Beef extract supplementation promotes myoblast proliferation and myotube growth in C2C12 cells.

PURPOSE: We previously determined that the intake of beef extract for 4 weeks increases skeletal muscle mass in rats. Thus, this study aimed to clarify whether beef extract has a hypertrophic effect on muscle cells and to determine the signaling pathway underlying beef extract-induced myotube hypertrophy. METHODS: We assessed the effects of beef extract supplement on mouse C2C12 skeletal muscle cell proliferation and differentiation and myotube growth. In addition, the phosphorylation of Akt, ERK1/2, and mTOR following beef extract supplementation was examined by western blotting. Furthermore, the bioactive constituents of beef extract were examined using amino acid analysis and dialysis. RESULTS: In the proliferative stage, beef extract significantly increased myoblast proliferation. In the differentiation stage, beef extract supplementation did not promote myoblast differentiation. In mature myotubes, beef extract supplementation increased myotube diameter and promoted protein synthesis. Although Akt and ERK1/2 levels were not affected, beef extract supplementation increased mTOR phosphorylation, which indicated that the mTOR pathway mediates beef extract-induced myotube hypertrophy. The hypertrophic activity was observed in fractions of > 7000 Da. CONCLUSIONS: Beef extract promoted C2C12 myoblast proliferation and C2C12 myotube hypertrophy. Myotube hypertrophy was potentially induced by mTOR activation and active components in beef extract were estimated to be > 7000 Da.

Antioxidative compounds from Garcinia buchananii stem bark.

An aqueous ethanolic extract of the stem bark of Garcinia buchananii showed strong antioxidative activity using H2O2 scavenging, oxygen radical absorbance capacity (ORAC), and Trolox equivalent antioxidant capacity (TEAC) assays. Activity-guided fractionation afforded three new compounds, isomanniflavanone (1), an ent-eriodictyol-(3α→6)-dihydroquercetin-linked biflavanone, 1,5-dimethoxyajacareubin (2), and the depsidone garcinisidone-G (3), and six known compounds, (2″R,3″R)-preussianon, euxanthone, 2-isoprenyl-1,3,5,6-tetrahydroxyxanthone, jacareubin, isogarcinol, and garcinol. All compounds were described for the first time in Garcinia buchananii. The absolute configurations were determined by a combination of NMR, ECD spectroscopy, and polarimetry. These natural products showed high in vitro antioxidative power, especially isomanniflavanone, with an EC50 value of 8.5 µM (H2O2 scavenging), 3.50/4.95 mmol TE/mmol (H/L-TEAC), and 7.54/14.56 mmol TE/mmol (H/L-ORAC).

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.

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).

Protocol for high-resolution separation of rodent myosin heavy chain isoforms in a mini-gel electrophoresis system.

Skeletal muscle comprises several fiber types classified based on their contractile and metabolic properties. Skeletal muscle fiber types are classified according to their myosin heavy chain isoforms (MyHC I, IIa, IIx, and IIb). We attained good separation of MyHC isoforms in a mini-gel system by modifying a previously developed electrophoresis protocol. Increased glycerol and decreased cross-linking agent concentrations improved the separation of MyHC isoforms. Sample preparation with dithiothreitol and protease inhibitors produced clear MyHC band boundaries. This protocol included silver staining, with a linear range. The protocol provided high resolution and a highly accurate assay of rodent MyHC isoforms.

Beef extract supplementation increases leg muscle mass and modifies skeletal muscle fiber types in rats.

The objective of this research was to investigate the effects of beef extract on fat metabolism, muscle mass and muscle fiber types in rats. We also investigated the synergetic effect of endurance exercise. Twenty-four male rats weighing about 270 g were assigned to two diets containing 0 or 6% beef extract (BE). Half the rats fed each diet were subjected to compulsory exercise (CE) for 30 min every other day. After 4 weeks feeding, the blood was collected and various organs were dissected. The muscle fiber type of the soleus and extensor digitorum longus (EDL) muscles were evaluated by histochemical and electrophoretical analyses. Rats supplemented with BE showed a decrease in fat content in liver and abdomen and an increase in the activity of carnitine palmitoyl transferase II in liver. BE as well as exercise increased the relative weights of both soleus and EDL. BE alone and BE plus CE did not affect the distribution of muscle fiber types in soleus. BE without exercise decreased in type IIb of EDL from 54% to 44% with compensatory increase in type IIa from 41% to 49% and type I from 5% to 7% compared with the nonsupplemented, nonexercised control group. No synergetic effect on a fast to slow fiber conversion due to the combination of BE and CE was detected. Thus, BE supplement increased muscle mass and slow type fiber in EDL. The effects of BE supplement on muscle characteristics were similar to those of exercise. beef extract, fat metabolism, muscle fiber type, muscle mass, L-carnitine

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.

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.

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.