Antibacterial activity of Dioscorea bulbifera Linn. extract and its active component flavanthrinin against skin-associated bacteria.

BACKGROUND: Dioscorea bulbifera Linn. has been used for wound care in Thailand. However, a comprehensive evaluation of its antibacterial activity is required. This study aimed to investigate the antibacterial efficacy of D. bulbifera extract against skin-associated bacteria and isolate and characterize its active antibacterial agent, flavanthrinin. METHODS: Air-dried bulbils of D. bulbifera were pulverised and extracted with hexane, dichloromethane, ethyl acetate, methanol, ethanol, and distilled water; vacuum filtered; concentrated; freeze-dried; and stored at -20 ºC. Antibacterial activity of the extracts was assessed using microdilution techniques against several skin-associated bacteria. Thin-layer chromatography (TLC) bioautography was used to identify the active compounds in the extract, which were fractionated by column chromatography and purified by preparative TLC. The chemical structures of the purified compounds were analysed using nuclear magnetic resonance (NMR). The cytotoxicity of the extract and its active compounds was evaluated in Vero cells. RESULTS: The ethyl acetate extract exhibited distinct inhibition zones against bacteria compared to other extracts. Therefore, the ethyl acetate extract of D. bulbifera in the ethyl acetate layer was used for subsequent analyses. D. bulbifera extract exhibited antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.78-1.56 mg/mL. An active compound, identified through TLC-bioautography, demonstrated enhanced antibacterial activity, with MICs of 0.02-0.78 mg/mL. NMR analysis identified this bioactive compound as flavanthrinin. Both D. bulbifera extract and flavanthrinin-containing fraction demonstrated potent antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and S. epidermidis. The flavanthrinin containing fraction demonstrated low cytotoxicity against Vero cells, showing CC50 values of 0.41 ± 0.03 mg/mL. These values are lower than the MIC value, indicating that this fraction is safer than the initial ethyl acetate extract. CONCLUSIONS: Dioscorea bulbifera extract and its bioactive component flavanthrinin demonstrated significant antibacterial activity against the skin-associated bacteria Staphylococci, including MRSA. Flavanthrinin has potential as a complementary therapeutic agent for managing skin infections owing to its potent antibacterial effects and low cytotoxicity.

Gene Combination of CD44 rs187116, CD133 rs2240688, NF-κB1 rs28362491 and GSTM1 Deletion as a Potential Biomarker in Risk Prediction of Breast Cancer in Lower Northern Thailand.

Background: Biomarkers play an important role in oncology, including risk assessment, treatment prediction, and monitoring the progression of disease. In breast cancer, many genes are used as biomarkers. Since, several SNP variations of hallmark ­ related genes have been reported to be of value in risk prediction in various cancers and populations, some genetic polymorphism loci were combined and reported as biomarkers for use in the risk assessment of breast cancer in Thai people. Methods: Twelve cancer gene hallmarks (15 polymorphic loci) were selected and genotyped in 184 breast cancer patients and 176 healthy individuals in Phitsanulok, Thailand. Results: AA genotype of CD44 rs187116 (c.67+4883G>A), the C allele of CD133 rs2240688 (c.*667A>C), the *2 allele (4 bp deletion) of NF-κB1 rs28362491 and the homozygous null allele genotype of GSTM1 were significantly associated with an increased risk of breast cancer (p<0.05). A combination of these 4 significant loci showed that AA-AA-*1*1-homozygous null allele genotype has the greatest correlation with increased risk of breast cancer (OR = 21.00; 95% CI: 1.77 to 248.11; p = 0.015), followed by GA-AA-*2*2- homozygous null allele genotype (p = 0.037) and GG-AC-*1*2- homozygous null allele genotype (p = 0.028). Conclusion: These findings suggest that the polymorphisms of CD44 rs187116 (c.67+4883G>A), CD133 rs2240688 (c.*667A>C), NF-κB1 rs28362491 and GSTM1 homozygous null allele genotype might be associated with an increased risk of breast cancer, and this gene combination could possibly be used as biomarkers for risk prediction, which would be of benefit in planning health surveillance and cancer prevention.

Antibacterial activity of cuminaldehyde on food-borne pathogens, the bioactive component of essential oil from Cuminum cyminum L. collected in Thailand.

