Main Glycosides Research Articles

A simple LC-ESI-MS/MS method for quantification of bacopaside I in rat plasma and its application to a pharmacokinetic study.

Bacopaside I is one of the main pseudojujubogenin glycosides isolated from Bacopa monniera. In the present study, a rapid and robust LC-ESI-MS/MS method was developed and validated to quantify bacopaside I in rat plasma. After plasma samples were deproteinized by methanol, the post-treatment samples were analyzed on a Zorbax Eclipse Plus C18 (2.1×50mm, 1.8μm) column using a mobile phase of acetonitrile and water (65:35, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with selected reaction monitoring (SRM) mode via electrospray ionization source. This method covered a linearity range of 10-2000ng/mL with the lower limit of quantification of 10ng/mL. The intra- and inter-day precisions of analysis were less than 10.2%, and the accuracies were between -11.1% and 8.4% at the concentrations of 25, 150 and 1800ng/mL. The total run time was 6.0min. This method was successfully applied to the preclinical pharmacokinetic study of bacopaside I following intravenous or oral administration to rats.

Determination of Rebaudioside A and Stevioside in Leaves of <i>S. rebaudiana</i> Bertoni Grown in México by a Validated HPLC Method

Stevia rebaudiana is a plant with high sweetening capacity due to its content of glycosides, mainly stevioside and rebaudioside A. Several techniques have been used to determine the concentrations of glycosides in Stevia, although an HPLC method is recommended by the FAO/WHO-JECFA. Varieties of Stevia have been recently grown in Mexico, with no previous report of glycosides by a validated method. The aim of this study was to validate an isocratic HPLC method for content determination of main glycosides in the leaves of Stevia cultivated in Mexico. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium-phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 mL/min. Rebaudioside A and stevioside were determined in two Stevia varieties: Morita II and Criolla, and also validation parameters were calculated. Rebaudioside A content (g/100g) in Morita II was 15.15 ± 0.02 while stevioside was 3.97 ± 0.003; in the case of Criolla they were 4.03 ± 0.01 and 8.80 ± 0.14, respectively (p < 0.001). The recoveries of fortified samples were 100% ± 10% and precision RSD was ≤6.27%. The criteria of validation showed accuracy, linearity (≥0.99), and precision; therefore, the determination of glycosides was performed with reliability.

Attenuation of Salt-Loading Induced Cardiomegaly and Dyslipidemia in Wistar Rats by Aqueous Leaf Extract of &amp;lt;i&amp;gt;Chromolaena odorata&amp;lt;/i&amp;gt;

The effect of aqueous extract of the leaves of Chromolaena odorata on body weight, organ sizes, lipid profiles and atherogenic indices was investigated in normal and sub-chronic salt-loaded rats. The normal and treatment control groups were fed 100% of commercial feed, while the test control, reference and test treatment groups received an 8% salt-loaded diet. The extract (at 100 and 200 mg/kg body weight) and moduretics (at 1 mg/kg body weight) were orally administered daily. The normal and test control groups orally received appropriate volumes of water. The extract was screened for bioactive components using gas chromatography-coupled-flame ionization detector. The main glycosides, saponins, allicins, alkaloids, benzoic acid derivatives, terpenes and lignans detected were arbutin, avenacin B-1 (and avenacin A-1), diallyl thiosulphinate, lupanine, ferulic acid (and vanillic acid), limonene and retusin, respectively. Compared to test control, the extract dose-dependently, significantly (P 0.05) lowered the heart size, plasma levels of triglyceride, total density lipoprotein, very low density lipoprotein, low density lipoprotein and non-high density lipoprotein cholesterol and atherogenic indices (cardiac risk ratio, atherogenic coefficient and atherogenic index of plasma). It also significantly increased plasma high density lipoprotein level. These results suggest a protective mechanism of the extract against hypertension induced cardiomegaly and dyslipidemia, thus suggesting that this may underlie its antihypertensive action.

Rapid HPLC Method for Determination of Rebaudioside D in Leaves of &amp;lt;i&amp;gt;Stevia rebaudiana&amp;lt;/i&amp;gt; Bertoni Grown in the Southeast of M&amp;#233;xico

Stevia leaves contain glycosides on which biological activity and sweetening capacity has been reported. Besides the main glycosides—stevioside and rebaudioside A—there are minor glycosides that may contribute to the activity and thus it is important to quantify them. Rebaudioside D is one of the minor glycoside present in S. rebaudiana leaves and there are no reports of a validated method to quantify it. Therefore a simple and sensitive high performance liquid chromatography (HPLC) method was validated for the determination of rebaudioside D in leaves of Stevia rebaudiana B. grown in the southeast of Mexico. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 ml/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The retention time of rebaudioside D was found to be 3.47 min ± 0.04 (S.D.). The calibration curves were linear over the working range (25 - 150 μg/ml), with correlation coefficient ≥0.99 and determination coefficient ≥0.98. The calculated limit of detection (LOD) was 8.53 μg/ml, while the limit of quantitation (LOQ) was 25.85 μg/ml. The percent recoveries of fortified samples were 100% ± 10% and precision relative standard deviation was ≤2.79%. The criteria of validation showed accuracy, linearity, and precision; therefore the method is suitable for quantitative analysis of rebaudioside D in Stevia rebaudiana leaves. Rebaudioside D content (g/100g) in Morita II and Criolla varieties grown in the southeast of Mexico were 0.43 and 0.46, respectively with no significant differences (p > 0.05) between them.

Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications.

Quantitative Estimation of Triterpenoids and Formononetin in Rhizomes of Black Cohosh (A. racemosa) and Dietary Supplements that Claim to Contain A. racemosa by Reversed Phase Ultra-Performance Liquid Chromatography and Evaporative Light Scattering Detection

Actaea racemosa (Black cohosh) is one of the most extensively studied herbal products, and is used as an herbal dietary supplement for the alleviation of menopausal symptoms [1]. The rhizomes/roots used in black cohosh products are often collected from the wild; correct identification is therefore crucial. Actein, 23-epi-26-deoxyactein (syn. 27-deoxyactein), acetylshengmanol 3-O-xyloside, cimigenol 3-O-arabinoside, and cimigenol 3-O-xyloside represent the major triterpene glycosides found in black cohosh [1]. Black cohosh has been described by previous studies to contain a phytoestrogen, formononetin. A UPLC-ELSD method has been developed for the analysis of major triterpenoids and formononetin in Actaea racemosa samples. The best results were obtained with Acquity UPLC™ BEH C18 (100 mm × 2.1 mm, i.d., 1.7 µm) column system using an isocratic elution with a mobile phase consisting of water and acetonitrile:methanol (7:3) at a constant flow rate of 0.3 mL/min. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. Within 5.5 minutes three main glycosides [cimiracemoside (2), 26-deoxyactein (3) and actein (4)] and the isoflavonoid formononentin (1), could be separated, with detection limits of 5, 5, 10 and 0.1 µg/mL, respectively. The method was successfully used to analyze different Actaea recemosa market products, as well as to distinguish between other A. species. For the products containing black cohosh and in rhizomes of black cohosh, there was a significant variability in the amounts of the selected triterpene glycosides and formononetin was not detected. Acknowledgements: This research is funded in part by “Science Based Authentication of Dietary Supplements” Funded by the Food and Drug Administration grant number 2 U01 FD 002071-07 and the United States Department of Agriculture, Agricultural Research Service Specific Cooperative Agreement Number 58-6408-06-067.

Profiling and quantification of isoflavones in soymilk from soy protein isolate using extracted ion chromatography and positive ion fragmentation techniques

Dietary supplements on soy based foods and beverages are increasingly gaining prominence all over the world. In this study, liquid chromatography coupled with positive electrospray ionisation tandem mass spectrometry (LC-ESI-MS) and diode array detection was used for the quantitation and characterisation of isoflavones in fermented and unfermented soymilk made from soy protein isolate SUPRO 590. Bifidobacterium animalis ssp. lactis Bb12 was used for the fermentation of soymilk. The isoflavones were found to produce characteristic radical ions as well as molecules of H 2O, CO 2, a sugar unit, and an alcohol through collision-induced fragmentation. Product ion fragments revealed unique fragmentation pathways for each isoflavone compound. Characteristic fragmentation of different isoflavones were unequivocally identified and differentiated. The occurrence of aldehydes such as pentanal, ethanal and methanal was shown to be specifically linked with isoflavone aglycones, daidzein, genistein and glycitein, respectively. Main glycosides such as genistin, daidzin and glycitin as well as the acetyl-, and malonyl forms also showed respective aglycone ions in their spectra fragmentation. Thus positive ion fragmentation was important in the absolute confirmation of isoflavones and to reveal the occurrence of other related compounds such as aldehydes in soymilk.

Dereplication of glycosides from Sapindus saponaria using liquid chromatography-mass spectrometry

Fruits of Sapindus saponaria (Sapindaceae), a widespread tree throughout the tropics, were collected each 30 days during six months. Liquid-chromatography with UV and MS detection (LC/UV/ESI-MS) and MS/MS fragmentation studies showed that the main glycosides present in these fruits are saponins (SAP) derived from the triterpenes hederagenin and oleanolic acid, and acyclic sesquiterpene oligoglycosides (ASOGs). Using these methods of analysis, we detected up to thirty SAPs and sixty-three ASOGs. The plant produces these compounds as a complex mixture of naturally non-regiosselective acetylated glycosides. Alkaline hydrolysis of the natural glycosides produced simplified mixtures of compounds formed of only four SAPs and five ASOGs. Quantitative analysis of the saponificated glycosides showed that the amount of SAPs accumulated during fruit maturation was almost constant at ca. 110 mg g-1 while the quantity of ASOGs are considerably higher and hits a maximum accumulation of ca. 540 mg g-1 at an age of ca. 3 months.

