Air-dried Material Research Articles

Determination of Shikonin and Rosmarinic Acid in Echium vulgare L. and Echium russicum J.F. Gmel. by Capillary Electrophoresis

A simple and rapid capillary zone electrophoresis (CZE) procedure for determination of shikonin and rosmarinic acid in roots of Echium vulgare L. and Echium russicum J.F. Gmel. is described. The separation was achieved in less than 4 min. The analysis was performed in fused silica capillary (i.d. 75 µm, 48.5 cm total length) using 50 mM borate, at pH 9.5 as the run buffer with applied voltage of 30 kV and constant temperature (27 °C). The method ensures good linearity and repeatability with high correlation coefficients (0.994 and 0.998 for shikonin and rosmarinic acid, respectively). The compounds were detected by UV absorption at 218 (shikonin) and 202 nm (rosmarinic acid). The limits of detection (LOD) for shikonin and rosmarinic acid were 0.603 ppm and 0.270 ppm, respectively. The method was used for estimation of both components in Echium plants. We found out that the content of shikonin and rosmarinic acid were 4.4–4.8 mg/kg air-dry material and 20.0–54.2 mg/kg air-dry material, respectively, in the Echium species analyzed.

Phytoecdysteroids from the Aerial Part of Silene colpophylla

The genus Silene L. includes about 700 species according to current data [1] and represents the richest source of ecdysteroids. Greater than 100 species of this genus have now been found to produce ecdysteroids [2]. Greater than 80 of the 476 identified ecdysteroids are synthesized by Silene species [3, 4]. The goal of the present work was to study the chemical composition of ecdysteroids from the aerial part of S. colpophylla Wrigley, which we recommend for the first time as a source of ecdysteroids. Ecdysteroids were isolated from the aerial part of S. colpophylla collected during flowering in 2009–2012. Starting extracts were obtained by exhaustive extraction of ground air-dried raw material by EtOH (70%). Lipophilic substances were removed by hexane. Subsequent multiple extraction by n-BuOH extracted terpenoid and phenolic compounds. The concentrated residual was fractionated successively using CHCl3–EtOH (9:1), EtOH (70%), and H2O. The CHCl3–EtOH fraction of the BuOH extract was separated by repeated chromatography over columns of silica gel. A total of 17 ecdysteroids were isolated from S. colpophylla for the first time. Six of these were identified by various physicochemical analytical methods (UV, HPLC, MS, NMR). HPLC-MS analysis was carried out on an Agilent 1200 LC with a diode-matrix detector, a hybrid quadrupole–timeof-flight micrOTOF-Q mass spectrometer (Bruker), and a Zorbax SB-C18 column (2.1 50 mm) using HCOOH–MeOH 2% eluent (linear gradient AcN content from 10 to 90% over 0–20 min) at flow rate 0.2 mL/min. The mass detector operated in chemical ionization and electrospray modes at atmospheric pressure (APCI pos. and API-ES). Positive and negative ions were scanned in the range m/z 100–3000. The drying gas (N2) flow rate was 4 mL/min at 220°C. HPLC-UV analysis was carried out on an Agilent 1100 LC with a diode-matrix detector and a Zorbax Eclipse XDB C8 chromatography column (4.6 150 mm, 5 m) using MeOH–TFA (0.1%) eluent (gradient from 2 to 100% MeOH) at flow rate 0.8 mL/min. The analytical wavelength for detecting phytoecdysteroids was max 254 nm. The principal compounds in chromatograms of the CHCl3–EtOH fraction had absorption maxima in the range 244– 252 nm and corresponded to ecdysteroids from plants of the genus Silene [2–4], the structural features of which included an , -unsaturated ketone. The following ecdysteroids were isolated for the first time. Compound 1, tR 14.00 min. UV spectrum (MeOH, max, nm): 248. Mass spectrum, m/z 497 [MH] +, 479, 461, 443, 425; 541 [M + HCOO]–, identified as integristerone A; C27H44O8 [2, 3]. Compound 2, tR 16.15 min. UV spectrum (MeOH, max, nm): 246. Mass spectrum, m/z 481 [MH] +, 463, 445, 427, 409; 525 [M + HCOO]–, identified as 20-hydroxyecdysone; C27H44O7 [2, 4]. Compound 3, tR 16.49 min. UV spectrum (MeOH, max, nm): 250. Mass spectrum, m/z 497 [MH] +, 481, 463, 445, 427; 541 [M + HCOO]–, identified as polipodine B; C27H44O8 [2, 3]. Compound 4, tR 19.08 min. UV spectrum (MeOH, max, nm): 248. Mass spectrum, m/z 465 [MH] +, 447, 429, 411; 509 [M + HCOO]–, identified as ecdysone; C27H44O6 [2, 3]. Compound 5, tR 20.03 min. UV spectrum (MeOH, max, nm): 246. Mass spectrum, m/z 465 [MH] +, 447, 429, 411, 393; 509 [M + HCOO]–, identified as 2-deoxy-20-hydroxyecdysone; C27H44O6 [3, 4].

