Secondary Metabolic Profiles Research Articles

Activation of secondary metabolite gene clusters in Chaetomium olivaceum via the deletion of a histone deacetylase

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1–A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1–A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources.Key points• Deletion of a class I histone deacetylase activated secondary metabolite gene clusters.• Three polyketides and one asterriquinone were isolated from HDAC deleted strain.• Two different PKSs were reported in C. olivaceum SD-80A.

Secondary metabolic profiling, antioxidant potential, enzyme inhibitory activities and in silico and ADME studies: a multifunctional approach to reveal medicinal and industrial potential of Tanacetum falconeri

Tanacetum falconeri is a significant flowering plant that possesses cytotoxic, insecticidal, antibacterial, and phytotoxic properties. Its chemodiversity and bioactivities, however, have not been thoroughly investigated. In this work, several extracts from various parts of T. falconeri were assessed for their chemical profile, antioxidant activity, and potential for enzyme inhibition. The total phenolic contents of T. falconeri varied from 40.28 ± 0.47 mg GAE/g to 11.92 ± 0.22 mg GAE/g in various extracts, while flavonoid contents were found highest in TFFM (36.79 ± 0.36 mg QE/g extract) and lowest (11.08 ± 0.22 mg QE/g extract) in TFSC (chloroform extract of stem) in similar pattern as found in total phenolic contents. Highest DPPH inhibition was observed for TFFC (49.58 ± 0.11 mg TE/g extract) and TFSM (46.33 ± 0.10 mg TE/g extract), whereas, TFSM was also potentially active against (98.95 ± 0.57 mg TE/g) ABTS radical. In addition, TFSM was also most active in metal reducing assays: CUPRAC (151.76 ± 1.59 mg TE/g extract) and FRAP (101.30 ± 0.32 mg TE/g extract). In phosphomolybdenum assay, the highest activity was found for TFFE (1.71 ± 0.03 mg TE/g extract), TFSM (1.64 ± 0.035 mg TE/g extract), TFSH (1.60 ± 0.033 mg TE/g extract) and TFFH (1.58 ± 0.08 mg TE/g extract), while highest metal chelating activity was recorded for TFSH (25.93 ± 0.79 mg EDTAE/g extract), TFSE (22.90 ± 1.12 mg EDTAE/g extract) and TFSC (19.31 ± 0.50 mg EDTAE/g extract). In biological screening, all extracts had stronger inhibitory capacity against AChE while in case of BChE the chloroform extract of flower (TFFC) and stem (TFSC) showed the highest activities with inhibitory values of 2.57 ± 0.24 and 2.10 ± 0.18 respectively. Similarly, TFFC and TFSC had stronger inhibitory capacity (1.09 ± 0.015 and 1.08 ± 0.002 mmol ACAE/g extract) against α-Amylase and (0.50 ± 0.02 and 0.55 ± 0.02 mmol ACAE/g extract) α-Glucosidase. UHPLC-MS study of methanolic extract revealed the presence of 133 components including sterols, triterpenes, flavonoids, alkaloids, and coumarins. The total phenolic contents were substantially linked with all antioxidant assays in multivariate analysis. These findings were validated by docking investigations, which revealed that the selected compounds exhibited high binding free energy with the enzymes tested. Finally, it was found that T. falconeri is a viable industrial crop with potential use in the production of functional goods and nutraceuticals.

Untargeted Metabolomics to Investigate the Influence of Epigenetic Modifiers on the Metabolism of Fusarium verticillioides.

Toxigenic fungi are capable of producing toxic metabolites, called mycotoxins. But the presence of silent and lowly expressed genes represents the main challenge for the discovery of novel mycotoxins, especially their lesser-known forms, commonly referred to as "emerging mycotoxins." Epigenetic modifiers (EMs) are compounds that are able to alter the production of metabolites through the induction of silent biosynthetic pathways leading to an enhanced chemical diversity. The aim of this study was to assess the effects of different chemical modulators on the metabolic profiles of the well-known toxigenic fungal species, Fusarium verticillioides. Four EMs, 5-azacytidine, sodium butyrate, nicotinamide (NIC), and sodium valproate (SV), were used. Following their addition to Fusarium verticillioides cultures, the metabolic profiles were analyzed by using UHPLC-HRMS/MS under targeted and untargeted metabolomics approaches. Metabolites were putatively annotated through the use of MS-DIAL and MS-FINDER. Our results show that the treatment with SV induced the most important alteration of the secondary metabolic profile of F. verticillioides, by promoting the expression of cryptic genes. Among the 50 most discriminating metabolites across five culture conditions, 12 were fusarins or fusarin analogs. In contrast, SB and NIC had little impact on these metabolites. The study highlights SV's ability to alter gene expression by inhibiting DNA deacetylation in fungal strains. This research could have significant implications for agriculture and food industry, especially in regions facing major mycotoxin challenges.

