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Main conclusionSA and H2O2, in single and mixed elicitation stimulate specialized metabolism and activate oxidative stress in C. tenuiflora plants.Single elicitation with salicylic acid (SA at 75 µM) and, hydrogen peroxide (at 150 µM), and mixed elicitation (75 µM SA + 150 µM H2O2) were evaluated on specialized metabolism in Castilleja tenuiflora Benth. plants. Total phenolic content (TPC), phenylalanine ammonia-lyase (PAL) activity, antioxidant enzymes and specialized metabolite profiles, as well as the expression levels of eight genes involved in phenolic (Cte-TyrDC, Cte-GOT2, Cte-ADD, Cte-AO3, Cte-PAL1, Cte-CHS1) and terpene pathways (Cte-DXS1 and Cte-G10H) and their correlation with major metabolite (verbascoside and aucubin) concentrations were investigated. TPC content (three-fold) and PAL activity (11.5-fold) increased with mixed elicitation, as well as catalase and peroxidase activity (11.3-fold and 10.8-fold, respectively), compared to single elicitation. Phenylethanoid accumulation was greatest under mixed elicitation, followed by SA and H2O2. Lignan accumulation was differential, depending on the plant part and the elicitor. Flavonoids only appeared after mixed elicitation. The high concentration of verbascoside under mixed elicitation was related to a high gene expression. Single elicitation induced iridoid accumulation in specific parts (H2O2 in aerial parts and SA in roots), whereas under mixed elicitation, it accumulated in both parts. A high concentration of aucubin in the aerial part was related to a high expression level of genes of the terpene pathway Cte-DXS1 and Cte-G10H, and in the root with Cte-G10H, while Cte-DXS1 was downregulated in this tissue in all treatments. Mixed elicitation with SA and H2O2 represents an interesting tool to increase the production of specialized metabolites in plants.

Pharmacodynamic interactions between plant isolated compounds are important to understand the mode of action of an herbal extract to formulate or create better standardized extracts, phytomedicines, or phytopharmaceuticals. In this work, we propose binary mixtures using a leader compound to found pharmacodynamic interactions in inhibition of the NF-κB/AP-1 pathway using RAW-Blue™ cells. Eight compounds were isolated from Castilleja tenuiflora, four were new furofuran-type lignans for the species magnolin, eudesmin, sesamin, and kobusin. Magnolin (60.97%) was the most effective lignan inhibiting the NF-κB/AP-1 pathway, followed by eudesmin (56.82%), tenuifloroside (52.91%), sesamin (52.63%), and kobusin (45.45%). Verbascoside, a major compound contained in wild C. tenuiflora showed an inhibitory effect on NF-κB/AP-1. This polyphenol was chosen as a leader compound for binary mixtures. Verbacoside-aucubin and verbascoside-kobusin produced synergism, while verbascoside-tenuifloroside had subadditivity in all concentrations. Verbascoside-kobusin is a promising mixture to use on NF-κB/AP-1 related diseases and anti-inflammatory C. tenuiflora-based phytomedicines.

Castilleja tenuiflora Benth. (Orobanchaceae) is a medicinal plant that synthetizes terpenes and phenolic compounds of therapeutic value. This study aimed to analyze the effect of methyl jasmonate (MeJA) at different concentrations on growth and content of total phenolic compounds (TPC) in C. tenuiflora in vitro plants. Changes in the concentration of secondary metabolites and gene expression patterns of four enzymes (DXS, G10H, PAL, and CHS) involved in metabolite biosynthesis were carried out under MeJA elicitation. MeJA did not decrease growth and 100 µM MeJA caused greater production of TPC. The increase in TPC was accompanied by an increase in PAL activity and in H2O2 content. MeJA elicitation had a greater effect on phenolic compounds than on iridoids, increasing the contents of verbascoside and aucubin to 82.1 ± 0.8 mg g−1 dry matter and 14.6 ± 0.2 mg g−1 dry matter, respectively. The highest accumulation of aucubin occurred after short exposure to the elicitor. MeJA treatment up-regulated the expression of metabolite biosynthesis related genes Cte-PAL1, Cte-CHS1, Cte-DXS1, and Cte-G10H when compared to control plants. The present work contributes to the understanding of the molecular basis of secondary metabolite biosynthesis in C. tenuiflora.Key messageMethyl jasmonate enhances accumulation of iridoid glycosides and phenolic compounds in Castilleja tenuiflora in vitro plants accompanied by the up-regulation of Cte-PAL1, Cte-CHS1, Cte-DXS1, and Cte-G10H.

