Triterpene saponin content in the roots of red beet (Beta vulgaris L.) cultivars.

Triterpene saponins in the roots of Beta vulgaris cultivars Red Sphere, Rocket, and Wodan were profiled and quantitated using reverse-phase liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI/MS/MS). Results obtained indicated that the roots of all three cultivars contained 11 saponins consisting of oleanolic acid or hederagenin aglycone and varying numbers of sugars, with the dominant triglycoside derivative of oleanolic acid. The relative proportions of derivatives of these two aglycones were similar in the three subspecies: cv. Red Sphere contained 99.1 and 0.9%; cv. Rocket, 98.2 and 1.8%; and Wodan 98.8 and 1.2% of oleanolic acid and hederagenin glycosides, respectively. To the best of our knowledge this is the first report on the occurrence, structure, and content of triterpenoid saponins in red beet.

Profiles of steroidal saponins from the aerial parts of Tribulus pentandrus, T. megistopterus subsp. pterocarpus and T. parvispinus by LC-ESI-MS/MS.

INTRODUCTION: Tribulus is a well-known pharmaceutical herb that has been used for a long time in the traditional Chinese and Indian systems of medicine for the treatment of various diseases. It has been found that the genus Tribulus is rich in biologically active furostane-, cholestane- and spirostane-type steroidal saponins. OBJECTIVE: To develop a rapid, sensitive and accurate method based on liquid-phase extraction followed by high-performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC-ESI-MS) to identify different saponins in three species of the genus Tribulus, and to quantify the compounds that are already known. METHODOLOGY: After extraction from the species studied, the extracts were subjected to HPLC analyses with an XTerra® MS C(18) -column and a binary mobile phase consisting of 0.05% formic acid in water and acetonitrile, and with an ESI-MS detection in the negative ion mode. Data were acquired and processed using the Xcalibur 1.3 software. RESULTS: The results exhibited that the profiles of native steroidal glycosides of both T. pentandrus and T. megistopterus subsp. pterocarpus were very similar to each other, but that of T. parvispinus was remarkably different. The fragmentation patterns provided evidence that the saponins possess spirostane-, cholestane- and furostane-type aglycones. Quantitative analyses suggested that these species are a rich source of steroidal saponins. CONCLUSION: HPLC-ESI-MS/MS allowed identification of the key compounds without preparative isolation of the components from the crude extract of Tribulus species.

Determination of polyphenols in Mentha longifolia and M. piperita field-grown and in vitro plant samples using UPLC-TQ-MS.

Nine polyphenols in the aerial parts of Mentha longifolia have been separated by chromatographic techniques. Their structures have been confirmed by HPLC/electrospray ionization-MS/MS. The compounds identified included rosmarinic acid, salvianolic acid L, dedihydro-salvianolic acid, luteolin-glucuronide, luteolin-diglucuronide, luteolin-glucopyranosyl-rhamnopyranoside, and eriodictyol-glucopyranosyl-rhamnopyranoside. The extracts of M. longifolia and M. piperita field plants, in vitro plants, callus tissues, and cell suspension cultures were profiled, and their polyphenol composition was compared in different tissues and quantified using ultra-performance column liquid chromatography (UPLC)/triple-quadrupole-MS in the selected-ion recording detection mode. Determination of desired compounds was based on calibration curves obtained for standards, which were previously isolated from M. longifolia aerial parts. The UPLC profiles revealed considerable differences in the synthesis of secondary metabolites among samples coming from field plants, in vitro plants, callus tissues, and cell suspension cultures. Plant tissues coming from field cultivation (for both M. piperita and M. longifolia) contained several phenolic compounds (flavonoids and phenolic acids), whereas plants from in vitro conditions, callus tissues, and suspension cultures contained only a few of them. Rosmarinic acid dominated in all of these samples. These results show that under in vitro conditions, the metabolism of phenolics undergoes a fundamental change.

Antimutagenic and anti-oxidant activities of isoflavonoids from Belamcanda chinensis (L.) DC.

The isoflavonoid fractions obtained from a methanolic extract of Belamcanda chinensis (L.) DC (syn. Iris domestica Goldblatt & Mabb.) rhizomes inhibited the chemically induced mutations in Salmonella typhimurium TA98 and TA100 in the Ames test. We have studied direct mutagenesis induced by N-nitroquinoline, and indirect mutation induction caused by metabolically activated 2-AF. The fractions enriched in isoflavonoids, obtained by sequential liquid-liquid extraction with diethyl ether and butanol, followed by ODS column separation, inhibited indirect mutagenesis in TA98 almost completely. In TA100 the maximum inhibition ranged between 80% and 100% depending on the test fraction. The inhibition of direct mutagenesis was lower, reaching about 50% in TA98 and in TA100, but it was dose-dependent only in the latter strain. Three in vitro anti-oxidant spectrophotometric assays-DPPH free-radical scavenging test, the phosphomolybdenum assay, and the linoleic acid peroxidation assay were also performed to support the process of bioactivity-guided fractionation and to provide more information about the potential mechanisms of action of the herb under study. The isoflavonoid fractions have the capability to scavenge free radicals, to reduce transition-metal ions and to protect polyunsaturated fatty acids from peroxidation. The analysis of the fractions obtained with high-performance liquid chromatography with photodiode-array and mass-spectrometric detection revealed several potentially bioactive isoflavones, either as glycosides or aglycones, depending on the polarity of the solvents used for fractionation. The main compounds were tectoridin and iridin in the glycoside fractions and the aglycones irigenin, tectorigenin, and 5,6,7,3'-tetrahydroxy-4'-methoxyisoflavone. The activities reported here can be regarded to be of additional value when using this plant as a phyto-estrogenic and chemopreventive agent.

