Evaluation of Nano-Wall Material for Production of Novel Lyophilized-Probiotic Product.

Lyophilization is one of the most used methods for bacterial preservation. In this process, the cryoprotectant not only largely decreases cellular damage but also plays an important part in the conservation of viability during freeze-drying. This study investigated using cryoprotectant and a mixture of the cryoprotectant to maintain probiotic activity. Seven probiotic strains were considered: (Limosilactobacillus reuteri KUKPS6103; Lacticaseibacillus rhamnosus KUKPS6007; Lacticaseibacillus paracasei KUKPS6201; Lactobacillus acidophilus KUKPS6107; Ligilactobacillus salivarius KUKPS6202; Bacillus coagulans KPSTF02; Saccharomyces cerevisiae subsp. boulardii KUKPS6005) for the production of a multi-strain probiotic and the complex medium for the lyophilized synbiotic production. Cholesterol removal, antioxidant activity, biofilm formation and gamma aminobutyric acid (GABA) production of the probiotic strains were analyzed. The most biofilm formation occurred in L. reuteri KUKPS6103 and the least in B. coagulans KPSTF02. The multi-strain probiotic had the highest cholesterol removal. All the probiotic strains had GABA production that matched the standard of γ-aminobutyric acid. The lyophilized synbiotic product containing complex medium as a cryoprotectant and wall material retained a high viability of 7.53 × 108 CFU/g (8.89 log CFU/g) after 8 weeks of storage. We found that the survival rate of the multi-strain probiotic after freeze-drying was 15.37% in the presence of complex medium that was used as high performing wall material. Our findings provided a new type of wall material that is safer and more effective and, can be extensively applied in relevant food applications.

In silico multiscale drug design to discover key structural features of potential JAK2 inhibitors.

Background: JAK2 inhibitors have been proposed as a new therapeutic option for thalassemia therapy. The objective of this study was to discover the key structural features for improving 2-aminopyrimidine derivatives as potential JAK2 inhibitors. Materials & methods: Quantitative structure-activity relationship (QSAR) approaches (hologram QSAR and comparative molecular similarity indices analysis), molecular dynamics simulations, binding energy calculations and pharmacokinetic predictions were employed. Results: Reliable QSAR models, binding mode and binding interactions of JAK2 inhibitors were obtained and these obtained results were used as the key information for rational design of highly potent JAK2 inhibitors. Conclusion: The concept of new potential JAK2 inhibitors integrated from the obtained results was proved, producing two newly designed compounds, D01 and D02, with potential for use as JAK2 inhibitors.

Quercetin-Rich Ethanolic Extract of Polygonum odoratum var Pakphai Leaves Decreased Gene Expression and Secretion of Pro-Inflammatory Mediators in Lipopolysaccharide-Induced Murine RAW264.7 Macrophages.

Polygonum odoratum var. Pakphai has been used in traditional Thai medicine for the treatment of flatulence and constipation and to relieve the inflammation caused by insect bites. Quercetin (Q), which is abundant in plant-based foods, has been found to exert anti-inflammatory properties. This study evaluated the anti-inflammatory activity of P. odoratum ethanolic extract in RAW264.7 macrophage cells. Leaves were extracted with 50% ethanol, phenolics and flavonoids were then analyzed using UHPLC-QTOF-MS and HPLC-DAD. RAW264.7 cells were induced with lipopolysaccharides (LPSs). They were then treated with the extract and prostaglandin E2 (PGE2), and interleukin-6 (IL-6) and tumor necrotic factor-alpha (TNF-α) concentrations were determined. Levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6 and TNF-α mRNAs were analyzed using qRT-PCR. Chemical analysis demonstrated that the extract was abundant with Q while also containing catechin, gallic acid, epicatechin gallate and coumarin. The extract increased the viability of RAW264.7 cells and dose-dependently decreased nitric oxide production, PGE2, IL-6 and TNF-α levels in the medium from the LPS-induced RAW264.7 cell culture. Consistently, COX-2, iNOS, IL-6 and TNF-α mRNA levels were decreased in a concentration-dependent manner (p < 0.05). Thus, the quercetin-rich ethanolic extract derived from P. odoratum var Pakphai leaves can exert anti-inflammatory activity in LPS-induced RAW264.7 cells through a reduction of the pro-inflammatory mediator response.

Five Variable Number of Tandem Repeats Loci (D17S5, APOB, TPO Intron 10, IL-1α Intron 6, and CIAS1) in Thais and Application in the Prenatal Diagnostic Laboratory.

