Plant-based zinc nanoflowers assisted molecularly imprinted polymer for the design of an electrochemical sensor for selective determination of abrocitinib.

The first electrochemical sensor application in the literature is described for the sensitive and selective determination of the selective Janus kinase (JAK)-1 inhibitor abrocitinib (ABR). ABR is approved by the U.S. Food and Drug Administration (FDA) for the treatment of atopic dermatitis. The molecularly imprinted polymer (MIP)-based sensor was designed to incorporate zinc nanoflower (ZnNFs)-graphene oxide (GO) conjugate (ZnNFs@GO), synthesized from the root methanolic extract (RME) of the species Alkanna cappadocica Boiss. et Bal. to improve the porosity and effective surface area of the glassy carbon electrode (GCE). Furthermore, the MIP structure was prepared using ABR as a template molecule, 4-aminobenzoic acid (4-ABA) as a functional monomer, and other additional components. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to characterize the surface and structure of the synthesized nanomaterial and MIP-based surface. Among the electrochemical methods, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were preferred for detailed electrochemical characterization, and differential pulse voltammetry (DPV) was preferred for all other electrochemical measurements using 5.0 mM [Fe(CN)6]3-/4- solution as the redox probe. The MIP-based sensor, which was the result of a detailed optimization phase, gave a linear response in the 1.0 × 10-13 - 1.0 × 10-12 M range in standard solution and serum sample. The obtained limit of detection (LOD) and limit of quantification (LOQ) values and recovery studies demonstrated the sensitivity, accuracy, and applicability of the sensor. Selectivity, the most important feature of the MIP-based sensor, was verified by imprinting factor calculations using ibrutinib, ruxolitinib, tofacitinib, zonisamide, and acetazolamide.

The effects of Alkanna tinctoria Tausch on split-thickness skin graft donor site management: a randomized, blinded placebo-controlled trial.

BACKGROUND: A prospective, randomized, placebo-controlled clinical trial was conducted to compare the healing effectiveness of Alkanna tinctoria (L.) Tausch (Boraginaceae) with standard dressing on wound healing at the donor site after removal of the skin graft. METHODS: Enrolled patients were randomly allocated to receive topicalA. tinctoria extract ointment (20%) or standard dressing (dressing with base ointment) daily. Wound healing was assessed using the Bates-Jenson assessment tool at the 2nd and 4th weeks after intervention. RESULTS: Decreases in wound score were significantly greater in the A. tinctoria group compared with the placebo group (P <0.05). The surface areas of graft donor sites in the A. tinctoria group were significantly reduced as compared with the control group at day 28 of the intervention (P < 0.05). The proportion of patients in the A. tinctoria group achieving complete wound healing within 2 to 4 weeks was 50% and 96.66%, respectively, significantly higher than in patients receiving standard care: 0% and 23.3%, respectively. CONCLUSIONS: This clinical study showed that A. tinctoria dressing accelerates wound healing after graft harvesting. TRIAL REGISTRATION: IRCT ID: IRCT201511165781N2 .

Quantitative determination of alkannins and shikonins in endemic Mediterranean Alkanna species.

The optical antipodes alkannin/shikonin (A/S) and their esters are potent pharmaceutical substances found in the roots of 150 Boraginaceous species. This study estimated and compared total and free A/S content and A/S enantiomeric ratio in roots of 11 Alkanna species (A. corcyrensis, A. tinctoria, A. pindicola, A. orientalis, A. methanaea, A. calliensis, A. graeca, A. primuliflora, A. stribrnyi, A. sieberi and A. noneiformis) growing wild in various Greek regions, to compare with cultivated species. It also re-characterized the chirality of A/S commercial samples, since most of them were misnamed by the providers. Several Alkanna species were collected (groups 1 and 3) and botanically identified, whereas some Alkanna species were cultivated from collected seeds (group 2). Free A/S and derivatives were extracted from the dried roots of Alkanna species and analyzed by high performance liquid chromatography-diode array detection (HPLC-DAD). For total A/S content the hexane extracts of Alkanna roots were hydrolyzed and analyzed by HPLC-DAD. Chirality determination and A/S enantiomeric ratio estimation was performed for several commercial samples by polarimetry,chiral LC-DAD and circular dichroism studies. Quantitative analysis revealed that A/S content varied from one region to another even within the same species. Most of the cultivated samples contained greater amounts of free and total A/S compared with the wild ones, wheras no difference was observed in A/S enantiomeric ratio. All the Alkanna samples tested contain mainly alkannin derivatives. Some of the examined Alkanna species of the Greek flora that are endemic to the Mediterranean area could serve as alternative sources for medicinally valuable A/S derivatives. Most of the commercial A/S samples tested were misnamed in terms of chirality and re-characterized.

