Plant In Vitro Culture Factories for Pentacyclic Triterpenoid Production.

Pentacyclic triterpenoids are a diverse subclass of naturally occurring terpenes with various biological activities and applications. These compounds are broadly distributed in natural plant resources, but their low abundance and the slow growth cycle of plants pose challenges to their extraction and production. The biosynthesis of pentacyclic triterpenoids occurs through two main pathways, the mevalonic acid (MVA) pathway and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which involve several enzymes and modifications. Plant in vitro cultures, including elicited and hairy root cultures, have emerged as an effective and sustainable system for pentacyclic triterpenoid production, circumventing the limitations associated with natural plant resources. Bioreactor systems and controlling key parameters, such as media composition, temperature, light quality, and elicitor treatments, have been optimized to enhance the production and characterization of specific pentacyclic triterpenoids. These systems offer a promising bioprocessing tool for producing pentacyclic triterpenoids characterized by a low carbon footprint and a sustainable source of these compounds for various industrial applications.

Transcriptional and Metabolic Profiling of Arabidopsis thaliana Transgenic Plants Expressing Histone Acetyltransferase HAC1 upon the Application of Abiotic Stress-Salt and Low Temperature.

Augmented knowledge of plant responses upon application of stress could help improve our understanding of plant tolerance under abiotic stress conditions. Histone acetylation plays an important role in gene expression regulation during plant growth and development and in the response of plants to abiotic stress. The current study examines the level of transcripts and free metabolite content in transgenic Arabidopsis thaliana plants expressing a gene encoding histone acetyltransferase from Medicago truncatula (MtHAC1) after its heterologous expression. Stable transgenic plants with HAC1 gain and loss of function were constructed, and their T5 generation was used. Transgenic lines with HAC1-modified expression showed a deviation in root growth dynamics and leaf area compared to the wild-type control. Transcriptional profiles were evaluated after the application of salinity stress caused by 150 mM NaCl at four different time points (0, 24, 48, and 72 h) in treated and non-treated transgenic and control plants. The content and quantity of free metabolites-amino acids, mono- and dicarbohydrates, organic acids, and fatty acids-were assessed at time points 0 h and 72 h in treated and non-treated transgenic and control plants. The obtained transcript profiles of HAC1 in transgenic plants with modified expression and control were assessed after application of cold stress (low temperature, 4 °C).

Sambucus ebulus (Elderberry) Fruits Modulate Inflammation and Complement System Activity in Humans.

Sambucus ebulus (SE) fruits are used for immune stimulation and amelioration of gastrointestinal inflammatory conditions. Currently, there is no scientific evidence of their effects on various aspects of the immune response mechanisms in humans. The purpose of this study was to evaluate the immunomodulatory potential of SE fruit infusion intake in healthy humans. Anthocyanin content was determined with UPLC-ESI-MS/MS. Fifty-three volunteers enrolled in a 4-week SE infusion intake intervention. Blood count, serum total protein, Interleukin 1 beta (IL-1ß), Interleukin 6 (IL-6), Tumor Necrosis Factor Alpha (TNFα), High-sensitivity C-reactive protein (hs-CRP), C3, and C4 levels were measured on automatic analyzers, and Interleukin 8 (IL-8) was measured manually with an ELISA kit. Cyanidin-3-O-galactoside (48.15 mg/g DW), followed by cyaniding-3-sambubioside (43.41 ± 1.07 mg/g DW), were the most abundant anthocyanins in SE samples. A significant decrease in total protein (2.82%), IL-6 (20.15%), TNFα (5.38%), IL-8 (5.50%), C3 (4.16%), and C4 (14.29%) was established in the whole group. Total protein, IL-8, TNFα, and C4 decreased in women (3.11%, 4.76%, 5.09%, and 11.11%), and IL-6 decreased (40.61%) in men. Hb (1.20%) and hematocrit (1.55%) levels decreased in the whole group and in the women group (1.61% and 2.20%). SE fruits exert immune-modulatory activity as revealed by decreased pro-inflammatory status and complement activity markers in healthy volunteers after a 4-week intervention.

New Insights into the Research of Bioactive Compounds from Plant Origins with Nutraceutical and Pharmaceutical Potential.

