Metabolome Profiling of Marrubium peregrinum L. and Marrubium friwaldskyanum Boiss Reveals Their Potential as Sources of Plant-Based Pharmaceuticals.

Marrubium species have been used since ancient times as food additives and curative treatments. Their phytochemical composition and various pharmacological activities were the focus of a number of scientific investigations but no comprehensive metabolome profiling to identify the numerous primary and secondary metabolites has been performed so far. This study aimed to generate a comprehensive picture of the total metabolite content of two Marrubium species-M. peregrinum and M. friwaldskyanum-to provide detailed information about the main primary and secondary metabolites. In addition, the elemental composition was also evaluated. For this purpose, non-targeted metabolomic analyses were conducted using GC-MS, UPLC-MS/MS and ICP-MS approaches. Nearly 500 compounds and 12 elements were detected and described. The results showed a strong presence of phenolic acids, flavonoids and their glucosides, which are generally of great interest due to their various pharmacological activities. Furthermore, tissue-specific analyses for M. friwaldskyanum stem, leaves and flowers were carried out in order to outline the sources of potentially important bioactive molecules. The results generated from this study depict the Marrubium metabolome and reveal its dual scientific importance-from one side, providing information about the metabolites that is fundamental and vital for the survival of these species, and from the other side, defining the large diversity of secondary substances that are a potential source of phytotherapeutic agents.

Genomic, transcriptomic, and metabolomic analysis of Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites.

Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.

Identification of a Hydroxygallic Acid Derivative, Zingibroside R1 and a Sterol Lipid as Potential Active Ingredients of Cuscuta chinensis Extract That Has Neuroprotective and Antioxidant Effects in Aged Caenorhabditis elegans.

We examined the effects of the extracts from two traditional Chinese medicine plants, Cuscuta chinensis and Eucommia ulmoides, on the healthspan of the model organism Caenorhabditis elegans. C. chinensis increased the short-term memory and the mechanosensory response of aged C. elegans. Furthermore, both extracts improved the resistance towards oxidative stress, and decreased the intracellular level of reactive oxygen species. Chemical analyses of the extracts revealed the presence of several bioactive compounds such as chlorogenic acid, cinnamic acid, and quercetin. A fraction from the C. chinensis extract enriched in zingibroside R1 improved the lifespan, the survival after heat stress, and the locomotion in a manner similar to the full C. chinensis extract. Thus, zingibroside R1 could be (partly) responsible for the observed health benefits of C. chinensis. Furthermore, a hydroxygallic acid derivative and the sterol lipid 4-alpha-formyl-stigmasta-7,24(241)-dien-3-beta-ol are abundantly present in the C. chinensis extract and its most bioactive fraction, but hardly in E. ulmoides, making them good candidates to explain the overall healthspan benefits of C. chinensis compared to the specific positive effects on stress resistance by E. ulmoides. Our findings highlight the overall anti-aging effects of C. chinensis in C. elegans and provide first hints about the components responsible for these effects.

Metabolomics-based profiling for quality assessment and revealing the impact of drying of Turmeric (Curcuma longa L.).

Turmeric, the rhizomes of Curcuma longa L., is one of the top selling spices, food preservatives, and food colorants. In addition, it exhibits health promoting benefits owing to its unique phytochemical composition. Nevertheless, it is commonly subjected to heat drying, hence, the dried powder is the most used form and can easily be adulterated with allied species. Therefore, our research aimed to profile the phytochemical composition and investigate the impact of drying of turmeric. Extraction and fractionation followed by LC- and GC-MS analysis resulted in the identification of a total of 161 metabolites belonged to various phytochemical classes. Moreover, multivariate data analysis identified curcuminoids, terpecurcumins, and organic acids as potential markers for drying. Based on the applied analytical techniques in combination with chemometrics, these investigations have succeeded to provide good coverage of the metabolome of turmeric in both fresh and dried forms.