Polysaccharides from Ferula sinkiangensis and Potent Inhibition of Protein Tyrosine Phosphatase 1B

Ferula sinkiangensis is known as a medicinal plant for thousands of years in China. It is a perennial plant species endemic in Xinjiang and is used in traditional Uyghur medicine for a long time 1 . There are many reports on the pharmacological activities of Ferula, which include treating dysentery, counteracting toxins killing parasitic worms, and resolving phlegm. It is also used as a deodorant. Previous investigations of the chemical composition of Ferula have identified essential oils, flavonoids, minerals, and ferulic acid in the extract [2]. Most reports of Ferula have concentrated on the pharmacological aspects of these compounds. Only a few reports have described methods for the extraction of the polysaccharides, and none has described the isolation or identification of these compounds. To the best of our knowledge, there is no report about polysaccharides from roots of Ferula.This study will provide a basis for future research and application of these polysaccharides. The goal of the current work was to establish a method to isolate and quantitate the polysaccharides from roots of Ferula, as well as to determine their monosaccharide compositions, elucidate their structures, and evaluate water-soluble polysaccharides (FSPs), a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B) in vitro. Lipids, pigments, and low-molecular-weight compounds were removed from air-dried raw material by extraction with petroleum ether, CHCl3, and MeOH [3]. The remaining raw material was dried and then extracted with hot water to obtain FSPs. The yield was 9.5%. The total sugar and protein were measured by anthrone-sulfuric acid and Bradford methods [4, 5] with D-glucose and bovine serum albumin (BSA) as standard. The analysis of total sugar and protein contents indicated that FSPs was composed of 45.1 3% polysaccharide and 12.5 3% protein. Furthermore, uronic acid measurement by the m-hydroxydiphenol method [6] with D-galacturonic acid as standard indicated that 28.1 3% of the polysaccharide moiety in FSPs was acidic. Consequently, the preliminary results demonstrated that FSPs was an acid proteoglycan. After removing proteins by the Savage method [7], the crude polysaccharides (FSPs) gave a negative response to the Bradford test, and no absorption was detected at 280 and 260 nm, indicating the absence of protein and nucleic acids. The neutral polysaccharide, eluted with distilled water, was named FSPs-n. The acidic polysaccharides, eluted by the stepwise addition of 0–1.0 M NaCl solutions, was named FSPs-a. The yields of the two fractions were 6.8 and 14.8% in the crude polysaccharides [8, 9]. According to the results of HPLC analysis [10, 11], the carbohydrate portion of FSPs contained ribose, arabinose, glucose, fucose, and galactose in a ratio of 8.9:3.3:2.1:1.5:0.3. FSPs-n was composed of glucose, xylose, arabinose, galactose, and mannose in a ratio of 3.9:4.0:1.8:1.4:0.8, and FSPs-a contained glucose, xylose, mannose, and arabinose in a ratio of 6.5:4.0:1.7:1.0. Furthermore, FSPs and FSPs-a were composed of glucuronic acid and galacturonic acid in a ratio of 1.1:0.3 and 2.3:5.5, respectively.

Antimicrobial Qualities of Senna Alata

The antimicrobial activity of the root and leaf extracts of the Senna alata plant against some infectious bacteria (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa etc) and fungi (spergillus flavus, Aspergillus niger, Candida albicans etc) as well as the physiochemical and microbiological quality of the plant was determined using the cup plate agar diffusion method. The freshly collected fresh mature leaves and roots were chopped into pieces and shade-dried at 32-35 0 C to constant weight for 5 days. 50g each of the plant parts was coarsely powdered using a mortar and pestle and finely powdered using an electric blender. Each of the powdered air-dried plant material was extracted with water, acetone and methanol. All the extracts demonstrated considerable activity against both Gram negative and Gram positive bacteria and some fungi with the organic extracts showing higher activity than the aqueous extracts. Stretococus pyogenes and S. aureus were the most susceptible to all the extracts followed by Salmonella typhi and Escherichia coli. The most susceptible fungi were Cryptococcus neoformans and Candida albicans while the least susceptible was Aspergillus flavus. The minimum inhibitory concentration and minimum bactericidal concentration of the methanol extracts ranged between 3-10 mg/ml and 25-50 mg/ml for bacteria and fungi respectively. Preliminary phytochemical analysis showed that the extracts contained tannins, saponins, glycosides, flavonoids and phenols. The results obtained show the basis for the local usage of S. alata Linn as an antimicrobial. Phytochemical result showed ethanol to be a better solvent for the extraction of the bioactive agents in Senna alata which include: glycosides, alkaloids, saponins, tannins, flavonoids and volatile oil. Despite the significant progress made in microbiology and the control of microorganisms, sporadic incidents of epidemics due to drug resistant microorganisms pose an enormous threat to public health. The use of medicinal plants for antimicrobial activities needs to be given more attention.

