Elicitor-enhanced steroidal sapogenin accumulation in hairy root cultures of Trigonella foenum-graecum.

In current work, we studied hairy root induction in Trigonella foenum graecum, which is an important medicinal plant, and examined the impact of different elicitors on some phytochemical characteristics and metabolites production in hairy root cultures. Accordingly, some factors such as five strain types of Agrobacterium rhizogenes (1724, 15834, A4, A13 and MSU) and three different explants, namely leaf, cotyledon and hypocotyl were studied. The results showed that different A. rhizogenes strains exhibited different infection efficiency. MSU and 15834 had highest efficiency of hairy root induction than other strains. Also, hairy root induction frequency in leaf explants was higher than in other explants. Salicylic acid (SA), nitric oxide (NO), CaCl2 and penconazole (PEN) were used in elicitation process. Hairy roots were treated with SA (0.1 and 0.5 mM), NO (10 and 50 µM), CaCl2 (5 and 10 mM) and PEN (5 and 10 mg/L). Applied elicitors enhanced antioxidant enzymes activities and reduced oxidative stress markers; this observation might be ascribed to regulation of the oxidative status of the elicited cells. Significant increase of antioxidant metabolites (total phenol, flavonoid and anthocyanin) in PEN-treated hairy roots was associated to phenylalanine ammonia lyase activity, indicating an up-regulation of phenylpropanoid/flavonoid metabolism. PEN and CaCl2 treatment enhanced steroidal sapogenin in hairy root cultures. These results suggested that use of elicitors can enhance the production of secondary metabolites in transformed hairy roots. Among the elicitors applied, CaCl2 and PEN were the most effective in increasing secondary metabolite production in transformed hairy roots of T. foenum graecum.

Iron nanoparticle regulate succinate dehydrogenase activity in canola plants under drought stress.

Application of nutrients as nanoparticle (NP) is an operative manner of nutrient supply for plants, especially under stress conditions. The present study was designed to highlight the role of iron NP on drought tolerance and elucidate the underlying mechanisms in drought-stressed canola plants. Drought stress was imposed by polyethylene glycol different concentrations (0, 10 and 15% (W/V)) with or without iron NP (1.5 and 3 mg/l). A comparative study of several physiological and biochemical parameters have been carried out in canola plants treated by drought and iron NP. Stressed-canola plants showed a reduction in growth parameters, whereas iron NP mostly stimulated growth of stressed plants, which was accompanied by reinforcement in defense mechanisms. Regarding impacts on compatible osmolytes, the data revealed that iron NP was able to regulate osmotic potential by increasing protein, proline and soluble sugar contents. The iron NP application was activated the enzymatic defense system (catalase and polyphenol oxidase) and promoted the non-enzymatic antioxidants (phenol, flavonol and flavonoid). Both of these adaptive responses declined free radicals as well as lipid peroxidation and enhanced the membrane stability and drought tolerance of the plants. Enhanced chlorophyll accumulation via induction of protoporphyrin, magnesium protoporphyrin and protochlorophyllide, by iron NP also contributed towards better stress tolerance. Enzymes of Krebs cycle, namely succinate dehydrogenase and aconitase, were induced by iron NP in canola plants grown under drought stress. These results propose a multifaceted involvement of iron NP, through regulation of activity of respiratory enzymes and antioxidant enzymes, production of reactive oxygen species, osmoregulation and secondary metabolites metabolism, in response to drought stress.

Cerium and samarium blocked antioxidant enzymes in wheat plants.

This work was conducted to study positive and negative impacts of cerium (Ce) and samarium (Sm) on two cultivars (Arta and Baharan) in wheat plant. Symbols of stress such as proline, malondialdehyde (MDA) and antioxidant enzymes, which may be complicated in the suppression responses of plants, were also studied. Wheat plants were exposed to 0, 2500, 5000, 7500, 10,000 and 15,000 µM of Ce and Sm for 7 days. The growth enhanced in plants treated with lesser Ce and Sm concentration (2500 µM) and declined in plants treated with upper concentrations as compared to untreated plants. The treatment with 2500 µM of Ce and Sm increased dry weigh in Arta by 68.42 and 20% and in Baharan by 32.14% and 27.3%. Thus, Ce and Sm had hormesis effect on growth in wheat plants. According to plant's growth parameter patterns, Arta cultivar had more sensitive to Sm than to Ce, whereas Baharan cultivar had sensitive to Ce than to Sm. Our results indicated impact of Ce and Sm on proline accumulation depended on the dosage of Ce and Sm. It was observed that Ce and Sm accumulated in wheat plants at higher exposure doses. Increment of MDA content by Ce and Sm treatments showed that these metals caused oxidative stress in wheat plants. Ce and Sm blocked enzymatic antioxidant system (superoxide dismutases, peroxidase and polyphenol peroxidase) in wheat. In wheat plants treated with lower Ce and Sm concentrations higher amounts of non-enzymatic antioxidant metabolites were detected. Thus, we showed the potential negative impact of unsuitable utilization of REEs in plants and suggested growth and interruption in physiological and biochemical mechanisms as a possible factor to recognize the underlying toxicological processes.

