Rapid Detection of Antioxidant Flavonoids in Azalea (Rhododendron mucronulatum) Flowers using On-line HPLC-ABTS+ System and Preparative Isolation of Three Flavonoids by Centrifugal Partition Chromatography

High-performance liquid chromatography coupled to an on-line ABTS⁺-based assay (on-line HPLC-ABTS⁺) system was used to determine the principle antioxidants in azalea flowers. Three flavonoids, myricetin, quercetin, and kaempferol, recovered in ethyl acetate extracts of azalea flowers were determined to have antioxidant activities. These three flavonoids were isolated and purified by successive centrifugal partition chromatography (CPC) using two different biphasic solvent systems, consisting of n-hexane-ethyl acetate-methanol-water (3:5:3:5 or 4:5:4:5, v/v). A total of 46.2 mg of myricetin, 28.9 mg of quercetin, and 10.6 mg of kaempferol with purities of 97.0%, 95.4%, and 93.9%, respectively, were purified from 500 mg of ethyl acetate soluble material from azalea flowers. The structures were identified by their retention time, UV spectra, and ESI-MS in the positive ion mode and were confirmed by NMR experiments.     

Copaifera langsdorffii Bark as a Source of Chemicals: Structural and Chemical Characterization

The chemical composition and the anatomy of Copaifera langsdorffii bark are reported here for the first time by studying trees grown in a native forest area in the Amazon region, Brazil. The bark is thin, dark reddish brown, and exfoliates in irregular flakes. It is very dense, showing highly lignified cells and abundant sclereids, and cellular fillings of phenolic nature. It includes a poorly developed rhytidome and a periderm with thin- and thick-walled phellem cells. The mean chemical composition was: ash 3.7%, total extractives 21.3%, mainly corresponding to polar compounds soluble in ethanol and water, suberin 0.8%, and lignin 36.6%. The polysaccharides showed a predominance of glucose and xylose (66.4% and 23.5% of total monosaccharides, respectively). The ethanol-water bark extract had a high content in phenolics: total phenolics 589.2 mg gallic acid/g extract, flavonoids 441.9 mg catechin/g extract, and tannins 54.8 mg catechin/g extract. The antioxidant activity was high, comparable to known antioxidant reference compounds: 720.3 mg Trolox per g of extract or 92.1 mg Trolox per g of bark. After bark grinding, the finest fraction was enriched in polar extractives (40.6%). C. langsdorffii bark is a potential source of functional extractives, therefore representing a valorization of the residual bark obtained during the industrial tree processing for timber.     

Lipophilic Extractives in Populus × euramericana “Guariento” Stemwood and Bark

Abstract

The lipophilic extractives in stemwood and bark from three different heights of Populus × euramericana “Guariento” were analyzed. The bark samples, especially from 4 and 8 meters height, contained much more extractives than the stemwood samples. The lipophilic extractives identified by Gas Chromatography–Mass Spectroscopy (GC-MS) were composed of five component groups (i.e. triglycerides, steryl esters, free fatty acids, sterols, and free fatty alcohols both in the stemwood and bark). Besides ferulic acid esters, α-amyrin and its esters, 4-hydroxycinnamic acid esters of fatty alcohols were also identified in the stemwood and bark. Small amounts of alkanes and oligomeric or polymeric material with higher molar mass than triglycerides were present only in the bark. Glycerides, mainly triglycerides, were the largest component group of the lipophilic extractives. The high proportion of short-chain fatty acids released after alkaline hydrolysis are beneficial when removing pitch particles or fatty acid soaps by dispersing and washing during pulping and papermaking.     

An Efficient Strategy Based on Macroporous Resins and Semi-Preparative High Performance Liquid Chromatography for Rapid Separation of Five Flavonoids Components From Flaveria Bidentis(L.) Kuntze

In this study, a simple, rapid, and efficient purification method of five major flavonoids from crude Flaveria bidentis extracts was established. Five flavonoids components were initially obtained by ethanol-water extraction of Flaveria bidentis (L.) Kuntze, followed by using D4020 resin-based column chromatography and semi-preparative high performance liquid chromatography. 9.3 mg hyperoside, 6.5 mg patuletin-3-O-glucoside, 1.7 mg isorhamnetin 3-sulphate, 8.4 mg astragalin, and 4.9 mg 6-methoxykaempferol-3-O-galactoside at high purity of over 94% were obtained from 580 mg Flaveria bidentis extracts. This method is more efficient than HSCCC comparing with the results of these two methods regarding analysts’ purity, recovery, and running time.     

