Safety and mechanism of appetite suppression by a novel hydroxycitric acid extract (HCA-SX)

A growing body of evidence demonstrates the efficacy of Garcinia cambogia-derived natural (–)-hydroxycitric acid (HCA) in weight management by curbing appetite and inhibiting body fat biosynthesis. However, the exact mechanism of action of this novel phytopharmaceutical has yet to be fully understood. In a previous study, we showed that in the rat brain cortex a novel HCA extract (HCA-SX, Super CitriMax) increases the release/availability of radiolabeled 5-hydroxytryptamine or serotonin ([³H]-5-HT), a neurotransmitter implicated in the regulation of eating behavior and appetite control. The aim of the present study was 2-fold: (a) to determine the effect of HCA-SX on 5-HT uptake in rat brain cortex in vitro; and (b) to evaluate the safety of HCA-SX in vivo. Isolated rat brain cortex slices were incubated in oxygenated Krebs solution for 20 min and transferred to buffer solutions containing [³H]-5-HT for different time intervals. In some experiments, tissues were exposed to HCA-SX (10 M – 1 mM) and the serotonin receptor reuptake inhibitors (SRRI) fluoxetine (100 M) plus clomipramine (10 M). Uptake of [³H]-5-HT was expressed as d.p.m./mg wet weight. A time-dependent uptake of [³H]-5-HT occurred in cortical slices reaching a maximum at 60 min. HCA-SX, and fluoxetine plus clomipramine inhibited the time-dependent uptake of [³H]-5-HT. At 90 min, HCA-SX (300 M) caused a 20% decrease, whereas fluoxetine plus clomipramine inhibited [³H]-5-HT uptake by 30%. In safety studies, acute oral toxicity, acute dermal toxicity, primary dermal irritation and primary eye irritation, were conducted in animals using various doses of HCA-SX. Results indicate that the LD₅₀ of HCA-SX is greater than 5000 mg/kg when administered once orally via gastric intubation to fasted male and female Albino rats. No gross toxicological findings were observed under the experimental conditions. Taken together, these in vivo toxicological studies demonstrate that HCA-SX is a safe, natural supplement under the conditions it was tested. Furthermore, HCA-SX can inhibit [³H]-5-HT uptake (and also increase 5-HT availability) in isolated rat brain cortical slices in a manner similar to that of SRRIs, and thus may prove beneficial in controlling appetite, as well as treatment of depression, insomnia, migraine headaches and other serotonin-deficient conditions.     

Inhibition of Helicobacter pylori in vitro by various berry extracts,with enhanced susceptibility to clarithromycin

The objective of this study was to evaluate the effects of various berry extracts, with and without clarithromycin on Helicobacter pylori. Resistance to clarithromycin by H. pylori has been reported, leading to interest in alternatives/adjuncts to therapy with clarithromycin. H. pylori American type culture collection (ATCC) strain 49503 was grown, cell suspensions were made in PBS and diluted 10-fold. One hundred microL of the suspension was then incubated for 18 h with extracts of raspberry, strawberry, cranberry, elderberry, blueberry, bilberry, and OptiBerry, a blend of the six berries, at 0.25-1% concentrations. Serially diluted cell suspensions were exposed for 1 h to clarithromycin at 15 microg/ml. Ten microl of bacterial samples from the 10(-7) dilution tube were plated and incubated for 18 h and the number of colonies were counted. Growth of H. pylori was confirmed by the CLO test. All berry extracts significantly (p < 0.05) inhibited H. pylori, compared with controls, and also increased susceptibility of H. pylori to clarithromycin, with OptiBerry demonstrating maximal effects.     

Antimicrobial effects of antioxidants with and without clarithromycin on Helicobacter pylori

Increasing resistance to currently used antimicrobials has resulted in the evaluation of other agents that have antimicrobial activity against Helicobacter pylori. H. pylori American Type Culture Collection (ATCC) strain 49503 (a toxin-producing strain known to be associated with gastric cancer) was grown, a cell suspension prepared in 2 mL PBS and diluted 10-fold. One hundred L of this cell suspension was added to vitamin C 0.5%, vitamin E 0.5%, garcinol 100 g/mL, Protykin® (containing 50% rans-resveratrol) 100 g/mL and garcinol + Protykin® 100 g/mL in Lennox broth, and incubated for 16 h under microaerophilic conditions. Three replicates of 10 L from each 10^–7 dilution tube were plated, colonies were counted after 16 h, and growth of H. pylori was confirmed by the CLO® test. These colony counts were compared to control cultures without the addition of any antioxidants. The experiments were then repeated with the addition of 15 g/mL of clarithromycin to experimental and control samples. Enhanced killing of H. pylori by 37.6% was noted when vitamin C was added, which increased to 66% when clarithromycin was added, compared to controls (p < 0.05). With garcinol and Protykin® alone there was 91.4 and 87% killing of H. pylori, respectively, while a combination of garcinol + Protykin® resulted in 90.8% killing compared to controls (p < 0.05). When clarithromycin was added, there was 76.3% increased killing with garcinol alone, 55.3% with Protykin® alone, and 73.7% with garcinol + Protykin® compared to controls (containing clarithromycin) (p < 0.05). Vitamin E had no effect on H. pylori growth compared to controls. We conclude from this study that some antioxidants such as vitamin C, garcinol and Protykin®, but not vitamin E, may have potential as antimicrobial agents against H. pylori. (Mol Cell Biochem 270: 125–130, 2005)     

