Chemical Profiling (HPLC-NMR & HPLC-MS), Isolation,and Identification of Bioactive Meroditerpenoids from the Southern Australian Marine Brown Alga Sargassum paradoxum

A phytochemical investigation of a southern Australian marine brown alga, Sargassum paradoxum, resulted in the isolation and identification of four new (5, 9, 10, and 15) and nine previously reported (1, 2, 6–8, and 11–14) bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight (1, 2, 5–7, and 10–12) other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 μL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration.     

A Chemoinformatics Approach to the Discovery of Lead-Like Molecules from Marine and Microbial Sources En Route to Antitumor and Antibiotic Drugs

The comprehensive information of small molecules and their biological activities in the PubChem database allows chemoinformatic researchers to access and make use of large-scale biological activity data to improve the precision of drug profiling. A Quantitative Structure–Activity Relationship approach, for classification, was used for the prediction of active/inactive compounds relatively to overall biological activity, antitumor and antibiotic activities using a data set of 1804 compounds from PubChem. Using the best classification models for antibiotic and antitumor activities a data set of marine and microbial natural products from the AntiMarin database were screened—57 and 16 new lead compounds for antibiotic and antitumor drug design were proposed, respectively. All compounds proposed by our approach are classified as non-antibiotic and non-antitumor compounds in the AntiMarin database. Recently several of the lead-like compounds proposed by us were reported as being active in the literature.     

Application of HPLC-NMR in the Identification of Plocamenone and Isoplocamenone from the Marine Red Alga Plocamium angustum

A combination of on-line HPLC-NMR and off-line chemical investigations has resulted in the identification of the previously reported polyhalogenated monoterpene plocamenone, together with the new structural analogue isoplocamenone from the crude extract of the marine alga Plocamium angustum. On-flow and stop-flow HPLC-NMR analyses (including the acquisition of WET 2D NMR spectra) rapidly assisted in the identification of the major component plocamenone and in the partial identification of its unstable double bond isomer isoplocamenone. Conventional off-line isolation and structural characterization techniques were employed to unequivocally confirm both structures, leading to a structural revision for plocamenone, as well as to obtain sufficient quantities for biological testing.     

Bioprospecting Red Sea Coastal Ecosystems for Culturable Microorganisms and Their Antimicrobial Potential

Microorganisms that inhabit unchartered unique soil such as in the highly saline and hot Red Sea lagoons on the Saudi Arabian coastline, represent untapped sources of potentially new bioactive compounds. In this study, a culture-dependent approach was applied to three types of sediments: mangrove mud (MN), microbial mat (MM), and barren soil (BS), collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL). The isolated bacteria were evaluated for their potential to produce bioactive compounds. The phylogenetic characterization of 251 bacterial isolates based on the 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity to other strain sequences in the NCBI database, being ≤98%. We demonstrate that 49 of the 251 isolates exhibit the potential to produce antimicrobial compounds. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 49 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards Staphylococcus aureus, Salmonella typhimurium and Pseudomonas syringae. We report the previously unknown antimicrobial activity of B. borstelensis, P. dendritiformis and M. salipaludis against all three indicator pathogens. Our study demonstrates the evidence of diverse cultured microbes associated with the Red Sea harbor/lagoon environments and their potential to produce antimicrobial compounds.     

Effect of photoperiod and temperature on resistance againstPhytophthora infestans in susceptible and resistant potato cultivars: Effect on deposition of structural phenolics on the cell wall and resistance to penetration