Background: Prenatal diagnosis of genetic disease requires DNA analysis of fetal tissue of a responsible gene. Accurate diagnosis is useful for the appropriate management of pregnancy. However, maternal contamination of fetal specimens poses a high preanalytical risk of prenatal misdiagnosis. We have examined five variable number of tandem repeat (VNTR) polymorphisms for use in monitoring potential maternal contamination. Materials and Methods: A study was conducted to examine the heterozygosities of five VNTR loci including, D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 in 200 unrelated Thai subjects and applied to the monitoring of maternal contamination in 22 families at risk of having fetuses with severe thalassemia. Results: The heterozygosities of D17S5, APOB, TPO intron 10, IL-1α intron 6, and CIAS1 VNTRs were 59.5, 19.5, 66.0, 35.5, and 42.0%, respectively. Therefore, the TPO intron 10 and D17S5 loci were chosen for prenatal diagnosis of thalassemia in 22 families. Analyses of these VNTRs demonstrated an increase of informative data from 59.1% provided by the routine D1S80 VNTR analysis to 90.9%. Conclusions: The VNTR diagnostic procedure described above is simple, cost-effective, rapid, and does not require the use of sophisticated instruments; it should prove useful in the prenatal diagnosis of thalassemia.

The Effects of Ibuprofen, Naproxen and Diclofenac on cell Apoptosis, Cell Proliferation and Histology Changes in Human Cholangiocarcinoma Cell Lines.

OBJECTIVE: To examine the effects of ibuprofen, naproxen and diclofenac, non-steroidal anti-inflammatory drugs (NSAIDs) on cell proliferation activity of the human CCA cell lines. METHODS: KKU-M139 and KKU-213B cell lines were used in this study. The cell viability was assessed by the MTT assay. Lipid synthesis determined by Oil red O staining and colorimetric assay. An inverted phase-contrast light microscope was used to investigate the histological change of the cells. Caspases 3/7 activity and AnnexinV/PI were used to assess apoptosis by multiple microplate reader. RESULTS: The results showed that ibuprofen, naproxen and diclofenac suppressed the viability of the KKU-M139 and KKU-213B cells in a dose-dependent manner, as measured especially diclofenac. However, these three NSAIDs slightly decreased lipid synthesis determined by Oil red O staining and colorimetric assay. The histological change observations showed the shrinking cell and become star-shaped in high dose treated groups. Interestingly, these NSAIDs exhibited in both of KKU-M139 and KKU-213B cell lines, the diclofenac-treated cells had the most injury cells. The cells exhibited cell injury features. In addition, the detection of caspase 3/7 and AnnexinV/PI in this investigation revealed early cell apoptotic characteristics. CONCLUSION: These finding suggest that ibuprofen, naproxen and diclofenac suppress cell viability. The results reveal that ibuprofen, naproxen and diclofenac, which induce the histological change and apoptosis. This study indicates that these NSAIDs may be used as an anti-proliferation agent for the treatment of CCA in the future.

Antioxidant Potential and Cytotoxic Effect of Isoflavones Extract from Thai Fermented Soybean (Thua-Nao).

Thua-nao, or Thai fermented soybeans, is a traditional Lanna fermented food in Northern Thailand. It is produced by using a specific bacterial species called Bacillus subtilis var. Thua-nao. We investigated the antioxidant activity and cytotoxic effect of isoflavones from Thua-nao. The phenolic compound contents and total flavonoid contents were determined by spectrophotometry. The antioxidant activity was examined using the ABTS, FRAP, and DPPH assays. The isoflavone contents and phenolic compositions were examined by the high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) techniques. The ability of isoflavones to inhibit human cancer cell growth was assessed by the MTT assay. The total phenolic content, total flavonoid content, and antioxidant activities of the isoflavones were 49.00 ± 0.51 mg GAE/g of dry extract (DE), 10.76 ± 0.82 mg QE/g of DE, 61.03 ± 0.97 µmol Trolox/g of DE, 66.54 ± 3.97 µM FeSO4/g of DE, and 22.47 ± 1.92% of DPPH inhibition, respectively. Additionally, the isoflavone extracts from Thua-nao had high isoflavone contents and polyphenolic compound compositions, especially daidzein and genistein. The isoflavone demonstrated a weak inhibition of MCF-7 and HEK293 cancer cell growth. It has a high antioxidant component, which is beneficial and can be developed for new therapeutic uses. However, further studies on the benefits of Thua-nao should be performed for realizing better and more effective uses soon.