The regulatory role of γ-aminobutyric acid in chickpea plants depends on drought tolerance and water scarcity level.

γ-Aminobutyric acid (GABA) is a non-protein amino acid with multifunctional roles in dynamic plant responses. To determine the effects of exogenous GABA application (0, 25 and 50 µM) on drought response, two chickpea cultivars with contrasting tolerance to water deficit were examined. Plants were exposed to four irrigation levels (irrigation to 100, 60, 40 and 20% field capacity). Water deficit decreased growth, chlorophyll content, and photosynthetic efficiency. It increased electrolyte leakage and lipid peroxidation owing to both higher ROS accumulation and lower antioxidant enzyme activity. These negative effects of water deficit and the alleviating role of GABA application were more prominent in the sensitive, as compared to the tolerant cultivar. Water deficit also increased proline and GABA contents more in the tolerant cultivar, whereas their content was more enhanced by GABA application in the sensitive one. This may confer an additional level of regulation that results in better alleviation of drought damage in tolerant chickpea cultivars. In conclusion, the stimulatory effect of GABA on growth and physiological modulation depends on both the water stress severity and the cultivar sensitivity to it, implying a probable unknown GABA-related mechanism established by tolerant chickpea cultivars; a lost or not gained mechanism in susceptible ones.

Nitric oxide induced carotenoid contents in Crocus sativus under salinity.

Crocus sativus possesses unique apocarotenoid compounds such as crocins and picrocrocein involved in color, taste, flavor and medicinal benefits of Saffron. Crocus sativus L. corms were treated with Nitric Oxide (NO) and salt. Crocins and picrocrocin contents were determined with high-performance liquid chromatography (HPLC) resulted a significant increase of crocins in treated plants with NO and salt that reveals the stimulating effect of NO in apocarotenoid biosynthesis besides the inductive role of salt stress. This raise can be attributed to expression of CsPDS, CsPSY, CsLYC, CsBCH, and CsCCD2 that were remarkably altered. Treating plants with NO caused more phenol production in oppose to less flavonoid content; however, salinity could increase both. Therefore, NO induced crocins and picrocrocin biosynthesis due to impressing gene expression. This increasing effect was enhanced when salinity was simultaneously imposed.

Penconazole and calcium ameliorate drought stress in canola by upregulating the antioxidative enzymes.

The aim of this research was to gauge the alternations in the lipid peroxidation and antioxidative enzyme activity in two cultivars (cv. RGS003 and cv. Sarigol) of canola under drought stress and drought tolerance amelioration by penconazole (PEN) and calcium (Ca). Plants were treated with different polyethylene glycol (PEG) concentrations (0, 5, 10 and 15%) without or with PEN (15 mg L-1) and Ca (15 mM). The Ca treatment prevented the negative effects of drought on fresh weight (FW) in RGS003 and Sarigol at 5 and 15% PEG respectively. Ca and PEN/Ca treatments caused significant induction in the proline content in Sarigol at 15% PEG; the latter treatment was accompanied by higher glycine betaine (GB), lower malondialdehyde (MDA) and growth recovery. Hydrogen peroxide (HO2) content in Sarigol was proportional to the severity of drought stress and all PEN, Ca and PEN/Ca treatments significantly reduced the H2O2 content. PEN and PEN/Ca caused alleviation of the drought-induced oxidative stress in RGS003. RGS003 cultivar exhibited significantly higher antioxidative enzymes activity at most levels of drought, which could lead to its drought tolerance and lower MDA content. In contrast to that of Sarigol, the activity of catalase and superoxide dismutase (SOD) increased with Ca and PEN/Ca treatments in RGS003 under low stress. The application of PEN and Ca induced significantly P5CS and SOD expression in RGS003 under drought stress after 24 h. Overall, these data demonstrated that PEN and Ca have the ability to enhance the tolerance against the drought stress in canola plants.