Deposition of phenolic compounds before and after inoculation withPhytophthora infestans was evaluated in two Mexican cultivars (Malinche and Tollocan) with major unknown R genes for resistance to potato late blight, two cultivars (393295.236=CIP1 and 391046.22=CIP2) without R genes from the International Potato Center (CIP) and the susceptible cultivar Atlantic. Before inoculation, plants were grown in growth chambers at two temperatures (16 or 24 C) and two photoperiods (PPD 12 or 16 h day length). Forty-eight hours after inoculation, the number of penetrations was recorded and depositions of phenolic compounds were classified according to detection and location in (a) the anticlinal cell wall, (b) the whole cell, (c) the stomatal cells, and (d) without detectable depositions of phenolic compounds. The concentration of phenolic compounds in the epidermal cells was slightly increased at 16 C and 16 h PPD and penetration frequency was lower at 16 C (12 h PPD). Concentration of phenolic compounds was not correlated with penetration frequency, but was correlated with the resistance level of the different potato cultivars. Atlantic had the highest number of penetrations followed by Tollocan, CIP1, CIP2, and Malinche. The cytological observations indicated that four types of deposition of phenolic compounds occurred in all five potato cultivars irrespective of their type and level of resistance. These results suggest that deposition of phenolic compounds on epidermal cells is a general resistance mechanism in potato leaves that does not have a specific relation with resistance to the penetration ofP. infestans. Phenolic depositions were intrinsically similar in potato cultivars with and without R genes, which stresses the difficulty in differentiating between horizontal and vertical resistance.     

Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

Evaluation of the GrazeIn model of grass dry‐matter intake and milk production prediction for dairy cows in temperate grass‐based production systems. 2 – Animal characteristics

This study evaluated the prediction accuracy of grass dry‐matter intake (GDMI) and milk yield predicted by the GrazeIn model using a large database representing 8787 per cow GDMI measurements. In this study, the animal input variables (age, parity, week of lactation, potential peak milk yield, milk fat content, milk protein content, bodyweight, body condition score (BCS), week of conception, BCS at calving and calf birth weight) were investigated. The mean actual GDMI of the database was 15·9 kg DM per cow d^?1 and GrazeIn predicted a mean GDMI for the database of 15·5 kg DM per cow d^?1. The mean bias was ?0·4 kg DM per cow d^?1. GrazeIn predicted GDMI for the total database with an RPE of 15·5% at cow level. The mean actual daily milk yield of the database was 21·3 kg per cow d^?1 and GrazeIn predicted a daily milk yield for the database of 22·2 kg per cow d^?1. The mean bias was +0·9 kg per cow d^?1. GrazeIn predicted milk yield for the total database with an RPE of 16·7% at cow level. From the evaluation, GrazeIn predicted milk yield of all cows in late lactation with a larger level of error than in early and mid‐lactation. This error appears to be due to the persistency of the lactation curve used by the model, which results in a higher predicted milk yield in late lactation compared with the actual milk yield.     

Isolation of Scalarane-Type Sesterterpenoids from the Marine Sponge Dysidea sp. and Stereochemical Reassignment of 12-epi-Phyllactone D/E

The chemical investigation of the marine sponge Dysidea sp., which was collected from Bohol province in the Philippines, resulted in the identification of 15 new scalarane-type sesterterpenoids (1–14, 16), together with 15 known compounds. The chemical structures of the new compounds were elucidated based on NMR spectroscopy and HRMS. The structure of 12-epi-phyllactone D/E (15) isolated during this study was originally identified in 2007. However, careful inspection of our experimental ¹³C NMR spectrum revealed considerable discrepancies with the reported data at C-9, C-12, C-14, and C-23, leading to the correction of the reported compound to the C-12 epimer of 15, phyllactone D/E. The biological properties of compounds 1–16 were evaluated using the MDA-MB-231 cancer cell line. Compound 7, which bears a pentenone E-ring, exhibits significant cytotoxicity with a GI₅₀ value of 4.21 μM.     

Physicochemical assessment and bioactive properties of condensed distillers solubles,a by-product from the sorghum bio-fuel industry

Large volumes of condensed distillers solubles (CDS) are generated as by-products, from the sorghum bioethanol industry. The objective was to assess the physico-chemical and bioactive properties of CDS. The unfractionated CDS showed the highest content of phenolic compounds (16 mg GAE/g), antioxidant (522 μM Trolox/g) and antimicrobial activity (MIC 1%(w/v) against Campylobacter spp.) compared to its extracts. The water and methanol extracts also showed high levels of phenolic compounds and antioxidant activity (11.6 and 9.2 mg GAE/g and 349 and 409 μM Trolox/g respectively), followed by ethanol and acetone extractions (7.5 and 6.6 mg GAE/g; 337 and 346 μM Trolox/g respectively). A positive correlation was revealed between total phenol and antioxidant activity. The main phenolic compounds found in the extracts were protocatechuic acid, 4-hydroxybenzoic acid, taxifolin, ferulic acid, cinnamic acid and p-coumaric acid. This study indicates the potential of using CDS as a functional ingredient for other food and feed applications.     