Co-Delivery of Dragon's Blood and Alkanna tinctoria Extracts Using Electrospun Nanofibers: In Vitro and In Vivo Wound Healing Evaluation in Diabetic Rat Model.

The increasing prevalence of diabetic wounds presents a significant challenge due to the difficulty of natural healing and various obstacles. Dragon's blood (DB) and Alkanna tinctoria (AT) are well recognized for their potent healing abilities, which include potent antibacterial and anti-inflammatory activities. In this study, electrospun nanofibers (NFs) based on polyvinyl pyrrolidone (PVP) were co-loaded with both DB and AT, aiming to magnify their efficacy as wound-dressing applications for diabetic wound healing. The evaluation of these NFs as wound dressings was conducted using a streptozotocin-induced diabetic rat model. Electrospun NFs were prepared using the electrospinning of the PVP polymer, resulting in nanofibers with consistent, smooth surfaces. The loading capacity (LC) of AT and DB into NFs was 64.1 and 70.4 µg/mg, respectively, while in the co-loaded NFs, LC was 49.6 for AT and 57.2 µg/mg for DB. In addition, X-ray diffraction (XRD) revealed that DB and AT were amorphously dispersed within the NFs. The loaded NFs showed a dissolution time of 30 s in PBS (pH 7.4), which facilitated the release of AT and DB (25-38% after 10 min), followed by a complete release achieved after 180 min. The antibacterial evaluation demonstrated that the DB-AT mixture had potent activity against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). Along with that, the DB-AT NFs showed effective growth inhibition for both P. aeruginosa and S. aureus compared to the control NFs. Moreover, wound healing was evaluated in vivo in diabetic Wistar rats over 14 days. The results revealed that the DB-AT NFs improved wound healing within 14 days significantly compared to the other groups. These results highlight the potential application of the developed DB-AT NFs in wound healing management, particularly in diabetic wounds.

Phytochemical Profiling and Biological Assessment of the Aerial Parts from Three Mediterranean Alkanna Species (A. orientalis, A. tinctoria, A. kotschyana) in the Boraginaceae Family.

This study focuses on the phytochemical analysis of the aerial parts of three Alkanna species: A. orientalis (L.) Boiss., A. tinctoria Tausch. and A. kotschyana A. DC. (Boraginaceae) growing wild in the Mediterranean basin, as mostly the roots of the genus have been widely researched. Their methanol extracts were subjected to qualitative LC-MS analyses, resulting in the annotation of 28 different secondary metabolites, with 27 originating from A. orientalis, 25 from A. tinctoria and 23 from A. kotschyana. The detected metabolites are categorized into three chemical types: organic acids (2), flavonoids and their glycosides (17), and caffeic acid derivatives (9). Furthermore, the chemical profiles of the three species are discussed chemotaxonomically. Caffeic acid and its derivatives, along with glucosides of quercetin and kaempferol, were identified in all three studied species. Additionally, their total phenolic and flavonoid contents were determined. The antioxidant capacity was evaluated through various chemical assays, as well as their in vitro enzyme inhibitory properties towards cholinesterases (AChE and BChE), α-amylase and α-glucosidase. The results showed that A. tinctoria exhibited the strongest antioxidant activity (211 mgTE/g extract in DPPH and 366 mgTE/g extract in ABTS), probably due to its high total phenolic (53.3 mgGAE/g extract) and flavonoid (20.8 mgRE/g extract) content, followed by A. kotschyana. These chemical and biological findings provide valuable insights for potential promising applications of the aerial parts of the species outside of the well-known uses of their roots.

