Characterization of the IAA-Producing and -Degrading Pseudomonas Strains Regulating Growth of the Common Duckweed (Lemna minor L.).

The rhizosphere represents a center of complex and dynamic interactions between plants and microbes, resulting in various positive effects on plant growth and development. However, less is known about the effects of indole-3-acetic acid (IAA) on aquatic plants. In this study, we report the characterization of four Pseudomonas strains isolated from the rhizosphere of the common duckweed (Lemna minor) with IAA-degradation and -utilization ability. Our results confirm previous reports on the negative effect of IAA on aquatic plants, contrary to the effect on terrestrial plants. P. putida A3-104/5 demonstrated particularly beneficial traits, as it exhibited not only IAA-degrading and -producing activity but also a positive effect on the doubling time of duckweeds in the presence of IAA, positive chemotaxis in the presence of IAA, increased tolerance to oxidative stress in the presence of IAA and increased biofilm formation related to IAA. Similarly, P. gessardii C31-106/3 significantly shortened the doubling time of duckweeds in the presence of IAA, while having a neutral effect in the absence of IAA. These traits are important in the context of plant-bacteria interactions and highlight the role of IAA as a common metabolite in these interactions, especially in aquatic environments where plants are facing unique challenges compared to their terrestrial counterparts. We conclude that IAA-degrading and -producing strains presented in this study might regulate IAA effects on aquatic plants and confer evolutionary benefits under adverse conditions (e.g., under oxidative stress, excess of IAA or nutrient scarcity).

Population-Related Variability in Qualitative and Quantitative Secondary Metabolite Profile of Gentianella austriaca (A. & J. Kern.) Holub.

Phytochemical profiling of six natural populations of Gentianella austriaca was performed by HPLC identification and quantification of a number of secondary metabolites, and evaluation of time series of peak areas by chemometric analysis. Phytochemical analysis of G. austriaca revealed the presence of iridoids, flavone-C-glucosides and xanthones. Twelve secondary metabolites were identified in the aerial parts, roots and seeds, including swertiamarin (SWM), gentiopicrin (GP), sweroside (SWZ), isoorientin (ISOOR), swertisin (SWE), demethylbellidifolin-8-O-glucoside (DMB-8-O-glc), bellidifolin-8-O-glucoside (BDF-8-O-glc), mangiferin (MGF), corymbiferin (CBF), corymbiferin-1-O-glucoside (CBF-1-O-glc), bellidifolin (BDF) and campestroside. Multivariate statistical analyses showed relatively low variability among populations according to secondary metabolite content. However, some pharmacologically important compounds were found in higher amounts in a few populations, which could be useful for conservation and future biotechnological procedures.

Xanthones Production in Gentiana dinarica Beck Hairy Root Cultures Grown in Simple Bioreactors.

The hairy root clones of Gentiana dinarica cl-B, cl-D, cl-3, and cl-14 were cultivated in parallel in diverse simple bioreactors, including temporary immersion systems RITA® (TIS RITA®), bubble column bioreactors (BCB), and Erlenmeyer flasks (EF), and evaluated for biomass production and xanthone content. The obtained results showed that TIS RITA® and BCB containing ½ MS medium with 4% sucrose provided equally good growth conditions in which the majority of the clones displayed the higher percentage of dry matter (DM%), and xanthones norswertianin-1-O-primeveroside (nor-1-O-prim) and norswertianin production than those cultivated in EF. Thin and well branched hairy root clone cl-B grown in BCB for 7 weeks was superior regarding all growth parameters tested, including growth index (19.97), dry weight (2.88 g), and DM% (25.70%) compared to all other clones. Cl-B cultured in TIS RITA® contained the highest amount of nor-1-O-prim (56.82 mg per vessel). In BCB with constant aeration, cl-B accumulated the highest norswertianin content reaching 18.08 mg/vessel. The optimized conditions for cultivation of selected G. dinarica hairy root clones in highly aerated TIS RITA® and BCB systems contribute to the development of bioreactor technology designed for the large scale commercial production of xanthones nor-1-O-prim and norswertianin.

Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production.

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L-1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L-1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones-mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside-in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 µM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2-3-fold compared to the control shoots. Elicitors at 100-300 µM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 µM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

Gentiana asclepiadea L. from Two High Mountainous Habitats: Inter- and Intrapopulation Variability Based on Species' Phytochemistry.

