The CENP-A nucleosome: where and when it happens during the inner kinetochore's assembly.

CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin.

Influence of Abiotic and Biotic Elicitors on Organogenesis, Biomass Accumulation, and Production of Key Secondary Metabolites in Asteraceae Plants.

The medicinal plants of the Asteraceae family are a valuable source of bioactive secondary metabolites, including polyphenols, phenolic acids, flavonoids, acetylenes, sesquiterpene lactones, triterpenes, etc. Under stressful conditions, the plants develop these secondary substances to carry out physiological tasks in plant cells. Secondary Asteraceae metabolites that are of the greatest interest to consumers are artemisinin (an anti-malarial drug from Artemisia annua L.-sweet wormwood), steviol glycosides (an intense sweetener from Stevia rebaudiana Bert.-stevia), caffeic acid derivatives (with a broad spectrum of biological activities synthesized from Echinacea purpurea (L.) Moench-echinacea and Cichorium intybus L.-chicory), helenalin and dihydrohelenalin (anti-inflammatory drug from Arnica montana L.-mountain arnica), parthenolide ("medieval aspirin" from Tanacetum parthenium (L.) Sch.Bip.-feverfew), and silymarin (liver-protective medicine from Silybum marianum (L.) Gaertn.-milk thistle). The necessity to enhance secondary metabolite synthesis has arisen due to the widespread use of these metabolites in numerous industrial sectors. Elicitation is an effective strategy to enhance the production of secondary metabolites in in vitro cultures. Suitable technological platforms for the production of phytochemicals are cell suspension, shoots, and hairy root cultures. Numerous reports describe an enhanced accumulation of desired metabolites after the application of various abiotic and biotic elicitors. Elicitors induce transcriptional changes in biosynthetic genes, leading to the metabolic reprogramming of secondary metabolism and clarifying the mechanism of the synthesis of bioactive compounds. This review summarizes biotechnological investigations concerning the biosynthesis of medicinally essential metabolites in plants of the Asteraceae family after various elicitor treatments.

Hollow spherical nucleic acid structures based on polymer-coated phospholipid vesicles.

A feasible one pot synthesis of hollow spherical nucleic acids (SNAs) using phospholipid liposomes is reported. These constructs are synthesized in a chemically straightforward process involving formation of unilamellar liposomes, coating the liposomes with a thin cross-linked polymeric layer, and grafting the latter with short (about 20 bases) DNA oligonucleotide strands. They consist of vesicular cores, composed of readily available phospholipid (1,2-dipalmitoyl-sn-glycero-phosphocholine), whereas the strands are deliberately arranged on the surface of the vesicular entities. The initial vesicular structure and morphology are preserved during the coating and grafting reactions. The novel hollow/vesicular SNAs are characterized with a hydrodynamic radius and radius of gyration of 78.3 and 88.5 nm, respectively, and moderately negative (-14.2 mV) ζ potential. They carry thousands (5868) of oligonucleotide strands per vesicle, which are not strongly radially oriented and adopt an unextended conformation as anticipated from the smaller value of the grafting density compared to the critical grafting density at the transition to brush conformation. The constructs are practically devoid of toxicity and exhibit high binding affinity to complementary nucleic acids. Unlike any other nucleic acid structural motif, they cross the cell membrane and enter cells without the need of transfection agents.

Radioulnar contrasts in fingerprint ridge counts: Searching for dermatoglyphic markers of early sex development.

OBJECTIVES: Using prenatally fixed dermatoglyphics features as markers of prenatal sex development is limited due to insufficient knowledge on their sex differences. This study aims to examine more thoroughly sex differences in radioulnar contrasts. METHODS: Fingerprints of 360 females and 331 males from four samples of different ethnic backgrounds (Czechs, Slovaks, Vietnamese and Lusatian Sorbs) were studied. On both hands, finger ridge counts were recorded, and all possible radioulnar contrasts were computed as a difference between ridge count at a radial position minus ridge count at a respective ulnar position on the hand. Radioulnar contrasts with population-congruent and numerically large dimorphism were selected and the dimorphism of the selected radioulnar contrasts was then tested using nonparametric analysis of variance. RESULTS: Greater dimorphism of radioulnar contrasts occurred on the right hand than on the left hand. Population congruent direction and relatively strong dimorphism (Cohen's d greater than 0.3) was found in six radioulnar contrasts on the right hand, all of which involved the radial ridge count of the 2nd finger. Of these, the highest average dimorphism was observed for the difference between the radial ridge count of the 2nd finger and the ulnar ridge count of the 4th finger (2r4u contrast), where the average effect size from all four population samples was comparable to a published average effect size of the 2D:4D finger length ratio. CONCLUSION: We propose that 2r4u contrast of ridge counts could serve as a marker of prenatal sexual development targeting a temporally narrow developmental window.

