(Bio)active Compounds in Daisy Flower (Bellis perennis).

The common daisy (Bellis perennis) belongs to the family Asteraceae and, in recent years, some new research has been published on the bioactive compounds and biological activities of its extracts. In 2014, the knowledge was partially summarized, but several new studies have been published in the last nine years. In addition, the substances were tabularly consolidated to give a comprehensive overview of over 310 individual components, compound classes, and bioactivities, as well as their accurate plant organ origin. The latest results have shown that the plant has antioxidative, antimicrobial, anticancerogenic, wound healing, antidepressive, anxiolytic, nephroprotective, and insulin mimetic effects, as well as an effect on lipid metabolism. Some studies in the field of homeopathy were also listed. Ideally, a biological effect and one or several compound(s) can be correlated. However, the compounds of the extracts used have often been qualified and quantified, but it remains unclear which of these substances have an activity. The works often stick at the level of the crude extract or a fraction, but not at a single purified and tested compound and, consequently, they are hampered by a missing comprehensive bioactivity workflow. This review provides a critical overview and gaps and offers a basis for further research in this area.

Garcinia buchananii stem bark extract and its bioactive constituents manniflavanone, GB-2 and buchananiflavanone attenuate intestinal inhibitory neuromuscular transmission.

Garcinia buchananii stem bark extract (GBB), commonly used for treating diarrhea in Africa, triggers ectopic aboral contractions, causing inhibition of propulsive motility in the colon ex vivo. To determine whether or not these effects were associated with decreased inhibitory neuromuscular transmission, the responsible constituent compounds, and mechanisms of action, we studied the effects of GBB and specific fractions and flavanones isolated from GBB on intestinal motility using pellet propulsion assays in guinea pig distal colons. In addition, microelectrode recordings were used to measure the effects on the inhibitory junction potentials (IJPs) in the porcine ileum and descending colon smooth muscle. Psychoactive Drug Screening Program secondary receptor functional assays were used to determine whether or not GBB and its constituent compounds act via purinergic (P2Y) and muscarinic receptors. GBB inhibited propulsive motility, but (2R,3S,2″R,3″R)-manniflavanone (MNF), (2R,3S,2″R,3″R)-GB-2 (GB-2) and (2R,3S,2″S)-buchananiflavanone (BNF), the main ingredients of GBB, did not affect motility. We discovered that, in the porcine descending colon, IJPs contained purinergic, nitrergic, and nonpurinergic nonnitrergic components. Furthermore, ileal IJPs were purely purinergic. GBB blocked all components of IJPs, while MNF and GB-2 inhibited purinergic IJPs only. BNF inhibited the purinergic and nonpurinergic components of IJPs. MRS2365, a Y1 (P2Y) agonist, did not evoke sustained membrane hyperpolarization in the presence of GBB. However, GBB, MNF, GB-2 and BNF did not affect P2Y or muscarinic receptors. In conclusion, inhibitory neuromuscular transmission in the porcine descending colon involves all components of IJPs. GBB decreases inhibitory neuromuscular transmission, likely by the actions of MNF, GB-2 and BNF. These effects do not involve P2Y or muscarinic receptors.

Rotenoids and Isoflavones from Xeroderris stuhlmannii (Taub.) Mendonça & E.P. Souza and Their Biological Activities.

The phytochemical study of the ethanolic extract of the leaf of Xeroderris stuhlmannii led to the isolation of five hitherto unreported compounds including two isoflavones (1-2), and three rotenoids (3-5), along with eight known isoflavonoid derivatives (6-13) and one pterocarpan derivative (14). The structures of the new compounds and those of the known ones were established by the spectroscopic (1D and 2D NMR) and spectrometric (HRESIMS) techniques as well as a comparison of their spectroscopic data with those reported in the literature. The leaf extract, fractions, and isolated compounds were tested for their antibacterial effects against nine bacterial strains. Compounds 3, 8, 11, and 12 showed a significant antibacterial effect, with a minimum inhibitory concentration (MIC) value of 62.5 µg/mL each, against Salmonella typhi, Staphylococcus aureus, Klessiella pneumonae, and Escherichia coli, respectively. In addition, the leaf extract, fractions, and isolated compounds were tested for their antifungal effects against four fungal strains. The hexane fraction showed a significant antifungal effect with an MIC value of 125 µg/mL against Candida parasilosis, whereas compounds 3, 8, and 12 showed significant antifungal activity with an MIC value of 62.5 µg/mL, each against Candida parasilosis, Candida albicans, and Candida krusei, respectively.

