Chitosan/Hyaluronate Complex-Coated Electrospun Poly(3-hydroxybutyrate) Materials Containing Extracts from Melissa officinalis and/or Hypericum perforatum with Various Biological Activities: Antioxidant, Antibacterial and In Vitro Anticancer Effects.

The present study aimed to fabricate innovative fibrous materials with various biological activities from poly(3-hydroxybutyrate), sodium hyaluronate (HA), chitosan (Ch), Melissa officinalis (MO), Hypericum perforatum (HP) extract, or a combination of both extracts. Electrospinning or electrospinning followed by dip coating and the subsequent formation of a polyelectrolyte complex were the methods used to prepare these materials. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) were applied for investigating the morphology of materials, their thermal characteristics, and their surface chemical composition. The composition and design of the mats had an influence on the in vitro release behavior of the main bioactive compounds present in the MO and HP extracts incorporated in the materials. It was found that as-created materials comprising a combination of both extracts and a Ch/HA complex exerted higher antioxidant activity than that of (non-)coated MO-containing mats and Ch/HA-coated mats containing HP. The novel materials manifested antibacterial efficacy towards the pathogenic bacteria S. aureus and E. coli, as evidenced by the performed microbiological screening. Furthermore, the mats possessed a great growth inhibitory effect on HeLa cancer cells but had a less pronounced effect on the growth of normal mouse BALB/3T3 fibroblasts. The loading of both extracts in the mats and the formation of coating led to the enhancement of the in vitro anticancer and antibacterial activities of the materials. Thus, the novel materials have potential for use in local cancer therapy as well as for use as wound dressings.

Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity.

In this study, UHPLC-HRMS analysis of the defatted methanol extract obtained from Inula salicina L. led to the identification of 58 compounds-hydroxycinnamic and hydroxybenzoic acids and their glycosides, acylquinic and caffeoylhexaric acids, and flavonoids and their glycosides. In addition, a new natural compound, N-(8-methylnepetin)-3-hydroxypiperidin-2-one was isolated and its structure was elucidated by NMR spectroscopy. The presence of a flavoalkaloid in genus Inula is described now for the first time. Chlorogenic acid was the main compound followed by 3,5-, 1,5- and 4,5-dicaffeoylquinic acids. The methanol extract was studied for its antioxidant potential by DPPH, ABTS, and FRAP assays and sun protective properties. In addition, a study was conducted to assess the effectiveness of the tested extract in inhibiting biofilm formation by Gram-positive and Gram-negative strains. Results from crystal violet tests revealed a notable decrease in biofilm mass due to the extract. The anti-biofilm efficacy was confirmed through the observation of the biofilm viability by live/dead staining. The obtained results showed that this plant extract could be used in the development of cosmetic products with antibacterial and sun protection properties.

Phytochemical Profile, Antioxidant Potential, Antimicrobial Activity, and Cytotoxicity of Dry Extract from Rosa damascena Mill.

Dry rose extract (DRE) obtained industrially by aqueous ethanol extraction from R. damascena flowers and its phenolic-enriched fraction, obtained by re-extraction with ethyl acetate (EAE) were the subject of this study. 1H NMR of DRE allowed the identification and quantitation of fructose and glucose, while the combined use of HPLC-DAD-ESIMS and HPLC-HRMS showed the presence of 14 kaempferol glycosides, 12 quercetin glycosides, 4 phenolic acids and their esters, 4 galloyl glycosides, 7 ellagitannins, and quinic acid. In addition, the structures of 13 of the flavonoid glycosides were further confirmed by NMR. EAE was found to be richer in TPC and TFC and showed better antioxidant activity (DPPH, ABTS, and FRAP) compared to DRE. Both extracts displayed significant activity against Propionibacterium acnes, Staphylococcus aureus, and S. epidermidis, but showed no activity against Candida albicans. Toxicity tests on normal human skin fibroblasts revealed low toxicity for both extracts with stronger effects observed at 24 hours of treatment that were compensated for over the following two days. Human hepatocarcinoma (HepG2) cells exhibited an opposite response after treatment with a concentration above 350 µg/mL for EAE and 500 µg/mL for DRE, showing increased toxicity after the third day of treatment. Lower concentrations were non-toxic and did not significantly affect the cell cycle parameters of either of the cell lines.