Fungicidal and plant growth-promoting traits of Lasiodiplodia pseudotheobromae, an endophyte from Andrographis paniculata.

Fungal endophytes are widespread and dwell inside plant cells for at least part of their life without producing any symptoms of infection. Distinct host plants may have different fungal endophyte rates and community compositions. Despite this, the endophytic fungi connected with the host plant and their hostile behaviors, remain unknown. The objective of the current research was to isolate and identify endophytic fungal species from the root of Andrographis paniculata. The effects of fungal isolate APR5 on the mycelial growth of phytopathogens and the production of plant-promoting traits were assessed. Endophytic fungal isolate APR5 showed higher inhibitory efficacy in dual and double plate assay against the tested phytopathogenic fungi. The scanning electron microscope analysis demonstrated that the phytopathogenic fungal hyphae were coiled by endophytes which makes them shrink and disintegrate. Further, an ethyl acetate crude extract effectively suppressed the mycelium growth of Rhizoctonia solani by 75 ± 0.1% in an agar well diffusion assay. The fungal isolate APR5 was identified as Lasiodiplodia pseudotheobromae using the nuclear ribosomal DNA internal transcribed spacer (ITS) region and qualitatively evaluated for their capacity to produce plant growth-promoting hormones. Gas chromatography-mass spectrometry was implemented to acquire a preliminary understanding of the secondary metabolic profile of ethyl acetate crude extract. 1-octadecene, erythritol, niacin, oleic acid, phenol, pantolactone, phenyl ethyl alcohol, p-cresol, and tbutyl hydroquinone are the metabolites analyzed in a crude extract of APR5 isolate and are reported to have antimicrobial properties.

The screening for marine fungal strains with high potential in alkaloids production by in situ colony assay and LC-MS/MS based secondary metabolic profiling.

Alkaloids are the second primary class of secondary metabolites (SMs) from marine organisms, most of which have antioxidant, antitumor, antibacterial, anti-inflammatory, and other activities. However, the SMs obtained by traditional isolation strategies have drawbacks such as highly reduplication and weak bioactivity. Therefore, it is significantly important to establish an efficient strategy for screening strains and mining novel compounds. In this study, we utilized in situ colony assay combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the strain with high potential in alkaloids production. The strain was identified by genetic marker genes and morphological analysis. The secondary metabolites from the strain were isolated by the combine use of vacuum liquid chromatography (VLC), ODS column chromatography, and Sephadex LH-20. Their structures were elucidated by 1D/2D NMR, HR-ESI-MS, and other spectroscopic technologies. Finally, these compounds bioactivity were assay, including anti-inflammatory and anti-β aggregation. Eighteen marine fungi were preliminarily screened for alkaloids production by in situ colony assay using Dragendorff reagent as dye, and nine of them turned orange, which indicated abundant alkaloids. By thin-layer chromatography (TLC), LC-MS/MS, and multiple approaches assisted Feature-Based Molecular Networking (FBMN) analysis of fermentation extracts, a strain ACD-5 (Penicillium mallochii with GenBank accession number OM368350) from sea cucumber gut was selected for its diverse alkaloids profiles especially azaphilones. In bioassays, the crude extracts of ACD-5 in Czapek-dox broth and brown rice medium showed moderate antioxidant, acetylcholinesterase inhibitory, anti-neuroinflammatory, and anti-β aggregation activities. Three chlorinated azaphilone alkaloids, compounds 1-3 (sclerotioramine, isochromophilone VI, and isochromophilone IX, respectively), were isolated from the fermentation products of ACD-5 in brown rice medium guided by bioactivities and mass spectrometry analysis. Compound 1 had shown remarkable anti-neuroinflammatory activity in liposaccharide induced BV-2 cells. In summary, in situ colony screening together with LC-MS/MS, multi-approach assisted FBMN can act as an efficient screening method for strains with potential in alkaloids production.

