Anti-inflammatory potential of a Thai traditional remedy called Prabchompoothaweep in an animal model of acute and sub-acute inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Prabchompoothaweep (PCT) is a Thai remedy which is composed of 23 herbs and has been added onto the National List of Essential Medicines (NLEM) of Thailand. This remedy has been used to treat allergic rhinitis and asthma in Thai traditional medicine for many years. Furthermore, a few studies have reported anti-allergic, anti-malarial, anti-inflammatory and antioxidant activities. AIM OF THE STUDY: This study aims to evaluate anti-inflammatory activity of PCT extract in an animal model. MATERIALS AND METHODS: The animal model of acute inflammation was studied over a 24-h period, utilizing the method of carrageenan-induced paw oedema. In addition, sub-acute inflammation was examined over 7 days, using the formalin-induced paw oedema method. The treatment groups received PCT extracts, via the oral route, at 1-h prior to injection and then the sub plantar of the rat right paw was injected with the named substances to generate paw oedema. The paw thickness was measured by vernier caliper at regular intervals after injection. At the end of experiment, the blood and paw tissues were collected for measurement of pro-inflammatory cytokines and histological examination respectively. RESULTS: In acute inflammation, all doses of PCT extract (250, 500 and 1000 mg/kg p.o.) significantly reduced paw thickness after the first 3 h in a dose-dependent manner and the percentage of inhibition was 38.7%, 47.8% and 49.5% respectively. The pro-inflammatory cytokines, including TNF-α and IL-1ß, statistically decreased with all doses of the extracts. However, the histological examination did not reveal significant results due to the short time duration. As regards to sub-acute inflammation, all doses of PCT extract significantly reduced paw thickness with 12.78%, 23.64% and 35.78%, in a dose dependent manner. Also, the pro-inflammatory cytokines (TNF-α and IL-1ß) significantly decreased at day 7. Interestingly, the histological examination of paw tissue demonstrated reductions of mononuclear infiltrations of inflammatory cells, this was observed in the group receiving PCT extracts, also in a dose-dependent manner. CONCLUSION: Therefore, PCT exerted anti-inflammatory activity in an animal model of acute and sub-acute inflammation, suggesting that it could be used as a new source for treatment of inflammatory diseases.

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.

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.

Protection of Iron-Induced Oxidative Damage in Neuroblastoma (SH-SY5Y) Cells by Combination of 1-(N-Acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one and Green Tea Extract.

Iron is a crucial trace element and essential for many cellular processes; however, excessive iron accumulation can induce oxidative stress and cell damage. Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, have been associated with altered iron homoeostasis causing altered iron distribution and accumulation in brain tissue. This study aims to investigate the protective effect of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in combination with green tea extract (GTE) on iron-induced oxidative stress in neuroblastoma (SH-SY5Y) cells. Cells were cultured in medium with or without ferric chloride loading. Their viability and mitochondrial activity were assessed using MTT and JC-1 staining methods. Levels of the cellular labile iron pool (LIP), reactive oxygen species (ROS), and lipid-peroxidation products were determined using calcein acetoxymethyl ester, 2',7'-dichlorohydrofluorescein diacetate, and TBARS-based assays, respectively. The viability of iron-loaded cells was found to be significantly increased after treatment with CM1 (10 µM) for 24 h. CM1 co-treatment with GTE resulted in a greater protective effect than their monotherapy. Combination of CM1 and GTE also reduced mitochondrial disruption and LIP content and ROS and TBARS production. In conclusion, the combination of CM1 and GTE exhibits protection against iron-induced oxidative stress in neuroblastoma cells.

Biofilm formation and transfer of a streptomycin resistance gene in enterococci from fermented pork.

OBJECTIVES: Multidrug-resistant (MDR) enterococci are found extensively in food samples. This study characterized the phenotypic virulence factors and the ability of horizontal gene transfer of a streptomycin resistance gene among enterococci isolated from fermented pork. METHODS: Thirty-six MDR enterococci were subjected to screening of gelatinase, biofilm formation at various temperatures (4 °C, 25 °C and 37 °C), clumping ability and conjugation. RESULTS: All gelatinase-positive and clumping-positive strains were Enterococcus faecalis (41.7% and 38.9%, respectively). None of Enterococcus faecium and Enterococcus hirae demonstrated both phenotypes. Moderate and strong biofilm formations were found mostly at optimal temperatures in all the three species tested. However, moderate and weak biofilm formations could be found in 52.8% at 4 °C. No association was observed between biofilm formation and asa1, efaA, gelE and esp genes. Surprisingly, our data revealed evidence of the streptomycin resistance gene (aadE) being transferred among meat E. faecalis isolates as characterized by the pheromone-clumping response. CONCLUSIONS: Here we report the co-existence of some virulence factors and MDR enterococci from fermented pork. Our data demonstrated for the first time that the aadE gene could be transferred via conjugation among enterococci isolated from meat, contributing to streptomycin resistance. This study highlights the importance of horizontal gene transfer within the food chain reservoir and that transfer to humans might be possible, causing harm or untreatable diseases.

