不同甜叶菊品种叶中绿原酸类成分的比较研究

该文以14个扦插培育的甜叶菊品种叶为材料,从8种不同型号的树脂中筛选出一种合适的大孔吸附树脂对甜叶菊叶中绿原酸类成分进行纯化前处理,采用HPLC法对不同甜叶菊品种叶中所含绿原酸类成分进行比较分析。结果表明:(1)在8种不同型号的树脂中,XAD~(-1)6对甜叶菊叶中绿原酸类成分吸附-解析性能最佳。(2)经优化,上样液浓度1.20 mg·mL~(-1)、样品溶液pH 3、解析液乙醇体积分数70%时XAD~(-1)6树脂对甜叶菊叶中绿原酸类成分具有较好的纯化效果。(3) HPLC检测分析表明,在14个品种中共检测出新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C六种绿原酸类成分,其中主要成分均为异绿原酸A、绿原酸、异绿原酸C,而在品种3、5、13、14中没有检测出异绿原酸B。(4) 14个品种中6个绿原酸类成分的含量分别为异绿原酸A 20.55~54.3 mg·g~(-1)、绿原酸17.96~32.93 mg·g~(-1)、异绿原酸C 4.15~19.49 mg·g~(-1)、新原酸0.61~4.61 mg·g~(-1)、隐绿原酸0.52~3.11 mg·g~(-1)、异绿原酸B 0.0~3.17 mg·g~(-1),6种绿原酸类成分总量为43.9~97.8 mg·g~(-1)。可见,不同品种甜叶菊叶中绿原酸类成分含量有明显差异,富含绿原酸类成分的甜叶菊品种可用于开发获取绿原酸类物质。     

Chlorogenic acid as an antiherbivore defence of willows against leaf beetles

In the leaves of 13 Finnish willow species, the content of a phenolic, chlorogenic acid, was found to vary from 0 up to 18 mg g^–1 D.W. Effects of pure chlorogenic acid on insect feeding behaviour were tested using four common leaf beetle species which are in the field mainly found on willows with low-chlorogenic acid leaves. One species, Lochmaea capreae L., was invariably deterred by pure chlorogenic acid applied in naturally occurring concentrations on the willow leaves. Accordingly, in 2-choice laboratory feeding trials L. capreae was found to prefer low-chlorogenic acid leaves of four willow species over high-chlorogenic acid leaves of Salix pentandra L. and S. myrsinifolia Salisb. When presented on the leaves of S. phylicifolia L, pure chlorogenic acid inhibited also the feeding by Phratora polaris Sp.-Schn. Instead, chlorogenic acid had no significant effect on Ph. polaris when it was presented on the leaves of another willow S. cinerea L. In laboratory, Ph. polaris did not show general preference for willow species with low chlorogenic acid content in their leaves. Thus, the response of Ph. polaris to chlorogenic acid seems to depend on the plant species. Apparently variation in other traits such as leaf hairyness may easily override the potential effect of chlorogenic acid content on Ph. polaris. To two other leaf beetle species, Galerucella lineola F. and Plagiodera versicolora Laich., chlorogenic acid is an ineffective deterrent even at unnaturally high concentrations. In laboratory, G. lineola and P. versicolora did not prefer willows with low chlorogenic acid content in their leaves. Thus, among four studied leaf beetle species, only L. capreae seems to be clearly affected by this phenolic. Therefore, overall importance of chlorogenic acid as a defence against willow-feeding leaf beetles appears to be very limited.     

Protective Effects of Chlorogenic Acid on Paraquat-induced Oxidative Stress in Rats

The protective effects of chlorogenic acid on paraquat-induced oxidative stress were examined in rats. The activities of erythrocytes and liver glutathione peroxidase, and of both liver catalase and glutathione reductase, which were increased by feeding paraquat, declined to the levels in the control rats by supplementing chlorogenic acid to the paraquat diet. The activity of superoxide dismutase was not changed by dietary paraquat or by supplementing chlorogenic acid to the paraquat diet. Paraquat in the diet markedly decreased the liver triacylglycerol and phospholipid concentrations, as well as the food intake and body weight gain, while chlorogenic acid protected against these decreases. These in vivo results and the in vitro superoxide anion scavenging activity of chlorogenic acid suggest that chlorogenic acid acted preventively against paraquat-induced oxidative stress.     

