A. N. Assimopoulou

The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products

Wound healing properties of plant extracts that contain the naphthoquinone natural products alkannin (1) and shikonin (2) have been known for many centuries. More recently, the biological properties of 1, 2, and related derivatives have been demonstrated experimentally, and their production both by cell cultures and chemical synthesis has been studied extensively.

Inhibitory Activity of Minor Polyphenolic and Nonpolyphenolic Constituents of Olive Oil Against In Vitro Low-Density Lipoprotein Oxidation

The minor polyphenolic and nonpolyphenolic constituents of olive oil were examined, in various doses, against copper ion-induced low-density lipoprotein (LDL) oxidation and were found, in optimal doses (final concentration, 10 microM or 20 microM), to have remarkable biological activity, contributing to that previously reported for the major phenolic compounds. The main phytosterols, beta-sitosterol, campesterol, and stigmasterol, were found to have 43.8%, 37.3%, and 33.4% LDL mean protection (MP) activity, respectively, while free cholesterol exhibited 43.2% MP. The triterpenoid derivative compounds, ursolic acid, uvaol, and oleanolic acid, had similar MP activities of 50.5%, 46.8%, and 46.0%, respectively. Tocopherol (Toc) isomers exhibited an increasing effect in the following order: alpha-Toc (33.6%) < beta-Toc (36.1%) < gamma-Toc (42.9%) < delta-Toc (46.0%). The flavonoid polyphenols, quercetin, luteolin, and rutin, exhibited the highest activities--46.8%, 49.5%, and 53.7% MP, respectively, comparable to the 49.0% MP activity found for oleuropein. These findings indicate the relative independence of LDL protection activity in regard to structural differences among the involved compounds. A relation to the Mediterranean diet is also demonstrated.

Alkannins and shikonins: a new class of wound healing agents.

Alkannins and Shikonins (A/S) are chiral-pairs of naturally occurring isohexenylnaphthazarins. They are found in the external layer of the roots of at least a hundred and fifty species that belong mainly to the genera Alkanna, Lithospermum, Echium, Onosma and Arnebia of the Boraginaceae family. Their occurrence in Jatropha glandulifera, a member of the Euphorbiaceae, should be considered as an exception. Pharmaceutical formulations with wound healing properties based on A/S have been in the market for many years. Although their wound-healing, anti-inflammatory, antimicrobial, antioxidant, antithrombotic and antitumor properties have been extensively documented, significant insight into their specific molecular pathways and mechanisms was hindered until recently. With the establishment of viable synthetic and biosynthetic routes of A/S and the synthesis of specific derivatives that were discovered the last few years, the effects of those compounds in the molecular-cell biology of human tissues in health and disease have just started being explored in depth, revealing a new class of drugs that hold promise as the basis for many valuable therapeutic targets. In the recent years, a wealth of new information arising from research efforts, on the wound healing properties of A/S has been accumulated. In this paper we review the findings and advances on the molecular and biological properties of A/S that promote wound healing.

Electrospun fiber mats containing shikonin and derivatives with potential biomedical applications.

Alkannin, shikonin (A/S) and their derivatives are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant and antitumor activity. Clinical studies over the years revealed that A/S derivatives-based wound healing preparations (such as HELIXDERM(®)) are among a very small group of therapeutics that modulate both the inflammatory and proliferative phases of wound healing and present significant tissue regenerative activity. The purpose of the present work was to combine the biological properties of A/S and the advantages of electrospun meshes to prepare a potent topical/transdermal biomaterial for A/S. Four biocompatible polymers (cellulose acetate, poly(L-lactide), poly(lactide-co-glycolide) LA/GA:50/50 and 75/25) were used for the first time, to produce electrospun fiber mats containing either shikonin or A/S mixture in various amounts. Both drugs were effectively loaded into the above biomaterials. The incorporation of drugs did not considerably affect fibers morphology and their mean diameter size varied from 315 to 670 nm. High drug entrapment efficiencies (ranged from 74% to 95%) and appropriate release profiles were achieved, that render these fibers as potential A/S topical/transdermal wound healing dressings. Given the multifunctional activity of the natural products alkannins and shikonins, their consideration as bioactive constituents for tissue engineering scaffolds seems a promising strategy for repairing and regenerating tissues and mainly skin.