Novel hybrids of sclareol and 1,2,4-triazolo[1,5-a]pyrimidine show collateral sensitivity in multidrug-resistant glioblastoma cells.

The synthesis of 24 hybrid molecules, consisting of naturally occurring sclareol (SCL) and synthetic 1,2,4-triazolo[1,5-a]pyrimidines (TPs), is described. New compounds were designed with the aim of improving the cytotoxic properties, activity, and selectivity of the parent compounds. Six analogs (12a-f) contained 4-benzylpiperazine linkage, while 4-benzyldiamine linkage was present in eighteen derivatives (12g-r and 13a-f). Hybrids 13a-f consist of two TP units. After purification, all hybrids (12a-r and 13a-f), as well as their precursors (9a-e and 11a-c), were tested on human glioblastoma U87 cells. More than half of the tested synthesized molecules, 16 out of 31, caused a significant reduction of U87 cell viability (more than 75% reduction) at 30 µM. The concentration-dependent cytotoxicity of these 16 compounds was also examined on U87 cells, corresponding multidrug-resistant (MDR) U87-TxR cells with increased P-glycoprotein (P-gp) expression and activity, and normal lung fibroblasts MRC-5. Importantly, 12l and 12r were active in the nanomolar range, while seven compounds (11b, 11c, 12i, 12l, 12n, 12q, and 12r) were more selective towards glioblastoma cells than SCL. All compounds except 12r evaded MDR, showing even better cytotoxicity in U87-TxR cells. In particular, 11c, 12a, 12g, 12j, 12k, 12m, 12n, and SCL showed collateral sensitivity. Hybrid compounds 12l, 12q, and 12r decreased P-gp activity to the same extent as a well-known P-gp inhibitor - tariquidar (TQ). Hybrid compound 12l and its precursor 11c affected different cellular processes including the cell cycle, cell death, and mitochondrial membrane potential, and changed the levels of reactive oxygen and nitrogen species (ROS/RNS) in glioblastoma cells. Collateral sensitivity towards MDR glioblastoma cells was caused by the modulation of oxidative stress accompanied by inhibition of mitochondria.

Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro.

Alchemilla vulgaris L. (lady's mantle) was used for centuries in Europe and Balkan countries for treatments of numerous conditions and diseases of the reproductive system, yet some of the biological activities of lady's mantle have been poorly studied and neglected. The present study aimed to estimate the potential of A. vulgaris ethanolic extract from Southeast Serbia to prevent and suppress tumor development in vitro, validated by antioxidant, genoprotective, and cytotoxic properties. A total of 45 compounds were detected by UHPLC-HRMS analysis in A. vulgaris ethanolic extract. Measurement of antioxidant activity revealed the significant potential of the tested extract to scavenge free radicals. In addition, the analysis of micronuclei showed an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes. A. vulgaris extract strongly suppressed the growth of human cell lines derived from different types of tumors (MCF-7, A375, A549, and HCT116). The observed antitumor effect is realized through the blockade of cell division, caspase-dependent apoptosis, and autophagic cell death. Our study has shown that Alchemilla vulgaris L. is a valuable source of bioactive compounds able to protect the subcellular structure from damage, thus preventing tumorigenesis as well as suppressing tumor cell growth.

A jojoba (Simmondsia chinensis) seed cake extracts express hepatoprotective activity against paracetamol-induced toxicity in rats.

This study aimed to investigate the hepatoprotective activity of jojoba seed cake extracts against an acute paracetamol (PC) intoxication. Two aqueous extracts from jojoba (Simmondsia chinensis) seed cake, a simmondsin-rich extract (WE), and a simmondsin-hydrolyzed extract (NE) using Viscozyme L enzyme have been prepared and characterized. After enzyme treatment, simmondsin content decreased from 33.0 % to 3.0 % and glucose content increased from 16.2 % to 27.3 % reflecting simmondsin hydrolysis. Both extracts were administered to different rat groups via gavage (0.6 g/kg b.w.) before PC treatment (2 g/kg b.w.) three times a week for 3 weeks. The PC intoxication altered the serum biomarkers, the oxidative status, and the Tumor necrosis factor alpha (TNF-α), Bax and Bcl-2 protein expressions of tested animals. In addition, the histological analysis of liver tissues proved significant injury and hepatocellular necrosis. WE and NE extract showed a relatively high in vitro radical scavenging (ORAC) and averting activities (HORAC) with a polyphenol content of 3.6 % and 2.9 %, respectively. Both extracts showed a powerful in vivo hepatoprotective activity against PC-induced toxicity by improving the hepatocellular antioxidant status and blocking proteins expression (TNF-α, Bax and Bcl-2), involved in inflammation and liver damage. However, the enzymatic treatment improved the hepatoprotective activity of NE despite its lower simmondsin content and lower in vitro antioxidant capacity. This enhancement could be linked to the synergetic effect between the antioxidant components and the new hydrolytic products as glucose, uronic acids, arabinose and simmondsin-aglycons. These results suggest that jojoba waste could be potentially valorized in developing hepatoprotective drugs.