Evolution des Teneurs en Acides Chlorogénique et "Isochlorogénique' au Cours du Traitement de Vernalisation des Racines de Cichorium intybus

In a cold-requiring variety of endive (chicory, Cichorium intybus L. ev. Witloof) we could demonstrate a relationship between the variations in the content of chlorogenic acid during the cold treatment of the roots and their disposition to produce flowers in vitro. An increase in the level of chlorogenic acid in the roots preceded the development of their aptitude to form flower buds. In addition, the three main forms of “isochlorogenic’ acid are present in the roots of this cultivar: 3,5-dicaffeoyl-quinic acid, 3,4-dicaffeoyl-quinic acid, and traces of 4,5-dicaffeoyl-quinic acid. The variations in the levels of chlorogenic and “isochlorogenic’ acids indicate that part of the chlorogenic acid (3-caffeoyl-quinic acid) is converted to 3,5-dicaffeoyl-quinic acid.     

Studies on a chlorogenic acid-producing endophytic fungi isolated from <Emphasis Type="Italic">Eucommia ulmoides</Emphasis> Oliver

Eucommia ulmoides Oliver is a traditional medicinal plant of China, and it is one of the main sources of chlorogenic acid. Chlorogenic acid is an ester of caffeic acid, quinic acid, and a phenolic compound that has antibacterial, antifungal, antioxidant, and antitumor activities. The purpose of this study was to determine whether endophytic fungi isolated from Eucommia ulmoides Oliver had the same ability to produce chlorogenic acid. Primary screening was done by antibacterial and antifungal reactions, and the strain reselection was done with high-performance liquid chromatography (HPLC) to identify the fermentation products of the selected strains. Extracts of the leaf and cortex of Eucommia ulmoides Oliver were also deteted by HPLC, then positive results of HPLC were analyzed by GC-MS and LC-MS. In this study, 29 strains were isolated from Eucommia ulmoides Oliver. Most of them had antibacterial activity, and a few of them had antifungal activity. One ingredient of the B5 extract had a retention time identical to that of authentic chlorogenic acid. With GC-MS, other ingredients, isocoumarin and p-chlorocinnamide, were found. With LC-MS, chlorogenic acid and geniposide related to Eucommia ulmoides Oliver were found. The strain B5 was identified as Sordariomycete sp. Thus, endophytic fungi may produce the bioactive compound chlorogenic acid, as their host plant does, and could be used for the production of chlorogenic acid by fermentation in the future.     

Pyrolysis of Chlorogenic Acid and Rutin

Chlorogenic acid and rutin, major polyphenols in tobacco, were pyrolysed with a furnace type pyrolyser connected directly to a gas Chromatograph and 22 compounds (including catechol, benzoic acid, 4-vinylcatechol and quinic acid γ-lactone) from chlorogenic acid and 24 compounds [including catechol, 5-methyl-2-furaldehyde, 4-methylcatechol and 1,6-anhydroglucopyranose (levoglucosan)] from rutin have been identified as pyrolysis products. The gas Chromatograph was also replaced by a capillary cold trap which allowed collection of the pyrolysis products prior to a quantitative determination using an internal standard. Comparison of the pyrolysis products produced from chlorogenic acid or rutin with those derived from tobacco and analysis of the pyrolysis products from a mixture of tobacco and chlorogenic acid or rutin indicated that fairly large proportions of catechol, 4-vinylcatechol and quinic acid γ-lactone produced by the pyrolysis of tobacco may originate from endogenous chlorogenic acid.     

Accumulation of chlorogenic acid in cell suspension cultures of Eucommia ulmoides

Suspension cultures of Eucommia ulmoides were developed and shown to accumulate chlorogenic acid. MS medium plus 2.0 mg l^–1 2,4-dichlorophenoxy acetic acid was used for the cell suspension cultures of Eucommia ulmoides. The chlorogenic acid content of suspension cells was analyzed by capillary electrophoresis, and the mean content was 2.15%, approximate to that of Eucommia ulmoides leaves.     

