Phytochemistry and Biological Activities of Agrostemma Genus-A Review.

The family Caryophyllaceae comprises more than 2600 species spread widely across all the continents. Their economic importance is mainly as ornamentals (carnation) and as weeds in agriculture. Some species have been used traditionally (and some are still) in herbal medicine or as emulsifiers in food processing. These applications are based on the high content of triterpenoid saponins. Typical for this family are also ribosome-inactivating proteins (RIPs), which are potentially highly toxic. Agrostemma githago L. (common corncockle) was historically considered a serious toxicological hazard owing to cereal grain contamination by its seeds. Notwithstanding, it was also recommended as a drug by various herbalists. In this review, the literature was searched in the PubMed, Google Scholar, and Scopus databases for papers focused on the chemical composition and bioactivity of the two accepted species of the Agrostemma genus. This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and MetaAnalysis (PRISMA) guidelines. Current research reports the cytotoxicity against neoplastic cells; the protection against oxidative stress; the suppression of Leishmania major culture growth; the inhibition of protein synthesis; and the antiviral, anti-angiogenic, and antihypercholesterolemic activities of common corncockle. The future prospects of using A. githago saponins as adjuvants in drug formulations and enhancing the cytotoxicity of RIPs are also discussed.

Protopine and Allocryptopine Interactions with Plasma Proteins.

A comprehensive study of the interactions of human serum albumin (HSA) and α-1-acid glycoprotein (AAG) with two isoquinoline alkaloids, i.e., allocryptopine (ACP) and protopine (PP), was performed. The UV-Vis spectroscopy, molecular docking, competitive binding assays, and circular dichroism (CD) spectroscopy were used for the investigations. The results showed that ACP and PP form spontaneous and stable complexes with HSA and AAG, with ACP displaying a stronger affinity towards both proteins. Molecular docking studies revealed the preferential binding of ACP and PP to specific sites within HSA, with site 2 (IIIA) being identified as the favored location for both alkaloids. This was supported by competitive binding assays using markers specific to HSA's drug binding sites. Similarly, for AAG, a decrease in fluorescence intensity upon addition of the alkaloids to AAG/quinaldine red (QR) complexes indicated the replacement of the marker by the alkaloids, with ACP showing a greater extent of replacement than PP. CD spectroscopy showed that the proteins' structures remained largely unchanged, suggesting that the formation of complexes did not significantly perturb the overall spatial configuration of these macromolecules. These findings are crucial for advancing the knowledge on the natural product-protein interactions and the future design of isoquinoline alkaloid-based therapeutics.

Comparative transcriptomics of two Salvia subg. Perovskia species contribute towards molecular background of abietane-type diterpenoid biosynthesis.

Tanshinones, are a group of diterpenoid red pigments present in Danshen - an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography-Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy.

Seasonal and Geographic Dynamics in Bioproperties and Phytochemical Profile of Limonium algarvense Erben.

This study delved into the influence of ecological and seasonal dynamics on the synthesis of secondary metabolites in the medicinal halophyte Limonium algarvense Erben, commonly known as sea lavender, and examined their antioxidant and anti-inflammatory properties. Aerial parts of sea lavender were systematically collected across winter, spring, summer, and autumn seasons from distinct geographic locations in southern Portugal, specifically "Ria de Alvor" in Portimão and "Ria Formosa" in Tavira. The investigation involved determining the total polyphenolic profile through spectrophotometric methods, establishing the chemical profile via liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), and evaluating in vitro antioxidant properties using radical and metal-based methods, along with assessing anti-inflammatory capacity through a cell model. Results unveiled varying polyphenol levels and profiles across seasons, with spring and autumn samples exhibiting the highest content, accompanied by the most notable antioxidant and anti-inflammatory capacities. Geographic location emerged as an influential factor, particularly distinguishing plants from "Ria de Alvor". Seasonal fluctuations were associated with environmental factors, including temperature, which, when excessively high, can impair plant metabolism, but also with the presence of flowers and seeds in spring and autumn samples, which also seems to contribute to elevated polyphenol levels and enhanced bioproperties of these samples. Additionally, genetic factors may be related to differences observed between ecotypes (geographical location). This study underscores sea lavender's potential as a natural source of antioxidant and anti-inflammatory agents, emphasizing the significance of considering both geographic location and seasonal dynamics in the assessment of phenolic composition and bioactive properties in medicinal plant species.