Mini Tensiometer-Time Domain Reflectometry Coil Probe for Measuring Soil Water Retention Properties

Time domain reflectometry (TDR) is used widely for measuring soil-water content. New TDR coil probe technology facilitates the development of small, nondestructive probes for simultaneous measurement of soil-water content (θ) and soil-water potential (ψ). In this study we developed mini tensiometer-time domain reflectometry (T-TDR) coil probes, 6-mm wide and 32-mm long. The coil probes were calibrated against a conventional three-rod probe and were used for measuring θ for a aggregated volcanic ash soil (VAS) and a uniform sand. A commonly-used dielectric mixing model did not accurately describe the measured relation between the dielectric constant of the T-TDR coil probe (εcoil) and θ, and a new calibration model for εcoil (θ) was proposed instead. The new model assumes single-region behavior for sand and two-region behavior for aggregated VAS, when plotting the normalized dielectric constant of the coil probe (εcoil–ε dry; where εdry is the dielectric constant of the T-TDR coil probe for air-dried material) as a function of θ. The new calibration model accurately described the (εcoil–ε dry)-θ relations measured by 7 T-TDR coil probes on both sand and VAS. Additionally, there was a good agreement between measured soil-water retention curves (ψ > –100 cm H2O) by the new T-TDR coil probes and independent measurements by the hanging water column method.

Nymphayol and Gerbolide A from Artemisia porrecta

The genus Artemisia L. is one of the largest taxa in the Asteraceae family and is interesting with respect to chemistry because it is the source of many secondary metabolites including terpenoids, flavonoids, and coumarins [1, 2]. We studied the aerial part of A. porrecta Krasch. ex Poljakov growing in South Kazakhstan Province, Republic of Kazakhstan [3]. The aerial part (leaves, flower heads, buds) of the plant (0.450 kg) that was collected during flowering in DombraSyrnai gorge in the vicinity of Karatau State Preserve of South Kazakhstan Province was extracted (3 ) with CHCl3 to afford total extracted substances (45.0 g). The condensed extract was worked up with aqueous EtOH (1:2). The filtrate was worked up with CHCl3 (1:1). The resulting total extracted substances (20.13 g) were chromatographed over a column of KSK silica gel (extract:sorbent ratio 1:25). Elution of the column by petroleum ether:EtOAc (95:5) isolated colorless crystalline compound 1, mp 139–141°C, C25H42O. The yield was 0.037 g (0.009% calculated for air-dried raw material), Rf 0.47 (petroleum ether:EtOAc, 4:2). Based on spectral data (IR, UV, PMR, and 13C NMR), physicochemical constants, and comparison with an authentic sample, 1 was identified as 25,26-dinorcholest-5-en-3 -ol, i.e., the steroid nymphayol, which was isolated previously from Nymphaea stallata Willd. [4]. Elution of the column by petroleum ether:EtOAc (9:1) isolated colorless crystalline compound 2, mp 168–170°C. The yield was 0.068 g (0.015% calculated for air-dried raw material), Rf 0.31 (petroleum ether:EtOAc, 2:3). The mass spectrum of 2 contained a peak for the molecular ion with m/z 292.2 [M]+. According to high-resolution mass spectrometry data, 2 had the formula C17H24O4. According to IR, UV, PMR, 13C NMR, and mass spectral data, 2 was the sesquiterpene lactone 9 -acetoxy6 ,7 ,11 (H)-germacr-1(10),4(5)-dien-12,6-olide (herbolide A), which was isolated previously from A. herba-alba subsp. valentine [5]. Thus, the chemical study of the aerial part of A. porrecta isolated the steroid nymphayol and the germacrane sesquiterpene lactone herbolide A, which were identified from this species for the first time.