Gentisic acid conjugates of Medicago truncatula roots.

Three phenolic glycosides 5-O-{[5''-O-E-(4'''-O-threo-guaiacylglycerol)-feruloyl]-beta-apiofuranosyl-(1-->2)-beta-xylopyranosyl} gentisic acid, 5-O-[(5''-O-vanilloyl)-beta-apiofuranosyl-(1-->2)-beta-xylopyranosyl] gentisic acid and 1-O-[E-(4'''-O-threo-guaiacylglycerol)-feruloyl]-3-O-beta-galacturonopyranosyl glycerol were isolated and identified from the roots of Medicago truncatula together with four known 5-O-beta-xylopyranosyl gentisic acid, vicenin-2, hovetrichoside C and pterosupin identified for the first time in this species. Structural elucidation was carried out on the basis of UV, mass, (1)H and (13)C NMR spectral data.

Phenolics in aerial parts of Persian clover Trifolium resupinatum.

The nutritional quality of Persian clover (Trifolium resupinatum), an important pasture crop, depends not only on a high protein content but also on the occurrence of animal health and welfare promoting phytochemicals. Nine phenolic constituents present in the aerial parts of this species were isolated and their structures confirmed by NMR and ESI-MS analyses. The compounds included two chlorogenic acids, four quercetin and two kaempferol glycosides, as well as the isoflavone formononetin-7-glucoside. The concentration of isoflavone was low, not exceeding 1.2 mg/g of dry matter. The concentration of flavonols ranged between 5.9 and 11.8 mg/g, depending on the sampling dates, with the highest concentration occurring in the first cut. A similar trend in the concentration was found for chlorogenic acids, which ranged from 2 mg/g in summer to 7.3 mg/g in spring.

Distribution of steroidal saponins in Tribulus terrestris from different geographical regions.

The steroidal saponins of Tribulus terrestris L. (Zygophyllaceae) are considered to be the factor responsible for biological activity of products derived from this plant. The activity depends on the concentration and the composition of active saponins, which in turn is influenced by the geographical origin of plant material. Samples of T. terrestris collected in Bulgaria, Greece, Serbia, Macedonia, Turkey, Georgia, Iran, Vietnam and India were analyzed by LC-ESI/MS/MS for the presence and the concentration of protodioscin (1), prototribestin (2), pseudoprotodioscin (3), dioscin (4), tribestin (5) and tribulosin (6). The flavonoid rutin (7) was also included in the comparison. The results revealed distinct differences in the content of these compounds depending on region of sample collection, plant part studied and stage of plant development. The samples from Bulgaria, Turkey, Greece, Serbia, Macedonia, Georgia and Iran exhibited similar chemical profile and only some quantitative difference in the content of 1-7 with protodioscin (1) and prototribestin (2) as main components. The Vietnamese and Indian samples exhibit totally different chemical profile. They lack 2 and 5, while tribulosin (6) is present in high amounts. Compounds different from 1 to 7 are dominating in these 3 samples. The presented results suggested the existence of one chemotype common to the East South European and West Asian regions. Most probably, the Vietnamese and Indian samples belong to other chemotypes which are still to be studied and characterized. No clear correlation between the burrs morphology and the chemical composition of the samples has been found.

Concentration of isoflavones and other phenolics in the aerial parts of Trifolium species.

Some species of the genus Trifolium are well-known for their content of isoflavones, which are natural compounds showing health-promoting activities. Until recently, only a few species of this genus have been characterized with respect to their composition. In the present study, 57 Trifolium species have been analyzed for their contents of isoflavones, phenolic acids, flavonoids, and clovamides. The cluster analysis of experimental data allowed us to identify a number of species, which should be of interest as potential sources of these metabolites. The isoflavone contents of the three species ( T. heldreichianum, T. scabrum, and T. subterraneum) had extremely high amounts of these compounds, reaching 7-9% of dry matter, and the concentration in a number of other species was higher or at least comparable to the amounts occurring in T. pratense, one of the major isoflavone sources for the nutraceutical industry. Several species contained high amounts of all four analyzed groups of phenolics (isoflavones, phenolic acids, flavonoids, and clovamides). These species may also be of great interest as the association of several groups of active molecules is highly desired for effective disease prevention.

