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.

Isolation and structure elucidation of highly antioxidative 3,8″-linked biflavanones and flavanone-C-glycosides from Garcinia buchananii bark.

The aim of this study was to identify antioxidants from Garcinia buchananii bark extract using hydrogen peroxide scavenging and oxygen radical absorbance capacity (ORAC) assays. LC-MS/MS analysis, 1D- and 2D-NMR, and circular dichroism (CD) spectroscopy led to the unequivocal identification of the major antioxidative molecules as a series of three 3,8″-linked biflavanones and two flavanone-C-glycosides. Besides the previously reported (2R,3R,2″R,3″R)-naringenin-C-3/C-8″ dihydroquercetin linked biflavanone (GB-2; 4) and (2R,3S,2″R,3″R)-manniflavanone (3), whose stereochemistry has been revised, the antioxidants identified for the first time in Garcinia buchananii were (2R,3R)-taxifolin-6-C-ß-D-glucopyranoside (1), (2R,3R)-aromadendrin-6-C-ß-D-glucopyranoside (2), and the new compound (2R,3S,2″S)-buchananiflavanone (5). The H2O2 scavenging and the ORAC assays demonstrated that these natural products have an extraordinarily high antioxidative power, especially (2R,3S,2″R,3″R)-manniflavanone (3) and GB-2 (4), with EC50 values of 2.8 and 2.2 µM, respectively, and 13.73 and 12.10 µmol TE/ µmol. These findings demonstrate that G. buchananii bark extract is a rich natural source of antioxidants.

Quantitation of resveratrol in red wines by means of stable isotope dilution analysis-ultra-performance liquid chromatography-Quan-time-of-flight mass spectrometry and cross validation.

A stable isotope dilution analysis (SIDA) was developed for the quantitative analysis of the health-promoting phytoalexin (E)-resveratrol in red wines by means of UPLC-QuanTOF-MS. After hemisynthetic preparation of (E)-3,5,4'-trihydroxy-2,4,6-trideuterostilbene ((E)-[(2)H(3)]-resveratrol) as the stable isotope labeled internal standard, validation experiments revealed recovery rate of 96.2 ± 0.8% RSD, thus demonstrating the robustness and accuracy of the SIDA-UPLC-QuanTOF-MS method. Repeatability and reproducibility expressed as RSD showed excellent values of 3.0% and 4.0% for (E)-[(2)H(3)]-resveratrol. Cross validation against a SIDA-HPLC-MS/MS analysis using a triple quadrupole mass spectrometer revealed comparable data, but the SIDA-UPLC-QuanTOF-MS was four times faster, thus making the latter method preferential for an accurate high-throughput analysis of wine samples. Comparison of the SIDA data to those obtained by quantitation using a standard addition method and external calibration, respectively, revealed 97.7% and 32.4% of the resveratrol concentration determined by means of SIDA-UPLC-QuanTOF-MS and 101.0% and 12.7% of the resveratrol levels found by using SIDA-HPLC-MS/MS.

Matrix-calibrated LC-MS/MS quantitation and sensory evaluation of oak Ellagitannins and their transformation products in red wines.

Aimed at investigating the concentrations and taste contribution of the oak-derived ellagitannins castalagin and vescalagin as well as their transformation products acutissimin A/B, epiacutissimin A/B, and beta-1-O-ethylvescalagin in red wine, a highly sensitive and accurate quantification method was developed on the basis of LC-MS/MS-MRM analysis with matrix calibration. Method validation showed good recovery rates ranging from 102.4 +/- 5.9% (vescalagin) to 113.7 +/- 15.2% (epiacutissimin A). In oak-matured wines, castalagin was found as the predominant ellagitannin, followed by beta-1-O-ethylvescalagin, whereas the flavano-C-ellagitannins (epi)acutissimin A/B were present in significantly lower amounts. In contrast to the high threshold concentration levels (600-1000 micromol/L) and the puckering astringent orosensation induced by flavan-3-ols, all of the ellagitannin derivatives were found to induce a smooth and velvety astringent oral sensation at rather low threshold concentrations ranging from 0.9 to 2.8 micromol/L. Dose/activity considerations demonstrated that, among all the ellagitannins investigated, castalagin exclusively exceeded its threshold concentration in various oak-matured wine samples.