A Development Strategy of Tailor-made Natural Deep Eutectic Solvents for the Enhanced Extraction of Hydroxynaphthoquinones from Alkanna tinctoria Roots

Natural hydroxynaphthoquinone enantiomers (HNQs) are well-described pharmaceutical and cosmeceutical agents especially present in the roots of Alkanna tinctoria (L.) Tausch, a species native to the Mediterranean region. In this work, eco-friendly natural deep eutectic solvents (NaDESs) were developed for the selective extraction of these compounds. An extensive screening was performed using more than sixty tailor-made NaDESs. The impact of the intrinsic physicochemical properties on the HNQs extraction efficiency as well as the specificity towards the different enantiomeric pairs was thoroughly investigated. As a result of a multivariate analysis and of the one factor-a-time solvent optimization, the eutectic mixture composed of levulinic acid and glucose (LeG) using a molar ratio of 5:1 (molHBA:molHBD) and 20% of water (w/w) was found as the most appropriate mixture for the highest extraction efficiency of HNQs. Further optimization of the extraction process was attained by response surface methodology, using a temperature of 45 °C, a solid-to-liquid ratio of 30 mg/mL, and an extraction time of 50 min. A maximum extraction output of 41.72 ± 1.04 mg/g was reached for HNQs, comparable to that of the commonly used organic solvents. A solid-phase extraction step was also proposed for the recovery of HNQs and for NaDESs recycling. Our results revealed NaDESs as a highly customizable class of green solvents with remarkable capabilities for the extraction of HNQs.

Biosynthesis of silver nanoparticles using Cynara scolymus, Lavandula angustifolia, Alkanna Tinctoria and its antimicrobial activities—A comparative study

Nanotechnology is a promising field of science and involves use of nanoparticle with size ranging between 1 nm–100 nm. The phenomenal size properties enable nanoparticles to anchor a prominent place in various biomedical applications. Silver is known for its antimicrobial nature. This study elucidates the qualitative phytochemical properties of three plant extracts, its biogenic synthesis of silver nanoparticles and their antimicrobial activity. Green Silver nanoparticles (AgNPs) were synthesized from 1mM Silver Nitrate (AgNO3) solution using leaf extracts of Cynara scolymus (Artichoke), Alkanna tinctoria (Alkanet), and Lavandula angustifolia (Lavender) respectively. The synthesized silver nanoparticles were characterized visually, via UV–vis spectrophotometer and X-ray Diffraction (XRD). The biogenic particles were tested against pathogenic bacterial strains (Staphylococcus aureus, and Escherichia coli) and Plant pathogenic fungal strains (Aspergillus flavus and Aspergillus niger). The formation of AgNPs with size <100 nm was ensured by UV vis spectrophotometer and XRD. Antibacterial activity of lavender mediated AgNP was highly significant, followed by artichoke mediated AgNP and finally, alkanet AgNP were effective. However, in contrast, Artichoke mediated AgNP showed significant activity against plant fungal strains, followed by Alkanet AgNP, and finally by Lavender mediated AgNPs. We concluded that the three plants have versatile biochemical molecules responsible for broad spectrum of AgNP against bacterial and fungal strains. Studies on combined use of AgNPs with other antimicrobial agents may solve the problem of toxicity and possible risk of drug resistance.

Spatial and Ecological Drivers of Genetic Structure in Greek Populations of Alkanna tinctoria (Boraginaceae), a Polyploid Medicinal Herb

Background and Aims: Quantifying genetic variation is fundamental to understand a species’ demographic trajectory and its ability to adapt to future changes. In comparison with diploids, however, genetic variation and factors fostering genetic divergence remain poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies.Methods: We collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing. In addition, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high-quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations, complemented by genome size measurements and chromosomal counts. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables.Key Results: We found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modeling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria.Conclusion: Current findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.

Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization.

The current state and prospects of the development of soft dosage forms with Boraginaceae species

Soft dosage forms make up a significant segment in the structure of medicines both pharmaceutical and industrial production. Herbal ointments are an integral part of this group. Species of the Boraginaceae family are the source of biologically active substances used to treat inflammatory processes of all layers of the skin, muscle tissue, tendons and joints.Aim. To determine data on the current state of the development of soft dosage forms based on species of the Boraginaceae family and identify further prospects for studies in this area.Materials and methods. The scientific databases, in particular Abdata Pharma-Daten-Service, BioMed Central, Scopus, scientific periodicals of Ukraine – Vernadsky National Library of Ukraine, etc., were analyzed.Results and discussion. As a result of the data analysis of the Information Fund “State Register of Medicines of Ukraine” it has been found that only medicines with comfrey (Symphytum officinale L.) are registered in Ukraine. In the EU countries, only soft dosage forms with comfrey are registered, with the exception of Greece where there are ointments with Alkanna tinctoria (L.) Taush, and France where there are substances for the production of ointments withPulmonaria obscura Dumort. and Anchusa officinalis L. There are many soft homeopathic dosage forms with comfrey at the US pharmaceutical market; however, there are ointments and suppositories with Borago officinalis L. and a gel with Myosotis arvensis (L.) Hill. At the Brazilian pharmaceutical market there is the cream “Acheflan” based on Cordia verbenacea DC. The European and Asian scientists study ointments with Cordia myxa L., Cordia obliqua Willd., Heliotropium indicum L., Arnebia euchroma Rolye. (Johnst.), Onosma dichroanthum Boiss, Arnebia densiflora (Nordm.) Ledeb, Ehretia microphylla Lam., Anchusa officinalis L.Conclusions. Homeopathic products predominate among the soft dosage forms based on Boraginaceae species. At the pharmaceutical market, along with comfrey drugs, there are medicines and active substances with Borago officinalis, Myosotis arvensis (L.) Hill, Alkanna tinctoria (L.) Taush, Pulmonaria obscura Dumort., Anchusa officinalis L. and Cordia verbenacea DC. There is an active research of the existing products with comfrey, as well as the development and study of new soft dosage forms with species of Arnebia, Heliotropium, Cordia, Onosma, Ehretia and Anchusa genus. Taking the above into account the further research for creating effective and safe drugs based on the Boraginaceae species is promising.