Antidiabetogenic activity of oleanolic acid glycosides from medicinal foodstuffs

Oleanolic acid glycosides from several medicinal foodstuffs were found to show potent inhibitory activity on the increase of serum glucose levels in oral glucose-loaded rats. By examination of the structure-activity relationships, the 3-O-glucuronide moiety and the 28-carboxyl group in oleanolic acid glycosides were required to exert the hypoglycemic activity. Oleanolic acid glycosides were found to have neither insulin-like nor insulin-releasing activity, but they inhibited gastric emptying and glucose-uptake in the small intestine. Investigation of the mode of action revealed that the inhibition of gastric emptying was mediated by capsaicin-sensitive sensory nerves and the central nervous system. Furthermore, oleanolic acid glycosides were suggested to suppress the gastric emptying by stimulating the release and/or production of dopamine to act through dopamine2 receptors, which in turn causes the release of prostaglandins.     

Changes in Amount of Polyphenols and Activity of Related Enzymes during Growth of Tobacco Flower and Capsule

Developmental stages of tobacco (Nicotiana tabacum L. cv. Burley 21) flower and capsule were correlated with tissue contents of polyphenols and activities of phenylalanine ammonialyase, polyphenoloxidase, and peroxidase. Chlorogenic acid, scopolin, and scopoletin were present in most tissues, whereas rutin and two dihydroxyphenolic glycosides concentrated primarily in the corolla and placenta, respectively. Ovules contained only chlorogenic acid. As development progressed, polyphenols accounted for nearly 15% of the dry weight in the green capsule of field-grown plants. Fertilization triggered a rapid increase of chlorogenic acid in the ovary. When l-phenylalanine-U-(14)C was fed to the detached green capsules and capsule parts, an incorporation of radioactivity into chlorogenic acid and dihydroxyphenolic glycosides occurred which suggested in situ synthesis of these compounds. This was subtantiated by a positive correlation between phenylalanine ammonia-lyase activity and polyphenol accumulation. High polyphenoloxidase activity was associated mainly with the ovary, whereas peroxidase activity was maximal during senescence of all tissues. Polyacrylamide gel slab electrophoresis revealed five cathodic bands and one diffuse zone with poly-phenoloxidase activity in flower extracts. Two anodic poly-phenoloxidase isozymes appeared only in the fertilized ovary. Among 17 peroxidase isozymes, six cathodic forms were present throughout floral development, and the anodic ones increased in number and activity at the later stages of capsule growth.     

Antioxidant Properties of the Major Polyphenolic Compounds in Broccoli

We have examined the antioxidant activity of the major phenolic compounds in Broccoli: two flavonol glycosides (quercetin 3-O-sophoroside and kaemp-ferol 3-O-sophoroside) and four hydroxycinnamic acid esters (1,2'-disinapoyl-2-feruloyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2'-trisinapoyl gentiobiose and 1,2-disinapoyl gentiobiose). The Trolox C equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid per-oxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the two flavonol glycosides were less active than their respective aglycones. TEAC values for the hydroxycinnamic acid esters were less than the sum of their constituent hydroxycinnamic acids on a molar basis. Quercetin 3-O-sophoroside was a potent inhibitor of lipid peroxidation, in contrast to kaempferol 3-O-sophoroside. The hydroxycinnamic acid esters were highly effective at preventing lipid damage with the exception of 1,2,2'-trisinapoyl gentiobiose. The six compounds analysed herein demonstrate the antioxidant activity of the major phenolics in broccoli and indicate the effect on antioxidant activity of sugar substitutions in the phenolic B ring.     

