Fractionation of plant extracts by thin-layer electrophoretic and chromatographic procedures

Methods are described for separating plant tissue extracts into amino acid, organic acid, and neutral fractions by means of thin-layer electrophoresis. The electrophoresis also provides the first-dimensional oeparation for both amino acid and organic acid fractions. Separation of amino acids and organic acids in the second dimension, and of sugars in both dimensions, is achieved by thin-layer chromatography. Suitable procedures for including the study of phospholipids and phosphate esters are suggested. Extracts from 20 mg tissue can be studied easily in this way.     

Fractionation of gibberellins in plant extracts by reverse phase high performance liquid chromatography

In studies on endogenous plant gibberellins (GAs), reverse phase (Bondapak C₁₈) high performance liquid chromatography (HPLC) has proved to be a useful method for the fractionation of plant extracts. The behavior of 18 authentic GAs in such a chromatographic system is described. The main factors determining chromatographic behavior are the degree and the position of hydroxylation of the GA. Generally, dihydroxylated GAs elute before monohydroxylated GAs, whereas 13-hydroxylated GAs elute before 3-hydroxylated GAs. The number of carboxyl groups and the degree of saturation of the A-ring have little effect. For 20-carbon GAs, the oxidation state at C-20 is only relevant insofar as GAs having a methyl group at this position elute later than those with other groups (lactone, aldehyde, or carboxyl).     

天然植物提取物对茄子黄萎病菌的抑制活性

以茄子黄萎菌为供试菌种,研究了54种天然植物的乙醇提取液对茄黄萎菌的抑菌活性. 结果表明, 有15种植物提取物对茄子黄萎菌菌丝生长抑制率大于50%,其中细辛、黄连、厚朴、儿茶、苦参、甘草、蛇床子、桔梗、洋葱的提取物对供试菌菌丝生长抑制作用较强,抑制效果均在65%以上,蛇床子的抑菌率达86.84%.有16种植物提取物对病菌孢子萌发的抑制作用大于70%,其中7种植物提取物对孢子萌发的抑制率大于95%以上,细辛、黄连、厚朴基本上抑制了孢子的萌发.儿茶提取物对菌丝生长的抑制效果较差,但却显著抑制了孢子萌发,抑制率达100%.蛇床子提取物虽然抑菌效果较高,但其处理的孢子萌发率为11.3%.研究发现天然植物提取物中存在着抑制黄萎菌的活性物质,这为生物防治黄萎病和进一步研究蔬菜抗病增产理论提供了新的思路与途径.     

Direct screening of natural product extracts using mass spectrometry

The authors describe first a proof-of-concept experiment to show direct affinity screening using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) is a rapid and informative approach for natural product extract screening. The study used 10 alkaloid-enriched plant extracts and 8 desalted marine extracts spiked with specific inhibitors of bovine carbonic anhydrase II (bCAII; EC4.2.1.1) as a model set. The spiked extracts were incubated with bCAII and then analyzed by ESI-FTICR-MS. The noncovalent complexes were detected, and the specific inhibitors were reidentified in the spiked natural product extracts. There was no interference from the desalted/alkaloid-enriched extracts to the formation of the noncovalent complexes. The method allowed quick identification of the molecular mass of the bound ligand. The authors then applied the screening to identify active compounds in natural product extracts. They employed direct infusion and online size exclusion chromatography (SEC) ESI-FTICR-MS to detect intact target-ligand complex. Eighty-five methanolic plant extracts were screened against bCAII by direct infusion ESI-FTICR-MS and by online SEC-ESI-FTICR-MS. One noncovalent complex was identified from the same plant extract by both methods. The molecular weight of the bound ligand from this extract was determined. Mass-directed purification gave 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one (1) as the active compound. Subsequently, the binding to bCAII was confirmed by ESI-FTICR-MS. The binding specificity was determined by competition experiments between 1 and furosemide, a specific ligand of bCAII.     

Antioxidative activities of aroma extracts isolated from natural plants

Natural leaves and flowers containing numerous aroma chemicals are widely used in aromatherapy since ancient times. In addition to their pleasant smells, aroma chemicals might have some beneficial health effects. Aroma extracts, isolated from coffee beans, soybeans, and mung beans by steam distillation under mild conditions (55 degrees C and 85 mm Hg) were examined for their antioxidative activities. The inhibitory effect of these extracts toward hexanal/hexanoic acid conversion was measured in the testing solution over prolonged time periods. The inhibitory effects of these extracts toward malonaldehyde formation from lipids oxidized by Fenton's reagent were also measured. The antioxidative activity of these extracts, in particular coffee bean extract, was consistent with that of BHT or alpha-tocopherol (vitamin E). Soybeans and mung beans extract contained maltol, which inhibits hexanal oxidation significantly. Eugenol, which is one of the major constituents of mung bean extract, exhibited potent antioxidative activity in an aldehyde/carboxylic acid assay. Antioxidants such as eugenol and maltol may play an important role in the pharmaceutical activities of natural plant extracts used for aromatherapy.     

Anti-oxidation activity of ethanol extracts from natural thalli of lichens

Screening test on anti-oxidation activity using 1,1-diphenyl-2-picrylhydrazyl(DPPH) was performed for 99 ethanol extracts of 85 species of natural thalli of lichens in order to find novel anti-oxidation compounds.The 17 extracts of natural thalli showed high anti-oxidation activity.Among them,the activities of extracts from Hypogymnia vittata,Peltigera aphthosa,Nephromopsis ornata,Pseudevernia furfuracea,Cladonia vulcani and Peltigera elizabethae were higher.Extracts of Peltigera spp.showed higher activity than those of other genera.The ethanol extract of P.aphthosa had been separated into ethyl acetate-soluble and water-soluble fractions.Two anti-oxidative spots were found only in the water-soluble fractions by thin-layer chromatography.The compound in the lower spot had the same Rf value,UV spectrum,and color as authentic solorinine that was previously found as a unique quaternary ammonium compound from Peltigera spp.We now report that the hydrophilic lichen substance,solorinine showed a nearly same anti-oxidation activity(EC50=120?mol/Lol/L) as standard antioxidant Trolox(EC50=150?mol/L).     

Action of plant extracts on the natural immunity indices of animals

The influence of extracts from oak bark, St. John's-wort leaves and pine buds on natural immunity characteristics of mice has been studied. The injection of these extracts into mice has been found to enhance their resistance to infection with Staphylococcus aureus and Bordetella pertussis virulent cultures, to decrease the enzymatic activity of 5'-nucleotidase in the peritoneal exudate macrophages of mice and to increase the level of lysozyme in their blood. The action of these extracts has proved to depend on their dosage and the time of observation.     

Towards large-scale purification of natural CSF-1 from human placenta tissue extracts

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.     

Bacterial model system for screening and determining optimal concentration of anti-caries natural extracts

In general, an antimicrobial test for screening anti-caries natural extracts was performed by measuring the minimum bactericidal concentration (MBC) against the type strains of mutans streptococci. However, it is unclear if the antimicrobial efficiency of natural extracts on the type strains of mutans streptococci is the same on the clinical strains. In this study, we introduced a bacterial model system for the screening of anti-caries and determining the optimal concentration of them to develop oral hygiene products for Korean populations.