Natural populations of Gentiana asclepiadea L., located at two mountainous sites, were HPLC-analyzed regarding the contents of six representative secondary metabolites. The contents of swertiamarin (SWM), gentiopicrin (GP), sweroside (SWZ), mangiferin (MGF), isoorientin (ISOOR), and isovitexin (ISOV) were determined in six populations (three per study site), and separately for aboveground and belowground plant parts. PCA showed a clear separation of four groups according to the contents of the analyzed secondary metabolites. Out of six analyzed compounds, five were present in all samples and only one (SWZ) was found in Golija populations (belowground parts) but not in Vlasina populations, and its presence can be indicative of the geolocation of populations. Clear separation of groups was mostly affected by the different contents of chemical compounds in plant parts (aboveground versus belowground) and by the differences related to population origin (higher content of SWM and GP in belowground parts of individuals from Vlasina populations and higher content of MGF and ISOOR of individuals from Golija populations). The results of this study contribute to the spatiochemical profiling of G. asclepiadea populations and a better understanding of inter- and intrapopulation variability of pharmacologically important compounds.

In vitro cultivation of tansy (Tanacetum vulgare L.): a tool for the production of potent pharmaceutical agents.

In this study, tansy (Tanacetum vulgare L.) in vitro culture was established from seeds collected from natural populations. The multiplication of plantlets was conducted through shoot tips that exhibited potent apical growth and regeneration capacities on basal medium (BM), without the addition of any plant growth regulators (PGRs). PGRs were also omitted for the establishment and cultivation of tansy root cultures. Both abaxial and adaxial leaf surfaces of in vitro micropropagated plantlets were covered with glandular biseriate trichomes. Histochemical staining showed that glandular secretions were rich in lipid and terpene compounds, confirmed by GC-MS analysis of essential oil (EO). In the total EO, similar portions of oxygenated monoterpenes (38.5% m/m) and oxygenated sesquiterpenes (22.6% m/m) were detected. Chemical profiles of methanol extracts of in vitro cultured tansy shoots and roots varied in quantity and quality from those obtained from wild-growingtansy. HPLC analysis indicated that the methanol extracts of in vitro cultured roots were the richest in 3,5-O-dicaffeoylquinic acid (3,5-O-DCQA), in which the concentration was 6 times higher (10.220 mg/g DW) than that in the extract obtained from roots of wild-growing tansy (1.684 mg/g DW). This result is noticeable in the manner of industrial production of biologically active 3,5-O-DCQA that has been shown to have antioxidant, hepatoprotective, antiviral, antimutagenic, and immunomodulatory activity. Biotechnological interventions on secondary metabolite production taking place in trichomes could further enhance the production of some important tansy metabolites and further investigation will be directed toward the elucidation of the pharmaceutical potential of tansy in vitro obtained metabolites, as mixtures or single moieties.

Gentiana clusii Perr.&Song.: Enhanced production of secondary metabolites by in vitro propagation.

Shoot and root in vitro culture of endemic European species Gentiana clusii was established for the first time. The effects of different concentrations of benzyl adenine (BA), 6-phurphurylaminopurine (KIN), indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) on shoot propagation and rooting of G. clusii were investigated. The optimal in vitro conditions for shoot propagation and long-term maintenance were achieved using woody plant medium (WPM) supplemented with 0.5 mg l-1 KIN, and subsequent application of IBA at 0.5 mg l-1 significantly improved rooting of these shoots. Root culture was established from excised root tips cultured in ½ MS liquid media with increasing concentrations of IBA (0.1-1.0 mg l-1). A high root growth rate and considerable biomass yield were obtained by addition of 1.0 mg l-1 IBA. HPLC analysis revealed that in vitro culture considerably promoted the production of secondary metabolites in G. clusii. The selected protocol for shoot propagation (WPM + 0.5 mg l-1 KIN) increased the content of sweroside, gentiopicrin and norswertianin-1-O-primeveroside (N-1-P) for more than 2-fold compared with the wild plants. IBA promoted N-1-P and norswertianin production in root cultures; their contents were enhanced 6.4- and 18.6-fold, respectively, compared with the wild plants. The extract of these roots displayed the highest antioxidant capacity (IC50 = 66.57 µg ml-1). The established shoot and root propagation protocols facilitate in vitro conservation of G. clusii, and provides a promising tool for the large scale production of valuable secoiridoids and xanthones.

