Antioxidant and Anti-Inflammatory Activities of Phenolic Acid-Rich Extract from Hairy Roots of Dracocephalum moldavica.

This study evaluates the antioxidant properties and anti-inflammatory potential of polyphenolic acid-rich fractions of 80% methanolic extract from the hairy roots of Dracocephalum moldavica. The fractionation of the crude extract yielded the following: a diethyl ether fraction rich in caffeic acid (DM1) (25.85 mg/g DWE), an n-butyl fraction rich in rosmarinic acid (DM3) (43.94 mg/g DWE) and a water residue rich in salvianolic acid B (DM4) (51.46 mg/g DWE). The content of these compounds was determined using high-performance liquid chromatography (HPLC). Their antioxidant activity was evaluated based on DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt) and FRAP assays. The anti-inflammatory activity of the fractions was determined by their effect on nuclear factor kappa-B (NF-κB) activation and interleukin-1ß (IL-1ß) production in LPS E. coli stimulated monocytes. The level of pro-inflammatory IL-1ß in cells was measured using ELISA. The activation of NF-κB in THP1-Blue™ cells, resulting in the secretion of SEAP (secreted embryonic alkaline phosphatase), was detected spectrophotometrically using Quanti-Blue reagent. Among the tested fractions, the diethyl ether fraction (DM1) showed the highest antioxidant potential, with an EC50 value of 15.41 µg/mL in the DPPH assay and 11.47 µg/mL in ABTS and a reduction potential of 10.9 mM Fe(II)/g DWE in FRAP. DM1 at a concentration of 10 mg/mL also efficiently reduced LPS-induced SEAP secretion (53% inhibition) and IL-1ß production (47% inhibition) without affecting the normal growth of L929 fibroblast cells.

Effect of Light Conditions on Polyphenol Production in Transformed Shoot Culture of Salvia bulleyana Diels.

Various strategies have been used to increase the efficiency of secondary metabolite production in Salvia plants. This report is the first to examine the spontaneous development of Salvia bulleyana shoots transformed by Agrobacterium rhizogenes on hairy roots and the influence of light conditions on the phytochemical profile of this shoot culture. The transformed shoots were cultivated on solid MS medium with 0.1 mg/L of IAA (indole-3-acetic acid) and 1 mg/L of m-Top (meta-topolin), and their transgenic characteristic was confirmed by PCR-based detection of the rolB and rolC genes in the target plant genome. This study assessed the phytochemical, morphological, and physiological responses of the shoot culture under stimulation by light-emitting diodes (LEDs) with different wavelengths (white, WL; blue, B; red, RL; and red/blue, ML) and under fluorescent lamps (FL, control). Eleven polyphenols identified as phenolic acids and their derivatives were detected via ultrahigh-performance liquid chromatography with diode-array detection coupled to electrospray ionization tandem mass spectrometry (UPLC-DAD/ESI-MS) in the plant material, and their content was determined using high-performance liquid chromatography (HPLC). Rosmarinic acid was the predominant compound in the analyzed extracts. The mixed red and blue LEDs gave the highest levels of polyphenol and rosmarinic acid accumulation (respectively, 24.3 mg/g of DW and 20.0 mg/g of DW), reaching two times greater concentrations of polyphenols and three times greater rosmarinic acid levels compared to the aerial parts of two-year-old intact plants. Similar to WL, ML also stimulated regeneration ability and biomass accumulation effectively. However, the highest total photosynthetic pigment production (1.13 mg/g of DW for total chlorophyll and 0.231 mg/g of DW for carotenoids) was found in the shoots cultivated under RL followed by BL, while the culture exposed to BL was characterized as having the highest antioxidant enzyme activities.

The Effect of the Stress-Signalling Mediator Triacontanol on Biochemical and Physiological Modifications in Dracocephalum forrestii Culture.

