齿瓣石斛化学成分的研究

通过硅胶、Sephadex LH-20反复柱层析、纯化,从齿瓣石斛的茎中分离得到7个化合物,利用理化性质和波谱分析分别鉴定为新甘草苷(1)、3,5-二羟基黄酮-7-O-葡萄糖醛酸苷(2)、芦丁(3)、芹菜素-7-O-芸香糖苷(4)、芹菜素-7-O-葡萄糖苷(5)、β-胡萝卜苷(6)和β-谷甾醇(7)。以上化合物均为首次从齿瓣石斛中分离得到。     

Study of the inclusion complexes of aromatic molecules with cyclodextrins using ionspray mass spectrometry

The formation of inclusion complexes between cyclodextrins (cyclohexa-, cyclohepta-, and cyclooctamylose) and either 1-anilinonaphthalene-8-sulfonate or 2-p-toluidinylnaphthalene-6-sulfonate was investigated by ionspray mass spectrometry operated both in the positive and in the negative ion mode. This soft ionisation technique allowed the detection of the inclusion complexes; the presence of false positives was excluded by increasing the voltage at the orifice which caused breakage of the electrostatic adducts and some fragmentation of the free cyclodextrin molecules, but left the inclusion complexes intact. The spectra recorded in the negative mode showed the presence of complexes formed by two cyclodextrin molecules and one aromatic molecule; such stoichiometry was not detected in the positive mode.     

A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers

Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.     

Coupling of fully automated chip-based electrospray ionization to high-capacity ion trap mass spectrometer for ganglioside analysis

NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, ultrafast ion detection, and multistage sequencing at superior sensitivity. The interface between the NanoMate and HCT mass spectrometer consists of an in-laboratory constructed mounting device that allows adjustment of the robot position with respect to the mass spectrometer inlet. The coupling was optimized for ganglioside (GG) high-throughput analysis in the negative ion mode and was implemented in clinical glycolipidomics for identification and structural characterization of anencephaly-associated species. By NanoMate HCT mass spectrometry (MS), data corroborating significant differences in GG expression in anencephalic versus age-matched normal brain tissue were collected. The feasibility of chip-based nanoESI HCT multistage collision-induced dissociation (CID MSⁿ) for polysialylated GG fragmentation and isomer discrimination was tested on a GT1 (d18:1/18:0) anencephaly-associated structure. MS²-MS⁴ obtained by accumulating scans at variable fragmentation amplitudes gave rise to the first fragmentation patterns from which the presence of GT1b structural isomer could be determined unequivocally without the need for supplementary investigation by any other analytical or biochemical methods.     

Lipid profiling of Synechococcus sp. PCC7002 and two related strains by HPLC coupled to ESI-(ion trap)-MS/MS

The lipid profiles of Synechococcus sp. PCC7002 and two related 16S rDNA (99% identity) strains were established by a new method of high-performance liquid chromatography coupled to electrospray-mass spectrometry (HPLC-MS). Lipids were analysed in the positive and negative ionization mode, and fragmentation patterns are reported. No differences in the lipid profile between the three strains could be observed, but the relative content of some species differed. Major lipid species were found to be 1-octadecatrienoyl-2-hexadecanoyl-3-(6'-sulfo-alpha-D-quinovosyl)-sn-glycerol [SQDG (18:3/16:0)] and 1-octadecatrienoyl-2-hexadecenoyl-3-beta-D-monogalactosyl-sn-glycerol [MGDG (18:3/16:1)]. Ten species of SQDG, six species of PG (phosphatidyl-glycerol), seven species of MGDG, and two species of DGDG (digalactosyl-diacyl-glycerol) were detected. A PG species (m/z 761) containing hydroxylinolenic acid or oxophytodienoic acid acyl ester (C18H32O3), and SQDG species containing C17:1 and C17:3 fatty acyl esters are reported for the first time in cyanobacteria. The method also allowed the separation of two pairs of closely related isobaric MGDG species (m/z 770 and m/z 772 in positive ionization).     

Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical “ladder” of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.     