The roots of olive cultivars differing in tolerance to Verticillium dahliae show quantitative differences in phenolic and triterpenic profiles

ABSTRACT Verticillium wilt of olive (VWO), caused by Verticillium dahliae, is a major concern in many olive-growing countries. An efficient VWO control measure is the use of tolerant/resistant cultivars. Low information is available about olive secondary metabolites and its relationship with VWO tolerance. In this study, a comprehensive metabolic profiling of the roots of six olive cultivars differing in their level of tolerance/susceptibility to VWO was addressed. Potential changes in the metabolite profiles due to the presence of the pathogen were also assessed. A strong relationship between the quantitative basal composition of the root secondary metabolic profile and VWO tolerance/susceptibility of olive varieties was found. Tolerant cultivars showed higher content of secoiridoids, while the susceptible ones presented greater amounts of verbascoside and methoxypinoresinol glucoside. The presence of V. dahliae only caused few significant variations mostly restricted to the earliest times after inoculation. Thus, a rapid activation of biochemical-based root defense mechanisms was observed. Key policy highlights Quantitative differences of secondary metabolites in roots contribute to explain the tolerance/susceptibility of olive cultivars to Verticillium dahliae. Higher basal content of secoiridoids correlate with tolerance, while greater concentration of verbascoside and methoxypinoresinol glucoside seem to be linked to susceptibility. Few alterations are observed in the olive root metabolic profiles in the presence of the pathogen. Changes in the root metabolic profile occur at early times after pathogen inoculation which suggests a rapid activation of a biochemical-based defense response against V. dahliae.

Deciphering the antimicrobial activity of multifaceted rhizospheric biocontrol agents of solanaceous crops viz., Trichoderma harzianum MC2, and Trichoderma harzianum NBG.

The Solanaceae family is generally known to be the third most economically important plant taxon, but also harbors a host of plant pathogens. Diseases like wilt and fruit rot of solanaceous crops cause huge yield losses in the field as well as in storage. In the present study, eight isolates of Trichoderma spp. were obtained from rhizospheric micro-flora of three solanaceous crops: tomato, brinjal, and chili plants, and were subsequently screened for pre-eminent biocontrol activity against three fungal (Fusarium oxysporum f. sp. lycopersicum, Colletotrichum gloeosporioides, and Rhizoctonia solani) and one bacterial (Ralstonia solanacearum) pathogen. Morphological, ITS, and tef1α marker-based molecular identification revealed eight isolates were different strains of Trichoderma. Seven isolates were distinguished as T. harzianum while one was identified as T. asperellum. In vitro antagonistic and biochemical assays indicated significant biocontrol activity governed by all eight isolates. Two fungal isolates, T. harzianum MC2 and T. harzianum NBG were further evaluated to decipher their best biological control activity. Preliminary insights into the secondary metabolic profile of both isolates were retrieved by liquid chromatography-mass spectrometry (LC-MS). Further, a field experiment was conducted with the isolates T. harzianum MC2 and T. harzianum NBG which successfully resulted in suppression of bacterial wilt disease in tomato. Which possibly confer biocontrol properties to the identified isolates. The efficacy of these two strains in suppressing bacterial wilt and promoting plant growth in the tomato crop was also tested in the field. The disease incidence was significantly reduced by 47.50% and yield incremented by 54.49% in plants treated in combination with both the bioagents. The results of scanning electron microscopy were also in consensus with the in planta results. The results altogether prove that T. harzianum MC2 and T. harzianum NBG are promising microbes for their prospective use in agricultural biopesticide formulations.