Triacontanol (TRIA) has been reported to influence signal transduction in the crosstalk triggered by various stress factors. As a signal player, it is also known to affect many physiological processes, including enhancing the biosynthesis of secondary metabolites. Such knowledge can be used to direct or boost the production of bioactive secondary compounds without stress induction. Therefore, the aim of this study is to evaluate the use of TRIA as a factor stimulating the growth and production of bioactive compounds in the shoot culture of Dracocephalum forrestii. TRIA was applied at three concentrations (2.5, 5, and 10 µM), alone or in combination with phytohormones (6-benzylaminopurine and indole-3-acetic acid). After five weeks, growth and physiochemical parameters (chlorophyll content, antioxidant enzyme activity, and phenolic acid level) were determined. The results indicate that TRIA application significantly increased shoot dry weight, chlorophyll content, antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase), and total polyphenol level; it also influenced the multiplication ratio in combination with growth regulators. The greatest antioxidant enzyme activity was observed for 5 µM TRIA in hormone-free medium, while the most significant secondary metabolite production was obtained for phytohormone-containing medium supplemented with 10 µM TRIA: total phenolic acid content (19.4 mg/g dry weight) was twice that of the control. Hence, the TRIA application appears to be a valuable biotechnology technique for modifying plant metabolite production.

Evaluation of Patient Experiences Regarding Pharmacist-Administrated Vaccination and Attitude towards Future Additional Pharmacy Services in Poland.

In order to increase the number of vaccinations performed during the COVID-19 pandemic in Poland, a significant change was introduced in the legislation allowing for the vaccination to also be performed in pharmacies. A cross-sectional survey was conducted among those who chose a pharmacy as a vaccination point during the pandemic COVID-19. The aim of the study was to determine the overall level of patient satisfaction with pharmacist-administered vaccination in pharmacies and to examine patient opinions regarding the further expansion of pharmacy services. A patient survey was conducted immediately after vaccination in the period from January to March 2022. A total of 398 questionnaires were completed. The respondents reported high satisfaction with pharmacist-administered vaccinations (94.5%). In addition, the majority of consumers felt safe during the vaccination procedure (98.5%), and 88.4% declared they would come for other vaccinations at the pharmacy. The two main reasons for choosing this vaccination place were easy access regarding location (94.2%) and proposed vaccination hours (95.2%). The participants reported high levels of satisfaction concerning the pre-vaccination interview (91.2%), information level before immunization (91.5%), injection technique (96.7%), adjusting the premises to the service (95%) and general care in pharmacy after with vaccination (87.7%). The majority of respondents supported the future expansion of pharmacist-administered services, although the support rate ranged from 52% to 83% depending on the type of service offered. The majority of patients supported the continuation of ongoing prescriptions, blood pressure and blood glucose measurement services by pharmacists. Our findings indicate that the involvement of pharmacies in vaccination programs and other public health services met high levels of patient acceptance.

The effect of different light treatments on morphogenesis, phenolic compound accumulation and antioxidant potential of Dracocephalum forrestii transformed shoots cultured in vitro.

The aim of this study was to evaluate the effects of wavelengths of light emitted from LEDs on cultured in vitro transformed shoots of Dracocephalum forrestii. The shoots were grown on MS agar medium with 0.5 mg/l BPA (N-benzyl-9-(tetrahydropyranyl)-adenine) and 0.2 mg/l IAA (indole-3-acetic acid) under four light environments: blue, red, red/blue (7:3) and white (control). After four weeks of culture, shoot multiplication rate, biomass and morphology were evaluated, as well as bioactive phenolic content, antioxidant capacities and antioxidant enzyme activities. The hydromethanolic extracts from shoots were analyzed using UHPLC method, and antioxidant potential was evaluated using radical scavenging (1,1-diphenyl-2-picrohydrazyl and superoxide anion), and ferric reducing antioxidant power (FRAP), and enzymatic methods, i.e. sodium dismutase (SOD), catalase (CAT) and peroxidase (POD) activity. It was found that the blue and red/blue light had the strongest effect on morphogenesis and shoot propagation; in these conditions, more than five new shoots were obtained per explant. The blue light cultures demonstrated the highest fresh (0.41 g/tube FW) and dry weights (0.045 g/tube DW), the highest levels of polyphenols (99.7 mg/g DW), i.e. almost three times greater than under white light (35.4 mg/g DW), as well as the highest antioxidant potential. Therefore, LED culture appears to be a beneficial strategy for enhancing the production of the medicinal value of transformed D. forrestii shoot culture.

