In Vivo Biochemical and Gene Expression Analyses of the Antioxidant Activities and Hypocholesterolaemic Properties of Tamarindus indica Fruit Pulp Extract

Background

Tamarindus indica (T. indica) is a medicinal plant with many biological activities including anti-diabetic, hypolipidaemic and anti-bacterial activities. A recent study demonstrated the hypolipidaemic effect of T. indica fruit pulp in hamsters. However, the biochemical and molecular mechanisms responsible for these effects have not been fully elucidated. Hence, the aims of this study were to evaluate the antioxidant activities and potential hypocholesterolaemic properties of T. indica, using in vitro and in vivo approaches.

Methodology/Principal Findings

The in vitro study demonstrated that T. indica fruit pulp had significant amount of phenolic (244.9±10.1 mg GAE/extract) and flavonoid (93.9±2.6 mg RE/g extract) content and possessed antioxidant activities. In the in vivo study, hamsters fed with high-cholesterol diet for ten weeks showed elevated serum triglyceride, total cholesterol, HDL-C and LDL-C levels. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters significantly lowered serum triglyceride, total cholesterol and LDL-C levels but had no effect on the HDL-C level. The lipid-lowering effect was accompanied with significant increase in the expression of Apo A1, Abcg5 and LDL receptor genes and significant decrease in the expression of HMG-CoA reductase and Mtp genes. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters also protected against oxidative damage by increasing hepatic antioxidant enzymes, antioxidant activities and preventing hepatic lipid peroxidation.

Conclusion/Significance

It is postulated that tamarind fruit pulp exerts its hypocholesterolaemic effect by increasing cholesterol efflux, enhancing LDL-C uptake and clearance, suppressing triglyceride accumulation and inhibiting cholesterol biosynthesis. T. indica fruit pulp has potential antioxidative effects and is potentially protective against diet-induced hypercholesterolaemia.     

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: an intra and interethnic comparative approach

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1 0/0, GSTT1 0/0, GSTP1 Ile(105)Val, and GSTA1 A/B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM10/0 and GSTT1 0/0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (A/A), 53.9% (A/B), and 21.4% (B/B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.     

Production of wine from mango (Magnifera indica L.) using Saccharomyces and Schizosaccharomyces species isolated from palm wine

Juice extracted from pulp of the mature ripe tropical fruit, mango ( Magnifera indica L.), containing 15.9% soluble solids, was fermented with four strains of yeast isolated from palm wine. Two of the strains belonged to the genus Schizosaccharomyces (T1 and T2) while the other two were Saccharomyces (B2 and M1). The two strains of Schizosaccharomyces were found to be suitable for the production of sweet, table mango wine with alcohol contents of 8.0 and 9.0% for T1 and T2, respectively. The two strains of Saccharomyces were found suitable for the production of dry mango fruit wines containing 10.0% alcohol.     

The liver X receptor ligand T0901317 down-regulates APOA5 gene expression through activation of SREBP-1c

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X receptor (LXR) ligand-mediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found that APOA5 mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5 promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease of APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.     

APOA1 gene polymorphisms in the South Asian immigrant population in the United States

BACKGROUND:

Coronary artery disease (CAD) is a leading cause of death in the United States. South Asian immigrants (SAIs) from the Indian subcontinent living in the US are disproportionately at higher risk of CAD than other immigrant populations. Unique genetic factors may predispose SAIs to increased risk of developing CAD when adopting a Western lifestyle including a higher-fat diet, more sedentary behavior and additional gene-environment interactions. SAIs are known to have low levels of the protective high density lipoprotein (HDL) and an altered function for Apo-lipoprotein A-1 (ApoA1), the main protein component of HDL cholesterol. One gene that may be genetically distinctive in this population is APOA1 which codes for ApoA-1 protein, a potentially important contributing factor in the development of CAD.

MATERIALS AND METHODS:

DNA sequencing was performed to determine the status of the seven single-nucleotide polymorphisms (SNPs) in the APOA1 gene from 94 unrelated SAI adults. Genotypes, allelic frequencies, and intragenic linkage disequilibrium of the APOA1 SNPs were calculated.

RESULTS:

Several polymorphisms and patterns were common among persons of south Asian ethnicity. Frequencies for SNPs T655C, T756C and T1001C were found to be different than those reported in European Caucasian individuals. Linkage disequilibrium was found to be present between most (13 of 15) SNP pairings indicating common inheritance patterns.

CONCLUSIONS:

SAIs showed variability in the sequence of the APOA1 gene and linkage disequilibrium for most SNPS. This pattern of APOA1 SNPs may contribute to decreased levels of HDL cholesterol reported in SAIs, leading to an increased risk for developing CAD in this population.     

