Ivan Atanassov

Industrial Cultivation of Oil Bearing Rose and Rose Oil Production in Bulgaria During 21ST Century, Directions and Challenges

ABSTRACT The rose growing and flower processing is a centuries-old industry in Bulgaria which was a subject of substantial structural changes during the transitional period from state-ruled to market driven economy in the 1990s. The industry successfully passed in the 21st century becoming more open and dynamic. Here we review the industrial cultivation of oil-bearing roses in Bulgaria, the recent developments, current challenges and opportunities for future development.

Rosa Damascena—Genetic Resources and Capacity Building for Molecular Breeding

ABSTRACT Rose genetics and genomics research has tremendously advanced during the last 10 years. This mini-review focuses on the current status of knowledge related to the worldwide genetic resources characterization of Rosa damascena, a species which belongs to the Damask group of roses and which has economic importance in several countries related to its cultivation for production of rose oil and rose water. We discuss the needs and options for R. damascena improvement and we present the research activities of the AgroBioInstitute related to R. damascena improvement in frame of the NSF funded DO02–105 “Centre for sustainable development of plant and animal genomics” project.

COMPARATIVE GC/MS ANALYSIS OF ROSE FLOWER AND DISTILLED OIL VOLATILES OF THE OIL BEARING ROSE ROSA DAMASCENA

ABSTRACT The accelerated and successful oil bearing rose breeding requires routine application of efficient procedure for analysis of flower volatiles, with capacity to extrapolate the obtained flower data to the volatile composition of the distilled rose oil. In the current study a procedure for solvent extraction and GC/MS analysis of rose flower and rose oil volatiles from oil bearing roses including Rosa damascena is presented. The procedure allows reliable identification of 68 volatiles in the rose flowers which are also detected in the distilled rose oil. The described procedure was further applied for comparative analysis of the flower and distilled rose oil volatiles from eight different genotypes of oil bearing roses. A data set consisting of ratios of the relative abundance of given volatile in the flower spectra to the relative abundance of the same volatile in the distilled rose oil spectra was generated. ANOVA test for a data subset of 27 volatiles detected in the flowers and rose oils of all analyzed oil bearing rose genotypes showed no significant influence of the genotype on the ratio of relative abundances of flower to rose oil volatiles. The average and relative standard deviation values of the obtained ratios between relative abundances of flower and rose oil volatiles for the analyzed genotypes were calculated for each identified flower compound. The results demonstrate that the described flower solvent extraction and GC/MS analysis procedure could be reliably applied for prediction of the volatile composition of distilled rose oils from wide range of oil bearing rose genotypes based on the extrapolation of GC/MS analysis data from single or few flowers from each studied plant. The possibilities for incorporation of the described procedure into oil bearing rose breeding and genetic resources characterization are discussed.

Reducing methyl eugenol content in Rosa damascena Mill rose oil by changing the traditional rose flower harvesting practices

Methyl eugenol (ME) is a naturally occurring carcinogenic compound found in a number of essential oils including rose oil distilled from Rosa damascena Mill flowers. In the current study, we evaluate the effect of flower harvesting practices on the ME content in the produced rose oil. The obtained results show nearly twice reduction in ME content in the rose oil distilled from petals of full-blown flowers. At the same time, GC/MS analysis of rose oils distilled from stages 3 and 4 rose flower buds (flower buds prior opening of petals) showed more than 5 times ME reduction and preservation of the relative content of the major rose oil compounds. Moreover, the comparative study of rose flower yield and rose oil content of rose buds and full-blown flowers showed that harvesting of rose flower buds results in above three times increase in the formed flower buds from the studied rose plants and more than twice increase in the rose flower and rose oil yields for the same rose plantation areas. The overall results from this study allow us to propose a change in the traditional full-blown rose flower harvesting to harvesting of rose flower buds at stages 3 and 4 during the entire flowering period.