Background Cuminum cyminum L., commonly known as cumin, has been traditionally used in Thai traditional medicine and traditional food flavoring. The present study investigated the chemical composition, antimicrobial activity against all tested major food-borne pathogenic bacteria, and bioactive components of essential oil extracted from C. cyminum L. collected in Thailand. Methods The main components of the essential oil were investigated by gas chromatography-mass spectrometry (GC-MS) technique. Antibacterial activities against Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Salmonella Typhi were investigated by disk diffusion and microdilution method. The presence of the biologically active antibacterial components was also confirmed by the thin-layer chromatography (TLC)-bioautography. Results The main components of the essential oil investigated by GC-MS were cuminaldehyde (27.10%), beta-pinene (25.04%) and gamma-terpinene (15.68%). The essential oil exhibited antibacterial activity against B. cereus, S. aureus, E. coli and S. Typhi. The essential oil showed the strongest antimicrobial activity against B. cereus with a comparable inhibition zone to tetracycline. TLC confirmed the presence of biologically active antibacterial component in the essential oil against all tested food-borne bacteria. It is further demonstrated that cuminaldehyde was the most active compound in TLC-bioautography which inhibited all of tested bacteria. Conclusions Essential oil extracted from C. cyminum L. exhibited antibacterial activity against all tested major food-borne pathogenic bacteria. Cuminaldehyde is a major bioactive component. Our results suggest that the essential oil extracted from C. cyminum L. could be applied as an alternative natural preservative to control food-borne disease and have the potential for further development of new antibacterial agents.

Decreased DNA methylation of a CpG site in the HBAP1 gene in plasma DNA from pregnant women.

OBJECTIVE: The objective of this study is to identify potential CpG site(s) or DNA methylation pattern(s) in the pseudo α-globin 1 gene (HBAP1 gene), the gene which locates in α-thalassemia-1 deletion mutation, to differentiate plasma DNA between pregnant and non-pregnant women. METHOD: DNA methylation profiles of placenta and peripheral blood from the MethBase database were compared to screen differentially methylated regions. This region was confirmed the differential by methylation-sensitive high resolution melt (MS-HRM) analysis. The differential region was used to compare DNA methylation profile of plasma DNA between pregnant and non-pregnant women by bisulfite amplicon sequencing in three levels: overall, individual CpG sites and individual molecules (DNA methylation patterns). RESULT: Using MethBase data, four consecutive CpG sites in the HBAP1 gene were identified as regions of differential DNA methylation between placenta and peripheral blood. The confirmation by MS-HRM showed the differential DNA methylation profile between the placenta and plasma from non-pregnant women. The comparison of DNA methylation profiles between the plasma of pregnant and non-pregnant women showed that, in the overall levels of the four CpG sites, DNA methylation of pregnant women was detected at lower levels than non-pregnant women. In the individual CpG site level, only the second CpG site showed differential DNA methylation between the groups. In the DNA methylation pattern level, there was no strongly significant differences in DNA methylation patterns between the pregnant and non-pregnant groups. CONCLUSION: Our result demonstrated that, in the plasma from pregnant women, only one of the four CpG sites displays a decrease in DNA methylation compared with non-pregnant women. It indicates that this CpG site might be useful for determining the presence or absence of fetal wild-type α-globin gene cluster allele in maternal plasma.

Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion.

In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5'-breakpoint of α-thalassemia Southeast Asian (SEA) deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA.

Changes in the NS1 gene of avian influenza viruses isolated in Thailand affect expression of type I interferon in primary chicken embryonic fibroblast cells.

The non-structural protein 1 (NS1) of avian influenza virus was defined as one of the virulent factors. To understand the effect of NS1 protein of influenza virus H5N1 isolated in Thailand on type I (α/ß) interferon (IFN) synthesis, five reverse genetic viruses were constructed and used as models. The viruses were generated using NS genomic segment from A/Peurto Rico/8/1934 (H1N1) and four avian influenza viruses isolated from the first outbreak in Thailand. All the viruses have the rest of the genome from A/Peurto Rico/8/1934 (H1N1). The constructed viruses were named (1) NS1 PR8/34, (2) NS1 wild type, (3) NS1 L15FD53G, (4) NS1 N171I and (5) NS1 E71K, respectively. The type I (α/ß) IFN gene expression in control and infected primary chicken embryonic fibroblast cells were analyzed by quantitative polymerase chain reaction. The results show that the inhibition of IFN-ß gene expression by NS1 wild type infected cells is stronger than NS1 N171I, NS1 E71K, NS1 PR8/34 and NS1 L15FD53G, respectively. The data suggest that the difference of amino acid sequence of NS1 protein contributes to the IFN-ß antagonist. In contrast, the difference of the NS1 protein does not influence in the IFN-α antagonistic activity.

Detection of distribution of avian influenza H5N1 virus by immunohistochemistry, chromogenic in situ hybridization and real-time PCR techniques in experimentally infected chickens.

Ten specific pathogen free (SPF) chickens were inoculated intranasally with avian influenza virus subtype H5N1. Evaluation revealed distribution of the virus in twelve organs: liver, intestine, bursa, lung, trachea, thymus, heart, pancreas, brain, spleen, kidney, and esophagus. Immunohistochemistry (IHC), chromogenic in situ hybridization (CISH), and real-time polymerase chain reaction (PCR) were developed and compared for detection of the virus from the organs. The distribution of avian influenza H5N1 in chickens varied by animal and detecting technique. The heart, kidneys, intestines, lungs, and pancreas were positive with all three techniques, while the others varied by techique. The three techniques can be used to detect avian influenza effectively, but the pros and cons of each technique need to be determined. The decision of which technique to use depends on the objective of the examination, budget, type and quality of samples, laboratory facilities and technician skills.