New Drimane Sesquiterpenes and Polyketides from Marine-Derived Fungus Penicillium sp. TW58-16 and Their Anti-Inflammatory and α-Glucosidase Inhibitory Effects

Marine fungi-derived natural products represent an excellent reservoir for the discovery of novel lead compounds with biological activities. Here, we report the identification of two new drimane sesquiterpenes (1 and 2) and six new polyketides (3–8), together with 10 known compounds (9–18), from a marine-derived fungus Penicillium sp. TW58-16. The planar structures of these compounds were elucidated by extensive 1D and 2D NMR, which was supported by HR-ESI-MS data. The absolute configurations of these compounds were determined by experimental and calculated electronic circular dichroism (ECD), and their optical rotations compared with those reported. Evaluation of the anti-inflammatory activity of compounds 1–18 revealed that compound 5 significantly inhibited the release of nitric oxide (NO) induced by lipopolysaccharide (LPS) in RAW264.7 cells, correlating with the inhibition of expression of inducible nitric oxide synthase (iNOS). In addition, we revealed that compounds 1, 3–6, 14, 16, and 18 showed strong α-glucosidase inhibitory effects with inhibition rates of 35.4%, 73.2%, 55.6%, 74.4%, 32.0%, 36.9%, 88.0%, and 91.1%, respectively, which were comparable with or even better than that of the positive control, acarbose. Together, our results illustrate the potential of discovering new marine-based therapeutic agents against inflammation and diabetes mellitus.     

Isolation,Structural Characterization and Antidiabetic Activity of New Diketopiperazine Alkaloids from Mangrove Endophytic Fungus Aspergillus sp. 16-5c

Six new DIKETOPIPERAZINE alkaloids aspergiamides A–F (1–6), together with ten known alkaloids (7–16), were isolated from the mangrove endophytic fungus Aspergillus sp. 16-5c. The structures of the new compounds were elucidated based on 1D/2D NMR spectroscopic and HR-ESIMS data analyses. The absolute configurations of aspergiamides A-F were established based on the experimental and calculated ECD data. All the compounds were evaluated for the antidiabetic activity against α-glucosidase and PTP1B enzyme. The bioassay results disclosed compounds 1 and 9 exhibited significant α-glucosidase inhibitory with IC₅₀ values of 18.2 and 7.6 μM, respectively; compounds 3, 10, 11, and 15 exhibited moderate α-glucosidase inhibition with IC₅₀ values ranging from 40.7 to 83.9 μM; while no compounds showed obvious PTP1B enzyme inhibition activity.     

Profiling of the Molecular Weight and Structural Isomer Abundance of Macroalgae-Derived Phlorotannins

Phlorotannins are a group of complex polymers of phloroglucinol (1,3,5-trihydroxybenzene) unique to macroalgae. These phenolic compounds are integral structural components of the cell wall in brown algae, but also play many secondary ecological roles such as protection from UV radiation and defense against grazing. This study employed Ultra Performance Liquid Chromatography (UPLC) with tandem mass spectrometry to investigate isomeric complexity and observed differences in phlorotannins derived from macroalgae harvested off the Irish coast (Fucus serratus, Fucus vesiculosus, Himanthalia elongata and Cystoseira nodicaulis). Antioxidant activity and total phenolic content assays were used as an index for producing phlorotannin fractions, enriched using molecular weight cut-off dialysis with subsequent flash chromatography to profile phlorotannin isomers in these macroalgae. These fractions were profiled using UPLC-MS with multiple reaction monitoring (MRM) and the level of isomerization for specific molecular weight phlorotannins between 3 and 16 monomers were determined. The majority of the low molecular weight (LMW) phlorotannins were found to have a molecular weight range equivalent to 4–12 monomers of phloroglucinol. The level of isomerization within the individual macroalgal species differed, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights. F. vesiculosus had the highest number of isomers of 61 at one specific molecular mass, corresponding to 12 phloroglucinol units (PGUs). These results highlight the complex nature of these extracts and emphasize the challenges involved in structural elucidation of these compounds.     

