Medicinal plants produce specialized metabolites (SM) that are used as drugs. However, due to low yields of field cultivation and the increasing market demand, this production method often failed to meet supply needs. Biotechnological alternatives, such as in vitro plant cultures, offer promising solutions. Nonetheless, SM production in these systems remains too low for industrial exploitation, necessitating an elicitation step to induce the plant defense metabolism. Traditional elicitation methods mimic environmental conditions that trigger plant-specialized metabolism, often with an artificial signal that mimics microbial interaction. Recent insights into the essential role of the plant microbiota, provides new opportunities for elicitation strategies by microbial coculture in a controlled environment. The successful co-culture of in vitro medicinal plants with synthetic microbial communities could enable sustainable production of pharmaceutically important SM.

Using stress elicitors in fruits and vegetables is considered a good strategy to increase the content of bioactive compounds in plant foods. However, bioactive compounds can affect the sensory characteristics of food products, and little is known about their shelf-life stability in fresh produce treated with elicitors. In the present work, carotenoids and polyphenols were quantified by spectrophotometric methods in red and green butterhead lettuce treated with elicitors that had previously been demonstrated to increase bioactive compounds: arachidonic acid (AA), methyl jasmonate (MJ), and Harpin protein (HP). The bioactive compounds were determined immediately and during three weeks after harvest. A descriptive sensory analysis was carried out, which included odor, taste, tactile, and visual attributes of control and elicitor-treated lettuce. Carotenoids showed greater shelf-life stability than polyphenols, and both were more stable in red than in green lettuce during the first two weeks of storage. The best elicitor was MJ, which increased phenolic compounds (red and green lettuce), anthocyanins, and carotenoids (red lettuce) through the storage period. Color intensity, crispness, wettability, and bitter taste were some of the primary sensory attributes in butterhead lettuce and were not affected by any treatment. Other organoleptic properties were also not affected by the elicitors. These results suggest that elicitation could improve the content of bioactive compounds, which is stable through the shelf-life of butterhead lettuce, without any adverse effect on the sensory properties.

In current work, we studied hairy root induction in Trigonella foenum graecum, which is an important medicinal plant, and examined the impact of different elicitors on some phytochemical characteristics and metabolites production in hairy root cultures. Accordingly, some factors such as five strain types of Agrobacterium rhizogenes (1724, 15834, A4, A13 and MSU) and three different explants, namely leaf, cotyledon and hypocotyl were studied. The results showed that different A. rhizogenes strains exhibited different infection efficiency. MSU and 15834 had highest efficiency of hairy root induction than other strains. Also, hairy root induction frequency in leaf explants was higher than in other explants. Salicylic acid (SA), nitric oxide (NO), CaCl2 and penconazole (PEN) were used in elicitation process. Hairy roots were treated with SA (0.1 and 0.5 mM), NO (10 and 50 µM), CaCl2 (5 and 10 mM) and PEN (5 and 10 mg/L). Applied elicitors enhanced antioxidant enzymes activities and reduced oxidative stress markers; this observation might be ascribed to regulation of the oxidative status of the elicited cells. Significant increase of antioxidant metabolites (total phenol, flavonoid and anthocyanin) in PEN-treated hairy roots was associated to phenylalanine ammonia lyase activity, indicating an up-regulation of phenylpropanoid/flavonoid metabolism. PEN and CaCl2 treatment enhanced steroidal sapogenin in hairy root cultures. These results suggested that use of elicitors can enhance the production of secondary metabolites in transformed hairy roots. Among the elicitors applied, CaCl2 and PEN were the most effective in increasing secondary metabolite production in transformed hairy roots of T. foenum graecum.

Plants plastically alter their metabolism in response to environmental stimuli, which induces changes in the accumulation of specialized metabolites. This ability can be utilized to manipulate plant phytochemistry in a desired direction. However, the abundance of secondary metabolites in the different plant species, especially medicinal, is enormous; therefore, it is difficult to establish a clear direction for the effects of metabolic modulators on phytochemical composition, especially given the possibility of using different types thereof. In order to gain insight into these changes, we investigated the effects of foliar-applied chitosan (ChL, 100 mg/L), selenium (Se, 10 mg/L), salicylic acid (SA, 150 mg/L), or an equal volume mixture thereof on Hypericum perforatum L. metabolism. Selenium and SA proved to be the more effective than ChL in enhancing the accumulation of phenolic compounds. The greatest increase was found in the concentration of neochlorogenic acid after Se-spraying. The treatment with the elicitors generally increased the concentration of identified flavonoids, but not the level of naphthodianthrone or phloroglucinol metabolites. The most pronounced response was observed on day 10 following the application of the compounds, and is likely the consequence of elevated levels of O2-˙, free proline, and modulated activity of enzymatic antioxidants.

