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The purpose of this work was to study the chemical composition and in vitro antioxidant, antimicrobial and anti-inflammatory activities of essential oil and carlina oxide isolated to Carlina hispanica roots. The in vitro combinatory effect of carlina oxide with BHT, EDTA and diclofenac to reduce their minimum effective dose and minimize their side effects was also tested. The essential oil obtained from the roots has been studied using GC and GC/MS. Antioxidant activity was assessed using radical scavenging activity (DPPH), the β-carotene bleaching test using the BHT as a positive control, and the ferrous Iron chelating using EDTA as a positive control. The activity of anti-inflammatory was assessed by the protein denaturation method using the diclofenac as a positive control. The antimicrobial activity was evaluated against six microbial strains using the Disc Diffusion Assay and the microplate method to determine the MIC. The essential oil of C. hispanica was represented mainly by an acetylenic compound carlina oxide (81.1 %) which was isolated and identified by spectroscopic 1H NMR, 13C NMR and IR methods. The results of the biological activities showed that both essential oil and carlina oxide had an interesting antioxidant and anti-inflammatory power. Likewise, the combinations of positive controls with carlina oxide showed better activities with a substantial reduction in inhibitory concentrations and dose of the antioxidants and anti-inflammatories synthetics. Further studies are needed to provide these positive control combinations with carlina oxide in the pharmaceutical industry. Keyworlds: Carlina hispanica, essential oil, carlina oxide, biological activities. [ABSTRACT FROM AUTHOR]

Abstract In this work, a fast and low-cost voltammetric methodology for determination of carlina oxide in plant extracts was developed. The best results were obtained using a boron-doped diamond electrode (BDDE). The voltammetric measurements of carlina oxide were performed in a 0.1 mol/L solution of sulphuric acid. After 30 s of stirring the solution, differential pulse voltammograms (DPVs) were recorded from 0.5 to 1.8 V. The amplitude was 75 mV and the scan rate was 175 mV/s. Measurements were recorded in non-deaerated solutions. The background current was subtracted from each registered voltammogram; then they were cut from 0.5 to 1.5 V. The detection and quantification limits were 0.28 and 0.93 μg/L, respectively, and repeatability expressed as the relative standard deviation of 0.1 mg/L of carlina oxide was 1.9% (n = 5). The results were compared with those obtained using gas chromatography with a flame ionization detector and high performance liquid chromatography with a photodiode array detector. Graphical abstract fx1 Highlights • Voltammetric determination of carline oxide; • Chromatographic quantitative analysis of carlina oxide; • Direct determination of carlin oxide in plant extracts; • Methodological aspects of the determination of carline oxide; [ABSTRACT FROM AUTHOR]

Essential oils (EOs) are widely studied as possible candidates for new eco-friendly herbicides for weed management due to their phytotoxicity. In this study we tested the phytotoxic potential of the EO obtained from the roots of Carlina acaulis L. (Apiaceae) against the weed Bidens pilosa L. This EO, containing 98% of the polyacetylene carlina oxide, showed strong phytotoxic effects on the plant metabolism, such as leaf necrosis, reduction of relative water content and total leaf area, and an increase in the dry weight/fresh weight ratio, suggesting a water status alteration. The EO also damaged the photosynthetic machinery, as evidenced by the significant reduction of the effective quantum yield of photosystem II (Φ II) and the maximum quantum yield of photosystem II (F v /F m). In addition, the non-photochemical quenching (Φ NPQ) significantly increased after spraying with C. acaulis EO. Damage to photosystem II was further demonstrated through the reduction of manganese and calcium concentrations, possibly due to an alteration in the correct functionality of the Mn 4 Ca cluster of the PSII. Metabolomics analysis revealed an accumulation of branched-chain amino acids, such as isoleucine and valine, which is commonly related to osmotic alterations under drought stress situations and a general reduction in sugar content (fructose, glucose, mannose, among others), suggesting reduction of the photosynthetic efficiency too. Overall, these findings suggest C. acaulis EO as a promising natural product with phytotoxic potential against weeds that deserves further investigation. [Display omitted] • The phytotoxicity of Carlina caulis essential oils (EO) was bioassayed on Bidens Pilosa. • The 98% of the EO was constituted by the polyacetylene carlina oxide. • Carlina oxide treatment altered plant growth and damaged the photosystem II. • Carlina oxide induced an accumulation of amino acids and sugars. • C. acaulis EO is a promising natural product for weed control. [ABSTRACT FROM AUTHOR]