The Protective Function and Modification of Secondary Metabolite Accumulation in Response to Light Stress in Dracocephalum forrestii Shoots.

The aim of this work was to determine the effect of stress conditions caused by different light sources, i.e., blue LED (λ = 430 nm), red LED (λ = 670 nm), blue and red LED (70%:30%) and white LED (430-670 nm) on the growth and morphology of cultivated in vitro Dracocephalum forrestii shoot culture. It also examines the effects on bioactive phenolic compound production and photosynthetic pigment content, as well as on antioxidant enzyme activity (CAT, SOD, POD) and antioxidant properties. The most beneficial proliferation effect was observed under white LEDs (7.1 ± 2.1 shoots per explant). The white and blue lights stimulated the highest fresh weight gain, while red light induced the highest dry weight gain. The total phenolic acid content ranged from 13.824 ± 1.181 to 20.018 ± 801 mg g DW-1 depending on light conditions. The highest content of rosmarinic acid was found in the control shoots (cultivated under fluorescent lamps), followed by culture grown under red light. All LED treatments, especially red and blue, increased salvianolic acid B content, and blue increased apigenin p-coumarylrhamnoside biosynthesis. The greatest ferric reduction activity was observed in shoots cultivated under red light, followed by blue; this is associated with the presence of the highest total phenol content, especially phenolic acids. Similarly, the highest DPPH radical scavenging potential was observed under red light followed by blue. This study proves that LEDs have emerged as significant support for directed in vitro propagation, taking advantage of specific stress responses on various light spectra. This study also showed how stress induced by different LED light spectra increases in Dracocephalum forrestii the synthesis of pharmacologically-active compounds. Hence, light stress may turn out to be a simpler alternative to metabolic engineering for improving the production of secondary metabolites of therapeutic value.

Cytokinin Signaling and De Novo Shoot Organogenesis.

The ability to restore or replace injured tissues can be undoubtedly named among the most spectacular achievements of plant organisms. One of such regeneration pathways is organogenesis, the formation of individual organs from nonmeristematic tissue sections. The process can be triggered in vitro by incubation on medium supplemented with phytohormones. Cytokinins are a class of phytohormones demonstrating pleiotropic effects and a powerful network of molecular interactions. The present study reviews existing knowledge on the possible sequence of molecular and genetic events behind de novo shoot organogenesis initiated by cytokinins. Overall, the review aims to collect reactions encompassed by cytokinin primary responses, starting from phytohormone perception by the dedicated receptors, to transcriptional reprogramming of cell fate by the last module of multistep-phosphorelays. It also includes a brief reminder of other control mechanisms, such as epigenetic reprogramming.

Transformed Shoots of Dracocephalum forrestii W.W. Smith from Different Bioreactor Systems as a Rich Source of Natural Phenolic Compounds.

