Influence of growing conditions on morphological and chemical properties and biological effects of extracts of Arnica montana L.

Arnica montana L. (Asteraceae) je višegodišnja planinska biljka čije se cvetne glavice (Arnicae flos) uglavnom koriste za spoljašnju upotrebu kod modrica i uganuća u fitofarmaceutskim preparatima kao što su masti, kreme i gelovi. Do pre par godina svetsko tržište pomenute sirovine je zadovoljavano isključivo iz spontanog resursa, ali su biljne populacije, zbog prekomernog sakupljanja, znatno degradirane, zbog čega je u većini evropskih zemalja stavljena zabrana za njeno dalje sakupljanje iz prirode. Obzirom da tražnja za ovom sirovinom na tržištu ne opada, kultivacija arnike se nameće kao jedino održivo rešenje zadovoljenja potreba farmaceutske industrije. Budući da A. montana (narodni naziv – arnika) nije autohtona biljna vrsta za područje Srbije, iako se spontano javlja u planinskim oblastima nekih susednih zemalja i Balkanskog poluostrva uopšte, cilj ovih istraživanja je bio da se iznađe tehnološko rešenje njivske proizvodnje na osnovu ispitivanja uticaja vremena zasnivanja, primene đubrenja i tipa sadnica. U istraživanju su pored cvetne glavice, kao glavne droge, posmatrani i podzemni organi arnike: rizom i koren, kao sporedne sirovine. Prva faza eksperimentalnog rada obuhvatila je trogodišnji tro-faktorijalni poljski ogled načina proizvodnje arnike, pri čemu su se kao faktori ispitivali: vreme zasnivanja useva (prolećna i jesenja sadnja), đubrenje (stajnjak i NPK) i način zasnivanja useva (iz semena i klonskom propagacijom – deobom bokora). U drugoj fazi eksperimenta, izvršena je karakterizacija sirovine u smislu njene brze identifikacije, hromatografskim tehnikama identifikovani su prisutni sekundarni metaboliti i određen njihov sadržaj u ispitivanim uzorcima i testirani su biološki efekti odabranih ekstrakata. Ispitivanja su vršena na ogledu postavljenom 2008. godine na lokalitetu Kaluđerskih bara (1008 m n.v.), na planini Tara, u krugu rasadnika Nacionalnog Parka „Tara“. Obzirom na to da arnika prvu vegetaciju provodi u fazi rozete sva merenja morfoloških parametara, prinosa i hemijskih karakterizacija sirovina vršena su tokom 2009. i 2010. godine. Na ogledu su praćeni sledeći parameri: prečnik rozete, visina cvetnog izdanka, broj cvetnih izdanaka, broj cvetnih glavica, prečnik cvetne glavice, broj sekundarnih rozeta. Određeni su sledeći prinosi: prinos cvetne glavice, prinos rizoma, prinos korena i prinos etarskog ulja (rizoma i korena)...Arnica (Arnica montana L., Asteraceae) is a perennial mountain plant whose flower heads (Arnicae flos) are mainly used for topical treatment of bruises and sprains in phytopharmaceutical preparations such as ointments, creams and gels. Until few years ago, the world market demand for this raw material was almost exclusively covered from the spontaneous resources, but plant populations are, due to overcollecting, significantly degraded, what caused prohibition of its collection from the wild in most of European countries. Since the demand for this raw material on the market is constant rise, cultivation arnica is imposed as the only sustainable solution to meet the needs of the pharmaceutical industry. Since the A. montana plant specie is not native to the region of Serbia, although it occurs spontaneously in some mountain areas of neighboring countries and the Balkans in general, the aim of this study was to find a suitable technology solution of field cultivation based on the examination of induced various ecological factor effects. In this research, beside observations connected with flower heads, as the main drug, underground parts of arnica: rhizome and roots, were also studied as side products. The first phase of the experimental work included a three-year three-factorial field experiment in different modes of production, where as the factors examined: time of plantation establishment (spring and autumn planting), fertilization (manure and NPK) and methods of propagation (from seed and clonal propagation - division of the tuft). In the second phase of the experiment, characterization of the raw material was carried out in the terms of its rapid identification, qualitative evaluations based on the content of secondary metabolites and testing of biological effects of extracts obtained. Tests were performed on the field experiment conducted in 2008 at locality of Kaludjerske Bare (1008 m) on mountain Tara, in area of Nursery production department of the National Park "Tara". Since arnica its first vegetation remains in the rosette phenophase, all measurements of morphological parameters, yields and chemical characterizations of raw materials was carried out in 2009 and 2010 year..

