Preparation of Biologically Transformed Raw Material of Woolly Foxglove (Digitalis lanata Ehrh.) and Isolation of Digoxin Therefrom

A biotechnological approach is proposed for anaerobic conservation of aerial parts of woolly foxglove, followed by air–sun drying of the biologically transformed raw material. During the conservation, primary glycosides of foxglove undergo complete conversion into secondary glycosides, with no further transformations. A simple method is described for preparing an enriched glycoside fraction from the transformed raw material (yield, 3.6%) and for isolating highly purified digoxin from this fraction (yield, 0.06% of the starting raw material); other secondary glycosides can also be isolated.     

Dilute acid hydrolysis of presscakes from silage and grass to recover hemicellulose-derived sugars

Grass and silage presscakes from various types of raw materials were hydrolyzed with dilute acid at moderate conditions to recover hemicellulose-derived sugars. Extracting the material with cold water prior to hydrolysis significantly increased the yield. The poor performance without extraction was due to the high buffer capacity of the material. The best results were obtained with extracted grass and silage from a permanent pasture and extracted clover/grass silage. The highest observed sugar yield was 16.43 g/100 g dry-matter, which represents approximately 25% of the available sugars and 60% of the hemicellulose fraction. Including the soluble sugar oligomers, the yields were even higher (up to 20 g/100 g dry-matter). A statistical experiment design with extracted clover/grass silage was performed to estimate the effects of temperature, time, and dry-matter concentration. Acid and dry-matter concentration had the highest effect on sugar yield, whereas temperature and acid concentration were mainly responsible for forming sugar degradation products. These findings agree with recent kinetic theories. Yields in this experiment were comparable to those of other lignocellulosic materials.     

Seed Maturity and the Effects of Different Drying Conditions on Desiccation Tolerance and Seed Longevity in Foxglove (Digitalis purpurea L.)

The effects of different drying methods on desiccation toleranceand longevity in seeds of foxglove (Digitalis purpurea L.) wereassessed from just prior to mass maturity (when seeds have attainedmaximum dry weight), and at intervals during the post-abscissionphase of development. Tolerance of drying under seed conservationconditions (15% relative humidity, RH, and 15 °C), was acquiredclose to mass maturity at 36 d after flowering (DAF). Increasesin desiccation tolerance were induced when drying was delayedfor 4 d by placing seeds in a near-saturated atmosphere (approx.100% RH), or if seeds were pre-dried for 7 d at either approx.32% or approx. 73% RH. Irrespective of the drying treatment, seed longevity increasedthroughout the sampling period, i.e. beyond the point of massmaturity and throughout the post-abscission phase, up to thepoint of incipient natural dispersal. At each developmentalstage, delayed drying or pre-drying led to an increase in seedlongevity under controlled ageing conditions compared with seedsdried directly under seed conservation conditions. Increasesin longevity were apparent as increases in the estimates forthe intercept of transformed seed survival curves (K_i) and forthe standard deviation of the normal distribution of seed lifespans,and also in the mean time to death of individuals in storage,consistent with a continuation of ripening events. The results are discussed in relation to the assessment of seedlongevity and to current post-harvest drying practices for seedsintended for long-term ex-situ conservation.Copyright 1995,1999 Academic Press Digitalis purpurea L., foxglove, seed development, seed drying, seed longevity     

Development of Desiccation Tolerance and Longevity in Seeds from Detached Capsules of Foxglove (Digitalis purpurea L)