Flavonoids in horse chestnut (Aesculus hippocastanum) seeds and powdered waste water byproducts.

Horse chestnut extracts are widely used in pharmacy and cosmetic industries. The main active constituents are saponins of oleane type, but seeds of horse chestnut also contain flavonoids, being glycosides of quercetin and kaempferol. Their contribution to the overall activity of the extracts was not clear. In the present work, the main flavonoids from horse chestnut seeds were isolated and their structures established with spectral methods. Seven glycosides were isolated, out of which six ( 2, 3, 4, 7, 11, 13) were previously reported and one ( 9) was identified as a new tamarixetin 3- O- [beta- d-glucopyranosyl(1-->3)]- O-beta- d-xylopyranosyl-(1-->2)- O-beta- d-glucopyranoside. The structures of three additional compounds 1, 10, and 12, not previously reported, were deduced on the basis of their LC-ESI/MS/MS fragmentation characteristics. A new ultraperformance liquid chromatographic (UPLC) method has been developed for profiling and quantitation of horse chestnut flavonoids. The method allowed good separation over 4.5 min. Thirteen compounds could be identified in the profile, out of which di- and triglycoisdes of quercetin and kaempferol were the dominant forms and their acylated forms occurred in just trace amounts. The total concentration of flavonoids in the powdered horse chestnut seed was 0.88% of dry matter. The alcohol extract contained 3.46%, and after purification on C18 solid phase, this concentration increased to 9.40% of dry matter. The flavonoid profile and their content were also measured in the horse chestnut wastewater obtained as byproduct in industrial processing of horse chestnut seeds. The total flavonoid concentration in the powder obtained after evaporation of water was 2.58%, while after purification on solid phase, this increased to 11.23% dry matter. It was concluded that flavonoids are present in a horse chestnut extract in a relatively high amount and have the potential to contribute to the overall activity of these extracts. Industrial horse chestnut wastewater can be used to obtain quercetine and kaempferol glycosides for cosmetic, nutraceutical, and food supplement industries.

Flavonoids from barrel medic (Medicago truncatula) aerial parts.

Twenty-three flavonoids have been identified in the aerial parts of barrel medic, and their structures were established by spectrometric and spectroscopic (ESI-MS/MS and NMR) techniques. Eight of the identified compounds, including apigenin 7-O-beta-D-glucuronopyranosyl-(1-->3)-O-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside, apigenin 7-O-[2'-O-sinapoyl-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside], apigenin 7-O-{2-O-feruloyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucopyranoside}, chrysoeriol 7-O-[beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside, chrysoeriol 7-O-{2'-O-p-coumaroyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside}, tricin 7-O-beta-D-glucuronopyranosyl-4'-O-glucopyranoside, tricin 7-O-[2'-O-feruloyl-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucopyranoside], and tricin 7-O-{2'-O-p-coumaroyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside}, have not been reported before in the plant kingdom. Additionally, the presence of two luteolin, three apigenin, one chrysoeriol, and six tricin glycosides, previously identified in alfalfa (Medicago sativa), was confirmed in M. truncatula. Moreover, besides the above flavones, the aerial parts of this species contained three flavonols including rutin, laricitrin 3,7,5'-triglucoside, and laricitrin 3,5'-diglucoside.

Growth of Penicillium verrucosum and production of ochratoxin A on nonsterilized wheat grain incubated at different temperatures and water content.

The results of two experiments with wheat grain inoculated with Penicillium verrucosum are reported. In Experiment I, wheat grain, containing 10, 20 and 30% water, was incubated for 2 weeks at 10, 15, 21 and 28 degrees C. In Experiment II, wheat grain, containing 14, 16, 18, 20 and 22% water, was incubated for 2 weeks at 10, 15, and 20 degrees C. At initial moisture content (IMC) of the wheat grain up to 16% neither P. verrucosum growth nor ochratoxin A (OTA) formation were observed. In the range of IMC from 18% to 22% both the fungal growth and OTA synthesis were distinct, and the parameters were higher at higher temperature in the range 10-21 degrees C. A temperature of 28 degrees C was probably too high for proper metabolism of the fungus, including OTA formation. OTA formation was distinctly related to P. verrucosum abundance in the temperature range 10-21 degrees C, expressed both as the counts of fungal colony forming units (CFU) on agar DYSG medium and diameters of the fungal colonies growing around the wheat kernels placed on the surface of DYSG medium. OTA formation and abundance of P. verrucosum were negatively correlated with the percentage of wheat kernels, placed on DYSG medium, with growing colonies of fungi different from P. verrucosum. CFU counts of P. verrucosum on the wheat grain were significantly related to the diameter of the fungal colonies growing around the wheat kernels placed on DYSG medium. The relationship is described by an exponential regression equation.

Determination of saponins in aerial parts of barrel medic (Medicago truncatula) by liquid chromatography-electrospray ionization/mass spectrometry.

Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.