Antifungal Activity of Lavandula angustifolia Essential Oil against Candida albicans: Time-Kill Study on Pediatric Sputum Isolates

The aim of our study was to determine the susceptibility of 15 Candida albicans sputum isolates on fluconazole and caspofungin, as well as the antifungal potential of Lavandula angustifolia essential oil (LAEO). The commercial LAEO was analyzed using gas chromatography-mass spectrometry. The antifungal activity was evaluated using EUCAST protocol. A killing assay was performed to evaluate kinetics of 2% LAEO within 30 min treatment. LAEO with major constituents’ linalool (33.4%) and linalyl acetate (30.5%) effective inhibited grows of C. albicans in concentration range 0.5–2%. Fluconazole activity was noted in 67% of the isolates with MICs in range 0.06–1 µg/mL. Surprisingly, 40% of isolates were non-wild-type (non-WT), while MICs for WT ranged between 0.125–0.25 µg/mL. There were no significant differences in the LAEO MICs among fluconazole-resistant and fluconazole-susceptible sputum strains (p = 0.31) and neither among caspofungin non-WT and WT isolates (p = 0.79). The 2% LAEO rapidly achieved 50% growth reduction in all tested strains between 0.2 and 3.5 min. Within 30 min, the same LAEO concentration exhibited a 99.9% reduction in 27% isolates. This study demonstrated that 2% solution of LAEO showed a significant antifungal activity which is equally effective against fluconazole and caspofungin susceptible and less-susceptible strains

Ethnomedicinal application of species from genus Thymus in the Pirot County (Southeastern Serbia)

The species from genus Thymus are polymorphous plants from the family Lamiaceae, spread in Serbia in many species, subspecies, varieties, and forms. The aerial parts of species from genus Thymus have a long tradition of being used worldwide. The subject of this study was the research of ethnomedicinal application of thyme in Pirot County (Southeastern Serbia). Ethnomedicinal surveys were conducted among the population in four municipalities: Pirot, Babušnica, Bela Palanka, and Dimitrovgrad. It was noticed that 56.9 % of interviewed persons use the species of genus Thymus in the Pirot County. They are usually used in the county’s folk medicine in the form of herbal tea against colds to relieve dry and spastic cough, especially in bronchitis and asthma, and for sedation

Microencapsulation of Oregano and Thyme essential oils with hydroxypropyl-β cyclodextrin

Among plant natural products, Oregano and Thyme essential oils (EOs) are well-known for their antimicrobial and antioxidant activity. The biological activities of EOs may be reduced due to oxidation and volatilization. Stability and preservation of EOs can be assured with the microencapsulation method based on complex coacervation technology. Cyclodextrins are suitable as carriers for volatile substances insoluble in water, and hydroxypropyl-β cyclodextrin (HPCD) leads the group considering the aqueous solubility and safe toxicity profile. The aim of this study was to find conditions for preparing Oregano and Thyme EOs microencapsulates with the best technological properties.Oregano and Thyme EOs were encapsulated by the freeze drying (lyophilization) method. Nine different combinations were prepared, where HPCD content (10, 15 and 20%) and EO:HPCD mass ratio (1:1, 1:5 and 1:10) were varied. After stirring (200 rpm) for 24 h at a room temperature, suspensions were filtered through 0.45 mm PTFE filters. Samples were evaporated under vacuum and frozen (- 80 °C for 1 h), then main drying was carried out (-60 °C, pressure of 0.011 mbar for 40 h), and final drying (-65 °C, pressure of 0.054 mbar for 1 h). To ensure the particle purity, lyophilizates were washed with acetonitrile and dried at 25 °C. The content of each EO was determined spectrophotometrically, encapsulation efficiency (EE%) and yield were calculated. Duncan’s post hoc test was used to evaluate the differences between samples. The best conditions for both EOs were obtained with 15% HPCD and EO:HPCD mass ratio of 1:10. Yields of EOs for chosen Oregano and Thyme EOs complexes were 86.81 ± 2.20% and 89.83 ± 2.80%, respectively, with the following EE% of 49.08 ± 1.80% and 49.29 ± 0.18%. Microencapsulation is a promising method for improving the EOs stability profile, and these results could be very useful in the pharmaceutical and food industry for the implementation of new products