Phenolic compounds from Serenoa repens fruit

Fruit of Serenoa repens (W. Bartram) Small (saw palmetto) (Arecaceae) is used in folk and scientific medicine to treat and prevent prostate hyperplasia and non-bacterial prostatitis [1]. Information about the chemical composition of the fruit is limited to data on the lipid complex and the presence in them of flavonoids, despite broad pharmacological studies of S. repens preparations [2]. The goal of the present work was to study the composition of phenolic compounds from S. repens fruit. Fruit of S. repens was supplied by Pacific Botanicals (USA). The species was determined by Cand. Pharm. Sci. G. B. Chekhirova (IGEB, SB, RAS, Ulan-Ude). A sample of the raw material was preserved in the herbarium of IGEB, SB, RAS (No. Asm-37/25-98/0912). Extraction and Fractionation. Fruit of S. repens (400 g) was separated beforehand into pericarp and seeds, the ratio of which was 1.34:1, in order to fractionate the extracted substances. Then, raw material was ground and defatted in a Soxhlet apparatus using hexane. The remaining raw material was dried and extracted with EtOH (96, 70, and 50%; 5 ; 90°C). The EtOH extracts were combined and concentrated in vacuo to an aqueous residue that was subjected to liquid–liquid extraction by CHCl3, EtOAc, and BuOH. This produced fractions of pericarp (FP) and seeds (FS) from CHCl3, FP-1 (0.25% yield of airdried raw material mass) and FS-1 (0.13%); EtOAc, FP-2 (0.43%) and FS-2 (0.18%); BuOH, FP-3 (1.99%) and FS-3 (0.73%); and H2O, FP-4 (9.79%) and FS-4 (2.45%). Fraction FP-1 (520 mg) was separated by column chromatography (CC) over polyamide (2 40 cm, eluent H2O:EtOH, 100:0 4:96) and Sephadex LH-20 (2.5 90 cm, eluent CHCl3:Me2CO, 100:0 70:30) to afford six compounds. These were protocatechoic (4 mg, 1), 4-hydroxybenzoic (2 mg, 2), pyrocatechoic (11 mg, 3), syringic (2 mg, 4), veratric (9 mg, 5) [3], and o-coumaric acids (4 mg, 6) [4]. Separation of fraction FS-1 (220 mg) under analogous conditions produced three compounds. These were 1 (4 mg), 2 (12 mg), and isovanillic acid (9 mg, 7) [3]. The minor components of the fractions were determined by GC-MS as TMS-derivatives (Table 1). As a result, 19 compounds were found in FP-1. These were 1–6; 3-hydroxybenzoic (7); 3-methoxybenzoic (m-anisic, 8); 4-methoxybenzoic (p-anisic, 9); 2,4-dihydroxybenzoic (10); 2,5-dihydroxybenzoic (gentisic, 11); 2,6-dihydroxybenzoic (12); 3,5-dihydroxybenzoic (13); 2-hydroxy-3-methoxybenzoic (14); 3-hydroxy4-methoxybenzoic (isovanillic, 15); 2-hydroxy-5-methoxybenzoic (16); 3-methoxy-4-hydroxybenzoic (vanillic, 17); p-coumaric (18); and ferulic acids (19). Fraction FS-1 yielded 10 compounds (1-5, 7, 11, 12, 15, 17). Distinguishing features of the seed fraction (FS-1) were the lack of phenylpropanoids, the content of which in the pericarp fraction (FP-1) was 2.50%, and the higher content of monohydroxy-substituted benzoic acids (6.86%) and lower content of methoxylated acids. The contents of the dominant components in the fractions (%) were 3 (3.96), 5 (3.27), and 6 (1.37) in FP-1 and 1 (2.74), 2 (6.46), and 15 (5.57) in FS-1.

Chemical composition of Artemisia hippolyti

Artemisia hippolyti A. But. sp. n. is an endemic species that grows on stony peaks in the Betpak-Dala desert and the southern part of Karaganda Province, Republic of Kazakhstan. The species A. hippolyti is similar to A. glabella Kar. et Kir. but differs sharply from the latter by the tomentose downiness of the leaves, the external spathe leaflets, and the thalamus shape. These species under natural conditions grow together but develop asynchronously because A. glabella flowers and fruits somewhat earlier than A. hippolyti [1]. We studied the constituent composition of the essential oil, determined the qualitative and quantitative contents of sesquiterpene lactones of A. hippolyti, and compared these results with those obtained earlier by us for A. glabella because these species were similar botanically. Raw material (flower heads, leaves, buds) of A. hippolyti was collected during flowering in the vicinity of Pystan mountain, Zhana-arkin Region, Karaganda Province. Essential oil was obtained by steam distillation of air-dried aerial part of the raw material in a Clevenger apparatus for 3 h. The qualitative composition and quantitative content of the constituents in essential oil were analyzed by GC-MS on an Agilent 7890/5975C GC with a mass-selective detector using an HP-5MS 5% Phenyl Methyl Silox column (30 m 0.25 mm) with He carrier gas flow rate 1 mL/min and vaporizer temperature 230°C. The GC column was held at 40°C for 5 min with the temperature programmed to 240°C at 5°C/min and then held isothermally for 10 min. Samples were introduced by flow division with sample volume 0.2 L. The mass spectra recording conditions were 70 eV and mass range m/z 10–350. The percent contents of constituents were calculated automatically from the peak areas of the total-ion chromatogram. Constituents were identified by mass spectra and RT using the Wiley GC/MS library. Essential oil of A. hippolyti had a pleasant aroma and was a blue liquid (yield 0.15%) (Table 1). According to previous results [2], essential oil of A. glabella contained quantitatively 1,8-cineol ~12%; linalool, up to 8%; terpineol-4, 6.5; and -terpineol, 5. Therefore, the constituent compositions of essential oils of both species were similar but differed in the quantitative contents of the main constituents with the minor constituents differing both qualitatively and quantitatively. Finely ground aerial part of A. hippolyti was extracted with CHCl 3 (3) for the study of extracted compounds. The obtained extract was evaporated in vacuo in a rotary evaporator. HPLC of the CHCl 3 extract detected the sesquiterpene lactones arglabin, ludartin, isoepoxyestafiatin, hanphyllin, and artefin, the quantitative contents of which were 0.103%, 0.012, 0.13, 0.705, and 0.333, respectively, calculated as air-dried raw material. These sesquiterpene lactones were identified for the first time from this species. Qualitative and quantitative analyses of the extract of A. hippolyti were performed by reversed-phase HPLC on a Hewlett–Packard Agilent 1100 Series instrument in isocratic mode with an analytical column packed with Zorbax CB-C 18 sorbent (9.4 250 mm, 5 m), mobile phase MeCN:H 2 O (50:50), detection at 204 nm, column at room temperature, mobile phase flow rate 1.5 mL/min, and injected sample volume 20 L. The quantitative contents of the samples (%) were determined by comparison with an internal standard. The data were processed using ChemStation software. The sesquiterpene lactones arglabin, ludartin, isoepoxyestafiatin, hanphyllin, and artefin were isolated as a result of the study. According to data previously obtained by us [3], A. glabella contained 0.34, 0.008, and 0.0007% of arglabin, argolide, and ketopelenolide B, respectively. A comparison of A. hippolyti and A. glabella found a difference in the qualitative composition of sesquiterpene lactones. However, arglabin was present in both species although its content in A. hippolyti was significantly less than in A. glabella.