Effects of fungal endophytes and arbuscular mycorrhizal fungi on growth of Echium vulgare and alkannin/shikonin and their derivatives production in roots.

Endophytic fungi as well as arbuscular mycorrhizal fungi (AMF) are known to stimulate plant growth and production of secondary metabolites in medicinal plants. Here, 10 endophytic fungi isolated from roots of wild Alkanna tinctoria plants and 5 AMF purchased from the Glomeromycota in vitro collection were evaluated, during two successive three-month greenhouse experiments, on the growth of Echium vulgare and alkannin/shikonin and their derivatives (A/Sd) production in the roots. Some of the endophytic fungi tested significantly increased plant growth parameters as compared to the control: Cladosporium allicinum, Cadophora sp., Clonostachys sp., Trichoderma hispanicum and Leptosphaeria ladina increased root volume, Plectosphaerella sp. And T. hispanicum root fresh weight and root water retention and T. hispanicum plant water retention. However, none of these fungi impacted A/Sd production. Conversely, none of the AMF strains tested impacted plant growth parameters, but those inoculated with Rhizophagus intraradices MUCL 49410 had a significantly higher concentration of alkannin/shikonin (A/S), acetyl-A/S, ß,ß- dimethylacryl-A/S, isovaleryl-A/S and total A/Sd, compared to the control plants. Further studies are needed to investigate the mechanisms involved in the production of A/Sd in plants associated to specific endophytic fungi/AMF and on the cultivation conditions required for optimal production of these compounds.

Ratanjot (Alkanna tinctoria L.) Root Extract, Rich in Antioxidants, Exhibits Strong Antimicrobial Activity against Foodborne Pathogens and Is a Potential Food Preservative.

Natural and sustainable plant-based antioxidants and antimicrobials are highly desirable for improving food quality and safety. The present investigation assessed the antimicrobial and antioxidant properties of active components from Alkanna tinctoria L. (herb) roots, also known as Ratanjot root. Two methods were used to extract active components: microwave-assisted hot water (MAHW) and ethanolic extraction. MAHW extract yielded 6.29%, while the ethanol extract yielded 18.27%, suggesting superior Ratanjot root extract powder (RRP) solubility in ethanol over water. The ethanol extract showed significantly higher antioxidant activity than the MAHW extract. Gas Chromatography-Mass Spectrometry analysis revealed three major phenolic compounds: butanoic acid, 3-hydroxy-3-methyl-; arnebin 7, and diisooctyl pthalate. The color attributes (L*, a*, b*, H°ab, C*ab) for the ethanolic and MAHW extracts revealed significant differences (p < 0.05) in all the above parameters for both types of extracts, except for yellowness (b*) and chroma (C*ab) values. The ethanol extract exhibited antimicrobial activity against 14 foodborne bacteria, with a significantly higher inhibitory effect against Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) than the Gram-negative bacteria (Salmonella enterica serovar Typhimurium and Escherichia coli). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were both 25 mg/mL for the Gram-negative bacteria, while the MIC and MBC concentrations varied for Gram-positive bacteria (0.049-0.098 mg/mL and 0.098-0.195 mg/mL) and the antimicrobial effect was bactericidal. The antimicrobial activities of RRP extract remained stable under broad temperature (37-100 °C) and pH (2-6) conditions, as well as during refrigerated storage for 30 days. Application of RRP at 1% (10 mg/g) and 2.5% (25 mg/g) levels in a cooked chicken meatball model system prevented lipid oxidation and improved sensory attributes and retarded microbial growth during refrigerated (4 °C) storage for 20 days. Furthermore, the RRP extract was non-toxic when tested with sheep erythrocytes and did not inhibit the growth of probiotics, Lacticaseibacillus casei, and Lactiplantibacillus plantarum. In conclusion, the study suggests that RRP possesses excellent antimicrobial and antioxidant activities, thus making it suitable for food preservation.

Profiling of pyrrolizidine alkaloids using a retronecine-based untargeted metabolomics approach coupled to the quantitation of the retronecine-core in medicinal plants using UHPLC-QTOF.

