Synergistic effects of polymyxin and vancomycin combinations on carbapenem- and polymyxin-resistant Klebsiella pneumoniae and their molecular characteristics.

IMPORTANCE: This study provides insights into the mechanisms of polymyxin resistance in K. pneumoniae clinical isolates and demonstrates potential strategies of polymyxin and vancomycin combinations for combating this resistance. We also identified possible mechanisms that might be associated with the treatment of these combinations against carbapenem- and polymyxin-resistant K. pneumoniae clinical isolates. The findings have significant implications for the development of alternative therapies and the effective management of infections caused by these pathogens.

Standardized Extract of Centella asiatica Prevents Fear Memory Deficit in 3xTg-AD Mice.

ECa 233 is a standardized extract of Centella asiatica (CA), an herb widely used in traditional Chinese and Ayurvedic medicine. Previous studies reported that ECa 233 enhanced memory retention and synaptic plasticity in the hippocampus of healthy rats. Because of this, we became curious whether ECa 233 has a therapeutic effect on the fear memory deficit in the triple transgenic Alzheimer's disease (3xTg-AD) model mice. Fear memory is a crucial emotional memory for survival that is found to be impaired in patients with early-onset Alzheimer's disease (AD). In this study, we orally administered ECa 233 (doses: 10, 30, and 100[Formula: see text]mg/kg) to 3xTg-AD mice, who were five months old, for 30 consecutive days. We found that ECa 233 prevented a cued fear memory deficit and enhanced hippocampal long-term potentiation (LTP) in 3xTg-AD mice. Subsequent proteomic and western blot analyses revealed increased expression levels of the molecules related to LTP induction and maintenance, including brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB) and its network proteins, and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2) in the hippocampi and amygdala of 3xTg-AD mice after ECa 233 pre-treatment. Our results indicate that ECa 233 is a promising potential herbal standardized extract that could be used in preventing the fear memory deficit and synaptic dysfunction before the early onset of AD.

Metabolite profiling and identification of novel umami compounds in the chaya leaves of two species using multiplatform metabolomics.

Chaya (Cnidoscolus chayamansa and C. aconitifolius) is a fast-growing medicinal plant, and its leaves exhibit a strong umami taste. Here metabolite variation and umami-related compounds in the leaves of two chaya species were determined using a multiplatform untargeted-metabolomics approach, electronic tongue, and in silico screening. Metabolite profiles varied between the leaves of the two species and among leaf maturation stages. Young leaves exhibited the highest umami taste intensity, followed by mature and old leaves. Partial least square regression and computational molecular docking analyses revealed five potent umami substances (quinic acid, trigonelline, alanyl-tyrosine, leucyl-glycyl-proline, and leucyl-aspartyl-glutamine) and three known umami compounds (l-glutamic acid, pyroglutamic acid, and 5'-adenosine monophosphate). The five substances were validated as novel umami compounds using electronic tongue assay; leucyl-glycyl-proline exhibited synergism with monosodium glutamate, thereby enhancing the umami taste. Thus, substances contributing to the taste of chaya leaves were successfully identified.

