Ivan Ivanov

The Jerusalem artichoke (Helianthus tuberosus L.) known also as topinambour is plant with high importance for human and animal nutrition during the last decade. Its tubers were consumed fresh, stewed or they were added as flour in food products to improve their functional properties. In the current research a comparative study of bioactive substance and free radical scavenging activities of flour obtained from tubers of different varieties and wild populations of Jerusalem artichoke (Helianthus tuberosus L.) grown on territory of Bulgaria was done. The ultrasonic irradiation (42 kHz) was performed with 70 % ethanol and distilled water as solvents to accelerate extraction process. The total fructans, phenolic content and radical scavenging activities of the extracts were investigated. The 70 % ethanol extracts possessed the highest total phenolic content (6-17 mg GAE/g dry weight) and antioxidant activity defined by ABTS and CUPRAC methods. The water extracts characterized with higher fructan levels - 32 to 69 g/100 g dry weight. The flour obtained from tubers of Scorospelcu variety and wild population of Helianthus tuberosus L. were evaluated as a valuable source of total polyphenols and soluble dietary fibers, because of the rich fructan content. The results from our study also revealed the potential application of these flours as radical scavengers in human and animal nutrition for preparation of foods with improve health benefits.

Consumption of the edible flowers forms new trend in human nutrition, especially in Bulgaria. Therefore, the determination of bioactive compounds content in their petals presents important tasks for their evaluation as natural source of antioxidants for human diet. In this study the quantity of natural pigments, total phenolic content and total flavonoids content, as well the antioxidant capacities in 95 % (v/v) ethanol, 70 % (v/v) ethanol, 80 % (v/v) methanol and distilled water extracts obtained from five edible flowers targets (Tagetes erecta L.), marigold (Calendula officinalis L.), geranium (Geranium macrorrhizum L.), bougainvillea (Bougainvillea spectabilis Willd.), Jerusalem artichoke (Helinathus tuberous L.) were invastigated. Total phenols and flavonoid quantification of the extracts was achieved by using Folin–Ciocalteau and AlCl3 reagents, respectively. Geranium 95 % ethanol extracts were evaluated as the richest source of total phenols-19.79 mg GAE/g fw, while 80% methanol extracts of Helinathus tuberous L. showed the highest values of total flavonoids content-8.89 mg QE/g fw. The highest antioxidant capacities using two methods, DPPH and FRAP assays were obtained for 95% ethanol geranium (Geranium macrorrhizum L.) extracts-242.9 and 106 mM TE/g fw, respectively. There was positive linear correlation between antioxidant activity and total phenolic content for all investigated edible flower extracts. All studied flowers could be a valuable source of antioxidants for addition to dishes or fresh consumption for preventing diets. These findings showed that the tested flowers could be considered as new sources of safe natural antioxidants and colorants for food industry.

Eco-friendly synthesis of methyl esters of two alginic acids with different ratio between M-block and G-blocks were performed by microwave irradiation. The alginates methyl esters have been characterized by degree of esterification (DE), HPLC-SEC, IR-FT and NMR spectroscopy and thermal analysis (TGA/DTA). The influence of reaction time on DE of alginic acids esters were investigated and compared with conventional and ultrasound–assisted synthesis. The highest DE (45 %) of methylated alginic acid (M/G ratio 61/39) was obtained by microwave-assisted synthesis (MAS) for 5 min. In addition, MAS accelerate esterification process as significantly reduced the reaction time from 60 min to 5 minutes. The microwave-assisted esterification did not degrade the alginic acid polymer chains as the coefficient of polydispersity remained constant 1.09 for alginic acid (M/G ratio 61/39) and 1.17 for alginic acid (M/G ratio 37/63), respectively. The ability of complexation of methyl esters of alginic acids with Ca 2+ ions was investigated and the promising results were obtained for future design of modified alginate-based micro particles as potential drug carriers.

The aim of the current research was to determine the ascorbic acid content, total tannins and total phenolic content in three extracts (water, 50 % v/v ethanol, 70 % (v/v) ethanol) obtained from dry wild growing rose hip fruits and to evaluate their antioxidant potential by four reliable methods: DPPH, ABTS, FRAP, CUPRAC. The highest ascorbic acid content was observed in 70 % (v/v) ethanol extract – 2404 mg/100 g dw, while the total tannins dominated in the water extracts – 3.86 g/100 g dw. The 50 % ethanol extracts of rose hip demonstrated the highest total phenolic content-6.9 g GAE /100 g and the antioxidant activity: DPPH assay – 295.0 ± 1.0; ABTS – 368.4±3.0; FRAP – 390.1±4.8; CUPRAC 1358.2±14.8 mM TE/g dw, respectively. The present study showed that the investigated wild growing Rosa canina L. from Bulgaria was evaluated as a rich source of antioxidants and revealed their potential application as food and herbal cosmetic preparations.

