Publications

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    ABSTRACT: Investigations have been made to study the production of phenolic compounds (total phenolics, flavonoids and phenylpropanoids) and total antioxidant capacity in 27 Macedonian traditional medicinal plants to improve its potential as a source of natural antioxidants. Antioxidant potential of plant extracts was analyzed by five different assays: cupric reducing antioxidant capacity (CUPRAC), phosphomolybdenum method (PM), reducing power (RP), 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2’-azinobis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS•+) radical scavenging activity. Origanum vulgare extract consistently exhibited the highest content of phenolic compounds and the strongest antioxidant capacity based on the tests performed, and can be proposed as a promising source of natural antioxidants. Melissa officinalis and Salvia ringens were also identified as valuable sources of antioxidant compounds. A positive linear correlation between antioxidant activity and total phenolics, flavonoids and phenylpropanoids indicates that these compounds are likely to be the main antioxidants contributing to the observed activities of evaluated plants. These findings suggest that the medicinal plants studied in this paper are good sources of bioactive compounds for the food and pharmaceutical industries.
    Central European Journal of Biology 08/2014; · 0.82 Impact Factor
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    ABSTRACT: Regeneration of transgenic shoots was achieved from Hypericum perforatum L. hairy roots on hormone-free MS/B5 medium for a period of 4 weeks under a photoperiod of 16-h light. A control experiment was set up with root segments obtained from in vitro grown seedlings. Investigations have been made to study the production of phenolic compounds in non transgenic and transgenic shoot cultures. Six groups of phenolic compounds such as phenolic acids, flavonols, flavan-3-ols, naphtodianthrones, phloroglucinols, and xanthones were recorded in the transgenic shoots. Chlorogenic acid was found as the most representative phenolic acid in shoot extracts. With regard to the class of quercetin derivatives in transformed shoots, quercetin 6-C-glucoside usually dominated among the glycosides followed by quercitrin and hyperoside. The analysis of flavan-3-ols in transgenic shoots resulted in the identification of epicatechin and proanthocyanidin dimers. One of the main achievements in this study was considerably enhanced hypericin and pseudohypericin production in transgenic shoots. The concentration of identified naphtodianthrones was about 12-fold higher in transformed shoots compared to control. Chromatographic analysis of phloroglucinols in transgenic shoots resulted in the identification of hyperforin, while its homolog adhyperforin was detected in traces. A twofold higher content of hyperforin was observed in transgenic shoots compared to control. Although mangiferin was found as the main representative xanthone in shoot extracts, several other xanthones identified as c-mangostin isomers, trihydroxy-1-methoxy-C-prenyl xanthone, garcinone E, and banaxathone E were de novo synthesized in transformed shoots. Therefore, H. perforatum transgenic shoots could be considered as a source for rapid and increased production of naphtodianthrones and other specific phenolic compounds.
    Acta Physiologiae Plantarum 07/2014; · 1.31 Impact Factor
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    ABSTRACT: Hypericum perforatum L. is a medicinal plant considered as an important natural source of secondary metabolites with a wide range of pharmacological attributes. Hairy roots (HR) were induced from root segments of in vitro grown seedlings from H. perforatum after cocultivation with Agrobacterium rhizogenes A4. Investigations have been made to study the production of phenolic compounds in dark-grown (HR1) and photoperiod-exposed (HR2) cultures. The chromatographic analysis of phenolic acids, flavonols, flavan-3-ols, and xanthones revealed marked differences between HR1 and HR2 cultures. The production of quinic acid, kaempferol, and seven identified xanthones was increased in HR2. Moreover, HR2 showed a capability for de novo biosynthesis of two phenolic acids (3-p-coumaroylquinic acid and 3-feruloylquinic acid), three flavonol glycosides (kaempferol hexoside, hyperoside, and quercetin acetylglycoside), and five xanthones (tetrahydroxy-one-methoxyxanthone, 1,3,5-trihydroxy-6-methoxyxanthone, 1,3,5,6-tetrahydroxy-2-prenylxanthone, paxanthone, and banaxanthone E). On the other side, HR1 cultures were better producers of flavan-3-ols (catechin, epicatechin, and proanthocyanidin dimers) than HR2. This is the first comparative study on phenolic profile of H. perforatum HR cultures grown under dark and photoperiod conditions.
    The Scientific World Journal 12/2013; 2013:602752. · 1.73 Impact Factor
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    Sonja Gadzovska-Simic, Oliver Tusevski
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    ABSTRACT: Investigations have been made to develop an efficient protocol for identification and quantification of phenolic acids and flavonoids in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR grew rapidly on hormone-free medium and had plagiotropic growth with vigorous production of lateral roots. Phenolic acids and flavonoids in control roots and HR were analyzed using HPLC/DAD/ESI-MSn. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots produced flavonol glycosides such as quercetin 6-C-glucoside, rutin and isorhamnetin O-hexoside. Chromatographic analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. H. perforatum HR represent promising experimental system for enhanced production of phenolic compounds.
    International Journal of Pharma and Bio Sciences. 07/2013; 4(3):737-748.
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    ABSTRACT: Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration postelicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.