Sclerotinia fructigena infection and chlorogenic acid content in relation to antifungal compounds in apple fruits

Several organic acids, known to occur in many apple varieties, were shown to be present in the juice of the variety Edward VII. When these were supplied separately to the brown rot pathogen, Sclerotinia fructigena, growing in culture, chlorogenic acid and quinic acid were both readily converted to compounds of higher antifungal activity, one of which was 4-hydroxybenzoic acid. The chlorogenic acid content of concentrated healthy fruit juice was ?6 mg/g, whereas the corresponding syrup from diseased fruit contained only ?2 mg/g. The possible significance of this metabolism of chlorogenic acid by the fungus is discussed.     

接骨草的显微结构及其绿原酸组织化学定位研究

运用石蜡切片法和荧光显微镜观察法研究了3个不同接骨草(Sambucus chinensis Lindl.)居群营养器官的显微结构及其绿原酸的分布规律。结果表明:(1)接骨草地上茎厚角组织明显,髓部由大小不等的两类薄壁细胞组成,且有单宁细胞分布;地下根状茎厚角组织细胞小,髓部薄壁细胞大小差异不明显,皮层及髓中有油细胞分布。(2)叶片为异面叶,栅栏组织细胞为短柱状,油细胞不明显。(3)绿原酸分布在根状茎皮层部分细胞、茎厚角组织部分细胞及叶片的海绵组织中,以海绵组织中含量最高。研究认为,髓部薄壁细胞大小的差异可作为接骨草的一个鉴别特征;荧光显微镜观察法可迅速准确显示绿原酸的分布;在所研究的3个接骨草居群中,怀化居群的绿原酸含量最高,若以绿原酸为有效成分来采收接骨草,可以只采收叶。     

Instant coffee extract with high chlorogenic acids content inhibits hepatic G‐6‐Pase in vitro,but does not reduce the glycaemia

Coffee is the main source of chlorogenic acid in the human diet, and it contains several chlorogenic acid isomers, of which the 5‐caffeoylquinic acid (5‐CQA) is the predominant isomer. Because there are no available data about the action of chlorogenic acids from instant coffee on hepatic glucose‐6‐phosphatase (G‐6‐Pase) activity and blood glucose levels, these effects were investigated in rats. The changes on G‐6‐Pase activity and liver glucose output induced by 5‐CQA were also investigated. Instant coffee extract with high chlorogenic acids content (37.8%) inhibited (p < 0.05) the G‐6‐Pase activity of the hepatocyte microsomal fraction in a dose‐dependent way (up to 53), but IV administration of this extract did not change the glycaemia (p > 0.05). Similarly, 5‐CQA (1 mM) reduced (p < 0.05) the activity of microsomal G‐6‐Pase by about 40%, but had no effect (p > 0.05) on glucose output arising from glycogenolysis in liver perfusion. It was concluded that instant coffee extract with high content of chlorogenic acids inhibited hepatic G‐6‐Pase in vitro, but failed to reduce the glycaemia probably because the coffee chlorogenic acids did not reach enough levels within the hepatocytes to inhibit the G‐6‐Pase and reduce the liver glucose output. Copyright © 2015 John Wiley & Sons, Ltd.     

Inhibition behaviours of some phenolic acids on rat kidney aldose reductase enzyme: an in vitro study

Aldose reductase (AR) inhibitors have vital importance in the treatment and prevention of diabetic complications. In this study, rat kidney AR was purified 19.34-fold with a yield of 3.49% and a specific activity of 0.88?U/mg using DE-52 Cellulose anion exchange chromatography, gel filtration chromatography and 2′5′ ADP Sepharose-4B affinity chromatography, respectively. After purification, the in vitro inhibition effects of some phenolic acids (tannic acid, chlorogenic acid, sinapic acid, protocatechuic acid, 4-hydroxybenzoic acid, p-coumaric acid, ferulic acid, vanillic acid, syringic acid, α-resorcylic acid, 3-hydroxybenzoic acid and gallic acid) were investigated on purified enzyme. We determined IC₅₀, K_i values and inhibition types of these phenolic acids. As a result, tannic and chlorogenic acid had a strong inhibition effect. On the other hand, gallic acid had a weak inhibition effect. In this study, all phenolic acids except for chlorogenic acid and p-coumaric acid showed non-competitive inhibition effects on rat kidney AR.     