Chemical characterization, antioxidant activity, -amylase and acetylcholinesterase inhibitory potential of Angelica pancicii Vandas ex Velen / Caracterización química, actividad antioxidante, potencial inhibidor de -amilasa y acetilcolinesterasa de Angelica pancicii Vandas ex Velen

Abstract: Comparative study GC - FID /M S of essential oils of fruits, leaves and roots of the endemic plant Angelica pancicii Vandas ex Velen. revealed a significant difference in their chemical composition. The enantiomeric purity of the main component in the fruit oil (+) - ß - phellandrene was a lso confirmed. In addition, imperatorin, isoimperatorin, oxypeucedanin, oxypeucedanin hydrate, angeloylpangelin and umbelliprenin were isolated from the fruit hexane extract. The content of these coumarins in the hexane extracts from different plant parts was further determined by HPLC. The essential oils and hexane extracts were assessed for their antioxidant potential and inhibitory effect towards  - amylase and acetylcholinesterase enzymes. The fruit and leaf essential oils (> 80%) as well as the fruit he xane extract (> 62%) significantly inhibited acetylcholinesterase enzyme. Distinguish free radical scavenging properties were detected for the leaf (Inh. 95.0 ± 2.2 %) and the root (Inh. 66.0 ± 2.4 %) extracts.

Resumen: Estudio comparativo GC - FID / MS de aceites esenciales de frutas, hojas y raíces de la planta endémica Angelica pancicii Vandas ex Velen revelaron una dife rencia significativa en su composición química. También se confirmó la pureza enantiomérica del componente principal del aceite de fruta (+) - ß - felandreno. Además, se aislaron imperatorina, isoimperatorina, oxipeucedanina, hidrato de oxipeucedanina, angeloi lpangelina y umbeliprenina del extracto de hexano del fruto. El contenido de estas cumarinas en los extractos de hexano de diferentes partes de la planta se determinó adicionalmente mediante HPLC. Los aceites esenciales y extractos de hexano se evaluaron p or su potencial antioxidante efecto inhibidor de las enzimas -  - amilasa y acetilcolinesterasa. Los aceites esenciales de frutas y hojas (> 80%), así como el extracto de hexano de frutas (> 62%) inhibieron significativamente la enzima acetilcolinesterasa. Se detectaron propiedades de captación de radicales libres diferenciadas para los extractos de hoja (Inh. 95,0 ± 2,2%) y de raíz (Inh. 66,0 ± 2,4%).

Essential Oil Composition of Five Thymus Species from Bulgaria.

In this study, the essential oil composition of five Thymus species, belonging to Sect. Hyphodromi (A. Kerner) Halácsy - Thymus atticus Celak., T. leucotrichus Halácsy, T. striatus Vahl, T. zygioides Griseb. and T. perinicus (Velen.) Jalas. was studied by GC/MS/FID. T. atticus, T. leucotrichus, and T. striatus were characterized by high amounts of sesquiterpenoids (57.7, 78.9 and 79.7 %, respectively) with ß-caryophyllene and caryophyllene oxide as the main constituents. Aromatic compounds (61.2 %) were the most abundant group in T. zygioides essential oil, with thymol (51.2 %) as the principal component. The essential oil from the endemic species T. perinicus contained almost equal amounts of monoterpenoids (37.8 %) and aromatic compounds (36.0 %) with borneol (17.9 %) and thymol (20.9 %) as the major components. The obtained results revealed the existence of new chemotypes of T. atticus (caryophyllene oxide/ß-caryophyllene), T. leucotrichus (ß-caryophyllene/elemol/germacrene D) and T. striatus (ß-caryophyllene/germacrene D/caryophyllene oxide). The essential oil content of endemic T. perinicus is reported for the first time. Principal component analysis (PCA) and cluster analysis (CA) were used to investigate the variations in the essential oils of different Thymus species from Sect. Hyphodromi (A. Kerner) Halácsy.