Maesa indica Roxb. Sweet is a shrub known for its richness in secondary metabolites. A callus culture protocol was established to enhance its chemical profile. Sixteen elicitation culture treatments were evaluated, and we confirmed that the treatment of 200 mg/L polyethylene glycol (4000) coupled with exposure to 30 W UV irradiation for 60 min (PEG4) resulted in the highest total phenolic and total flavonoid contents, which were 4.1 and 4.9 times those of the plant ethanolic extract and 4.9 and 4.8 times those of a control sample, respectively. The phenolic compounds in the different treatments were identified qualitatively and quantitatively using the LC-ESI-MS/MS-MRM technique. Molecular docking studies of the phenolic compounds were conducted using MOE software and revealed that rutin showed the highest binding affinity toward the anti-cancer target (p38α MAPK). The cytotoxicity of the ME and PEG 4 treatment was tested against colon, breast, prostate, lung, and liver cell lines using an MTT assay. The highest cytotoxic effect of PEG4 was against prostate cancer with an IC50 value of 25.5 µg/mL. Hence, this study showed enhanced secondary metabolite accumulation and identified the phenolic compounds in the 16 treatments. The cytotoxicity assay highlighted the possible cytotoxic effect of the PEG4 treatment, and we recommend further investigations into its activity.

Galanthamine is an immensely valuable alkaloid exhibiting anti-cancer and antiviral activity. The cultivation of plant tissues in in vitro conditions is a good source for the synthesis and enrichment of secondary metabolites of commercial interest. In this study, the Amaryllidaceae alkaloid galanthamine was quantified in three Zephyranthes species, such as Zephyranthes candida, Zephyranthes grandiflora, and Zephyranthes citrina, and the impact of the methyl jasmonate (MJ) signaling molecule on galanthamine accumulation was monitored in in vitro-derived plant tissues. This is the first ever study of the MJ-regulated accumulation of galanthamine in in vitro-grown Zephyranthes tissues. Shoot regeneration was obtained in all three Zephyranthes species on Murashige and Skoog (MS) medium containing 2.0 mgL-1 benzylaminopurine (BAP) + 0.5 mgL-1 naphthalene acetic acid (NAA). The regenerated shoots were rooted on a medium containing 2.0 mgL-1 indole butyric acid (IBA). A GC-MS study of Zephyranthes extracts revealed the presence of 34 phyto-compounds of varied levels with therapeutic activities against diseases. The galanthamine content was quantified in plant parts of the three Zephyranthes species using high-performance thin layer chromatography (HPTLC); the maximum was found in Z. candida bulb (2.41 µg g-1 dry wt.), followed by Z. grandiflora (2.13 µg g-1 dry wt.), and then Z. citrina (2.02 µg g-1 dry wt.). The galanthamine content showed bulb > leaf > root source order. The in vitro-generated plantlets were treated with different MJ concentrations, and the galanthamine yield was measured in bulb, leaf, and root tissues. The highest galanthamine content was recorded in bulbs of Z. candida (3.97 µg g-1 dry wt.) treated with 150 µM MJ, showing an increase of 64.73% compared to the control. This accumulation may be attributed to MJ-induced stress, highlighting the potential commercial synthesis of galanthamine in vitro.

The development and production of secondary metabolites from priceless medicinal plants are restricted by drought stress. Mentha pulegium L. belongs to the Lamiaceae family and is a significant plant grown in the Mediterranean region for its medicinal and aesthetic properties. This study investigated the effects of three polyethylene glycol (PEG) (0, 5, and 10%) as a drought stress inducer and four silicon nanoparticle (SiNP) (0, 25, 50, and 100 ppm) concentrations as an elicitor to overcome the adverse effect of drought stress, on the growth parameters and bioactive chemical composition of M. pulegium L. plants grown in vitro. The experiment was performed as a factorial experiment using a completely randomized design (CRD) consisting of 12 treatments with two factors (3 PEG × 4 SiNPs concentrations), 6 replicates were used for each treatment for a total of 72 experimental units.The percentage of shoot formation was inversely proportional to the PEG concentration; for the highest PEG concentration, the lowest percentage of shoot formation (70.26%) was achieved at 10% PEG. SiNPs at 50 ppm enhanced shoot formation, the number of shoots, shoot height, fresh and dry weight, rosmarinic acid, total phenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. The methanol extract from M. pulegium revealed the presence of significant secondary metabolites using gas chromatography‒mass spectrometry (GC-MS). The principal constituents of the extract were limonene (2.51, 2.99%), linalool (3.84, 4.64%), geraniol (6.49, 8.77%), menthol (59.73, 65.43%), pulegone (3.76, 2.76%) and hexadecanoic acid methyl ester or methyl palmitate (3.2, 4.71%) for the 0 ppm SiNPs, PEG 0% and 50 ppm SiNPs, and PEG 10%, respectively. Most of the chemical components identified by GC‒MS in the methanol extract were greater in the 50 ppm SiNP and 10% PEG treatment groups than in the control group. SiNP improves drought tolerance by regulating biosynthesis and accumulating some osmolytes and lessens the negative effects of polyethylene glycol-induced drought stress.Based on the results, the best treatment for most of the parameters was 50 ppm SiNPs combined with 10% PEG, the morphological and chemical characteristics were inversely proportional to the PEG concentration, as the highest PEG concentration (10%) had the lowest results. Most parameters decreased at the highest SiNP concentration (100 ppm), except for the DPPH scavenging percentage, as there was no significant difference between the 50 and 100 ppm SiNPs.