The combination of pro-oxidative acting vanicosides and GLUT1 inhibitor (WZB117) exerts a synergistic cytotoxic effect against melanoma cells.

Vanicosides A and B isolated from Reynoutria sachalinensis rhizomes are disaccharide phenylpropanoid esters with proven antioxidant activity. Our earlier study showed the cytotoxic activity of vanicosides against melanoma cells, but the mechanism of cell death has not been elucidated. Based on the chemical structure of vanicosides, we proposed that they may induce cell death by generating reactive oxygen species (ROS) into melanoma cells. Moreover, the glucose molecule in their structure can affect the glucose transporters (GLUTs), upregulated in cancer cells. The A375 (melanotic) and C32 (amelanotic) melanoma cell lines were applied. Cell viability assay and ROS-Glo™ assay were performed before and after blocking of Glucose Transporter Type 1 (GLUT1) by WZB117. Fibroblasts and the SKOV-3 line were included in the study to test selectivity in the action of vanicosides and help to elucidate the mechanism of action. Upon incubation with vanicosides, high production of ROS occured, especially inside C32 cells, which was significantly reduced after GLUT-1 blocking. The A375 cells produced less ROS. Melanoma cells were simillary sensitive to the cytotoxic effects of vanicosides, which was clearly enhanced when vanicosides were used together with the WZB117 (GLUT1 inhibitor). The SKOV-3 line and the fibroblasts showed much less sensitivity to the cytotoxicity of vanicosides, also used together with WZB117. Moreover, no significant ROS formation was observed in these lines. The study proved that vanicosides generate ROS inside melanoma cells. These findings suggest that the combination of pro-oxidative acting vanicosides and GLUT1 inhibitors exerts a synergistic cytotoxic effect on melanoma cells.

Metabolic and Transcriptomic Approaches of Chitosan and Water Stress on Polyphenolic and Terpenoid Components and Gene Expression in Salvia abrotanoides (Karl.) and S. yangii.

In this research, a HPLC analysis, along with transcriptomics tools, was applied to evaluate chitosan and water stress for the prediction of phenolic flavonoids patterns and terpenoid components accumulation in Salvia abrotanoides Karel and S. yangii. The results indicated that the tanshinone contents under drought stress conditions increased 4.2-fold with increasing drought stress intensity in both species. The rosmarinic acid content in the leaves varied from 0.038 to 11.43 mg/g DW. In addition, the flavonoid content was increased (1.8 and 1.4-fold) under mild water deficit conditions with a moderate concentration of chitosan (100 mg L-1). The application of foliar chitosan at 100 and 200 mg L-1 under well-watered and mild stress conditions led to increases in hydroxyl cryptotanshinone (OH-CT) and cryptotanshinone (CT) contents as the major terpenoid components in both species. The expressions of the studied genes (DXS2, HMGR, KSL, 4CL, and TAT) were also noticeably induced by water deficit and variably modulated by the treatment with chitosan. According to our findings, both the drought stress and the application of foliar chitosan altered the expression levels of certain genes. Specifically, we observed changes in the expression levels of DXS and HMGR, which are upstream genes in the MEP and MVA pathways, respectively. Additionally, the expression level of KSL, a downstream gene involved in diterpenoid synthesis, was also affected. Finally, the present investigation confirmed that chitosan treatments and water stress were affected in both the methylerythritol phosphate pathway (MEP) and mevalonate (MVA) pathways, but their commitment to the production of other isoprenoids has to be considered and discussed.

Bioassay-guided isolation in Salvia abrotanoides Karel. stem based on its anti-fungal and anti-trichomonas activity.