Anti-inflammatory and Antioxidant Activities of the Extracts from Leaves and Stems of Polygonum odoratum Lour.

BACKGROUND: Polygonum odoratum is an indigenous vegetable that has been used as a favoring agent and also used as a Thai traditional medicine to treat flatulence. OBJECTIVE: To analyze active ingredients, total phenolic and total flavonoid contents, antiinflammatory and antioxidant activities from leaf and stem extracts of P. odoratum. METHODS: Leaves and stems were dried and extracted by using methanol, dichloromethane and water for obtaining Methanolic Leaf Extract (MLE), Methanolic Stem Extract (MTE), Dichloromethane Leaf Extract (DLE), Dichloromethane Stem Extract (DTE), Water Leaf Extract (WLE) and Water Stem Extract (WTE). The extracts were quantified for total phenolic and total flavonoid contents by spectrophotometry and active compounds were analyzed by using GC-MS. Antioxidant activity was determined by ABTS and DPPH radicals scavenging assays. Anti-inflammatory activity was tested by the inhibition of nitric oxide production in RAW 264.7 macrophage cells induced by lipopolysaccharide. RESULTS: The DLE exhibited the most potent anti-inflammatory effect by inhibiting nitric oxide production in a concentration-dependent manner (IC50 = 53.75±0.72 µg/mL). MLE exhibited strong antioxidant activity and contained the highest concentration of phenolic compounds (52.59±0.58 mg gallic acid equivalent/g extract) and flavonoid (19.97+0.11 mg quercetin equivalent/g extract). E-15-Heptadecenal and 3, 7, 11, 15-tetramethyl-2- hexadecen-1-ol were found predominantly in the methanol extracts. CONCLUSION: The leaf extract of P. odoratum showed potent anti-inflammatory and antioxidant activities, mediated by DLE and MLE, respectively.

Antimicrobial resistance of Enterococcus spp. isolated from Thai fermented pork in Chiang Rai Province, Thailand.

OBJECTIVES: The aim of this study was to evaluate the prevalence of antimicrobial-resistant phenotypes and genes of Enterococcus spp. in order to explore the range of resistance profiles from Thai traditional fermented pork. METHODS: A total of 120 Thai fermented pork specimens were collected in Chiang Rai, Thailand. Antimicrobial resistance among isolated enterococci to 11 antimicrobial agents was determined by the agar disk diffusion method. Antibiotic resistance genes from resistant phenotypes and virulence genes were observed. RESULTS: A total of 119 enterococci were found contaminating the collected samples. The most prevalent species was Enterococcus faecalis (68.9%), followed by Enterococcus hirae (16.0%), Enterococcus faecium (13.4%) and Enterococcus gallinarum (1.7%). The highest percentage of resistance was to ciprofloxacin (97.5%), followed by erythromycin (78.2%) and tetracycline (67.2%), whilst high-level gentamicin- and streptomycin-resistant isolates were of lower frequency (7.6% and 22.7%, respectively). All isolates were susceptible to the clinically important agents vancomycin and teicoplanin. Overall, a relatively high frequency of multidrug-resistant (MDR) enterococci was observed (76.2%). Antimicrobial-resistant phenotypes were found to carry aacA-aphD, addE, erm(B), mefA/E, cat, tet(L) and tet(M) resistance genes. Virulence genes were also evaluated and the gelE gene was found to be the most common (37.8%). CONCLUSIONS: These data highlight the importance of MDR enterococci in fermented pork in Thailand. This is the first report to detect the unusual species E. hirae carrying the mefA/E macrolide resistance gene. These clinically important and unusual enterococci isolates from Thai fermented pork could be a source of transferable resistance genes to other bacteria.

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.

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.