Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste Material from Thymus vulgaris L., Lamiaceae

Medicinal plants have a long tradition of use in folk and conventional medicine. In recent years numerous studies confirm various bioactivities of natural products, among them antibacterial activity. Natural antibacterial agents such are essential oils and isolated compounds now represent a notable source for pharmaceutical and food industry and are widely used in cosmetology. They meet standards of \u27green consumerism\u27 together with excellent antibacterial activity. Aromatic plants such is Thymus vulgaris L. are the major sources of essential oils. Thyme essential oil, as well as dominant compounds thymol and carvacrol are generally recognised as safe and have been registered by European Commission for use as flavouring agents in foodstuffs. However, essential oil is present in very low amount (0,8-2,6%) in thyme leaves. Thus, the majority of plant material remains unused after the isolation. Nowadays, the biological potential of various plant waste materials are in focus of numerous studies. These investigations also include the antimicrobial activity considering the fact that waste material extracts represent the valuable source of different phenolic compounds. Regarding all this, the aim of the present study was to determine antibacterial potential of chemically characterised extracts obtained from waste material remaining after the preparation of drug (stems) and isolation of thyme essential oil (deodorised leaves, postdistillation decoction) on selected bacterial strains. Also, in order to determine safety of waste extracts their cytotoxicity was investigated. All extracts were prepared with maceration using 45% or 75% ethanol (EtOH) for 24 h at room temperature (1:10 w/v). Total phenolic compounds and flavonoids were determined spectrophotometrically. Extracts were chemically characterized by HPLC/DAD analysis. Antibacerial testing was done with broth dilution method against several bacterial strains (Staphylococcus aureus, Bacillus cereus, Salmonella infantis, Escherichia coli and Campylobacter jejuni). Cytotoxicity and cytoprotection studies were performed by XTT assay. Result of HPLC analysis showed that investigated extracts, especially those obtained from deodorised leaves represent a valuable source of rosmarinic acid and luteolin 7-O-glucuronide. Antibacterial testing indicated that all waste material extracts, except the extract T2, possess similar or even stronger bacteriostatic activity than T1. No cytotoxicity nor cytoprotection were determined. In conclusion, results of this study confirmed antibacterial potential investigated thyme extracts. High concentrations of rosmarinic acid and luteolin 7-O-glucuronide, which both have numerous pharmacological activities, were determined. This indicates that thyme postdistillation waste material extracts could be used for isolation of dominant compounds or as addities in pharmaceutical and food industry

MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options—especially during pregnancy

SK119, a Novel Shikonin Derivative, Leads to Apoptosis in Melanoma Cell Lines and Exhibits Synergistic Effects with Vemurafenib and Cobimetinib

Melanoma is a complex and heterogenous disease, displays the deadliest form of skin cancer, and accounts for approx. 80% of all skin cancer deaths. In this study, we reported on the synthesis and pharmacological effects of a novel shikonin derivative (SK119), which is active in a nano-molar range and exhibits several promising in vitro effects in different human melanoma cells. SK119 was synthesized from shikonin as part of our search for novel, promising shikonin derivatives. It was screened against a panel of melanoma and non-tumorigenic cell lines using XTT viability assays. Moreover, we studied its pharmacological effects using apoptosis and Western blot experiments. Finally, it was combined with current clinically used melanoma therapeutics. SK119 exhibited IC50 values in a nano-molar range, induced apoptosis and led to a dose-dependent increase in the expression and protein phosphorylation of HSP27 and HSP90 in WM9 and MUG-Mel 2 cells. Combinatorial treatment, which is highly recommended in melanoma, revealed the synergistic effects of SK119 with vemurafenib and cobimetinib. SK119 treatment changed the expression levels of apoptosis genes and death receptor expression and exhibited synergistic effects with vemurafenib and cobimetinib in human melanoma cells. Further research indicates a promising potential in melanoma therapy

Phytochemical and pharmacological investigations on constituents of medicinal plants with potential anti-cancer activity