Wulfenia carinthiaca Jacq., antioxidant and pharmacological activities

Relative antioxidant activities of a methanolic extract of three phenylpropanoid glycosides and three iridoid glycosides from Wulfenia carinthiaca were evaluated using the Briggs-Rauscher (BR) reaction method. This method is based on the inhibitory effects by antioxidants on oscillations of the BR reaction. The total extract showed a certain antioxidant activity with respect to resorcinol chosen as standard. The three phenylpropanoid glycosides showed a very high relative antioxidant activity while iridoid glycosides had practically no activity. These experimental results were confirmed by empirical calculations based on the BDE (Bond Dissociation Enthalpy) theory. The total phenolic content was also measured for the phenylpropanoid glycosides using the Folin-Ciocalteu reagent. The obtained values as gallic acid equivalents were in perfect agreement with the relative antioxidant activities. From a pharmacological point of view the results obtained demonstrate that the methanolic extract of W. carinthiaca have antinociceptive and antiedematogenic effects in the different models adopted. The plant extract produced a significant inhibition, dose related, of the rat paw edema induced by carrageenin. The anti-inflammatory activity is probably due to the phenylpropanoid compounds present in the plant. The histological sections of paw tissue in animals treated with Wulfenia carinthiaca extract confirmed the anti-inflammatory effects. The results of the antinociceptive assay indicated a significant reduction on the number of abdominal writhes of mice, induced by acetic acid.     

Fluorogenic sialic acid glycosides for quantification of sialidase activity upon unnatural substrates

Herein we report the synthesis of N-acetyl neuraminic acid derivatives as 4-methylumbelliferyl glycosides and their use in fluorometrically quantifying human and bacterial sialidase activity and substrate specificities. We found that sialidases in the human promyelocytic leukemic cell line HL60 were able to cleave sialic acid substrates with fluorinated C-5 modifications, in some cases to a greater degree than the natural N-acetyl functionality. Human sialidases isoforms were also able to cleave unnatural substrates with bulky and hydrophobic C-5 modifications. In contrast, we found that a bacterial sialidase isolated from Clostridium perfringens to be less tolerant of sialic acid derivatization at this position, with virtually no cleavage of these glycosides observed. From our results, we conclude that human sialidase activity is a significant factor in sialic acid metabolic glycoengineering efforts utilizing unnatural sialic acid derivatives. Our fluorogenic probes have enabled further understanding of the activities and substrate specificities of human sialidases in a cellular context.     

Hydrolysis of black soybean isoflavone glycosides by Bacillus subtilis natto

Hydrolysis of isoflavone glycosides by Bacillus subtilis natto NTU-18 in black soymilk is reported. At the concentration of 3–5% (w/v), black soymilk in flask cultures, the isoflavones, daidzin, and genistin were highly deglycosylated within 24 h. Deglycosylation of isoflavones was further carried out in a 7-l fermenter with 5% black soymilk. During the fermentation, viable cells increased from 10³ to 10⁹ CFU ml⁻¹ in 15 h, and the activity of β-glucosidase appeared at 8 h after inoculation and reached a maximum (3.3 U/ml) at 12 h, then decreased rapidly. Deglycosylation of isoflavone glycosides was observed at the same period, the deglycosylation rate of daidzin and genistin at 24 h was 100 and 75%, respectively. It is significantly higher than the previous reports of fermentation with lactic acid bacteria. In accordance with the deglycosylation of isoflavone glycosides, the estrogenic activity of the 24 h fermented black soymilk for ERβ estrogen receptor increased to threefold; meanwhile, the fermented broth activated ERα estrogen receptor to a less extent than ERβ. These results suggest that this fermentation effectively hydrolyzed the glycosides from isoflavone in black soymilk and the fermented black soymilk has the potential to be applied to selective estrogen receptor modulator products.     

Correlation between Chlorophyll and Chlorogenic Acid Content in Tobacco Leaves

Developmental stages of tobacco (Nicotiana tabacum L. cv. Burley 21) flower and capsule were correlated with tissue contents of polyphenols and activities of phenylalanine ammonialyase, polyphenoloxidase, and peroxidase. Chlorogenic acid, scopolin, and scopoletin were present in most tissues, whereas rutin and two dihydroxyphenolic glycosides concentrated primarily in the corolla and placenta, respectively. Ovules contained only chlorogenic acid. As development progressed, polyphenols accounted for nearly 15% of the dry weight in the green capsule of field-grown plants. Fertilization triggered a rapid increase of chlorogenic acid in the ovary. When l-phenylalanine-U-¹⁴C was fed to the detached green capsules and capsule parts, an incorporation of radioactivity into chlorogenic acid and dihydroxyphenolic glycosides occurred which suggested in situ synthesis of these compounds. This was subtantiated by a positive correlation between phenylalanine ammonia-lyase activity and polyphenol accumulation. High polyphenoloxidase activity was associated mainly with the ovary, whereas peroxidase activity was maximal during senescence of all tissues. Polyacrylamide gel slab electrophoresis revealed five cathodic bands and one diffuse zone with poly-phenoloxidase activity in flower extracts. Two anodic poly-phenoloxidase isozymes appeared only in the fertilized ovary. Among 17 peroxidase isozymes, six cathodic forms were present throughout floral development, and the anodic ones increased in number and activity at the later stages of capsule growth.     