Comparative analysis of DNA polymorphisms and phylogenetic relationships among Syzygium cumini Skeels based on phenotypic characters and RAPD technique

The Indian black berry (Syzygium cumini Skeels) has a great nutraceutical and medicinal properties. As in other fruit crops, the fruit characteristics are important attributes for differentiation were also determined for different accessions of S. cumini. The fruit weight, length, breadth, length: breadth ratio, pulp weight, pulp content, seed weight and pulp: seed ratio significantly varied in different accessions. Molecular characterization was carried out using PCR based RAPD technique. Out of 80 RAPD primers, only 18 primers produced stable polymorphisms that were used to examine the phylogenetic relationship. A sum of 207 loci were generated out of which 201 loci found polymorphic. The average genetic dissimilarity was 97 per cent among jamun accessions. The phylogenetic relationship was also determined by principal coordinates analysis (PCoA) that explained 46.95 per cent cumulative variance. The two-dimensional PCoA analysis showed grouping of the different accessions that were plotted into four sub-plots, representing clustering of accessions. The UPGMA (r = 0.967) and NJ (r = 0.987) dendrogram constructed based on the dissimilarity matrix revealed a good degree of fit with the cophenetic correlation value. The dendrogram grouped the accessions into three main clusters according to their eco-geographical regions which given useful insight into their phylogenetic relationships.     

Novel polymorphisms of the APOA2 gene and its promoter region affect body traits in cattle

Apolipoprotein A-II (APOA2) is one of the major constituents of high-density lipoprotein and plays a critical role in lipid metabolism and obesity. However, similar research for the bovine APOA2 gene is lacking. In this study, polymorphisms of the bovine APOA2 gene and its promoter region were detected in 1021 cows from four breeds by sequencing and PCR-RFLP methods. Totally, we detected six novel mutations which included one mutation in the promoter region, two mutations in the exons and three mutations in the introns. There were four polymorphisms within APOA2 gene were analyzed. The allele A, T, T and G frequencies of the four loci were predominant in the four breeds when in separate or combinations analysis which suggested cows with those alleles to be more adapted to the steppe environment. The association analysis indicated three SVs in Nangyang cows, two SVs in Qinchun cows and the 9 haplotypes in Nangyang cows were significantly associated with body traits (P < 0.05 or P < 0.01). The results of this study suggested the bovine APOA2 gene may be a strong candidate gene for body traits in the cattle breeding program.     

Molecular identification of Mango, Mangifera indica L.var. totupura

Mango (>Mangifera indica) belonging to Anacardiaceae family is a fruit that grows in tropical regions. It is considered as the King of fruits. The present work was taken up to identify a tool in identifying the mango species at the molecular level. The chloroplast trnL-F region was amplified from extracted total genomic DNA using the polymerase chain reaction (PCR) and sequenced. Sequence of the dominant DGGE band revealed that Mangifera indica in tested leaves was Mangifera indica (100% similarity to the ITS sequences of Mangifera indica). This sequence was deposited in NCBI with the accession no. GQ927757. ABBREVIATIONS: AFLP - Amplified fragment length polymorphism , cpDNA - Chloroplast DNA, DDGE - Denaturing gradient gel electrophoresis, DNA - Deoxyribo nucleic acid, EDTA - Ethylenediamine tetraacetic acid, HCl - Hydrochloric acid, ISSR - Inter simple sequence repeats, ITS - Internal transcribed spacer, MATAB - Methyl Ammonium Bromide, Na(2)SO(3) - Sodium sulphite, NaCl - Sodium chloride, NCBI - National Centre for Biotechnology Information, PCR - Polymerase chain reaction, PEG - Polyethylene glycol, RAPD - Randomly amplified polymorphic DNA, trnL-F - Transfer RNA genes start codon- termination codon.     

Prediction of genetic risk for dyslipidemia

The purpose of the present study was to identify genetic variants that confer susceptibility to dyslipidemia. A total of 5213 individuals from two independent populations were examined: Subject panel A comprised 3794 individuals who visited participating hospitals; subject panel B comprised 1419 community-dwelling elderly individuals. The genotypes for 100 polymorphisms of 65 candidate genes were determined. The chi(2) test and multivariable logistic regression analysis revealed that seven polymorphisms of APOA5, APOC3, APOA1, ACAT2, and LPL were significantly associated with hypertriglyceridemia, six polymorphisms of APOA5, LIPC, and CYP3A4 with low HDL-cholesterol, and three polymorphisms of APOE and CCR2 with high LDL-cholesterol in subject panel A. For validation of these associations, the same polymorphisms were examined in subject panel B. Six polymorphisms of APOA5, APOC3, APOA1, and LPL were again significantly associated with hypertriglyceridemia, three polymorphisms of APOA5 with low HDL-cholesterol, and two polymorphisms of APOE with high LDL-cholesterol. Serum triglyceride, HDL-cholesterol, and LDL-cholesterol concentrations differed significantly among genotypes of these corresponding polymorphisms in both subject panels. These results indicate that polymorphisms of APOA5, APOC3, APOA1, and LPL are determinants of hypertriglyceridemia and that those of APOA5 and APOE are determinants of low HDL-cholesterol and high LDL-cholesterol, respectively, in Japanese individuals.     

Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T1 seedlings of tomato exposed to metal ions

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T₀). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T₁) produced the highest GUS activity when treated with 150 μM Cu²⁺ compared to the control (without Cu²⁺). However, Zn²⁺ and Fe²⁺ treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T₁ seedlings of tomato when subjected to Cu²⁺ ions.     

Gene expression profiles in genetically different mice infected with Toxoplasma gondii: ALDH1A2, BEX2, EGR2, CCL3 and PLAU

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.