Habitat fragmentation differentially shapes neutral and immune gene variation in a tropical bird species

Habitat fragmentation is a major cause of biodiversity loss, responsible for an alteration of intraspecific patterns of neutral genetic diversity and structure. Although neutral genetic variation can be informative for demographic inferences, it may be a poor predictor of adaptive genetic diversity and thus of the consequences of habitat fragmentation on selective evolutionary processes. In this context, we contrasted patterns of genetic diversity and structure of neutral loci (microsatellites) and immune genes (i.e., toll-like receptors) in an understorey bird species, the wedge-billed woodcreeper Glyphorynchus spirurus. The objectives were (1) to investigate forest fragmentation effects on population genetic diversity, (2) to disentangle the relative role of demography (genetic drift and migration) and selection, and (3) to assess whether immunogenetic patterns could be associated with variation of ectoparasite (i.e., ticks) pressures. Our results revealed an erosion of neutral genetic diversity and a substantial genetic differentiation among fragmented populations, resulting from a decrease in landscape connectivity and leading to the divergence of distinct genetic pools at a small spatial scale. Patterns of genetic diversity observed for TLR4 and TLR5 were concordant with neutral genetic patterns, whereas those observed for TLR3 and TLR21 were discordant. This result underlines that the dominant evolutionary force shaping immunogenetic diversity (genetic drift vs. selection) may be different depending on loci considered. Finally, tick prevalence was higher in fragmented environments. We discussed the hypothesis that pathogen selective pressures may contribute to maintain adaptive genetic diversity despite the negative demographic effect of habitat fragmentation on neutral genetic diversity.Subject terms: Tropical ecology, Genetic variation     

Comparison of a preliminary procedure for the general unknown screening of drugs and toxic compounds using a quadrupole-linear ion-trap mass spectrometer with a liquid chromatography-mass spectrometry reference technique

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) might be a complement to GC-MS and HPLC-diode array detection for the general unknown screening (GUS) of drugs and toxic compounds, particularly when using information- or data-dependent acquisition (IDA or DDA), an auto-adaptive MS-MS product-ion scan mode where, at each unit time, the m/z ratios above a given intensity threshold are selected for fragmentation. A new quadrupole-linear ion-trap mass spectrometer (LC-QqQlinear ion-trap) was evaluated for GUS using IDA. For the first detection step (so-called "survey scan") the single quadrupole "enhanced" MS mode (EMS), where ions are accumulated then filtered in the Q3-linear ion-trap, was used. The so-called "enhanced" parent ion scan mode (EPI) used at two alternated fragmentation energies gave the best signal intensity and the best mass spectral information when adding mass spectra obtained in low and high fragmentation conditions, respectively, both in the positive (+20 and +50 eV) and negative (-15 and -40 eV) modes. Solid-phase extracts of serum spiked with eight test compounds (chosen for their retention times distributed along the 30-min long chromatogram and for ionising in both the positive and negative modes) were analysed in parallel with this LC-MS-MS technique and with a reference LC-MS method run on a single-quadrupole instrument where low and high in-source fragmentation conditions in the positive and the negative ion modes are alternated. A C(18), 5 microm (150 x 1 mm I.D.) column and a gradient elution of acetonitrile in pH 3, 2 mM ammonium formate, were used for both. Higher signal-to-noise ratios were obtained with the LC-QqQlinear ion-trap instrument than with the reference technique, resulting in mass spectra devoid of contaminant ions and at least as informative as the reconstructed single-MS spectra. After optimisation of the IDA intensity threshold for the detection of tiny chromatographic peaks in noise, five out of the eight compounds (milrinone, lorazepam, fluometuron, piretanide and warfarin) could be unambiguously identified at the concentration of 0.1 mg/l in serum, in the positive or negative modes, or in both, versus only two by LC-MS. All of them could be identified at 1 mg/l by both techniques. These preliminary results show that the sensitivity and mass structural information brought by this new LC-QqQlinear ion-trap instrument may help design an efficient toxicological GUS procedure.     