Stevia rebaudiana Bertoni is widеly used as a source of natural sweetening agent in human nutition. The aim of present study was to characterise the leaves extracts as evaluate the polyphenol and carbohydrate contents. The effect of different particle size and various solvents on the antioxidant activity of leaves extract were also studied. The content of fructans, polyphenols, radical scavenge activity (DPPH), metal reducing activity (FRAP) in the extracts were established. It was found that the fructans amount did not depend significantly from the size of grinding (2.8 % DW). The type of the solvent had a highest effect only to the yields of the extract (from 254 mg/g DW to 377 mg/g DW). Additionally, the total polyphenols content (from 12.7 mg GAE/g DW to 15.6 mg GAE /g DW), radical scavenge activity - DPPH (from 135.8 mM TE/g DW to 221.4 mM TE/g DW) and metal reducing activity-FRAP DPPH (from 117.7 mM TE/g DW to 149.5 mM TE/g DW) were influenced mainly from the particle size and degree of grinding. The highest values of the presented parameters concerning to antioxidant activity were obtained when the dried leaves of stevia were finely ground and water were used as extracting solvent. Radical scavenge activity and metal–reducing activity correlated very well with total polyphenol content.

Hairy root cultures of Salvia tomentosa were initiated by transformation with Agrobacterium rhizogenes. To prevent necrosis in the explants and to protect young hairy roots, Amberlite XAD-4 resin, in combination with a temporary immersion cultivation system, was applied. HPLC analyzes showed that the resin adsorbed more than 93% of the released phenolic acids and 100% of the released flavonoids. The decreased content of the released phenolics significantly reduced their destructive effects on the plant tissues, prevented, and speeded up the appearance of hairy roots.

Plant cells contain a wide range of interesting secondary metabolites, which are used as natural pigments and flavoring agents in foods and cosmetics as well as phyto-pharmaceutical products. However, conventional industrial extraction from whole plants or parts of them is limited due to environmental and geographical issues. The production of secondary metabolites from in vitro cultures can be considered as alternative to classical technologies and allows a year-round cultivation in the bioreactor under optimal conditions with constant high-level quality and quantity. Compared to plant cell suspensions, differentiated plant in vitro systems offer the advantage that they are genetically stable. Moreover, the separation of the biomass from culture medium after fermentation is much easier. Nevertheless, several investigations in the literature described that differentiated plant in vitro systems are instable concerning the yield of the target metabolites, especially in submerged cultivations. Other major problems are associated with the challenges of cultivation conditions and bioreactor design as well as upscaling of the process. This article reviews bioreactor designs for cultivation of differentiated plant in vitro systems, secondary metabolite production in different bioreactor systems as well as aspects of process control, management, and modeling and gives perspectives for future cultivation methods.

The constituents of essential oils, obtained from different aerial parts (leaves and flowers) of Salvia ringens Sibth. et Sm. growing wild in Bulgaria, have been analyzed by GC-MS. The yields of leaves and flowers oils were similar (0.03 % v/w and 0.04 % v/w). 60 compounds were identified in both oils, which correspond to 97.7 % and 97.3 % of the total oils constituents for leaves and flower oils, respectively. Both leaves and flowers oils showed similar chemical compositions, with predominance of camphor (17.2 % and 18.8 %) and borneol (7.2 % and 8.7 %). Further analyses of minor component distributions proves that the both leaves and flowers could be equally used as a raw material for obtaining of camphor reach essential oil from Salvia ringens Sibth. et Sm.