The first partial derivatives of λ with respect to demographic parameters quantify linear responses of population growth to vital rate perturbations, but sensitivity itself is situational and can show non-linear responses. We use the second derivative of λ with respect to the demographic parameters to explore this type of responses in a population of a root hemiparasite (Castilleja tenuiflora). For plants growing with and without hosts, population transition matrices were built for the 2016–2018 period, and sensitivities and elasticities together with pure derivatives of the Hessian matrix were calculated, together with the second derivatives to survival and fecundity. We transformed the Hessian matrix to a logarithmic scale to avoid values with no biological meaning. Germination showed high sensitivity values, while permanence of individuals with dry aerial tissue that re-sprout had the highest elasticity. Negative values on the second derivative and on the logarithmic representation were obtained for germination with and without hosts in both years. Survival and fecundity have a similar behaviour in both conditions and periods, with negative values for survival for the first category and no negative values for fecundity. High negative values of the second derivative of the transition from seed to seedling indicate an evolutionary stable strategy and imply seeds are important to the permanence of C. tenuiflora population. A stochastic analysis of the second derivative of the transition from seed to seedling was performed with a Gamma distribution analysis, and showed negative values in all cases, corroborating this result.

In this study, an in vitro co-culture system of Castilleja tenuiflora and its host, Baccharis conferta, was used, and the impact of their interaction on specialized metabolite content was analyzed. After 4 weeks of co-culture, haustoria formation was verified through environmental scanning electron and confocal microscopy, confirming the successful establishment of the plant–plant interaction. Shoot height and biomass of the aerial part of the hemiparasite were not affected significantly by co-culture. However, root biomass increased by 53% compared to individually grown plants. Co-culture significantly reduced the host’s root length without negatively affecting its overall growth or survival. Phytochemical profile alterations were observed in both species. For C. tenuiflora, the lignans sesamin and eudesmin are proposed as differentially accumulated metabolites, while in B. conferta, the caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and the flavonoid acacetin were expressed differently. The development and chemical profiles of B. conferta and C. tenuiflora change when they grow in a co-culture because of the host–parasite interaction. Here, we report the feasibility of using a hemiparasite–host system to investigate more profound research questions. Future biotechnological applications of this system include elucidating the genetic regulators involved in haustorium formation, as well as optimizing environmental and physiological conditions to enhance its biosynthetic capacity for the production of specialized metabolites with therapeutic value.

Stress is an important factor in the etiology of some illnesses such as gastric ulcers and depression. Castilleja tenuiflora Benth. (Orobanchaceae) is used in Mexican traditional medicine for the treatment of gastrointestinal diseases and nervous disorders. Previous studies indicated that organic extracts from C. tenuiflora had gastroprotective effects and antidepressant activity. In this study, we aimed to evaluate the gastroprotective and antidepressant activity of fractions and isolated compounds from the methanolic extract (MECt) of C. tenuiflora in stressed mice. Chromatographic fractionation of MECt produced four fractions (FCt-1, FCt-2, CFt-3, and FCt-4) as well as four bioactive compounds which were identified using TLC, HPLC and NMR analyses. The cold restraint stress (CRS)-induced gastric ulcer model followed by the tail suspension test and the forced swim test were used to evaluate the gastroprotective effect and antidepressant activity of the extract fractions. FCt-2 and FCt-3 at 100 mg/kg had significant gastroprotective and antidepressant effects. All isolated compounds (verbascoside, teniufloroside and mixture geniposide/ musseanoside) displayed gastroprotective effects and antidepressant activity at 1 or 2 mg/kg. The above results allow us to conclude that these polyphenols and iridoids from C. tenuiflora are responsible for the gastroprotective and antidepressant effects.