Plant bioactive compounds are essential for human health due to their multiple biological effects, such as antioxidant, anticarcinogenic, antiallergenic, anti-inflammatory, antimutagenic, and antimicrobial activities, which can have beneficial effects on various noncommunicable diseases, such as autoimmune, inflammatory, cardiovascular, cancer, metabolic, and neurodegenerative diseases [...].

Comparative Phytochemical Analysis of Aronia melanocarpa L. Fruit Juices on Bulgarian Market.

Aronia melanocarpa L. (black chokeberry), belonging to the Rosaceae family, contains high amounts of polyphenolics and therefore exhibits one of the highest antioxidant and anti-inflammatory activities among berry fruits. Chokeberries are used in the food industry for juice, nectar, and wine production and as colorants. We aimed to compare the phytochemical composition of three chokeberry juices commercially available in the local market as sources of beneficial phytochemicals. Using GC-MS and LC-MS/MS, we performed the identification and quantitation of polar compounds and polyphenolics. The concentrations of 13 amino acids, including 6 essential amino acids, 10 organic acids, 20 sugar alcohols and derivatives, 14 saccharides, 12 fatty acids and esters, and 38 polyphenols, were estimated. One of the analyzed juices had the highest polyphenolic content (5273.87 ± 63.16 µg/mL), possibly due to 2.9 times higher anthocyanin concentration compared to anthocyanins in other tested juices. This study provides new data concerning phytochemical composition in terms of amino acids, organic acids, sugar acids, fatty acids and their esters, and polyphenols as phytocomponents of commercially available chokeberry juices. Results show that after all processing techniques and possibly different plant growth conditions, chokeberry juices are a valuable source of health-promoting phytochemicals such as phenolic acids, pro-anthocyanins, and anthocyanins, thus considering them as functional foods. We demonstrated a diversity of the active substances in bioactive foods marketed as "same"; therefore, the standardized therapeutic effect could be expected only by the utilization of food supplements with guaranteed constant content.

A common F-box gene regulates the leucine homeostasis of Medicago truncatula and Arabidopsis thaliana.

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein-protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

High resolution LC-MS/MS characterization of polyphenolic composition and evaluation of antioxidant activity of Sambucus ebulus fruit tea traditionally used in Bulgaria as a functional food.

The purpose of this study was to perform phytochemical analysis of tea from Sambucus ebulus fruits concerning hydroxycinnamic acids, flavonol glucosides, stilbenes and proanthocyanidin mono-, di- and trimers content. In total, 33 compounds were identified and quantified using UPLC-DAD-ESI/MS/MS system and the results are presented in mg/g dry weight (DW). Among analyzed hydroxycinnamic acids, 5-Caffeoylquinic acid (114.17 mg/g) was most abundant, followed by 3-p-Coumaroylquinic acid (50.33 mg/g) and 3-p-Feruloylquinic acid, p-Coumaric acid glucoside and 4-p-Coumaroylquinic acid (31.36 mg/g, 29.78 mg/g and 27.70 mg/g, respectively). Flavonol glucosides were represented predominantly by Quercetin-3-O-galactoside, Quercetin-3-O-rhamnosyl-galactoside Quercetin-3-O-glucoside and Quercetin-3-O-rhamnosyl-glucoside (3.68 mg/g, 3.22 mg/g, 2.87 mg/g and 2.56 mg/g, respectively). trans-Resveratrol-3-O-glucoside, epicatechin (40.62 mg/g) and proanthocyanidin di- and -trimers (19.90 mg/g - 31.42 mg/g) also were present in the tea. ABTS cation decolorization assay revealed 1.248 mM UAE activity and the percent of DPPH radical scavenging was 14.25%, corresponding to 39.07 µM Trolox equivalents.

Phytochemical Composition, Anti-Inflammatory and ER Stress-Reducing Potential of Sambucus ebulus L. Fruit Extract.