Indication that starch and sucrose are biomarkers for oil yield in oil palm (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis Jacq.) is the most productive oil-producing crop per hectare of land. The oil that accumulates in the mesocarp tissue of the fruit is the highest observed among fruit-producing plants. A comparative analysis between high-, medium-, and low-yielding oil palms, particularly during fruit development, revealed unique characteristics. Metabolomics analysis was able to distinguish accumulation patterns defining of the various developmental stages and oil yield. Interestingly, high- and medium-yielding oil palms exhibited substantially increased sucrose levels compared to low-yielding palms. In addition, parameters such as starch granule morphology, granule size, total starch content, and starch chain length distribution (CLD) differed significantly among the oil yield categories with a clear correlation between oil yield and various starch parameters. These results provide new insights into carbohydrate and starch metabolism for biosynthesis of oil palm fruits, indicating that starch and sucrose can be used as novel, easy-to-analyze, and reliable biomarker for oil yield.

A Solanum lycopersicoides reference genome facilitates insights into tomato specialized metabolism and immunity.

Wild relatives of tomato are a valuable source of natural variation in tomato breeding, as many can be hybridized to the cultivated species (Solanum lycopersicum). Several, including Solanum lycopersicoides, have been crossed to S. lycopersicum for the development of ordered introgression lines (ILs), facilitating breeding for desirable traits. Despite the utility of these wild relatives and their associated ILs, few finished genome sequences have been produced to aid genetic and genomic studies. Here we report a chromosome-scale genome assembly for S. lycopersicoides LA2951, which contains 37 938 predicted protein-coding genes. With the aid of this genome assembly, we have precisely delimited the boundaries of the S. lycopersicoides introgressions in a set of S. lycopersicum cv. VF36 × LA2951 ILs. We demonstrate the usefulness of the LA2951 genome by identifying several quantitative trait loci for phenolics and carotenoids, including underlying candidate genes, and by investigating the genome organization and immunity-associated function of the clustered Pto gene family. In addition, syntenic analysis of R2R3MYB genes sheds light on the identity of the Aubergine locus underlying anthocyanin production. The genome sequence and IL map provide valuable resources for studying fruit nutrient/quality traits, pathogen resistance, and environmental stress tolerance. We present a new genome resource for the wild species S. lycopersicoides, which we use to shed light on the Aubergine locus responsible for anthocyanin production. We also provide IL boundary mappings, which facilitated identifying novel carotenoid quantitative trait loci of which one was likely driven by an uncharacterized lycopene ß-cyclase whose function we demonstrate.

Potential Valorization of Edible Nuts By-Products: Exploring the Immune-Modulatory and Antioxidants Effects of Selected Nut Shells Extracts in Relation to Their Metabolic Profiles.

The immune system is a potent army that defends our body against various infections and diseases through innate and adaptive immunity. Herbal medicine is one of the essential sources for enhancing immunity because of affordability, availability, minor side effects, and consumers' preferences. Hazelnuts, walnuts, almonds, and peanuts are among the most widespread edible nuts that are rich in phenolics, fats, fibers, vitamins, proteins, and minerals. The potential of nut shells in phytoremediation has attracted increasing attention as a sustainable solution for waste recycling. Here, we determined the in vitro immune-modulatory activity as well as the metabolite profile of the four nut shell extracts. The addition of the extracts to LPS-stimulated macrophages, especially peanut and walnut shells, has downregulated the gene expression of AP-1, TNF-α, IL-8, iNOS, and COX-2 expression levels. Significant antioxidant capabilities and immune-modulatory effects have been traced for peanut shells. UPLC-MS metabolic profiling of the four nut shell extracts allowed the detection of a relatively high level of phenolic compounds in peanut shells. Intriguingly, a significant correlation between the antioxidant capacity and the total phenolic content was found, indicating the contribution of the phenolic compounds to the antioxidant properties and hence the immune-modulatory activity. Furthermore, molecular docking and structure-activity relationship (SAR) studies revealed kaempferol rutinoside and proanthocyanidin A5' as potential iNOS inhibitors.

