Chimeric advanced drug delivery nano systems (chi-aDDnSs) for shikonin combining dendritic and liposomal technology

The interest of drug delivery has focused on the creation of new formulations with improved properties, taking much attention to the drug release from the carrier. Liposomes have already been commercialized, while dendrimers and hyperbranched polymers are emerging as potentially ideal drug delivery vehicles. Chimeric advanced drug delivery nano systems (chi-aDDnSs) are mixed nanosystems combining different biomaterials that can offer advantages as drug carriers. Alkannin and shikonin (A/S) are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant and recently established antitumor activity. In this work three generations of hyperbranched aliphatic polyesters were used for the first time to form complexes with shikonin, as well as liposomal chi-aDDnSs. Characterization of the shikonin-loaded chi-aDDnSs was performed by measuring their particle size distribution, ζ-potential, drug encapsulation efficiency and the in vitro release profile. The analysis revealed sufficient drug encapsulation and appropriately featured release profiles. Chi-aDDnSs were also examined for their physical stability at 4°C. The results are considered promising and could be used as a road map for designing in vivo experiments. © 2011 Elsevier B.V

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

Advanced Drug Delivery Nanosystems for Shikonin : a Calorimetric and Electron Paramagnetic Resonance Study

Drug delivery is considered a mature scientific and technological platform for producing innovative medicines with nanosystems composed of intelligent bio-materials that carry active pharmaceutical ingredients forming advanced drug delivery nanosystems (aDDnSs). Shikonin and its enantiomer alkannin are natural products that have been extensively studied in vitro and in vivo for, among others, their antitumor activity, and various efforts have been made to prepare shikonin-loaded drug delivery systems. This study is focused on chimeric aDDnSs and specifically on liposomal formulations combining three lipids (egg-phosphatidylcholine; dipalmitoyl phosphatidylcholine; and distearoyl phosphatidylcholine) and a hyperbranched polymer (PFH-64-OH). Furthermore, PEGylated liposomal formulations of all samples were also prepared. Calorimetric techniques and electron paramagnetic resonance were used to explore and evaluate the interactions and stability of the liposomal formulations, showing that the presence of hyperbranched polymers promote the overall stability of the chimeric aDDnSs based on the drug release profile enhancement. Furthermore, results showed that polyethylene glycol enhances drug stabilization inside the liposomes, forming a stable and promising carrier for shikonin with improved characteristics

Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells

Leukemia remains life-threatening despite remarkable advances in chemotherapy. The poor prognosis and drug resistance are challenging treatment. Novel drugs are urgently needed. Shikonin, a natural naphthoquinone, has been previously shown by us to be particularly effective towards various leukemia cell lines compared to solid tumors. However, the underlying mechanisms are still poorly understood. Here, we investigated shikonin and 14 derivatives on U937 leukemia cells. Four derivatives (isobutyrylshikonin, 2-methylbutyrylshikonin, isovalerylshikonin and ß,ß-dimethylacrylshikonin) were more active than shikonin. AnnexinV-PI analysis revealed that shikonins induced apoptosis. Cell cycle G1/S check point regulation and the transcription factor c-MYC, which plays a vital role in cell cycle regulation and proliferation, were identified as the most commonly down-regulated mechanisms upon treatment with shikonins in mRNA microarray hybridizations. Western blotting and DNA-binding assays confirmed the inhibition of c-MYC expression and transcriptional activity by shikonins. Reduction of c-MYC expression was closely associated with deregulated ERK, JNK MAPK and AKT activity, indicating their involvement in shikonin-triggered c-MYC inactivation. Molecular docking studies revealed that shikonin and its derivatives bind to the same DNA-binding domain of c-MYC as the known c-MYC inhibitors 10058-F4 and 10074-G5. This finding indicates that shikonins bind to c-MYC. The effect of shikonin on U937 cells was confirmed in other leukemia cell lines (Jurkat, Molt4, CCRF-CEM, and multidrug-resistant CEM/ADR5000), where shikonin also inhibited c-MYC expression and influenced phosphorylation of AKT, ERK1/2, and SAPK/JNK. In summary, inhibition of c-MYC and related pathways represents a novel mechanism of shikonin and its derivatives to explain their anti-leukemic activity

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. © Copyright © 2021 Rat, Naranjo, Krigas, Grigoriadou, Maloupa, Alonso, Schneider, Papageorgiou, Assimopoulou, Tsafantakis, Fokialakis and Willems