Comparison of neuraminidase activity of influenza A virus subtype H5N1 and H1N1 using reverse genetics virus.

Neuraminidase (NA) is an envelope surface glycoprotein of influenza A viruses. It cleaves alpha-(2,3) or alpha-(2,6) glycosidic linkage between a terminal sialic acid residue of the host cell receptor and hemagglutinin of the viral envelope, thus releasing viral progeny from the infected cell. In this study, a reassortant virus (H1N1-NA-H5N1) containing the NA gene from A/duck/Phitsanulok/ NIAH6-5-0001/2007 (H5N1) virus and seven remaining genetic segments from A/ Puerto Rico/8/1934 (H1N1) was constructed using reverse genetic technique. NA activity of H1N1-NA-H5N1 virus was lower than that of A/Puerto Rico/8/1934 (H1N1), and NA activity of A/duck/Phitsanulok/NIAH6-5-0001/2007 study (H5N1) was the lowest among them (p < 0.05). To our knowledge, this is the first comparative study of NA activity of H1N1 and H5N1 virus using reverse genetic technique. It also indicates that the NA gene may be expressed at a higher level in the H1N1 infected cell than the H5N1 infected cell.

Genetic characterization of nonstructural genes of H5N1 avian influenza viruses isolated in Thailand in 2004-2005.

The outbreak of highly pathogenic avian influenza (HPAI) viruses has severely disrupted poultry production and trade. Humans have been infected with HPAI H5N1 viruses and many have died. The nonstructural (NS) proteins of the virus are a factor that determines virulence. In this report, 80 NS genes of H5N1 HPAI viruses isolated from Thailand were completely sequenced and phylogenically analyzed. The percentages of identity and variable site NS1 genes were similar to NS2/nuclear export protein (NEP) genes. All NS1 genes from the samples were located in allelic group A. The NS1 and NS2/NEP proteins possess 225 and 121 amino acids, respectively. All NS1 protein samples had five amino acid deletions typical of avian influenza viruses isolated since 2002. An amino acid substitution at position 92 (G92E) of the NS1 protein, known to promote the inhibition of host immune responses, was also found in the samples.

Whole genome sequences of H5N1 influenza a virus isolated from a little grebe in Thailand.

This is the first report of the whole genome sequence of influenza A virus in an aquatic resident bird of Thailand. It was categorized into genotype Z according to its characteristics of a 20 amino acid deletion in neuraminidase and a five amino acid deletion in the nonstructural protein. The indicator for a highly pathogenic trait of the virus is the presence of a polybasic amino acid sequence at the cleavage site of HA0. The feature of resistance to the antiviral drug amantadine is found at the 31st amino acid position of M2 (serine to asparagine). Phylogenic analyses revealed that virus A/little grebe/Thailand/Phichit-01/2004 (H5N1) is closely related to the chicken and human isolates recovered from Thailand. The high degrees of similarity among the sequences and phylogenic trees indicate there was no difference between the viruses isolated from poultry and aquatic birds in Thailand at the time of study. The results also suggest the source of H5N1 avian influenza virus in the little grebe and others in Thailand may have the same origin.

Homogeneity of beta(0)-thalassemia codon 17 (A-->T) alleles in Northern Thailand using a direct DNA sequencing method.

The aim of this study was to characterize beta-globin gene micro-haplotype polymorphisms (frameworks) associated with a beta-thalassemia mutations common in Northern Thailand using a direct DNA sequencing method. A total of 11 beta-thalassemia major patients homozygous for the codon 17 (A-->T) mutation admitted to Chiang Mai University Hospital were examined. All 22 alleles were found to contain the Asian framework 3A. The homogeneity of the framework associated with the codon 17 (A-->T) mutation indicates a relatively recent origin of the codon 17 (A-->T) mutation. Similar studies in other East Asian populations may provide information concerning the origin and the migrational spread of this beta-thalassemia mutation.

Spontaneous mutation of the hemoglobin Leiden (beta 6 or 7 Glu-->0) in a Thai girl.

We report a case of 12-year old Thai girl suffering from mild non-transfusion-dependent thalassemia intermedia. She is the single child in her family. On examination she looked pale; there was no hepatosplenomegaly. The Hb concentration was 9 g/dL. Hb typing and molecular mutation study revealed compound heterozygosity for HbE and Hb Leiden (alpha2beta26/7-Glu, codon 6/7-GAG). The proportion of HbE was 47% whereas that of Hb Leiden was 39%. The patient had no HbA. Hb typing of her father and mother revealed HbE trait, and no Hb Leiden was demonstrated. As the paternity test confirmed the parenthood, we assume that Hb Leiden has arisen by spontaneous mutation. A study of the beta< or= -globin gene framework by molecular cloning and subsequent DNA sequencing of the beta-globin gene in the members of the family indicated that the Hb Leiden mutation occurred on a maternal inherited chromosome. The deletion of codon 6 or 7 (-GAG) of the beta-globin gene in the patient may be due to an unequal crossing over during the mother's oogenesis.