Total Choline and Choline-Containing Moieties of Commercially Available Pulses

Estimating dietary choline intake can be challenging due to missing foods in the current United States Department of Agriculture (USDA) database. The objectives of the study were to quantify the choline-containing moieties and the total choline content of a variety of pulses available in North America and use the expanded compositional database to determine the potential contribution of pulses to dietary choline intake. Commonly consumed pulses (n?=?32) were analyzed by hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) and compared to the current USDA database. Cooking was found to reduce the relative percent from free choline and increased the contribution of phosphatidylcholine to total choline for most pulses (P?<?0.05). Using the expanded database to estimate choline content of recipes using pulses as meat alternatives, resulted in a different estimation of choline content per serving (±30 %), compared to the USDA database. These results suggest that when pulses are a large part of a meal or diet, the use of accurate food composition data should be used.     

Bioactivity Potential of Marine Natural Products from Scleractinia-Associated Microbes and In Silico Anti-SARS-COV-2 Evaluation

Marine organisms and their associated microbes are rich in diverse chemical leads. With the development of marine biotechnology, a considerable number of research activities are focused on marine bacteria and fungi-derived bioactive compounds. Marine bacteria and fungi are ranked on the top of the hierarchy of all organisms, as they are responsible for producing a wide range of bioactive secondary metabolites with possible pharmaceutical applications. Thus, they have the potential to provide future drugs against challenging diseases, such as cancer, a range of viral diseases, malaria, and inflammation. This review aims at describing the literature on secondary metabolites that have been obtained from Scleractinian-associated organisms including bacteria, fungi, and zooxanthellae, with full coverage of the period from 1982 to 2020, as well as illustrating their biological activities and structure activity relationship (SAR). Moreover, all these compounds were filtered based on ADME analysis to determine their physicochemical properties, and 15 compounds were selected. The selected compounds were virtually investigated for potential inhibition for SARS-CoV-2 targets using molecular docking studies. Promising potential results against SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and methyltransferase (nsp16) are presented.     

Smenamides A and B,Chlorinated Peptide/Polyketide Hybrids Containing a Dolapyrrolidinone Unit from the Caribbean Sponge Smenospongia aurea. Evaluation of Their Role as Leads in Antitumor Drug Research

An in-depth study of the secondary metabolites contained in the Caribbean sponge Smenospongia aurea led to the isolation of smenamide A (1) and B (2), hybrid peptide/polyketide compounds containing a dolapyrrolidinone unit. Their structures were elucidated using high-resolution ESI-MS/MS and homo- and heteronuclear 2D NMR experiments. Structures of smenamides suggested that they are products of the cyanobacterial metabolism, and 16S rRNA metagenomic analysis detected Synechococcus spongiarum as the only cyanobacterium present in S. aurea. Smenamides showed potent cytotoxic activity at nanomolar levels on lung cancer Calu-1 cells, which for compound 1 is exerted through a clear pro-apoptotic mechanism. This makes smenamides promising leads for antitumor drug design.     

An Anti-Inflammatory 2,4-Cyclized-3,4-Secospongian Diterpenoid and Furanoterpene-Related Metabolites of a Marine Sponge Spongia sp. from the Red Sea

