Evaluation of Polyphenols Synthesized in Mature Seeds of Common Bean (Phaseolus vulgaris L.) Advanced Mutant Lines.

This study aimed to investigate the availability of flavonoids, anthocyanins, and phenolic acids in mutant bean seeds, focusing on M7 mutant lines, and their corresponding initial and local cultivars. HPLC-DAD-MS/MS and HPLC-MS/MS were used to analyze twenty-eight genotypes of common bean. The obtained results suggest that the mutations resulted in four newly synthesized anthocyanins in the mutant bean seeds, namely, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside, and petunidin 3-O-glucoside, in 20 accessions with colored seed shapes out of the total of 28. Importantly, the initial cultivar with white seeds, as well as the mutant white seeds, did not contain anthocyanins. The mutant lines were classified into groups based on their colors as novel qualitative characteristics. Five phenolic acids were further quantified: ferulic, p-coumaric, caffeic, sinapic, and traces of chlorogenic acids. Flavonoids were represented by epicatechin, quercetin, and luteolin, and their concentrations in the mutant genotypes were several-fold superior compared to those of the initial cultivar. All mutant lines exhibited higher concentrations of phenolic acids and flavonoids. These findings contribute to the understanding of the genetics and biochemistry of phenolic accumulation and anthocyanin production in common bean seeds, which is relevant to health benefits and might have implications for common bean breeding programs and food security efforts.

Metabolomics and health: from nutritional crops and plant-based pharmaceuticals to profiling of human biofluids.

During the past decade metabolomics has emerged as one of the fastest developing branches of "-omics" technologies. Metabolomics involves documentation, identification, and quantification of metabolites through modern analytical platforms in various biological systems. Advanced analytical tools, such as gas chromatography-mass spectrometry (GC/MS), liquid chromatography-mass spectroscopy (LC/MS), and non-destructive nuclear magnetic resonance (NMR) spectroscopy, have facilitated metabolite profiling of complex biological matrices. Metabolomics, along with transcriptomics, has an influential role in discovering connections between genetic regulation, metabolite phenotyping and biomarkers identification. Comprehensive metabolite profiling allows integration of the summarized data towards manipulation of biosynthetic pathways, determination of nutritional quality markers, improvement in crop yield, selection of desired metabolites/genes, and their heritability in modern breeding. Along with that, metabolomics is invaluable in predicting the biological activity of medicinal plants, assisting the bioactivity-guided fractionation process and bioactive leads discovery, as well as serving as a tool for quality control and authentication of commercial plant-derived natural products. Metabolomic analysis of human biofluids is implemented in clinical practice to discriminate between physiological and pathological state in humans, to aid early disease biomarker discovery and predict individual response to drug therapy. Thus, metabolomics could be utilized to preserve human health by improving the nutritional quality of crops and accelerating plant-derived bioactive leads discovery through disease diagnostics, or through increasing the therapeutic efficacy of drugs via more personalized approach. Here, we attempt to explore the potential value of metabolite profiling comprising the above-mentioned applications of metabolomics in crop improvement, medicinal plants utilization, and, in the prognosis, diagnosis and management of complex diseases.

Biotechnologically-Produced Myconoside and Calceolarioside E Induce Nrf2 Expression in Neutrophils.

The pathological manifestation of various diseases can be suppressed by the activation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a transcriptional regulator of the cellular redox balance. Haberlea rhodopensis Friv. is a resurrection plant species endemic for Bulgaria, containing biologically active phenylethanoid glycosides that might possess antioxidant or redox activity. This study aimed to analyze the metabolic profile of in vitro cultured H. rhodopensis and to identify molecules that increase Nrf2 expression in bone marrow neutrophils. Fractions B, D, and E containing myconoside, or myconoside and calceolarioside E in ratios 1:0.6 and 0.25:1 were found to be the most active ones. Fraction B (200 µg/mL) improved neutrophil survival and strongly increased the Nrf2 intracellular level, while D and E, as well as, myconoside and calceolarioside E at the same ratios had a superior effect. Calceolarioside E (32 µg/mL) had stronger activity than myconoside, the effect of which was very similar to that of 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me), used as a positive control. These data indicate that both molecules, used alone or in combination have stimulatory activity on the endogenous Nrf2 level, indicating their therapeutic potential to regulate the cellular redox homeostasis oxidative stress-associated pathologies.