Phytochemical and molluscicidal investigations of Fagonia arabica

The aqueous methanolic extract of the aerial parts of Fagonia arabica L. (family Zygophyllaceae) was successively fractionated using certain organic solvents. From the ethyl acetate fraction, two flavonoid glycosides were isolated and identified as kaempferol-7-O-rhamnoside and acacetin-7-O-rhamnoside. Four triterpenoidal glycosides were isolated from the butanolic layer. Their structures were elucidated on the basis of the spectral and chemical data as 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranoside oleanolic acid (1), 3-O-alpha-L-arabinopyranosyl quinovic acid 28-O-beta-D-glucopyranoside (2), 3-O-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinosyl oleanolic acid (3) and 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabino-pyranosyl quinovic acid 28-O-beta-D-glucopyranoside (4). The two monodesmosidic saponins 1 and 3 were found to possess strong molluscicidal activity against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni in Egypt (LC90 = 13.33 and 16.44 microM), whereas the other two bidesmosidic saponins 2 and 4 as well as the two flavonoid glycosides were inactive up to 50 microM.     

Synthesis of sulfated oligosaccharide glycosides having high anti-HIV activity and the relationship between activity and chemical structure.

Sulfated laminara-oligosaccharide glycosides having high anti-human immunodeficiency virus (HIV) activities were synthesized from laminara-tetraose, -pentaose and -hexaose. The oligosaccharide glycosides were synthesized by treating peracetylated beta-oligosaccharides with various alcohols and Lewis acid catalysts. The effects of the number of glucose residues and the alkyl chain-length on anti-HIV activity were examined. The anti-HIV activity of sulfated dodecyl laminara-pentaosides and -hexaosides increased with increasing degree of sulfation (DS) and the pentaoside having an almost fully-sulfated saccharide portion had the highest activity, whereas for the hexaoside a somewhat lower DS manifested the highest activity. Sulfated laminara-oligosaccharide glycosides having fluoroalkyl-containing aglycons of high hydrophobicity showed potent inhibitory effects against HIV infection. In contrast, hydrophilic substituents containing oligo(ethyleneoxy) groups as aglycons in the sulfated oligosaccharides did not show high anti-HIV activity.     

Effect of triterpenoid glycosides on alpha- and beta-amylase activity and total protein content in wheat seedlings

Influence of the aleanolic acid glycosides from Silphium perfoliatum L. (silphioside B, C, E and G) and their progenins on the amylase activity and total protein content in wheat seedlings was studied. Treatment of the Triticum aestivum L. seeds with 1-10 microM water solutions of mono- and diglycosides (mono- and bisdesmosines) elevated the alpha-amylase and total amylase activities in seedlings. Silphioside E containing three glucose moieties in its molecule did not change alpha-amylase activity, but it did if bis-triglycoside acetylated carbohydrate (as in silphioside C). Effects of 5-10 microM solutions of the active glycosides was comparable with that of exogenous gibberellin A3 and 6-benzylaminopurine.     

Isolation and characterization of new limonoid glycosides from Citrus unshiu peels.

Three limonoid glycosides were isolated from Citrus unshiu peels, and their structures were determined based on MS and NMR spectroscopic data as nomilinic acid 17-O-beta-D-glucopyranoside (1), methyl nomilinate 17-O-beta-D-glucopyranoside (2), and obacunone 17-O-beta-D-glucopyranoside (3). In particular, the location of the sugar moiety was clearly determined by the B/E constant linked scan FABMS method. No limonoid glycosides obtained here were found to have antitumor activity in NCI-H292 and EL-4 cell lines.     