Antioxidant Activity and Cytotoxic Effect of Ventilago denticulata Willd Leaves Extracts.

BACKGROUND: Oxidative stress is characterized by an imbalance between the antioxidant defense systems and the formation of reactive oxygen species (ROS). The excess of ROS can damage biomolecules and leading to several chronic conditions and diseases such as diabetes, antherosclerosis, ischemic injury, inflammation and carcinogenesis. Plant extracts and their constituents as a natural source of antioxidants have been extensively studied. OBJECTIVE: The study aimed to investigate the antioxidant and cytotoxicity of aqueous and ethanolic Rhang Dang (entilago denticulata Willd) leaves extract. MATERIAL AND METHOD: The aqueous and ethanolic extracts of Rhang Dang leaves were preliminary analyzed for their phenolic profile (total phenolics and total flavonoids). These extracts were evaluated for their antioxidant properties by different methods such as DPPH radical scavenging andperoxyl radical scavenging activity generated by AAPH (2,2'-Azobis (2-methylpropionamidine) dihydrochloride). Their cytotoxic effects on hepatocellular carcinoma cell line (HepG2) and peripheral blood mononuclear cells (PBMC) were determined by MTT assay. Anti-hemolytic activity was examined using spectrophotometrical method. RESULTS: The ethanolic extract from Rhang Dang leaves exhibited a strong antioxidant activity and prevented hemolysis. It showed the highest amount of phenolics (91.03 ± 12.43 mg of gallic acid equivalents/g extract) and flavonoid compound (69.76 ± 10.84 mg of catechin equivalents/g). Interestingly, this extract was more cytotoxic to HepG2 cells than PBMC. CONCLUSION: The ethanolic extract from Rhang Dang leaves had strong antioxidant activity and cytotoxic effect on cancer cells.

Iron-Chelating and Anti-Hemolytic Properties of Ethanolic Extract of Lotus (Nelumbonucifera Gaertn) Leaves.

BACKGROUND: Iron overload is the major consequence of blood transfusion in ß-thalassemia patients. Redox iron plays a critical role in the formation of reactive oxygen species and subsequently leads to oxidative stress damage in many cells, especially red blood cells and hepatocytes. Iron deposition in hepatocytes is associated with fibrosis and cirrhosis. Polyphenolic compounds found in natural products are interesting iron chelators and antioxidants. OBJECTIVE: This study aims to evaluate the iron-chelating properties and free-radical scavenging activities of lotus leaf extract in iron-loaded HepG2 cells. MATERIAL AND METHOD: Lotus (Nelumbonucifera Gaertn) leaves were extracted with 80% (v/v) ethanol. The extract was examined for free-radical scavenging activity by using 2, 2-diphenyl-1-picrylhydrazyl assay (DPPH assay); iron-binding and anti-hemolytic activities using spectrophotometrical method. Iron-depriving activity of the extract was determined in iron loaded human hepatocellular (HepG2) cells using fluorescence technique. RESULTS: The lotus extract showed antioxidant and anti-hemolytic activities in a concentration-dependent manner. Furthermore, it was able to bind iron rapidly and was saturated within 10 minute. With 24-hour treatment, this extract dose dependently decreased the level of labile iron pool in iron loaded HepG2 cells. CONCLUSION: Lotus leaf extract had strong antioxidant activities, iron chelating properties on iron loaded HepG2 cells and anti-hemolytic activity.

Characterisation of a novel oral iron chelator: 1-(N-Acetyl-6-Aminohexyl)-3-Hydroxy-2-Methylpyridin-4-one.

OBJECTIVES: Desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) are iron chelators currently in clinical use for the treatment of iron overload. Due to difficulties with administration and associated side effects with these three molecules, the search continues for an efficient nontoxic orally active iron chelator. This communication describes the properties of one such candidate, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1). METHODS: Physicochemical characterisation techniques, including partition coefficient, pKa values and logK values for iron(III). Iron scavenging assays, from iron citrate, nontransferrin bound iron and iron-loaded rats. Cytotoxicity studies using white cells, hepatocytes and cardiomyocytes. KEY FINDINGS: CM1 possesses high affinity and selectivity for iron(III) and a suitable partition coefficient to permeate membranes. CM1 forms a neutral 3 : 1 iron(III) complex under physiological conditions and so, it is predicted to be capable of entry into mammalian cells to scavenge excess intracellular iron and to efflux from cells as the neutral 3 : 1 complex. CM1 is demonstrated to be orally active and to possess a higher efficacy than DFP in rats. CM1 displays no toxicity to a range of cell types. CONCLUSION: The above promising studies will be extended to monitor the pharmacokinetics and metabolism of CM1. CM1 is an excellent candidate for phase 1 clinical trials.