Rat uterine polyamine biosynthetic decarboxylase activities following multiple injections of estradiol-17 beta and/or estriol

A single injection of 0.5 micrograms estradiol-17 beta (E2) plus 0.5 micrograms estriol (E3) stimulated a different pattern in 22-24 day-old rat uterine ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMDC) activities than was induced by either a single injection of 0.5 micrograms E2 or multiple injections of 0.5 micrograms E3. Differences included alterations in enzyme activity peak timing as well as activity duration. Every 3 hour injections of 0.05 micrograms E2 induced maximum uterine ODC activity at 4, 24, 32, and 40 hours, intermediate activity at 48, 64, and 72 hours as well as a small peak by 56 hours. When 0.05 micrograms E2 plus 0.05 micrograms E3 were injected simultaneously every 3 hours, the ODC activity pattern was similar except that activity fell to intermediate levels by 40 hours. It is suggested that E3 alterations of E2 induced uterine enzyme activities (when monitored at frequent intervals) could be physiological alterations in uterine growth responses due to E2-E3 hormone interactions. However, there appeared to be no differences between E2 or E2 plus E3 induction of DNA synthesis and luminal epithelial cell height and cross-sectional area or ODC and SAMDC activities when measured at 24, 48, or 72 hours.     

Concanavalin A distorts the beta-GlcNAc-(1-->2)-Man linkage of beta- GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-- >6)]-Man upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.      

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.      

Glycosylated collagen

Collagen was separated from segments of thoracic aorta excised from normal and streptozotocin-induced diabetic rats. The extent of collagen glycosylation was determined using a colorimetric chemical procedure specific for the detection of ketoamine-linked hexoses in proteins. Diabetic rats exhibited a significant increase in glycosylated collagen as compared to normal animals. Glycosylated collagen may contribute to the development of diabetic vascular complications.     

MARCH6 and TRC8 facilitate the quality control of cytosolic and tail‐anchored proteins

Misfolded or damaged proteins are typically targeted for destruction by proteasome‐mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome‐mediated degradation of the soluble misfolded reporter, mCherry‐CL1, involves two ER‐resident E3 ligases, MARCH6 and TRC8. mCherry‐CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail‐anchored protein heme oxygenase‐1 (HO‐1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO‐1 following intramembrane proteolysis. Our results highlight how ER‐resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail‐anchored proteins.     

Poor physical fitness is independently associated with mild cognitive impairment in elderly Koreans

The purpose of this study was to investigate the association between physical fitness and mild cognitive impairment (MCI) in elderly Koreans. This was a cross-sectional study that involved 134 men and 299 women aged 65 to 88 years. Six senior fitness tests were used as independent variables: 30 s chair stand for lower body strength, arm curl for upper body strength, chair-sit-and-reach for lower body flexibility, back scratch for upper body flexibility, 8-ft up-and-go for agility/dynamic balance, and 2-min walk for aerobic endurance. Global cognitive function was assessed using the Korean version of the Mini-Mental State Examination (MMSE). Potential covariates such as age, education levels, blood lipids, and insulin resistance (IR) markers were also assessed. Compared to individuals without MMSE-based MCI, individuals with MMSE-based MCI had poor physical fitness based on the senior fitness test (SFT). There were significant positive trends observed for education level (p=0.001) and MMSE score (p<0.001) across incremental levels of physical fitness in this study population. Individuals with moderate (OR=0.341, p=0.006) and high (OR=0.271, p=0.007) physical fitness based on a composite score of the SFT measures were less likely to have MMSE-based MCI than individuals with low physical fitness (referent, OR=1). The strength of the association between moderate (OR=0.377, p=0.038) or high (OR=0.282, p=0.050) physical fitness and MMSE-based MCI was somewhat attenuated but remained statistically significant even after adjustment for the measured compounding factors. We found that poor physical fitness was independently associated with MMSE-based MCI in elderly Koreans.