Quantitation of Toxic Steroidal Glycoalkaloids and Newly Identified Saponins in Post-Harvest Light-Stressed Potato (Solanum tuberosum L.) Varieties.

Although domesticated potatoes contain a large variety of steroidal glycoalkaloids (SGAs) and saponins, in the past, many research projects mainly focused on the two major SGAs, α-solanine and α-chaconine. This study investigates the quantitative changes, induced by post-harvest LED light exposure, of six SGAs and four saponins in 12 potato cultivars at three different time points (1, 7, and 16 days), by using ultra-performance liquid chromatography tandem mass spectrometry. Altogether, SGA contents of 3.0-17.1 mg/100 g fresh weight (FW) could be observed in the analyzed tubers with potato varieties highly exceeding the newly discussed safety limit of 10 mg/100 g. The overall contents of 0.1-5.4 mg/100 g FW of the so far barely studied saponins, like protoneodioscin or barogenin-solatrioside, highly differed between the assayed potato cultivars. Furthermore, cultivar-specific regulations of SGAs and saponins could be observed due to light exposure.

Impact of Phytochemicals on Viability and Cereulide Toxin Synthesis in Bacillus cereus Revealed by a Novel High-Throughput Method, Coupling an AlamarBlue-Based Assay with UPLC-MS/MS.

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure at low doses remain largely unexplored. Natural substances, such as plant-based secondary metabolites, are widely known for their effective antibacterial potential, which makes them promising as ingredients in food and also as a surrogate for antibiotics. In this work, we tested a range of structurally related phytochemicals, including benzene derivatives, monoterpenes, hydroxycinnamic acid derivatives and vitamins, for their inhibitory effects on the growth of B. cereus and the production of cereulide toxin. For this purpose, we developed a high-throughput, small-scale method which allowed us to analyze B. cereus survival and cereulide production simultaneously in one workflow by coupling an AlamarBlue-based viability assay with ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). This combinatory method allowed us to identify not only phytochemicals with high antibacterial potential, but also ones specifically eradicating cereulide biosynthesis already at very low concentrations, such as gingerol and curcumin.

Comprehensive structure-activity-relationship studies of sensory active compounds in licorice (Glycyrrhiza glabra).

Licorice saponins, the main constituents of Glycyrrhiza glabra L. roots, are highly appreciated by the consumer for their pleasant sweet and long lasting licorice taste. The objective of the present study was to understand the molecular features that contribute to bitter, sweet and licorice sensation of licorice roots, and whether individual compounds elicit more than one of these sensations. Therefore, a sensomics approach was conducted, followed by purification of the compounds with highest sensory impact, and by synthesis as well as full characterization via HRESIMS, ESIMS/MS and 1D/2D-NMR experiments. This led to the discovery and structure determination of 28 sweet, bitter and licorice tasting key phytochemicals, including two unknown compounds. A combination of sensorial, cell-based and computational analysis revealed distinct structural features, such as spatial arrangement of functional groups in the triterpenoid E-ring, driving to different taste sensations and sweet receptor hTAS1R2/R3 stimulation.

Fast and Sensitive LC-MS/MS Method for the Quantitation of Saponins in Various Sugar Beet Materials.

An LC-MS/MS method was developed for the simultaneous quantitative analysis of the following 11 triterpene saponins within different sugar beet materials and plant compartments: betavulgaroside I (1), betavulgaroside II (2), betavulgaroside III (3), betavulgaroside IV (4), betavulgaroside VIII (5), boussingoside A2 (6), 3-O-[ß-d-glucopyranosyl-(1 → 2)-(ß-d-xylopyranosyl-(1 → 3))-ß-d-glucuronopyranosyl]-28-O-ß-d-glucopyranosyl-3ß-hydroxyolean-12-en-28-oic acid (7), betavulgaroside V (8), chikusetsusaponin IVa (9), calenduloside E (10), and ginsenoside R0 (11). Our results showed highly varying amounts of saponins within different varieties, roots, and leaves as well as different plant compartments. The amounts for sugar beet roots were in the range of 862 mg/kg to 2 452 mg/kg. They were mostly higher for leaves compared to roots of the same variety with amounts ranging from 907 mg/kg to 5 398 mg/kg. Furthermore, the occurrence of sugar beet saponins within different side streams was examined; in this context, sugar beet fiber contained the highest amounts of saponins for all investigated plant constituents and byproduct streams with a total amount of 12.7 g/kg. Finally, this is the first publication about the occurrence of individual saponins in sugar beets.

