Chemistry,physiological properties,and microbial production of hydroxycitric acid

The tropical plants Garcinia cambogia and Hibiscus subdariffa produce hydroxycitric acid (HCA), of which the absolute configurations are (2S,3S) and (2S,3R), respectively. (2S,3S)-HCA is an inhibitor of ATP-citrate lyase, which is involved in fatty acid synthesis. (2S,3R)-HCA inhibits pancreatic α-amylase and intestinal α-glucosidase, leading to a reduction in carbohydrate metabolism. In this study, we review current knowledge on the structure, biological occurrence, and physiological properties of HCA. The availability of HCA is limited by the restricted habitat of its source plants and the difficulty of stereoselective organic synthesis. Hence, in our recent study, thousands of microbial strains were screened and finally two bacterial strains were, for the first time, found to produce trace amounts of HCA. The HCA variants produced were the Hibiscus-type (2S,3R) enantiomer. Subsequent genome shuffling rapidly generated a mutant population with improved HCA yield relative to the parent strain of bacteria. These bacteria are a potential alternative source of natural HCA.     

Effect of Dietary Garcinia cambogia Extract on Serum Essential Minerals (Calcium, Phosphorus, Magnesium) and Trace Elements (Iron, Copper, Zinc) in Rats Fed with High-Lipid Diet

The aim of the study was to investigate the effect of Garcinia cambogia extract on serum calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), zinc (Zn) and copper (Cu) concentrations in rats fed with the normal or the high-lipid and -cholesterol diet. Thirty 1-year-old female Sprague-Dawley rats (pathogen-free), weighing an average of 229 g, were randomly assigned to three experimental groups of ten animals each. Diets and tap water were given ad libitum for 75 days. Group 1 (control group) was fed with basal diet (2 % liquid vegetable oil, 0 % cholesterol), while the diets of groups 2 and 3 contained vegetable oil (2 % liquid vegetable oil and 5 % hydrogenated vegetable oil) and cholesterol (3 %) in high levels. 4,5 % G. cambogia extract containing 65 % HCA was added to the diet of group 3 as from day 45. Blood samples were withdrawn on days 0, 45 and 75. Serum mineral levels were analyzed using standard enzymatic colorimetric methods with a spectrophotometer. All significant differences were p<0.05. Serum Ca levels were not significantly different between all groups on days 45 and 75. Serum P level was significantly higher in the group fed with high-lipid diet and G. cambogia extract than in the control group on day 45. Serum Mg level was significantly higher in group 2 than in the control group on day 45. Serum Fe levels were significantly lower in the control group than in the other groups on days 45 and 75. Serum Zn level of the group fed with high-lipid diet and G. cambogia extract was significantly higher than in the control group on day 75. Serum Cu levels were significantly higher in group 2 than in the control group, and in group 3 than in group 2 on day 75. In conclusion, a diet containing the high fat amounts may lead to the increase in circular levels of some minerals due to the short-chain fatty acid production lowering the luminal pH which increases mineral solubility, or serving as a fuel for mucosal cells and stimulating cell proliferation in the large intestine. G. cambogia extract may be used in the P and Cu deficiencies due to increases resulting in the present P and Cu amounts in G. cambogia extract, or the use of phytate P in diet. It was hoped that with further evidence-based study this product will enter to mainstream medicines.     

Metabolomics reveals the mechanism of (−)-hydroxycitric acid promotion of protein synthesis and inhibition of fatty acid synthesis in broiler chickens

(−)-Hydroxycitric acid (HCA), a major component of Garcinia cambogia extracts, has been shown to suppress BW gain and fat accumulation in animals and humans. However, the mechanism remains unknown. In this study, gas chromatography-mass spectrometry was used to analyse serum metabolites, and principal component analysis and partial least-squares-discriminant analysis models were generated to analyse serum metabolite changes in broiler chickens after the administration of (−)-HCA at 0, 1000, 2000 and 3000 mg/kg diets for 28 days. Metabolites showing significant changes were screened by ‘variable importance in the projection’ plots. The results showed that 20 metabolites in the 1000 mg/kg (−)-HCA treatment group and 16 metabolites in 3000 mg/kg (−)-HCA treatment group were significantly altered. Metabolites pathway enrichment analysis indicated that these metabolites were mainly associated with metabolism of amino acids, protein synthesis, citric acid cycle, and uric acid and fatty acid synthesis. The data indicated that (−)-HCA promoted protein synthesis by regulating the metabolic directions of amino acids. At the same time, (−)-HCA treatment inhibited fatty acid synthesis by promoting the citric acid cycle, resulting in reduced cytosolic acetyl-CoA content in broiler chickens. The present study identified global changes in metabolites and analysed the main canonical metabolic pathways in broiler chickens supplemented with (−)-HCA. These results will deepen our understanding of the mechanism of (−)-HCA’s effects in animals.     