Tyrosinase inhibitory constituents from a polyphenol enriched fraction of rose oil distillation wastewater

During the water steam distillation process of rose flowers, the non-volatile phenolic compounds remain in the waste. We recently developed a strategy to separate rose oil distillation water (RODW) into a polyphenol depleted water fraction and a polyphenol enriched fraction (RF20-SP207). Bioassay-guided investigation of RF20-SP207 led to the isolation of quercetin, kaempferol and ellagic acid. Their structures were elucidated by spectroscopic analysis as well as by comparison with literature data. Tyrosinase inhibition studies were performed with RF20-SP207, fractions I-IV, and the isolated compounds of the most active fraction. RF20-SP207 strongly inhibited the enzyme with an IC50 of 0.41 μg/mL. From the tested fractions only fraction IV (IC50=5.81 μg/mL) exhibited strong anti-tyrosinase activities. Quercetin, kaempferol and ellagic acid were identified in fraction IV and inhibited mushroom tyrosinase with IC50 values of 4.2 μM, 5.5 μM and 5.2 μM, respectively, which is approximately 10 times more potent than that of the positive control kojic acid (56.1μM). The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated that RF20-SP207 and fraction IV are uncompetitive inhibitors of tyrosinase when l-tyrosine is used as a substrate. A mixed inhibition was determined for ellagic acid, and a competitive inhibition for quercetin and kaempferol. In conclusion, the recovered polyphenol fraction RF20-SP207 from RODW was found to be a potent tyrosinase inhibitor. This value-added product could be used as an active ingredient in cosmetic products related to hyperpigmentation.

Recovery of Polyphenols from Rose Oil Distillation Wastewater Using Adsorption Resins – A Pilot Study

The production of rose oil from rose flowers by water steam distillation leaves a water fraction of the distillate as main part of the waste. Therefore, the rose oil distillation wastewater represents a serious environmental problem due to the high content of polyphenols which are difficult to decompose and have to be considered as biopollutants when discarded into the drainage system and rivers. On the other hand, natural polyphenols are valuable compounds with useful properties as bioactive substances. Until now there is no established practice for processing of rose oil distillation wastewater and utilization of contained substances. Thus, it was the aim of this study to develop a strategy to separate this wastewater into a polyphenol depleted water fraction and a polyphenol enriched fraction which could be developed into innovative value-added products. In a first step, the phytochemical profile of rose oil distillation wastewater was determined. Its HPLC-PDA-MS analysis revealed the presence of flavan-3-ols, flavanones, flavonols and flavones. In a second step, the development of a stepwise concentration of rose oil distillation wastewater was performed. The concentration process includes a filtration process to eliminate suspended solids in the wastewater, followed by adsorption of the contained phenolic compounds onto adsorption resins (XAD and SP). Finally, desorption of the polyphenol fraction from the resin matrix was achieved using ethanol and/or aqueous ethanol. The result of the process was a wastewater low in soluble organic compounds and an enriched polyphenol fraction (RF20 SP-207). The profile of this fraction was similar to that of rose oil distillation wastewater and showed the presence of flavonols such as quercetin and kaempferol glycosides as major metabolites. These compounds were isolated from the enriched polyphenol fraction and their structures confirmed by NMR. In summary, a pilot medium scale system was developed using adsorption resins for the recovery of polyphenols from rose oil distillation wastewater suggesting an industrial scalability of the process.

LESSONS FROM THE MICROSATELLITE CHARACTERIZATION OF A SEGREGATING POPULATION DERIVED FROM SEEDS OF OPEN POLLINATED ROSA DAMASCENA MILL. F. TRIGINTIPETALA PLANTS

ABSTRACT A population of 24 plants derived from seeds of open pollinated Rosa damascena plants were analyzed at 22 microsatellite loci. The comparison of the obtained microsatellite profiles with those of the mother plants revealed that the seeds of the open pollinated oil rose plants are a result of self-pollination of the mother plants or cross-pollination with neighboring R. damascena plants possessing the same genotype. Analysis of the allele segregation demonstrated that R. damascena is a segmental allotetraploid with polysomic or disomic type of inheritance depending on the chromosomal location of the corresponding locus. The most probable allele configurations for the studied loci in R. damascena were determined. The possibility for development of a large segregating population from oil rose is discussed.