Chemical compounds of esential oils from Xyloselinum vietnamense Pimenov & Kljuykov and Xyloselinum leonidii Pimenov & Kljuykov in Vietnam

Xyloselinum Pimenov & Kljuykov, an edemic genus for Vietnam, consisting of two species, namely Xyloselinum vietnamense Pimenov & Kljuykov and Xyloselinum leonidii Pimenov & Kljuykov. X. vietnamense is a perennial shrub, 1.0-1.5 m tall; leaves are alternate, tripinnate. X. leonidii is also a perennial shrub, 0.8-1.8 m tall; leaves are alternate, tripinnate. Materials for study of X. vietnamense and X. leonidii were collected in Bat Dai Son Nature Reserve, Quan Ba district and Sinh Lung commune, Dong Van district, Ha Giang province. The essential oil yields from leaves and roots of X. vietnamense were different, viz. 0.16% in leaves and 0.6% in roots (by air-dry material). Main compounds of essential oil from leaves of X. vietnamense were dominated by sabinene (75.0%), santalone (5,1%), γ-terpinene (2.5%). Main compounds of essential oil from roots of X. vietnamense were sabinen (36.5%), terpinene-4-ol (10.3%), Z-β-ocimene (9.7%). The essential oil yields from leaves and roots of X. leonidii were 0.15% and 0.7% respectively (by air-dry material). Main compounds of essential oil from leaves of X. leonidii were sabinene (29.3%), β-phellandrene (17.8%), myrcene (12.9%), α-pinene (7.6%). Main compounds of essential oil from roots of X. leonidii were β-pinene (13.7%), Z-β-ocimene (12.9%), sabinene (10,0%) and β-thujene (9.5%).

Content rating of chosen trace elements in plants and soil after application of newspaper waste

The research was conducted to determine the content of Cr, Cd, Ni and Pb in soil and plant biomass after application of newspaper waste. The pot experiment was conducted in the years 2010 and 2011 in the greenhouse and included the following objects: NP-0 soil without addition of newspaper, NP-1 the soil with addition of 1 % of newspaper and NP-3 the soil with addition of 3 % of newspaper. Addition of newspaper was calculated relative to the dry weight of the soil material. The study was conducted in pots containing 5.50 kg of air-dried soil material. Two plants were grown in the experiment: spring rape, ‘Felix’ c.v., in the first year and naked oats, ‘Siwek’ c.v., in the second. Rape was collected at the end of the flowering stage (73-day vegetation period), and oats at grain milk stage (75 days of vegetation). After preparation of plant material and soil, content of trace elements was determined by ICP-OES method on a Perkin Elmer Optima 7300DV apparatus. The results were converted to absolute dry weight of the material. Application of newspaper to soil did not increase (except for cadmium) the contents of lead, chromium or nickel in the aboveground parts of the plant. Studied trace elements were accumulated mainly in the plant roots. Roots contained on average from 51 to 82 % more of the test elements in relation to the aboveground parts. Higher amount of newspaper (3 %) added to the soil led to a higher accumulation of studied trace elements in the roots and lower in the plant aboveground parts. The addition of newspaper to the soil caused a reduction in acidity. Newspaper introduced to the soil generally did not increase the total content of trace elements.

Иммуноферментный анализ усниновой кислоты в лишайниках

An enzyme immunoassay for usnic acid in lichens was developed, the sensitivity of which was 0.1 microg/g of air-dried material (0.00001%). Polyclonal rabbit antibodies against bovine serum albumin conjugated to (+)-usnic acid under the conditions of formaldehyde condensation made it possible to determine the analyzed substance in solutions at concentrations from 1 ng/mL when it interacts with an immobilized gelatin conjugate homologous in the binding mode. Usnic acid in 2-26600 microg/g (0.0002-2.6%) amounts was found in all 236 studied samples of lichens belonging to 53 species and 8 families.

Chemical composition of the essential oil of Acorus macrospadiceus (Yam.) F. N. Wei & Y. K. Li from Vietnam

Acorus macrospadiceus (Yam.) F. N. Wei & Y. K. Li (Acoraceae) is a rare plant which distributes in some provinces of Vietnam such as Phu Tho, Thai Nguyen, Vinh Phuc and Bac Giang. This species is listed as endangered in the Red Data Book of Vietnam (1996, 2007). Up to now, there has been no research on Acorus macrospadiceus in Vietnam. The essential oil yielded were 0.2% from air-dried material. GC/MS analysis of the oil resulted in the identification of 31 constituents. The major constituents found in the oil were cis asaron (45.14%), trans asaron (15.49%), b-caryophyllen (12.08%) and neronidol (6.70%).