Sambucus ebulus L. (SE) fruits are used for their immunostimulation, hematopoietic and antiviral potential. Recently, we focused on analyzing the mechanism underlying SE fruit aqueous extract's (FAE) immunomodulation and anti-inflammatory activities, with attention to its endoplasmic reticulum (ER) stress-reducing potential. J774A.1 macrophages were treated with SE FAE alone or in conditions of lipopolysaccharides (LPS) stimulation. Using GC-MS and LC-MS/MS, its phytochemical composition was analyzed. To measure transcription and protein levels, we used qPCR and Western blot, respectively. The prevailing phytochemicals in SE FAE were hydroxycinnamic acids, proanthocyanidins and anthocyanins. The content of some amino acids, organic acids, alcohols, fatty acids and esters were newly reported. Extracts exerted an immunostimulation potential by stimulating IL-6, TNFα, Ccl2, COX2 and iNOS transcription, without inducing ER stress. SE FAE suppressed the LPS-induced transcription of inflammation related genes (IL-1ß, IL-6, TNFα, Ccl2, Icam-1, Fabp4, COX2, iNOS, Noxo1, IL-1ra, Sirt-1) and reduced the protein levels of iNOS, peIF2α, ATF6α and CHOP. The effects were comparable to that of salicylic acid. SE suppresses LPS-stimulated inflammatory markers on the transcription and translation levels. Targeting ER stress is possibly another mechanism underlying its anti-inflammatory potential. These findings reveal the potential of SE fruits as a beneficial therapeutic of inflammation and ER stress-related pathological conditions.

Triterpenoids and Other Non-Polar Compounds in Leaves of Wild and Cultivated Vaccinium Species.

The purpose of the current study was to identify and quantify triterpenoids and other non-polar compounds in the leaves of three high bush blueberry cultivars (Vaccinium corymbosum L. var. Bluegold, var. Bluecrop and var. Elliott) and three natural populations of Vaccinium species (Vaccinium uliginosum L., Vaccinium myrtillus L. and Vaccinium vitis-idaea L.) by means of gas chromatography mass spectrometry (GC-MS) and high-performance liquid chromatography with diode array detector (HPLC-DAD). Metabolite profiles differed significantly among the Vaccinium species analyzed, as well as among the populations of the same species. The populations of V. vitis-idaea predominantly contained relative concentrations of phytosterols (varying between 10.48% of total ion current (TIC) and 22.29% of TIC) and almost twice the content of triterpenes (from 29.84% of TIC to 49.62% of TIC) of the other berry species investigated. The leaves of V. corymbosum varieties biosynthesized the highest relative amount of fatty acids, while the leaves of the populations of V. uliginosum had the highest relative concentrations of fatty alcohols. The results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that the diverse populations of each berry species analyzed differed from each other, most likely due to variations in the climatic and geographical conditions of their localities.

Chemical Composition, In Vitro Antioxidant Potential, and Antimicrobial Activities of Essential Oils and Hydrosols from Native American Muscadine Grapes.

Essential oils and hydrosols of two cultivars of muscadine grapes (Muscadinia rotundifolia (Michx.) Small.) were obtained by hydro-distillation of flowers and berry skins. Twenty-three volatile compounds were identified in essential oils from the muscadine flowers, and twenty volatiles in their corresponding hydrosols. The composition of volatiles in berry skins differed significantly from that of the vine flowers. The antioxidant potential of investigated essential oils and hydrosols was evaluated using five in vitro assays: DPPH (2,2-diphenyl-1-picrylhydrazyl) method, TEAC (Trolox equivalent antioxidant capacity), FRAP (Ferric reducing antioxidant power), CUPRAC (cupric ion reducing antioxidant capacity), and NO (nitric oxide radical scavenging assay). The essential oils from the flowers of both cultivars showed the strongest antioxidant power, whereas the hydrosols were the significantly less active. All investigated essential oils showed very weak antibacterial activities against Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. However, the essential oils from the flowers of both cultivars showed moderate antifungal activities against Candida albicans, which were stronger for the oil from "Carlos" (white muscadine cultivar). To the best of our knowledge, this is the first report on obtaining and characterizing essential oils and hydrosols from muscadine grapes. This study demonstrated the variations in aromatic compounds accumulated in flowers and mature berry skins of muscadine grapes, and evaluated their possible antioxidant and antimicrobial activities. The presented results will be the base for future research, focused on a better understanding of the molecular and regulatory mechanisms involved in aromatic compound biosynthesis and accumulation in muscadine grapes.

GC-MS characterization of n-hexane soluble fraction from dandelion (Taraxacum officinale Weber ex F.H. Wigg.) aerial parts and its antioxidant and antimicrobial properties.