A Comparative Study of the Antihypertensive and Cardioprotective Potentials of Hot and Cold Aqueous Extracts of Hibiscus sabdariffa L. in Relation to Their Metabolic Profiles.

Ethnopharmacological relevance: Since ancient times, Hibiscus sabdariffa L. calyces have been used as a folk remedy for the treatment of hypertension. However, it is questionable as to whether there is a difference in the antihypertensive activity of the hot or cold aqueous extracts. Aim of the study: We designed this study to specify the best method for water extraction of the antihypertensive metabolites of H. sabdariffa and to confirm their in vivo antihypertensive capabilities. Materials and methods: The powdered dried calyces of H. sabdariffa were independently extracted with cold and hot water. A comparative study was performed between the cold and hot aqueous extracts of H. sabdariffa based on evaluation of the in vitro renin and angiotensin-converting enzyme (ACE) inhibition activities. Additionally, both extracts were subjected to an in vivo study for the evaluation of their antihypertensive activities in L-Nw-Nitro arginine methyl ester (L-NAME)-induced hypertensive rats. Further, a metabolomics study was also performed for both extracts to identify their chemical constituents. Results: The cold and hot extracts significantly reduced the angiotensin II, ACE, and aldosterone levels in the plasma. Furthermore, in the myocardium and aorta, decreased iNOS (inducible nitric oxide synthase) levels and elevated eNOS (endothelial nitric oxide synthase), as well as the rise in plasma NO levels, were reported with both extracts, but better results were displayed with the hot extract, leading to a potential antihypertensive effect. Additionally, the cold and hot Hibiscus extracts induced a cardioprotective effect through reducing necrosis, inflammation, and vacuolization that results from the induction of hypertension, an effect that was more prominent with the hot extract. Moreover, a comprehensive metabolomics approach using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was able to trace the metabolites in each extraction. Conclusion: The extracts showed different anthocyanin and phenolic compounds, but the hot extract showed higher contents of specific phenolics to which the superior antihypertensive and cardioprotective activities could be related.

The integration of MS-based metabolomics and multivariate data analysis allows for improved quality assessment of Zingiber officinale Roscoe.

Ginger (Zingiber officinale Roscoe) is consumed for health-promoting effects and as a food condiment. Comprehensive phytochemical analysis, other than gingerols and shogaols, has not yet been deeply investigated. Hence, the current research aimed to establish a non-targeted metabolomics approach for the discrimination between fresh ginger rhizome samples collected from four different producing countries, i.e., China, India, Pakistan, and Peru. In addition, lab-dried samples were analyzed to trace drying-induced metabolites. A comprehensive extraction procedure was carried out resulting in production of polar and non-polar fractions. The polar fraction was analyzed by ultra-performance liquid chromatography coupled with Fourier transform tandem mass spectrometry (UPLC-C18-FT-MS/MS) and gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) post derivatization. UPLC-C8-FT-MS/MS was used for analysis of non-polar fraction. Results revealed for identification of a total of 253 metabolites. In addition, multivariate data analysis (MVDA), including principal component analysis (PCA) demonstrated clustering of Asian specimens. Several metabolites with a characteristic pattern for the origin revealing the highest contents of bioactive metabolites in the Peruvian product. Moreover, chemical markers identified, including [6]-gingerol and [6]-shogaol discriminating between fresh and dried samples. Furthermore, abundances of some primary metabolites, including amino acids and cinnamic acid, have confirmed the biosynthetic pathway of gingerols and their transformation upon drying to shogaols. The proposed approach can be applied as a potential candidate for quality assessment of ginger and other medicinal plants.

Metabolomic Analysis of Natural Variation in Arabidopsis.

