Antioxidant Activity, Inhibition of Intestinal Cancer Cell Growth and Polyphenolic Compounds of the Seagrass Posidonia oceanica's Extracts from Living Plants and Beach Casts.

The aim of the present study was to investigate the use of Posidonia oceanica for making products beneficial for human health. Firstly, we demonstrated that the antioxidant defense (i.e., SOD and APX activity) of P. oceanica's living leaves (LP) has low efficacy, as they partly neutralize the produced H2O2. However, high H2O2 levels led LP to produce, as a response to oxidative stress, high phenolic content, including chicoric acid, p-coumaric acid, caftaric acid, trans-cinnamic and rutin hydrate, as shown by UHPLC-DAD analysis. In addition, LP extracts inhibited intestinal cancer cell proliferation. Moreover, P. oceanica's beach casts consisting of either Wet 'Necromass' (WNP) or Dry 'Necromass' (DNP) were used for preparing extracts. Both DNP and WNP exhibited antioxidant and antiproliferative activities, although lower as compared to those of LP extracts. Although both P. oceanica's meadows and beach casts are considered priority habitats in the Mediterranean Sea due to their high ecological value, legislation framework for beach casts forbidding their removal is still missing. Our results suggested that both LP and DNP could be utilized for the production of high-added value products promoting human health, provided that a sustainability management strategy would be applied for P. oceanica's meadows and beach casts.

Occurrence of Luteolin in the Greek Flora, Isolation of Luteolin and Its Action for the Treatment of Periodontal Diseases.

Higher plants possess the ability to synthesize a great number of compounds with many different functions, known as secondary metabolites. Polyphenols, a class of flavonoids, are secondary metabolites that play a crucial role in plant adaptation to both biotic and abiotic environments, including UV radiation, high light intensity, low/high temperatures, and attacks from pathogens, among others. One of the compounds that has received great attention over the last few years is luteolin. The objective of the current paper is to review the extraction and detection methods of luteolin in plants of the Greek flora, as well as their luteolin content. Furthermore, plant species, crop management and environmental factors can affect luteolin content and/or its derivatives. Luteolin exhibits various biological activities, such as cytotoxic, anti-inflammatory, antioxidant and antibacterial ones. As a result, luteolin has been employed as a bioactive molecule in numerous applications within the food industry and the biomedical field. Among the different available options for managing periodontitis, dental care products containing herbal compounds have been in the spotlight owing to the beneficial pharmacological properties of the bioactive ingredients. In this context, luteolin's anti-inflammatory activity has been harnessed to combat periodontal disease and promote the restoration of damaged bone tissue.

A Review of the Role of Natural Products as Treatment Approaches for Xerostomia.

Xerostomia, commonly known as dry mouth, is a widespread oral health malfunction characterized by decreased salivary flow. This condition results in discomfort, impaired speech and mastication, dysphagia, heightened susceptibility to oral infections, and ultimately, a diminished oral health-related quality of life. The etiology of xerostomia is multifaceted, with primary causes encompassing the use of xerostomic medications, radiation therapy to the head and neck, and systemic diseases such as Sjögren's syndrome. Consequently, there is a growing interest in devising management strategies to address this oral health issue, which presents significant challenges due to the intricate nature of saliva. Historically, natural products have served medicinal purposes, and in contemporary pharmaceutical research and development, they continue to play a crucial role, including the treatment of xerostomia. In this context, the present review aims to provide an overview of the current state of knowledge regarding natural compounds and extracts for xerostomia treatment, paving the way for developing novel therapeutic strategies for this common oral health issue.

Bacterial responses to plant antimicrobials: the case of alkannin and shikonin derivatives.

Alkannin, shikonin and their derivatives (A/S) are secondary metabolites produced in the roots of certain plants of the Boraginaceae family such as Lithospermum erythrorhizon Siebold & Zucc. and Alkanna tinctoria (L.) Tausch. These naphthoquinones express anti-cancer, wound healing, and antimicrobial activities. To study the interactions between endophytic bacteria isolated from A. tinctoria and the antimicrobials A/S, endophytic bacteria known to be resistant to the compounds were screened for their effect on A/S in liquid medium. Thereafter, the strain Pseudomonas sp. R-72008, was selected and tested for its ability to modify A/S in nutrient medium and minimal medium with A/S as sole carbon source. Bacterial growth was recorded, and high performance liquid chromatography-diode array and ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry analyses were performed to detect and quantify metabolites. In nutrient medium inoculated with R-72008, a decrease in the amount of A/S monomers initially present was observed and correlated with an increase of A/S oligomers. Moreover, a significant decrease of initial A/S monomers in minimal medium was correlated with bacterial growth, showing for the first time that a bacterial strain, Pseudomonas sp. R-72008, was able to use the naphthoquinones A/S as sole carbon source. This study opens new perspectives on the interactions between bacteria and plant antimicrobials.