Background and purpose: Salvia abrotanoides is considered a medicinal plant and has a broad distribution in Iran. In Iran's traditional medicine, it is also used to treat leishmaniasis, malaria, atherosclerosis, cardiovascular disease, and as a disinfectant. This research aimed to determine the anti-Candida component from S. abratonoides and anti-Trichomonas natural compounds from the stems of this plant. Experimental approach: The plant shoots were collected, dried, and after removing the leaves, grounded. Dried plant material was extracted in a maceration tank, concentrated by a Rotavap, degreased, and fractionated by normal column chromatography. Based on anti-fungal screening against Candida species, Fr. 4, with more anti-fungal activity, was selected for phytochemical analysis, by different chromatographic methods on the silica gel column and Sephadex LH-20. Isolated compounds were elucidated by NMR analysis, mass spectrum, and ultraviolet spectroscopy. Anti-fungal effects were investigated using the fungal suspension, incubation, and parasite-counting methods on purified compounds. Antibacterial effects were assessed using the Broth dilution test and reported according to the MIC parameter. Findings/Results: Two diterpenoid compounds named carnosol (compound 1), 11-hydroxy-12-methoxy-20-norabiata-8, 11, 13-trien (compound 2), and a flavonoid: 6,7-dimethoxy-5, 4'-dihydroxyflavone (compound 3) were isolated and identified. Compound 1 had selective anti-fungal effects against C. albicans, C. glabrata, and C. parapsilosis, but weak toxicity against Trichomonas vaginalis with IC50 of 675.8 µg/mL, less than metronidazole with an IC50 of 13.2 µg/mL. Conclusion and implications: Carnosol as the main component was assayed against Candida, Aspergillus, Rhizopus, and Trichomanas species. The results confirmed its effect on Candida compared to standard drugs.

Oral Wound Healing Potential of Polygoni Cuspidati Rhizoma et Radix Decoction-In Vitro Study.

Polygoni Cuspidati Rhizoma et Radix (syn. rhizomes of Reynoutria japonica Houtt.) is a pharmacopoeial raw material in Europe and China. In traditional medicine, one of the applications for Reynoutria japonica rhizomes is wound healing. In a recent in vitro study, we demonstrated that ethanol and acetone extracts from this herbal drug have the potential to heal oral gum wounds. However, considering that a majority of herbal medicines have been traditionally administered as water decoctions, in the present study, a decoction of Reynoutria japonica rhizomes was prepared and detailed tests to determine its in vitro gingival wound healing activity were conducted. We used the primary human gingival fibroblasts (HGF) incubated with a decoction to determine cell viability (MTT assay), cell proliferation (the confocal laser scanning microscope-CLSM), and cell migration (wound healing assay). Moreover, the collagen type III expression was examined using immunocytochemical staining. The studied decoction was qualitatively and quantitatively characterized using the validated HPLC/DAD/ESI-HR-QTOF-MS method. The Folin-Ciocalteu assay was used to determine the total phenols and tannins content. Additionally, HPLC-RI analysis of decoction and the previously obtained ethanol and acetone extracts was used to determine the composition of saccharides. Low concentration (from 50 to 1000 µg/mL) of decoction after 24 h incubation caused a significant increase in HGF cell viability. No cytotoxic effect was observed at any tested concentration (up to 2000 µg/mL). The lowest active concentration of decoction (50 µg/mL) was selected for further experiments. It significantly stimulated human gingival fibroblasts to proliferate, migrate, and increase the synthesis of collagen III. Phytochemical analysis showed significantly fewer polyphenols in the decoction than in the ethanol and acetone extracts tested earlier. In contrast, high levels of polysaccharides were observed. In our opinion, they may have a significant effect on the oral wound healing parameters analyzed in vitro. The results obtained encourage the use of this raw material in its traditional, safe form-decoction.

Identification of Polyphenolic Compounds Responsible for Antioxidant, Anti-Candida Activities and Nutritional Properties in Different Pistachio (Pistacia vera L.) Hull Cultivars.

The use of by-products from the agri-food industry is a promising approach for production of value-added, polyphenol-rich dietary supplements or natural pharmaceutical preparations. During pistachio nut processing, a great amount of husk is removed, leaving large biomass for potential re-use. The present study compares antiglycative, antioxidant, and antifungal activities as well as nutritional values of 12 genotypes belonging to four pistachio cultivars. Antioxidant activity was measured using DPPH and ABTS assays. Antiglycative activity was evaluated as inhibition of advanced glycation end product (AGE) formation in the bovine serum albumin/methylglyoxal model. HPLC analysis was performed to determine the major phenolic compounds. Cyanidin-3-O-galactoside (120.81-181.94 mg/100 g DW), gallic acid (27.89-45.25), catechin (7.2-11.01), and eriodictyol-7-O-glucoside (7.23-16.02) were the major components. Among genotypes, the highest total flavonol content (14.8 mg quercetin equivalents/g DW) and total phenolic content (262 mg tannic acid equivalent/g DW) were in KAL1 (Kaleghouchi) and FAN2 (Fandoghi), respectively. The highest antioxidant (EC50 = 375 µg/mL) and anti-glycative activities were obtained for Fan1. Furthermore, potent inhibitory activity against Candida species was recorded with MIC values of 3.12-12.5 µg/mL. The oil content ranged from 5.4% in Fan2 to 7.6% in Akb1. The nutritional parameters of the tested cultivars were highly variable: crude protein (9.8-15.8%), ADF (acid detergent fiber 11.9-18.2%), NDF (neutral detergent fiber, 14.8-25.6%), and condensed tannins (1.74-2.86%). Finally, cyanidin-3-O-galactoside was considered an effective compound responsible for antioxidant and anti-glycative activities.