Pyrrolizidine alkaloids (PA) are secondary metabolites of high toxicological relevance. Several PA quantitative methodologies were developed based on a limited number of certified standards, including time consuming solid phase extraction (SPE) purification steps. Herein, we shed light on the variability of PA in herbal extracts and propose a quantification methodology based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) for the evaluation of the total PA content as retronecine-equivalents (RE) directly from crude matrices. Particularly in the focus of the investigation were Alkanna spp. (Boraginaceae), which possess a wide range of pharmaceutical properties. In addition, a comparative PA screening of crude and SPE enriched extracts was performed and PA-containing plants from Fabaceae and Compositae families were included to demonstrate universal applicability. In total, 105 PA were identified using HRMSe experiments, specific MS/MS fragmentation PA patterns, a customized in-house library and literature data. Among them, 18 glycosidic PA derivatives were reported for the first time in literature. Using a hierarchical clustering approach, PA distribution in herbal extracts was shown to be family-dependent and significantly different among species. This was further supported by the results of the total PA concentrations, obtained using a retronecine/heliotridine/internal standard-based targeted UHPLC-HRMS quantification method, which varied from 8.64 ± 0.08-3096.28 ± 273.72 µg RE/g extract dry weight in shoots extracts of Alkanna spp. and leaves extracts of Crotalaria retusa L. respectively. Worth mentioning is that the procedure allowed to quantify PA in Alkanna spp. If the procedure based on 35 specific PA recommended by European regulations had been used, results would have been equal to zero for the four species since none were observed in Alkanna spp. Finally, by combining the RE results with the corresponding dereplication results, a customized correction factor for each extract (ranging from 2.12 to 2.48) was assessed leading to a more accurate estimate of the PA content regardless of the molecular weight of each PA. The present methodology will facilitate PA quantification directly from crude extracts and avoid the underestimation the real PA content due to limited availabilty of authentic reference compounds in botanical extracts used in phytomedicines or food supplements/cosmetics.

Using extract from alkanet (Alkanna tinctoria) as a source of both a red lipid stain and a blue counterstain for histology.

Alkanet (Alkanna tinctoria) is a plant native to and cultivated in parts of Europe, Asia and the Middle East. It has been used for thousands of years as a medicinal agent and as a colorant for textiles, food and cosmetics. An extract from the root of this plant has been used with a mordant to stain nuclei. We describe here the versatility of different extracts from this plant to stain lipids red and to counterstain certain other tissue elements blue. The color variation and selective differential staining is due to solvent polarity and pH. Extracts contain numerous chemical species, all of which are derivatives of the indicator dye, naphthazurin. Our red extract is nonionic below pH 7 and partitions from its somewhat polar solvent of 100% isopropanol to nonpolar lipids. The blue extract is dianionic at high pH in 70% isopropanol and binds ionically to cationic tissue structures such as collagen, muscle and cytoplasm of other cells.

Alkanna tinctoria-based sustainable alkanin natural colorant for eco-dyeing of wool.

The awareness of sustainability and widespread utilization of green technologies in textile dye houses are revolutionizing not only textile industries but related fields. The current study is concerned with ultrasonic (US)-assisted utilization of extracts of Alkanna tinctoria (a source of natural alkanin dye) for wool dyeing. The extracts are obtained in various media, and both extracts and wool fabrics have been  US treated for 15-60 min. Dyeing is performed by applying variable parameters and utilizing herbal-based extracts as a source of bio-mordant, hence improving the fastness rating and enhancing color strength. Good color strength and fastness ratings are obtained using irradiated extract at 4 pH when the US-treated wool fabric is dyed at 65 °C for 60 min before and after chemical and bio-mordanting. For comparative studies, chemical mordants are also employed. In contrast to chemical mordants, the bio-mordants have made the dyeing process more sustainable with good to excellent fastness rating.

Arbuscular mycorrhizal fungi impact the production of alkannin/shikonin and their derivatives in Alkanna tinctoria Tausch. grown in semi-hydroponic and pot cultivation systems.