Aragoside and iridoid glucosides from Aragoa cundinamarcensis

From the water-soluble part of an extract of Aragoa cundinamarcensis were isolated seven iridoid glucosides, namely aucubin, catalpol, rehmannioside D, globularin, gardoside methyl ester, epiloganin and mussaenoside. The main glycoside isolated, however, was a new caffeoyl phenylethanoid triglycoside, named aragoside, containing two β-gluco- and one α-arabinopyranosyl moieties which constituted almost 5% of the dry weight of the plant. Finally, sorbitol was found to be the main carbohydrate constituent of the plant. This distinctive combination of compounds is very similar to that reported from some species of Plantago. The present findings therefore support the results from a recently published molecular phylogenetic study of plastid and nuclear ribosomal DNA sequences, where Aragoa was found to be the closest relative to Plantago so far discovered.

Simultaneous determination of oleuropein and hydroxytyrosol in rat plasma using liquid chromatography with fluorescence detection

Oleuropein, the main glycoside present in olives, and hydroxytyrosol, the principal degradation product of oleuropein present in olive oil, have been linked to reduction of coronary heart disease and certain cancers. In the present study a direct and sensitive reversed-phase high-performance liquid chromatographic assay was developed for simultaneous quantification of both oleuropein and hydroxytyrosol. The plasma protein was precipitated with acetonitrile, samples were then centrifuged and supernatants were dried, and reconstituted with water prior to injection. The chromatographic analysis was carried out using a phenyl column and an isocratic elution of acidified water and acetonitrile with fluorescence detection at 281 and 316 nm for excitation and emission, respectively. The calibration curve was linear and limits of quantification were 30 ng/ml and 3 μg/ml for hydroxytyrosol and oleuropein, respectively. The method has been successfully applied to monitor oleuropein and hydroxytyrosol plasma levels in the rat.

Separation ofCimicifuga racemosa triterpene glycosides by reversed phase high performance liquid chromatography and evaporative light scattering detection

The main triterpene glycosides ofCimicifuga racemosa were separated by reversed phase HPLC, using a C-18 column, Evaporative Light Scattering (ELS) detection and a grient system consisting of water, acetonitrile and reagent alcohol. Within 35 min three main glycosides could be separated and quantified in the methanolic root extract with detection limits of 10.5, 15.6 and 31.6 μg·mL−1 respectively. The method was successfully used, to analyzed differentCimicifuga racemosa market products, as well as to distinguish between otherCimicifuga samples from China.

A steroid glycoside from the seeds ofYucca macrocarpa

glycosides, of which the more polar one greatly predominated, and also trace amounts of still more polar glycosides. The isolation of the main steroid glycoside (SG) was achieved by defardng the carefully ground seeds with hexane, followed by exlraction with 80% isopropyl alcohol. Evaporation of the alcoholic extract gave a 20% total yield of unpurified glycosides. Crystallization of this mixture from ethanol enabled the main glycoside to be obtained in the practically pure state with a yield of I0%, and two recrystaUizations of this permitted the complete elimination of a contaminating weakly polar glycoside. By TLC, in a complete acid hydrolysate of the SG we identified glucose, galactose, and an aglycon close in chromatographic mobility to authentic specimens of smilagenin and tigogenin. The further determination of the structure of the SG was made with the use of IH and t3C NMR spectroscopy.

Flavonoids in the leaves ofTrillium undulatum willdenow

Three flavonol glycosides were identified in the leaves ofTrillium undulatum. The main glycoside was kaempferol 3-O-α-rhamnosyl-(1→2)-O-[α-rhamnosyl-(1→6)]-β-glucoside; the glycosidic sugars and their linkage pattern were quite different from those of the leaf flavonoids ofT. tschonoskii, T. apetalon, T. Kamtschaticum, T. erectum andT. grandiflorum. Two minor compounds were kaempferol/quercetin 3-O-rutinoside.

Phytochemical Reinvestigation ofXysmalobium undulatumRoots (Uzara)1

Four major cardenolide glycosides of Xysmalobium undulatum (L.) R. Br. roots (Uzara) have been isolated for the first time in pure form. The structures were elucidated by spectral analyses and determined as sophorosides and their 17-epimers, respectively. Thus, the structural elucidation of uzarin and xysmalorin, the two main glycosides, has been completed. The corresponding H-17beta isomers, allouzarin and alloxysmalorin, were characterized as genuine compounds.