Fragaria vesca L. (wild strawberry) belongs to the Rosaceae family. The leaves and roots from wild strawberry are
herbal materials applied in traditional medicine. Fragaria vesca are rich source of biologically active substances like
tannins, procyanidins, anthocyanidins, flavonoids and phenolic acids. The aim of this study was to compare the value of
phytochemical compounds and antioxidant activities in infusion and decoction obtained from the wild strawberry
leaves. The extracts were analyzed regarding their secondary metabolite content (total polyphenols, total flavonoids
and total proanthocyanidins) and antioxidant activities (DPPH and CuPRAC methods). The analysis of decoction
extracts from the leaves harvested in blooming period revealed the highest level of total polyphenols (46.1 mg GAE/g
DW), total flavonoids (4.7 mg QE/ g DW), total proantocyanidines (22.3 mg/ g DW) and antioxidant activities – radical
scavenging activity (DPPH – 325.0 mM TE/ g DW) and metal reducing ability (CuPRAC – 1257.9 mM TE/ g DW). The
results showed that the water extracts from leaves of Fragaria vesca are appropriate additives for preparation of
functional foods and natural cosmetic products with improved biological activity.

Chemical composition and bioactivity of essential oil from Salvia tomentosa Mill. natively grown in Bulgaria were investigated. GC-MS analysis identified 60 compounds which represented 98% of the oil constituents. The prevalent constituents were monoterpenes with eight dominant compounds being identified: borneol (10.3%), β-pinene (9%), camphor (7.9%), α-pinene (6%), camphene (4%), 1.8-cineole (3.8%), α-limonene (3.5%) and β-caryophyllene (3%). The essential oil showed considerable acetylcholinesterase inhibitory activity (IC50=0.28±0.06 μg/mL), comparable with that of galanthamine. Study of antioxidant activity strongly suggested that the hydrogen atom transfer reaction was preferable over the electron transfer (ORAC=175.0±0.40 μM Trolox equivalents/g oil and FRAP=1.45±0.21 mM Trolox equivalents/g oil). The essential oil showed moderate antifungal and antibacterial activities against Candida albicans and Gram-positive bacteria, whereas it was almost inactive against the investigated Gram-negative strains. The results suggested that the essential oil of Bulgarian S. tomentosa could be considered as a prospective active ingredient for prevention of oxidative stress-related and neurodegenerative disorders in aromatherapy. Because of the high antioxidant capacity, the oil could be considered as natural supplement or antioxidant in cosmetics and food products.

In vitro antioxidant activity, total phenolic and total dihydroxycinnamic derivatives contents of different herbal infusions (green tea, black tea, peppermint, yarrow, rooibos and red dates) were evaluated. The total phenolic contents (TPC) and total dihydroxycinnamic derivatives contents were determined by Folin–Ciocalteau method and Arnow‘s reagent, respectively. In vitro antioxidant activity of the extracts was analyzed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay. From the investigated herbal infusions, the green tea water extracts demonstrated the highest TPC 106 - 172 mg GAE/g and antioxidant activity 1100 - 1222 mM TE/g. From the results obtained, only peppermint and yarrow water extracts contained more total dihydroxycinnamic derivatives contents 25 and 19 mg chlorogenic acid/g, respectively. As all, these infusions possessed the highest antioxidant capacity, which positively correlated with the phenolic content. The current study demonstrated that investigated plants are valuable source of natural antioxidants and could be successively used in foods, nutrition formula with improved healthy effect.

Ficus carica L. (fig) belongs to mulberry tree (Moraceae) which is one of the oldest fruits in the world. The extracts from fig possess anthelmintic, anticarcinogenic, purgative, aphrodisiac, anti-inflammatory, expectorant, diuretic, anti-anxiety (mild sedative) activity. Phytoconstituents as umbelliferone, scopoletin, psoralens, bergapten, xanthotoxin, xanthotoxol, marmesin have been isolated from fig leaves. The current study was conducted to evaluate the total polyphenols, total flavonoids (TF), and to investigate the antioxidant capacities of aqueous, methanolic, ethanolic, acetone and n-hexane extracts prepared from Ficus carica leaves. Total phenolic content (TFC) was determined by using the Folin-Ciocalteau method. The antioxidant capacities were evaluated by DPPH and FRAP spectrophotometric methods. The results indicated that TPC, TF, DPPH and FRAP values were higher in aqueous extract of Ficus carica leaves: 6.6 mg GAE / g DW, 2.1 mg QE / g DW, 59.6 mM TE / g DW (DPPH method) and 53.9 mM TE / g DW (FRAP method), respectively. The results clearly demonstrated that these extracts had antioxidant capacity and it could be find significant correlation between total phenolic content and antioxidant activity. Therefore, Ficus carica leaves were evaluated as a rich source of polyphenols and they possessed high antioxidant properties.