Secondary metabolic profiling of Serratia marcescens NP10 reveals new stephensiolides and glucosamine derivatives with bacterial membrane activity

Secondary metabolic profiling, using UPLC-MSE and molecular networking, revealed the secondary metabolites produced by Serratia marcescens NP10. The NP10 strain co-produced cyclic and open-ring stephensiolides (i.e., fatty acyl chain linked to Thr–Ser–Ser–Ile/Leu–Ile/Leu/Val) and glucosamine derivatives (i.e., fatty acyl chain linked to Val–glucose–butyric/oxo-hexanoic acid), with the structures of sixteen new stephensiolides (L–Y) and three new glucosamine derivatives (L–N) proposed. Genome mining identified sphA (stephensiolides) and gcd (glucosamine derivatives) gene clusters within Serratia genomes available on NBCI using antiSMASH, revealing specificity scores of the adenylation-domains within each module that corroborates MSE data. Of the nine RP-HPLC fractions, two stephensiolides and two glucosamine derivatives exhibited activity against Staphylococcus aureus (IC50 of 25–79 µg/mL). 1H NMR analysis confirmed the structure of the four active compounds as stephensiolide K, a novel analogue stephensiolide U, and glucosamine derivatives A and C. Stephensiolides K and U were found to cause membrane depolarisation and affect the membrane permeability of S. aureus, while glucosamine derivatives A and C primarily caused membrane depolarisation. New members of the stephensiolide and glucosamine derivative families were thus identified, and results obtained shed light on their antibacterial properties and mode of membrane activity.

Metabolomic Strategy to Characterize the Profile of Secondary Metabolites in Aspergillus aculeatus DL1011 Regulated by Chemical Epigenetic Agents.

Chemical epigenetic regulation (CER) is an effective method to activate the silent pathway of fungal secondary metabolite synthesis. However, conventional methods for CER study are laborious and time-consuming. In the meantime, the overall profile of the secondary metabolites in the fungi treated by the CER reagent is not well characterized. In this study, suberohydroxamic acid (SBHA), a histone deacetylase inhibitor, was added to a culture of Aspergillus aculeatus DL1011 and a new strategy based on LC-MS/MS analysis integrated with various metabolomic tools (MetaboAnalyst, MS-DIAL, SIRIUS and GNPS) was developed to characterize the profile of induced metabolites. As a result, 13.6%, 29.5% and 27.2% of metabolites were identified as newly biosynthesized, increasing and decreasing in abundance by CER, respectively. The structures of the 18 newly induced secondary metabolites were further identified by the new strategy to demonstrate that 72.2% of them (1 novel compound and 12 known compounds) were first discovered in A. aculeatus upon SBHA treatment. The accuracy of the new approach was confirmed by purification and NMR data analysis of major newly biosynthesized secondary metabolites. The bioassay showed that the newly biosynthesized compounds, roseopurpurin analogues, showed selective activities against DPPH scavenging, cytotoxicity and SHP1 inhibition. Our research demonstrated that CER was beneficial for changing the secondary metabolic profile of fungi and was an effective means of increasing the diversity of active metabolites. Our work also supplied a metabolomic strategy to characterize the profile changes and determine the newly induced compounds in the secondary metabolites of fungi treated with the chemical epigenetic regulator.

Secondary metabolic profile of rose-scented geranium: A tool for characterization, distinction and quality control of Indian genotypes

Rose-scented geranium ( Pelargonium graveolens L'Herit. ex Aiton), belonging to the family Geraniaceae , has gained considerable attention because of its fragrance and high economic value. The compositional complexity of rose-scented geranium essential oil (GEO) has been challenging for quality control. In this study, the chemical profiles of GEOs extracted from Indian cultivars/genotypes were developed using polarity-based fractionation, followed by characterization using a combination of gas chromatography (GC-FID), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy (NMR). The analysis led to the identification of 121 compounds. Major components of the GEOs were citronellol (22.4–42.2 %), geraniol (5.7–30.5 %), linalool (2.1–9.8 %), citronellyl formate (4.0–9.5 %), isomenthone (5.3–7.5 %), geranyl formate (2.0–5.1 %), 6,9-guaiadiene (0.0–4.3 %), 10- epi - γ -eudesmol (0.2–6.4 %), 2-phenyl ethyl tiglate (1.0–2.1 %), geranyl tiglate (1.4–2.7 %), germacrene D (0.8–1.7 %) and geranial (0.4–1.6 %). This study identified effective markers which could help determine the authenticity of Indian cultivars/genotypes. Physical properties and chromatographic profiles of GEOs and terpeneless GEOs (TLOs) derived from Indian cultivar/genotype were compared. The two-step chromatographic separation coupled with principal component analysis allowed the identification of possible markers. The presence or absence of the sesquiterpenes, namely 10- epi - γ -eudesmol, 6,9-guaiadiene, and some other minor constituents, physical, and organoleptic properties , could effectively distinguish and define the quality of Indian cultivars/genotypes. Besides, the GEOs and TLOs prepared from different cultivars exhibited significant antimicrobial activity against Staphylococcus aureus and Candida albicans . • Rose-scented geranium essential oil is known for its chemical complexity. • Chemical characterization and quality control of Indian genotypes were done. • Fractionation followed by GC-MS analysis led to the separation of co-eluting components. • The compounds identified could be viable tools for genotypes authentication. • Indian genotypes meet international standards.