Carlina genus plants e.g. Carlina acanthifolia subsp. utzka have been still used in folk medicine of many European countries and its biological activity is mostly associated with root essential oils. In the present paper, Raman spectroscopy (RS) was applied for the first time for evaluation of essential oil distribution in root of C. acnthifolia subsp. utzka and identification of root structures containing the essential oil. Furthermore, RS technique was applied to assess chemical stability of oil during drying of plant material or distillation process. Gas chromatography–mass spectrometry was used for qualitative and quantitative analysis of the essential oil. The identity of compounds was confirmed using Raman, ATR-IR and NMR spectroscopy. Carlina oxide was found to be the main component of the oil (98.96% ± 0.15). The spectroscopic study showed the high stability of essential oil and Raman distribution analysis indicated that the oil reservoirs were localized mostly in the structures of outer layer of the root while the inner part showed nearly no signal assigned to the oil. Raman spectroscopy technique enabled rapid, non-destructive direct analysis of plant material with minimal sample preparation and allowed straightforward, unambiguous identification of the essential oil in the sample. [ABSTRACT FROM AUTHOR]

Carlina acaulis L., belonging to the Asteraceae (Compositae) family, is a perennial herb native to the Alps, which has a long history as a food and traditional remedy , being one of the most important medicinal plants in Europe. Its reported uses go from diuretic, cholagogic, and antibacterial agent for the treatment of gastritis, skin diseases, acne, eczema, and ulcers. Even if this plant presents a rich phytocomplex, its root essential oil (EO) is the product that surely raised most research interest. It has the exceptional feature of having as main constituent the polyacetylene carlina oxide, and is currently listed among botanicals employable in food supplements in Europe. This EO and carlina oxide showed an encouraging bioactivity on a variety of bacteria and fungi, such as Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa , Proteus vulgaris , Candida albicans and C. glabrata. Moreover, these products showed marked insecticidal and acaricidal activities against insect pests and vectors of medical, agricultural, and economic importance, revealing to be promising in the horizon of botanical insecticides and acaricides. Its EO as well as carlina oxide resulted to be active on vectors, e.g. Culex quinquefasciatus, urban and livestock pests e.g. Musca domestica , stored product pests e.g. Acarus siro , Trogoderma granarium and Prostephanus truncatus , and agricultural pests, e.g. Lobesia botrana , Bactrocera oleae and Ceratitis capitata, among others. These insecticidal properties are also accompanied by low-to-moderate toxicity on non-target organisms such as earthworms and aquatic microcrustaceans, as well as on vertebrate cells and mammals. Innovative formulations have been developed and tested for applying these products in real-world conditions with encouraging results. Different cultivation systems of C. acaulis have been carried out, with classic field cultivation having the greatest advantage over other systems, such as hydroponic and in vitro micropropagation in terms of insecticidal and acarcicidal compound yield. This review puts a focus for the first time on C. acaulis , its active compounds and its potential against vectors, pests, and pathogens, and on current issues related to its cultivation on a large-scale level. Moreover, its biological properties and related encapsulation techniques developed are extensively reviewed. [Display omitted] • Carlina acaulis potential as a green insecticide and acaricide was reviewed. • Carlina oxide was examined as the main responsible for the insecticidal action. • Non-target toxicity was investigated. • Challenges and perspectives for the plant cultivation were considered. • The crucial role of nanoformulations for essential oil delivery was discussed. [ABSTRACT FROM AUTHOR]