Functional Polyglycidol-Based Block Copolymers for DNA Complexation.

Gene therapy is an attractive therapeutic method for the treatment of genetic disorders for which the efficient delivery of nucleic acids into a target cell is critical. The present study is aimed at evaluating the potential of copolymers based on linear polyglycidol to act as carriers of nucleic acids. Functional copolymers with linear polyglycidol as a non-ionic hydrophilic block and a second block bearing amine hydrochloride pendant groups were prepared using previously synthesized poly(allyl glycidyl ether)-b-polyglycidol block copolymers as precursors. The amine functionalities were introduced via highly efficient radical addition of 2-aminoethanethiol hydrochloride to the alkene side groups. The modified copolymers formed loose aggregates with strongly positive surface charge in aqueous media, stabilized by the presence of dodecyl residues at the end of the copolymer structures and the hydrogen-bonding interactions in polyglycidol segments. The copolymer aggregates were able to condense DNA into stable and compact nanosized polyplex particles through electrostatic interactions. The copolymers and the corresponding polyplexes showed low to moderate cytotoxicity on a panel of human cancer cell lines. The cell internalization evaluation demonstrated the capability of the polyplexes to successfully deliver DNA into the cancer cells.

Evaluation of Hepcidin and Atherosclerosis in Dialysis Patients.

BACKGROUND: Chronic kidney disease (CKD) is a condition that involves 10% - 15% of population worldwide, which increases the risk of cardio-vascular diseases (CVD). Chronic kidney disease is one of the main reasons for illness and mortality in the world. Chronic kidney disease is a serious health problem caused by involvement of a large number of patients with kidney injury, especially in industrial countries. Among the main reasons for this are population living longer and the number of diseases in elderly persons, such as diabetes mellitus type 2, hypertension, and cardio-vascular diseases. METHODS: We evaluated 63 patients on chronic dialysis at the Dialysis Centre at University "Aleksandrovska" Hospital; the average age was 49.9 ± 7.8. Their results were compared to 63 age matched controls. Blood samplings were taken before dialysis procedure. In the included groups, we measured CBC, serum iron (by Ferrozine method), ferritin, soluble transferrin receptors and hsCRP (by nephelometric method), hepcidin (by ELISA method), and homocysteine (by CLIA method). IMT was measured by using electronic calipers and evaluated by automated software programs. RESULTS: We established elevated serum hepcidin levels in CKD patients (205.1 ± 29.9 µg/L) compared to the control group (20.8 ± 3.1 µg/L), p < 0.001. Serum homocysteine and hsCRP concentrations were elevated in CKD cases (48.7 ± 6.8 µmol/L; 29.7 ± 4.1 mg/L) compared to controls (7.9 ± 1.8 µmol/L; 1.1 ± 0.4 mg/L), p < 0.005. In patients with CKD we found a strong positive correlation between serum hepcidin and homocysteine concentrations, r = 0.879, p < 0.001. In patients with impaired kidney function soluble transferrin receptors correlated negatively to hepcidin: r = -0.799, p < 0.001. In dialysis, the transferrin concentration correlated highly positive to hepcidin: r = 0.691, p < 0.001. IMT in CKD patients correlated positively to hepcidin and homocysteine levels: r = 0.788 and r = 0.841, respectively, p < 0.005. CONCLUSIONS: Chronic kidney disease is connected to cardio-vascular disease risk factors. CKD might be an independent CVD risk factor. In early kidney injury stages, increased morbidity is found from CVD. The risk of fatal and non-fatal cardio-vascular incidents is connected to kidney injury. For clinical practice, early evaluation of hepcidin and atherosclerosis in chronic kidney disease patients is very important.

A Copper-Nitroxide Adduct Exhibiting Separate Single Crystal-to-Single Crystal Polymerization-Depolymerization and Spin Crossover Transitions.