Optimization of microwave assisted extraction of simmondsins and polyphenols from Jojoba (Simmondsia chinensis) seed cake using Box-Behnken statistical design.

A closed-vessel microwave-assisted extraction (MAE) of simmondsins and polyphenols from defatted Jojoba cake using Box-Benkhen design with four independent variables (solvent/cake ratio, ethanol concentration, extraction time and microwave power) was investigated. ANOVA results showed that the obtained models were significant at 95% confidence level. Optimal extraction conditions were found for highest values of microwave power (500 W) and extraction time (15 min) and for moderate values of solvent to cake ratio (41 - 45 mL/g). Optimum simmondsins yield (23.35%) was obtained with pure water as solvent. However, optimum polyphenols yield (2.33%) and ORAC antioxidant activity (656 µmol TE/g) were obtained with 46.79% and 42.04% ethanol in water, respectively. ORAC antioxidant activity was found to be well correlated to polyphenol and simmondsin contents. These results indicate that MAE is an effective technique for recovery of bioactive compounds for food and pharmaceutical industries from Jojoba by-products.

Caffeoylquinic Acids, Cytotoxic, Antioxidant, Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria.

Chlorogenic (5-CQA), 1,5-, 3,5-, 4,5- and 3,4-dicaffeoylquinic (DCQA) acids were identified and quantified in the methanol extracts of Inula oculus-christi L., I. bifrons L., I. aschersoniana Janka var. aschersoniana, I. ensifolia L., I. conyza (Griess.) DC. and I. germanica L. by HPLC analysis. The amount of 5-CQA varied from 5.48 to 28.44 mg/g DE and the highest content was detected in I. ensifolia. 1,5-DCQA (4.05-55.25 mg/g DE) was the most abundant dicaffeoyl ester of quinic acid followed by 3,5-DCQA, 4,5-DCQA and 3,4-DCQA. The extract of I. ensifolia showed the highest total phenolic content (119.92±0.95 mg GAE/g DE) and exhibited the strongest DPPH radical scavenging activity (69.41±0.55 %). I. bifrons extract was found to be the most active sample against ABTS.+ (TEAC 0.257±0.012 mg/mL) and the best tyrosinase inhibitor. The studied extracts demonstrated a low inhibitory effect towards acetylcholinesterase and possessed low cytotoxicity in concentration range from 10 to 300 µg/mL toward non-cancer (MDCK II) and cancer (A 549) cells.

Phenolic profile and antioxidant, acetylcholinesterase, and tyrosinase inhibitor activities of Jurinea tzar-ferdinandii Davidov, an endemic plant of the Balkans / Perfil fenólico y actividad antioxidante, acetilcolinesterasa e inhibidora de tirosinasa de Jurinea tzar-ferdinandii Davidov, planta endémica de los Balcanes

The methanol extract of the Balkan endemic species Jurinea tzar-ferdinandii Davidov demonstrated weak antioxidant activity against DPPH• and ABTS+• and low inhibitory potential against acetylcholinesterase (8.3% Inh.) and tyrosinase (IC50 = 208 ± 8 µg/mL) enzymes. Phytochemical investigation of the extract led to isolation and identification of apigenin, luteolin, apigenin-7-O-glucoside, apigenin-4'-O-glucoside, apigenin-7-O-gentiobioside, luteolin-4'-O-glucoside, rutin, narcissin, chlorogenic and 1,5-dicaffeoylquinic acid. With exception of apigenin and rutin, all isolated compounds are reported for the first time in the representatives of genus Jurinea. The distribution of flavonoids was discussed from chemotaxonomic point of view.