CO2 biofixation and fatty acid composition of two indigenous Dunaliella sp. isolates (ABRIINW-CH2 and ABRIINW-SH33) in response to extremely high CO2 levels.

Global warming, as a result of atmospheric CO2 increase, is regarded as an important universal concern. Microalgae are considered as appropriate microorganisms for CO2 assimilation. Here we aimed to investigate carbon biofixation ability of two indigenous isolates of Dunaliella spp. (ABRIINW-CH2 and ABRIINW-SH33) under elevated CO2 concentrations of 10, 20, and 30% (v/v) as well as their lipid content, productivity, and fatty acid profile under adjusted pH conditions. The maximum biomass production and CO2 biofixation rates were obtained under 10% CO2. High CO2 concentrations were favorable for the accumulation of lipids, lipid productivity, and polyunsaturated fatty acids formation. The highest lipid content and lipid productivity was obtained at 10% CO2. The highest fraction of the fatty acids (FA) profile was allocated to omega-3 FAs at 20% CO2. Accordingly, these isolates were able to tolerate extremely high CO2 concentrations and present even enhanced growth as well as formation of valuable products.

Differential proteomics: Effect of growth regulators on salt stress responses in safflower seedlings.

Productivity of safflower (Carthamus tinctorius L.) is markedly reduced by salt stress. This study is based on analysis of proteins profile of safflower plants treated with 200 mM NaCl, with exogenously applied salicylic acid (SA) and penconazole (PEN), as growth regulators. Plants were investigated through a gel-based proteomic approach, which resulted in the identification of 17 salt-responsive proteins related to different metabolic modifications. Of these, seven different proteins were up or down regulated by both SA and PEN, suggesting the synergistic and antagonistic effects of SA and PEN. The classification of differentially expressed proteins showed that salt-responsive proteins were mainly involved in photosynthesis, ion homeostasis, and oxidative stress response, as well as nitrogen, protein, and carbohydrate metabolism. The identified stress-responsive proteins in this study could pave the way to develop salt tolerance in safflower, thus sustaining its productivity under salinity. In addition, SA and PEN may be considered as a foliar application to ameliorate salinity effects, due to their low price and availability.

Antioxidative enzymes activities and accumulation of steroids in hairy roots of Trigonella.

Steroidal sapogenins and phytosterols are a group of secondary metabolites which are very considerable in the pharmaceutical industry. Fenugreek (Trigonella foenum-graecum L.) is the good source of these compounds. In recent decades, there is a great interest to production of these compounds by cultivation of transformed roots. In present study, hairy roots induction in two Trigonella species (T. foenum-graeceum, T. monantha) with three strains of Agrobacterium rhizogenes (15,834, A4 and wt) was investigated. Transgenic status of roots was confirmed by PCR using rolB specific primers. Virulence of these strains was examined on explants of leaf, leaf cotyledone and hypocotyle in both species. The best strain was wt for hairy root induction in hypocotyle and leaf explants of T. foenum-graeceum and T. monantha. Significant quantitative differences were showed between shoot, root and hairy roots in both species. Protein content in root and hairy root of both species was significantly lower in comparison with shoot. Superoxide dismutase (SOD) and peroxidase (POX) activities in hairy roots of both species were higher as compared to other organs. The hairy roots of both species showed an ability to synthesize steroidal sapogenins. These results presented that hairy roots could be a suitable procedure for producing sapogenins compounds that have medicinal value in Trigonella.

A genome-wide identification, characterization and functional analysis of salt-related long non-coding RNAs in non-model plant Pistacia vera L. using transcriptome high throughput sequencing.

Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression in response to plant stresses. Given the importance regulatory roles of lncRNAs, providing methods for predicting the function of these molecules, especially in non-model plants, is strongly demanded by researchers. Here, we constructed a reference sequence for lncRNAs in P. vera (Pistacia vera L.) with 53220 transcripts. In total, we identified 1909 and 2802 salt responsive lncRNAs in Ghazvini, a salt tolerant cultivar, after 6 and 24 h salt treatment, respectively and 1820 lncRNAs in Sarakhs, a salt sensitive cultivar, after 6 h salt treatment. Functional analysis of these lncRNAs by several hybrid methods, revealed that salt responsive NAT-related lncRNAs associated with transcription factors, CERK1, LEA, Laccase genes and several genes involved in the hormone signaling pathways. Moreover, gene ontology (GO) enrichment analysis of salt responsive target genes related to top five selected lncRNAs showed their involvement in the regulation of ATPase, cation transporter, kinase and UDP-glycosyltransferases genes. Quantitative real-time PCR (qRT-PCR) experiment results of lncRNAs, pre-miRNAs and mature miRNAs were in accordance with our RNA-seq analysis. In the present study, a comparative analysis of differentially expressed lncRNAs and microRNA precursors between salt tolerant and sensitive pistachio cultivars provides valuable knowledge on gene expression regulation under salt stress condition.