Ethyl m-Digallate from Red Maple, Acer rubrum L., as the Major Resistance Factor to Forest Tent Caterpillar, Malacosoma disstria Hbn.

An ethanolic extract of red maple (Acer rubrum L.) leaves (RME) applied to trembling aspen (Populus tremuloides Michx.) leaves reduced feeding in choice test assays with forest tent caterpillar larvae (Malacosoma disstria Hbn.) (FTC), whereas a trembling aspen foliage extract, similarly applied, stimulated feeding. Compounds isolated from the RME were gallic acid, methyl gallate, ethyl gallate, m-digallate, ethyl m-digallate, 1-O-galloyl--D-glucose, 1-O-galloyl--L-rhamnose, kaempferol 3-O--D-glucoside, kaempferol 3-O--D-galactoside, kaempferol 3-O--L-rhamnoside, kaempferol-3-O-rhamnoglucoside, quercetin 3-O--D-glucoside, quercetin 3-O--L-rhamnoside and quercetin 3-O-rhamnoglucoside, (–)-epicatechin, (+)-catechin and ellagic acid. All of the gallates, (–)-epicatechin, and kaempferol 3-O--L-rhamnoside deterred feeding on trembling aspen leaf disks when applied at 0.28 mg/cm². The two digallates deterred feeding by 90% and were the most effective. HPLC analysis indicated that ethyl m-digallate is present in amounts 10–100 × higher in RME (2.5–250 mg/g) than any other compound. Thus, ethyl m-digallate appears to be the major compound protecting red maple from feeding by FTC, with a minor contribution from other gallates.     

Unexploited Thapsia garganica,Orlaya maritima,and Retama raetam Seeds: Potential Sources of Unsaturated Fatty Acid and Natural Antioxidants

Total lipid contents, fatty acid compositions, phenolic profiles and antioxidants activities of seeds from Thapsia garganica, Orlaya maritima, and Retama raetam were investigated. The oil values were more than 26 %, except seeds of R. raetam (ca. 3 %). Unsaturated fatty acids accounted for the majority of the fatty acids (more than 75 %). Oleic and linoleic acid were the predominant fatty acids. Total phenolic compounds (24–104 mg GAE g^?1 DR), total flavonoids (4–102 mg QE g^?1g DR), total tannins (28–85 mg GAE g^?1 DR) and condensed tannins (0.62–131 mg CE g^?1 DR) were also determined. The antioxidant activities using different assays were evaluated. The predominant detected classes were the phenolic acids (42–85 %) and the flavonoids (11–48 %). The major phenolic acids were caffeic, trans‐4‐hydroxy‐3‐methoxycinnamic, p‐coumaric, and gallic acid. The predominant flavonoids were quercetin, luteolin, naringin, apigenin, and kaempferol. This study brings attention to the medicinal importance of these species as a source of oil and antioxidant molecules.     

Response Surface Optimized Ultrasonic-Assisted Extraction of Quercetin and Isolation of Phenolic Compounds From Hypericum perforatum L. by Column Chromatography

The effects of ultrasonic-assisted extraction factors for the main phenolic compound (quercetin) from Hypericum perforatum L. were optimized using the Box–Behnken design (BBD) combined with response surface methodology. The BBD was employed to evaluate the effects of extraction temperature (30–70°C), extraction time (20–80 min), methanol concentration (20–80%, v/v), and HCl concentration (0.8–2.0 M) on the content of one of the major phenolic compounds of quercetin. The extracts were analyzed by high performance liquid chromatography (HPLC). The major phenolic compounds of H. perforatum were isolated and the antioxidant capacity and total phenol content were determined in crude extract and fractions. The optimum conditions were determined as extraction temperature 67°C, extraction time 67 min, methanol concentration 77% (v/v), and HCl concentration 1.2 M. The predicted content of quercetin was 10.81 mg/g dried plant under the optimal conditions and the subsequent verification experiment with 11.09 mg/g dried plant confirmed the validity of the predicted model. The isolated compounds were identified as quercetin, cyanidin, protocatechuic acid, and kaempferol.     