El extracto de metanol de la especie endémica de los Balcanes Jurinea tzar-ferdinandii Davidov demostró una actividad antioxidante débil contra DPPH• y ABTS+• y un bajo potencial inhibidor contra las enzimas acetilcolinesterasa (8.3% Inh.) tirosinasa (IC50 = 208 ± 8 µg/mL). La investigación fitoquímica del extracto condujo al aislamiento e identificación de apigenina, luteolina, apigenina-7-Oglucósido, apigenina-4'-O-glucósido, apigenina-7-O-gentiobiósido, luteolina-4'-O-glucósido, rutina, narcissin, clorogénico y ácido 1,5- dicafeoilquinico. Con excepción de la apigenina y la rutina, todos los compuestos aislados se informan por primera vez en el género Jurinea. La distribución de flavonoides se discute desde el punto de vista quimiotaxonómico.

Research Advances on Health Effects of Edible Artemisia Species and Some Sesquiterpene Lactones Constituents.

The genus Artemisia, often known collectively as "wormwood", has aroused great interest in the scientific community, pharmaceutical and food industries, generating many studies on the most varied aspects of these plants. In this review, the most recent evidence on health effects of edible Artemisia species and some of its constituents are presented and discussed, based on studies published until 2020, available in the Scopus, Web of Sciences and PubMed databases, related to food applications, nutritional and sesquiterpene lactones composition, and their therapeutic effects supported by in vivo and clinical studies. The analysis of more than 300 selected articles highlights the beneficial effect on health and the high clinical relevance of several Artemisia species besides some sesquiterpene lactones constituents and their derivatives. From an integrated perspective, as it includes therapeutic and nutritional properties, without ignoring some adverse effects described in the literature, this review shows the great potential of Artemisia plants and some of their constituents as dietary supplements, functional foods and as the source of new, more efficient, and safe medicines. Despite all the benefits demonstrated, some gaps need to be filled, mainly related to the use of raw Artemisia extracts, such as its standardization and clinical trials on adverse effects and its health care efficacy.

Essential oil composition of the Balkan endemic Thymus longedentatus (Degen & Urum. ) Ronniger / Composición del aceite esencial de Thymus longedentatus (Degen & Urum. ) Ronniger endemic de los Balcanes

The chemical composition of essential oil obtained from the aerial parts of the Balkan endemic species Thymus longedentatus was studied by GC-MS. Twenty eight compounds, consisting 98.2% of the total components were detected in amounts exceeding 0.1%. The oil was characterized by relatively high concencentration of oxygentated monoterpenes (78.7%), among which citral isomers neral (27.5%) and geranial (30.3%) were the principal ones. In addition, 1,8-cineole (7.8%), trans-ß-ocimene (7.5%), and ß-myrcene (3.7%) were detected in significant ammounts. Germacrene D (4.4%) was found to be the main sesquiterpene in the essential oil.

Se estudió mediante GC-MS la composición química del aceite esencial obtenido de las partes aéreas de Thymus longedentatus, especie endémica de los Balcanes. Se detectaron veintiocho compuestos, que representaban un 98,2% del total de compuestos y que se encontraban en cantidades superiores al 0,1%. El aceite se caracterizó por una concentración relativamente alta de monoterpenos oxigenados (78.7%), entre los cuales los isómeros de citral, neral (27.5%) y geranial (30.3%) eran los principales. Además, se detectó 1,8-cineol (7,8%), trans-ß-ocimeno (7,5%) y ß-mirceno (3,7%) en cantidades significativas. Se encontró que Germacreno D (4.4%) es el principal sesquiterpeno en el aceite esencial.

Essential oil composition of Inula britannica L. from Bulgaria.

The separately distilled flowers (F) and leaves' (L) essential oils of Inula britannica L. were investigated using capillary gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 83 constituents, representing 96.91% (F) and 96.73% (L) of the total oils, were registered. The oils were rich in terpenoids (57.85% and 77.28%), of which sesquiterpenoids dominated. The main constituents of the essential oils were viridiflorol (7.17%-8.20%) and himachalol (3.45%-8.71%) followed by 6,10,14-trimethyl-2-pentadecanone (5.43%-2.95%), 13-tetradecanolide (3.93%-4.87%) and 3-methyl-4-propyl-2,5-furandione (4.06%-0.29%).

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.

Variability in the essential-oil composition of Sideritis scardica GRISEB. from native Bulgarian populations.