A complex cascade of solid-state processes initiated by variation of temperature was found for the heterospin complex [Cu(hfac)2L(Me/Et)] formed in the reaction of copper(II) hexafluoroacetylacetonate [Cu(hfac)2] with stable nitronyl nitroxide 2-(1-methyl-3-ethyl-1H-pyrazol-4-yl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl (L(Me/Et)). The cooling of the compound below 260 K initiated a solid-state chemical reaction, which led to a depolymerization of chains and formation of a pair heterospin complex [Cu(hfac)2L(Me/Et)2][[Cu(hfac)2]3L(Me/Et)2]. Further decrease in temperature below 144 K led to a spin transition accompanied by a drastic decrease in the effective magnetic moment from 2.52 to 2.24 µB. When the compound was heated, the order of effects was reversed: at first, the magnetic moment abruptly increased, and then the molecular fragments of the pair cluster united into polymer chains. Two hysteresis loops correspond to this cascade of temperature-induced structural transformations on the experimental dependence µeff(T): one at high (T↑ = 283 K and T↓ = 260 K) and the other at low (T↑ = 161 K, T↓ = 144 K) temperature. The spin transitions were also recorded for the [[Cu(hfac)2]3L(Bu/Et)2] and [[Cu(hfac)2]5L(Bu/Et)4] molecular complexes, which are models of the trinuclear fragment of the {[Cu(hfac)2]3L(Me/Et)2} pair cluster.

Field experiment estimate of electoral fraud in Russian parliamentary elections.

Electoral fraud is a widespread phenomenon, especially outside the developed world. Despite abundant qualitative and anecdotal evidence on its existence from around the world, there is very limited quantitative evidence on the extent of electoral fraud. We exploit random assignment of independent observers to 156 of 3,164 polling stations in the city of Moscow to estimate the effect of electoral fraud on the outcome of the Russian parliamentary elections held on December 4, 2011. We estimate the actual share of votes for the incumbent United Russia party to be at least 11 percentage points lower than the official count (36% instead of 47%). Our results suggest that the extent of the fraud was sufficient to have had a substantial impact on the outcome of the elections; they also confirm that the presence of observers is an important factor in ensuring the integrity of the procedure.

Developmental and Environmental Effects on Sesquiterpene Lactones in Cultivated Arnica montana L.

The amount of sesquiterpene lactones and the lactone profile of Arnica montana L. in flowering and seed formation stages in vitro and in vivo propagated from seeds of German, Ukrainian, and Austrian origin and grown in two experimental fields were studied. It was found that in vitro propagated 2-year plants in full flowering stage accumulated higher amount of lactones in comparison to in vivo propagated 3-year plants and to the seed formation stage, respectively. Helenalins predominated in in vivo propagated 2-year or in vitro propagated 3-year plants. 2-Methylbutyrate (2MeBu) was the principal ester in the samples with prevalence of helenalins, while isobutyrate (iBu) was the major one in the samples with predominance of 11,13-dihydrohelenalins. The results revealed that the environmental conditions on Vitosha Mt. are more suitable for cultivation of A. montana giving higher content of lactones.

Comparison of antioxidant activity of the fruits derived from in vitro propagated and traditionally cultivated tayberry plants.

BACKGROUND: Tayberry is a hybrid between Rubus fruticosus L. and Rubus idaeus L. These fruits contain valuable vitamins and antioxidants. An effective protocol for micropropagation of tayberry plants is here described. Different concentrations of cytokinins (6-benzylaminopurine, zeatin, and 6-(γ,γ-dimethylallylamino)purine) were added in Murashige and Skoog, 1962 (MS) medium to micropropagation using stem tip and nodal explants. RESULTS: The highest propagation rate was recorded on MS medium containing 2 mg L(-1) zeatin, where the shoot formation resulted in 3.4 shoots per stem tip explant after 4 weeks of culture. It was found that half-strength MS medium with 0.1 mg L(-1) indole-3-butyric acid was the best for plant rooting. For ex vitro acclimatization of plants, the mixture of peat, soil, and perlite (1:1:1 v/v/v) was the most suitable planting substrate for hardening. The micropropagation protocol described in this study might be useful for the production of healthy plant materials. Tayberry fruits from in vitro propagated plants and adapted to the field conditions possessed higher antioxidant capacity in comparison to traditionally cultivated plants. CONCLUSION: Fruit extracts of micropropagated tayberry plants and adapted to field conditions can be used as a rich source of natural antioxidants. © 2015 Society of Chemical Industry.

Anion selectivity in ion exchange reactions with surface functionalized ionosilicas.