Introduction: Alkanna tinctoria Tausch. is a medicinal plant well-known to produce important therapeutic compounds, such as alkannin/shikonin and their derivatives (A/Sd). It associates with arbuscular mycorrhizal fungi (AMF), which are known, amongst others beneficial effects, to modulate the plant secondary metabolites (SMs) biosynthesis. However, to the best of our knowledge, no study on the effects of AMF strains on the growth and production of A/Sd in A. tinctoria has been reported in the literature. Methods: Here, three experiments were conducted. In Experiment 1, plants were associated with the GINCO strain Rhizophagus irregularis MUCL 41833 and, in Experiment 2, with two strains of GINCO (R. irregularis MUCL 41833 and Rhizophagus aggregatus MUCL 49408) and two native strains isolated from wild growing A. tinctoria (R. irregularis and Septoglomus viscosum) and were grown in a semi-hydroponic (S-H) cultivation system. Plants were harvested after 9 and 37 days in Experiment 1 and 9 days in Experiment 2. In Experiment 3, plants were associated with the two native AMF strains and with R. irregularis MUCL 41833 and were grown for 85 days in pots under greenhouse conditions. Quantification and identification of A/Sd were performed by HPLC-PDA and by HPLC-HRMS/MS, respectively. LePGT1, LePGT2, and GHQH genes involved in the A/Sd biosynthesis were analyzed through RT-qPCR. Results: In Experiment 1, no significant differences were noticed in the production of A/Sd. Conversely, in Experiments 2 and 3, plants associated with the native AMF R. irregularis had the highest content of total A/Sd expressed as shikonin equivalent. In Experiment 1, a significantly higher relative expression of both LePGT1 and LePGT2 was observed in plants inoculated with R. irregularis MUCL 41833 compared with control plants after 37 days in the S-H cultivation system. Similarly, a significantly higher relative expression of LePGT2 in plants inoculated with R. irregularis MUCL 41833 was noticed after 9 versus 37 days in the S-H cultivation system. In Experiment 2, a significant lower relative expression of LePGT2 was observed in native AMF R. irregularis inoculated plants compared to the control. Discussion: Overall, our study showed that the native R. irregularis strain increased A/Sd production in A. tinctoria regardless of the growing system used, further suggesting that the inoculation of native/best performing AMF is a promising method to improve the production of important SMs.

Structure and activity of a novel robust peroxidase from Alkanna frigida cell culture.

Demands for peroxidases (POX)s with diverse physicochemical properties have steadily grown as more applications of POXs are demonstrated. Plants are among the best sources of versatile POXs, and plant biotechnology, as an agricultural hassle-free technology, promises to circumvent the limitations of natural resource exploitation and to address the demands. Following this trend, it was shown that POX production steadily increased during the 31-day subculture of Alkanna frigida (from Boraginaceae) callus on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The purified cationic enzyme (POXalf) maintained its optimal activity over pH 4-7 for 2 years. It was resistant to H2O2 high concentrations (IC50 = 543.7 mM) and showed high specific activity in the reaction with phenol (4320.5 AU mg-1 > 20-fold of HRP AU). Furthermore, the specificity constant ratio of guaiacol to phenol indicated a 100 times faster reaction of POXalf with guaiacol. However, in contrast to HRP, it had little effect on diazo derivatives of aniline and meta-diaminobenzene. Based on the resulting primary structure from the tandem mass analysis, the POXalf 3D structure was constructed via homology modelling. Despite the high topological similarity between the HRP and POXalf structures, there were important differences between the active site pockets that could explain the observed differences in the corresponding substrate spectra and the specific activities. Considering the dynamics of POXalf production, its inactivity towards IAA and its high affinity for guaiacol, POXalf may have associated roles with A. frigida cell wall construction and monolignol metabolism.

Evaluation and HPLC characterisation of a new herbal ointment for the treatment of full-thickness burns in rats.