Essential Oil Composition and Antioxidant Activity of Oregano and Marjoram as Affected by Different Light-Emitting Diodes.

Oregano and marjoram are important aromatic spices in the food industry, as well as medicinal plants with remarkable antioxidant properties. Despite their popularity, little is known about treatments that would influence the antioxidant capacity of essential oils. In this study, different spectra of LED light, namely blue, red, white, blue-red, and natural ambient light as a control, were applied to assess the essential oil content, composition, flavonoid, phenolic, and antioxidant capacity of oregano and marjoram. GC-MS analysis revealed thymol, terpinen-4-ol, sabinene, linalool, p-cymene, and γ-terpinene as the main compounds. In oregano, the thymol content ranged from 11.91% to 48.26%, while in marjoram it varied from 17.47% to 35.06% in different samples. In oregano and marjoram, the highest phenolic contents were in blue (61.26 mg of tannic acid E/g of DW) and in white (65.18 mg of TAE/g of DW) light, respectively, while blue-red illumination caused the highest increase in total flavonoids. The antioxidant activity of oregano and marjoram extract was evaluated using two food model systems, including DPPH and ß-carotene bleaching. The highest antioxidant capacity was obtained in control light in oregano and blue-red light in marjoram. The results provide information on how to improve the desired essential oil profile and antioxidant capacity of extracts for industrial producers.

Electrostimulation improves plant growth and modulates the flavonoid profile in aeroponic culture of Scutellaria baicalensis Georgi.

Scutellaria baicalensis Georgi is a valuable medicinal plant of the Lamiaceae family. The roots, Scutellariae baicalensis radix, are valued in the traditional medicine of East Asia and are also listed in several pharmacopeias, such as the Chinese and European versions. The roots contain a high amount of flavones, such as baicalein, wogonin and their glucuronides, baicalin and wogonoside, respectively, with rare structures of unsubstituted B-ring. These major constituents are responsible for its pharmacological activity, mainly anti-inflammatory, antiviral, and antitumor, as well as BDZ-receptor modulating. There is a fast-growing demand for both the crude drug and the individual flavonoids obtained from it. However, the variability of content and composition of flavonoids in the roots is significant and affects pharmaceutical use, and little is known about the influence of various factors on root quality. In our experiments, we use aeroponics to determine the effect of electroporation as an abiotic stressor on plant growth, development, and root mass, as well as on its metabolic profile. Results: Electroporation significantly impacted plant growth and the content of flavonoids, especially baicalein and wogonin, depending on the treatment parameters. Concentrations of aglycones were increased in at least half of the treatment conditions. The greatest amounts (a 2.5-fold increase compared to controls) were recorded after applying an electrical field characterized by the following parameters: E = 3 kV/cm, t = 100 µs, and N = 10. In conclusion, electrostimulation is an innovative and efficient way to increase plant growth and yield in an aeroponic system, as well as modulate the profile and content of bioactive flavones in the roots. However, the fine-tuning of these parameters, such as the electrical field strength (E), length (t), and number (N) of impulses delivered, is of great importance. It was also shown that cultivation of the experimental plants in aeroponics had a positive impact on their survival and development while being a sustainable and efficient horticultural practice.

Changes in polyphenolic composition, physiological characteristics, and yield-related traits of Moshgak (Ducrosia anethifolia Boiss.) populations in response to drought stress.