Inhibition of topoisomerase I by naphthoquinone derivatives

Alkannin and shikonin are naturally occurring naphthoquinones. We have tested several derivatives of the title compounds and we have found that naphthoquinones bearing at least one phenolic hydroxyl group are potent inhibitors of topoisomerase I. The ability of the tested compounds to complex Zn++ parallels with a few exceptions their topoisomerase I inhibition properties while their intercalation and redox properties do not.

Alkannin and shikonin: effect on free radical processes and on inflammation - a preliminary pharmacochemical investigation.

Alkannin and shikonin, two natural products from Alkanna tinctoria and Lithospermum erhythrorhizon (Boraginaceae), are used in folk medicine where they are claimed to possess, among other properties, wound healing and anti‐inflammatory activity. We investigated, together with the structurally related naphthazarin, their in vitro antioxidant and hydroxyl radical scavenging activity as well as their in vivo antiinflammatory activity. I was found that all examined compounds significantly inhibited in vitro lipid peroxidation of ra hepatic microsomal membranes, competed with DMSO for free hydroxyl radicals, and reduced inflammation (mouse paw edema induced by FCA) very efficiently.The examined compounds proved equal or superior to the common reference compounds for each of these properties. I is concluded that the claimed and/or proven actions of alkannin and shikonin are attributable at least partly to their intervention in free radical processes.

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.

Pharmacophore-driven identification of PPARγ agonists from natural sources

In a search for more effective and safe anti-diabetic compounds, we developed a pharmacophore model based on partial agonists of PPARγ. The model was used for the virtual screening of the Chinese Natural Product Database (CNPD), a library of plant-derived natural products primarily used in folk medicine. From the resulting hits, we selected methyl oleanonate, a compound found, among others, in Pistacia lentiscus var. Chia oleoresin (Chios mastic gum). The acid of methyl oleanonate, oleanonic acid, was identified as a PPARγ agonist through bioassay-guided chromatographic fractionations of Chios mastic gum fractions, whereas some other sub-fractions exhibited also biological activity towards PPARγ. The results from the present work are two-fold: on the one hand we demonstrate that the pharmacophore model we developed is able to select novel ligand scaffolds that act as PPARγ agonists; while at the same time it manifests that natural products are highly relevant for use in virtual screening-based drug discovery.

Preparative isolation and purification of alkannin/shikonin derivatives from natural products by high-speed counter-current chromatography†

Alkannin and shikonin (A/S) and their derivatives have been found in the roots of several Boraginaceous species and are also produced through plant tissue cultures. The chiral compounds A/S are potent pharmaceutical substances with a wide spectrum of biological and pharmacological activities like wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant activity. High-speed counter-current chromatography (HSCCC) was applied for the first time to the separation, preparative isolation and purification of A/S and their esters from extracts of Alkanna tinctoria roots, as well as commercial samples. The constituents of HSCCC fractions and their purity were determined by high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS), since DAD cannot detect oligomeric A/S derivatives that are present in most of the samples containing the respective monomeric derivatives. The purity of HSCCC fractions was compared with the one of fractions isolated by column chromatography (CC) using as stationary phases silica gel and Sephadex LH-20. As shown, the purity of monomeric alkannin/shikonin was greater by HSCCC than CC separation of commercial A/S samples.

Determination of Naturally Occurring Hydroxynaphthoquinone Polymers by Size-Exclusion Chromatography

SummaryPolymerization of alkannin, shikonin, and their derivatives, potent pharmaceutical substances, crucially affects their use in pharmaceuticals, cosmetics, and as food colorants, because it leads to loss of their antimicrobial activity, reduction of the lustre of their red coloration, and a decrease in their solubility.In this study size-exclusion chromatography (SEC) has been used for the first time for qualitative and quantitative analysis of monomeric and polymeric hydroxynaphthoquinone alkannin and shikonin derivatives. The purity and degree of polymerization has been determined to evaluate severalAlkanna tinctoria root samples from different geographical sources, and commercial samples of alkannin and shikonin, as pharmaceutical raw materials. Conditions for extraction of hydroxynaphthoquinones fromAlkanna tinctoria roots with olive oil were optimized in terms of polimerization, aiming to improve the biological activity of the final pharmaceutical product, Helixderm.