Chemical investigation of a Red Sea Spongia sp. led to the isolation of four new compounds, i.e., 17-dehydroxysponalactone (1), a carboxylic acid, spongiafuranic acid A (2), one hydroxamic acid, spongiafuranohydroxamic acid A (3), and a furanyl trinorsesterpenoid 16-epi-irciformonin G (4), along with three known metabolites (−)-sponalisolide B (5), 18-nor- 3,17-dihydroxy-spongia-3,13(16),14-trien-2-one (6), and cholesta-7-ene-3β,5α-diol-6-one (7). The biosynthetic pathway for the molecular skeleton of 1 and related compounds was postulated for the first time. Anti-inflammatory activity of these metabolites to inhibit superoxide anion generation and elastase release in N-formyl-methionyl-leucyl phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophil cells and cytotoxicity of these compounds toward three cancer cell lines and one human dermal fibroblast cell line were assayed. Compound 1 was found to significantly reduce the superoxide anion generation and elastase release at a concentration of 10 μM, and compound 5 was also found to display strong inhibitory activity against superoxide anion generation at the same concentration. Due to the noncytotoxic activity and the potent inhibitory effect toward the superoxide anion generation and elastase release, 1 and 5 can be considered to be promising anti-inflammatory agents.     

Molecular encapsulation of flavours as helical inclusion complexes of amylose

The formation of helical inclusion compounds of amylose with different typical flavour compounds was optimised. Maximal guest molecule contents between 5.7 and 10.4 wt% in the complex were obtained. This corresponds to segments of 8–16 anhydroglucose units in the amylose helix per included guest molecule. Contents of this order of magnitude approach the maximum content of guest molecules possible in the core of the amylose helix. A method for determining the association constants for complexing using headspace gas chromatography was applied. Constants ranging from K=29–1080 l/mol were obtained. The association constant with the same guest molecule is strongly dependent on the origin and the chain length of the amylose.Flavours with low boiling points such as hexanal (b.p. 128 °C) form complexes which are completely stable under freeze-drying conditions and show long-term stability. Flavour release was only observed at high temperatures (80 °C). On addition of water, guest molecules are quickly released but with water activities of a_w=0.3—as found in normal dry food—the complexes are stable at room temperature.Complexes with two or more guest molecules show a remarkable influence on complexing ability induced by the co-inclusion compounds. The overall content of guest molecules is lower compared to that obtained with individual flavour compounds.     

Anti-Chikungunya Viral Activities of Aplysiatoxin-Related Compounds from the Marine Cyanobacterium Trichodesmium erythraeum

Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC₅₀ values of 1.3 μM and 2.7 μM, respectively, and selectivity indices of 10.9 and 9.2, respectively.     

Presence of Caffeic Acid in Flaxseed Lignan Macromolecule

Phenolic compounds were extracted from defatted flaxseeds using ethanol-dioxane (1:1, v/v). The crude extract obtained was purified using Amberlite XAD-16 column chromatography with water and methanol as mobile phases. RP-HPLC and SE-HPLC showed a lignan macromolecule (LM) as a dominant phenolic compound in the purified extract. After the alkaline hydrolysis of LM caffeic acid glucoside (CaAG) was isolated using a semi-preparative HPLC and its structure was confirmed by LC-ESI-MS. In LM of the investigated flaxseed, one molecule of caffeic acid corresponded with five molecules of p-coumaric acid and two molecules of ferulic acid. The presence of caffeic acid in the lignan molecule might be very beneficial due to its high antioxidant activity.     

Fate of polyphenols and antioxidant activity of barley throughout malting and brewing

Phenolic contents of barley and malt extracts and their corresponding antioxidant activities were investigated using a chromatographic online antioxidant detection system. Ethyl acetate extracts of barley and malt were separated using reverse phase HPLC and compounds eluting from the column were submitted to two UV–visible detections: one for the phenolic compounds; and the other for the reduced form of the radical cation 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) after the compounds were allowed to react online with it. Prodelphinidin B3 and procyanidin B3 were identified as two major contributors in the antioxidant activity of barley, in addition to catechin. Malting had a dramatic impact on these three compounds by resulting in a sharp decrease in their detected amounts and the associated antioxidant activities. Two other antioxidants, ferulic and sinapic acids, showed a better ability to withstand not only malting but also brewing steps. As for the overall phenolic content and antioxidant capacity, the study showed that malting allowed a better release and/or extraction of phenolic compounds, while the first brewing step caused the most significant damage by drastically decreasing the total polyphenols and their activity. Hopping however did not significantly affect neither the phenolic content nor the antioxidant activity.