A radiosensitizer, gallotannin-rich extract from Bouea macrophylla seeds, inhibits radiation-induced epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: Radioresistance can pose a significant obstacle to the effective treatment of breast cancers. Epithelial-mesenchymal transition (EMT) is a critical step in the acquisition of stem cell traits and radioresistance. Here, we investigated whether Maprang seed extract (MPSE), a gallotannin-rich extract of seed from Bouea macrophylla Griffith, could inhibit the radiation-induced EMT process and enhance the radiosensitivity of breast cancer cells. METHODS: Breast cancer cells were pre-treated with MPSE before irradiation (IR), the radiosensitizing activity of MPSE was assessed using the colony formation assay. Radiation-induced EMT and stemness phenotype were identified using breast cancer stem cells (CSCs) marker (CD24-/low/CD44+) and mammosphere formation assay. Cell motility was determined via the wound healing assay and transwell migration. Radiation-induced cell death was assessed via the apoptosis assay and SA-ß-galactosidase staining for cellular senescence. CSCs- and EMT-related genes were confirmed by real-time PCR (qPCR) and Western blotting. RESULTS: Pre-treated with MPSE before irradiation could reduce the clonogenic activity and enhance radiosensitivity of breast cancer cell lines with sensitization enhancement ratios (SERs) of 2.33 and 1.35 for MCF7 and MDA-MB231cells, respectively. Pretreatment of breast cancer cells followed by IR resulted in an increased level of DNA damage maker (γ-H2A histone family member) and enhanced radiation-induced cell death. Irradiation induced EMT process, which displayed a significant EMT phenotype with a down-regulated epithelial marker E-cadherin and up-regulated mesenchymal marker vimentin in comparison with untreated breast cancer cells. Notably, we observed that pretreatment with MPSE attenuated the radiation-induced EMT process and decrease some stemness-like properties characterized by mammosphere formation and the CSC marker. Furthermore, pretreatment with MPSE attenuated the radiation-induced activation of the pro-survival pathway by decrease the expression of phosphorylation of ERK and AKT and sensitized breast cancer cells to radiation. CONCLUSION: MPSE enhanced the radiosensitivity of breast cancer cells by enhancing IR-induced DNA damage and cell death, and attenuating the IR-induced EMT process and stemness phenotype via targeting survival pathways PI3K/AKT and MAPK in irradiated breast cancer cells. Our findings describe a novel strategy for increasing the efficacy of radiotherapy for breast cancer patients using a safer and low-cost natural product, MPSE.

Influence of collagen and some proteins on gel properties of jellyfish gelatin.

Marine gelatin is one of the food proteins used in food and non-food products, offering desirable functionalities such as gelling, thickening, and binding. Jellyfish has been chosen for this gelatin research, in view of the benefits of its main collagen protein and lower fat content, which may reduce the amounts of chemicals used in the preparative steps of gelatin production. To date, the lack of identified proteins in gelatin has limited the understanding of differentiating intrinsic factors quantitatively and qualitatively affecting gel properties. No comparison has been made between marine gelatin of fish and that of jellyfish, regarding protein type and distribution differences. Therefore, the study aimed at characterizing jellyfish gelatin extracted from by-products, that are i.e., pieces that have broken off during the grading and cleaning step of salted jellyfish processing. Different pretreatment by hydrochloric acid (HCl) concentrations (0.1 and 0.2 M) and hot water extraction time (12 and 24 h) were studied as factors in jellyfish gelatin extraction. The resultant jellyfish gelatin with the highest gel strength (JFG1), as well as two commercial gelatins of fish gelatin (FG) and bovine gelatin (BG), were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results show that the jellyfish gelatin (JFG1) extracted with 0.1 M HCl at 60°C for 12 h delivered a maximum gel strength of 323.74 g, which is lower than for FG and BG, exhibiting 640.65 and 540.06 g, respectively. The gelling and melting temperatures of JFG1 were 7.1°C and 20.5°C, displaying a cold set gel and unstable gel at room temperature, whereas the gelling and melting temperatures of FG and BG were 17.4°C, 21.3°C, and 27.5°C, 32.7°C, respectively. Proteomic analysis shows that 29 proteins, of which 10 are types of collagen proteins and 19 are non-collagen proteins, are common to all BG, FG, and JFG1, and that JFG1 is missing 3 other collagen proteins (collagen alpha-2 (XI chain), collagen alpha-2 (I chain), and collagen alpha-2 (IV chain), that are important to gel networks. Thus, the lack of these 3 collagen types influences the inferior gel properties of jellyfish gelatin.

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines.

Despite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

Black rice cultivar from Java Island of Indonesia revealed genomic, proteomic, and anthocyanin nutritional value.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.

Chemosensitizing activity of peptide from Lentinus squarrosulus (Mont.) on cisplatin-induced apoptosis in human lung cancer cells.