Exploring the industrial importance of a miracle herb Withania somnifera (L.) Dunal: Authentication through chemical profiling, in vitro studies and computational analyses

Withania somnifera (L.) Dunal has been identified as a miracle herb, thus the present study was carried out to investigate W. somnifera growing in Cholistan Desert for its secondary metabolic profile and various biological activities. The WS-C fraction of methanolic extract (WS-M) of W. somnifera was found rich in phenolics (39.96 ± 0.90 mg GAE/g extract), and it exhibited significant DPPH (30.69 ± 0.78 mg TE/g extract) and ABTS (113.60 ± 2.41 mg TE/g extract) free radical inhibitory activities, cupric (Cu+2) and ferric (Fe+3) reducing potential as 157.51 ± 2.10 and 124.60 ± 0.34 mg TE/g extract. The same fraction also exhibited strong total antioxidant capacity (1.39 ± 0.01 mmol TE/g extract) in phosphomolybdenum assay. Other fractions displayed relatively lower potential. In metal-chelating assay, WS-M, WS-E and WS-B showed nearly equal potential with values of 22.35 ± 0.62, 20.96 ± 0.12 and 22.10 ± 0.75 mg EDTAE/g of the extract, while in AChE, BChE and tyrosinase inhibitory assay, again WS-C fraction was most active (7.50 ± 0.21, 8.01 ± 0.53 mg GALAE/g extract and 36.81 ± 0.20 mg KAE/g extract, respectively). In α-amylase and α-glucosidase inhibitory assay, WS-C showed significant potential with values as 0.74 ± 0.01 and 1.14 ± 0.004 mmol ACAE/g extract, respectively. UHPLC-MS analysis of WS-C fraction leads to identify 100 secondary metabolites of phenolic, withanolide, terpenoid, steroid, lignin, flavonoid and limonoid classes of compounds. This profile makes W. somnifera a fascinating plant. These findings were validated by computational studies, which revealed that selected compounds exhibited high binding free energy and inhibition constants with the enzymes tested. Pearson correlation analysis clearly established that the phenols in the tested extracts were the main players as antioxidants and enzyme inhibitors, which was also substantiated through Principal Component Analysis (PCA). These results suggest that chloroform might be suitable for preparing further applications with W. somnifera extracts, and this plant can be placed in the list of top-selling herbs, and may become source of commercial assets.

Metabolite profiling of Bruguiera cylindrica reveals presence of potential bioactive compounds

Bruguiera cylindrica parts are commonly used in Chinese and Indian traditional medicine to treat diarrhea, fever, and many ailments. The present study aims non targeted analysis of key secondary metabolites of B. cylindrica by gas chromatography mass spectrometry (GC-MS) and ultra-high performance liquid chromatography hybrid quadrupole-Exactive-Orbitrap high resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS). GC-MS and UHPLC-Q-Exactive Orbitrap HRMS were utilized for metabolic profiling of ethyl acetate extract of B. cylindrica leaves. Key metabolites in the extract were identified and predicted based on chemical similarity using online databases such as ChemSpider and mzCloud. Thirty-six compounds belonging to different classes of secondary metabolites viz. flavonoids, fatty acids, fatty acid amides, carboxylic acids, and alkaloids were identified in the extract. Pentacyclic triterpenes like betulin, ursolic acid and a tropine, an alkaloid with potential pharmacological and therapeutic activities such as anticancer properties, neuromuscular blockers and antioxidants, were also identified. This study combined GC-MS and UHPLC-Q-Exactive Orbitrap HRMS with available online database for effective and rapid identification of bioactive metabolites in the ethyl acetate extract of mangrove without individual standard application. This is the first report on the HRMS based secondary metabolic profiling of B. cylindrica, with comprehensive map of its biologically important metabolites.