OBJECTIVE: Burn injuries are among the most common accidental health problems worldwide, frequently leading to health and socio-economic challenges. Despite this, no standard protocol for managing burn injuries can overcome the adverse effects of currently used drugs. The present study sets out to develop and evaluate the efficacy of new herbal ointments in providing synergistic anti-inflammatory, antimicrobial, antioxidant, and cell-proliferating activities. It also investigates the high-performance liquid chromatography (HPLC) characterisation of these new herbal ointments. METHOD: Three different concentrations of the new herbal ointment, which incorporates extracts of Matricaria aurea flower heads, arial parts of Calendula tripterocarpa, Rosmarinus officinalis leaves, Alkanna tinctoria roots, and myrrh were developed and evaluated. Ointments designed to promote burn-wound healing were prepared and compared with ß-sitosterol ointment and silver sulfadiazine cream, as a commercial standards. RESULTS: According to statistical and histopathological analyses and visual inspections, the new herbal formulas showed faster wound healing, more tolerability, and less toxicity than the commercial standards. CONCLUSION: The new herbal ointments, developed in our study, have shown promising results. The formula offers mechanical protection without any release of non-biodegradable particles. It maintains the optimum moisture and pH of the skin, while minimising scar-tissue formation. These advantages, in addition to availability, low costs, and easy handling, may support the use of this new herbal formula as an effective and safe alternative treatment, designed to promote the healing of burn injuries.

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 Activity of Alkanna Species in vitro Culture and Intact Plant Extracts Against Antibiotic Resistant Bacteria.

Overcoming the antibiotic resistance is nowadays a challenge. There is still no clear strategy to combat this problem. Therefore, the urgent need to find new sources of antibacterial agents exists. According to some literature, substances of plant origin are able to overcome bacterial resistance against antibiotics. Alkanna species plants are among the valuable producers of these metabolites. But there is a problem of obtaining the standardized product. So, this review is focused on the discussion of the possibilities of biotechnological production of antimicrobial agents from Alkanna genus species against some microorganisms including antibiotic resistant bacterial strains.

Development of novel microsatellite markers for Alkanna tinctoria by comparative transcriptomics.

PREMISE: Alkanna tinctoria (Boraginaceae) is an important medicinal herb with its main distribution across the Mediterranean region. To reveal its genetic variation and population structure, microsatellite markers were developed and validated in four Greek populations. METHODS AND RESULTS: RNA-Seq data of the related species Arnebia euchroma and Echium plantagineum were assembled and mined to identify conserved ortholog sets containing simple sequence repeat motifs. Fifty potential loci were identified and then tested on A. tinctoria, of which 17 loci were polymorphic. The number of alleles ranged from one to nine, and the levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and 0.000 to 0.820, respectively. Most of these loci could be successfully amplified in eight other species of Boraginaceae (Alkanna graeca, A. hellenica, A. sfikasiana, Echium vulgare, E. plantagineum, Lithospermum officinale, Borago officinalis, and Anchusa officinalis). CONCLUSIONS: This study provides the first set of microsatellite loci for studying the genetic variation and population structure of A. tinctoria and shows their potential usefulness in other Boraginaceae species.

Esmaltes de uñas ecológicos y medición de la saturación periférica de oxígeno en pacientes quirúrgicos. Presentación de casos / Ecological nail polishes and measurement of peripheral oxygen saturation in surgical patients. Presentation of cases

Resumen: Introducción: Inicialmente en la anestesia, el médico dependía de sus propios sentidos, en la actualidad la ingeniería electrónica optimiza la medición de funciones vitales. El oxímetro de pulso permite la identificación temprana de episodios de hipoxemia por medición de la saturación periférica de oxígeno (SpO2). Múltiples factores interfieren en su lectura, por ejemplo, los esmaltes en las uñas. En África es común que las mujeres se apliquen esmaltes ecológicos de henna natural y de Alkanna tinctoria (phudan). Objetivo: Evaluar la influencia del esmalte de uñas ecológico en la medición de la saturación periférica de oxígeno en pacientes sometidos a cirugía. Casos clínicos: 1) Paciente programada para practicar histerectomía total abdominal, con uñas pintadas con esmalte de henna de un color castaño rojizo, al medir saturación periférica de oxígeno se observó 92% y al retirar el esmalte, 98%. 2) Situación similar se presentó en paciente con hernia epigástrica gigante, uñas pintadas de colorante rojo de Alkanna tinctoria, se monitorizó (SpO2), se observó 94%. Se retiró el esmalte y se observó 99%. Conclusiones: Los esmaltes ecológicos de uñas alteran la medición de la saturación periférica de oxígeno en pacientes sometidos a cirugía en las condiciones que se practica la anestesiología en este país.