Hairy root culture as a valuable tool for allelopathic studies in apple.

Allelopathic plants exploit their chemical 'weapons' to prevail over the competition, suppress neighboring plants and consequently use the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficult to perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root cultures of apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation of apple cultivars Melrose, Golden Delicious, Cadel and Gloster using Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain 15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be used as a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidate the possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin, kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected in untransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry (UHPLC/(+/-)HESI-MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production in apple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicals exuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantly affected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days of treatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsis shoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanent in vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species.

Chemical and Morphological Inter- and Intrapopulation Variability in Natural Populations of Gentiana pneumonanthe L.

Inter- and intrapopulation variability in six natural populations of the rare species Gentiana pneumonanthe was examined based on morphological and chemical data. Population size and linear morphometric parameters differed significantly among populations, but without a clear connection to habitat conditions, i. e. water supply and light availability. Leaf shape varied from ovate to lanceolate in all populations, and one population was distinctive in having the largest number of leaves of transitional shape. HPLC analyses of six secondary metabolites were performed separately for belowground parts, and aboveground vegetative and reproductive parts of individual plants (6 populations ×7 individuals ×3 plant parts, n=126) in order to examine differences at the population and individual levels. Three secoiridoids (swertiamarin (SWM), sweroside (SWZ), and gentiopicrin (GP)), one xanthone (mangiferin (MGF)), and two flavones (isoorientin (IO) and isovitexin (IV)) were detected and quantified in the analyzed samples: sweroside dominated in the aboveground reproductive part, mangiferin in the aboveground vegetative part, and gentiopicrin in the belowground part. At the population level, differences in contents of the analyzed chemicals among populations were significant only for a few metabolites. At the individual level, a pronounced organ-dependent distribution of secondary metabolites was revealed. The results of this study contribute to a better understanding of natural variability within populations of the rare and threatened G. pneumonanthe, and provide data on the contents and within-plant distribution of secondary metabolites, which are important as pharmacologically active compounds and may be useful for further biotechnological procedures regarding this species.

Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders.

Gastrointestinal disturbances, nutritional deficiencies, and food intolerances are frequently observed in children with neurodevelopmental disorders (NDD). To reveal possible association of celiac disease risk variants (HLA-DQ), lactose intolerance associated variant (LCT-13910C>T) as well as variant associated with vitamin D function (VDR FokI) with NDD, polymerase chain reaction-based methodology was used. Additionally, intestinal peptide permeability was estimated in NDD patients and healthy children by measuring the level of peptides in urine using high-performance liquid chromatography. Levels of opioid peptides, casomorphin 8, and gluten exorphin C were significantly elevated in urine samples of NDD patients (P = 0.004 and P = 0.005, respectively), but no association of genetic risk variants for celiac disease and lactose intolerance with NDD was found. Our results indicate that increased intestinal peptide permeability observed in analyzed NDD patients is not associated with genetic predictors of celiac disease or lactose intolerance. We have also found that FF genotype of VDR FokI and lower serum levels of vitamin D (25-OH) showed association with childhood autism (CHA), a subgroup of NDD. We hypothesize that vitamin D might be important for the development of CHA.

Xanthone-rich extract from Gentiana dinarica transformed roots and its active component norswertianin induce autophagy and ROS-dependent differentiation of human glioblastoma cell line.