Methodological advances in coupled-mass spectrometry (gas chromatography and liquid chromatography; GC-MS and LC-MS) have rendered the profiling of highly complex plant extracts relatively facile and allowed that their high-throughput use aids the investigation of a range of biological questions. Among these is the elucidation of the genetic factors underlying metabolite abundance. For this purpose genome-wide association studies (GWAS) are being widely adopted in Arabidopsis with the resultant quantitative trait loci being subjected to cross-validation by the use of recombinant inbred lines, introgression lines, and T-DNA insertional knockout lines.

Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties.

Wild teas are valuable genetic resources for studying domestication and breeding. Here we report the assembly of a high-quality chromosome-scale reference genome for an ancient tea tree. The further RNA sequencing of 217 diverse tea accessions clarifies the pedigree of tea cultivars and reveals key contributors in the breeding of Chinese tea. Candidate genes associated with flavonoid biosynthesis are identified by genome-wide association study. Specifically, diverse allelic function of CsANR, CsF3'5'H and CsMYB5 is verified by transient overexpression and enzymatic assays, providing comprehensive insights into the biosynthesis of catechins, the most important bioactive compounds in tea plants. The inconspicuous differentiation between ancient trees and cultivars at both genetic and metabolic levels implies that tea may not have undergone long-term artificial directional selection in terms of flavor-related metabolites. These genomic resources provide evolutionary insight into tea plants and lay the foundation for better understanding the biosynthesis of beneficial natural compounds.

An improved extraction method enables the comprehensive analysis of lipids, proteins, metabolites and phytohormones from a single sample of leaf tissue under water-deficit stress.

Phytohormones play essential roles in the regulation of growth and development in plants. Plant hormone profiling is therefore essential to understand developmental processes and the adaptation of plants to biotic and/or abiotic stresses. Interestingly, commonly used hormone extraction and profiling methods do not adequately resolve other molecular entities, such as polar metabolites, lipids, starch and proteins, which would be required to comprehensively describe the continuing biological processes at a systematic level. In this article we introduce an updated version of a previously published liquid:liquid metabolite extraction protocol, which not only allows for the profiling of primary and secondary metabolites, lipids, starch and proteins, but also enables the quantitative analysis of the major plant hormone classes, including abscisic acid, auxins, cytokinins, jasmonates and salicylates, from a single sample aliquot. The optimization of the method, which uses the introduction of acidified water, enabling the complete purification of major plant hormones into the organic (methyl-tert-butyl-ether) phase, eliminated the need for solid-phase extraction for sample clean-up, and therefore reduces both sampling time and cost. As a proof-of-concept analysis, Arabidopsis thaliana plants were subjected to water-deficit stress, which were then profiled for hormonal, metabolic, lipidomic and proteomic changes. Surprisingly, we determined not only previously described molecular changes but also significant changes regarding the breakdown of specific galactolipids, followed by the substantial accumulation of unsaturated fatty-acid derivatives and diverse jasmonates in the course of adaptation to water-deficit stress.

Conservation and diversification of flavonoid metabolism in the plant kingdom.

Flavonoids are by far the largest class of polyphenols with huge structural and functional diversity. However, the mystery regarding the exact evolutionary pressures which lead to the amazing diversity in plant flavonoids has yet to be completely uncovered. Here we review recent advances in understanding the conservation and diversification of flavonoid pathway from algae and early land plants to vascular plants including the model plant Arabidopsis and economically important species such as cereals, legumes, and medicinal plants. Studies on the origin and evolution of R2R3-MYB regulatory system demonstrated its highly conserved function of regulating flavonoid production in land plants and this innovation appears to have been crucial in boosting the overall levels of these compounds in land plants. Convergent evolution has occurred as different flavonoids independently which emerged in distant taxa resulting in similar defense and tolerance characteristics against environmental stresses. Future studies on an increasing number of plant species taking advantage of newly developed genomic and metabolite profiling technologies are envisaged to provide comprehensive insight into flavonoid biosynthesis as well as pathway diversification and the underlying evolutionary mechanisms.

Metabolomics in the Context of Plant Natural Products Research: From Sample Preparation to Metabolite Analysis.