Cyanogenic glycosides and their metabolic enzymes in barley, in relation to nitrogen levels

The possible role for cyanogenic glycosides as nitrogen storage compounds was studied in barley, Hordeum vulgare (cv. Golf), cultivated under different nitrogen regimes. Cyanogenic glycosides were absent in seeds and roots but were synthesized in seedlings where they accumulated at a level of about 150 nmol shoot⁻¹ in control plants and 110 nmol shoot⁻¹ in nitrogen-starved plants. An enzyme involved in the breakdown of cyanogenic glycosides, β-glucosidase (EC 3.2.1.-) exhibited high activity in seeds and was also detected in roots and shoots. The activity of β-cyanoalanine synthase (EC 4.4.1.9), which is involved in the metabolism of HCN, was low in seeds but very high in roots and shoots. There was no correlation between the activities of the two enzymes and the content of cyanogenic glycosides or nitrogen. The relative content of nitrogen in cyanogenic glycosides never exceeded 0.3% of total nitrogen, and the amount of cyanogenic glycosides decreased at a low rate even at a stage when nitrogen limitation inhibited growth.     

Xylanase treatment of plant cells induces glycosylation and fatty acylation of phytosterols

Treatment of tobacco suspension cells ( Nicotiana tabacum cv. KY 14) with a purified β -1,4-endoxylanase from Trichoderma viride [1 μg enzyme (ml cells)⁻¹] caused a 13-fold increase in the levels of acylated sterol glycosides and elicited the synthesis of phytoalexins. A commercial preparation of xylanase from Trichoderma viride caused an identical shift in sterols. In contrast, a commerical xylanase from Aureobasidium pullaulans had no effect on the levels of acylated sterol glycosides, but did elevate the levels of sterol esters. Treatment of the cells with Cu²⁺ or Ag⁺ also evoked a severalfold increase in the levels of acylated sterol glycosides. Analysis of the various sterol lipid classes revealed that the large xylanase-induced increase in acylated sterol glycosides occurred at the expense of sterol esters, free sterols and sterol glycosides. Further analyses revealed that the most abundant phytosterol in each of the four classes of sterol lipids was β -sitosterol. Linoleic acid was the most abundant fatty acid in the sterol esters, and palmitic and linoleic acids were the most abundant fatty acids in the acylated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acvlated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acylated sterol glycoside fractions. The results of the present study demonstrate that xylanase from Trichoderma viride induces a dramatic shift in the level of acylated sterol glycosides, indicating that endoxylanase was probably the active component in the cellulase enzyme preparations used in our previous study.     

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.     

Phenolics during early development of <Emphasis Type="Italic">Betula pubescens</Emphasis> seedlings: inhibition of phenylalanine ammonia lyase

We studied phenolic metabolism and plant growth in birch seedlings at the beginning of their development by inhibiting phenylalanine ammonia lyase (PAL), which is the first committed step in phenylpropanoid metabolism. Betula pubescens (Ehrh.) seeds were germinated in inhibitor-free media and the seedlings were transferred to hydroponic culture at the cotyledon stage. They were 6 days old at the start of the experiment, which lasted for 3 weeks. PAL activity was inhibited by three different concentrations of 2-aminoindane-2-phosphonic acid monohydrate (AIP) in the growing media. At the end of 3 weeks, phenolics in all plant parts (roots, stem, cotyledons, first, second and third true leaves) were determined. AIP inhibited strongly the accumulation of phenolic acids, salidroside, rhododendrins, ellagitannins and their precursors, flavan-3-ols, and soluble condensed tannins. The accumulation of lignin and flavonol glycoside derivatives was moderately inhibited. The accumulation of flavonol glycosides, such as quercetin glycosides and kaempferol glycosides, was not generally inhibited, even in leaves that emerged during the experiment, while the accumulation of insoluble condensed tannins was inhibited only slightly and not in all plant parts. This suggests that flavonol glycosides, which may have a UV-B protective role, and insoluble condensed tannins, which may have structural functions, are prioritized in seedling development. Inhibition of PAL with AIP decreased seedling growth and possible reasons for this are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acid hydrolase activity of cultured bovine oligodendrocytes

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8–11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.     

