Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

Uncovering the Interrelation between Metabolite Profiles and Bioactivity of In Vitro- and Wild-Grown Catmint (Nepeta nuda L.).

Nepeta nuda L. is a medicinal plant enriched with secondary metabolites serving to attract pollinators and deter herbivores. Phenolics and iridoids of N. nuda have been extensively investigated because of their beneficial impacts on human health. This study explores the chemical profiles of in vitro shoots and wild-grown N. nuda plants (flowers and leaves) through metabolomic analysis utilizing gas chromatography and mass spectrometry (GC-MS). Initially, we examined the differences in the volatiles' composition in in vitro-cultivated shoots comparing them with flowers and leaves from plants growing in natural environment. The characteristic iridoid 4a-α,7-ß,7a-α-nepetalactone was highly represented in shoots of in vitro plants and in flowers of plants from nature populations, whereas most of the monoterpenes were abundant in leaves of wild-grown plants. The known in vitro biological activities encompassing antioxidant, antiviral, antibacterial potentials alongside the newly assessed anti-inflammatory effects exhibited consistent associations with the total content of phenolics, reducing sugars, and the identified metabolic profiles in polar (organic acids, amino acids, alcohols, sugars, phenolics) and non-polar (fatty acids, alkanes, sterols) fractions. Phytohormonal levels were also quantified to infer the regulatory pathways governing phytochemical production. The overall dataset highlighted compounds with the potential to contribute to N. nuda bioactivity.

Exploring the Phytochemical Composition and Biological Potential of Balkan Endemic Species Stachys scardica Griseb.

Stachys scardica Griseb. is a Balkan endemic species listed in The Red Data Book of Bulgaria with the conservation status "endangered". Successful micropropagation was achieved on MS medium supplemented with 1.5 mg/L benzyladenine (BA), followed by a subsequent ex vitro adaptation in an experimental field resulting in 92% regenerated plants. Using nuclear magnetic resonance (NMR), phenylethanoid glycosides (verbascoside, leucosceptoside A), phenolic acids (chlorogenic acid), iridoids (allobetonicoside and 8-OAc-harpagide), and alkaloids (trigonelline) were identified, characteristic of plants belonging to the genus Stachys. High antioxidant and radical scavenging activities were observed in both in situ and ex vitro acclimated S. scardica plants, correlating with the reported high concentrations of total phenols and flavonoids in these variants. Ex vitro adapted plants also exhibited a well-defined anti-inflammatory potential, demonstrating high inhibitory activity against the complement system. Employing a disk diffusion method, a 100% inhibition effect was achieved compared to positive antibiotic controls against Staphylococcus epidermidis and Propionibacterium acnes, with moderate activity against Bacillus cereus. The induced in vitro and ex vitro model systems can enable the conservation of S. scardica in nature and offer future opportunities for the targeted biosynthesis of valuable secondary metabolites, with potential applications in the pharmaceutical and cosmetic industries.

Catmint (Nepeta nuda L.) Phylogenetics and Metabolic Responses in Variable Growth Conditions.

Nepeta nuda (catmint; Lamiaceae) is a perennial medicinal plant with a wide geographic distribution in Europe and Asia. This study first characterized the taxonomic position of N. nuda using DNA barcoding technology. Since medicinal plants are rich in secondary metabolites contributing to their adaptive immune response, we explored the N. nuda metabolic adjustment operating under variable environments. Through comparative analysis of wild-grown and in vitro cultivated plants, we assessed the change in phenolic and iridoid compounds, and the associated immune activities. The wild-grown plants from different Bulgarian locations contained variable amounts of phenolic compounds manifested by a general increase in flowers, as compared to leaves, while a strong reduction was observed in the in vitro plants. A similar trend was noted for the antioxidant and anti-herpesvirus activity of the extracts. The antimicrobial potential, however, was very similar, regardless the growth conditions. Analysis of the N. nuda extracts led to identification of 63 compounds including phenolic acids and derivatives, flavonoids, and iridoids. Quantification of the content of 21 target compounds indicated their general reduction in the extracts from in vitro plants, and only the ferulic acid (FA) was specifically increased. Cultivation of in vitro plants under different light quality and intensity indicated that these variable light conditions altered the content of bioactive compounds, such as aesculin, FA, rosmarinic acid, cirsimaritin, naringenin, rutin, isoquercetin, epideoxyloganic acid, chlorogenic acid. Thus, this study generated novel information on the regulation of N. nuda productivity using light and other cultivation conditions, which could be exploited for biotechnological purposes.