Green (cell) factories for advanced production of plant secondary metabolites.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant in vitro systems. The unique structural features of plant-derived secondary metabolites, such as their safety profile, multi-target spectrum and "metabolite likeness," have led to the establishment of many plant-derived drugs, comprising approximately a quarter of all drugs approved by the Food and Drug Administration and/or European Medicinal Agency. However, there are still many challenges to overcome to enhance the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant secondary metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this review, we present an integrated overview of the possible avenues for enhancing the biosynthesis of high-value marketable molecules produced by plant in vitro systems. These include metabolic engineering and CRISPR/Cas9 technology for the regulation of plant metabolism through overexpression/repression of single or multiple structural genes or transcriptional factors. The use of NMR-based metabolomics for monitoring metabolite concentrations and additionally as a tool to study the dynamics of plant cell metabolism and nutritional management is discussed here. Different types of bioreactor systems, their modification and optimal process parameters for the lab- or industrial-scale production of plant secondary metabolites are specified.

Authenticity and quality evaluation of different Rhodiola species and commercial products based on NMR-spectroscopy and HPLC.

INTRODUCTION: The main concern regarding the authenticity and quality of Rhodiola rosea L. (Sedum rosea (L.) Scop.) products is their adulteration with other Rhodiola species. OBJECTIVE: The aim of the study was the development of a reliable and practical analytical platform for quality and quantity assessment of the characteristic molecules in three Rhodiola species (R. rosea, R. kirilowii (Regel) Maxim and R. crenulata (Hook. f. & Thomson) H. Ohba), commercial products and their possible application as markers for the authentication of R. rosea based products. MATERIAL AND METHODS: The major molecules were identified by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR)-based metabolomics and quantitatively determined by high-performance liquid chromatography ultraviolet (HPLC-UV) analysis. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) revealed the specific patterns in the metabolite profiles of R. rosea and R. crenulata. RESULTS: The coumarin crenulatin was only identified in R. crenulata and can be used as a marker to detect potential adulteration of the commercial products. Crenulatin was identified in two of the four analysed products by NMR-spectroscopy. According to the HPLC data, in less than a quarter of all products, the labelled amounts of salidroside and total rosavins were confirmed. CONCLUSIONS: The developed analytical platform was found to be useful in the investigations of the phytochemical diversity of different Rhodiola species, the recognition of the unique metabolites between them and the identification of adulterated products. Therefore, this approach could be applied from the earliest to the latest stages of the value chain in the manufacturing of R. rosea based products.

Plant In Vitro Systems as a Sustainable Source of Active Ingredients for Cosmeceutical Application.

Cosmeceuticals are hybrids between cosmetics and pharmaceuticals which are being designed for a dual purpose: (1) To provide desired esthetical effects and (2) simultaneously treat dermatological conditions. The increased demand for natural remedies and the trends to use natural and safe ingredients resulted in intensive cultivation of medicinal plants. However, in many cases the whole process of plant cultivation, complex extraction procedure, and purification of the targeted molecules are not economically feasible. Therefore, the desired production of natural cosmetic products in sustainable and controllable fashion in the last years led to the intensive utilization of plant cell culture technology. The present review aims to highlight examples of biosynthesis of active ingredients derived through plant in vitro systems with potential cosmeceutical application. The exploitation of different type of extracts used in a possible cosmeceutical formulation, as well as, their activity tested in in vitro/in vivo models is thoroughly discussed. Furthermore, opportunities to manipulate the biosynthetic pathway, hence engineering the biosynthesis of some secondary metabolites, such as anthocyanins, have been highlighted.

Veronica austriaca L. Extract and Arbutin Expand Mature Double TNF-α/IFN-γ Neutrophils in Murine Bone Marrow Pool.