Wood Fiber Quality and Kraft Pulping Efficiencies of Trembling Aspen (Populus tremuloides Michx) Clones

Abstract

The natural variation in wood and pulp fiber quality of 15 aspen (Populus tremuloides Michx) clones, represented by 47 trees, was assessed from 4 different sites in British Columbia, Canada. Kraft pulping trials revealed substantial variation in the pulping efficiencies, illustrated by differences of 6% in total pulp yield, ～30% differences in H‐factor required to attain a target kappa of 21, and differences of up to 2 ISO brightness units in bleachability of kappa 21 pulp. Clearly, enormous variation exists in the natural stands of aspen, and presents some exciting opportunities for selecting clonal aspen for targeted end‐product applications. A comprehensive characterization of wood chemical composition, wood density, and fiber properties indicated that pulp yield is directly related to syringyl lignin monomer composition, and not inherent wood density, regardless of geographic locations, whereas pulp bleachability and viscosity appear to be associated with the inherent cell wall thickness of the starting wood resources (fiber coarseness). These findings suggest that geographic location imparts influences on wood fiber coarseness traits, while substantial genetic variability exists on all sites.     

Flavonoids released by carrot (Daucus carota) seedlings stimulate hyphal development of vesicular-arbuscular mycorrhizal fungi in the presence of optimal CO2 enrichment

Carbon dioxide has been previously identified as a critical volatile factor that stimulates hyphal growth ofGigaspora margarita, a vesiculararbuscular mycorrhizal fungus, and we determined the optimal concentration at 2.0%. The beneficial effect of CO₂ on fungal development is also visible in the presence of stimulatory (quercetin, myricetin) or inhibitory (naringenin) flavonoids. Sterile root exudates from carrot seedlings stimulate the hyphal development ofG. margarita in the presence of optimal CO₂ enrichment. Three flavonols (quercetin, kaempferol, rutin or quercetin 3-rutinoside) and two flavones (apigenin, luteolin) were identified in carrot root exudates by means of HPLC retention time. Flavonols like quercetin and kaempferol are known to have stimulatory effects on hyphal growth ofG. margarita.     

Plant Community Chemical Composition Influences Trembling Aspen (<Emphasis Type="Italic">Populus tremuloides</Emphasis>) Intake by Sheep

Nutrients and plant secondary compounds in aspen (Populus tremuloides) may interact with nutrients in the surrounding vegetation to influence aspen use by herbivores. Thus, this study aimed to determine aspen intake and preference by sheep in response to supplementary nutrients or plant secondary compounds (PSC) present in aspen trees. Thirty-two lambs were randomly assigned to one of four molasses-based supplementary feeds to a basal diet of tall fescue hay (N = 8) during three experiments. The supplements were as follows: (1) high-protein (60% canola meal), (2) a PSC (6% quebracho tannins), (3) 25% aspen bark, and (4) control (100% molasses). Supplements were fed from 0700 to 0900, then lambs were fed fresh aspen leaves collected from stands containing high (Experiment 1, 2) or low (Experiment 3) concentrations of phenolic glycosides (PG). In Experiment 2, lambs were simultaneously offered aspen, a forb (Lathyrus pauciflorus), and a grass (Bromus inermis) collected from the aspen understory. Animals supplemented with high protein or tannins showed greater intake of aspen leaves than animals supplemented with bark or the control diet (P < 0.05), likely because some condensed tannins have a positive effect on protein nutrition and protein aids in PSC detoxification. Overall, animals supplemented with bark showed the lowest aspen intake, suggesting PSC in bark and aspen leaves had additive inhibitory effects on intake. In summary, these results suggest that not only the concentration but also the types and proportions of nutrients and chemical defenses available in the plant community influence aspen use by herbivores.     

Quantitative determination of conjugated linoleic acid isomers in beef fat

The amounts of 14 conjugated linoleic acid (CLA) isomers (t12t14, t11t13, t10t12, t9t11, t8t10, t7t9, t6t8; 12,14 c/t, t11c13, c11t13, t10c12, 9,11 c/t, t8c10, t7c9‐18:2) in 20 beef samples were determined by triple‐column silver‐ion high‐performance liquid chromatography (Ag⁺‐HPLC). Quantitation was performed using an external CLA reference standard consisting of cis9,trans11‐18:2,trans9,trans11‐18:2 and cis9,cis11‐18: 2. Linearity was checked as being r > 0.9999 between 0.02 × 10^‐3 to 2 mg/ml. The determination limit (5‐fold signal/noise ratio) of the CLA reference was estimated to be 0.25, 0.50, 1.0 ng/injection for the cis/trans, trans,trans and cis,cis isomers, respectively. As expected, cis9,trans11‐18:2 was the predominant isomer (1.95 ± 0.54 mg/g fat) in beef, followed by trans7,cis9‐18:2 (0.19 ± 0.04 mg/g fat); cis,cis isomers were below the determination limit in most beef samples. Total CLA amounts determined by Ag⁺‐HPLC were compared to total CLAs determined by gas chromatography (GC, 100 m CPSil^TM 88 column). The amounts obtained by GC were generally higher than those determined by Ag⁺ ‐HPLC due to co‐eluting compounds.     