Influence of cryopreservation on the antioxidative activity of in vitro cultivated Hypericum species.

Antioxidative activity of two in vitro cultivated Hypericum species - H. rumeliacum Boiss. and H. tetrapterum Fr. - was estimated after cryopreservation. Both species were successfully regenerated after a cryopreservation procedure performed by the vitrification method. H. tetrapterum did not manifest any significant oxidative stress-induced changes caused by low-temperature treatment. Conversely, a decrease in green pigments' content of H. rumeliacum was measured, particularly pronounced in chlorophyll b, which was accompanied by an increase of carotenoids in the regenerated plants. A strong increase of malone dialdehyde and H2O2 levels in H. rumeliacum tissues was detected. Superoxide dismutase activity was enhanced by 170%, as well as the catalase activity, which was 220% above the control. The same trend was observed in H. tetrapterum, although less pronounced - 143% increase of superoxide dismutase and 112% of catalase. Cryopreservation did not influence the phenol content in the examined plants, but it led to an increase of flavonoid content, especially in H. tetrapterum, by 237%. Total antioxidant activity in regenerated H. tetrapterum varied around the control level, but it was increased in H. rumeliacum. The free proline content in H. tetrapterum remained almost unaffected after freezing, as opposed to H. rumeliacum, where a strong increase of proline content (208% above the control) occurred. An electrolyte leakage from the cells of H. rumeliacum regenerated after cryopreservation was also registered, albeit not significant.

Antimicrobial activity of extracts from in vivo and in vitro propagated Lamium album L. plants.

The antimicrobial activity of 18 different extracts from in vivo and in vitro grown L. album L. plants was evaluated against clinical bacteria and yeasts using the well diffusion method. All the used extracts demonstrated antibacterial activity, whereas only the water extracts from leaves (in vivo) possessed antifungal activity against Candida albicans NBIMCC 72 and Candida glabrata NBIMCC 8673 (14 and 20 mm diameter of inhibition zones and MIC 10 mg/ml, respectively). The methanol and ethanol extracts obtained from the in vitro propagated plants had a broader spectrum of antibacterial activity than those from in vivo plants, while the opposite tendency was observed for the chloroform extracts. All tested flower extracts possessed antimicrobial activity. The chloroform extract from in vivo flowers demonstrated the highest activity against E. faecalis NBIMCC 3915, S. aureus NBIMCC 3703, P. hauseri NBIMCC 1339 and P. aeruginosa NBIMCC 3700 (22 mm, 13 mm, 11 mm, 23 mm zone diameter of inhibition and MIC 0.313 mg/ml, respectively). The water extracts from leaves (both in vivo and in vitro) possessed higher antibacterial activity than extract from flowers. The obtained results showed that both in vivo and in vitro propagated L. album L. could be used as a source of antibacterial substances.

Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties.

A thermophilic actinomycete strain Thermoactinomyces sp. 21E producing a highly thermostable serine collagenase was isolated from Bulgarian soil. The collagenase, produced extracellular by Thermoactinomyces sp. 21E, was purified to homogeneity by heat treatment, ultrafiltration, saturation with ammonium sulfate and gel filtration chromatography with a 101-fold increase in specific activity and 58% recovery. The collagenase has a relative molecular mass of 50000 by SDS-PAGE. The optimum temperature for the enzyme activity was 60-65 degrees C in the absence of Ca(2+) and 70-75 degrees C in the presence of Ca(2+). About 40% of the original activity remaining after incubation at 85 degrees C for 30 min in the presence of Ca(2+). The optimum pH for the enzyme activity was 9.0-9.5 and the enzyme was stable for 1h at 70 degrees C in the pH range from 7.5 to 12.5. The collagenase was strongly inhibited by active-site inhibitors of serine protease PMSF and DFP, which indicated that the enzyme is serine protease. The enzyme activity was completely inhibited by Hg(2+), Cu(2+) and Fe(2+). However, Ca(2+ )strongly activated the collagenase activity. The collagenase from Thermoactinomyces sp. 21E showed high activity toward type I collagen, acid-soluble collagen, gelatin and Pz-PLGPR. However, elastin for collagenase was inert as substrate. The properties of the collagenase from strain 21E suggest that this enzyme is a new collagenolytic protease that differs from the collagenases and serine proteases reported so far.