Ducrosia anethifolia (DC.) Boiss. is an aromatic medicinal plant that has been traditionally used as an analgesic to treat headaches, backaches, colic, and cold. This study evaluated the yield, physiological, and phytochemical traits of 24 populations for 2 consecutive years under the water stress condition. The seed yield and physiological traits demonstrated the highest values in the first and second year, respectively. Hydrogen peroxide (H2O2), proline, malondialdehyde (MDA), and antioxidant activity enzymes were elevated, while chlorophyll, carotenoids, relative water content (RWC), and yield decreased under drought stress. High-performance liquid chromatography (HPLC) was also applied to assess the changes in some polyphenolic compounds in response to water stress. The increase in some phenolic compounds, such as p-coumaric acid, was recorded due to drought stress, while there was a decrease in flavonoids, that is luteolin and quercetin. Among the populations, Abarkuh2 indicated the highest increase in p-coumaric acid (96%) in response to drought stress. In general, high diversity among the studied populations provides new insights into choosing the beneficial populations for medicinal and food purposes. HIGHLIGHTS: • Changes in polyphenolics of Moshgak populations were obtained in response to water stress. • Gallic acid, ferulic acid, p-coumaric acid and vanillic acid were the major components. • The phenolic compounds was increased due to drought stress while flavonoids were decreased High variation was obtained between Moshgak populations.

Mycorrhizal Colonization Modulates the Essential Oil Profile and Enzymatic and Non-Enzymatic Antioxidants to Mitigate the Adverse Effects of Water Deficit in Salvia subg. Perovskia.

Among traditional Iranian herbs, Perovskia species (a subgenus of Salvia), while being valued ornamentals, are also studied for numerous potential pharmacological and therapeutic aspects. The current study was conducted to assess the effectiveness of two species of arbuscular mycorrhizal fungi (AMF), Funneliformis mosseae and Rhizophagus intraradices, separately and in combination, in terms of the essential oil content and compositions along with the enzymatic and non-enzymatic antioxidants in Salvia abrotanoides and S. yangii in response to three levels of irrigation, including 100% FC as well-watered, 75% FC (moderate irrigation deficit), and 50% FC (severe irrigation deficit). In both species, essential oil content, enzyme antioxidant activities, total phenolics, and flavonoids were increased significantly with the severity of stress; this increase was more pronounced in mycorrhizal inoculated herbs. Furthermore, leaf phosphorus concentration, relative water content, chlorophylls a and b, and total carotenoids decreased in parallel with reducing soil moisture; albeit, AMF inoculation improved the stress symptoms under increasing severity of water restriction compared with their control conditions. In addition, the percentage of root colonization was positively correlated with the relative water content (RWC) and leaf phosphorus concentration. Taking into account the essential oil groups, AMF colonization elevated some essential oil components, such as oxygenated monoterpenes, 1,8-cineol, camphor, and borneol, whereas the main sesquiterpenes, including E-ß-caryophyllene and α-humulene, remarkably decreased. Taken together, these findings highlighted the role of symbiosis with AMFs in increasing the tolerance of water deficit stress in S. abrotanoides and S. yangii and improving their essential oil composition.

Metabolic and physiological effects of water stress on Moshgak (Ducrosia anethifolia Boiss) populations using GC-MS and multivariate analyses.

Ducrosia anethifolia Boiss is a medicinal and aromatic plant that is used as a food additive and drug in the areas of natural distribution. In this study, eight populations from different geographical regions of Iran were evaluated for their essential oil composition, total phenolics and flavonoids as well as for free radical scavenging activity during two consecutive years under water deficit. Analysis of variance was performed using a randomized block design at three levels of irrigation and 2 years, using the GLM procedure of SAS software and cluster analysis was used according to Ward's method using squared Euclidean dissimilarity. The highest essential oil and total phenolics/flavonoids content were obtained in the second year. GC-MS analysis was used to analyze the essential oil components. In normal conditions, cis-chrysanthenyl acetate (39.1-66.4%) and α-pinene (1.02-4.7%) were recognized as major compounds. The essential oil components were elevated under water stress. The highest increase in cis-chrysanthenyl acetate (21.32%) in response to water stress was observed in Abarkuh1. Elevation in total phenolic, flavonoid and antioxidant activity was also observed in plants subjected to water deficit. The highest content of phenolic acids was measured in Semirom (136.74 mg TAE/g DW), while the highest flavonoid content was in Kerman (6.05 mg QUE/g DW) in severe water stress condition. Finally, a high diversity in the studied populations can be used to select advantageous populations for other pharmaceutical and food purposes.

Changes in Essential Oil Composition, Polyphenolic Compounds and Antioxidant Capacity of Ajowan (Trachyspermum ammi L.) Populations in Response to Water Deficit.

