols, were phosphorus is scarce. Traditional fertilization (TF) and organic fertilization (OF) have both been established there. This study examined their impact on the polyphenolic content of strawberries. RESULTS: Two anthocyanins were identified by high performance liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-ESI-MS/MS). The average total anthocyanin concentrations were found to be 651 mg kg-1 fresh weight in 100% OF, which represents a 56% increase compared to fruits that were not fertilized. In the case of flavonols, only quercetin-rhamnoside was identified, and its concentration reached 14.6 mg kg-1 with 100% OF. The ascorbic acid concentration reached 0.54 g kg-1 in 50% TF (a 20% increase over fruits without fertilization, WF). The antioxidant activities slightly increased in the fruits subjected to TF and OF in comparison with WF treatment. CONCLUSION: These results support a management strategy for obtaining the best quality and potential beneficial effects in health by increasing anthocyanins and other polyphenols under OF. © 2018 Society of Chemical Industry.