Analysis of Antiretroviral Nucleosides by Electrospray Ionization Mass Spectrometry and Collision Induced Dissociation

Abstract

Antiretroviral nucleoside drugs used against the human immunodeficiency virus (HIV) infection have been analyzed using negative ion electrospray ionization (ESI) mass spectrometry and collision-induced dissociation (CID-MS/MS). Mass fragmentation of azidothymidine (AZT), didanosine (ddI), dideoxycytidine (ddC) and dideoxythiacytidine (3TC) were obtained at different cone voltages and collision energies. Fragmentation of purines and pyrimidines occurred by different pathways. For purines (ddI), the fragmentation was similar to those found in endogenous nucleosides; mainly the pseudo molecular ion is present (M-H) and a cleavage through the glycosidic bond forming (B) was observed. For pyrimidines (AZT, ddC, 3TC), the fragmentation pathways were different from endogenous nucleosides; for AZT, the fragmentation occurred primarily through the elimination of the azido group in the 3′-position (M-H₂-N₃), whereas ddC and 3TC presented more complex fragmentation patterns. For ddC, fragmentation appeared to be dominated by a retro Diels-Alder mechanism (M-CONH). For 3TC, the sulfur atom in the sugar moiety provided greater stability to the charge, producing fragments where the charge resided initially in the dideoxyribose (M-C₂O₂H₆).     

Gone with the wind: Negative genetic and progeny fitness consequences of habitat fragmentation in the wind pollinated dioecious tree Brosimum alicastrum

Premise

Habitat fragmentation negatively affects population size and mating patterns that directly affect progeny fitness and genetic diversity; however, little is known about the effects of habitat fragmentation on dioecious, wind pollinated trees. We assessed the effects of habitat fragmentation on population sex ratios, genetic diversity, gene flow, mating patterns, and early progeny vigor in the tropical dioecious tree, Brosimum alicastrum.

Methods

We conducted our study in three continuous and three fragmented forest sites in a Mexican tropical dry forest. We used eight microsatellite loci to characterize the genetic diversity, gene flow via pollen distances, and mean relatedness of progeny. We compared early progeny vigor parameters of seedlings growing under greenhouse conditions.

Results

Sex ratios did not deviate from 1:1 between habitat conditions except for one population in a fragmented habitat, which was female biased. The genetic diversity of adult trees and their offspring was similar in both habitat conditions. Pollen gene flow distances were similar across habitat types; however, paternity correlations were greater in fragmented than in continuous habitats. Germination rates did not differ between habitat conditions; however, progeny from fragmented habitats produced fewer leaves and had a lower foliar area, total height, and total dry biomass than progeny from continuous habitats.

Conclusions

Changes in mating patterns because of habitat fragmentation have negative effects on early progeny vigor. We conclude that negative habitat fragmentation effects on mating patterns and early progeny vigor may be a serious threat to the long-term persistence of tropical dioecious trees.

     

Characterisation of glucosinolates using electrospray ion trap and electrospray quadrupole time-of-flight mass spectrometry

Twelve naturally occurring glucosinolates displaying alkenyl, hydroxylated, methylsulphinyl, aromatic and indole side chains were investigated by both negative and positive ion electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained from the anion and cation molecular ions of glucosinolates, the different fragments were investigated by MSn experiments using an ion trap spectrometer. The MS3 spectra obtained permitted possible fragmentation schemes to be proposed. These were supported by accurate mass measurements of some characteristic diagnostic ions with the help of a quadrupole time-of-flight instrument. The negative ion ESI-MS/MS behaviour of the different glucosinolates investigated in this study confirmed previously described patterns and revealed new interesting structural informative fragments. Some are common to all the glucosinolates and others are highly specific for a type of variable side chain. The positive ion ESI-MS/MS fragments obtained from the [MNa+Na]+ or [MK+K]+ molecular ions did not provide complementary specific diagnostic ions. Nevertheless, when compared with the negative ion mode, the daughter ions appeared more homogenous and with a better relative abundance for all of the 12 compounds studied. Moreover, the positive ion mode appeared to be more efficient than the negative mode for the study of methoxylated glucosinolates and should be useful to detect the glucosinolates present as organic salts in crude plant extracts.     