Molecularization of Foam-Active Saponins from Sugar Beet Side Streams (Beta vulgaris ssp. vulgaris var. altissima).

This work focuses on the isolation and characterization of saponins with a very low bitter intensity originating from sustainable plant materials, in particular the sugar beet pulp by-product stream. Via a concise foam activity screening of saponin-containing materials, which gives indications for their emulsifying ability, sugar beet root extract was selected and examined for low bitter saponins by means of activity guided fractionation. Individual saponins were isolated from sugar beet pulp, which was identified as the most convenient sugar beet saponin source. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy led to the unequivocal identification of the major, slightly bitter tasting compounds as a series of eight saponins. The complete assignment of 1H and 13C NMR signals for several saponins was carried out for the first time. A small-scale foam activity assay was established and applied to a broad spectrum of the isolated and commercially available saponins. Additionally, orosensory recognition thresholds were determined. Not only high recognition thresholds were determined (thresholds >1000 µmol/L) but also fundamental information about the foaming behavior of mono- and bidesmosidic saponins was collected. The obtained results are relevant to the utilization of saponins from other plant materials or by-product streams and for the use of sugar beet saponins as food additives.

Formation and Characterization of Polyphenol-Derived Red Chromophores. Enhancing the Color of Processed Cocoa Powders: Part 2.

Very recently, we described the formation of (+)-catechin- and (-)-epicatechin-derived polar chromophores by means of a cocoa alkalization process. In this study we focus on the generation of unpolar chromophores using model reactions via Dutching with softer alkaline conditions. UPLC-HR-ESI-TOF-MSe analysis and one-dimensional and two-dimensional NMR spectroscopy led to the unequivocal identification of dehydrodicatechin- and hydroxyxanthene-derived chromophores. The previously unknown 6- C-linked constitutional isomers of C-6'B/C-6D-(2 R,3 S)-dehydrodicatechin (3, 5) were first described, and furthermore, the dimers dehydrocatechin-C-6'B/C-8D-(2 S,3 S)-epicatechin (2) and dehydrocatechin-C-6'B/C-6D-(2 S,3 S)-epicatechin (4, 6) as well as the reddish-colored chromophores 8- C-xanthenocatechin (7), 8- C-xanthenoepicatechin (8), 6- C-xanthenocatechin (9), and 6- C-xanthenoepicatechin (10) were identified as new compounds. A LC-MS/MS method was developed to screen and quantify both classes of chromophores as well as their precursors in alkalized cocoa powders. The dehydrocatechin dimers showed degradation via the alkalization process; in contrast, the xanthenocatechins highlighted an increase in concentration with stronger alkalization, and, therefore, contribute to cocoa reddening. These results, together with those previously published, give a clear insight into the chemistry of polyphenol-derived chromophores generated by cocoa powder alkalization and enable a better understanding of chromophore formation mechanisms toward a more comprehensive color design of cocoa powders.

Formation and Characterization of Polyphenol-Derived Red Chromophores. Enhancing the Color of Processed Cocoa Powders: Part 1.

Although cocoa powder alkalization (Dutching) is a widely used industrial process to improve taste, dispersibility, and coloring of the final product, nevertheless knowledge about the compounds causing a change in coloring is fragmentary. By means of alkaline model reactions starting from the major cocoa polyphenol monomers, (+)-catechin or (-)-epicatechin, eight chromophores were derived from the first rearrangement product catechinic acid. LC-MS/MS analysis, one- and two dimensional-NMR, and electron paramagnetic resonance (EPR) spectroscopy led to the unequivocal identification of 6'-hydroxycatechinic acids (1a, 2a) and their radical states (1b, 2b), which were highlighted as main red chromophores. Six new dehydrocatechinic acid dimers (dehydrocatechinic acid-C-6'B/C-8D-(2 R,3 S)-catechin (3), dehydrocatechinic acid-C-6'B/C-6D-(2 R,3 S)-catechin (4, 5), dehydrocatechinic acid-C-6'B/C-8D-(2 R,3 R)-epicatechin (6), and dehydrocatechinic acid-C-6'B/C-6D-(2 R,3 R)-epicatechin (7, 8)) were also characterized as chromophores. 1-8 as well as their precursors were detected and quantified in alkalized cocoa powders via LC-MS/MS. With the increasing grade of alkalization, a decrease in catechin and epicatechin together with an increase in catechinic acid was observed. Compounds 1b, 2b, and 3-8 also showed a decrease in concentration by Dutching, which correlates to the accumulation of/to higher ordered chromophore oligomers and underlined the increase of the high molecular weight fraction. These findings give a first insight into the formation of structures causing the red coloring of cocoa, which offers the opportunity to optimize the alkalization process toward a better color design of cocoa powders.