QUANTIFICATION OF GALLIC ACID AND ELLAGIC ACID FROM THE SEED OF CORNUS OFFICINALIS BY UHPLC METHOD AND THEIR ANTIOXIDANT ACTIVITY

Gallic acid and ellagic acid have been identified in the seed of Cornus officinalis by the use of an ultrahigh performance liquid chromatography (UHPLC) method coupled with a diode array detector (DAD). The water extract of C. officinalis seed contained the highest gallic acid content (3.03 ± 0.10 mg/g seed), which was followed by aqueous methanol extract (2.43 ± 0.10 mg/g seed) and aqueous ethanol extract (1.53 ± 0.25 mg/g seed). But a higher concentration (12.32 ± 0.45 mg/g seed) of ellagic acid was obtained from extraction with aqueous methanol than with aqueous ethanol (11.03 ± 0.42 mg/g seed) and water (7.28 ± 0.16 mg/g seed). After heat treatment and acid hydrolysis, C. officinalis seed had higher concentrations of gallic acid and ellagic acid, contributing to more potent antioxidant activity. The results demonstrated a rich source of gallic acid and ellagic acid in C. officinalis seed, which might provide a novel source of these natural antioxidants.     

Red maple (Acer rubrum) inhibits feeding by beaver (Castor canadensis)

At many beaver (Castor canadensis) sites at Allegany State Park in New York State, red maple (Acer rubrum) is the only or one of the few tree species left standing at the ponds' edges. The relative palatability of red maple (RM) was studied in three ways. (1) At seven beaver sites, the available and utilized trees were recorded and an electivity index (E) computed. Of 15 tree species, RM ranked second or fourth lowest. (2) In experiment I, RM, sugar maple (A. saccharum, SM), and quaking aspen (Populus tremuloides) logs were presented cafeteria style at 10 colonies. RM was the least preferred. (3) Bark of RM was extracted with solvents. Aspen logs were painted (experiment II) or soaked (experiment III) with this RM extract and presented to beaver cafeteria-style, along with aspen and RM controls. This treatment rendered aspen logs less palatable, indicating that a chemical factor had been transferred.     

Extraction Kinetics and Anethole Content of Fennel (Foeniculum vulgare) and Anise Seed (Pimpinella anisum) Extracts Obtained by Soxhlet,Ultrasound, Percolation,Centrifugation, and Steam Distillation

Foeniculum vulgare (fennel) and Pimpinella anisum (anise) extracts were obtained by Soxhlet, cold percolation, ultrasound assisted extraction, and centrifugal extraction using ethanol as solvent; anise extracts were also obtained by steam distillation. Soxhlet presented the highest yields for both fennel and anise seed (16.8% and 23.3%, respectively). The highest anethole content among ethanolic extracts was obtained for centrifugal extraction (6.8 mg/g and 143 mg/g for fennel and anise extracts, respectively). Steam distillation presented low yield (0.26%), but high anethole content (68–98%, area). Anise ethanolic extracts also tested positive for flavonoids.     

Coniferyl benzoate in quaking aspen A ruffed grouse feeding deterrent

Quaking aspen (Populus tremuloides Michx.) staminate flower buds and catkins are important food resources for ruffed grouse (Bonasa umbellus); however, ruffed grouse select only certain quaking aspen to feed upon. Earlier studies indicate that the primary difference between quaking aspen that ruffed grouse feed upon and those not used is the level of coniferyl benzoate in the flower buds. Bioassays show that coniferyl benzoate is a feeding deterrent for ruffed grouse; its effect on ruffed grouse after ingestion has not been tested. Possible physiological effects, based on the chemical properties of coniferyl benzoate and its oxidation products, include inhibition of protein digestion, toxic effects, and antiestrogenic effects.Minnesota Agricultural Experiment Station Journal Series No. 17,079.     