Abstract: Introduction: Initially in anesthesia, the doctor relied on his own senses, currently by electronic engineering better measurement of vital functions. The pulse oximeter allows early identification of hypoxemic episodes, by measuring peripheral oxygen saturation (SpO2). Multiple factors interfere with its reading, for example nail polishes. In Africa, it is common for women to apply organic enamels of natural henna and Alkanna tinctoria (phudan). Objective: To evaluate the influence of ecological nail polish in the measurement of peripheral oxygen saturation in patients undergoing surgery. Clinical cases: 1) Patient scheduled to perform a total abdominal hysterectomy, with nails painted with henna enamel of a reddish-brown color, when measuring peripheral oxygen saturation, 92% is observed, and when removing the enamel 98% of saturation was observed. 2) A similar situation occurred in a patient with a giant epigastric hernia, nails painted with Alkanna tinctoria red dye, it was monitored (SpO2), 94% were observed. The enamel was removed and 99 % of saturation was observed. Conclusions: Ecological nail polishes alter the measurement of peripheral oxygen saturation in patients undergoing surgery, under the conditions that anesthesiology is practiced in this country.

In vitro antioxidant and free radical scavenging activity of four Alkanna species growing in Iran.

BACKGROUND: Medicinal plants are recognized as sources of natural antioxidants that can protect biological system from oxidative stress. OBJECTIVE: In this study, the antioxidant activities of four species of the Alkanna genus (Alkanna bracteosa, Alkanna frigida, Alkanna orientalis and Alkanna tricophila) were evaluated. MATERIALS AND METHODS: The dried roots of plant samples (100 g) were extracted separately by percolation method with MeOH/H2O (80/20) and four fractions were provided respectively with hexane, chloroform, ethyl acetate, butanol and water. Antioxidant activities were investigated by using 1,1-diphenyl-2-picrylhydrazyl, ferric thiocyanate and thiobarbituric acid methods and compared with quercetin (as positive control). RESULTS: The results indicated that the butanol fractions of four species had the highest antioxidant activity and radical scavenging activity compared with the other fractions studied (P < 0.05). The 50% effective concentration (half - effective doses) values of butanol fractions are less than quercetin and other fractions, so, these fractions showed potent antioxidant activity. This indicated that the active compounds in the root parts of Alkanna species dissolved in the butanol. All the fractions of four species of Alkanna had antioxidant activity, while, there were no significant differences (P > 0.05) between the total antioxidant activities of same fractions. The total antioxidant activity values of Alkanna fractions in a descending order are as follows: Butanol fraction > ethyl acetate fraction > total extract > hexane fraction > chloroform fraction > aqueous fraction. CONCLUSION: The antioxidant activities of butanol fractions of samples were higher than quercetin. This may be because most of the active compounds of Alkanna species dissolved in the butanol.

In vitro activity of Alkanna frigida extracts in comparison with glucantime against Leishmania major.

BACKGROUND AND OBJECTIVES: Current chemotherapies of cutaneous leihmaniasis have faced to some problems and limitations; Development of new leishmanicidal drugs from different sources like herbal plants, are crucially important. The objective of the present study was evaluation of in vitro activity of Alkanna frigida extracts in comparison with glucantime against Leishmania major. MATERIALS AND METHODS: L. major promastigotes were exposed to different concentrations of the A. frigida extracts, processed by ethyl acetate, ethanol, hexane and chloroform. The inhibitory effect, as the IC50, were calculated after 24, 48 and 72 hours by linear regression analysis values of the concentrations employed. RESULTS: The significant inhibition was observed after 24 and 48 hours with different concentrations of compounds (p < 0.05 in all tests). All extracts had potent activity against proliferation of the promastigotes, comparing to the untreated negative control. It could compete with the glucantime efficacy in some concentrations. Ethyl acetate and ethanol extractions showed potent IC50 value, 106 µg/ml and 86 µg/ml, respectively. Hexane and chloroform extractions had poor efficacy after 24 hours; however, the efficacy increased after 48 and 72 hours. CONCLUSION: The results indicated that the A. frigida has appropriate inhibitory effects on the growth of L. major promastigotes in vitro and can be of herbal targets for further investigation in vivo.