Antioxidative Maillard Reaction Products Generated in Processed Aged Garlic Extract.

A powder formulation of aged garlic extract was heated at 100 °C for 1 day to obtain higher antioxidant activity determined with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging (ARS) and oxygen radical absorbance capacity (ORAC) assays. Activity-guided fractionation afforded 12 new in vitro antioxidative Maillard-type products, α-[(2-formyl-5-hydroxymethyl)pyrrol-1-yl]arginine (3), 4-[7-hydroxy-6-(hydroxymethyl)-7,8-dihydro-6 H-pyrano[2,3- b] pyrazine-3-yl]butane-1,2,3-triol (4), 4-[6-(1,2-dihydroxyethyl)-6,7-dihydro-furo[2,3- b]pyrazin-3-yl]-butane-1,2,3-triol (5), α-[(2-formyl-5-hydroxymethyl)-pyrrol-1-yl] aspartic acid (12), 1-[5-(1,2-dihydroxyethyl)-2-oxotetrahydrofuran-3-yl]-5-(hydroxymethyl)-1 H-pyrrole-2-carbaldehyde (14), 4-(6-ethyl-2-pyrazinyl)-1,2,3-butanetriol (17), α-[(2-formyl-5-hydroxymethyl)pyrrol-1-yl] glutamic acid (19), ( S)-1-[(5-hydroxymethyl)furan-2-yl]methyl]-5-oxopyrrolidine-2-carboxylic acid (20), 3-hydroxy-1 H-[{5-(hydroxymethyl)furan-2-yl}methyl]-2,5-dioxo-3-pyrrolidine acetic acid (21), ( E)-4-(5-methylpyrazin-2-yl)but-3-ene-7,2-diol (23), 4-acetyl-6-(hydroxymethyl)picolinic acid (24), ( E)-4-(6-methylpyrazin-2-yl)but-3-ene-1,2-diol (26) and 14 known compounds, 1, 2, 6-11, 13, 15, 16, 18, 22 and 25, which were characterized via 1D/2D-NMR, CD spectroscopy, and mass spectrometry. ARS and ORAC activities of these antioxidants ranged from 0.01 to 0.49 µmol TE/µmol and from 0.01 to 3.50 µmol TE/µmol, respectively. Additionally, plausible formation pathways for the new organic acid-type products (15, 20, and 21) were proposed based on proving their generation in model reactions detected via liquid chromatography-mass spectrometry (LC-MS/MS).

Construction and Application of a Database for a Five-Dimensional Identification of Natural Compounds in Garcinia Species by Means of UPLC-ESI-TWIMS-TOF-MS: Introducing Gas Phase Polyphenol Conformer Drift Time Distribution Intensity Ratios.

Thirty-four reference compounds from G. buchananii were analyzed by means of UPLC-ESI-IMS-TOF-MS to build a database consisting of retention time, accurate m/ z of precursors and fragment ions, and rotationally averaged collision cross-sectional area (CCS). The CCS value of six selected compounds analyzed in bark extract in different concentrations and solvent systems showed excellent intra- and interday precision (RSD ≤ 0.9%). The established database was applied on different organs of G. buchananii as well as G. kola, G. mangostana, and G. cambogia enabling a fast and reliable identification of these natural bioactives. For several compounds, more than one drift time species could be highlighted, which we propose to be hydrogen bond stabilized rotational isomers transferred from solution to gas phase. We used all CCS values of one compound, and we propose to add also the intensity ratio of the conformers as a new and additional characteristic compound parameter in compound identification/screening/database applications to reduce dereplication and false positives and to strengthen the identification.