The purpose of the present study was to investigate the antioxidant activity and fructans content in ethanol and water extracts of roots of elecampane (Inula helenium L.), a Bulgarian medicinal plant. The extraction procedure included 95% (v/v) ethanol extraction and subsequent water treatment. The antioxidant activity was evaluated by several reliable methods such as DPPH-, ABTS-, FRAP- and CUPRAC-assays, as well as the total phenolic content. In addition, the total fructans and sugar content were determinated by spectrophotomeric, TLC and HPLC-RID methods. The level of fructans in ethanol extracts was 14.1 g / 100 g dry weight, as nystose and 1-kestose were only 0.3 g / 100 g dry weight, and 0.5 g / 100g dry weight, respectively. The absence of fructooligosacharides and sugars in water extracts after the ethanol pretreatment was established. Inulin content was evaluated to be 32 g / 100 g dry weight. The metabolites profile of roots revealed their potential application as radical scavengers due to the presence of polyphenols. Therefore, the root extracts of elecampane could be assumed as a rich source of biologically active substance, in particular dietary fiber with potential prebiotic effect, due to the presence of polysaccharide inulin and fructooligosacharides.

Ficus carica L. belongs to the family Moraceae. It is commonly known as fig, which is a medium sized
deciduous tree widely distributed in sub-tropical and tropical countries. It has been used as fruit and medicine
for several centuries. Phytoconstituents like flavonoids, phenolics, fatty acis, proanthocyanidine, anthocyanins,
phytosteroles (campesterol, stigmasterol, sitosterol), xanthotoxin, psoralens, bergapten, xanthotoxol have been
extracted from leaves and fruits, and peptides from latex. Traditionally the fruit extracts possessed
hypoglycemic, diuretic, expectorant, antioxidant, immunity, anthelmintic, hepetoprotective activities, latex has
anthelmintic and anticarcinogenic. The present review is therefore, an effort to give a detailed survey of the
literature on its pharmacognosy, phytochemistry, and pharmacological properties.

The aim of this study was profiling of primary metabolites of five Bulgarian Fumaria species (F. officinalis L., F. thuretii Boiss., F. kralikii Jord., F. rostellata Knaf. and F. schrammii Velen.) by GC-MS analyses. In polar fractions ten carbohydrates, one polyol, ten amino acids and six organic acids were identified. Apolar (lipid) fractions showed the presence of four free fatty acids, two esters of fatty acids with glycerol and two fatty alcohols. Sucrose and fructose were in the highest relative concentrations of identified carbohydrates. Citric acid was the dominant organic acid in polar fractions of five Fumaria species. Predominant compounds in lipid fractions were palmitic acid and 1-stearoyl-glycerol. Principal component analysis (PCA) of GC-MS data of polar and apolar fractions of five Bulgarian Fumaria species differentiates them in two groups (F. officinalis and F. thuretii; F. rostellata and F. schrammii, respectively), while F. kralikii had phythochemical similarity with plants of both distinguished groups. The obtained results of PCA of primary metabolites could be proposed as chemotaxonomic markers for plants of the genus Fumaria.

Fumaria plants have been traditionally used against skin diseases, as diuretics, laxatives, hepatoprotectants and etc. Their biological activity is associated with the presence of isoquinoline alkaloids, while polyphenols are not investigated well in this context. Polyphenols could increase the added value of medicinal plants extracts by enhancing their antioxidant activity or improving overall biological activities. The presented study evaluated the polyphenolic contents and antioxidant activities of extracts of five Bulgarian Fumaria species (Fumariaceae): Fumaria officinalis L., Fumaria thuretii Boiss., Fumaria kralikii Jord., Fumaria rostellata Knaf. and Fumaria schrammii (Asch) Velen. Qualitative and quantitative determinations of phenolic acids and flavonoids were performed by HPLC system with UV-detection. Antioxidant activity of extracts was evaluated by four popular spectrophotometric methods (DPPH, ABTS, FRAP and CUPRAC). The total phenolic content was the highest in extract of F. officinalis with quercetin (0.49 ± 0.03 mg/g DW), p-coumaric (1.10 ± 0.03 mg/g DW) and ferulic (2.35 ± 0.04 mg/g DW) acids as major compounds. Theses extracts also showed the highest antioxidant activity among the investigated plants. Our data reveal new possibilities to expand applications of Fumaria extracts from a local ethnomedicine drug to new industrial fields such as foods and cosmetics preservation.