Developing capsules of foxglove (Digitalis purpurea L.) weredetached at 4-d intervals between 12 and 28 d after flowering(DAF) and attached to canes within a natural foxglove standsuch that they were experiencing field conditions identicalto those experienced by normally developing, on-plant capsules.Seeds were subsequently harvested at 4-d intervals until 40total d after flowering (tDAF). Capsule detachment resultedin the cessation of dry matter accumulation; the mean dry weightof seeds from 12, 16, 20, 24, and 28 DAF-detached capsules was21, 32, 51, 60, and 79% respectively, of the mean dry weightof seeds during the post-abscission phase of normal, on-plantdevelopment. Nonetheless, seeds from detached capsules acquiredthe ability to germinate at harvest and tolerance to dryingunder seed conservation conditions (15% relative humidity and15 °C). The capability to withstand storage also arose followingcapsule detachment. Seed longevity increased the longer theperiod of detachment but, in the earlier-detached capsules (12,16, and 20 DAF) longevity subsequently declined. Only seedsfrom later detached capsules (24 and 28 DAF) acquired longevitieswhich were comparable with seeds from on-plant capsules, however,no seeds from detached capsules were as long lived as seedsfrom on-plant capsules harvested at 40 DAF. Digitalis purpurea L.; foxglove; capsule detachment; seed development; desiccation tolerance; longevity     

Extraction of crude polysaccharides from Duchesnea indica (Andrews) Focke: optimization by response surface methodology

A full set of optimization procedure was applied to the extraction of anti-viral polysaccharides from Duchesnea indica (Andrews) Focke. By Plackett–Burman factorial design, three parameters (extraction time, extraction temperature, and ratio of water to raw material) were identified as significant to the extraction yield. However, no significant parameters had been identified for antiviral activity. A three-level-three-factor Box–Behnken factorial design was then employed to further optimize the extraction condition. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also examined using appropriate statistical methods. This led to the construction of a response surface indicating the optimal values for each parameter and response studied. Concerning the extraction yield, an extraction at 98.51?ºC for 6.16 h with a ratio of water to raw material of 30.94 mL/g was found to be optimal. Under the optimized conditions, the experimental yield was 6.430 ± 0.078%, which was well matched with the predicted yield of 6.509%.     

Experimental studies for levulinic acid production from whole kernel grain sorghum.

Levulinic acid has potential as an important basic chemical material. This study proposed a method of making levulinic acid using abundant and low cost whole kernel sorghum grain as the raw material. Flour made from grinding whole kernel sorghum grains was blended with 2%, 5% and 8% aqueous solutions of sulfuric acid. Mixtures were heated to 160 or 200 degrees C in a pressurized reactor. A stepwise heating scheme helped improve the yield of levulinic acid. Levulinic acid yield was determined based on sorghum flour content, as opposed to total sorghum mass. Levulinic acid yield increased as reaction temperature increased. Higher sulfuric acid concentration also significantly increased the levulinic acid yield. However, flour loading had an adverse effect on levulinic acid yield. A maximum yield of 32.6% levulinic acid was achieved at 200 degrees C, 8% sulfuric acid concentration and 10% flour loading. A linear regression model was capable of predicting the levulinic acid yield with respect to effects of reaction temperature, mineral acid concentration and flour loading (R2 = 0.88).     

Search for optimum conditions of wheat straw hemicelluloses cold alkaline extraction process

A method for the selective extraction of hemicellulose from wheat straw involving cold alkaline extraction and subsequent separation by precipitation with ethanol is proposed. Wheat straw affords selective separation of the hemicellulose fraction from the cellulose and lignin fractions with the proposed method. The hemicellulose yield was optimized by using a 2n factor design to examine the influence of temperatures (temperature was designed between 20 and 40 °C), operation times (operation time was designed between 30 and 60 min) and alkali concentrations (alkali concentration was designed between 80 and 120 g L⁻¹). These conditions allowed 56.1% of all hemicellulose initially present in the raw material, and 59.1% of the lignin, to be extracted. Subsequent separation of hemicellulose in the liquid phase from the cold alkaline extraction by precipitation with ethanol provided a fraction containing 39.4% of all hemicellulose (45.2% hemicellulose in extract/total extract) and only 12% of all lignin in the raw material.     