Antioxidant Activity and Inhibition of Liver Cancer Cells' Growth of Extracts from 14 Marine Macroalgae Species of the Mediterranean Sea.

Macroalgae exhibit beneficial bioactivities for human health. Thus, the aim of the present study was to examine the antioxidant and anticancer potential of 14 macroalgae species' extracts, namely, Gigartina pistillata, Gigartina teedei, Gracilaria gracilis, Gracilaria sp., Gracilaria bursa pastoris, Colpomenia sinuosa, Cystoseira amentacea, Cystoseira barbata, Cystoseira compressa, Sargassum vulgare, Padina pavonica, Codium fragile, Ulva intestinalis, and Ulva rigida, from the Aegean Sea, Greece. The antioxidant activity was assessed using DPPH, ABTS•+, •OH, and O2•- radicals' scavenging assays, reducing power (RP), and protection from ROO•-induced DNA plasmid damage assays. Moreover, macroalgae extracts' total polyphenol contents (TPCs) were assessed. Extracts' inhibition against liver HepG2 cancer cell growth was assessed using the XTT assay. The results showed that G. teedei extract's IC50 was the lowest in DPPH (0.31 ± 0.006 mg/mL), ABTS•+ (0.02 ± 0.001 mg/mL), •OH (0.10 ± 0.007 mg/mL), O2•- (0.05 ± 0.003 mg/mL), and DNA plasmid breakage (0.038 ± 0.002 mg/mL) and exhibited the highest RP (RP0.5AU 0.24 ± 0.019 mg/mL) and TPC (12.53 ± 0.88 mg GAE/g dw). There was also a significant correlation between antioxidant activity and TPC. P. pavonica (IC50 0.93 ± 0.006 mg/mL) exhibited the highest inhibition against HepG2 cell growth. Conclusively, some of the tested extracts exhibited significant chemopreventive properties, and so they may be used for food products.

Expanding the Biological Properties of Alkannins and Shikonins: Their Impact on Adipogenesis and Life Expectancy in Nematodes.

Alkannin, shikonin (A/S) and their derivatives are naturally occurring hydroxynaphthoquinones biosynthesized in some species of the Boraginaceae family. These natural compounds have been extensively investigated for their biological properties over the last 40 years, demonstrating a plethora of activities, such as wound healing, regenerative, anti-inflammatory, antitumor, antimicrobial and antioxidant. This study aims to extend the current knowledge by investigating the effects of various A/S compounds on two model systems, namely on 3T3-L1 pre-adipocytes and the nematode Caenorhabditis elegans. The former constitutes an established in vitro model for investigating anti-obesity and insulin-mimetic properties, while the latter has been widely used as a model organism for studying fat accumulation, lifespan and the anthelmintic potential. A set of chemically well-defined A/S derivatives were screened for their effect on pre-adipocytes to assess cell toxicity, cell morphology, and cell differentiation. The differentiation of pre-adipocytes into mature adipocytes was examined upon treatment with A/S compounds in the presence/absence of insulin, aiming to establish a structure-activity relationship. The majority of A/S compounds induced cell proliferation at sub-micromolar concentrations. The ester derivatives exhibited higher IC50 values, and thus, proved to be less toxic to 3T3-L1 cells. The parent molecules, A and S tested at 1 µM resulted in a truncated differentiation with a reduced number of forming lipids, whereas compounds lacking the side chain hydroxyl group projected higher populations of mature adipocytes. In C. elegans mutant strain SS104, A/S enriched extracts were not able to inhibit the fat accumulation but resulted in a drastic shortage of survival. Thus, the set of A/S compounds were tested at 15 and 60 µg/ml in the wild-type strain N2 for their nematocidal activity, which is of relevance for the discovery of anthelmintic drugs. The most pronounced nematocidal activity was observed for naphthazarin and ß,ß-dimethyl-acryl-shikonin, followed by isovaleryl-shikonin. The latter 2 A/S esters were identified as the most abundant constituents in the mixture of A/S derivatives isolated from Alkanna tinctoria (L.) Tausch. Taken together, the findings show that the structural variations in the moiety of A/S compounds significantly impact the modulation of their biological activities in both model systems investigated in this study.

Headspace gas chromatography-mass spectrometry in the analysis of lavender's essential oil: Optimization by response surface methodology.

A static headspace gas chromatography - mass spectrometry (HS-GC/MS) method was developed and optimized with the aim to be applied in the analysis of lavender essential oil. To obtain a comprehensive profile of the essential oil, the optimum HS-GC/MS method parameters were selected based on a Design of Experiments (DοE) process. Plackett-Burman experimental design was applied by utilizing seven parameters of the HS injection system. Incubation equilibration temperature and time, agitator's vortex speed, post injection dwell time, inlet temperature, split ratio and injection flow rate were screened to select the optimum conditions on the basis of the number and the intensity of the identified compounds. Other parameters, such as sample volume and dilution solvent ratio, were also examined to achieve a comprehensive profile in a chromatographic run of 55 min. With the obtained optimum method, more than 40 volatile compounds were identified in lavender's essential oils from different geographical regions in Greece. The method can be utilized for the quality assessment of lavender's essential oil and provide information on its characteristic aroma and discrimination among species based on the acquired GC-MS profiles.

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.

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.

Pistacia lentiscus Oleoresin: Virtual Screening and Identification of Masticadienonic and Isomasticadienonic Acids as Inhibitors of 11ß-Hydroxysteroid Dehydrogenase 1.

In traditional medicine, the oleoresinous gum of Pistacia lentiscus var. chia, so-called mastic gum, has been used to treat multiple conditions such as coughs, sore throats, eczema, dyslipidemia, and diabetes. Mastic gum is rich in triterpenes, which have been postulated to exert antidiabetic effects and improve lipid metabolism. In fact, there is evidence of oleanonic acid, a constituent of mastic gum, acting as a peroxisome proliferator-activated receptor γ agonist, and mastic gum being antidiabetic in mice in vivo. Despite these findings, the exact antidiabetic mechanism of mastic gum remains unknown. Glucocorticoids play a key role in regulating glucose and fatty acid metabolism, and inhibition of 11ß-hydroxysteroid dehydrogenase 1 that converts inactive cortisone to active cortisol has been proposed as a promising approach to combat metabolic disturbances including diabetes. In this study, a pharmacophore-based virtual screening was applied to filter a natural product database for possible 11ß-hydroxysteroid dehydrogenase 1 inhibitors. The hit list analysis was especially focused on the triterpenoids present in Pistacia species. Multiple triterpenoids, such as masticadienonic acid and isomasticadienonic acid, main constituents of mastic gum, were identified. Indeed, masticadienonic acid and isomasticadienonic acid selectively inhibited 11ß-hydroxysteroid dehydrogenase 1 over 11ß-hydroxysteroid dehydrogenase 2 at low micromolar concentrations. These findings suggest that inhibition of 11ß-hydroxysteroid dehydrogenase 1 contributes to the antidiabetic activity of mastic gum.

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.

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.

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.

Analysis of antioxidant compounds in sweet orange peel by HPLC-diode array detection-electrospray ionization mass spectrometry.

HPLC-diode array detection-electrospray ionization mass spectrometry was used to determine qualitatively and quantitatively the flavonoid content of several fractions and residues of extracts of Greek navel sweet orange peel (Citrus sinensis) from the region of southern Greece (Leonidi-Tripoli). The main groups of flavonoids found according to HPLC retention times, spectral data and literature references were polymethoxylated flavones, C-glycosylated flavones, O-glycosylated flavones, O-glycosylated flavanones, flavonols and phenolic acids and their derivatives. The ethyl acetate fraction which has been shown in previous work to possess the best radical scavenging activity among the others was found to contain C-glycosylated flavones, polymethoxylated flavones, O-glycosylated flavones, O-glycosylated flavanones, two phenolic acid derivatives and two unknown compounds, all in low concentrations. The group of C-glycosylated flavones was reported for the first time in the peel of Navel sweet orange. The C-glycosylated flavones found according to their spectral characteristics and literature were 6-C-beta-glucosyldiosmin, 6,8-di-C-glucopyranosylapigenin, 6,8-di-C-beta-glucosyldiosmin and two unknown. The results suggest that the ethyl acetate fraction of navel Citrus sinensis peel consists of significant antioxidant compounds and can be used as a food additive of natural origin or a pharmaceutical supplement using as a source of peel the byproducts of the orange juice industry.

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.