Structural characterization of trace stilbene glycosides in Lysidice brevicalyx Wei using liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry

The mass fragmentation patterns of stilbene glycosides isolated from the genus Lysidice were investigated by negative ion electrospray ionization tandem mass spectrometry, and the influence of collision energy on their fragmentation behavior is discussed. It is found that the presence of the Y₀⁻ and B₀⁻ ions in the MS² spectra is characteristic for 1 → 6 linked diglycosyl stilbenes, while the Y₀⁻, Y₁⁻, and Z₁⁻ ions are representative ions of 1 → 2 linked diglycosyl stilbenes. These results indicate that ESI-MSⁿ in the negative ion mode can be used to differentiate 1 → 6 and 1 → 2 linked diglycosyl stilbenes. Based on the fragmentation rules, 9 new trace constituents were identified or tentatively characterized in a fraction of Lysidice brevicalyx by using HPLC/HRMS and HPLC-DAD/ESI-MSⁿ. The results of the present study can assist in on-line structural identification of analogous constituents and targeted isolation of novel compounds from crude plant extracts.     

Thermospray HPLC/MS: a new mass spectrometric technique for the profiling of steroids

The analysis of various steroid classes by thermospray HPLC-MS using solvent systems containing 0.1 M ammonium acetate has been described. For simple unconjugated 3-oxo-4-ene steroids the positive ion spectra are dominated by a parent ion M + H+ and with increasing numbers of hydroxyl group intense ions formed by sequential losses of water (M + H- n18)+ become important. Steroids with dihydroxyacetone side-chains readily lose these side-chains and the resulting (M + H-60)+ fragment is the base peak in their spectra. The (M + H-60)+ ion is not important for most steroids with glycerol-type side-chains. Although competition between thermal degradation and vaporization was observed at lower concentrations, the effect was minimized after optimizing conditions and the protonated molecular ion was easily detected when as little as 1-10 pmol of material were injected on-column. Steroid glucuronides when analyzed in the negative ion mode give simple spectra with base peak and parent ion (M-H)-. Lack of fragmentation permits facile and sensitive measurement of individual glucoronides by selected-ion-monitoring. Extensive fragmentation is seen in the positive ion mode with sequential losses of H2O from the molecular ions (M + NH4)+ and from the aglycone fragment ion. For simple unconjugated steroids the sensitivity of HPLC-MS in selected-ion-monitoring mode can be excellent. When the protonated molecular ion of testosterone was monitored the signal/noise ratio for 30 pg testosterone was about 10.     

Diterpenes and other components of Croton hirtus (Euphorbiaceae)

The roots of Croton hirtus (Euphorbiaceae) collected in Paraiso, Cartago (Costa Rica) afforded, in addition to the few known metabolites, 20 new compounds: the bis-nor dolabradane 1, the dolabradanes 2 and 3, the kauranes 4-7, the cyclopropakauranes 8 and 9, the hirtusanes 10-15, the germacradiene esters 16-18 and the C-25 compound 21, presumably formed by a Diels-Alder reaction between compound 18 and a monoterpene. All structures were elucidated using high field 1D and 2D NMR techniques. MS fragmentation patterns are here reported. The absolute configurations of 4 and 9 were elucidated by using circular dichroism. The separation was performed with modern chromatographic technics.     

Meat tenderizing effect of injecting encapsulated Ca2+ in liposome into rabbit before slaughter

Encapsulated calcium in liposome (L-Ca) produced by using egg phosphatidyl choline in the laboratory was injected into rabbit to evaluate the effect of calcium injection on the ageing of meat. After injecting L-Ca into the blood vessels of rabbit to increase the Ca2+ concentration in the body for 24 h, the fragmentation rate of myofibrils was observed. The fragmentation rates in the loin from the control group and L-Ca injected group were 2.56% and 3.10% in 2 days, 12.27% and 16.18% in 6 days, and 33.56% and 49.60% in 10 days, respectively (p<0.05). SDS-PAGE patterns of connectin and nebulin show that the total degradation of connectin by the control group took longer than 2-3 days, while it was within 1 day for the L-Ca-injected group. The control group took 8-10 days for nebulin, while the L-Ca-injected group took 2-3 days for total degradation. These results indicate that, injecting L-Ca into rabbit was effective for reducing the ageing period of meat without resulting in any physical shock or contamination.     