(2R,3S,2″R,3″R)-Manniflavanone Protects Proliferating Skeletal Muscle Cells against Oxidative Stress and Stimulates Myotube Formation.

We investigated the antioxidative properties of (2R,3S,2″R,3″R)-manniflavanone (MF) using in vitro assays and examined its effects on myogenesis and lactate-induced oxidative stress in C2C12 cells. MF was purified from Garcinia buchananii stem bark. H2O2 and oxygen radical absorbance capacity assays demonstrated that MF is a powerful antioxidant. This finding was supported by diphenylpicrylhydrazine radical scavenging activity of MF. MF was less cytotoxic to C2C12 cells compared to ascorbic acid and myricetin. Moreover, MF accelerated myotube formation in the differentiated C2C12 cells by up-regulating myogenic proteins such as MyoG and myosin heavy chain. Furthermore, MF rescued late differentiation of myoblast suppressed by lactate treatment and up-regulated the expression levels of Nrf2 in lactate-induced oxidative stress, indicating that MF stimulates antioxidative activity inside C2C12 cells. Collectively, MF is a potent antioxidant with a higher safety profile than ascorbic acid and myricetin. It reduces oxidative stress-induced delaying of skeletal muscle differentiation by scavenging reactive oxygen species and regulating myogenic proteins factors.

Taste-Active Maillard Reaction Products in Roasted Garlic (Allium sativum).

In order to gain first insight into candidate Maillard reaction products formed upon thermal processing of garlic, mixtures of glucose and S-allyl-l-cysteine, the major sulfur-containing amino acid in garlic, were low-moisture heated, and nine major reaction products were isolated. LC-TOF-MS, 1D/2D NMR, and CD spectroscopy led to their identification as acortatarin A (1), pollenopyrroside A (2), epi-acortatarin A (3), xylapyrroside A (4), 5-hydroxymethyl-1-[(5-hydroxymethyl-2-furanyl)methyl]-1H-pyrrole-2-carbalde-hyde (5), 3-(allylthio)-2-(2-formyl-5-hydroxymethyl-1H-pyrrol-1-yl)propanoic acid (6), (4S)-4-(allylthiomethyl)-3,4-dihydro-3-oxo-1H-pyrrolo[2,1-c][1,4]oxazine-6-carbaldehyde (7), (2R)-3-(allylthio)-2-[(4R)-4-(allylthiomethyl)-6-formyl-3-oxo-3,4-dihydropyrrolo-[1,2-a]pyrazin-2(1H)-yl]propanoic acid (8), and (2R)-3-(allylthio)-2-((4S)-4-(allylthiomethyl)-6-formyl-3-oxo-3,4-dihydropyrrolo-[1,2-a]pyrazin-2(1H)-yl)propanoic acid (9). Among the Maillard reaction products identified, compounds 5-9 have not previously been published. The thermal generation of the literature known spiroalkaloids 1-4 is reported for the first time. Sensory analysis revealed a bitter taste with thresholds between 0.5 and 785 µmol/kg for 1-5 and 7-9. Compound 6 did not show any intrinsic taste (water) but exhibited a strong mouthfullness (kokumi) enhancing activity above 186 µmol/kg. LC-MS/MS analysis showed 1-9 to be generated upon pan-frying of garlic with the highest concentration of 793.7 µmol/kg found for 6, thus exceeding its kokumi threshold by a factor of 4 and giving evidence for its potential taste modulation activity in processed garlic preparations.

A new NMR approach for structure determination of thermally unstable biflavanones and application to phytochemicals from Garcinia buchananii.