Plants from the Veronica genus are used across the world as traditional remedies. In the present study, extracts from the aerial part of the scarcely investigated Veronica austriaca L., collected from two habitats in Bulgaria-the Balkan Mountains (Vau-1) and the Rhodopi Mountains (Vau-2), were analyzed by nuclear magnetic resonance (NMR) spectroscopy. The secondary metabolite, arbutin, was identified as a major constituent in both extracts, and further quantified by high-performance liquid chromatography (HPLC), while catalpol, aucubin and verbascoside were detected at lower amounts. The effect of the extracts and of pure arbutin on the survival of neutrophils isolated from murine bone marrow (BM) were determined by colorimetric assay. The production of cytokines-tumor necrosis factor (TNF)-α and interferon (IFN)-γ was evaluated by flowcytometry. While Vau-1 inhibited neutrophil vitality in a dose-dependent manner, arbutin stimulated the survival of neutrophils at lower concentrations, and inhibited cell density at higher concentrations. The Vau-1 increased the level of intracellular TNF-α, while Vau-2 and arbutin failed to do so, and expanded the frequency of mature double TNF-α+/IFN-γhi neutrophils within the BM pool.

Genetic transformation of rare Verbascum eriophorum Godr. plants and metabolic alterations revealed by NMR-based metabolomics.

OBJECTIVES: To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. RESULTS: Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. CONCLUSION: Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances.

Extracts of Salvia species are used in traditional medicine to treat various diseases. The economic importance of this genus has increased in recent years due to evidence that some of its secondary metabolites have valuable pharmaceutical and nutraceutical properties.The bioactivity of sage extracts is mainly due to their content of terpenes and polyphenols. The increasing demand for sage products combined with environmental, ecological and climatic limitations on the production of sage metabolites from field-grown plants have led to extensive investigations into biotechnological approaches for the production of Salvia phytochemicals. The purpose of this review is to evaluate recent progress in investigations of sage in vitro systems as tools for producing important terpenoids and polyphenols and in development of methods for manipulating regulatory processes to enhance secondary metabolite production in such systems.

Large-Scale Production of Specialized Metabolites In Vitro Cultures.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

The Cytotoxic Properties of Extreme Fungi's Bioactive Components-An Updated Metabolic and Omics Overview.

Fungi are the most diverse living organisms on planet Earth, where their ubiquitous presence in various ecosystems offers vast potential for the research and discovery of new, naturally occurring medicinal products. Concerning human health, cancer remains one of the leading causes of mortality. While extensive research is being conducted on treatments and their efficacy in various stages of cancer, finding cytotoxic drugs that target tumor cells with no/less toxicity toward normal tissue is a significant challenge. In addition, traditional cancer treatments continue to suffer from chemical resistance. Fortunately, the cytotoxic properties of several natural products derived from various microorganisms, including fungi, are now well-established. The current review aims to extract and consolidate the findings of various scientific studies that identified fungi-derived bioactive metabolites with antitumor (anticancer) properties. The antitumor secondary metabolites identified from extremophilic and extremotolerant fungi are grouped according to their biological activity and type. It became evident that the significance of these compounds, with their medicinal properties and their potential application in cancer treatment, is tremendous. Furthermore, the utilization of omics tools, analysis, and genome mining technology to identify the novel metabolites for targeted treatments is discussed. Through this review, we tried to accentuate the invaluable importance of fungi grown in extreme environments and the necessity of innovative research in discovering naturally occurring bioactive compounds for the development of novel cancer treatments.

Crocus sativus Extract as a Biological Agent for Disease-Modifying Therapy of Collagenase-Induced Mouse Model of Osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is an age-related joint disease that involves the degeneration of cartilage and is the most prevalent form of arthritis, affecting a large part of the population. OA is a multifactorial disorder, and no single etiological mechanism has been found to be common to all forms of the disease. Currently used therapies for control of the disease are mainly nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid medications. The aim of this study was to investigate the extract from Crocus sativus as a biological disease-suppressing therapy agent. METHODS: Balb/c mice were injected intra-articularly with Clostridium histolyticum type IA for induction of osteoarthritis. The mice were randomized to five groups: control group, I group (CIOA untreated), II group (CIOA + 100 mg/kg/daily saffron), III group (CIOA + 50 mg/kg/daily saffron), IV group (CIOA + 25 mg/kg/daily saffron). Flow-cytometry analysis was used to study the splenocytes' phenotype isolated from the treated animals. The serum levels of inflammatory and anti-inflammatory cytokines were analyzed with ELISA. The histological assessment was used to analyze the saffron extract effect on histopathological alterations. RESULTS: Saffron treatment significantly decreased osteoarthritis-associated joint histological manifestations and decreased serum TNFα levels. The flow-cytometry analysis showed a decrease in pro-inflammatory immune cell subtypes in the spleen. CONCLUSIONS: The results obtained suggest that saffron affected the disease progression and could be a potential therapeutic approach in osteoarthritic patients' therapy.