Ajowan (Trachyspermum ammi L.) is considered a valuable spice plant with a high thymol content. Seed yield, essential oil constituents, polyphenolic composition, and antioxidant capacity of ajowan (Trachyspermum ammi L.) populations were evaluated in three (normal, moderate, and severe) water irrigation regimes. The highest essential oil content (5.55%) was obtained under normal condition in the Yazd population. However, both essential oil and seed yield showed significant reductions as a result of water stress. According to gas chromatography-mass spectrometry (GC-MS) analysis, thymol (61.44%), γ-terpinene (26.96%), and p-cymene (20.32%) were identified as the major components of the oil. The highest (89.01%) and the lowest (37.54%) thymol contents were in Farsmar and Hamadan populations in severe stress condition, respectively. Based on HPLC analysis, chlorogenic (3.75-47.35 mg/100 g), caffeic (13.2-40.10 mg/100 g), and ferulic acid (11.25-40.10 mg/100 g) were identified as the major phenolic acids, while rutin was determined as the major flavonoid (11.741-20.123 mg/100 g). Moreover, total phenolic and flavonoid contents were elevated under drought stress treatment, while antioxidants responded inconsistently to stress based on two model systems. Overall, the Yazd population exhibited a superior response to water stress, as evidenced by its less reduced thymol and oil yield content, while Arak and Khormo had the highest accumulation of polyphenolic compounds.

Use of natural cysteine protease inhibitors in limiting SARS-Co-2 fusion into human respiratory cells.

Specific antibodies that humans acquire as a result of disease or after vaccination are needed to effectively suppress infection with a specific variant of SARS CoV-2 virus. The S protein of the D614G variant of coronavirus is used as an antigen in known vaccines to date. It is known that COVID-19 disease resulting from infection with this coronavirus can often be very dangerous to the health and lives of patients. In contrast, vaccines produce antibodies against an older version of the protein S-D614G (January 2020) and therefore have difficulty recognizing new variants of the virus. In our project we propose to obtain specific and precise antibodies by means of so-called controlled infection against specific infectious variants of the SARS-CoV-2 virus "here and now". Currently, several variants of this pathogen have already emerged that threaten the health and lives of patients. We propose to reduce this threat by partially, but not completely, blocking the fusion mechanism of the SARS-CoV-2 virus into human respiratory cells. According to our plan, this can be achieved by inhibiting cathepsin L activity in respiratory cells, after introducing natural and non-toxic cysteine protease inhibitors into this area. We obtain these inhibitors by our own method from natural, "human body friendly" natural resources. We hypothesize that blocking cathepsin L will reduce the number of infecting viruses in cells to such an extent that COVID-19 developing in infected individuals will not threaten their health and life. At the same time, the number of viruses will be sufficient for the body's own immune system to produce precise antibodies against a specific version of this pathogen.

A first approach for the micropropagation of the medicinal halophyte Polygonum maritimum L. and phenolic profile of acclimatized plants.

Polygonum maritimum L. (sea knotgrass) belongs to a genus commonly used in folk medicine to treat inflammation-related disorders. In vitro pharmacological studies have confirmed these properties that were ascribed to bioactive flavonoids, such as myricetin and quercetin glycosides. Therefore, this study aimed at establishing a micropropagation procedure for sea knotgrass for obtaining standardized materials for its potential commercial cultivation. For that, a complete plant regeneration protocol was developed by improving shoot multiplication from nodal explants, rooting and acclimatization procedures, followed by the assessment of the phenolic profile of the in vitro-produced plants. The combination of 3 mg/L 6-benzylaminopurine (BA) + 0.1 mg/L indole-3-acetic acid (IAA) induced the maximum shoot formation (10.3), which was significantly increased from the first to the second cycle (18.3). The best rooting capacity was observed on shoots derived from the control medium (100%), followed by 2 mg/L kinetin (KIN) (97%) and 3 mg/L BA + 0.1 mg/L IAA (90%); however, the shoot number at the end of the rooting phase was higher on shoots derived from 3 mg/L BA + 0.1 mg/L IAA (6.16). The plant growth regulators used in the multiplication phase influenced survival in the acclimatization process, and plants derived from the control medium had the highest survival percentage (63.1%). Acetone extracts made from aerial organs of micropropagated sea knotgrass showed a predominance of the flavonoid myricetin-3-O-rhamnoside (8.135 mg/g). Overall, the halophyte sea knotgrass was successfully micropropagated showing its potential as a medicinal crop for the extraction of bioactive molecules.