Previous activity-guided phytochemical studies on Garcinia buchananii stem bark, which is traditionally used in Africa to treat various gastrointestinal and metabolic illnesses, revealed xanthones, polyisoprenylated benzophenones, flavanone-C-glycosides, biflavonoids, and/or biflavanones as bioactive key molecules. Unequivocal structure elucidation of biflavonoids and biflavanones by means of NMR spectroscopy is often complicated by the hindered rotation of the monomers around the C-C axis (atropisomerism), resulting in a high spectral complexity. In order to facilitate an unrestricted rotation, NMR spectra are usually recorded at elevated temperatures, commonly over 80 °C, which effects in a single set of resonance signals. However, under these conditions, one of the target compounds of this investigation, (2R,3S,2″R,3″R)-manniflavanone (1), undergoes degradation. Therefore, we demonstrated in the present study that the 1,1-ADEQUATE could be successfully used as a powerful alternative approach to confirm the C-C connectivities in 1, avoiding detrimental conditions. However, a moderate increase in temperature up to 50 °C was sufficient to deliver sharp signals in the proton NMR experiment of (2R,3S,2″R,3″R)-isomanniflavanone (2) and (2″R,3″R)-preussianone (3). In addition, two new compounds could be isolated, namely (2R,3S,2″R,3″R)-GB-2 7″-O-ß-D-glucopyranoside (4) and (2R,3S,2″R,3″R)-manniflavanone-7″-O-ß-D-glucopyranoside (5), and whose structures were elucidated by spectroscopic analysis including 1D and 2D NMR and mass spectrometry methods. The absolute configurations were determined by a combination of NMR and electronic circular dichroism (ECD) spectroscopy. The aforementioned compounds exhibited high anti-oxidative capacity in the H2O2 scavenging, hydrophilic Trolox equivalent antioxidant capacity (H-TEAC) and hydrophilic oxygen radical absorbance capacity (H-ORAC) assays.

UPLC-ESI-TOF MS-Based Metabolite Profiling of the Antioxidative Food Supplement Garcinia buchananii.

Comparative antioxidative analyses of aqueous ethanolic extracts from leaf, root, and stem of Garcinia buchananii revealed high activity of all three organs. To investigate the metabolite composition of the different parts of G. buchananii, an untargeted metabolomics approach using UPLC-ESI-TOF MS with simultaneous acquisition of low- and high-collision energy mass spectra (MS(e)) was performed. Unsupervised statistics (PCA) highlighted clear differences in the metabolomes of the three organs. OPLS-DA revealed (2R,3S,2″R,3″R)-GB-1, (2R,3S)-morelloflavone, and (2R,3S)-volkensiflavone as the most decisive marker compounds discriminating leaf from root and stem extract. Leaves represent the best source to isolate GB-1, morelloflavone, and volkensiflavone. Root extract is the best organ to isolate xanthones and stem bark extract the best source to isolate (2R,3S,2″R,3″R)-manniflavanone; the identified polyisoprenylated benzophenones are characteristic compounds for the leaf organ. Morelloflavone, volkensiflavone, and garcicowin C were isolated for the first time from G. buchananii, identified via MS, NMR, and CD spectroscopy, and showed in H2O2 scavenging, H/L-TEAC, and H/L-ORAC assays moderate to strong in vitro antioxidative activities.

All-trans-configuration in Zanthoxylum alkylamides swaps the tingling with a numbing sensation and diminishes salivation.

The methanol soluble prepared from a supercritical fluid extract of Szechuan pepper (Zanthoxylum piperitum) was screened for its key tingling and numbing chemosensates by application of taste dilution analysis. Further separation of fractions perceived with the highest sensory impact, followed by LC-TOF-MS, LC-MS, and 1D/2D NMR experiments, led to the structure determination of the known alkylamides hydroxy-γ-sanshool (1), hydroxy-α-sanshool (2), hydroxy-ß-sanshool (3), bungeanool (4), isobungeanool (5), and hydroxy-γ-isosanshool (6), as well as hydroxy-ε-sanshool (7), the structure of which has not yet been confirmed by NMR, and hydroxy-ζ-sanshool (8), which has not been previously reported in the literature. Psychophysical half-tongue experiments using filter paper rectangles (1 × 2 cm) as the vehicle revealed amides 1, 2, 4, 5, 7, and 8, showing at least one cis-configured double bond, elicited the well-known tingling and paresthetic orosensation above threshold levels of 3.5-8.3 nmol/cm(2). In contrast, the all-trans-configured amides 3 and 6 induced a numbing and anesthetic sensation above thresholds of 3.9 and 7.1 nmol/cm(2), respectively. Interestingly, the mono-cis-configured major amide 2 was found to induce massive salivation, whereas the all-trans-configuration of 3 did not.

(2R,3S,2'' R,3''R)-manniflavanone, a new gastrointestinal smooth muscle L-type calcium channel inhibitor, which underlies the spasmolytic properties of Garcinia buchananii stem bark extract.