Ziel dieser Dissertation war die phytochemische und pharmakologische Untersuchung chinesischer Heilkräuter mit potenziell krebshemmender Wirkung. Dafür wurden 12 Pflanzen aufgrund eines vorausgegangenen Screenings ausgewählt. Diese wurden sukzessiv mit verschiedenen Lösungsmitteln und verschiedenen Methoden extrahiert. Alle Extrakte wurden in in vitro Vitalitätstests gegen verschiedene Krebszelllinien untersucht. Sechs Extrakte zeigten hohe, vier mäßige Aktivität.Mittels aktivitätsgerichteter Fraktionierung wurden vier hochaktive Shikonin Derivate aus Onosma paniculata isoliert. Der aktive Extrakt sowie Dimethylacryl- und Epoxyshikonin beeinflussten die Zellmorphologie, die Zellzyklusverteilung und die Spaltung von Caspase-3 in verschiedenen Melanomzellen. Dimethylacrylshikonin veränderte auch die mRNA Expression einiger proapoptotischer Gene. Die Ergebnisse lassen vermuten, dass Dimethylacrylshikonin nicht nur Caspase abhängig Apoptose induziert, sondern auch Caspase unabhängig. Zuletzt wurden die Aktivitäten der isolierten Substanzen mit den anderer Shikonin und Alkannin Derivate verglichen.Costunolid und Dehydrocostus lacton wurden als aktive Substanzen aus Saussurea lappa isoliert. Untersuchungen an einigen Weichteilsarkomzelllinien zeigten, dass beide die Zellzyklusverteilung und Expression verschiedener Metalloproteinasen änderten. Dehydrocostus Lacton verringerte auch die Expression des ABCB1 Transporters. Allerdings konnten keine apoptotische Ereignisse (Veränderung des mitochondrialen Membranpotenzial oder Caspase-3 Spaltung) festgestellt werden.Aus Helianthus angustifolius wurden vier Furanogermacranolide (8-Methacrylyl-4,15-iso-atriplicolid, 8-Isobutyryl-4,15-iso-atriplicolid, 8-(2-Methylbutyryl)-4,15-iso-atriplicolid und 8-Isovaleryl-4,15-iso-atriplicolid) isoliert und als aktive Substanzen identifiziert. Letzteres wurde bisher noch nicht beschrieben. Die anderen drei wurden bisher noch nicht aus dieser Pflanzenart isoliert.The goal of this doctoral thesis was the phytochemical and pharmacological investigation of Chinese herbs with potential anti-cancer activity. 12 plants were chosen for investigation based on a previous study. The plant material was successively extracted with different solvents and different extraction methods. All extracts were subjected to in vitro viability assays using several cancer cell lines. Six extracts showed high activity, four of them moderate activity. Using activity-guided fractionation, four active shikonin derivatives were isolated from Onosma paniculata. The active extract as well as dimethylacryl- and epoxyshikonin were shown to affect cell morphology, cell cycle distribution and cleavage of caspase-3 in different melanoma cell lines. Dimethylacrylshikonin also changed the mRNA expression levels of several proapoptotic genes. The results suggest that dimethylacrylshikonin can not only lead to caspase-dependent apoptosis but also to caspase-independent. Finally, the activity of the isolated compounds was compared to several other shikonin and alkannin derivatives.Costunolide and dehydrocostus lactone were identified as active principles in Saussurea lappa. Investigations of their effects on several soft tissue sarcomas revealed that they changed cell cycle distribution and inhibited the expression of different metalloproteinases. Dehydrocostus lactone also decreased the expression levels of ABCB1 transporter. However, apoptotic events (disruption of the mitochondrial membrane potential or cleavage of caspase-3) were not observed.From Helianthus angustifolius, four furanogermacranolides (8-methacrylyl-4,15-iso-atriplicolide, 8-isobutyryl-4,15-iso-atriplicolide, 8-(2-methylbutyryl)-4,15-iso-atriplicolide and 8-isovaleryl-4,15-iso-atriplicolide) were isolated and identified as active principles. The last compound has not been reported before. The other three have not yet been reported in this plant species.vorgelegt von Nadine KretschmerZsfassung in dt. SpracheGraz, Univ., Diss., 2011(VLID)21621

Novel Pathway for Catabolism of the Organic Sulfur Compound 3,3′-Dithiodipropionic Acid via 3-Mercaptopropionic Acid and 3-Sulfinopropionic Acid to Propionyl-Coenzyme A by the Aerobic Bacterium Tetrathiobacter mimigardefordensis Strain DPN7▿

The hitherto unstudied microbial degradation of the organic disulfide 3,3′-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7T (DSM 17166T; LMG 22922T), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis

SK119, a Novel Shikonin Derivative, Leads to Apoptosis in Melanoma Cell Lines and Exhibits Synergistic Effects with Vemurafenib and Cobimetinib