Light Intensity and Temperature Effect on Salvia yangii (B. T. Drew) Metabolic Profile in vitro.

Plant in vitro culture is a feasible system for the testing influence of an environmental factor on the accumulation and chemodiversity of specialized metabolites, especially in medicinal plants. Light and temperature are among the most important factors affecting the physiology of plant organisms but their influence on specific metabolic pathways is not completely understood. Here, we examined the morphogenetic response, photosynthetic pigments content, lipid peroxidation level, DPPH radical scavenging activity, and the production of volatile and non-volatile constituents in Salvia yangii B. T. Drew (syn. Perovskia atriplicifolia Benth.) in vitro cultures kept under different light intensities (70, 130, and 220 µmol m-2 s-1) and at two selected temperatures (25 and 30°C). The experiment was continued for 7 months to monitor the changes in the treatment response in time. Phytochemical analysis was performed using chromatographic (GC-MS and UHLPC) and spectrophotometric techniques. The light intensity significantly influenced metabolic response in a non-linear manner, whereas temperature-induced adaptive modifications varied within the long cultivation. Significant differences were noted in the content of carnosic and rosmarinic acid, as well as in several sesquiterpenes (alloaromadendrene, ß-caryophyllene, α-humulene). At elevated (30°C) temperature, a trend of differently modulated content of two major antioxidants-rosmarinic acid (RA, a phenylpropanoid pathway derived phenolic acid) and carnosic acid (CA, an abietane diterpenoid) was observed, where RA, but not CA, was depending on the light intensity. At 25°C, both compounds depended on light but in various ways. Among the volatile terpenoid compounds, the influence of light was pronounced, leading to modulation of proportions between individual mono- and sesquiterpenes as well as between hydrocarbon and oxygenated compounds. The study provided new information on the metabolic profile plasticity in S. yangii and added to the existing knowledge on the chemical adaptations in plant species from severe habitats.

In vitro activity of juglone (5-hydroxy-1,4-naphthoquinone) against both fluconazole-resistant and susceptible Candida isolates.

BACKGROUND: The rise in antifungal resistance and drug class limitations are causing higher morbidity and mortality rates all over the world. This issue highlights the urgent need for new and improved antifungal drugs with a novel target. AIMS: In order to evaluate whether juglone can be served as an alternative antifungal to cure drug-resistant Candida infections, we studied the in vitro susceptibility of juglone against fluconazole-susceptible and -resistance Candida isolates, alone and in combination. METHODS: Antifungal susceptibility testing was performed according to the CLSI (Clinical and Laboratory Standards Institute) guidelines. RESULTS: Juglone exhibited the highest minimal inhibitory concentration (MIC) values, followed by fluconazole and nystatin. Voriconazole showed significantly better antifungal activity than juglone, fluconazole, and nystatin, with MIC50 and MIC90 of 0.031 and 0.5µg/mL. There were significant differences in MICs of fluconazole (p<0.001) and juglone (p<0.0003) between Candidaalbicans and the rest of the species. Combination of juglone with fluconazole revealed insignificant effects against fluconazole-susceptible and -resistant Candida isolates. Juglone increased the antifungal activity of fluconazole; however, no synergism effects were observed for any combination, and only an insignificant effect was found against all tested Candida species. CONCLUSIONS: Although obtaining new antifungal drugs is a critical point, a completely novel approach should be implemented.

Non-psychotropic cannabinoids as inhibitors of TET1 protein.

Non-psychotropic cannabinoids (e.g., cannabidiol, cannabinol and cannabigerol) are contained in numerous alimentary and medicinal products. Therefore, predicting and studying their possible side effects, such as changes in DNA methylation, is an important task for assessing the safety of these products. Interference with TET enzymes by chelating ferrous ions can contribute to the altered methylation pattern. All tested cannabinoids displayed a strong affinity for Fe(II) ions. Cannabidiol and cannabinol exhibited potent inhibitory activities (IC50 = 4.8 and 6.27 µM, respectively) towards the TET1 protein, whereas cannabigerol had no effect on the enzyme activity. An in silico molecular docking study revealed marked binding potential within the catalytic cavity for CBD/CBN, but some affinity was also found for CBG, thus the total lack of activity remains unexplained. These results imply that cannabinoids could affect the activity of the TET1 protein not only due to their affinity for Fe(II) but also due to other types of interactions (e.g., hydrophobic interactions and hydrogen bonding).