Chicoric and Chlorogenic Acids in Plant Species from Georgia

Chicoric acid was isolated from dandelion (Taraxacum officinaleWigg.) leaves by column chromatography. Conditions for HPLC analysis of chicoric and chlorogenic acids were optimized. These acids were assayed in some plants growing in Georgia. The optimum conservation temperature for the preservation of chicoric and chlorogenic acids in the leaves of dandelion and bilberry (Vaccinium arctostaphylosL.) was determined.     

Caffeoylquinic Acids: Separation Method,Antiradical Properties and Cytotoxicity

Twelve chlorogenic acid derivatives and two flavones were isolated from Moquiniastrum floribundum (Asteraceae, other name: Gochnatia floribunda). Compounds were evaluated in relation to their cytotoxicity and antiradical properties. Cytotoxicity was not observed for compounds, however, chlorogenic acid derivatives showed antiradical activity and were more active than the Trolox standard. Quinic acid esterified with caffeoyl group at C‐4 position showed higher antiradical activity compared to acylation at C‐3 or C‐5 positions. Additional caffeoyl groups esterified in quinic acid increase the antiradical activity observed for 4‐caffeoylquinic acid. Excepted to 3,4‐dicaffeoylquinic acid methyl ester, methyl ester derivatives show higher capacity of trapping radicals than their respective acids. Consequently, the presence of caffeoyl group at C‐4 position of quinic acid is suggested as fundamental to obtain the highest antiradical activity.     

EFFECT OF FLUOROPHENYLALANINE ON L-PHENYLALANINE AMMONIA-LYASE ACTIVITY IN AVENA COLEOPTILES

Treatment of etiolated A vena coleoptile apices with several concentrations of DL-p-fluorophenylalanine, previously reported to stimulate coleoptile elongation, caused marked reductions in chlorogenic acid content. Levels of this analogue which had no growth effect caused no detectable alteration of chlorogenic acid accumulation. Evidence is presented which indicates that chlorogenic acid levels may reflect extractable L-phenylalanine ammonia-lyase activity in this tissue. Incubation of coleoptiles with 5 mm DL-p-fluorophenylalanine resulted in significantly lowered extractable ammonia-lyase activity compared to controls. Similar treatment of tissues with the ortho isomer also caused a reduction of enzyme activity but to a lesser extent. Treatment with the meta isomer had no effect on extractable enzyme activity. These findings provide further evidence for the hypothesis that DL-p-fluorophenylalanine stimulates coleoptile elongation by lowering L-phenylalanine ammonia-lyase activity and the subsequent biosynthesis of potentially inhibitory low molecular weight phenols.     

THE INDUCTION OF FLOWERING IN NICOTIANA. II. PHOTOPERIODIC ALTERATION OF THE CHLOROGENIC ACID CONCENTRATION

Chlorogenic acid and its 2 isomers, believed to be the 4– and 5–0-caffeoylquinic acids, have been extracted from leaves of photoperiodic species of Nicotiana, separated by column chromatography (gradient elution) and measured quantitatively during the course of photoperiodic induction. In both N. tabacum cultivar ‘Maryland Mammoth’ (a short-day plant) and N. sylveslris (a long-day plant), the change of the meristem from the vegetative to the flowering shape is preceded by a rise in the chlorogenic acid content of the leaves. During the differentiation of the flower primordia in the shoot apex, there is a drop in the concentration of the chlorogenic acid in the leaves, which is especially marked in the short-day species. The levels of the 2 isomers decrease also at that time. There is no change in the ratios of the 3 caffeoylquinic acids during photoperiodic induction. In both the long- and the short-day species, the 3-isomer (chlorogenic acid) is present at the highest concentration. In the short-day species, there is more of the 4-isomer than of the 5-isomer, whereas in the long-day species, the level of the 5-isomer equals or surpasses that of the 4–0-caffeoylquinic acid.     

Preparative separation of chlorogenic acid by centrifugal partition chromatography from highbush blueberry leaves (Vaccinium corymbosum L.)

