OBJECTIVE: To evaluate the cytotoxic, apoptotic, mutagenic and immunomodulatory activities of Kaempferia galanga Linn. (KG) extract and ethyl-p-methoxycinnamate (EPMC) in vitro. METHODS: The present study investigated the cytotoxic [using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test], apoptotic (using a mitochondrial membrane potential assay), mutagenic (using a micronucleus test) and immunomodulatory (using flow cytometry) activities of the ethanolic extract of KG and its bioactive component, EPMC, against two cholangiocarcinoma (CCA) cell lines, CL-6 and HuCCT1, and one normal human cell line, OUMS-36T-1F. RESULTS: Both KG extract and EPMC exhibited moderate cytotoxic activity against both CCA cells. The cytotoxic activity was supported by their concentration-dependent induction of apoptosis. CL-6 was most sensitive (3-4 fold) and selective to 5-fluorouracil (5-FU), compared with KG extract and EPMC [median half inhibiting concentration (IC) and selectivity index (SI) were 23.01 μg/mL and 17.32; 78.41 μg/mL and 4.44; 100.76 μg/mL and 2.20, respectively for 5-FU vs. KG extract vs. EPMC]. HuCCT1 was relatively more sensitive and selective to 5-FU and EPMC than KG extract [median IC and SI were 66.03 μg/mL and 6.04; 60.90 μg/mL and 3.65; 156.60 μg/mL and 2.23, respectively for 5-FU vs. EPMC vs. KG extract]. EPMC produced relatively potent cytotoxic activity against polymorphonuclear cells (IC = 92.20 μg/mL). KG extract and EPMC exhibited concentration-dependent mutagenic activity, as well as inhibition of tumor necrosis factor-α and interleukin-6. CONCLUSION Considering cytotoxic, apoptotic, immunomodulatory and mutagenic activities, further development of KG as a drug candidate is likely to focus on the oral pharmaceutical formulation of a standardized KG extract rather than isolated compounds.

Malaria is one of the most important public health problems in tropical areas on the globe. Several factors are associated with susceptibility to malaria and disease severity, including innate immunity such as blood group, hemoglobinopathy, and heme oxygenase-1 (HO-1) polymorphisms. This study was carried out to investigate association among ABO blood group, thalassemia types and HO-1 polymorphisms in malaria. The malarial blood samples were collected from patients along the Thai-Myanmar border. Determination of ABO blood group, thalassemia variants, and HO-1 polymorphisms were performed using agglutination test, low pressure liquid chromatography and polymerase chain reaction, respectively. Plasmodium vivax was the major infected malaria species in the study samples. Distribution of ABO blood type in the malaria-infected samples was similar to that in healthy subjects, of which blood type O being most prevalent. Association between blood group A and decreased risk of severe malaria was significant. Six thalassemia types (30%) were detected, i.e., hemoglobin E (HbE), β-thalassemia, α-thalassemia 1, α-thalassemia 2, HbE with α-thalassemia 2, and β-thalassemia with α-thalassemia 2. Malaria infected samples without thalassemia showed significantly higher risk to severe malaria. The prevalence of HO-1 polymorphisms, S/S, S/L and L/L were 25, 62, and 13%, respectively. Further study with larger sample size is required to confirm the impact of these 3 host genetic factors in malaria patients.
Agglutination Tests ; Blood Group Antigens ; Chromatography, Liquid ; Healthy Volunteers ; Heme Oxygenase (Decyclizing) ; Heme Oxygenase-1 ; Heme ; Hemoglobin E ; Hemoglobinopathies ; Hemoglobins ; Humans ; Immunity, Innate ; Malaria ; Plasmodium vivax ; Polymerase Chain Reaction ; Prevalence ; Public Health ; Sample Size ; Thalassemia