Carlina acaulis is a medicinal plant that has shown antioxidant activity in in vitro studies, but to date no corresponding in vivo data is available. Therefore, in the present study the antioxidant activity and its impact in counteracting Aβ toxicity were studied in the Caenorhabditis elegans model. A dichloromethane extract of the roots of C. acaulis was prepared and characterised via gas-liquid-chromatography/mass-spectrometry (GLC-MS). The in vitro antioxidant activity was confirmed via 2,2-diphenyl-1-picrylhydracyl assay. The extract was further separated by thin layer chromatography into two fractions, one of which was a fraction of the dichloromethane extract of C. acaulis containing mostly Carlina oxide (CarOx). Different strains of C. elegans were employed to study the expression of hsp-16.2p::GFP as a marker for oxidative stress, delocalisation of the transcription factor DAF-16 as a possible mechanism of antioxidant activity, the effect of the drug under lethal oxidative stress, and the effect against beta-amyloid (Aβ) toxicity in a paralysis assay. The C. acaulis extract and CarOx showed high antioxidant activity (stress reduction by 47% and 64%, respectively) in C. elegans and could activate the transcription factor DAF-16 which directs the expression of anti-stress genes. In paralysis assay, only the total extract was significantly active, delaying paralysis by 1.6 h. In conclusion, in vivo antioxidant activity was shown for C. acaulis for the first time in the C. elegans model. The active antioxidant compound is Carlina oxide. This activity, however, is not sufficient to counteract Aβ toxicity. Other mechanisms and possibly other active compounds are involved in this effect. [ABSTRACT FROM AUTHOR]

Context: Carlinae radix is an herbal drug, commonly used by the locals in southeastern Serbia for the treatment of respiratory and urogenital diseases and, externally, for various skin conditions. There still seems to be no detailed studies correlating the chemical composition of this drug and its ethnopharmacological uses. Objective: Chemical composition, antimicrobial activity and mode of action of C. radix essential oil, isolated from commercial samples (confirmation of whose true biological identity was also the aim of this work) were analyzed. Antimicrobial potential of decoctions (extracts prepared by boiling plant material in a given solvent), used in ethnomedicine preferentially to the pure essential oil, was also investigated. Materials and methods: The essential oil obtained by hydrodistillation was screened for antimicrobial activity by disc diffusion and broth microdilution methods. Effects of the oil on the growth of Staphylococcus aureus cells were investigated using turbidimetric measurements and visualized using scanning electron microscopy. Analyses of the chemical composition of the oils were done using gas chromatography and gas chromatography/mass spectrometry. Results and discussion: Both the essential oil and the decocts exhibited a very high antimicrobial activity against all tested strains, with S. aureus as the most sensitive one [e.g., for the oil sample the values for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 0.02, 0.04 µL/mL, respectively]. Growth curves of S. aureus demonstrated a significant decrease in turbidity (for the MIC concentration this amounted to ca. 70%) showing a concentration-dependent lysis of the cells, confirmed by scanning electron microscopy. Chemical composition, anatomical and morphological features of the sample pointed to Carlina acanthifolia L. (Asteraceae) instead of Carlina acaulis L. (Asteraceae). Conclusion: The results showed significant antimicrobial effect of the essential oil and the decoctions and support the use of this plant in ethnomedicine for the treatment of various human infections, especially those caused by S. aureus. Adulteration of the drug would not cause significant differences in its biological activity, since chemical composition of the sample showed high similarity with those containing C. acaulis roots. [ABSTRACT FROM AUTHOR]