Absolute configuration of hydroxycitric acid produced by microorganisms

Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer.     

Genome shuffling of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. U121 for improved production of hydroxycitric acid

(2S, 3R)-Hydroxycitric acid (HCA) from Hibiscus subdariffa inhibits pancreatic α-amylase and intestine α-glucosidase, leading to reduction of carbohydrate metabolism. In our previous study, Streptomyces sp. U121 was identified as a producer of (2S, 3R)-HCA [Hida et al. (2005) Bioscience, Biotechnology, and Biochemistry 69:1555–1561]. Here, we applied genome shuffling of Streptomyces sp. U121 to achieve rapid improvement of HCA production. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing fivefold more HCA over wild type was obtained by three rounds of genome shuffling. For efficient screening of the mutant library, trans-epoxyaconitic acid (EAA), an antibiotic analog of HCA, was utilized. EAA inhibited the regeneration of nonfused protoplasts, resulting in selective screening of shuffled strains. Mutant strains with enhanced EAA resistance exhibited significantly higher HCA production in liquid media. Furthermore, the best mutant showed increased cell growth in flask culture, as well as increased HCA production.     

Integrated analysis of proteomics-delineated and metabolomics-delineated hepatic metabolic responses to (−)-hydroxycitric acid in chick embryos

(−)-Hydroxycitric acid [(−)-HCA] is widely used as a nutritional supplement to control body weight and fat accumulation in animals and humans, whereas the underlying biochemical mechanism is unclear. Broiler chicken was used as a model for studies of obesity due to its natural hyperglycemia and being insulin resistant. The current study aimed to obtain a systematic view of serum metabolites and hepatic proteins and well understand the mechanism of hepatic metabolic response to (−)-HCA treatment in chick embryos. The results showed that 22, 90, and 82 of differentially expressed proteins were identified at E14d, E19d, and H1d in chick embryos treated with (−)-HCA, respectively. Meanwhile, 5, 83, and 88 of serum metabolites significantly changed at E14d, E19d, and H1d in chick embryos after (−)-HCA treatment. Bioinformatics analysis showed that the key proteins and metabolites, which were significantly altered in chick embryos treated with (−)-HCA, were mainly involved in the citrate cycle, glycolysis/gluconeogenesis, fatty acid metabolism, and pyruvate metabolism. Our data indicated that (−)-HCA treatment might promote fat metabolism via regulating the key protein expression levels and metabolite contents in the citrate cycle, glycolysis/gluconeogenesis, and oxidative phosphorylation during chicken embryonic development. These results will deepen our understanding of the mechanism of fat reduction by (−)-HCA and provide substantial information for (−)-HCA as a nutritional supplement to control body weight gain and curb obesity-related diseases.     

Comparison of solution and crystal properties of Co(II)-substituted human carbonic anhydrase II

The visible absorption of crystals of Co(II)-substituted human carbonic anhydrase II (Co(II)-HCA II) were measured over a pH range of 6.0-11.0 giving an estimate of pK_a 8.4 for the ionization of the metal-bound water in the crystal. This is higher by about 1.2 pK_a units than the pK_a near 7.2 for Co(II)-CA II in solution. This effect is attributed to a nonspecific ionic strength effect of 1.4 M citrate in the precipitant solution used in the crystal growth. A pK_a of 8.3 for the aqueous ligand of the cobalt was measured for Co(II)-HCA II in solution containing 0.8 M citrate. Citrate is not an inhibitor of the catalytic activity of Co(II)-HCA II and was not observed in crystal structures. The X-ray structures at 1.5-1.6 Å resolution of Co(II)-HCA II were determined for crystals prepared at pH 6.0, 8.5 and 11.0 and revealed no conformational changes of amino-acid side chains as a result of the use of citrate. However, the studies of Co(II)-HCA II did reveal a change in metal coordination from tetrahedral at pH 11 to a coordination consistent with a mixed population of both tetrahedral and penta-coordinate at pH 8.5 to an octahedral geometry characteristic of the oxidized enzyme Co(III)-HCA II at pH 6.0.     