Soil dynamics modulation of stress enzymes and secondary metabolites in allium cepa (L.) under salinity stress

In Allium cepa (L.) cultivation, salinity stress is the most important environmental factor worldwide that affects plant growth, protection as well as yield stability. The present study was carried out to examine the alterations in soil dynamics, antioxidants, biochemical and secondary metabolic profile of A. cepa under alkaline stress. Various parameters of A. cepa were studied under salinity stress by exposing them to different concentrations of sodium carbonate (SC) and sodium bicarbonate (SBC) salts (0, 10 and 100 mM). Treatment of both SC and SBC in A. cepa increases superoxide dismutase, glutathione peroxidase and catalase concentrations. These were attributed to increase in peroxide, lipid peroxides as well as carbonyl groups. The salinity stress was driven by the alterations in the soil dynamics. Of note, the salt stress induces secondary metabolites changes in A. cepa. The compounds like phenol 2 4-bis (1 1-dimethylethyl), hexadecane, 7,9-di‑tert‑butyl‑1-oxaspiro(4,5) deca-6,9-diene-2,8‑dione, 1,2-benzenedicarboxylic acid, butyl octyl ester, 1,2-benzenedicarboxylic acid, diisooctyl ester are elevated. In addition, the level of total proteins is reduced in all the groups except 10 mM and Proline amino acid in the shoot is elevated. Reversing all the parameters tested to achieve control levels in A. cepa indicates that significant cellular mechanism appeared in stress condition that brings back the altered parameters to normal levels. The biochemical and secondary metabolite profile analysis of the petroleum ether extracts of A. cepa under various alkaline stress conditions reveals that the composition of secondary metabolites and their concentration in A. cepa varies depending on the extent of alkaline stress.

Growth and metabolism of basil grown in a new-concept microcosm under different lighting conditions

To simulate a typical market-oriented cultivation in laboratory, plants of basil (Ocimum basilicum L.) were grown from seedling to flowering stages in a new-concept microcosm device that enables roots and aerial parts to grow as under real crop conditions. To test the device efficacy, two microcosms were used with the same lighting architecture, temperature and photoperiodic conditions and with two different light spectra, white (W) or blue-red (BR), displaying a similar spectral power in the blue region. Plant growth, biomass yield, photosynthetic efficiency and nutrient uptake were determined. An innovative analytical approach for secondary metabolic profile was also developed to determine basil quality. The plants grew vigorous and healthy for the whole cultivation period and under both the lighting regimes, giving a biomass yield similar to those of basil grown under conventional greenhouse and field conditions. The two lighting regimes differently affected plant growth and yield, with the BR light, that was characterized by a higher photosynthetic photon flux density (PPFD), associated to higher plants, earlier flowering and greater yield. In the average, fresh and dry aerial biomasses per plant were about 250 g and 41 g under BR and about 114 g and 9 g under W light. Higher concentrations of major nutrients were detected in plants under W light, thus indicating that yield levels and major nutrient concentrations are not necessarily related to each other. Similar Fv/Fm (0.76–0.78) and ETR values were observed under the two light regimes, possibly indicating that plants under long lasting cultivation can adapt to different light regimes to reach similar photosynthetic efficiency levels. Different secondary metabolic profiles were detected in tissues sampled at the end of cultivation period and previously unreported profiles for basil were also recorded, possibly indicating that the extent of plant growth affects secondary metabolism in basil, in addition to light spectrum and PPFD level. This is the first report of basil grown under microcosm conditions from seedlings to adult plants. Our results indicate that the microcosm based-technology is effective in simulating a typical market-oriented cultivation and that long lasting cultivation emphasizes the effects of different environmental conditions on plant growth and metabolism.

Secondary metabolic profiles and anticancer actions from fruit extracts of immature pomegranates.