片段化景观中壳斗科植物种子捕食和扩散模式

生境片段化伴随的面积效应和边缘效应, 可改变分散贮食动物的竞争强度、觅食行为以及隐蔽条件, 影响种子捕食和扩散模式。阐明生境片段化对多物种种子捕食和扩散的影响, 对理解片段化生境中的植物更新和生物多样性维持十分重要。该研究在浙江省千岛湖地区的岛屿和大陆上开展了针对6种壳斗科植物的种子捕食和扩散实验, 分析了物种、分散贮食动物相对多度、种子产量、岛屿大小和边缘效应如何共同影响种子命运和种子扩散距离。主要结果: (1)种子命运和扩散距离在物种间存在显著差异; (2)大陆比岛屿有更长的种子留存时间, 小岛种子留存时间最短, 岛屿内部比岛屿边缘有更长的种子留存时间; (3)物种和岛屿大小对种子原地取食率存在交互作用, 白栎(Quercus fabri)种子在大岛上有更高的原地取食率; (4)种子在小岛上有最高的扩散率, 分散贮食动物相对多度对种子扩散后贮藏率有负效应。表明在千岛湖地区, 生境片段化改变了种子捕食和扩散模式, 且面积效应对不同物种的种子捕食和扩散模式产生了不同作用, 从而影响森林群落更新和生物多样性维持。     

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.     

Synthesis and Biological Activity of Some Dibromoalkadienyl Ether Analogs of Juvenile Hormone

Encapsulated calcium in liposome (L-Ca) produced by using egg phosphatidyl choline in the laboratory was injected into rabbit to evaluate the effect of calcium injection on the ageing of meat. After injecting L-Ca into the blood vessels of rabbit to increase the Ca²⁺ concentration in the body for 24 h, the fragmentation rate of myofibrils was observed. The fragmentation rates in the loin from the control group and L-Ca injected group were 2.56% and 3.10% in 2 days, 12.27% and 16.18% in 6 days, and 33.56% and 49.60% in 10 days, respectively (p<0.05). SDS–PAGE patterns of connectin and nebulin show that the total degradation of connectin by the control group took longer than 2–3 days, while it was within 1 day for the L-Ca-injected group. The control group took 8–10 days for nebulin, while the L-Ca-injected group took 2–3 days for total degradation. These results indicate that, injecting L-Ca into rabbit was effective for reducing the ageing period of meat without resulting in any physical shock or contamination.     

Lack of the Consensus Sequence Necessary for Tryptophan Prenylation in the ComX Pheromone Precursor

Recently we found that a single administration of T-2 toxin (T-2), a trichothecene mycotoxin, into mice induced DNA fragmentation, a biochemical hallmark of apoptosis, in the thymus.¹⁾ In this study, we investigated the effective chemical structure(s) of T-2-derived metabolites capable of inducing thymic apoptosis in vivo in mice. Metabolic conversion of T-2 to 3′-hydroxy-T-2 toxin (3′-OH-T-2) (Fig. 1) did not diminish the apoptosis-inducing activity, since essentially the same level of fragmented DNA was detected in the thymus taken from mice injected with either T-2 or 3′-OH-T-2. In contrast, hydrolysis of T-2 and 3′-OH-T-2 at the carbon-4 (C-4) position to HT-2 toxin (HT-2) and 3′-hydroxy-HT-2 toxin (3′-OH-HT-2), respectively, greatly decreased the level of DNA fragmentation. Similarly, hydrolysis of T-2 at the carbon-8 (C-8) position to neosolaniol strongly diminished its ability to induce DNA fragmentation. T-2 tetraol, having no ester groups, was unable to induce apoptosis. Based on the data presented in this study, we concluded that both the acetyl group at the C-4 position and the isovaleryl or 3′-hydroxyisovaleryl group at the C-8 position of the T-2 molecule are important for inducing cell death through apoptosis in the thymus.     

Acylated flavonol glycosides from Eugenia jambolana leaves.

Two acylated flavonol glycosides and 15 known polyphenols have been isolated and identified from the leaves of Eugenca jambolana Lam. The structures of the new compounds were identified as 3-O-(4"-O-acetyl)-alpha-L-rhamnopyranoside of mearnsetin (myricetin 4'-methyl ether) and myricetin 3-O-(4"-O-acetyl-2"-O-galloyl)-alpha-L-rhamnopyranoside. The complete structure elucidation of all isolated metabolites based on chemical and spectroscopic methods of analysis (UV, 1D and 2D NMR) as well as negative ESI-MS with and without CID in-source fragmentation.