Plant-derived natural products have long been considered a valuable source of lead compounds for drug development. Natural extracts are usually composed of hundreds to thousands of metabolites, whereby the bioactivity of natural extracts can be represented by synergism between several metabolites. However, isolating every single compound from a natural extract is not always possible due to the complex chemistry and presence of most secondary metabolites at very low levels. Metabolomics has emerged in recent years as an indispensable tool for the analysis of thousands of metabolites from crude natural extracts, leading to a paradigm shift in natural products drug research. Analytical methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) are used to comprehensively annotate the constituents of plant natural products for screening, drug discovery as well as for quality control purposes such as those required for phytomedicine. In this review, the current advancements in plant sample preparation, sample measurements, and data analysis are presented alongside a few case studies of the successful applications of these processes in plant natural product drug discovery.

Using an UPLC/MS-based untargeted metabolomics approach for assessing the antioxidant capacity and anti-aging potential of selected herbs.

Aging is an unavoidable fate that afflicts all life, during this process in mammals reactive oxygen species (ROS) are generated which stimulate tyrosinase, elastase and collagenase activities that actively participate in skin aging. Therefore, the maintenance of antioxidant homeostasis is an important anti-aging strategy for skin. Nature has excellent anti-aging remedies that act externally as well as internally to delay the visual signs of aging. In view of this fact, the present study investigates the in vitro anti-aging activity of five medicinal plants belonging to phenolic rich families namely Rosmarinus officinalis, Lavandula officinalis, Matricaria chamomilla, Camellia sinensis and Pelargonium graveolens. The selected plants are those most frequently used in the preparation of ethnomedicinal recipes for the prevention or treatment of aging. The inhibitory effects of the ethanolic and aqueous extracts of the five selected plants on the activity of tyrosinase, elastase, and collagenase enzymes were investigated. Furthermore, the chemical composition of the plants and the antioxidant capacity of their extracts were assessed. The results showed that R. officinalis had the highest total phenolics content which was correlated with its potent antioxidant and anti-aging activities. To pinpoint the active metabolites in the tested extracts, we evaluated the metabolite variations using ultra-performance liquid chromatography coupled with high resolution electrospray ionization-tandem mass spectrometry (UPLC-HR-ESI-MS/MS). Multivariate data analysis (MVDA) revealed that R. officinalis significantly accumulated metabolites from the aromatic diterpenoid, flavonoid and phenolic acid classes. These results indicate that rosemary can be used for further development of topical preparations with anti-aging properties.

A Solanum neorickii introgression population providing a powerful complement to the extensively characterized Solanum pennellii population.

We present a complementary resource for trait fine-mapping in tomato to those based on the intra-specific cross between cultivated tomato and the wild tomato species Solanum pennellii, which have been extensively used for quantitative genetics in tomato over the last 20 years. The current population of backcross inbred lines (BILs) is composed of 107 lines derived after three backcrosses of progeny of the wild species Solanum neorickii (LA2133) and cultivated tomato (cultivar TA209) and is freely available to the scientific community. These S. neorickii BILs were genotyped using the 10K SolCAP single nucleotide polymorphism chip, and 3111 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs harbor on average 4.3 introgressions per line, with a mean introgression length of 34.7 Mbp, allowing partitioning of the genome into 340 bins and thereby facilitating rapid trait mapping. We demonstrate the power of using this resource in comparison with archival data from the S. pennellii resources by carrying out metabolic quantitative trait locus analysis following gas chromatography-mass spectrometry on fruits harvested from the S. neorickii BILs. The metabolic candidate genes phenylalanine ammonia-lyase and cystathionine gamma-lyase were then tested and validated in F2 populations and via agroinfiltration-based overexpression in order to exemplify the fidelity of this method in identifying the genes that drive tomato metabolic phenotypes.

Quantitative Trait Loci Analysis Identifies a Prominent Gene Involved in the Production of Fatty Acid-Derived Flavor Volatiles in Tomato.