Anti-adipogenic activity of maackiain and ononin is mediated via inhibition of PPARγ in human adipocytes.

Obesity is a global health burden for which we do not yet have effective treatments for prevention or therapy. Plants are an invaluable source of bioactive leads possessing anti-adipogenic potential. Ethnopharmacological use of Ononis spinosa L. roots (OSR) for treatment of obesity and metabolic disorders requires а scientific rationale. The current study examined the anti-adipogenic capacity of OSR and its secondary metabolites ononin (ONON) and maackiain (MACK) in human adipocytes as an in vitro model of obesity. Both ONON and MACK diminished lipid accumulation during adipocyte differentiation. Molecular docking analysis exposed the potential interactions between MACK or ONON and target regulatory adipogenic proteins. Furthermore, results from an RT-qPCR analysis disclosed significant upregulation of AMPK by MACK and ONON treatment. In addition, ONON increased SIRT1, PI3K and ACC mRNA expression, while MACK notably downregulated CEBPA, AKT, SREBP1, ACC and ADIPOQ. The protein level of PI3K, C/EBPα, PPARγ and adiponectin was reduced upon MACK treatment in a concentration-dependent manner. Similarly, ONON suppressed PI3K, PPARγ and adiponectin protein abundance. Finally, our study provides evidence that ONON exerts anti-adipogenic effect by upregulation of SIRT1 and inhibition of PI3K, PPARγ and adiponectin, while MACK induced strong inhibitory effect on adipogenesis via hampering PI3K, PPARγ/C/EBPα signaling and anti-lipogenic effect through downregulation of SREBP1 and ACC. Even though OSR does not hamper adipogenic differentiation, it could be exploited as a source of natural leads with anti-adipogenic potential. The multidirectional mechanism of action of MACK warrant further validation in the context of in vivo obesity models.

Two-phase temporary immersion system for Agrobacterium rhizogenes genetic transformation of sage (Salvia tomentosa Mill.).

Hairy root cultures of Salvia tomentosa were initiated by transformation with Agrobacterium rhizogenes. To prevent necrosis in the explants and to protect young hairy roots, Amberlite XAD-4 resin, in combination with a temporary immersion cultivation system, was applied. HPLC analyzes showed that the resin adsorbed more than 93% of the released phenolic acids and 100% of the released flavonoids. The decreased content of the released phenolics significantly reduced their destructive effects on the plant tissues, prevented, and speeded up the appearance of hairy roots.

Usnic Acid Treatment Changes the Composition of Mycobacterium tuberculosis Cell Envelope and Alters Bacterial Redox Status.

Mycobacterium tuberculosis developed efficient adaptation mechanisms in response to different environmental conditions. This resulted in the ability to survive in human macrophages and in resistance to numerous antibiotics. To get insight into bacterial responses to potent antimycobacterial natural compounds, we tested how usnic acid, a lichen-derived secondary metabolite, would influence mycobacteria at transcriptomic and metabolomic levels. The analysis of expression of sigma factors revealed a profound impact of usnic acid on one of the primary genetic regulatory systems of M. tuberculosis Combined liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses allowed us to observe the perturbations in metabolic pathways, as well as in lipid composition, which took place within 24 h of exposure. Early bacterial response was related to redox homeostasis, lipid synthesis, and nucleic acid repair. Usnic acid treatment provoked disturbances of redox state in mycobacterial cells and increased production of structural elements of the cell wall and cell membrane. In addition, to increase the number of molecules related to restoration of redox balance, the rearrangements of the cell envelope were the first defense mechanisms observed under usnic acid treatment.IMPORTANCE The evaluation of mechanisms of mycobacterial response to natural products has been barely studied. However, it might be helpful to reveal bacterial adaptation strategies, which are eventually crucial for the discovery of new drug targets and, hence, understanding the resistance mechanisms. This study showed that the first-line mycobacterial defense against usnic acid, a potent antimicrobial agent, is the remodeling of the cell envelope and restoring redox homeostasis. Transcriptomic data correlated with metabolomics analysis. The observed metabolic changes appeared similar to those exerted by antibiotics.