A Polyphenol-Enriched Fraction of Rose Oil Distillation Wastewater Inhibits Cell Proliferation, Migration and TNF-α-Induced VEGF Secretion in Human Immortalized Keratinocytes

Water steam distillation of rose flowers separates the essential oil from the polyphenol-containing rose oil distillation wastewater. Recently, a strategy was developed to separate rose oil distillation wastewater into a polyphenol depleted water fraction and a polyphenol-enriched fraction [RF20-(SP-207)]. The objective of the present study was to investigate RF20-(SP-207) and fraction F(IV), augmented in quercetin and ellagic acid, for possible antiproliferative effects in immortalized human keratinocytes (HaCaT) since rose petals are known to contain compounds with potential antiproliferative activity.RF20-(SP-207) revealed dose-dependent antiproliferative activity (IC50 of 9.78 µg/mL). In a nontoxic concentration of 10 µg/mL, this effect was stronger than that of the two positive controls LY294002 (10 µM, PI3 K-inhibitor, 30 % inhibition) and NVP-BEZ235 (100 nM, dual PI3 K/mTOR inhibitor, 30 % inhibition) and clearly exceeded the antiproliferative action of quercetin (50 µM, 25 % inhibition) and ellagic acid (1 µM, 15 % inhibition). Time-lapse microscopy detected a significant impairment of cell migration of RF20-(SP-207) and F(IV). At concentrations of 10 µg/mL of both, extract and fraction, cell migration was strongly suppressed (51 % and 28 % gap closure, respectively, compared to 95 % gap closure 24 hours after control treatment). The suppression of cell migration was comparable to the positive controls LY294002, NVP-BEZ235, and quercetin. Furthermore, basal and TNF-α-stimulated VEGF-secretion was significantly reduced by RF20-(SP-207) and F(IV) at 10 µg/mL (44 % vs. untreated control).In conclusion, RF20-(SP-207) showed promising antiproliferative and antimigratory effects and could be developed as a supportive, therapy against hyperproliferation-involved skin diseases.

Mitochondrial Control Region DNA Variation in Turbot Populations from the Bulgarian and Romanian Black Sea Coasts

ABSTRACT The mitochondrial control region/CR/of 76 turbot specimens collected from the south and north Bulgarian and north Romanian regions of the west Black Sea coastal area was characterized. A total of 36 haplotypes were identified. Twenty-seven of these haplotypes were new and nine haplotypes were identical to previously reported Pmax haplotypes. The constructed haplotype parsimony network showed star-like phylogeny of the identified haplotypes, supporting the suggested Black Sea fish population bottleneck during the height of the last glacial period. The analysis of the haplotype sequences data did not provide clear indications on the existence of phylogeographic differentiation among the studied turbot populations inhabiting the west coast of the Black Sea. At the same time the haplotype phylogenetic analysis provided further support to the earlier proposed existence of two distinct turbot mitochondrial lineages, ‘western Mediterranean ‘and’ eastern secluded Mediterranean basins’. The present study offers an essential background for long term monitoring of the changes of the Black Sea turbot populations.

A Simple Procedure for Comparative GC-MS Analysis of Lavender (Lavandula Angustifolia Mill.) Flower Volatile Composition.

The efficient lavender (Lavandula angustifolia Mill.) breeding for essential oil production requires routine and accurate comparison the flower volatile composition of larger number individual plants from genetic resource pools and segregating populations. Here we describe the performance testing of a simple procedure for comparative GC-MS analysis of flower volatile composition of individual lavender plants. It involves collection of fixed number flowers at selected stage of development and direct solvent extraction and GC-MS analysis of the extracts. The summarized data on relative standard deviations for the analyzed compound abundancies determined from three replica analysis of individual plants from Bulgarian lavender varieties are presented. Further application of this comparative GC-MS analysis procedure demonstrates significant changes in the accumulation of certain volatiles during flower development. The comparative analysis also showed no significant daytime variation of the flower volatile composition and minor variations after testing the same plants within five consecutive days. The possibilities for routine applications of the tested procedure for comparative GC-MS analysis of flower volatiles in larger number of plants from lavender segregating populations or genetic resources collections are discussed.