Activation of cardiac ryanodine receptors by cardiac glycosides

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.     

Characterisation of new oligoglycosidic compounds in two Chinese medicinal herbs

A series of caffeic acid derivatives (3,5-dicaffeoyl-quinic acid, 3,4-dicaffeoyl-quinic acid, and 4,5-dicaffeoyl-quinic acid), and the new compound beta,3,4-trihydroxyphenethyl-O-[beta-apiofuranosyl-(1-->4)-alpha- rhamnopyranosyl-(1-->3)]-(4-O-caffeoyl)-beta-glucopyranoside (wedelosin), as well as three known flavonoid glycosides (quercetin 3-O-beta-glucoside, kaempferol 3-O-beta-apiosyl-(1-2)-beta-glucoside, and astragalin or kaempferol 3-O-beta-glucoside) were isolated from the Chinese medicinal herb Wedelia chinensis. Wedelosin showed an inhibitory activity on both the classical and the alternative activation pathway of the complement system. Another Chinese medicinal herb, Kyllinga brevifolia, yielded two known flavonoid glycosides [kaempferol 3-O-beta-apiosyl-(1-2)-beta-glucoside and isorhamnetin 3-O-beta-apiosyl-(1-2)-beta-glucoside], and a new quercetin triglycoside [quercetin 3-O-beta-apiofuranosyl-(1-->2)-beta-glucopyranoside 7-O-alpha-rhamnopyranoside]. The latter compound showed a moderate anti-viral activity.     

Structurally different flavonol glycosides and hydroxycinnamic acid derivatives respond differently to moderate UV-B radiation exposure

The aim of this study was to investigate the modifying influence of moderate ultraviolet-B (UV-B) radiation exposure on structurally different flavonol glycosides and hydroxycinnamic acid derivatives during pre-harvest using kale, a leafy Brassica species with a wide spectrum of different non-acylated and acylated flavonol glycosides. Juvenile kale plants were treated with short-term (1 day), moderate UV-B radiation [0.22-0.88 kJ m?2 day?1 biologically effective UV-B (UV-B(BE))]. Twenty compounds were quantified, revealing a structure-specific response of flavonol glycosides and hydroxycinnamic acid derivatives to UV-B radiation. A dose- and structure-dependent response of the investigated phenolic compounds to additional UV-B radiation was found. The investigated quercetin glycosides decreased under UV-B; for kaempferol glycosides, however, the amount of sugar moieties and the flavonol glycoside hydoxycinnamic acid residue influenced the response to UV-B. Monoacylated kaempferol tetraglucosides decreased in the investigated UV-B range, whereas the monoacylated kaempferol diglucosides increased strongly with doses of 0.88 kJ m?2 day?1 UV-B(BE) . The UV-B-induced increase in monoacylated kaempferol triglucosides was dependent on the acylation pattern. Furthermore, the hydroxycinnamic acid glycosides disinapoyl-gentiobiose and sinapoyl-feruloyl-gentiobiose were enhanced in a dose-dependent manner under UV-B. While UV-B radiation treatments often focus on flavonol aglycones or total flavonols, our investigations were extended to structurally different non-acylated and acylated glycosides of quercetin and kaempferol.     

Glycoside modification of oleanolic acid derivatives as a novel class of anti-osteoclast formation agents

Oleanolic acid, a natural product, possesses an anti-osteoclast formation activity. Targeting at discovery of novel and potent anti-bone resorption agents, 22 glycosides of oleanolic acid derivatives (including d-galactopyranosides, d-glucopyranosides, d-xylopyranoses, d-arabopyranoses and d-glycuronic acids) were synthesized at phase-transfer-catalyzed conditions (K₂CO₃, Bu₄NBr, CH₂Cl₂-H₂O) and their inhibitory activity on the formation of osteoclast-like multinucleated cells (OCLs) induced by 1α, 25-dihydroxy vitamin D₃ was evaluated in a co-culture assay system. The structure-activity relationships of these compounds were also discussed.