Mesoporous imidazolium functionalized surface functionalized ionosilicas have been investigated as anion exchange materials for the adsorption of oxo-anions in aqueous media. We studied particularly pertechnetate adsorption and could show that solids bearing long chain substituted imidazolium groups are highly efficient anion exchange materials often displaying high distribution coefficients between solid and liquid phases. We observed that the distribution coefficient of pertechnetate is a function of the presence of competing anionic species. As a consequence, our experiments allowed reproducing experimentally Hofmeister's series. However, pertechnetate adsorption on the material can completely be inhibited in the presence of highly fluorinated anions such as bis(trifluoromethylsulfonyl)amide (NTf2). This behaviour indicates a particularly imidazophilic behaviour of these anions, which have a particular importance due to their use in water immiscible ionic liquids. Finally, the adsorption process has been shown to be reversible. This feature is of importance in view of the regeneration of the anion exchange material.

The female reproductive system of the sea spider Phoxichilidium femoratum (Rathke, 1799).

Sea spiders (Pycnogonida) are marine chelicerates. Current pycnogonid phylogeny based on molecular data remains uncertain and contradicts traditional morphological perspectives. To resolve this conflict, understanding their inner anatomy is crucial. The reproductive system of sea spiders shows promise as a source of phylogenetic signal, yet our knowledge in this area is limited. This study presents the first description of the whole female reproductive system of a sea spider at the ultrastructural level. We suggest a more detailed functional regionalization of the ovary based on the ovarian wall ultrastructure and distribution of oocyte developmental stages. Meiosis begins in the germarium, and oocytes progress to the vitellarium through a transportational zone. Vitellogenic oocytes extend through the vitellarium wall, connected with it by a stalk - specialized cells. Balbiani bodies are present in early vitellogenic oocytes but dissipate later. The formation of the vitelline envelope, yolk, and fertilization envelope involves functionally diverse RER vesicles. The study also identifies a reproductive sinus as a separate haemocoel compartment that may enhance nutrient concentration near vitellogenic oocytes. Additionally, oviduct and gonopore glands are described in the female of P. femoratum, although their specific functions and prevalence in other sea spider species remain unclear.

pH-responsive niosome-based nanocarriers of antineoplastic agents.

Differences in pH between the tumour interstitium and healthy tissues can be used to induce conformational changes in the nanocarrier structure, thereby triggering drug release at the desired site. In the present study, novel pH-responsive nanocarriers were developed by modifying conventional niosomes with hexadecyl-poly(acrylic acid)n copolymers (HD-PAAn). Niosomal vesicles were prepared by the thin film hydration method using Span 60, Span 60/Tween 60 and cholesterol as main constituents, and HD-PAA modifiers of different concentrations (0.5, 1, 2.5, 5 mol%). Next, two model substances, a water-soluble fluorescent dye (calcein) and a hydrophobic agent with pronounced antineoplastic activity (curcumin), were loaded in the aqueous core and hydrophobic membrane of the elaborated niosomes, respectively. Physicochemical properties of blank and loaded nanocarriers such as hydrodynamic diameter (Dh), size distribution, zeta potential, morphology and pH-responsiveness were investigated in detail. The cytotoxicity of niosomal curcumin was evaluated against human malignant cell lines of different origins (MJ, T-24, HUT-78), and the mechanistic aspects of proapoptotic effects were elucidated. The formulation composed of Span 60/Tween 60/cholesterol/2.5% HD-PAA17 exhibited optimal physicochemical characteristics (Dh 302 nm; ζ potential -22.1 mV; high curcumin entrapment 83%), pH-dependent drug release and improved cytotoxic and apoptogenic activity compared to free curcumin.

Design of luminescent complexes with different Cu4I4 cores based on pyridyl phenoxarsines.

A series of luminescent Cu4I4 clusters with stair-step, cubane, and octahedral geometries supported by a novel type of cyclic As,N-ligand, pyridyl-containing 10-phenoxarsines, were synthesized and characterized by NMR spectroscopy, mass spectrometry, elemental analysis, and single-crystal X-ray diffraction analysis. An unusual arrangement of As,N-bidentate and µ2-iodo ligands was found in the octahedral cluster. The structural diversity of the Cu(I) complexes is reflected in their photophysical properties: the phosphorescence spectra of the compounds display emission in a broad spectral range of 495-597 nm. The complex with the Cu4I4L2 stoichiometry bearing a stair-step Cu4I4 core demonstrates temperature-dependent dual emission. The luminescence properties of all complexes were rationalized by DFT calculations.