Physico-chemical properties of a novel (-)-hydroxycitric acid extract and its effect on body weight, selected organ weights, hepatic lipid peroxidation and DNA fragmentation, hematology and clinical chemistry, and histopathological changes over a period of 90 days

Garcinia cambogia-derived (-)-hydroxycitric acid (HCA) is a popular and natural supplement for weight management. HCA is a competitive inhibitor of the enzyme ATP citrate lyase, which catalyzes the conversion of citrate and coenzyme A to oxaloacetate and acetyl coenzyme A (acetyl CoA) in the cytosol. Acetyl CoA is used in the synthesis of fatty acids, cholesterol and triglycerides, and in the synthesis of acetylcholine in the central nervous system. Studies have demonstrated the efficacy of a novel 60% calcium-potassium salt of HCA derived from Garcinia cambogia (HCA-SX, Super CitriMax) in weight management. Results have shown that HCA-SX promotes fat oxidation, enhances serotonin release and availability in the brain cortex, normalizes lipid profiles, and lowers serum leptin levels in obese subjects. Acute oral, acute dermal, primary dermal irritation and primary eye irritation toxicity, as well as Ames bacterial reverse mutation studies and mouse lymphoma tests have demonstrated the safety of HCA-SX. However, no detailed long-term safety of HCA-SX or any other HCA extract has been previously assessed. We evaluated the dose- and time-dependent effects of HCA-SX in Sprague-Dawley rats on body weight, selected organ weights, hepatic lipid peroxidation and DNA fragmentation, hematology and clinical chemistry over a period of 90 days. Furthermore, a 90-day histopathological evaluation was conducted. The animals were treated with 0, 0.2, 2.0 and 5.0% HCA-SX of feed intake and were sacrificed on 30, 60 or 90 days of treatment. The body weight and selected organ weights were assessed and correlated as a % of body weight and brain weight at 90 days of treatment. A significant reduction in body weight was observed in treated rats as compared to control animals. An advancing age-induced marginal increase in hepatic lipid peroxidation was observed in both male and female rats, while no such difference in hepatic DNA fragmentation was observed as compared to the control animals. Furthermore, selected organ weights individually and as a % of body weight and brain weight at 90 days of treatment exhibited no significant difference between the groups. No difference was observed in hematology and clinical chemistry or the histopathological evaluation. Taken together, these results show that 90 day treatment of HCA-SX results in a reduction in body weight, and does not cause any changes in major organs or in hematology, clinical chemistry, and histopathology.     

Bioactive flavonoid p-hydroxycinnamic acid stimulates osteoblastogenesis and suppresses adipogenesis in bone marrow culture

The bioactive flavonoid p-hydroxycinnamic acid (HCA), which is an intermediate-metabolic substance in plants and fruits, is synthesized from tyrosine. The biological effect of HCA is poorly understood. Among cinnamic acid and its related compounds, HCA has a specific-anabolic effect on bone, being found to stimulate osteoblastogenesis and to inhibit osteoclastogenesis through the suppression of NF-κB signaling, thereby preventing bone loss. Bone marrow mesenchymal stem cells give rise to ostoblasts and adipocytes. HCA might therefore have effects on osteoblastogenesis and adipogenesis in bone marrow culture. This study demonstrates (1) that HCA has stimulatory effects on osteoblastogenesis and mineralization and suppressive effects on adipogenesis in mouse bone marrow culture and (2) that HCA depresses adipogenesis in mouse 3T3-L1 preadipocytes in vitro. Such effects of HCA might be involved in the differentiation of mesenchymal stem cells.     

Cadmium stimulates the accumulation of salicylic acid and its putative precursors in maize (Zea mays) plants

The effect of 10, 25 and 50 µ M Cd(NO₃)₂ on the salicylic acid (SA) metabolism was investigated in young maize seedlings ( Zea mays L., hybrid Norma). Cadmium (Cd) was translocated into the leaves and induced oxidative damage, as indicated by the reduced chlorophyll content, the decreased quantum efficiency of photosystem II and the enhanced malondialdehyde (MDA) content, especially after 7 days. The activity of glutathione reductase (EC 1.6.4.2) increased from the fourth day and that of guaiacol peroxidase (EC 1.11.1.7) after 7 days of Cd stress compared with the control leaves. These effects of Cd exhibited a correlation with the concentration. Under these conditions, Cd did not affect the MDA content or the antioxidant enzyme activities in the roots. After 7 days, Cd increased the levels of free and bound forms of benzoic acid (BA), O -coumaric acid ( O -hydroxy-cinnamic) ( O- HCA) and SA in the leaves, but in the roots, only the 50 µ M rate of Cd caused changes in the free O -HCA acid and bound BA content.     