Tanshinones from Salvia miltiorrhiza inhibit Mycobacterium tuberculosis via disruption of the cell envelope surface and oxidative stress.

The unique structure of Mycobacterium tuberculosis cell envelope provides impermeable barrier against environmental stimuli. In the situation that this barrier is disturbed Mycobacteria react at the transcriptional and translational level to redirect metabolic processes and to maintain integrity of the cell. In this work we aimed to explore the early metabolic response of M. tuberculosis to tanshinones, which are active antimycobacterial compounds of Salvia miltiorrhiza Bunge root. The investigation of the expression of sigma factors revealed the significant shifts in the general bacterial regulatory network, whereas LC-MS metabolomics evidenced the changes in the composition of bacterial cell envelope and indicated altered metabolic pathways. Tanshinones acted via the disruption of the cell envelope surface and generation of reactive oxygen species. Bacteria responded with overproduction of inner region of outer membrane, fluctuations in the production of glycerophosphoinositolglycans, as well as changes in the levels of mycobactins, accompanied by enrichment of metabolic pathways related to redox balance and repair of damages caused by tanshinones.

Biotechnologically Produced Lavandula angustifolia Mill. Extract Rich in Rosmarinic Acid Resolves Psoriasis-Related Inflammation Through Janus Kinase/Signal Transducer and Activator of Transcription Signaling.

Psoriasis is a common skin pathology, characterized by dysregulation of epidermal keratinocyte function attended by persistent inflammation, suggesting that molecules with anti-inflammatory potential may be effective for its management. Rosmarinic acid (RA) is a natural bioactive molecule known to have an anti-inflammatory potential. Here we examined the effect of biotechnologically produced cell suspension extract of Lavandula angustifolia Mill (LV) high in RA content as treatment for psoriasis-associated inflammation in human keratinocytes. Regulatory genes from the nuclear factor kappa B (NF-κB) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways were upregulated upon stimulation with a combination of interferon gamma (IFN-γ), interleukin (IL)-17A and IL-22. We also observed that both LV extract and RA could inhibit JAK2, leading to reduced STAT1 phosphorylation. Further, we demonstrated that LV extract inhibited phosphoinositide 3-kinases (PI3K) and protein kinase B (AKT), which could be implicated in reduced hyperproliferation in keratinocytes. Collectively, these findings indicate that the biotechnologically produced LV extract resolved psoriasis-like inflammation in human keratinocytes by interfering the JAK2/STAT1 signaling pathway and its effectiveness is due to its high content of RA (10%). Hence, both LV extract and pure RA possess the potential to be incorporated in formulations for topical application as therapeutic approach against psoriasis.

Anti-Adipogenic Effect of Alchemilla monticola is Mediated Via PI3K/AKT Signaling Inhibition in Human Adipocytes.