Walking leg regeneration in the sea spider Nymphon brevirostre Hodge, 1863 (Pycnogonida).

Regeneration is widespread across all animal taxa, but patterns of its distribution and key factors determining regeneration capabilities stay enigmatic. A comparative approach could shed light on the problem, but its efficacy is limited by the fact that data is only available on a few species from derived taxa. Pycnogonida are nested basally within the Chelicerata. They can shed and replace their walking legs and have a high regeneration capacity. In this work, we carried careful observation on leg appendotomy and regeneration processes in a sea spider under laboratory settings. The limb structure and in vivo observation reveal autotomy as the most likely appendotomy mechanism. High regeneration capabilities were ascertained: an anatomically normal but small leg appeared in a single molting cycle and the full functionality regained in 2-3 cycles. Wound closure after appendotomy in N. brevirostre primarily relies on hemolymph coagulation, which apparently differs from both xiphosurans and crustaceans. Regeneration is provided by proliferation in the leg cutpiece. Regenerative morphogenesis resembles the normal ontogenetic morphogenesis of a walking leg, but accelerated. Unlike in most arthropods, in N. brevirostre, regeneration does not necessarily correspond to the molting cycle, inferring a plesiomorphic state.

Chromatin regulators in DNA replication and genome stability maintenance during S-phase.

The duplication of genetic information is central to life. The replication of genetic information is strictly controlled to ensure that each piece of genomic DNA is copied only once during a cell cycle. Factors that slow or stop replication forks cause replication stress. Replication stress is a major source of genome instability in cancer cells. Multiple control mechanisms facilitate the unimpeded fork progression, prevent fork collapse and coordinate fork repair. Chromatin alterations, caused by histone post-translational modifications and chromatin remodeling, have critical roles in normal replication and in avoiding replication stress and its consequences. This text reviews the chromatin regulators that ensure DNA replication and the proper response to replication stress. We also briefly touch on exploiting replication stress in therapeutic strategies. As chromatin regulators are frequently mutated in cancer, manipulating their activity could provide many possibilities for personalized treatment.

Micellar Form of a Ferrocene-Containing Camphor Sulfonamide with Improved Aqueous Solubility and Tumor Curing Potential.

The discovery of new anticancer drugs with а higher, more specific activity and diminished side effects than the conventional chemotherapeutic agents is a tremendous challenge to contemporary medical research and development. To achieve a pronounced efficacy, the design of antitumor agents can combine various biologically active subunits in one molecule, which can affect different regulatory pathways in cancer cells. We recently demonstrated that a newly synthesized organometallic compound, a ferrocene-containing camphor sulfonamide (DK164), possesses promising antiproliferative activity against breast and lung cancer cells. However, it still encounters the problem of solubility in biological fluids. In this work, we describe a novel micellar form of DK164 with significantly improved solubility in aqueous medium. DK164 was embedded in biodegradable micelles based on a poly(ethylene oxide)-b-poly(α-cinnamyl-ε-caprolactone-co-ε-caprolactone)-b-poly(ethylene oxide) triblock copolymer (PEO113-b-P(CyCL3-co-CL46)-b-PEO113), and the physicochemical parameters (size, size distribution, zeta potential, encapsulation efficiency) and biological activity of the obtained system were studied. We used cytotoxicity assays and flow cytometry to determine the type of cell death, as well as immunocytochemistry to assess the influence of the encapsulated drug on the dynamics of cellular key proteins (p53 and NFkB) and the process of autophagy. According to our results, the micellar form of the organometallic ferrocene derivate (DK164-NP) exhibited several advantages compared to the free substance, such as higher metabolic stability, better cellular uptake, improved bioavailability, and long-term activity, maintaining nearly the same biological activity and anticancer properties of the drug.

Antitumor Activity of Bioactive Compounds from Rapana venosa against Human Breast Cell Lines.