高速逆流色谱法制备灵芝萜烯酮醇

本研究建立一种从灵芝子实体提取物中快速制备灵芝萜烯酮醇的方法.以沪农灵芝1号子实体为原料,经乙醇提取、D101大孔树脂富集后,再经一次正相色谱柱层析,获得富含灵芝萜烯酮醇的流分.采用高速逆流色谱法对该流分进行分离,优化分离条件,获得的最佳条件为:溶剂体系为正己烷-乙酸乙酯-甲醇-水(V/V/V/V,12∶24∶18∶9...     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

Flavonoid glycosides extracted from hop (Humulus lupulus L.) as inhibitors of chemical mediator release from human basophilic KU812 cells

The antiallergic properties of hop water extract (HWE) were studied by evaluating histamine release from human basophilic KU812 cells induced by calcium ionophore A23187. HWE significantly inhibited histamine release, but boiling water extract and chloroform-methanol extract did not show any inhibitory effect on it. A 50% methanol-eluted fraction separated from HWE by XAD-4 column chromatography (MFH) had a strong inhibitory effect as compared with HWE. Quercetin glycosides and kaempherol glycosides were identified in MFH, of which quercetin glycosides contributed to the inhibition of histamine release. Most quercetin in HWE existed in glycoside form and its quercetin content, obtained by acid hydrolysis, was about 200 mug/g. HWE and MFH significantly inhibited protein kinase C, which plays a pivotal role in the degranulation of chemical mediators. These results indicate that HWE can inhibit type-I allergic reactions.     

Flavonoid Glycosides Extracted from Hop (Humulus lupulus L.) as Inhibitors of Chemical Mediator Release from Human Basophilic KU812 Cells

The antiallergic properties of hop water extract (HWE) were studied by evaluating histamine release from human basophilic KU812 cells induced by calcium ionophore A23187. HWE significantly inhibited histamine release, but boiling water extract and chloroform–methanol extract did not show any inhibitory effect on it. A 50% methanol-eluted fraction separated from HWE by XAD-4 column chromatography (MFH) had a strong inhibitory effect as compared with HWE. Quercetin glycosides and kaempherol glycosides were identified in MFH, of which quercetin glycosides contributed to the inhibition of histamine release. Most quercetin in HWE existed in glycoside form and its quercetin content, obtained by acid hydrolysis, was about 200 μg/g. HWE and MFH significantly inhibited protein kinase C, which plays a pivotal role in the degranulation of chemical mediators. These results indicate that HWE can inhibit type-I allergic reactions.     

Enzymatic hydrolysis of cod (Gadus morhua) by-products: Optimization of yield and properties of lipid and protein fractions

The main products of hydrolysis of fish by-products are hydrolysed protein and oil. The aim of this work was to study the effect of initial heat inactivation of endogenous enzymes, addition of water prior to hydrolysis, use of different commercial enzymes and combination of enzymes on the yield and purity of the protein and oil fractions after enzymatic hydrolysis of cod by-products. This study was designed to examine how all these factors were effective for destroying protein–lipid complexes in order to obtain pure oil and protein fractions and reduce the insoluble fraction.

Initial heating of raw material changed both raw material properties and inactivated endogenous enzymes thereby influencing the following hydrolysis. High amount of lipids in raw material combined with initial heating caused formation of protein–lipid complexes which was found in all protein containing fractions. The main constituents of the lipids in the complexes were phospholipids and other polar lipids. Insoluble protein–lipid complexes formed lead to increased amount of sludge, reduced FPH yield and high amount of lipids in FPH. The highest amount of separated oil was obtained in the experiments after initial heating without added water. These treatments also reduce amount of emulsion, which is not a desirable product after hydrolysis. Initial heating caused denaturation of protein, which decreased their emulsifying properties.

Results showed that it is not possible to obtain all desirable quality indicators such as: maximum oil and FPH yield, minimum emulsion and sludge yield and the highest protein recovery in FPH with the lowest amount of lipids in FPH fraction by using only one hydrolysis process. Therefore, the aim and requirements for the final products should be prioritised and defined very clearly before the process is designed taking into account the composition of raw material. Hydrolysis of unheated raw material with Alcalase and addition of water was the best compromise taking into account the mentioned quality indicators.     