Chemical composition of the essential oil of Asarum glabrum Merr. in Vietnam

Asarum glabrum (Aristolochiaceae) distributes in many provinces of Vietnam such as Ha Giang, Lai Chau, Lao Cai, Tuyen Quang, Vinh Phuc and Hanoi. This species is listed as vulnerable in the Red Data Book of Vietnam (1996, 2007) and in the Governmental Decree 32/2006/NĐ/CP. Up to now, there has been no research on Asarum spp. in Vietnam. The essential oil from aerial parts of the plant was obtained by steam distillation and yielded 0.36% by air dry material. This essential oil was analysed by GC/MS and 31 constituents were identified. The major constituents found in the oil were safrol (42.24%), apiole (27.11%) and myristicin (6.13%).

Phenylpropanoids from subterranean organs of Inula helenium

Inula helenium L. is a plant species of the family Asteraceae. Its rhizomes and roots are widely used in medical practice. Glucofructans [1], sesquiterpenes [2], thymol derivatives [3], and dammaradienyl acetate [4] were observed earlier in roots of I. helenium. No information is available on phenolic compounds from roots of I. helenium. Preliminary studies showed that subterranean organs of I. helenium contained phenylpropanoids, the chemical study of which is the focus of the present work. Subterranean organs of I. helenium were collected at the experimental plantation in the vicinity of Goryachinsk (Pribaikal Region, Republic of Buryatiya, 30 Aug., 2009, 52°48 98 N, 108°29 01 E). The species was determined by Dr. T. A. Aseeva (IGEB, SB, RAS). Extraction and Fractionation. Ground raw material (1.5 kg) was extracted (5 ) with EtOH (60%, 1:10, 2 h) on a boiling-water bath. The combined EtOH extracts were concentrated to a watery residue that was extracted with C6H14, CHCl3, EtOAc, and BuOH to produce C6H14 (61.8 g, 4.12% of air-dried raw material mass), CHCl3 (24.2 g, 1.61%), EtOAc (7.8 g, 0.52%), and BuOH fractions (260.9 g, 17.39%) and an aqueous residue (166.2 g, 11.08%). The EtOAc fraction (5 g) was chromatographed over Sephadex LH-20 (3 120 cm) using an EtOH:H2O gradient (96:4 0:100) with subsequent column chromatography (CC) of subfractions over polyamide (5 10, H2O:EtOH eluent, 0:100 96:4) and preparative TLC on cellulose (solvent systems 1 and 2) and SiO2 (solvent system 3). The separation isolated from the EtOAc fraction nine compounds that were identified by MS, UV, IR and 13C NMR spectroscopy as the methyl ester of caffeic acid (1, 12 mg) [5]; caffeic (2, 31 mg) [6]; 3,5-di-O-caffeoylquinic (3, 17 mg); 1,5-di-O-caffeoylquinic (4, 16 mg) [7]; 2,3-di-O-caffeoyltartaric (chicoric, 5, 11 mg) [8]; 2-O-caffeoyltartaric (caftaric, 6, 37 mg) [9]; 5-O-caffeoylquinic (7, 12 mg) [6]; and 3-O-caffeoylquinic acids (8, 46 mg) [10]; and echinacoside (9, 11 mg) [11]. Echinacoside (9), C35H46O20. UV spectrum (MeOH, max, nm): 221, 246 sh, 290 sh, 330. FAB-MS (m/z): 785 [M – H]–. 13C NMR spectrum (125 MHz, DMSO-d6): dihydroxyphenylethanol: 146.27 (C-4), 144.31 (C-3), 134.22 (C-1), 121.24 (C-6), 117.10 (C-5), 116.21 (C-2), 72.00 (C-7), 36.21 (C-8); caffeate: 168.92 (C-9), 149.93 (C-4), 148.21 (C-8), 146.63 (C-3), 127.56 (C-1), 123.24 (C-6), 116.11 (C-2), 115.11 (C-7), 114.69 (C-5); glucose I: 104.15 (C-1), 81.50 (C-3), 74.61 (C-2), 73.60 (C-5), 70.43 (C-4), 69.52 (C-6); glucose II: 104.08 (C-1), 77.80 (C-3), 76.16 (C-5), 74.83 (C-2), 71.39 (C-4), 62.60 (C-6); rhamnose: 102.57 (C-1), 73.63 (C-4), 72.17 (C-2, C-3), 70.27 (C-5), 18.27 (C-6).