Eritadenine from shiitake mushroom is a secondary metabolite with hypocholesterolemic, hypotensive and antiparasitic properties, thus promising for pharmaceutical and agricultural applications. Eritadenine is obtained from submerged mycelial cultures of shiitake, but the actual yields remain unsatisfactory to explore potential applications or industrial-scale production. In this study, green and blue LED lights were tested to increase yields of eritadenine in submerged cultures of shiitake. Notably, blue LEDs increased yields by 13-14 times, reaching 165.7 mg/L, compared to darkness (11.2 mg/L) and green light (12.1 mg/L) (p < 0.05, Tukey test). Nitrogen sources yeast extract (YE) and peptone (at 2 g/L) increased eritadenine production. YE promoted 22.6 mg/L, while peptone 18.3 mg/L. The recovery of eritadenine was evaluated using amberlite and activated charcoal (AC) adsorption isotherms. AC demonstrated the highest adsorption rate, with 75 mg of eritadenine per gram of AC, according to the Freundlich isotherm. The desorption rate reached 93.95% at pH 10. The extract obtained from submerged cultures had eritadenine content of 63.31%, corresponding to 87.86% of recovery, according to HPLC analysis. Furthermore, the novel bioherbicidal potential of eritadenine was tested on in vitro Chrysanthemum morifolium plants. The cultures extract containing eritadenine had a detrimental impact on plant development, generating mortality of 100% at 3%, 0.5%, and 0.25%. Moreover, pure eritadenine exhibited a phytotoxic effect similar than glyphosate on leaves, stems and roots. These findings highlight the significant bioherbicidal properties of eritadenine. Further studies are needed to understand the biosynthetic pathway of eritadenine and its bioherbicidal properties on weeds and illicit crops.

Chlorogenic acid is one of the most prominent bioactive phenolic acids with great pharmacological, cosmetic and nutritional value. The potential of Berula erecta in tissue culture was investigated for the production of chlorogenic acid and its elicitation combined with light of different wavelengths and low temperature. The content of chlorogenic acid in the samples was determined by HPLC-UV, while the content of total phenolic compounds and the antioxidant activity of their ethanol extracts were evaluated spectrophotometrically. The highest fresh and dry biomasses were obtained in plants grown at 23 °C. This is the first study in which chlorogenic acid has been identified and quantified in Berula erecta. The highest chlorogenic acid content was 4.049 mg/g DW. It was determined in a culture grown for 28 days after the beginning of the experiment at 12 °C and under blue light. The latter also contained the highest content of total phenolic compounds, and its extracts showed the highest antioxidant activity. Berula erecta could, potentially, be suitable for the in vitro production of chlorogenic acid, although many other studies should be conducted before implementation on an industrial scale.

Identification of Endocrine-Disrupting Chemicals (EDCs) in a regulatory context requires a high level of evidence. However, lines of evidence (e.g. human, in vivo, in vitro or in silico) are heterogeneous and incomplete for quantifying evidence of the adverse effects and mechanisms involved. To date, for the regulatory appraisal of metabolism-disrupting chemicals (MDCs), no harmonised guidance to assess the weight of evidence has been developed at the EU or international level. To explore how to develop this, we applied a formal Expert Knowledge Elicitation (EKE) approach within the European GOLIATH project. EKE captures expert judgment in a quantitative manner and provides an estimate of uncertainty of the final opinion. As a proof of principle, we selected one suspected MDC -triphenyl phosphate (TPP) - based on its related adverse endpoints (obesity/adipogenicity) relevant to metabolic disruption and a putative Molecular Initiating Event (MIE): activation of peroxisome proliferator activated receptor gamma (PPARγ). We conducted a systematic literature review and assessed the quality of the lines of evidence with two independent groups of experts within GOLIATH, with the objective of categorising the metabolic disruption properties of TPP, by applying an EKE approach. Having followed the entire process separately, both groups arrived at the same conclusion, designating TPP as a \"suspected MDC\" with an overall quantitative agreement exceeding 85%, indicating robust reproducibility. The EKE method provides to be an important way to bring together scientists with diverse expertise and is recommended for future work in this area.