Chavicol β- d-glucoside, a phenylpropanoid heteroside, benzyl-β- d-glucoside and glycosidically bound volatiles from subspecies of Cedronella canariensis

The volatile substances obtained after enzymatic hydrolysis of purified polar extracts from the aerial parts of the two subspecies of Cedronella canariensis were shown to be, among others, benzyl alcohol (29.4%), chavicol (11.6%), cis-3-hexenol (9.3%), 2-phenylethanol (8.6%), cis-pinocarveol (5.1%), myrtenol (2.2%), 1-phenylethanol (2.0%, tentatively), 1-octen-3-ol (1.8%), 1-hexanol (1.1%) for ssp. canariensis and chavicol (85.1%), benzyl alcohol (2.5%), cis-3-hexenol (2.5%), 1-octen-3-ol (2.3%); 1-hexanol (0.8%) for ssp. anisata. Sugars detected in the polar fraction after hydrolysis were glucose, rhamnose and fructose. The main glycosides obtained from the polar fraction before hydrolysis were chavicol glucoside (6 ppm, ssp. anisata) and benzyl alcohol glucoside (2 ppm, ssp. canariensis).

HPLC and MEKC determination of major flavonoids in selected food pools

The main flavone and flavonol glycosides were identified in four different food pools (soup, legumes/vegetables, salads, fruit) typical of the mediterranean diet. The analysis was performed by RP-HPLC or micellar electrokinetic chromatography (MEKC) coupled with diode array detection. For the quantitative evaluation the glycosidic fraction of each pool was hydrolyzed under controlled conditions and among the resulting aglycones quercetin and apigenin were detected as the most relevant. The mean content of these aglycones in the examined pools ranges from 12 to 43 mg/kg and from 3 to 15 mg/kg of dried sample for quercetin and apigenin, respectively.

Analysis of glycosides of Allochruza gypsophiloides in the preparation ?Allokhrozid? by chromatospectrophotometry

The possibility has been shown of using a readily available method for estimating the level of the main glycoside of the organs of the plant soapwort — acanthophylloside B — in commercial products used in the food and medical industries, and also in samples of the preparation Allokhrozid. The amount of the individual glycoside was calculated in relation to a standard sample of acanthophylloside B.

Flavone-C-Glycosides from the Mosses Plagiomnium elatum and Plagiomnium cuspidatum

In Plagiomnium elatum the new di-C-glycosides 6-C-β-D-glucopyranosyl-8-C-α-L-rhamnopyranosyl- luteolin (Elatin) and 6-C-hexosyl-8-C-rhamnosyl-chrysoeriol were detected. Furthermore four known flavone-C-glycosides were identified. The main glycosides of Plagiomnium cuspidatum are 6-C-glucosyl-7-O-glucosides of apigenin, luteolin and chrysoeriol respectively.

Ovarian Failure and Autoimmunity

We developed an ELISA system for the detection of human anti-ovarian antibodies. Bovine corpora lutea were extracted in PBS (pH 7.2) and fractionated by ultracentrifugation. Both the soluble fraction obtained after 80,000 g (S80) and the Triton-extracted membrane fraction (ST288) were used as antigens. Additionally, the luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor was isolated by affinity chromatography (wheat germ agglutinin and LH-Sepharose) and also used as an antigen. In 7 of 14 patients with primary sterility and endometriosis a positive reaction was observed. Similarly, 6 of 16 patients with secondary sterility and endometriosis were also positive. Patients being stimulated for in vitro fertilization and presenting either primary or secondary sterility were positive in 5 of 22 and 6 of 16 cases, respectively. In the S80 test 41 of 60 sera presented IgG2 antibodies, whereas in the ST288 test 38 of 60 belonged to the IgG1 subclass. Kappa and lambda chains were equally distributed. Some patients could recognize the unoccupied LH/hCG receptor as an antigen, while others recognized only the complex formed by the hormone plus the hormone receptor. The S80 and ST288 antigens were isolated by affinity chromatography. Gel permeation of the purified antigens revealed in each case the presence of an antigen complex. The apparent molecular weight was between 2,000 and 36,000 D. Cross-reactivity studies using affinity-purified antibodies demonstrated an antigenic relationship of the membrane, soluble, and extractable fractions. NAc-(beta-1----4)-D-glucosaminide and -D-galactopyranoside were the main terminal glycosides.

Cardiac glycosides and pregnanes from Adenium obesum (Studies on the constituents of Adenium. I).

Cardiac glycosides and pregnanes from the roots and the stems of Adenium obesum Roem. et Schult. were investigated. Among 30 cardiac glycosides including 15 known glycosides and 15 new combinations of the known aglycones and sugars, the structures of 11 glycosides were elucidated. Oleandrigenin beta-gentiobiosyl-beta-D-thevetoside was the main glycoside. Neridienone A and 16,17-dihydroneridienone A, common pregnanes in Apocynaceae, were also isolated.