Analysis of warfarin enantiomers: Chromatographic separation of six stereoisomeric esters prepared from (−)-(1S,2R,4R)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2- carboxylic acid

Reaction of rac-warfarin, (?)-(1S,2R,4R)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2- carboxylic acid [(?)-HCA] and carbodiimide reagents gave two noncyclic ketonic diastereoisomeric derivatives whereas rac-warfarin and (?)-HCA acid chloride with 4-(dimethylamino)pyridine gave four cyclic hemiketal diastereoisomeric ester derivatives. The structure and stereochemistry of diastereoisomeric esters prepared from warfarin and p-chlorowarfarin were determined from ¹H- and ¹³C-NMR spectra, mass spectra, and hydrolysis to warfarin and p-chlorowarfarin enantiomers. The structure and stereochemistry of one of the cyclic hemiketal diastereoisomeric derivatives of warfarin are supported by an X-ray crystallographic determination. Mechanisms for the formation of all products are proposed. © 1994 Wiley-Liss, Inc.     

Inflammation stimulates niacin receptor (GPR109A/HCA2) expression in adipose tissue and macrophages

Many of the beneficial and adverse effects of niacin are mediated via a G protein receptor, G protein-coupled receptor 109A/hydroxycarboxylic acid 2 receptor (GPR109A/HCA2), which is highly expressed in adipose tissue and macrophages. Here we demonstrate that immune activation increases GPR109A/HCA2 expression. Lipopolysaccharide (LPS), TNF, and interleukin (IL) 1 increase GPR109A/HCA2 expression 3- to 5-fold in adipose tissue. LPS also increased GPR109A/HCA2 mRNA levels 5.6-fold in spleen, a tissue rich in macrophages. In peritoneal macrophages and RAW cells, LPS increased GPR109A/HCA2 mRNA levels 20- to 80-fold. Zymosan, lipoteichoic acid, and polyinosine-polycytidylic acid, other Toll-like receptor activators, and TNF and IL-1 also increased GPR109A/HCA2 in macrophages. Inhibition of the myeloid differentiation factor 88 or TIR-domain-containing adaptor protein inducing IFNβ pathways both resulted in partial inhibition of LPS stimulation of GPR109A/HCA2, suggesting that LPS signals an increase in GPR109A/HCA2 expression by both pathways. Additionally, inhibition of NF-κB reduced the ability of LPS to increase GPR109A/HCA2 expression by ∼50% suggesting that both NF-κB and non-NF-κB pathways mediate the LPS effect. Finally, preventing the LPS-induced increase in GPR109A/HCA2 resulted in an increase in TG accumulation and the expression of enzymes that catalyze TG synthesis. These studies demonstrate that inflammation stimulates GPR109A/HCA2 and there are multiple intracellular signaling pathways that mediate this effect. The increase in GPR109A/HCA2 that accompanies macrophage activation inhibits the TG accumulation stimulated by macrophage activation.     

Citrate-permeable channels in the plasma membrane of cluster roots from white lupin

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (-P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3- than Cl- (PCit/PCl = 26.3), while ORAC was selective for Cl- over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of -P plants, as well as from normal (noncluster) roots of plants grown with 25 microm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of -P plants. However, the IRAC current from +P plants occurred less frequently than in the -P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.     

Gas chromatography/mass spectrometry method to quantify blood hydroxycitrate concentration

Hydroxycitrate (HCA), a popular dietary supplement for weight loss, is a competitive inhibitor of ATP-citrate lyase, an extramitochondrial enzyme involved in the initial steps of de novo lipogenesis (DNL). Although animal studies have shown that HCA effectively inhibits DNL and induces weight loss, these findings have not been consistent in humans. This raises the possibility that the bioavailability of HCA may differ among species. We developed a new GC/MS method to measure HCA levels in blood, using [U-(13)C]citrate (CA*) as internal standard to account for losses associated with the isolation, derivatization, and measurement of HCA. HCA and CA* were derivatized with BSTFA + 10% TMCS and analyzed using PCI/GC/MS (CA*, m/z 471; and HCA, m/z 553). The plasma HCA concentration was measured over a 3.5-h period in four subjects having ingested 2 g of HCA. Their plasma HCA concentration ranged from 0.8 to 8.4 microg/ml 30 min and 2 h after ingestion, respectively. These results demonstrate that when taken acutely, HCA is absorbed, yet present in small quantities in human plasma. This simple method requiring minimal sample preparation is able to measure trace amounts of HCA with accuracy and precision.     