Leptolyngbya fragilis ISC 108 is the most effective strain for dodecane biodegradation in contaminated soils.

One of the major environmental problems nowadays is petroleum hydrocarbons contamination. Bioremediation is widely used for cleaning ecosystems contaminated with petroleum hydrocarbons. This study was carried out to investigate the response of five microalgae strains isolated from different regions in Iran for 1% n-dodecane (DOD) degradation. The results revealed that Leptolyngbya fragilis ISC 108 is the most effective strain to utilize n-DOD as growth substrate under a mixotrophic condition. Currently, there is little information about mechanisms involved in microalgae response against DOD. The activity of antioxidant enzymes and total lipid and carbohydrate contents were observed to be greater in DOD-treated L. fragilis ISC 108. Lower values of lipid peroxidation and H2O2 along with an increase of dry weight and specific growth rate in L. fragilis ISC 108 under DOD treatment shows that at the cellular level this strain is better equipped with an efficient oxygen radical scavenging system. In conclusion, this study proposes that L. fragilis ISC 108 can be considered an ideal candidate for use in bioremediation of DOD contaminated sites.

Different effects of calcium and penconazole on primary and secondary metabolites of Brassica napus under drought.

The effects of penconazole (PEN) and calcium (Ca2+) on physiological and biochemical parameters were investigated in two canola cultivars (RGS003 and Sarigol) under water stress. Drought increased protein content in RGS003, but PEN, Ca2+ and PEN-Ca2+ treatment induced protein content in Sarigol. PEN, Ca2+ and PEN-Ca2+ treatment enhanced soluble sugar content in RGS003. In contrast to Sarigol, drought and PEN treatment induced total phenol content in RGS003. Flavonoid content increased by drought, but Ca2+ and PEN-Ca2+ treatment decreased it in both cultivars. Ca2+ and PEN-Ca2+ treatment enhanced tocopherol content in both cultivars under drought stress. Drought stress increased Phenylalanine ammonia-lyase (PAL) activity in Sarigol. PEN-Ca2+ treatment increased relative expression of PAL and its activity in RGS003. Fatty acid composition was modified by drought, PEN and Ca2+. Saturated fatty acid (stearic acid) content declined but unsaturated fatty acid (oleic acid) content enhanced in both cultivars under drought. The application of PEN and Ca2+ decreased unsaturated fatty acids (linoleic and linolenic acid) in RGS003 under drought. According to our results, PEN and Ca2+ changed physiological and biochemical parameters and therefore these compounds are suggested for reduction of the negative effects of drought stress in canola.

Diverse role of γ-aminobutyric acid in dynamic plant cell responses.

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is found in most prokaryotic and eukaryotic organisms. Although, ample research into GABA has occurred in mammals as it is a major inhibitory neurotransmitter; in plants, a role for GABA has often been suggested as a metabolite that changes under stress rather than as a signal, as no receptor or motif for GABA binding was identified until recently and many aspects of its biological function (ranging from perception to function) remain to be answered. In this review, flexible properties of GABA in regulation of plant responses to various environmental biotic and abiotic stresses and its integration in plant growth and development either as a metabolite or a signaling molecule are discussed. We have elaborated on the role of GABA in stress adaptation (i.e., salinity, hypoxia/anoxia, drought, temperature, heavy metals, plant-insect interplay and ROS-related responses) and its contribution in non-stress-related biological pathways (i.e., involvement in plant-microbe interaction, contribution to the carbon and nitrogen metabolism and governing of signal transduction pathways). This review aims to represent the multifunctional contribution of GABA in various biological and physiological mechanisms under stress conditions; the objective is to review the current state of knowledge about GABA role beyond stress-related responses. Our effort is to place findings about GABA in an organized and broader context to highlight its shared metabolic and biologic functions in plants under variable conditions. This will provide potential modes of GABA crosstalk in dynamic plant cell responses.

Oxidative damage and antioxidative system in algae.