Castilleja tenuiflora is a native perennial plant used in traditional Mexican medicine. In June 2022, leaf blight symptoms were observed in a wild population of C. tenuiflora plants. Disease incidence was 80% and disease intensity reached up to 5% of the leaf area. Currently, there are no reports of pathogens causing leaf blight in this plant; therefore, this work aimed to identify the fungi responsible for the disease. The fungi recovered from the diseased tissue were characterized by means of pathogenicity tests and cultural, morphological, and molecular characterization. The information obtained revealed that Alternaria alternata and Alternaria gossypina are the pathogens responsible for the disease. This is the first report implicating species of Alternaria in causing leaf blight of C. tenuiflora in Mexico, as well as the first report of Alternaria gossypina also in Mexico. These pathogens may threaten the in situ conservation of native C. tenuiflora populations and limit their in vitro propagation. Future research lines should focus on determining the effect of these pathogens on metabolite production.

Castilleja tenuiflora (Orobanchaceae) has been used in Mexican traditional medicine as a treatment for cough, dysentery, anxiety, nausea and vomiting as well as hepatic and gastrointestinal diseases. The ethanolic extract of the aerial parts of Castilleja tenuiflora was separated by silica gel column chromatography. The fractions were evaluated using the induced edema acetate 12-O-tetradecanoylphorbol (TPA) anti-inflammatory activity model. The most active fraction was subjected to medium-pressure liquid chromatography (MPLC) with UV detection at 206 and 240 nm. The following iridoids were isolated: geniposidic acid, aucubin, bartioside, 8-epi-loganin, mussaenoside, and the phenylpropanoid verbascoside. The most active iridoid was geniposidic acid, which was more active than the control (indomethacin), and the least active iridoid was mussaenoside. 8-epi-Loganin, and mussaenoside have not been previously reported to be anti-inflammatory compounds. The results of these investigations confirm the potential of Mexican plants for the production of bioactive compounds and validate the ethnomedical use of Castilleja tenuiflora-like anti-inflammatory plants.

Castilleja tenuiflora has been used for the treatment of several Central Nervous System (CNS) diseases. Herein we report the antidepressant activity of the methanol extract from the leaves of this medicinal plant. The oral administration of MeOH extract (500 mg/kg) induced a significant (p < 0.05) decrement of the immobility parameter on Forced Swimming Test (FST) and an increment in the latency and duration of the hypnosis, induced by administration of sodium pentobarbital (Pbi, 40 mg/kg, i.p.). Chemical analysis of this antidepressant extract allowed the isolation of (+)-piperitol-4-O-xylopyranosyl-(1→6)-O-glucopyranoside. This new furofuran lignan diglycoside was named tenuifloroside (1) and its complete chemical structure elucidation on the basis of 1D and 2D NMR spectra analysis of the natural compound 1 and its peracetylated derivative 1a is described. This compound was found together with two flavones—apigenin and luteolin 5-methyl ether—a phenylethanoid—verbascoside—and three iridoids—geniposide, caryoptoside and aucubin. All these compounds were purified by successive normal and reverse phase column chromatography. Tenuifloroside, caryoptoside and luteolin 5-methyl ether were isolated from Castilleja genus for the first time. These findings demonstrate that C. tenuiflora methanol extract has beneficial effect on depressive behaviors, and the knowledge of its chemical constitution allows us to propose a new standardized treatment for future investigations of this species in depressive illness.

Castilleja tenuiflora Benth. (Orobanchaceae) in vitro cultures are alternative sources of phenylethanoid glycosides (PhGs), promising natural products for chronic diseases treatment because of their extensive range of biological activities. To increase the yields of these bioactive compounds, the identification of factors affecting their biosynthesis is required. Here, we show that N deficiency stimulates PhGs biosynthesis by increasing the activity of PAL. The contents of two PhGs, verbascoside and isoverbascoside, were enhanced under N deficiency compared with control conditions. The maximum PAL activity in shoots cultured under N deficiency was also increased. Furthermore, we found de novo anthocyanin synthesis under N deficiency suggesting an additional impact of this stress factor on flavonoid metabolism. N deficiency negatively affected the shoot growth (length, biomass, and total chlorophyll content) and multiplication rate, and inhibited the root formation of C. tenuiflora shoots. Our results demonstrate that N deficiency increases PhGs biosynthesis in C. tenuiflora.