3D Numerical Simulation of Thawing Frozen Wood Using Microwave Energy: Frequency Effect on the Applicability of the Beer–Lambert Law

In this article, the frequency effect on the applicability of Beer–Lambert's law for thawing frozen wood using the microwave energy was analyzed. To this end, we use Maxwell's equations to determine the absorbed power and characterize the critical slab thickness L _crit of three Canadian eastern wood species: trembling aspen (Populus tremuloides Michx), yellow birch (Betula alleghaniensis), and sugar maple (Acer saccharum). The critical thickness L _crit above which the Beer–Lambert law is valid is estimated as a hyperbolic function in the frequency domain: L _crit = m/f ⁿ (f is the frequency of microwave radiation; m and n are adjustment constants). The nonlinear heat conduction problem involving phase changes such as wood freezing is solved by a three-dimensional volumetric specific enthalpy-based finite element method. The dielectric and thermophysical properties are functions of temperature and moisture content. The specific volumetric enthalpy approach is validated by experimental testing. For instance, we studied the frequency effect on the thawing of frozen trembling aspen wood.     

Determination of tocopherols and phytosterols in sunflower seeds by NIR spectrometry

The objective of this work was to develop a near‐infrared reflectance spectrometry (NIRS) calibration estimating the tocopherol and phytosterol contents in sunflower seeds. Approximately 1000 samples of grinded sunflower kernels were scanned by NIRS at 2‐nm intervals from 400 to 2500 nm. For each sample, standard measurements of tocopherol and phytosterol contents were performed. The total tocopherol content was obtained by high‐performance liquid chromatography coupled with a fluorescence detector, while the total phytosterol content was assessed by gas chromatography. For tocopherol, the calibration data set ranged from 175 to 1005 mg/kg oil (mean value around 510 ± 140 mg/kg oil), whereas for the phytosterol content, the calibration data set ranged from 180 to 470 mg/100 g oil (mean value of 320 ± 50 mg/100 g oil). The NIRS calibration showed a relatively good correlation (R² = 0.64) between predicted by NIRS and real values for the total tocopherol content but a poor correlation for the total phytosterol content (R² = 0.27). These results indicate that NIRS could be useful to classify samples with high and low tocopherol content. In contrast, the estimation of phytosterol contents by NIRS needs further investigation. Moreover, in this study, calibration was obtained by a modified partial least‐squares method; the use of other mathematical treatments can be suitable, particularly for total phytosterol content estimation.     

Optimization of Enzymatic Synthesis of n-Propyl Acetate (Fruit Flavor Ester) – Effect of the Support on the Properties of Biocatalysts

The present study deals with the optimization of the enzymatic synthesis of n-propyl acetate using response surface methodology (RSM). The biocatalysts were prepared by physical adsorption of lipase from Thermomyces lanuginosus (TLL) on mesoporous hydrophobic supports – poly-hydroxybutyrate (PHB) and poly-methacrylate (PMA) particles. Their catalytic properties were also assayed in the hydrolysis of olive oil emulsion. The biocatalyst TLL–PMA presented the highest immobilization yield (IY, 90.6 ± 2.8%), immobilized protein concentration (36.5 ± 0.4 mg/g of PMA), and catalytic activity in esterification reaction. TLL–PHB was more active in the hydrolysis of olive oil emulsion by a four-fold factor (1240.5 ± 29.2 IU/g of PHB). Maximum acid consumption percentage of 84.3% was observed after 50 min of reaction catalyzed by TLL–PMA using 2000 mM of each reactant (n-propanol and acetic acid) in heptane medium. The purified ester was confirmed by gas chromatography mass spectrometry (GC–MS) analysis. After six consecutive cycles of esterification reaction, the biocatalyst retained around 50% of its initial activity. The results show that PMA may be an interesting support to prepare active biocatalysts in the synthesis of fruit flavor ester by esterification reaction.     

Separation and Purification of α-Cyperone from Cyperus rotundus with Supercritical Fluid Extraction and High-Speed Counter-Current Chromatography

Abstract

α-Cyperone was separated and purified for the first time from essential oil of the traditional Chinese medicinal plant Cyperus rotundus L. by high-speed counter-current chromatography (HSCCC). Essential oil was obtained by extraction with supercritical carbon dioxide under the pressure of 20 MPa and temperature of 40°C. The separation was performed in one step with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.2:1.1:0.2, v/v), in which the lower phase was used as the mobile phase at a flow-rate of 2.0 ml/min in the head-to-tail elution mode. A total of 60 mg α-cyperone at 98.8% purity was yielded from 0.9 g essential oil as determined by HPLC analysis. The structure of the target compound was performed by electron impact ionization mass spectrometry (EI-MS) and further conformed by comparison with an authentic sample (National Institute of the Control of Pharmaceutical and Biological Products, Beijing, China).     

Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain

Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3′,4′-dihydroxylated leucocyanidin. 3′-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3′H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3′H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.