OBJECTIVE To formulate atractylodin-loaded poly (lactic- co- glycolic acid) (PLGA) nanoparticles and characterize the prepared nanoparticle formulation.METHODS The nanoparticle formu-lation was developed using solvent displacement method. The encapsulation and loading efficiency were characterized and particle size, and zeta potential were determined by dynamic light scattering technique.Drug release was assessed in vitro.RESULTS The size(mean±SD of diameter)of the prepared atractylodin-loaded PLGA nanoparticles were (161.27 ± 1.87)nm with narrow size distribution (mean PDI: 0.068±0.015)and zeta potential(28.83±0.35)mV.The encapsulation and loading efficiency were (48.31±0.83)% and(2.15±0.04)%,respectively.Drug release from atractylodin-loaded PLGA nanoparticles was observed up to (87.70 ± 0.47)% in 72 h with biphasic manner. Moreover, the nanoparticles were found to be freely dispersible in water without aggregation. CONCLUSION Results suggest that PLGA nanoparticles may be used as an effective drug delivery system for atractylodin.The anti-cholangiocar-cinoma activity of this nanoparticle formulation is required.

OBJECTIVE To identify potential cell signaling pathways and protein targets of the active compound isolated from Atracylodes lancea "atractylodin" in cholangiocarcinoma, using proteomics approach. METHODS The holangiocar- cinoma cell line was exposed with atractylodin for 3 and 6 h and the proteins from both intra- and extra- cellular components were extracted. The extract proteins were separated by SDS-PAGE and digested with trypsin.The LC-MS/MS was applied to identify proteins. Signaling pathways and protein expression were analyzed by MASCOT and STITCH software.RESULTS A total of 4,323 and 4,318 proteins were identified from intra-and extracellular components,respectively. Six intracellular proteins were linked with the signaling pathways (apoptosis, cell cycle control, and PI3K-AKT).Four extracellular proteins were linked with the signaling pathways(NF-κB and PI3K-AKT). CONCLUSION All these proteins will further study to confirm the link to the anticholangiocarcinoma ac-tivity of actractylodin.

Objective: To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma (CCA) cell line (CL-6) in comparison with human embryonic fibroblast cell line (OUMS). Methods: Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and CellEvent™ Caspase 3/7 assay. Results: Based on results of the cytotoxicity test in CL-6 cells, 50% inhibitory concentration (IC

Objective To investigate the role of human host heme-oxygenase-1 (HO-1) in pathogenesis of cerebral malaria in the in vitro model. Methods The effect of human host HO-1 [human brain microvascular endothelial cell (HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells (iRBC) through measurement of the enzymatic products iron and bilirubin. Results Following exposure to the HO-1 inducer CoPPIX at all concentrations, the HBMEC cells apoptosis occurred, which could be prominently observed at 15 μM of 3 h exposure. In contrast, there was no significant change in the morphology in the non-exposed iRBC at all concentrations and exposure time. This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin, of which the highest levels (106.03 and 1753.54% of baseline level, respectively) were observed at 15 μM vs. 20 μM at 3 h vs. 24 h exposure. For the effect of the HO-1 inhibitor ZnPPIX, HBMEC cell morphology was mostly unchanged, but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h (37.17% of baseline level). The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed (highest effect at 10 μM and 3 h exposure). Conclusions Results provide at least in part, insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.

Objective To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model. Methods Nine Syrian hamsters were divided into 3 groups as follows (n = 3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavalin A (ConA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS. Results Among the 24 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform ×3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups. Conclusions NDRG1, FETUB and NSFL1C glycoproteins isolated by ConA-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.

Objective To preliminarily investigate the possible role of prostaglandin D

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and alpha-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and alpha-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.
Female ; Hemoglobins/genetics/metabolism ; Humans ; Malaria, Falciparum/blood/complications/*genetics/parasitology ; Malaria, Vivax/blood/complications/*genetics/parasitology ; Male ; Middle Aged ; Plasmodium falciparum/physiology ; Plasmodium vivax/physiology ; Thailand/epidemiology ; Thalassemia/blood/complications/epidemiology/*genetics

Genetic polymorphisms of pvdhfr and pvdhps genes of Plasmodium vivax were investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal. The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP. Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia. All samples carried mutant combination alleles of pvdhfr and pvdhps. The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively. Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles. New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.
Amino Acid Sequence ; Bangladesh ; Base Sequence ; Dihydropteroate Synthase/*genetics ; Humans ; Malaria, Vivax/*parasitology ; Molecular Sequence Data ; Nepal ; Philippines ; Plasmodium vivax/*enzymology/*genetics/isolation & purification ; *Polymorphism, Genetic ; Tetrahydrofolate Dehydrogenase/*genetics