Traditional knowledge on the medicinal use of plants from genus Gentiana in the Pirot County (Serbia)

The paper provides an insight into the traditional use of medicinal plant species from the genus Gentiana in the Pirot County in Southeastern Serbia. The ethnopharmacological study was conducted in the form of interviews among the population in four municipalities: Pirot, Babušnica, Bela Palanka, and Dimitrovgrad. Among the population of Pirot County, it was noticed the use of two species from the genus Gentiana: G. cruciata and G. lutea. It was recorded that cross gentian (G. cruciata) has great popularity and versatile aspects on traditional usage, that was mentioned by 53 respondents, and that includes the treatment of cancer diseases, improving the immune system, diabetes, blood purification, for appetite, gastric and duodenal ulcer, for high blood pressure, inflammation, lung diseases. cold, cough, disease prevention, diseases of internal organs, high cholesterol, and leaking breast. The usage of the root of yellow gentian (G. lutea) was mentioned by 30 respondents, and that includes improving the immune system, appetite, for the stomach, blood purification, circulation, as an aphrodisiac, against cancer diseases, diabetes, and gastric and duodenal ulcer. It is important to note that yellow gentian, recorded in the study area, is the first species on the list of illegally collected species in Serbia

Polyphenolic Content and Biological Activities of Post-Distillation Waste of Three Sage Species from the Republic of Macedonia

This research was aimed at investigating polyphenolic content, antioxidant and anti-neurodegenerative activities of post-distillation waste extracts of Macedonian Salvia amplexicaulis, S. jurisicii and S. ringens, for the first time. Total phenolic and flavonoid contents varied in a broad range (3.92-146.49 mg GAE/g and 7.11-67.51 mg QE/g, respectively), with the highest values obtained for S. amplexicaulis and S. ringens extracts. Certain S. amplexicaulis and S. ringens extracts neutralized more than 80% of DPPH radicals at the highest concentration, while S. amplexicaulis extracts showed up to ≈50% inhibition of β-carotene oxidation in β-carotene/linoleic acid assay. Post-distillation waste extracts inhibited acetylcholinesterase (25.94-38.15%) and tyrosinase (18.84-59.52%), with the strongest inhibition of S. amplexicaulis extracts. The obtained results suggest that post-distillation waste of tested species, especially of S. amplexicaulis, show antioxidant and anti-neurodegenerative activities and could be considered as potential raw material rich in polyphenols

Cultivation cost-benefit analysis of some important medicinal plants in Serbia

Driven by frequent media misinformation about the level of profitability of growing certain medicinal plants, in this paper we presented a cost-benefit analysis based on twenty years of experience in field production. The observed costs and profits for peppermint, chamomile, lemon balm, marshmallow, valerian and pot marigold are based on the average values of production elements within the current prices of labor, energy and raw materials. Fixed costs in this paper were deliberately neglected and the discussion was based on the assumption of the existence and availability of infrastructure. In the cost analysis, we divided them into four main groups, which had different shares in total costs such as labor 45-79 %, drying 5 – 37 %, material, 9-16 % and machinery use 4-13 %. Regarding the level of profitability of cultivation of the six observed medicinal plants valerian was the most profitable with an estimated profit of over 4000 €/ha. Next best earning plants were lemon balm and marshmallow with about 3500 €/ha, while the income from peppermint and chamomile was more than twice lower and it was around 1500 €/ha. The lowest profit was realized by cultivating marigold (about 600 €/ha) due to the high labor consumption on the flower picking operation. In terms of labor consumption marshmallow, pot marigold and valerian are the most demanding with 365, 285 and 150 working days per hectare, respectively. The general conclusion of this observation of the profitability of growing medicinal plants would be that the producer must be aware of the costs and scope of labor engagement he expects per unit area before embarking on the calculation of production