Plant essential oil-based insecticides, with special reference to those that may be obtained from largely available biomasses, represent a valuable tool for Integrated Pest Management. However, the sublethal effects and the potential effects on aggressive insect traits of these green insecticides are understudied. Herein, the lethal and sub-lethal effects of the carlina oxide, constituting more than 97% of the whole Carlina acaulis (Asteraceae) root essential oil (EO), were determined against an invasive polyphagous tephritid pest, Ceratitis capitata (medfly). The carlina oxide was formulated in a mucilaginous solution containing carboxymethylcellulose sodium salt, sucrose, and hydrolysed proteins, showing high ingestion toxicity on medfly adults. The behavioural effects of carlina oxide at LC 10 and LC 30 were evaluated on the medfly aggressive traits, which are crucial for securing reproductive success in both sexes. Insecticide exposure affected the directionality of aggressive actions, but not the aggression escalation intensity and duration. The EO safety to mammals was investigated by studying its acute toxicity on the stomach, liver, and kidney of rats after oral administration. Only the highest dose (1000 mg/kg) of the EO caused modest neurological signs and moderate effects on the stomach, liver, and kidney. The other doses, which are closer to the practical use of the EO when formulated in protein baits, did not cause side effects. Overall, C. acaulis -based products are effective and safe to non-target mammals, deserving further consideration for eco-friendly pesticide formulations. [Display omitted] • Carlina acaulis essential oil represents green insecticides for fruit fly control. • Carlina oxide is the main constituent of the essential oil (>97%). • Carlina oxide influences the aggression directionality of C. capitata adults. • A "lure & kill" formulation based on carlina oxide has been developed against C. capitata. • Oral toxicity tests on rats showed that the C. acaulis essential oil is safe for mammals. [ABSTRACT FROM AUTHOR]

• Carlina acaulis plants can be cultivated in the field on an industrial scale. • Nitrogen fertilisation reduces the yield of Carlina acaulis biomass. • Nitrogen fertilisation does not affect the level of metabolites in C. acaulis plants. • The yield of metabolites is determined by the yield of biomass. • Carlina acaulis crops do not require intensive nitrogen fertilisation. Carlina acaulis L. can provide many valuable phytochemicals. However, the plant has a limited range and is under partial protection in many countries. Therefore, it is necessary to develop a cultivation methodology, including the mode of fertilisation. This study presents answers to the following research hypotheses: 1) C. acaulis plants can be successfully cultivated outside the natural habitat; 2) Nitrogen fertilisation of C. acaulis plants affects the yield of valuable metabolites. A cultivation experiment was carried out in a two-factor scheme with the random block method in five replicates. The research factors included the nitrogen dose (0, 4, and 8 g/m2) and the type of the nitrogen fertiliser, i.e. ammonium sulphate and calcium nitrate. The biomass yield and the metabolite content were determined with the gravimetric method and high performance liquid chromatography, respectively. It was found that C. acaulis plants were able to grow outside their natural habitat and nitrogen fertilisation negatively affected the yield of metabolites per plantation area. One hectare of cultivation without nitrogen fertilisation can provide approx. 18 kg of ursolic acid, 5 kg of oleanolic acid, 70 kg of chlorogenic acid, and 10 kg of carlina oxide. However, more research is needed to determine the effects of plant density, harvest time, and requirements for other nutrients. [ABSTRACT FROM AUTHOR]

Simple Summary: In recent years, botanical insecticides based on essential oils (EOs), or their main components, have received much attention as promising tools for Integrated Pest Management, due to their environmental safety and low side effects on non-target organisms. In this study, Carlina acaulis EO and its main component, carlina oxide, recently proven to be effective larvicidal and adulticidal agents against some insect pests, were analyzed for their toxicity, electroantennographic (EAG), and behavioral responses to adult olive fruit fly (Bactrocera oleae). The C. acaulis EO was more toxic to the tephritid than the carlina oxide, and both were more toxic to the same insect pest than EOs from other plant species tested in previous studies. The EAG responses evoked by the EO were significantly higher than those elicited by the carlina oxide. Carlina oxide did not lead to attraction or repellency responses in B. oleae males and females. Overall, our results highlight the potential employ of C. acaulis-borne products in the development of effective eco-friendly "lure and kill" formulations to be used in tephritid management. Among botanical insecticides based on essential oils (EOs) or their main components, Carlina acaulis EO and the aromatic polyacetylene carlina oxide, constituting more than 90% of its EO, were recently proven to be effective against the larvae and adults of some insect vectors and pests. In this study, the toxicity of C. acaulis EO and carlina oxide were tested on Bactrocera oleae adults using a protein bait formulation. The LC50 values of the C. acaulis EO and carlina oxide were 706 ppm and 1052 ppm, respectively. Electroantennographic (EAG) tests on B. oleae adults showed that both carlina EO and oxide elicited EAG dose-dependent responses in male and female antennae. The responses to the EO were significantly higher than those to carlina oxide, indicating that other compounds, despite their lower concentrations, can play a relevant role. Moreover, Y-tube assays carried out to assess the potential attractiveness or repellency of carlina oxide LC90 to B. oleae adults showed that it was unattractive to both males and females of B. oleae, and the time spent by both sexes in either the control or the treatment arm did not differ significantly. Overall, this study points out the potential use of C. acaulis EO and carlina oxide for the development of green and effective "lure-and-kill" tools. [ABSTRACT FROM AUTHOR]