Melanoma is a complex and heterogenous disease, displays the deadliest form of skin cancer, and accounts for approx. 80% of all skin cancer deaths. In this study, we reported on the synthesis and pharmacological effects of a novel shikonin derivative (SK119), which is active in a nano-molar range and exhibits several promising in vitro effects in different human melanoma cells. SK119 was synthesized from shikonin as part of our search for novel, promising shikonin derivatives. It was screened against a panel of melanoma and non-tumorigenic cell lines using XTT viability assays. Moreover, we studied its pharmacological effects using apoptosis and Western blot experiments. Finally, it was combined with current clinically used melanoma therapeutics. SK119 exhibited IC50 values in a nano-molar range, induced apoptosis and led to a dose-dependent increase in the expression and protein phosphorylation of HSP27 and HSP90 in WM9 and MUG-Mel 2 cells. Combinatorial treatment, which is highly recommended in melanoma, revealed the synergistic effects of SK119 with vemurafenib and cobimetinib. SK119 treatment changed the expression levels of apoptosis genes and death receptor expression and exhibited synergistic effects with vemurafenib and cobimetinib in human melanoma cells. Further research indicates a promising potential in melanoma therapy

Synthesis of Tetrahydrohonokiol Derivates and Their Evaluation for Cytotoxic Activity against CCRF-CEM Leukemia, U251 Glioblastoma and HCT-116 Colon Cancer Cells

Biphenyl neolignans such as honokiol and magnolol, which are the major active constituents of the Asian medicinal plant Magnolia officinalis, are known to exert a multitude of pharmacological and biological activities. Among these, cytotoxic and tumor growth inhibitory activity against various tumour cell lines are well-documented. To further elucidate the cytotoxic effects of honokiol derivatives, derivatizations were performed using tetrahydrohonokiol as a scaffold. The derivatizations comprised the introduction of functional groups, e.g., nitro and amino groups, as well as alkylation. This way, 18 derivatives, of which 13 were previously undescribed compounds, were evaluated against CCRF-CEM leukemia cells, U251 glioblastoma and HCT-116 colon cancer cells. The results revealed no significant cytotoxic effects in any of the three tested cell lines at a test concentration of 10 µM

Synthesis of Tetrahydrohonokiol Derivates and Their Evaluation for Cytotoxic Activity against CCRF-CEM Leukemia, U251 Glioblastoma and HCT-116 Colon Cancer Cells

Biphenyl neolignans such as honokiol and magnolol, which are the major active constituents of the Asian medicinal plant Magnolia officinalis, are known to exert a multitude of pharmacological and biological activities. Among these, cytotoxic and tumor growth inhibitory activity against various tumour cell lines are well-documented. To further elucidate the cytotoxic effects of honokiol derivatives, derivatizations were performed using tetrahydrohonokiol as a scaffold. The derivatizations comprised the introduction of functional groups, e.g., nitro and amino groups, as well as alkylation. This way, 18 derivatives, of which 13 were previously undescribed compounds, were evaluated against CCRF-CEM leukemia cells, U251 glioblastoma and HCT-116 colon cancer cells. The results revealed no significant cytotoxic effects in any of the three tested cell lines at a test concentration of 10 µM

Antimicrobial and Cytotoxic Isohexenylnaphthazarins from Arnebia euchroma (Royle) Jonst. (Boraginaceae) Callus and Cell Suspension Culture

The phytochemical investigation of the n-hexane extract from callus and cell suspension culture of Arnebia euchroma (Royle) Jonst. resulted in the isolation of nine isohexenylnaphthazarins: deoxyalkannin (1), alkannin (2), acetylalkannin (3), isobutyrylalkannin (4), β-hydroxyisovalerylalkannin (5), 2\u27\u27-(S)-α-methylbutyrylalkannin (6), propionylalkannin (7), teracrylalkannin (8) and acetylshikonin (9). Their structures were determined by MS and NMR spectroscopy. Pigments 2–8 are isolated for the first time from Arnebia in vitro cultures, 4 and 7 are reported in the present work as novel metabolites within the Arnebia genus, while 9 is a known constituent of both natural roots and in vitro cultures of A. euchroma. Moreover, methyl jasmonate and 1-monoglyceryl olate, palmitate and stearate are reported for the first time within the Boraginaceae family. The antimicrobial and cytotoxic activities of all isolated pigment compounds were tested, revealing a very interesting profile

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGloTM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma