Untargeted metabolomic analysis reveals a potential role of saponins in the partial resistance of pea (Pisum sativum) against a root rot pathogen, Aphanomyces euteiches.

In soil-borne diseases, the plant-pathogen interaction begins as soon as the seed germinates and develops into a seedling. Aphanomyces euteiches, an oomycete, stays dormant in soil and gets activated by sensing the host through chemical signals present in the root exudates. The composition of plant exudates may, thus, play an important role during the early phase of infection. To better understand the role of root exudates in plant resistance, we investigated the interaction between partially resistant lines (PI660736 and PI557500) and susceptible pea cultivars (CDC Meadow and AAC Chrome) against Aphanomyces euteiches during the pre-invasion phase. The root exudates of two sets of cultivars clearly distinguished from each other in inducing oospore germination. PI557500 root exudate not only had diminished induction but also inhibited the oospore germination. The contrast between the root exudates of resistance and susceptible cultivars was reflected in their metabolic profiles. Data from fractionation and oospore germination inhibitory experiments identified a group of saponins that accumulated differentially in susceptible and resistant cultivars. We detected 56 saponins and quantified 44 of them in pea root and 30 from root exudate; the majority of them, especially Soyasaponin I and dehydrosoyasaponin I with potent in vitro inhibitory activities, were present in significantly higher amounts in both roots and root exudates of PI660736 and PI557500 as compared to Meadow and Chrome. Our results provide evidence for saponins as deterrents against Aphanomyces euteiches, which might have contributed to the resistance against root rot in the studied pea cultivars.

Characterization of Neutral Lipids of the Oleaginous Alga Micractinum inermum.

An oleaginous microalga Micractinum inermum isolated from Mariana Lake, AB, Canada was cultured in a 1000 L photobioreactor with an f/2 medium to study its lipid content and neutral lipid profile. Algal biomass was collected at the stationary phase contained a significant amount of lipids (44.2%), as determined by Folch's method. The lipid was fractionated into neutral lipid, glycolipid and phospholipid fractions. The neutral lipid constitutes almost 77.3% of the total lipid species and is mainly composed of triacylglycerols (TAGs) determined by a proton NMR study. UHPLC-HRMS analysis allows us for the first time to identify 81 TAGs in the neutral lipid fraction of M. inermum. The fatty acid acyl side chains were identified based on fragment ions observed in MSMS analysis. TAGs with fatty acid acyl chains 18:1/18:1/18:1, 18:1/18:1/16:0, 18:2/18:1/16:0, and 18:2/18:2/18:0 were the major ones among the identified TAGs. Fatty acid analysis further supports the fact that oleic acid was the major fatty acid present in the neutral lipid fraction of M. inermum constituting 41.7%, followed by linoleic acid at 21.5%, and palmitic acid at 21.2%. The saturated and monounsaturated fatty acids were 67.8% or higher in the lipid fraction. Long-chain fatty acids were only present in a minor quantity. The results clearly demonstrate that M. inermum is an excellent source for TAGs.

Analysis of Polar Lipids in Hemp (Cannabis sativa L.) By-Products by Ultra-High Performance Liquid Chromatography and High-Resolution Mass Spectrometry.

Polar lipids were extracted from residual biomass of hemp (Cannabis sativa L.) by-products with EtOH and partitioned into aqueous and chloroform fractions. The chloroform fractions were studied for their lipid composition using solid-phase extraction (SPE) followed by UHPLC/HRMS and NMR analyses. The 1H NMR and gravimetric yield of SPE indicated triacylglycerols covered ≥ 51.3% of the chloroform fraction of hemp seed hulls and hemp cake. UHPLC/HRMS analyses of remaining polar lipids led to the identification of nine diacylglycerols (DAGs), six lysophosphatidylcholines (LPCs), five lysophosphatidylethanolamines (LPEs), eight phosphatidylethanolamines (PEs), and thirteen phosphatidylcholines (PCs) for the first time from hemp seed hulls. The regiospecificity of fatty acyl substitutes in glycerol backbone of individual phospholipids were assigned by analyzing the diagnostic fragment ions and their intensities. The heat-map analysis suggested that DAG 18:2/18:2, 1-LPC 18:2, 1-LPE 18:2, PE 18:2/18:2, and PC 18:2/18:2 were the predominant molecules within their classes, supported by the fact that linoleic acid was the major fatty acid covering > 41.1% of the total fatty acids determined by GC-FID analysis. The 31P NMR analysis confirmed the identification of phospholipids and suggested PC covers ≥ 37.9% of the total phospholipid present in hemp by-products. HPLC purification led to the isolation of 1,2-dilinoleoylphosphatidylcholine and 1-palmitoyl-2-linoleoylphosphatidylcholine. These two major PCs further confirmed the UHPLC/HRMS finding.

Biochemical Characterization and In Vitro Digestibility of Protein Isolates from Hemp (Cannabis sativa L.) By-Products for Salmonid Feed Applications.

Hemp (Cannabis sativa L.) processing by-products (hemp cake and hemp seed hulls) were studied for their protein content, extraction of protein isolates (PIs), and their in vitro protein digestibility (IVPD). Crude protein contents of hemp cake and hemp seed hulls were 30.4% and 8.6%, respectively, calculated based on generalized N-to-P conversion factor (N × 5.37). Extraction efficiency of PIs from defatted biomass ranged from 56.0 to 67.7% with alkaline extraction (0.1 M NaOH) followed by isoelectric precipitation (1.0 M HCl). Nitrogen analysis suggested that the total protein contents of PIs extracted using three different alkaline conditions (0.5 M, 0.1 M, and pH 10.0 with NaOH) were >69.7%. The hemp by-product PIs contained all essential amino acids (EAAs) required for fish with leucine, valine, and phenylalanine belonging to the five dominant amino acids. Overall, glutamate was the dominant non-EAA followed by aspartate. Coomassie staining of an SDS-PAGE gel revealed strong presence of the storage protein edestin. High IVPD of >88% was observed for PIs extracted from hemp seeds and by-products when evaluated using a two-phase in vitro gastric/pancreatic protein digestibility assay. PIs extracted from by-products were further tested for their antioxidant activities. The tested PIs showed dose-dependent DPPH radical scavenging activity and possessed strong ORAC values > 650 µM TE/g.

Triacylglycerols and Other Lipids Profiling of Hemp By-Products.

Hemp seed by-products, namely hemp cake (hemp meal) and hemp hulls were studied for their lipid content and composition. Total lipid content of hemp cake and hemp hulls was 13.1% and 17.5%, respectively. Oil extraction yields using hexane, on the other hand, were much lower in hemp cake (7.4%) and hemp hulls (12.1%). Oil derived from both hemp seeds and by-products were primarily composed of neutral lipids (>97.1%), mainly triacylglycerols (TAGs), determined by SPE and confirmed by NMR study. Linoleic acid was the major fatty acid present in oils derived from hemp by-products, covering almost 55%, followed by α-linolenic acid, covering around 18% of the total fatty acids. For the first time, 47 intact TAGs were identified in the hemp oils using UPLC-HRMS. Among them, TAGs with fatty acid acyl chain 18:3/18:2/18:2 and 18:3/18:2/18:1 were the major ones, followed by TAGs with fatty acid acyl chain of 18:3/18:3/18:2, 18:2/18:2/16:0, 18:2/18:2/18:1, 18:3/18:2.18:0, 18:2/18:2/18:0, 18:2/18:1/18:1 and 18:3/18:2:16:0. Besides TAGs, low levels of terpenes, carotenoids and cannabidiolic acid were also detected in the oils. Moreover, the oils extracted from hemp by-products possessed a dose-dependent DPPH radical scavenging property and their potencies were in a similar range compared to other vegetable oils.

Synthesis and Characterization of ß-Cyclodextrin-Essential Oil Inclusion Complexes for Tick Repellent Development.

Essential oils (EOs) are used in several pest management applications. Due to their volatility, EOs may experience bioactivity reduction, thus requiring protection to extend their properties. In the present study, we investigated the inclusion complex formation (IC) of ß-cyclodextrin (ß-CD) with selected EOs with known tick repellent activity using two co-precipitation methods. ICs were characterized by evaluating EO mass concentration and inclusion efficiency (% IE) and other instrumental methods. Co-precipitation method 2 yielded the highest EO mass concentration (88 ± 6 µg/mg ß-CD) for the 1:1 molar ratio geranium Egyptian EO IC. The EO volatile release over time from the ICs was investigated by headspace SPME/GC-MS analysis. ICs were also tested in tick repellency bioassays. ICs reported significant tick repellent activity, with lemongrass IC performing best overall. Method 1 showed the best combination of high mass concentration EO, controlled volatile release, and tick repellency with lemongrass EO. The results demonstrated that ß-CD had selectively encapsulated different EOs. Moreover, the formation of ICs may improve EO tick repellent properties protecting the active ingredients and providing a better, long-lasting repellent action. These findings will allow the development of more effective naturally derived repellent products to protect individuals from tick bites and prevent tick-borne illnesses.