Reactive oxygen species (ROS) typically produce in algae and act as secondary messengers in numerous cellular processes. Under abiotic stresses, the balance between production and suppression of ROS disappears and causes increase of ROS. Increasing excessive ROS can cause damage to various cellular components comprising cell membranes, proteins and lipids. Algae have an antioxidant defense system to overcome on oxidative damage. Antioxidant defense mechanisms are of two types, namely enzymatic and non-enzymatic antioxidants. The enzymatic antioxidants include superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The non-enzymatic antioxidants include carotenoids, tocopherol, ascorbic acid, glutathione, flavonoids and phenolic compounds. In this review, we describe the various types of ROS and their production, and antioxidant defense mechanisms for ROS suppression.

Manganese-induced changes in glandular trichomes density and essential oils production of Mentha aquatica L. at different growth stages.

Production and accumulation of essential oils in plants are influenced by intrinsic and environmental factors. Here, we attempted to elucidate the effect of manganese (Mn) supply on the density of glandular trichomes and the production of essential oils in Mentha aquatica (water mint; syn. Mentha hirsuta Huds.) at the different growth stages. To this aim, plants were treated with 100 µM of Mn (supplied as MnSO4·H2O) at early and late vegetative stages of growth. Then, the control and treated plants were harvested, and biochemical, morphological and molecular analyses indicated that Mn supply has affected M. aquatica at the different growth stages. The biomass, Mn accumulation, glandular trichomes density, essential oils yield and expression levels of the genes encoding enzymes involved in terpenoid biosynthesis pathway (1-Deoxy d-xylulose-5-phosphate synthase (Dxs), geranyl diphosphate synthase (Gpps), isopentenyl diphosphate isomerase (Ippi), ß-caryophyllene synthase (Cps), limonene synthase (Ls) and menthofuran synthase (Mfs)) were increased by Mn supply at both growth stages. However, the increased rates of the assayed parameters were varied between the early and late vegetative stages. Moreover, the content and chemical composition of terpenoid components were affected by Mn supply and plant growth stage. There were positive and weak correlations among the study variables under the Mn supply at the different growth stages. Given these findings, we propose that the application of Mn supply at both early and late vegetative stages elevates the growth, density of glandular trichomes and production of essential oils in M. aquatica.

COP1 plays a prominent role in drought stress tolerance in Arabidopsis and Pea.

Constitutively photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that has been studied extensively in the photomorphogenesis- and light-related processes in Arabidopsis. However, the possible role of COP1 in plant drought stress response remains unknown. Hence, in the present study, the stomatal behavior as one of the key elements in plant dehydration response was investigated in Arabidopsis cop1-4 and pea light-independent photomorphogenesis (lip1) mutants. We observed that water loss rate in the cop1-4 and lip1 detached leaves was significantly much faster than wild-type, resulting from failing to reduce the stomatal aperture by the mutants. But, interestingly, the cop1-4 and lip1 isolated leaves treated with abscisic acid (ABA) as well as cop1-4 and lip1 soil-grown under drought stress could close their stomata as wild-type. Hence, COP1 plays a fundamental role in the regulation of stomatal movements in response to dehydration and its function was conserved during evolution in both Arabidopsis and pea. Further evaluations showed the cop1-4 mutant was not significantly damaged from the oxidative stress derived from soil water limiting conditions when compared to wild-type. Similarly, the up-regulation level of several key stress-responsive genes was relatively lower in cop1-4 than wild-type. Therefore, COP1 might be considered as a potential key regulator of both short-and long-term dehydration response. Multiple stress-related cis-elements were also detected in the COP1 promoter region, which supported its up-regulation in response to drought, salt, and cold stresses. Besides, we figured out the constitutively open stomata of cop1-4 in darkness can be as a result of the reduced AtMYB61 expression.

Improving tolerance against drought in canola by penconazole and calcium.

Drought stress is one of the most important environmental factors that limit plant growth. Canola is an important agricultural crop grown primarily for its edible oil. In this study, penconazole (PEN), a triazole growth regulator, and calcium (Ca2+), a secondary messenger, were used to analyses their role in decease sensitivity and induce tolerance to drought stress in canola. Plants were treated by various PEG concentrations (0, 5, 10, and 15%) without or with PEN (15 mg l-1) and Ca2+ (15 mM). According to the obtained results in two cultivars of canola (RGS003 and Sarigol), improvement of growth by PEN and Ca2+observed at low concentration of drought (5%). Betterment of growth by PEN can be explained by induction in DPPH (1, 1-diphenyl-2- picrylhydrazyl), chlorophyll and P content and reduction in Electrolyte leakage in cultivar RGS003. Growth recovery by PEN is concomitant by increase in DPPH, succinate dehydrogenase, chlorophyll, carotenoid and K+ content in cultivar Sarigol. Ca2+ treatment by induction in Relative water content, DPPH, chlorophyll, carotenoid, indole-3-acetic acid content and the content of Ca2+ and K+ causes increase in growth in cultivar Sarigol. Overall, application of PEN and Ca2+ improved the performance in canola under drought stress. These results provide novel insights about the physiological and molecular roles of PEN and Ca2+ in canola plant tolerance against drought stress.