BACKGROUND: Glioblastoma multiforme (GMB) is the most malignant of all brain tumors with poor prognosis. Anticancer potential of xanthones, bioactive compounds found in Gentiana dinarica, is well-documented. Transformation of G. dinarica roots with Agrobacterium rhizogenes provides higher xanthones accumulation, which enables better exploitation of these anticancer compounds. HYPOTHESIS/PURPOSE: The aim of this study was to investigate antiglioma effect of three different G. dinarica extracts: E1-derived from untransformed roots, E2-derived from roots transformed using A. rhizogenes strain A4M70GUS, and E3-derived from roots transformed using A. rhizogenes strain 15834/PI. Further, mechanisms involved in anticancer potential of the most potent extract were examined in detail, and its active component was determined. METHODS: The cell viability was assessed using MTT and crystal violet test. Cell cycle analysis, the expression of differentiation markers, the levels of autophagy, and oxidative stress were analyzed by flow cytometry. Autophagy and related signaling pathways were assessed by immunoblotting. RESULTS: E3, in contrast to E1 and E2, strongly reduced growth of U251 human glioblastoma cells, triggered cell cycle arrest in G2/M phase, changed cellular morphology, and increased expression of markers of differentiated astrocytes (glial fibrillary acidic protein) and neurons (ß-tubulin). E3 stimulated autophagy, as demonstrated by enhanced intracellular acidification, increased microtubule-associated light chain 3B (LC3-I) conversion to autophagosome associated LC3-II, and decreased level of selective autophagy target p62. Induction of autophagy was associated with Akt-dependent inhibition of main autophagy suppressor mammalian target of rapamycin (mTOR). Both genetic and pharmacological inhibition of autophagy suppressed the expression of differentiation markers, but had no effect on cell cycle arrest in E3-treated cells. E3 stimulated oxidative stress, and antioxidants vitamin E and N-acetyl cysteine inhibited autophagy and differentiation of E3-treated U251 cells. The most prevalent compound of E3, xanthone aglycone norswertianin, also arrested glioblastoma cell proliferation in G2/M phase and induced glioblastoma cell differentiation through induction of autophagy and oxidative stress. CONCLUSION: These results indicate that E3 and its main active component norswertianin may serve as a potential candidate for differentiation therapy of glioblastoma.

Sorbus aucuparia and Sorbus aria as a Source of Antioxidant Phenolics, Tocopherols, and Pigments.

Due to its nutritive and medicinal properties, berries of some Sorbus species are used for the preparation of jams and jelly as well as in traditional medicine. On the other hand, their chemical composition is not much studied especially of those grown in Balkan Peninsula. We have analyzed individual phenolics, tocopherols, carotenoids and chlorophylls using HPLC in berries from Sorbus aucuparia and Sorbus aria collected in different localities in Serbia and Montenegro together with the amounts of total phenolics and proanthocyanidins as well as their radical scavenging activity against DPPH radical. Berries of S. aucuparia were richer source of polyphenolics in comparision with S. aria and, regardless the species and locality, caffeoylquinic acids such as neochlorogenic and chlorogenic acid were the most abundant compounds. Among analyzed tocopherols the most abundant in all samples was α-tocopherol (0.48 - 19.85 µg/g dw) as it was ß-carotene among carotenoids (mean concentration of 0.98 µg/g dw in S. aucuparia and 0.40 µg/g dw in S. aria, respectively). Correlation between total phenolics and DPPH radical scavenging activity was noticed. Our study represents comprehensive report on chemical composition of S. aucuparia and S. aria which could contribute to a better understanding of their quality.

Crataegus orientalis Leaves and Berries: Phenolic Profiles, Antioxidant and Anti-inflammatory Activity.

The present study was designed to define the phenolic content, antioxidant and anti-inflammatory activity of Crateagus orientalis Pall. ex M. Bieb., traditionally used by local people in southern parts of F.Y.R. Macedonia. The presence and content of 7 phenolics in ethanolic extracts of leaves and berries were studied using HPLC-DAD, where the most dominant compounds were hyperoside, isoquercitrin and chlorogenic acid. The leaf extract was more effective as a DPPH radical scavenger (IC50 = 29.7 µg/g) than the berry extract, as well as in the relative reducing power on Fe³âº. Anti-inflammatory potential was studied by means of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibitory activity; both extracts evinced activity. Furthermore, C. orientalis leaf extract showed a concentration dependent inhibition of COX-1 pathway products 12-HHT and TXB2, reaching IC50 values below the lowest applied concentration (68.9% and 55.2% of 12-HHT and TXB2 production inhibition, respectively, at concentration of 0.4 mg/mL). Although inhibitors such as acetylsalicylic acid and quercetin showed higher activity, this study demonstrates that the investigated extracts are potential anti-inflammatory agents.

Reduction of anxiety-like and depression-like behaviors in rats after one month of drinking Aronia melanocarpa berry juice.