Correlation of the chemical composition of essential oils from Origanum vulgare subsp. virens with their in vitro activity against pathogenic yeasts and filamentous fungi

Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens and its essential oil (EO) are widely used in the treatment of respiratory and cutaneous infections in traditional medicine. In order to establish a basis for its traditional use, the antimicrobial activity of the EO of O. vulgare subsp. virens was evaluated against human fungal pathogens. Different oil samples were studied in order to elucidate the intraspecific chemical variability and its impact on the biological activity. Flowering aerial parts of three samples of O. vulgare subsp. virens were collected in different geographical locations and EOs were isolated from air-dried plant material by hydrodistillation. The oils were analysed by GC and GC-MS. Minimal inhibitory and minimal lethal concentrations were measured by broth macrodilution methods for the oils and their main constituents against human pathogenic fungi and the influence of the oils on the filamentation in Candida albicans was assayed. The effect of the oil samples on cell metabolism and cell membrane integrity was studied by flow cytometry. Significant quantitative differences in chemical composition were found between the EO samples and, while the three samples generally displayed potent fungicidal activity, their antifungal potencies varied and appeared to be intensified by increasing carvacrol content. The inhibition of filamentation, on the other hand, may correlate more with γ-terpinene content. The flow cytometry results confirmed the occurrence of damage to the plasma membrane, although not necessarily as a direct effect of the oil on the membrane. The EO of O. vulgare subsp. virens is a broad-spectrum fungicide, thus justifying its potential for use in the treatment of superficial or mucosal fungal infections. The EO shows significant variability in chemical composition between samples, which, in turn, affects its biological activity.

Ion Induced Changes in the Structure of Bordered Pit Membranes

Ion-mediated changes in xylem hydraulic resistance are hypothesized to result from hydrogel like properties of pectins located in the bordered pit membranes separating adjacent xylem vessels. Although the kinetics of the ion-mediated changes in hydraulic resistance are consistent with the swelling/deswelling behavior of pectins, there is no direct evidence of this activity. In this report we use atomic force microscopy (AFM) to investigate structural changes in bordered pit membranes associated with changes in the ionic concentration of the surrounding solution. When submerged in de-ionized water, AFM revealed bordered pit membranes as relatively smooth, soft, and lacking any sharp edges surface, in contrast to pictures from scanning electron microscope (SEM) or AFM performed on air-dry material. Exposure of the bordered pit membranes to 50 mM KCl solution resulted in significant changes in both surface physical properties and elevation features. Specifically, bordered pit membranes became harder and the fiber edges were clearly visible. In addition, the membrane contracted and appeared much rougher due to exposed microfibers. In neither solution was there any evidence of discrete pores through the membrane whose dimensions were altered in response to the ionic composition of the surrounding solution. Instead the variable hydraulic resistance appears to involve changes in the both the permeability and the thickness of the pit membrane.

Towards an Efficient Protocol for the Determination of Triterpenic Acids in Olive Fruit: A Comparative Study of Drying and Extraction Methods

Triterpenic acids, such as maslinic acid and oleanolic acid, are commonly found in olive fruits and have been associated with many health benefits. The drying and extraction methods, as well as the solvents used, are critical factors in the determination of their concentration in plant tissues. Thus, there is an emerging need for standardisation of an efficient extraction protocol that determines triterpenic acid content in olive fruits. To evaluate common extraction methods of triterpenic acids from olive fruits and to determine the effect of the drying method on their content in order to propose an optimum protocol for their quantification. The efficacy of different drying and extraction methods was evaluated through the quantification of maslinic acid and oleanolic acid contents using the reversed-phase HPLC technique. Data showed that ultrasonic assisted extraction with ethanol or a mixture of ethanol:methanol (1:1, v/v) resulted in the recovery of significantly higher amounts of triterpenic acids than other methods used. The drying method also affected the estimated triterpenic acid content; frozen or lyophilised olive fruit material gave higher yields of triterpenic acids compared with air-dried material at both 35°C and 105°C. This study provides a rapid and low-cost extraction method, i.e. ultrasonic assisted extraction with an eco-friendly solvent such as ethanol, from frozen or lyophilised olive fruit for the accurate determination of the triterpenic acid content in olive fruit.

Enzyme immunoassay of the secondary metabolites of micromycetes as components of lichen substances

The composition of low-molecular biologically active metabolites typical of microscopic fungi has been studied in blastemas of fruticose lichens of the genera Cladonia, Cetraria, Evernia, Bryoria, and Usnes. The enzyme immunoassay method showed the presence of sterigmatocystin, emodin, mycophenolic acid, citrinin, alternariol, and diacetoxyscirpenol, which occurred regularly and, in most cases, at a frequency of 55 to 100%. The highest levels of accumulation were 0.001-0.003% for emodin, 0.0002% for alternariol and citrinin, 0.0001% for sterigmatocystin and mycophenolic acid, and 0.00005% of the weight of air-dry material for diacetoxyscirpenol. Other metabolites (cyclopiazonic acid, ergot alkaloids, ochratoxin A, PR toxin, deoxynivalenol, zearalenone, and fumonisins) were detected in these lichens less frequently (sometimes only upon the expansion of the territory of sampling), and their content was no more than 0.00005%. The peculiarities of the component composition and the levels of accumulation of fungal metabolites in lichens of different taxonomic affiliation were discussed.