Obesity is a persistent and continuously expanding social health concern. Excessive fat mass accumulation is associated with increased risk of chronic diseases including diabetes, atherosclerosis, non-alcoholic steatohepatitis, reproductive dysfunctions and certain types of cancer. Alchemilla monticola Opiz. is a perennial plant of the Rosaceae family traditionally used to treat inflammatory conditions and as a component of weight loss herbal mixtures. In the search for bioactive leads with potential anti-adipogenic effect from A. monticola extract (ALM), we have employed nuclear magnetic resonance (NMR) based metabolomics to obtain data for the phytochemical profile of the extract. Further, molecular docking simulation was performed against key adipogenic targets for selected pure compounds, present in the ALM extract. Evaluation of the biological activity was done in human adipocytes exposed to ALM (5, 10 and 25 µg/ml), pure astragalin (AST) or quercitrin (QUE) both at the concentrations of 5, 10 and 25 µM. Investigation of the molecular pathways involved was performed through real-time quantitative PCR and Western blot analyses. According to the docking predictions strong putative affinity was revealed for both AST and QUE towards peroxisome proliferator-activated receptor gamma (PPARγ) and phosphoinositide 3-kinase (PI3K). Assessment of the intracellular lipid accumulation revealed anti-adipogenic activity of ALM. Correspondingly, the expression of the adipogenic genes CCAAT/enhancer-binding protein alpha (CEBPA) and PPARG was downregulated upon ALM and AST treatment. The Western blotting results exposed protein kinase B (AKT), PI3K and PPARγ as targets for the inhibitory effect of ALM and AST on adipogenesis. Collectively, we provide a broader insight of the phytochemical composition of A. monticola. Additionally, we demonstrate the anti-adipogenic effect of ALM and its active compound AST in human adipocytes. Furthermore, PI3K/AKT signaling pathway is identified to mediate the ALM anti-adipogenic action. Hence, the ALM extract and its secondary metabolite AST are worth further exploration as potentially active agents in obesity management.

Clinopodium vulgare L. (wild basil) extract and its active constituents modulate cyclooxygenase-2 expression in neutrophils.

Clinopodium vulgare L. (wild basil) has a wide range of ethnopharmacological applications and accumulates a broad spectrum of phenolic compounds, recognized for their anti-inflammatory and anticancer properties. The triggered cyclooxygenase-2 (COX-2) expression is creating an immunosuppressive microenvironment in the inflamed tissue and considered to be the main cause of failure of even new anticancer-/immune-therapies. Nowadays, selective and novel plant-derived COX-2 inhibitors with safe profile are subject of profound research interest. This study aimed to analyze the metabolic profile of C. vulgare and search for phenolic molecules with potential biological properties. By application of 1H and 2D-NMR (Nuclear Magnetic Resonance) profiling, caffeic, chlorogenic acids and catechin were identified along with a bunch of primary and secondary metabolites. Further, the biological effect of C. vulgare extract (CVE) and its constituents on zymosan-induced COX-2 expression and apoptosis of murine neutrophils have been studied. The CVE, caffeic and chlorogenic acids inhibited zymosan-induced COX-2 expression in bone marrow neutrophils, in vitro and in vivo activated. The obtained data indicate that CVE may have a good potential to manipulate neutrophil functions, however, its action may depend on the cellular state, the inflammatory milieu and the relative content of caffeic and chlorogenic acid in the extract.

Transformed Root Culture: From Genetic Transformation to NMR-Based Metabolomics.

Hairy root (HR) culture is considered as "green factory" for mass production of bioactive molecules with pharmaceutical relevance. As such, HR culture has an immense potential as a valuable platform to elucidate biosynthetic pathways and physiological processes, generate recombinant therapeutic proteins, assist molecular breeding, and enhance phytoremediation efforts. However, some plant species appear recalcitrant to the classical Agrobacterium rhizogenes transformation techniques. Sonication-assisted Agrobacterium-mediated transformation (SAArT) is a highly effective method to deliver bacteria to target plant tissues that includes exposure of the explants to short periods of ultrasound in the presence of the bacteria.Nuclear magnetic resonance (NMR)-based metabolomics is one of the most powerful and suitable platforms for identifying and obtaining structural information on a wide range of compounds with a high analytical precision. In terms of plant science, NMR metabolomics is used to determine the phytochemical variations of medicinal plants or commercial cultivars in certain environments and conditions, including biotic stress and plant biotic interaction, structural determination of natural products, quality control of herbal drugs or dietary supplements, and comparison of metabolite differences between plants and their respective in vitro cultures.In this chapter, we attempt to summarize our knowledge and expertise in induction of hairy roots from rare and recalcitrant plant species by SAArT technique and further methodology for extraction of secondary metabolites of moderate to high polarity and their identification by using NMR-based metabolomics.