Studies on reactions of oxidizing sulfur-sulfur three-electron-bond complexes and reducing alpha-amino radicals derived from OH reaction with methionine in aqueous solution

The technique of pulse radiolysis with spectrophotometric detection has been used to investigate the possibility of electron transfer reactions between oxidizing sulfur-sulfur three-electron-bond complexes (Met2/S thereforeS+), or reducing alpha-amino radicals (CH3SCH2CH2CH.NH2) derived from reaction of methionine with OH radicals and hydroxycinnamic acid (HCA) derivatives, riboflavin (RF) or flavin adenine dinucleotide (FAD), respectively. The HCA derivatives, such as caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, widely distributed phenolic acids in fruit and vegetables, have been identified as good antioxidants previously can rapidly and efficiently repair oxidizing three-electron-bond complexes via electron transfer. RF and FAD can oxidize reducing alpha-amino radicals derived from methionine. The electron transfer rate constants approximately 10(9) dm3 x mol(-1)x s(-1) were determined by following the build-up kinetics of species produced.     

Nutrigenomic Analysis of Diet-Gene Interactions on Functional Supplements for Weight Management

Recent advances in molecular biology combined with the wealth of information generated by the Human Genome Project have fostered the emergence of nutrigenomics, a new discipline in the field of nutritional research. Nutrigenomics may provide the strategies for the development of safe and effective dietary interventions against the obesity epidemic. According to the World Health Organization, more than 60% of the global disease burden will be attributed to chronic disorders associated with obesity by 2020. Meanwhile in the US, the prevalence of obesity has doubled in adults and tripled in children during the past three decades. In this regard, a number of natural dietary supplements and micronutrients have been studied for their potential in weight management. Among these supplements, (–)-hydroxycitric acid (HCA), a natural extract isolated from the dried fruit rind of Garcinia cambogia, and the micronutrient niacin-bound chromium(III) (NBC) have been shown to be safe and efficacious for weight loss. Utilizing cDNA microarrays, we demonstrated for the first time that HCA-supplementation altered the expression of genes involved in lipolytic and adipogenic pathways in adipocytes from obese women and up-regulated the expression of serotonin receptor gene in the abdominal fat of rats. Similarly, we showed that NBC-supplementation up-regulated the expression of myogenic genes while suppressed the expression of genes that are highly expressed in brown adipose tissue in diabetic obese mice. The potential biological mechanisms underlying the observed beneficial effects of these supplements as elucidated by the state-of-the-art nutrigenomic technologies will be systematically discussed in this review.Key Words: Insulin resistance, glucose tolerance factor, supplemental chromium, Garcinia cambogia, (-)-hydroxycitric acid, overweight, obesity, diabetes, cardiovascular disease, nutritional interventions, microarrays, nutrigenomics.     

A comparative study on the aluminium- and copper-induced organic acid exudation from wheat roots

It is well established that aluminium (Al) and some heavy metals can elicit organic acid exudation from a range of species. In the present research we found that copper (Cu) can also induce organic acid exudation from the roots of wheat, rye, triticale, maize and soybean. Using intact wheat plants, we made a comparative study of Al- and Cu- induced organic acid exudation. In 5-day-old wheat seedlings, severe Cu stress (40 µ M CuCl₂) mainly induced the exudation of malate and citrate, and Al-tolerant genotypes could release significantly greater amounts of malate than Al-sensitive genotypes. The time course of the exudation of malate and citrate from the roots of 5-day-old seedlings of wheat (cv. Atlas) in 200 µ M AlCl₃ was similar to that in 40 µ M CuCl₂. In older wheat plants (15-day-old), moderate Cu stress (12 µ M CuCl₂) induced the exudation of large amounts of citrate and addition of Al or La sharply reduced Cu-induced citrate exudation, while Cu or La did not affect Al-induced malate efflux. When half of the root system of Atlas wheat was immersed in Al- or Cu-containing solution and the remaining half in Al- or Cu-free solution, organic acids were only exuded into the solution containing Al or Cu. This suggests that no long distance signal transport is involved in organic acid exudation induced by Al or Cu, and that direct contact of Al or Cu with plant roots is a prerequisite for the induction of organic acid exudation. The anion-channel inhibitor niflumic acid (NIF) significantly stimulated the exudation of both citrate and malate from 5-day-old wheat seedlings under severe Al or Cu stress. Our results suggest that Cu-induced organic acid efflux may be a common response, which may play a role in alleviating Cu toxicity in plants.