Microbiological and isotopic geochemical research in the arid steppe lakes and sor solonchaks of Western Transbaikalia

The rates of microbial processes in the sor solonchaks and the silts of the Western Transbaikalean saline lakes were investigated. The rate of deep CO2 fixation in the silt of Lake Beloe was as high as 0.19 mg C dm−3 day−1. The content of cellulose and protein in the sor solonchaks (for air-dry material) were up to 5.0 and 0.42 mg g−1, respectively. The rates of cellulose decomposition and proteolysis in the silt were 1.08 and 0.96 % day−1, respectively. Sulfate reduction (up to 1.82 mg S dm−3 day−1) prevails at the terminal stages of anaerobic decomposition of organic matter in the silt. During the flooding period, methanogenesis producing isotopically light CH4 probably prevails in the sor solonchaks and arid soils of the region. This suggestion is supported by the surface foaming of the local arid soils after abundant monsoon rains, an increase in the δ13C value for soil carbonates to -5.94‰ along the solonchak profile, and a decrease in the ·13C value for the soil carbonates formed by methane-oxidizing bacteria to −11.41‰. The seasonal peaks of isotopically heavier (to −16.60‰) organic matter in the bottom sediments of the lake may be explained as the result of activity of heterotrophic sulfate reducers, which exhibited the highest growth rates at the peak sulfate concentrations in the silt.

Flavonoids from Tanacetum vulgare flowers

Flowers of tansy (Tanacetum vulgare L.) are used in various medicinal forms as antihelminthic and cholegogic agents [1–5]. The pharmacological properties of tansy flower preparations are due mainly to the essential oil (thujone and other terpenoids) and flavonoids. According to various literature sources [3–5], apigenin, acacetin, luteolin, cinaroside, eupatilin, jaceidin, and jaceoside are the main flavonoids in tansy flowers. However, reports in the domestic and foreign literature are somewhat contradictory. The goal of the present work was to study the flavonoid composition of tansy flowers growing in Samara Oblast (Russia). We studied tansy flowers collected near Nizhnee Sancheleevo village, Samara Oblast (July, 2008). Flowers (150 g) were extracted exhaustively with EtOH (70%) by combining maceration (24 h) with subsequent thermal extraction at 85–90°C. The aqueous alcohol extracts were evaporated in vacuo to a thick residue (~50 mL). The condensed extract was dried over L 40/100 silica gel. The resulting powder (extract + silica gel) was placed on a layer of silica gel that was formed in CHCl3 and eluted by CHCl3 and CHCl3:EtOH in various ratios (97:3, 95:5, 93:7, 90:10, 88:12, 85:15, 80:20, 70:30). The separation of the compounds was monitored by TLC on Silufol UV 254 and Sorbfil PTSKh-AF-A-UV plates using CHCl3:EtOH (4:1) and CHCl3:MeOH:H2O (26:14:3). Fractions containing the dominant compound 1 were combined. The precipitate that formed in these was separated and recrystallized from EtOH to afford 1 in 0.2% yield of the air-dried raw material mass. Fractions containing compounds 2–4 (each separately) were transferred to a chromatographic column (sorbent height 4.0 cm; diameter, 5 cm). The respective chromatographic column was eluted by H2O and aqueous EtOH (20%, 40, 70, and 96). Purification over polyamide columns afforded 2 (96% EtOH eluent), 3 (40% EtOH eluent), and 4 (70% EtOH eluent). The compounds were purified additionally by recrystallization from aqueous EtOH. The chemical structures of flavonoids 1–4 were elucidated using PMR and UV spectroscopy and mass spectrometry in addition to chemical transformations. Flavonoids 1 and 3 were cleaved by acid hydrolysis (10% HCl, 100°C, 2 h) and -glucosidase (Fluka, Hungary) into glucose and the aglycons, which were identified by TLC as acacetin (5,7-dihydroxy-4 methoxyflavone) (2) and apigenin (5,7,4 -trihydroxyflavone) (4), respectively. Flavonoids 2 and 4 were also isolated from tansy raw material in the free state. The PMR spectrum of 1 showed two 2H doublets at 8.07 and 7.14 ppm with SSCC (J) 9 Hz that were assigned to H-2 ,6 and H-3 ,5 , respectively; two 1H doublets at 6.84 and 6.45 ppm with J = 2.5 Hz that were characteristic of ring A protons (H-8 and H-6), and a singlet for H-3 at 6.95 (flavone compound). A 3H singlet at 3.87 for the C-4 OCH3 group and a singlet for the 5-OH of the flavonoid were observed in the PMR spectrum. The carbohydrate was placed on the 7-OH group on the basis of these results and those from enzymatic hydrolysis, and UV spectroscopy (lack of a bathochromic shift of the short-wavelength absorption band in the presence of NaOAc) [6]. The glucose was bonded as -D-glucopyranosyl (characteristic doublet for the anomeric proton at 5.35 ppm with J = 7.2 Hz). The chemical studies and spectra data suggested that 1 had the structure 5,7-dihydroxy-4 -methoxyflavone 7-O-Dglucopyranoside (tilianin) [7]. The chemical structure of 3 was studied analogously. Based on UV, NMR, and mass spectral data in addition to results from chemical transformations it was identified as 5,7,4 -trihydroxyflavone 7-O-D-glucopyranoside (cosmosiin).