Immature fruits from Punica granatum L. thinning are a neglected side product of pomegranate production with cumbersome disposal costs for farmers. To explore value potential of immature fruits from pomegranate ‘Wonderful’ cultivars, the compositional landscapes and antitumorigenic activities of pomegranate extracts from two different stages of maturation were assessed. Cancer cell proliferation and cytotoxicity was quantified in human lung H1299 and colon HCT116 adenocarcinomas by crystal violet staining, MTS assay and caspase-3 activity. High performance liquid chromatography—diode array detector (HPLC/DAD) and high performance liquid chromatography—electrospray ionization—mass spectrometry (HPLC/ESI-MS) analyses indicate that immature fruits are rich sources of gallotannins and ellagitannins, with the highest amounts contained in immature fruit peels. Biological investigations reveal a robust anticancer activity by those immature P. granatum fruit extracts, which reflected induction of tumor cytotoxicity and cell death mechanisms. Together, present observations suggest P. granatum byproducts from the thinning process may provide unexplored values for virtuous circular economy.

Salinity Stress Alters the Secondary Metabolic Profile of M. sativa, M. arborea and Their Hybrid (Alborea).

Increased soil salinity, and therefore accumulation of ions, is one of the major abiotic stresses of cultivated plants that negatively affect their growth and yield. Among Medicago species, only Medicago truncatula, which is a model plant, has been extensively studied, while research regarding salinity responses of two important forage legumes of Medicago sativa (M. sativa) and Medicago arborea (M. arborea) has been limited. In the present work, differences between M. arborea, M. sativa and their hybrid Alborea were studied regarding growth parameters and metabolomic responses. The entries were subjected to three different treatments: (1) no NaCl application (control plants), (2) continuous application of 100 mM NaCl (acute stress) and (3) gradual application of NaCl at concentrations of 50-75-150 mM by increasing NaCl concentration every 10 days. According to the results, M. arborea maintained steady growth in all three treatments and appeared to be more resistant to salinity. Furthermore, results clearly demonstrated that M. arborea presented a different metabolic profile from that of M. sativa and their hybrid. In general, it was found that under acute and gradual stress, M. sativa overexpressed saponins in the shoots while M. arborea overexpressed saponins in the roots, which is the part of the plant where most of the saponins are produced and overexpressed. Alborea did not perform well, as more metabolites were downregulated than upregulated when subjected to salinity stress. Finally, saponins and hydroxycinnamic acids were key players of increased salinity tolerance.

A Metabolomics and Molecular Networking Approach to Elucidate the Structures of Secondary Metabolites Produced by Serratia marcescens Strains.

An integrated approach that combines reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry, untargeted ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MSE) and molecular networking (using the Global Natural Products Social molecular network platform) was used to elucidate the metabolic profiles and chemical structures of the secondary metabolites produced by pigmented (P1) and non-pigmented (NP1) Serratia marcescens (S. marcescens) strains. Tandem mass spectrometry-based molecular networking guided the structural elucidation of 18 compounds for the P1 strain (including 6 serratamolides, 10 glucosamine derivatives, prodigiosin and serratiochelin A) and 15 compounds for the NP1 strain (including 8 serratamolides, 6 glucosamine derivatives and serratiochelin A) using the MSE fragmentation profiles. The serratamolide homologues were comprised of a peptide moiety of two L-serine residues (cyclic or open-ring) linked to two fatty acid chains (lengths of C10, C12, or C12:1). Moreover, the putative structure of a novel open-ring serratamolide homologue was described. The glucosamine derivative homologues (i.e., N-butylglucosamine ester derivatives) consisted of four residues, including glucose/hexose, valine, a fatty acid chain (lengths of C13 – C17 and varying from saturated to unsaturated) and butyric acid. The putative structures of seven novel glucosamine derivative homologues and one glucosamine derivative congener (containing an oxo-hexanoic acid residue instead of a butyric acid residue) were described. Moreover, seven fractions collected during RP-HPLC, with major molecular ions corresponding to prodigiosin, serratamolides (A, B, and C), and glucosamine derivatives (A, C, and E), displayed antimicrobial activity against a clinical Enterococcus faecalis S1 strain using the disc diffusion assay. The minimum inhibitory and bactericidal concentration assays however, revealed that prodigiosin exhibited the greatest antimicrobial potency, followed by glucosamine derivative A and then the serratamolides (A, B, and C). These results provide crucial insight into the secondary metabolic profiles of pigmented and non-pigmented S. marcescens strains and confirms that S. marcescens strains are a promising natural source of novel antimicrobial metabolites.

Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State, Brazil.

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography–mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5–90.9%) in the cultivars “Cingapura”, “Equador”, “Guajarina”, “Iaçará”, and “Kottanadan”, and “Bragantina”, “Clonada”, and “Uthirankota” displayed oxygenated sesquiterpenoids (50.6–75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene (“Equador”/“Guajarina”, I); δ-elemene (“Iaçará”/“Kottanadan”/“Cingapura”, II); elemol (“Clonada”/“Uthirankota”, III) and α-muurolol, bicyclogermacrene, and cubebol (“Bragantina”, IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, β-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75–140.53 mg GAE·g−1 FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19–57.22 µU·mL−1), and carotenoids (0.21–2.31 µg·mL−1) displayed significant variations. Due to black pepper’s susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar’s volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.

Enhanced Production of Aromatic Amino Acids in Tobacco Plants Leads to Increased Phenylpropanoid Metabolites and Tolerance to Stresses.

Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precursors for a wide range of secondary metabolites that are important for plant defense. The goals of the current study were to test the effect of increased AAAs on primary and secondary metabolic profiles and to reveal whether these plants are more tolerant to abiotic stresses (oxidative, drought and salt) and to Phelipanche egyptiaca (Egyptian broomrape), an obligate parasitic plant. To this end, tobacco (Nicotiana tabacum) plants were transformed with a bacterial gene (AroG) encode to feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. Two sets of transgenic plants were obtained: the first had low expression of the AroG protein, a normal phenotype and minor metabolic changes; the second had high accumulation of the AroG protein with normal, or deleterious morphological changes having a dramatic shift in plant metabolism. Metabolic profiling analysis revealed that the leaves of the transgenic plants had increased levels of phenylalanine (up to 43-fold), tyrosine (up to 24-fold) and tryptophan (up to 10-fold) compared to control plants having an empty vector (EV) and wild type (WT) plants. The significant increase in phenylalanine was accompanied by higher levels of metabolites that belong to the phenylpropanoid pathway. AroG plants showed improved tolerance to salt stress but not to oxidative or drought stress. The most significant improved tolerance was to P. aegyptiaca. Unlike WT/EV plants that were heavily infected by the parasite, the transgenic AroG plants strongly inhibited P. aegyptiaca development, and only a few stems of the parasite appeared above the soil. This delayed development of P. aegyptiaca could be the result of higher accumulation of several phenylpropanoids in the transgenic AroG plants and in P. aegyptiaca, that apparently affected its growth. These findings indicate that high levels of AAAs and their related metabolites have the potential of controlling the development of parasitic plants.

Comparison of Secondary Metabolite Contents and Metabolic Profiles of Six Lycoris Species

Quantitative HPLC analysis was performed on six different species of Lycoris herbs to investigate variation in phytochemical content, especially galantamine and phenylpropanoid-derived compounds. The contents of these compounds differed widely among the Lycoris species, with L. radiata and L. chinensis containing the lowest and highest galantamine contents, respectively. Specifically, the galantamine content of L. radiata was 62.5% higher than that of L. chinensis. Following L. radiata, L. sanguinea contained the next highest galantamine content, which was 59.1% higher than that of L. chinensis. Furthermore, a total of 12 phenylpropanoid-derived compounds were found in the different Lycoris species, where L. sanguinea, L. squamigera, and L. uydoensis had the largest accumulation of these compounds. The total phenylpropanoid content of L. sanguinea was the highest, while that of L. radiata was the lowest. Seven of the phenylpropanoid-derived compounds, rutin, quercetin, catechin, epicatechin gallate, chlorogenic acid, benzoic acid, and kaempferol, were dominant. L. sanguinea, L. uydoensis, and L. squamigera showed amounts of these seven compounds that were 5–6 times greater than those of the other species in the study. To the best of our knowledge, our results provide the most detailed phytochemical information on these species to date, which is valuable for future applications using these medicinal plants.