The current research is dedicated to following the accumulation of prebiotics inulin and fructooligosaccharides in the roots of Bulgarian medicinal plants common chicory (Cichorium intybus L.), dandelion (Taraxacum officinale Web.) and meadow salsify (Tragopogon pratensis L.) in some areas of the Bulgaria for a period of two years. The roots of each plant were subjected to exhausting ethanol and subsequent water extraction. The contents of inulin-type fructans in the obtained extracts have been determined by a spectrophotometric method based on resorcinol reaction and TLC analysis. It has been found that the amounts of fructooligosaccharides and inulin were the highest in roots of the plants collected in the autumn season as in taproots of common chicory reached to 23 g/100 g dw and up to 34 g/100 g d.w in dandelion roots, respectively. The underground parts of the studied medicinal plants collected in the spring have been characterized by very high levels of fructo-oligosaccharides and sugars. Meadow salsify showed relatively constant levels of fructans in its taproots (18 g/100g d.w).
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The content of polyphenols in Fumaria officinalis L. and Fumaria thuretii Bioss. from two different
populations, naturally grown in Bulgaria has been studied. Significant differences between concentrations of
main polyphenols (rutin, hyperoside and syringic acid) and antioxidant activity in two different populations of
both analyzed species were not established (RSD 2-14%). Stability of secondary metabolism of F. officinalis
and F. thuretii indicates for genetic stability of Bulgarian Fumaria species growing in different populations.

"HPLC methods for determination of fat soluble vitamins and biological activities carotenoids have been
improved and adapted for application to different varieties of tomatoes. HPLC separation was achieved using a
reversed phase C18 column Discovery® with gradient system with acetonitril : methanol (8:2) and MTBE
(methyl tert-butyl ether) flow rate 1.0 mL/min for carotenoids (luteolin, lycopene and β-carotene), and column
Symmetry® with isocratic solvent system methanol: water (98:2) for quantitative determination of α-, β-and δ-
tocopherols. The calibration curves were linear from 10 to 500 μg/mL (r2>0.99) for tocopherols and from 5 to
50 μg/mL (r2>0.99) for carotenoids. The reliability of the proposed systems ware proved through
reproducibility test with different extracts from lyophilized tomatoes samples. Critical for the success of
determination was adding to mobile phases and during the extraction procedure synthetic antioxidant BHT (butylhydroxytoluene, 227 μmoll)."

Biomass accumulation and tropane alkaloids production by diploid and tetraploid hairy root cultures of Datura stramonium L. cultivated in stirred tank bioreactor at different aeration rates were investigated. The maximal growth for both hairy root cultures (ADB = 8.3 g/L and 6.8 g/L for diploid and tetraploid line, respectively) was achieved at aeration rate of 15.0 L/(L.h). The corresponding growth indexes were remarkably high (GIDW = 9.0 and 7.8 for diploid and tetraploid line, respectively) compared to the values, usually reported for other hairy root cultures. The optimal aeration rate for biomass accumulation was also optimal for alkaloids biosynthesis. According to our survey, the achieved maximal amounts of accumulated hyoscyamine (35.0 mg/L and 27.0 mg/L for diploid and tetraploid line) were the highest reported in the scientific literature for D. stramonium L. hairy roots. During the cultivation in stirred tank bioreactor, the hairy roots biosynthesized pharmaceutically important alkaloid scopolamine in minor concentrations. This is an important observation since scopolamine was not detected during submerged cultivation of these hairy root lines in other bioreactor types. However, the ploidy level was found to be the most important factor concerning scopolamine production by D. stramonium L. hairy roots cultures. The present work demonstrated the effect of ploidity levels on biomass accumulation and tropane alkaloids production by D. stramonium L. hairy roots cultivated in stirred tank bioreactor. This investigation show that the stirred tank bioreactor could be successfully applied for both maximal biomass accumulations, as well as for manipulation of tropane alkaloids production by diploid and tetraploid D. stramonium L. hairy root cultures.

The ultrasonic and microwave assisted extractions of tubers from the Jerusalem artichoke (Helianthus tuberosus
L.) with water, methanol and 70 % ethanol were carried out. The fructan contents (40-60 % dw) were analyzed by the
resorcinol method and TLC analysis. The total phenolic contents and the total flavonoid concentration of the extracts
were estimated, as their antioxidant activities were determinated by DPPH, ABTS, FRAP and CUPRAC methods. The
ethanol extracts obtained by the microwave extraction showed the greatest antioxidant activity, which was probably
due to the highest content of total phenols.