Transformed shoots of the Tibetan medicinal plant Dracocephalum forrestii were cultured in temporary immersion bioreactors (RITA and Plantform) and in nutrient sprinkle bioreactor (NSB) for 3 weeks in MS (Murashige and Skoog) liquid medium with 0.5 mg/L BPA (N-benzyl-9-(2-tetrahydropyranyl)-adenine) and 0.2 mg/L IAA (indole-3-acetic acid). The greatest biomass growth index (GI = 52.06 fresh weight (FW) and 55.67 dry weight (DW)) was observed for shoots in the RITA bioreactor, while the highest multiplication rate was found in the NSB (838 shoots per bioreactor). The levels of three phenolic acids and five flavonoid derivatives in the shoot hydromethanolic extract were evaluated using UHPLC (ultra-high performance liquid chromatography). The predominant metabolite was rosmarinic acid (RA)-the highest RA level (18.35 mg/g DW) and total evaluated phenol content (24.15 mg/g DW) were observed in shoots grown in NSB. The NSB culture, i.e., the most productive one, was evaluated for its antioxidant activity on the basis of reduction of ferric ions (ferric reducing antioxidant power, FRAP) and two scavenging radical (O2•- and DPPH, 1,1-diphenyl-2-picrylhydrazyl radical) assays; its antibacterial, antifungal, and antiproliative potential against L929 cells was also tested (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test). The plant material revealed moderate antioxidant and antimicrobial activities and demonstrated high safety in the MTT test-no cytotoxicity at concentrations up to 50 mg/mL was found, and less than a 20% decrease in L929 cell viability was observed at this concentration.

Computer-Aided Saturation Mutagenesis of Arabidopsis thaliana Ent-Copalyl Diphosphate Synthase.

Ent-copalyl diphosphate synthase controls the biosynthesis of gibberellin plant hormones, which in turn coordinate the expression of numerous enzymes. Some gibberellin-dependent genes encode enzymes coordinating the biosynthesis of tanshinones: diterpene derivatives with broad medical applications. New biotechnological approaches, such as metabolic engineering using naturally occurring or mutated enzymes, have been proposed to meet the growing demand for tanshinones which is currently met by the Chinese medicinal plant Salvia miltiorrhiza Bunge. These mutants may be prepared by directed evolution, saturation mutagenesis or rational enzyme design. In the presented paper, 15,257 non-synonymous variants of Arabidopsis thaliana ent-copalyl diphosphate synthase were obtained using the SNAP2 tool. The obtained forms were screened to isolate variants with potentially improved biological functions. A group of 455 mutants with potentially improved stability was isolated and subjected to further screening on the basis of ligand-substrate affinity, and both secondary structure and active site structure stability. Finally, a group of six single mutants was obtained, which were used to construct double mutants with potentially improved stability and ligand affinity. The potential influence of single mutations on protein stability and ligand affinity was evaluated by double mutant cycle analysis. Finally, the procedure was validated by in silico assessment of the experimentally verified enzyme mutants with reduced enzymatic activity.

The effect of purine-type cytokinin on the proliferation and production of phenolic compounds in transformed shoots of Dracocephalum forrestii.

Dracocephalum forrestii is a perennial, endemic to China plant with a number of pharmaceutical properties. Transformed shoots of the species spontaneously regenerated from hairy roots induced by Agrobacterium rhizogenes. The transgenic nature of the shoots was confirmed by polymerase chain reaction (PCR). The shoot culture was multiplied on Murashige and Skoog (MS) medium with 0.2 mg/l IAA and 0.2, 0.5, 1.0, 2.0 or 5.0 mg/l purine-type cytokinins (mT, BAR, BPA or BAP). The highest multiplication rate (about thirteen shoot or buds per explant) was obtained on MS medium with 0.2 mg/l mT after four weeks of culture. The phenolic compounds present in the hydromethanolic extracts from the D. forrestii transgenic shoots were characterized using UPLC-PDA-ESI-MS. The shoots were found to biosynthesize three phenolic acids and five flavonoid glycosides. UHPLC analysis of the hydromethanolic extracts found the predominant phenolic acid to be rosmarinic acid, with its highest content observed in shoots cultivated with 5.0 mg/l BPA. In contrast, the greatest production of flavonoid derivatives (especially acacetin derivatives) was observed in the medium supplemented with 2 mg/l BPA.