The treatment of mood and anxiety disorders by nutraceuticals is gaining growing awareness. Berries of Aronia melanocarpa (Black chokeberry) and their extracts, exceptionally abundant in diverse phenolic compounds, have become famous for the highest in vitro antioxidant activity among fruits and notable health benefits (e.g. anti-diabetic, anti-inflammatory, cardioprotective). This study was designed to investigate the behavioral effects of month-long unlimited consumption of Aronia master juice (AJ) and/or juice reconstruct without polyphenols (RJ), in young male rats. AJ was initially evaluated for its content of phenolic compounds by spectrophotometric assays and HPLC-DAD. Rats that were supplied with three various water concentrations of AJ and RJ, respectively: 20% + 0% (ARO group), 5% + 15% (RAJ) and 0 + 20% (PLC), were compared with those which consumed only water (CTL). Daily drinking of AJ solution was significantly elevated from the second or third week onward, which was most expressed in the ARO group. Only this group displayed behavioral variations, manifested by certain hyperactivity in open field tests and prominent reductions of anxiety-like behaviors in the elevated plus maze. The ARO rats also expressed an alleviation of depression-like behavior in forced swimming tests. These findings demonstrate the beneficial behavioral effects of the one-month-long free drinking of phenolic-rich AJ in rats (>20 ml per kg b. mass daily) that may be recognized as stimulating, anxiolytic-like and antidepressant-like. The in vitro assays suggested that MAO-A/MAO-B inhibitions by the phenolic compounds of AJ might be the possible in vivo mechanisms for such behavioral actions.

Metabolic plasticity and the energy economizing effect of ibogaine, the principal alkaloid of Tabernanthe iboga.

ETHNOPHARMACOLOGICAL RELEVANCE: The root bark of iboga plant-Tabernanthe iboga has been used traditionally in Central Africa as a psychoactive substance in religious rituals, while in smaller doses it is appreciated due to its stimulant properties. The iboga root bark, iboga extract or pure ibogaine are being recognized in the West as an anti-addiction remedy and their use is increasing. AIM OF THE STUDY: Our previous studies have demonstrated a transient ATP pool reduction under ibogaine accompanied by the induction of energy metabolism related enzymes. The present study aimed to find the cause of this energy deprivation and to foresee its immediate and long-term impact on metabolism. The overall project is designed to disclose the common mechanism of action at these seemingly diverse indications for iboga use, to predict eventual adverse effects and to build the grounds for its safe and beneficial utilization. MATERIALS AND METHODS: The rate of carbon dioxide (CO(2)) as a marker of energy metabolism in stationary yeast model under aerobic conditions in the presence of ibogaine at concentration of 1, 4 and 20mg/l was measured for 5h by gas chromatography. The overall oxidative load was determined fluorimetrically by 2',7'-dichlorofluorescein diacetate (H(2)DCFDA) and in vitro antioxidant properties of ibogaine were defined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) test. RESULTS: The CO(2) production under ibogaine was temporarily increased in a dose dependent manner. The increased energy consumption as an early effect of ibogaine was proven by the fact that in spite of energy mobilization, the ATP pool has been simultaneously decreased. Although increased cellular respiration co-produces reactive oxygen species (ROS), the overall oxidative load was significantly lowered by ibogaine. Since ibogaine does not show any significant in vitro antioxidant properties, the results indicate its stimulating influence on physiological oxidative stress defence system. CONCLUSION: Ibogaine triggers remodeling of the housekeeping metabolism. Under the initial energy cost it results in increased efficacy of physiological antioxidative systems, which reduce oxidative damage and lowers basal metabolic needs. Together with induced catabolic enzymes they set a new metabolic equilibrium that saves energy and makes it easily available in case of extra needs. While healthy organism profits from improved fitness and mental performance and can withstand higher stress without risking a disease, due to the same principle ibogaine provides beneficial support at the recovery after diseases including addiction syndrome.

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays.

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 µmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.

Phytochemical re-investigation of Gentiana utriculosa.

Xanthone-O-glycosides with 1,3,7,8-oxidation pattern and flavone isoorientin-3'-O-glucoside were isolated from the aerial parts of Gentiana utriculosa. Xanthone-C-glucoside lancerin was detected in Gentiana species for the first time. The distribution of these compounds within the section Calathianae is discussed.