Significantly increased fractions of transformed to total alpha2-macroglobulin concentrations in plasma from patients with multiple sclerosis

We examined the proteinase inhibitor alpha2-macroglobulin (alpha2M) in plasma from patients with multiple sclerosis (MS); a neurological disease of the central nervous system. The plasma concentrations of native and transformed alpha2M were measured in 90 patients with clinically definite MS, 73 with relapsing-remitting and 17 with secondary progressive MS, and 132 healthy individuals. Significantly lower concentrations of native alpha2M and significantly higher concentrations of transformed alpha2M were found in MS patients. A significant correlation between the concentrations of native and transformed alpha2M was found. The fraction of transformed to total alpha2M in the MS patients was 36% higher than in the healthy individuals. The results suggest an important involvement of alpha2M in regulation of increased proteolytic activity occurring in MS disease.     

Efficient enzymatic production of rebaudioside A from stevioside

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30?h with 2.4?mM stevioside, 7.2?mM sucrose, and 0.006?mM UDP was 78%.     

Formation of phenolic compounds in the roots of <Emphasis Type="Italic">Hedysarum theinum</Emphasis> cultured in vitro

A steadily growing culture of genetically transformed roots of a valuable medicinal Altaic plant Hedysarum theinum Krasnob. was established using a Ri-plasmid (pRi) T-DNA of A4 wild strain of Agrobacterium rhizogenes. The composition of secondary substances accumulated in the in vitro roots and the roots of H. theinum intact seedlings was investigated. Isoflavonoids were found to represent by their main low-molecular metabolites. Preparative HPLC made it possible to isolate four substances from the methanolic extract of H. theinum cultured roots. Using ¹H- and ¹³C-NMR-spectrometry, these substances were identified as formononetin, ononin (formononetin glycoside), malonyl ononin, and texasin glucoside. The qualitative composition of secondary metabolites of the genetically transformed roots and the roots of H. theinum seedlings was essentially the same, except that malonyl ononin was not found in the latter. The technique of producing artificial seeds on the basis of H. theinum roots cultured in vitro was tested, and the possibility of their use as a rhizogenic inoculum was substantiated. The culture of H. theinum roots is considered as a potential source of ecologically pure raw material for medicinal preparations, and the artificial seeds with root inoculum are a promising vehicle for propagation and conservation of this valuable plant.     

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non‐chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30–40‐fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re‐dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans‐membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by‐product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra‐/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642–653, 2017     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Formation of 6 beta-hydroxydexamethasone from dexamethasone by A6 cells.

A6 cells, a continuous cell line derived from kidney of Xenopus laevis, were incubated with [3H]-dexamethasone for 24 h. When radioactive compounds in media were separated by reversed phase high pressure liquid chromatography, two radioactive fractions were found. The less polar fraction which contained 91-93% of total radioactivity cochromatographed with dexamethasone, whereas the polar fraction contained 5% of total radioactivity in media. In order to rigorously identify the polar metabolite, large scale cultures were carried out and the polar compound was separated and purified by reversed phase high pressure liquid chromatography. The purified material was analyzed by secondary ion mass spectrometry and nuclear magnetic resonance spectroscopy. By these procedures, this material was identified as 6 beta-hydroxydexamethasone. To our knowledge these are the first data indicating that dexamethasone can be metabolized by transporting epithelia such as A6 cells.     

D-氨基酰化酶拆分D,L-苯丙氨酸制备D-苯丙氨酸

进行了以D,L-苯丙氨酸为原料经D-氨基酰化酶制备D-苯丙氨酸的研究。乙酰-D,L-苯丙氨酸浓度为0.5mol.L-1,给酶量为3×104U.L-1时,24 h拆分率可达到97%。采用阳离子交换树脂进行了拆分液中的D-苯丙氨酸的分离,D-苯丙氨酸的收率为95.4%。采用醋酸酐作为催化剂,在145℃的条件下,乙酰-L-苯丙氨酸可以消旋成乙酰-D,L-苯丙氨酸继续拆分。