This study is the first report describing the promising antitumor activity of biologically active compounds isolated from the hemolymph of marine snail Rapana venosa-a fraction with Mw between 50 and 100 kDa and two structural subunits (RvH1 and RvH2), tested on a panel of human breast cell lines-six lines of different molecular subtypes of breast cancer MDA-MB-231, MDA-MB-468, BT-474, BT-549, SK-BR-3, and MCF-7 and the non-cancerous MCF-10A. The fraction with Mw 50-100 kDa (HRv 50-100) showed good antitumor activity manifested by a significant decrease in cell viability, altered morphology, autophagy, and p53 activation in treated cancer cells. An apparent synergistic effect was observed for the combination of HRv 50-100 with cis-platin for all tested cell lines. The combination of HRv 50-100 with cisplatin and/or tamoxifen is three times more effective compared to treatment with classical chemotherapeutics alone. The main proteins in the active fraction, with Mw at ~50 kDa, ~65 kDa, ~100 kDa, were identified by MALDI-MS, MS/MS analyses, and bioinformatics. Homology was established with known proteins with antitumor potential detected in different mollusc species: peroxidase-like protein, glycoproteins Aplysianin A, L-amino acid oxidase (LAAO), and the functional unit with Mw 50 kDa of RvH. Our study reveals new perspectives for application of HRv 50-100 as an antitumor agent used alone or as a booster in combination with different chemotherapies.

Antioxidant and Antitumor Potential of Micropropagated Balkan Endemic Sideritis scardica Griseb.

Sideritis scardica Griseb. is a critically endangered Balkan endemic species, known for its antioxidant, neuroprotective and anti-inflammatory properties. The aim of the present study was to detail an efficient protocol for the micropropagation of S. scardica. In vitro cultures were initiated from the shoot tips of 40 days-old in vivo seedlings and the effects of different plant growth regulator treatments were examined. A Murashige and Skoog nutrient medium (MS) containing 1 mg/L zeatin and 0.1 mg/L indole-3-acetic acid (IAA) proved to be the most efficient for shoot multiplication as it produced quality, vigorous shoots with a mean number of six shoots per explant. For the first time, the antioxidant and antitumor activities of extracts from in vitro-obtained plants were evaluated. In vitro cultivated plants grown in the field revealed a higher total polyphenol content (3929.1 ± 112.2 mg GAE/100 g vs. 3563.5 ± 52.8 mg GAE/100 g) and higher ORAC antioxidant activity (1211.6 ± 27.3 µmol TE/g vs. 939.9 ± 52.4 µmol TE/g) than in situ cultivated plants. A comparison of the antitumor activities of extracts from in vitro propagated shoots, field-grown in vitro-obtained plants and in situ plants on HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) human cancer cell lines showed that in vitro propagated shoots had a significant concentration-dependent cytotoxic effect on the cervical adenocarcinoma cell line HeLa, while the field-grown in vitro-obtained and in situ-collected samples induced the highest reduction in the viability of the mammary carcinoma cell line MCF-7. In both cases, the cells of the control non-tumor cell line, BALB/3T3, were significantly less affected. The results showed that the in vitro multiplication protocol ensured the obtainment of numerous plants with antioxidant and antitumor potential.

Antitumor and Antioxidant Activities of In Vitro Cultivated and Wild-Growing Clinopodium vulgare L. Plants.

Clinopodium vulgare L. is a valuable medicinal plant used for its anti-inflammatory, antibacterial and wound-healing properties. The present study describes an efficient protocol for the micropropagation of C. vulgare and compares, for the first time, the chemical content and composition and antitumor and antioxidant activities of extracts from in vitro cultivated and wild-growing plants. The best nutrient medium was found to be Murashige and Skoog (MS) supplemented with 1 mg/L BAP and 0.1 IBA mg/L, yielding on average 6.9 shoots per nodal segment. Flower aqueous extracts from in vitro plants had higher total polyphenol content (29,927.6 ± 592.1 mg/100 g vs. 27,292.8 ± 85.3 mg/100 g) and ORAC antioxidant activity (7281.3 ± 82.9 µmol TE/g vs. 7246.3 ± 62.4 µmol TE/g) compared to the flowers of wild plants. HPLC detected qualitative and quantitative differences in phenolic constituents between the in vitro cultivated and wild-growing plants' extracts. Rosmarinic acid was the major phenolic constituent, being accumulated mainly in leaves, while neochlorogenic acid was a major compound in the flowers of cultivated plants. Catechin was found only in cultivated plants, but not in wild plants or cultivated plants' stems. Aqueous extracts of both cultivated and wild plants showed significant in vitro antitumor activity against human HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) cell lines. The best cytotoxic activity against most of the cancer cell lines, combined with the least detrimental effects on a non-tumor human keratinocyte cell line (HaCaT), was shown by the leaf (250 µg/mL) and flower (500 µg/mL) extracts of cultivated plants, making cultivated plants a valuable source of bioactive compounds and a suitable candidate for anticancer therapy.