The essential-oil composition of six native populations of Sideritis scardica from Bulgaria was studied by GC-FID and GC/MS analyses. Altogether, 37 components, representing 73.1 to 79.2% of the total oil content were identified. Among them, α-pinene (4.4-25.1%), ß-pinene (2.8-18.0%), oct-1-en-3-ol (2.3-8.0%), phenylacetaldehyde (0.5-9.5%), ß-bisabolene (1.3-11.0%), benzyl benzoate (1.1-14.3%), and m-camphorene (1; 0.3-12.4%) were the main compounds. All samples were characterized by low contents of oxygenated mono- and sesquiterpenes (≤1.6 and 2.3%, resp.). Principal component analysis (PCA) and cluster analysis (CA) showed a significant variability in the chemical composition of the studied samples as well as a correlation between the oil profiles and the ecological conditions of the natural habitats of S. scardica.

Cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of Artemisia alba.

The influence of plant growth regulators (PGR) on the essential oil composition and in vitro development of A. alba shoot cultures was studied. Two types of oils were determined, based on their terpenoid content. Close relations between the morphogenetic effects of PGR and the essential oil profile of the species were observed Predominance of root over shoot development was connected with a drop in the amounts of sesquiterpenoids and the direction of biosynthesis towards oxygenated monoterpenoids in the PGR-free control medium, as well in those with 1.0 mg L(-1) indole-3-butyric acid (IBA) supplementation. On the contrary, lack of root formation and stimulated callusogenesis caused by the addition of 0.2 mg L(-1) 6-benzyladenine (BA), irrespectively of the presence of IBA (either 0.5 mg L(-1) or 1.0 mg L(-1)), resulted in the strong prevalence of sesquiterpenoids in the oils. These results are indicative that the morphological development ofA. alba shoot cultures affects the terpenoid biosynthetic pathway bringing out the hypothesis for a possible root to shoot signaling.

Influence of nutrient medium composition on in vitro growth, polyphenolic content and antioxidant activity of Alchemilla mollis.

Alchemilla mollis (Buser) Rothm. (Rosaceae) is a high-mountain medicinal plant growing in the Balkan Peninsula, with only one population in Bulgaria. Alchemilla plants (Lady's mantle) are commonly used in traditional medicine for treatment of many gynecological diseases. The commercial drugs "Herba Alchemillae" induce a rapid regeneration of skin epithelium and have styptic and anti-inflammatory actions. Because of the high content of phenolic compounds (tannins and flavonoids) and the ecological plasticity of the species, field cultivation or in vitro biomass production of A. mollis are possible alternatives to its collection from nature. Four MS based nutrient media differing in the concentration of the minerals and supplemented with alpha-naphthaleneacetic acid (NAA) and benzylaminopurine (BAP) were tested in order to examine their influence on the shoot multiplication effectiveness and the antioxidant activity of A. mollis, and also the possible relation between these parameters under the conditions of in vitro culture. The cultures grown for two months on these media differed significantly in their multiplication rates (p < 0.001), as well as in their morphological features--height, leaf color and root development. Methanol extracts of in vitro cultivated and ex vitro adapted and acclimated on Vitosha Mt. (1500 m a. s. l.) plants were analyzed for tannin and flavonoid content and for free radical scavenging activity. The contents of flavonoids and tannins in the in vitro cultures of A. mollis cultivated on the four tested media differed significantly (p < 0.05). The highest flavonoid content was found in the shoots cultivated on the control MS medium, as well as in the ex vitro adapted plants. The antioxidant activity of the in vitro cultures correlated positively with the concentrations of the PGRs in the respective media, and the ex vitro adapted plants had the highest antioxidant activity (IC50 13.1 +/- 1.9 microg/mL) commensurable with that of the commercial antioxidant butylated hydroxytoluene (BHT) used as a positive control, with an IC50 of 12.65 microg/mL.

Essential oil composition of Achillea clusiana from Bulgaria.

The essential oil compositon of Achillea clusiana Tausch from Bulgaria has been studied by GC and GC/MS. Fifty-four components were registered, representing 92.5% of the oil. The oil was characterized by the presence of oxygenated mono- and sesquiterpenoids. The main components were beta-thujone (17.2%), 1,8-cineole (11.2%), camphor (11.1%) and alpha-thujone (7.8%). Farnesol (3.1%), nerolidol (2.7%) and oxygenated nerolidol derivatives (cabreuva oxides A-D, isohumbertiols A-D, bejarol and 7-hydroxy-6,7-dihydro-5,6E-dehydronerolidol) were the main sesquiterpenoids in the oil.