Effect of a novel oral active iron chelator: 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in iron-overloaded and non-overloaded mice.

OBJECTIVE: To evaluate efficacy and toxicity of a novel orally active bidentate iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in mice under normal and iron overload conditions. METHODS: Wild type C57BL/6 mice were fed with normal and 0.2% (w/w) ferrocene-supplemented (Fe) diets, respectively for 240 d and orally given the CM1 (50, 100 and 200 mg/kg) for 180 d. Blood iron profiles, hematological indices, liver enzymes and histopathology were determined. RESULTS: CM1 treatment lowered plasma levels of labile plasma iron and non-transferrin bound iron, but not ferritin in the Fe-fed mice. However, the treatment did not impact blood hemoglobin level, white blood cell and platelet numbers in both normal diet and Fe diet-fed mice. Interestingly, CM1 treatment did not markedly elevate plasma aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities in the normal diet-fed mice but it tended to increase the levels of the liver enzymes slightly in the Fe-fed mice. Hematoxylin and eosin staining result showed no abnormal pathological changes in heart, liver and spleen tissues. CONCLUSIONS: It is clear that CM1 would not be toxic to bone marrow and liver cells under normal and iron-overload conditions.

Iron-chelating and anti-lipid peroxidation properties of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in long-term iron loading ß-thalassemic mice.

OBJECTIVE: To evaluate the iron-chelating properties and free-radical scavenging activities of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) treatment in chronic iron-loaded ß-thalassemic (BKO) mice. METHODS: The BKO mice were fed with a ferrocene-rich diet and were orally administered with CM1 [50 mg/(kg.day)] for 6 months. Blood levels of non-transferrin bound iron, labile plasma iron, ferritin (Ft) and malondialdehyde were determined. RESULTS: The BKO mice were fed with an iron diet for 8 months which resulted in iron overload. Interestingly, the mice showed a decrease in the non-transferrin bound iron, labile plasma iron and malondialdehyde levels, but not the Ft levels after continuous CM1 treatment. CONCLUSIONS: CM1 could be an effective oral iron chelator that can reduce iron overload and lipid peroxidation in chronic iron overload ß-thalassemic mice.

Iron-chelating, free radical scavenging and anti-proliferative activities of Azadirachta indica.

BACKGROUND: Excessive production of reactive oxygen species leads to oxidative stress which occurs in various diseases, such as diabetes, cancer, neurodegenerative diseases, and secondary iron overload in thalassemia. Antioxidants are compounds that inhibit the oxidative processes and delay or suppress oxidative stress. Phytochemicals in herbs are interesting sources of natural antioxidants with cancer preventive properties. The use of natural products is beneficial for the prevention and/or treatment of oxidative stress mediated diseases. OBJECTIVE: The study aimed to investigate the antioxidant and anti-proliferative properties of ethanolic extract from Azadirachta indica (neem) leaves. MATERIAL AND METHOD: Neem leaves were extracted by 80% ethanol (v/v). The ethanolic extract was tested for free radical scavenging activity by 2,2'-azino-bis-3-ethylbenzothiaziline-6-sulfonic acid (ABTS) and for the reduction of the power of ferric ion (Fe3+) to ferrousion (Fe2+) by ferric reducing antioxidant plasma (FRAP) assay. Furthermore, the ability of iron-binding activity was investigated by the spectrophotometry technique. The inhibitory effect on the growth of human promyelocytic leukemic cell line (HL-60 cells) was determined by MTT assay. RESULTS: The ethanolic extract from neem leaves exhibited free radical scavenging activities and reduced the power of ferric ion (Fe3+) to ferrous ion (Fe2+) in dose responses. Furthermore, it was able to bind with iron rapidly within 5 minutes. Interestingly, this extract inhibited human promyelocytic leukemic cell line (HL-60 cells) growth in concentration response (0-500 microg/ml) for 24 hour treatment. CONCLUSION: The ethanolic extract from neem leaves had strong antioxidant activity and an anti-proliferative effect on cancer cells.