To gain insight into the genetic regulation of lipid metabolism in tomato, we conducted metabolic trait loci (mQTL) analysis following the lipidomic profiling of fruit pericarp and leaf tissue of the Solanum pennellii introgression lines (IL). To enhance mapping resolution for selected fruit-specific mQTL, we profiled the lipids in a subset of independently derived S. pennellii backcross inbred lines, as well as in a near-isogenic sub-IL population. We identified a putative lecithin:cholesterol acyltransferase that controls the levels of several lipids, and two members of the class III lipase family, LIP1 and LIP2, that were associated with decreased levels of diacylglycerols (DAGs) and triacylglycerols (TAGs). Lipases of this class cleave fatty acids from the glycerol backbone of acylglycerols. The released fatty acids serve as precursors of flavor volatiles. We show that LIP1 expression correlates with fatty acid-derived volatile levels. We further confirm the function of LIP1 in TAG and DAG breakdown and volatile synthesis using transgenic plants. Taken together, our study extensively characterized the genetic architecture of lipophilic compounds in tomato and demonstrated at molecular level that release of free fatty acids from the glycerol backbone can have a major impact on downstream volatile synthesis.

Tecia solanivora infestation increases tuber starch accumulation in Pastusa Suprema potatoes.

In response to infestation with larvae of the Guatemalan tuber moth (Tecia solanivora), some Solanum tuberosum (potato) varieties exhibit an overcompensation response, whereby the total dry mass of uninfested tubers is increased. Here, we describe early responses, within the first few days, of T. solanivora feeding, in the Colombian potato variety Pastusa Suprema. Non-targeted metabolite profiling showed significant secondary metabolism changes in T. solanivora-infested tubers, but not in uninfested systemic tubers. In contrast, changes in primary metabolism were greater in uninfested systemic tubers than in the infested tubers, with a notable 80% decline in systemic tuber sucrose levels within 1 d of T. solanivora infestation. This suggested either decreased sucrose transport from the leaves or increased sink strength, i.e., more rapid sucrose to starch conversion in the tubers. Increased sucrose synthesis was indicated by higher rubisco activase and lower starch synthase gene expression in the leaves of infested plants. Elevated sink strength was demonstrated by 45% more total starch deposition in systemic tubers of T. solanivora-infested plants compared to uninfested control plants. Thus, rather than investing in increased defense of uninfested tubers, Pastusa Suprema promotes deposition of photoassimilates in the form of starch as a response to T. solanivora infestation.

The Integration of Metabolomics and Next-Generation Sequencing Data to Elucidate the Pathways of Natural Product Metabolism in Medicinal Plants.

Plants have always been used as medicines since ancient times to treat diseases. The knowledge around the active components of herbal preparations has remained nevertheless fragmentary: the biosynthetic pathways of many secondary metabolites of pharmacological importance have been clarified only in a few species, while the chemodiversity present in many medicinal plants has remained largely unexplored. Despite the advancements of synthetic biology for production of medicinal compounds in heterologous hosts, the native plant species are often the most reliable and economic source for their production. It thus becomes fundamental to investigate the metabolic composition of medicinal plants to characterize their natural metabolic diversity and to define the biosynthetic routes in planta of important compounds to develop strategies to further increase their content. We present here a number of case studies for selected classes of secondary metabolites and we review their health benefits and the historical developments in their structural elucidation and characterization of biosynthetic genes. We cover the cases of benzoisoquinoline and monoterpenoid indole alkaloids, cannabinoids, caffeine, ginsenosides, withanolides, artemisinin, and taxol; we show how the "early" biochemical or the more recent integrative approaches-based on omics-analyses-have helped to elucidate their metabolic pathways and cellular compartmentation. We also summarize how the knowledge generated about their biosynthesis has been used to develop metabolic engineering strategies in heterologous and native hosts. We conclude that following the advent of novel, high-throughput and cost-effective analytical technologies, the secondary metabolism of medicinal plants can now be examined under the lens of systems biology.