Garcinia buchananii Baker stem bark extract (GBB) is a traditional medication of diarrhea and dysentery in sub-Saharan Africa. It is believed that GBB causes gastrointestinal smooth muscle relaxation. The aim of this study was to determine whether GBB has spasmolytic actions and identify compounds underlying these actions. Calcium (Ca(2+)) imaging was used to analyze the effect of GBB on Ca(2+) flashes and Ca(2+) waves in guinea pig gallbladder and distal colon smooth muscle. Intracellular microelectrode recording was used to determine the effect of GBB, six fractions of GBB, M1-5 and M7, and (2R,3S,2'' R,3''R)-manniflavanone, a compound isolated from M3 on action potentials in gallbladder smooth muscle. The technique was also used to analyze the effect of GBB, M3, and (2R,3S,2'' R,3''R)-manniflavanone on action potentials in the circular muscle of mouse and guinea pig distal colons, and the effect of GBB and (2R,3S,2''R,3'' R)-manniflavanone on slow waves in porcine ileum. GBB inhibited Ca(2+) flashes and Ca(2+) waves. GBB, M3 and (2R,3 S,2''R,3''R)-manniflavanone inhibited action potentials. L-type Ca(2+) channel activator Bay K 8644 increased the discharge of action potentials in mouse colon but did not trigger or increase action potentials in the presence of GBB and (2R,3S,2''R,3'' R)-manniflavanone. GBB and (2R,3S,2'' R,3''R)-manniflavanone inhibited action potentials in the presence of Bay K 8644. GBB and (2R,3 S,2''R,3''R)-manniflavanone reduced the amplitude but did not alter the frequency of slow waves in the porcine ileum. In conclusion, GBB and (2R,3S,2'' R,3''R)-manniflavanone relax smooth muscle by inhibiting L-type Ca(2+) channels, thus have potential for use as therapies of gastrointestinal smooth muscle spasms, and arrhythmias.

New highly in vitro antioxidative 3,8″-linked Biflav(an)ones and Flavanone-C-glycosides from Garcinia buchananii stem bark.

Very recently, we described highly antioxidative polyphenols isolated from the stem bark extract of the Garcinia buchananii tree. In this study, we describe additional antioxidants from Garcinia buchananii bark extract using hydrogen peroxide scavenging, oxygen radical absorbance capacity (ORAC), and trolox equivalent antioxidant capacity (TEAC) assays. UPLC-HR-ESI-TOF-MS(e) analysis, 1- and 2D-NMR, and circular dichroism (CD) spectroscopy led to the unequivocal identification of the antioxidative molecules as a series of five 3,8″-linked biflav(an)ones and two flavanone-C-glycosides. (2S,3R)-Taxifolin-6-C-ß-d-glucopyranoside (2), (2R,3S,2″S,3″S)-manniflavanone (3), (2R,3S)-buchananiflavonol (4), and (2S,3R,2″R,3″R)-GB-1 (6) are new compounds, and (2S,3S)-taxifolin-6-C-ß-d-glucopyranoside (1) was described so far only in one other plant. The structure of (2R,3S,2″R,3″R)-GB-1 (5) and (2R,3S,2″S)-GB2a (7) were confirmed. The H2O2 scavenging, TEAC, and the ORAC assays demonstrated that these natural products have an extraordinarily high antioxidative power, especially (2R,3S,2″S,3″S)-manniflavanone (3) with an EC50 value of 3.0 µM, 4.00 mmol TE/mmol, and 10.30 µmol TE/ µmol.

In vitro activity-guided identification of antioxidants in aged garlic extract.

Activity-guided fractionation was applied on an aged garlic extract (AGE), reported to show strong antioxidant activity, in order to locate the key in vitro antioxidant ingredients by means of the hydrogen peroxide scavenging (HPS) assay as well as the ORAC assay. Besides the previously reported four tetrahydro-ß-carbolines, (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid and (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-1,3-dicarboxylic acid, LC-MS/MS, LC-TOF-MS, and 1D/2D-NMR experiments led to the identification of coniferyl alcohol and its dilignols (-)-(2R,3S)-dihydrodehydrodiconiferyl alcohol, (+)-(2S,3R)-dehydrodiconiferyl alcohol, erythro-guaiacylglycerol-ß-O-4'-coniferyl ether, and threo-guaiacylglycerol-ß-O-4'-coniferyl ether as the major antioxidants in AGE. The purified individual compounds showed high antioxidant activity, with EC50 values of 9.7-11.8 µM (HPS assay) and 2.60-3.65 µmol TE/µmol (ORAC assay), respectively.