A comparative investigation of n-hexane soluble compounds from aerial parts of dandelion (Taraxacum officinale Weber ex F.H. Wigg.) collected during different vegetative stages was carried out. The GC-MS analysis of the n-hexane (unpolar) fraction showed the presence of 30 biologically active compounds. Phytol [14.7% of total ion current (TIC)], lupeol (14.5% of TIC), taraxasteryl acetate (11.4% of TIC), ß-sitosterol (10.3% of TIC), α-amyrin (9.0% of TIC), ß-amyrin (8.3% of TIC), and cycloartenol acetate (5.8% of TIC) were identified as the major components in n-hexane fraction. The unpolar fraction exhibited promising antioxidant activity - 46.7 mmol Trolox equivalents/g extract (determined by 1,1-diphenyl-2-picrylhydrazyl method). This fraction demonstrated insignificant antimicrobial activity and can be used in cosmetic and pharmaceutical industries.

Alkaloid profiles and acetylcholinesterase inhibitory activities of Fumaria species from Bulgaria.

GC-MS analysis of alkaloid profiles of five Fumaria species, naturally grown in Bulgaria (F. officinalis, F. thuretii, F. kralikii, F. rostellata and F. schrammii) and analysis of acetylcholinesterase inhibitory activity of alkaloid extracts were performed. Fourteen isoquinoline alkaloids were identified, with the principle ones being protopine, cryptopine, sinactine, parfumine, fumariline, fumarophycine, and fumaritine. Protopine contents, defined by HPLC analysis varied between 210.6 ± 8.8 µg/g DW (F. schrammii) and 334.5 ± 7.1 µg/g DW. (F. rostellata). While all of the investigated alkaloid extracts significantly inhibited acetylcholinesterase activity, the F. kralikii demonstrated the highest level of inhibition (IC(50) 0.13 ± 0.01 mg extract/mL).

In vitro antiviral activity of a series of wild berry fruit extracts against representatives of Picorna-, Orthomyxo- and Paramyxoviridae.

Wild berry species are known to exhibit a wide range of pharmacological activities. They have long been traditionally applied for their antiseptic, antimicrobial, cardioprotective and antioxidant properties. The aim of the present study is to reveal the potential for selective antiviral activity of total methanol extracts, as well as that of the anthocyanins and the non-anthocyanins from the following wild berries picked in Bulgaria: strawberry (Fragaria vesca L.) and raspberry (Rubus idaeus L.) of the Rosaceae plant family, and bilberry (Vaccinium myrtillis L.) and lingonberry (Vaccinium vitis-idaea L) of the Ericaceae. The antiviral effect has been tested against viruses that are important human pathogens and for which chemotherapy and/or chemoprophylaxis is indicated, namely poliovirus type 1 (PV-1) and coxsackievirus B1 (CV-B1) from the Picornaviridae virus family, human respiratory syncytial virus A2 (HRSV-A2) from the Paramyxoviridae and influenza virus A/H3N2 of Orthomyxoviridae. Wild berry fruits are freeze-dried and ground, then total methanol extracts are prepared. Further the extracts are fractioned by solid phase extraction and the non-anthocyanin and anthocyanin fractions are eluted. The in vitro antiviral effect is examined by the virus cytopathic effect (CPE) inhibition test. The results reveal that the total extracts of all tested berry fruits inhibit the replication of CV-B1 and influenza A virus. CV-B1 is inhibited to the highest degree by both bilberry and strawberry, as well as by lingonberry total extracts, and influenza A by bilberry and strawberry extracts. Anthocyanin fractions of all wild berries strongly inhibit the replication of influenza virus A/H3N2. Given the obtained results it is concluded that wild berry species are a valuable resource of antiviral substances and the present study should serve as a basis for further detailed research on the matter.

Two-phase temporary immersion system for Agrobacterium rhizogenes genetic transformation of sage (Salvia tomentosa Mill.).

Hairy root cultures of Salvia tomentosa were initiated by transformation with Agrobacterium rhizogenes. To prevent necrosis in the explants and to protect young hairy roots, Amberlite XAD-4 resin, in combination with a temporary immersion cultivation system, was applied. HPLC analyzes showed that the resin adsorbed more than 93% of the released phenolic acids and 100% of the released flavonoids. The decreased content of the released phenolics significantly reduced their destructive effects on the plant tissues, prevented, and speeded up the appearance of hairy roots.