Changes in primary and secondary metabolites of Mentha aquatica L. exposed to different concentrations of manganese.

This experiment was conducted in order to determine the effects of different concentrations of manganese (Mn) on the levels and correlations of multiple primary and secondary metabolites in Mentha aquatica. With this aim, four levels of Mn concentrations were used as follows: basic Hoagland's solution (control), 40, 80, and 160 µM of Mn supplied as MnSO4.H2O. The results indicated that the biomass and the contents of photosynthetic pigments and soluble carbohydrates were higher in the plants that were treated with the moderate concentrations of Mn (40 and 80 µM) than the control and 160 µM-treated plants. On the other hand, the contents of flavonoids, anthocyanins, malonaldehyde (MDA), hydrogen peroxide (H2O2), and the activities of antioxidant enzymes (total superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX)) were progressively increased with the enhancement of Mn concentration in the nutrient solution. In addition, there were clear differences in the content and chemical composition of essential oils among the control and treatment groups. In this study, 1,8-cineole, menthofuran, and ß-caryophyllene were the most abundant constituents of essential oils in both the control and Mn-treated plants. The correlation analysis between pairs of the primary and secondary metabolites showed that there were positive and negative correlations among the variables when the Mn concentration was increased in the nutrient solution. These findings clearly display a positive effect of Mn up to 80 µM in the nutrient solution on the growth of M. aquatica.

A Multivariate Analysis of the Composition and Properties of Extra Virgin Olive Oils Produced from Different Cultivars Grown in Iran.

The present study investigated variations in extra virgin olive oils in relation to fatty acid (FA) composition and the characteristics of 10 olive cultivars. The findings demonstrated that their oil yield properties, including refractive index, acid value, peroxide value, saponification value, iodine value, and composition, were significantly different. Moreover, based on GC-MS analysis, the presence of oleic acid [C18:1(9)] was identified as one of the major components. The highest amount of 18:1(9) was found in four major varieties of cultivars, namely Zard, Roghani, Karidolia, and Korfolia. Hierarchical cluster analysis of principal component analysis revealed two distinct categories of cultivars based on their FAs. The first category (cluster I), consisted of Arbequina, Karydolia, Roghani, and Zard cultivars, which can be considered cultivars with good commercial cultivation potential due to their high contents of unsaturated FAs and oil quantities produced.

Ultrasound-assisted extraction process of phenolic antioxidants from Olive leaves: a nutraceutical study using RSM and LC-ESI-DAD-MS.

In recent years, there has been an ever growing interest in finding new natural sources of food antioxidants. As a main fruit crop, olive is also valued due to its phenolic-containing leaves. Mathematically based optimization methods are used as powerful tools to extract different antioxidant compounds. The present study is aimed to provide an efficient extraction method for total phenol content (TPC), total flavonoid content (TFC) and antioxidant ability (DPPH scavenging assay and FRAP). The effects of ultrasonic temperature (35-65 °C), ultrasonic time (5-15 min), and ethanol to water ratio (Et: W) (25-75%) were evaluated. Second-order polynomial models were used through a rotatable Box-Behnken design (BBD) consisting of 15 experimental runs with three replicates at the center point. Interactional effects of the studied factors were significant in most cases for all responses. The highest extraction efficiency was found to be fifty-one percent of ethanol (65 °C, 15 min) to water ratio. Under optimal conditions, values for TPC, TFC, DPPHsc and FRAP assay were 183.4 (mg GAE. g-1 DW), 696.77 (mg Quercetin. g-1 DW), 78.98 (DPPHsc %) and 1942 µmol Fe+2/g DW, respectively. R2 values (R2 > 0.92) showed that RSM models could efficiently predict the yield of all responses. In the LC-ESI-MS-DAD profiling of the optimized extract, 27 compounds were identified with oleuropein as the main compound. In the present study, olive leaf is introduced as a promising source of natural antioxidant and can be used in food industries following further studies.