GC/MS chemical analysis of lavandin (Lavandula x intermedia) hydrolat: successive extraction fractions

Hydrolats are valuable co-products of the essential oil distillation process, whose volatile compounds can be quantified by various methods. In this paper, we have tried to estimate the liquid-liquid extraction cycle number threshold for volatile compounds quantification of lavandin (Lavandula x intermedia) hydrolat. For this purpose, ten consecutive hydrolat extractions with \textit{n}-hexane were analyzed GC/MS with hexadecane (C16) as an internal standard and compared with the lavandin essential oil. The chemical composition of the lavandinhydrolat showed similarity with its essential oil to the great extent, while volatile compounds dissolved in hydrolat exclusively belonged to the class of oxygenated monoterpenes. The total amount of extracted compounds has been estimated to 2174.2 mg/L, where the most dominant compounds in lavandinhydrolat were cis- and trans-furanoidlinalool oxide (676.3 and 634.1 mg/L, respectively), followed by much smaller amounts of linalool, camphor, and 1,8-cineole (167.6, 157.0, and 148.2 mg/L, respectively). Cumulative recoveries of total compounds yield after the third, fifth, and eighth extractions were 88 %, 96 %, and 99 %, respectively. Combined fraction analysis confirmed that in the first 5 cycles more than 95 % of the total yield (from 10 cycles) of extracted volatile compounds can be collected. Based on the results of this study, for volatile compounds quantification in lavandinhydrolat, 5 cycles of \textit{n}-hexane liquid-liquid extraction can be recommended

Microwave-assisted extraction of essential oil from ginger (Zingiber officinale Rosc.)

Comparison between conventional and microwave-assisted hydrodistillation at different power levels has been applied for the extraction of essential oil from Zingiber officinale Rosc. rhizome. In addition to the collected essential oils, as the main products, hydrolats were also collected, as valuable by-products from both extraction techniques. A comparison of two applied techniques was done in terms of extraction time, extraction yield, the chemical composition of essential oils and hydrolats, and environmental impact. Microwave-assisted hydrodistillation achieved a higher extraction yield (1.70, 1.70, and 1.85% for power levels of 180, 360, and 600 W, respectively) compared to hydrodistillation (1.50%). Furthermore, distillation time related to the energy consumption has been reduced from 144 min and 1.44 kWh for conventional hydrodistillation to 37, 32, 27 min and 0.11, 0.19, 0.27 kWh for power levels of 180, 360, and 600 W, respectively. Content of α-zingiberene in essential oil and hydrolat obtained by hydrodistillation was 29.89 and 6.87%, while content of α-zingiberene in essential oils and hydrolats obtained by microwave-assisted hydrodistillation was higher and amounts 34.12, 34.43, and 42.00% and 18.70, 22.60, and 32.92% for power level 180, 360, and 600 W, respectively. Microwave-assisted hydrodistillation has proven to be promising technique for the isolation of ginger’s essential oil regarding improved yield, reduced time and energy requirements, as well CO2 emissions while maintains oil quality

Screening of yarrow (Achillea millefolium Agg.) populations in Serbia for yield components and essential oil composition

Yarrow (Achillea millefolium Agg.) is well-known medicinal plant, with a wide spectrum of applications and it is one of the most frequently used plant drug in Serbia. In this study, we have observed cultivation and essential oil chemical properties of 28 A. millefolium populations collected from Serbian sites. In the second vegetation, the yield of useful part (upper 15 cm) ranged from 925 – 3630 kg/ha, while the yield of essential oil ranged from 0.40 – 0.82%. %. The most dominant compounds in monoterpene fraction were b-pinene (max. 36.3%), sabinene (max. 35.7%), 1,8-cineol (max. 26.6%) and borneol (max. 20.2%), while in the sesquiterpene fraction the most abundant compounds were trans-caryophyllene (max. 18.6%) and lavandulyl acetate (max. 18.1%). Among aromatic compounds, the most abundant was chamazulene (max. 29.1%).  This screening has shown that only 10 populations out of 28 satisfied official quality requirement of 0.02% of chamazulene in the dried drug. Four populations had higher yield than commercial variety ProA, while one of them had even higher level of chamazulene