There are several reports indicating that the roots of the Carlina acaulis L. used to be commonly applied as a treatment measure in skin diseases and as an antiparasitic agent, starting from antiquity to the 19th century; however, nowadays, it has lost its importance. Currently, numerous studies are being conducted assessing the possibility of reintroducing C. acaulis-derived extracts to phytotherapy. Determining the safety profile of the main constituents of the plant material is crucial for achieving this goal. Here, we aimed to determine the toxicity profile of carlina oxide, one of the most abundant components of the C. acaulis root extract. We obtained the carlina oxide by distillation of C. acaulis roots in the Deryng apparatus. The purity of the standard was evaluated using GC-MS, and the identity was confirmed by IR, Raman, and NMR spectroscopy. In vitro cytotoxicity was assessed using a panel of human cell lines of skin origin, including BJ normal fibroblasts and UACC-903, UACC-647, and C32 melanoma cells. This was accompanied by an in vivo zebrafish acute toxicity test (ZFET). In vitro studies showed a toxic effect of carlina oxide, as demonstrated by an induction of apoptosis and necrosis in both normal and melanoma cells. Decreased expression of AKT kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) was noted in the UACC-647 melanoma cell line. It was also observed that carlina oxide modified the expression of programmed cell death-ligand 1 (PD-L1) in tested cell lines. Carlina oxide exhibited high in vivo toxicity, with LC50 = 10.13 µg/mL upon the 96 h of exposure in the ZFET test. Here, we demonstrate that carlina oxide displays toxic effects to cells in culture and to living organisms. The data indicate that C. acaulis-based extracts considered for therapeutic use should be completely deprived of carlina oxide. [ABSTRACT FROM AUTHOR]

Carlina acaulis (Compositae) is traditionally used for food and medicinal purposes in central and southern Europe. Its root essential oil (EO), mainly composed by carlina oxide, is included in the BELFRIT botanical list of food supplements. It is also recognized as a potent mosquito larvicide. It is matter of concern whether this EO could be endowed with intrinsic toxicity to limit its use on a food level. Focusing on the insecticidal activity of this EO, we investigated the acute toxicity and sublethal effects on Musca domestica. In topical assays, the EO was extremely effective (LD 50 = 2.74 and 5.96 μg fly−1, on males and females, respectively). The exposure to a sublethal dose (LD 30) led to significant reductions of female longevity (LT 50 = 6.7–9.0 days vs. control LT 50 = 12.9–13.7 days). Treated females laid 2.5 times fewer eggs over control ones. F 1 vitality decreased: F 1 larvae and pupae showed high mortality, 2-4-fold higher over the control. The EO also showed high cytotoxicity on normal human fibroblasts (NHF-A12, IC 50 = 9.4–14.2 μg mL−1 after 6–48 h). Overall, our findings support the employ of this EO for developing botanical insecticides. At the same time, they encourage food safety authorities to perform a full toxicological assessment for possible restrictions at food level. Image 1 • Carlina acaulis root essential oil (EO) is included in the BELFRIT list of botanical food supplements. • Herein, its acute toxicity and sublethal effects on Musca domestica were studied. • EO was highly toxic to males and females (LD 50 = 2.74 and 5.96 μg fly−1, respectively). • Exposure to the EO sublethal dose (LD 30) reduced female longevity, fecundity and F 1 vitality. • The EO can be used to develop new insecticides; restriction of its employ as food may be considered. [ABSTRACT FROM AUTHOR]