    Plant Cell Tissue and Organ Culture 11/2012; · 2.61 Impact Factor
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    Conference on Medicinal and Aromatic plants of South East European Countries, 7th CMAPSEEC; 05/2012
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    ABSTRACT: We investigated the production of phenylpropanoids (phenolic compounds, flavanols, flavonols and anthocya-nins) and naphtodianthrones (hypericins) in elicited Hypericum perforatum L. cell suspensions. To determine whether secondary metabolite production could be enhanced, Hypericum cell suspensions were exposed to mycelia extract from the fungus Aspergillus flavus. Elicited Hypericum cell suspension cultures displayed reduced growth and viability and a modification of secondary metabolites production. Anthocyanins were only stimulated in fungal-elicited cell suspensions. Secondary metabolite production in elicited Hypericum cells revealed an antagonism between the flavonoid/naphtodian-throne and anthocyanin pathways. The data suggest a modification of the channeling of the phenylpropanoid compounds. Together, these results represent useful data for monitoring the channeling in different secondary metabolite pathways during the scaled-up production of naphtodianthrones for medicinal uses.
    Archives of Biological Sciences 01/2012; 64:113-121. · 0.79 Impact Factor
  • Sonja Gadzovska-Simic
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    ABSTRACT: Hypericum perforatum L. in vitro cultures with different morphological characteristics referred to shoots, calli and cells were used as experimental models for studying of secondary metabolite productions. To determine whether production of secondary metabolites could be enhanced, in vitro cultures were exposed to exogenous application of phytohormones and chemical elicitors (jasmonic acid, salicylic acid, pectin and chitin). To assess responses to biotic elicitors, cells were treated with mycelia extracts from three fungi Fusarium, Phoma and Botrytis. Shoot cultures exhibited a higher yield of secondary metabolites, but calli and cells showed remarkably fast and strong response to applied elicitors. Secondary metabolite production in Hypericum cells can be partially changed by supplementation of these elicitors and well controlled cultures could be used as a source for rapid and increased production of hypericin and pseudohypericin. This book is intended for graduated students and researchers in Plant Cell and Tissue Culture and Plant Molecular Biology. Students and researchers who are just beginning to work in this field will also find much valuable information in this book.
    08/2010; LAP Lambert Academic Publishing, Saarbrücken, Germany., ISBN: 978-3-8383-8304-0
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    ABSTRACT: Hypericum perforatum L. cell suspensions were evaluated for their viability, growth, dark gland formation and ability to produce phenylpropanoids and naphtodiantrones after elicitation with different jasmonic acid (JA) concentrations. Phenolic compounds were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization mass spectrometry (ESI-MS). The activities of two key enzymes of the phenylpropanoid/flavonoid pathways, phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) were also monitored to estimate general channeling in the different metabolic pathways. A 6-fold increase of phenolic compounds, flavanols and flavonols after JA elicitation was observed in cells. In contrast, anthocyanins were in lower amounts in JA treated cells suggesting a modification of the channeling in the phenylpropanoid pathway. Similar accumulations with maxima after 4 days of elicitation were found for naphtodianthrones (2.4-fold) such as hypericin and pseudohypericin in cells. At least a 6–8-fold increase of PAL and CHI activities was observed in JA elicited cells confirming a strong activation of the phenylpropanoid pathway. JA elicitation increased production of phenylpropanoids and naphtodianthrones in H. perforatum cell suspension without differentiation of dark glands under 16 h photoperiod.
    Plant Cell Tissue and Organ Culture 02/2007; · 2.61 Impact Factor
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    ABSTRACT: Investigations have been made to develop an efficient protocol for micropropagation allowing to improve hypericin and pseudohypericin productions in Hypericum perforatum L. in vitro cultures. The role of growth regulator treatments has been particularly studied. Three in vitro culture lines with different morphological characteristics were obtained during H. perforatum micropropagation and referred to shoots, calli and plantlets according to their appearance. Multiplication and callogenesis from apical segments from sterile germinated seedlings were obtained on solid MS/B5 culture medium in the presence of N6-benzyladenine (BA) (0.1–5.0 mg/l BA). Regenerative potential of shoots was assessed on medium supplemented with auxins (0.05–1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The main goal of the research was to summarize the influence of plant growth regulators on hypericin and pseudohypericin productions in in vitro cultures of Hypericum. A rapid method for naphtodianthrone quantification was developed. The use of a reversed-phase high performance liquid chromatography (HPLC) method with fluorescence detection was used. Identification of the compounds was confirmed by electrospray ionizationmass spectrometry (ESI-MS) with electrospray in negative ion mode [M–H]¯. Calli, shoots and plantlets of H. perforatum produced hypericin and pseudohypericin. The concentration range of BA from 0.1 to 2.0 mg/l improved the production of hypericin (25–50 μg/g dry mass (DM)) and pseudohypericin (170–350 μg/g DM) in shoots. In callus cultures, BA (4.0–5.0 mg/l) did not changed hypericin contents (15–20 μg/g DM) but influenced pseudohypericin productions (120–180 μg/g DM). In the presence of auxins (IAA and IBA), Hypericum plantlets produced hypericin (30–100 μg/g DM) and pseudohypericin (120–400 μg/g DM). The presence of IAA did not influence naphtodianthrone productions in plantlets, but IBA decreased hypericin and pseudohypericin amounts in plantlets. The specific accumulation of the naphtodianthrones in in vitro cultures was influenced by phytohormonal supplementation of the medium. Results indicated that the production of hypericin and pseudohypericin could be increased by carefully adapted in vitro cultures. Hypericum in vitro cultures represent promising systems for hypericin and pseudohypericin productions.
    Plant Physiology and Biochemistry 01/2005; · 2.78 Impact Factor
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    ABSTRACT: Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones.
    Central European Journal of Biology 8(10). · 0.82 Impact Factor

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