Introduction – Blueberries (genus Vaccinium) have gained worldwide focus because of the high anthocyanin content of their fruits. In contrast, the leaves of blueberry have not attracted any attention, even though they contain large quantities of chlorogenic acid, a strong antioxidant compound. Objective – The aim of this investigation was the quantification and preparative isolation of chlorogenic acid (5‐caffeoylquinic acid, 5‐CQA) from blueberry leaves using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – A water fraction containing a high concentration of 5‐CQA (14.5% of dry weight extract) was obtained by defatting a crude methanol extract from blueberry leaves. CPC was applied to isolate 5‐CQA from this water fraction using a two‐phase solvent system of ethyl acetate–ethanol–water at a volume ratio 4:1:5 (v/v/v). The flow‐rate of mobile phase was 2 mL/min with the ascending mode while rotating at 1200 rpm. The eluate was monitored at 330 nm. The structure of chlorogenic acid in the CPC fraction was confirmed with HPLC, UV, ESI/MS and NMR spectra. Results – The HPLC chromatogram showed that the fractions collected by CPC contained chlorogenic acid with 96% purity based on peak area percentage. The total amount of chlorogenic acid isolated from 0.5 g of a water fraction was 52.9 mg, corresponding to 10.6% of the water fraction. The isolated compound was identified successively as 5‐CQA with MS (parent ion at m/z 355.1 [M + H]⁺) and ¹H NMR spectra [caffeoyl moiety in the down field (δ 6.0–8.0 ppm) and quinic acid moiety in the up field (δ 2.0–5.5 ppm)]. Conclusion – 5‐CQA was successfully isolated from blueberry leaves by the CPC method in a one‐step procedure, indicating a further potential use for blueberry leaves. Copyright © 2010 John Wiley & Sons, Ltd.     

Interaction of 3’-O-caffeoyl D-quinic acid with multisubunit protein helianthinin

Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 10⁴M^−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK _ais 1.0 ± 0.1 × 10⁴M⁻¹ as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral titration at 28°C, theK _avalue is 1.38 ± 0.1 × 1 0 4M⁻¹ resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature,T _mof the protein decreases from 76°C to 72°C at 1 × 10⁻³M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature onK _aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin. Publication No. 324.     

Studies on Flavor Components in Soybean

Ethanol (1:1) extract of defatted soybean flour was fractionated systematically and the resulting phonolic acid fraction was investigated. This fraction had strong phenol-like flavor and contained at least seven phenolic acids including syringic, vanillic, ferulic, gentisic, salicylic, p-coumaric, and p-hydroxybenzoic acids. The main component among these was syringic acid, which was isolated as 3,5-dinitrobenzoate.

In addition, two isomers of chlorogenic acids, presumably isochlorogenic and chlorogenic acids approximately in a ratio of 1 : 10, were found in this extract. These substances have sour, bitter and astringent flavors.     

Modification of Tobacco Leaf Glycolate Oxidase Activity by Chlorogenic Acid and Other Polyphenols

Glycolate oxidase extracted from tobacco leaves (Nicotiana tabacum L. cv. NC-95) and assayed by the 2,6-dichlorophe-nolindophenol reduction method was stimulated by chlorogenic acid and other o-diphenols but not by p-diphenols such as hydroquinone. Chlorogenic acid also protected the enzyme against certain enzyme antagonists. A novel assay utilizing horseradish peroxidase with the chromogen o-dianisidine was developed for detecting glycolate oxidase in conjunction with disc electrophoresis. Dissociation of glycolate oxidase into an active monomer during ammonium sulfate fractiona-tion was confirmed electrophoretically. After electrophoresis, flavin mononucleotide was required for monomer activity whereas chlorogenic acid was inhibitory to enzyme band development.     

Intensity of radicle fluorescence as related to the resistance of seedlings of lettuce to the lettuce root aphid and carrot to the carrot fly

A technique is described which indicates by u.v. fluorescence the concentration of caffeoylquinic, chlorogenic and isochlorogenic acids in roots of germinating lettuce and carrot. The surface fluorescence of radicles from lettuce root aphid (Pemphigus bursarius) resistant seedlings was more intense than for susceptible seedlings, attributable to the higher concentration of isochlorogenic acid. In contrast, radicles of carrot seedlings resistant to carrot fly (Psila rosae) were less fluorescent than susceptible seedlings, corresponding to the lower concentration of chlorogenic acid in resistant seedlings. The technique is non-destructive and has been developed to distinguish between cultivars or breeding lines potentially resistant to these insect pests.