The limitations of cisplatin, a standard chemotherapy for lung cancer, have been documented with serious adverse effects and drug resistance. To address the need for novel therapy, this study firstly reveals the potential of peptide from Lentinus squarrosulus (Mont.) as a chemotherapeutic adjuvant for cisplatin treatment. The purified peptide from L. squarrosulus aqueous extracts was obtained after eluting with 0.4 M NaCl through FPLC equipped with anion exchange column. Preincubation for 24 h with 5 µg/mL of the peptide at prior to treatment with 5 µM cisplatin significantly diminished %cell viability in various human lung cancer cells but not in human dermal papilla and proximal renal cells. Flow cytometry indicated the augmentation of cisplatin-induced apoptosis in lung cancer cells pretreated with peptide from L. squarrosulus. Preculture with the peptide dramatically inhibited colony formation in lung cancer cells derived after cisplatin treatment. Strong suppression on integrin-mediated survival was evidenced with the diminution of integrins (ß1, ß3, ß5, α5, αV) and down-stream signals (p-FAK/FAK, p-Src/Src, p-Akt/Akt) consequence with alteration of p53, Bax, Blc-2 and Mcl-1 in cisplatin-treated lung cancer cells preincubated with peptide from L. squarrosulus. These results support the development of L. squarrosulus peptide as a novel combined chemotherapy with cisplatin for lung cancer treatment.

A novel peptide isolated from garlic shows anticancer effect against leukemic cell lines via interaction with Bcl-2 family proteins.

Leukemia is a group of cancer caused by the abnormal proliferation and differentiation of hematopoietic stem cells. Efforts geared toward effective therapeutic strategies with minimal side effects are underway. Peptides derived from natural resources have recently gained special attention as alternative chemotherapeutic agents due to their minimal adverse effects. In the present study, the aim was to isolate peptides from garlic (Allium sativum) and investigate their anticancer activity against leukemic cell lines. The protein extract of A. sativum was pepsin-digested to obtain protein hydrolysate followed by sequential purification methods. A novel anticancer peptide, VKLRSLLCS (VS-9), was identified and characterized by mass spectrometric analysis. The peptide was demonstrated to significantly inhibit the cell proliferation of MOLT-4 and K562 leukemic cell lines while exhibiting minimal inhibition against normal PBMC. Particularly, VS-9 could induce apoptosis and upregulate mRNA levels of caspase 3, caspase 8, caspase 9, and Bax while downregulating Bcl-2, Bcl-xL, and Bcl-w. Molecular docking of VS-9 with the anti-apoptotic Bcl-2 protein family suggested that VS-9 could bind the binding groove of the BH3 domain on target proteins. Protein-peptide interaction analysis by affinity chromatography and LC-MS/MS further showed that VS-9 could bind Bcl-2 proteins. Results suggest VS-9 as a potential garlic-derived novel anticancer peptide possessing apoptosis-inducing properties against leukemic cell lines via anti-apoptotic Bcl-2 protein family.

Antifungal Activity and Molecular Mechanisms of Partial Purified Antifungal Proteins from Rhinacanthus nasutus against Talaromyces marneffei.

Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, Rhinacanthus nasutus, against the human pathogenic fungus Talaromycesmarneffei. This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The R. nasutus crude protein extract inhibited the growth of T. marneffei. The anti-T. marneffei activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from R. nasutus was partially purified by size fractionation to ≤10, 10-30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti-T. marneffei activity with the MIC and MFC values of 32 to 128 µg/mL and >128 µg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with T. marneffei mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti-T. marneffei activity of the 10-30 kDa fraction was abrogated by deletion of gasA and gasC, the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from R. nasutus represent sources for novel drug development.

Protein expression profiles that underpin the preventive and therapeutic potential of Moringa oleifera Lam against azoxymethane and dextran sodium sulfate-induced mouse colon carcinogenesis.

Previous studies in a mouse model have indicated the anticancer potential of boiled Moringa oleifera pod (bMO)-supplemented diets; however, its molecular mechanisms are still unclear. Therefore, the present study aimed to explore the protein expression profiles responsible for the suppressive effect of bMO supplementation on azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced mouse colon carcinogenesis. Analysis by gel electrophoresis and liquid chromatography-tandem mass spectrophotometry demonstrated that there were 125 proteins that were differentially expressed in mouse colon tissues between 14 experimental groups of mice. The differentially expressed proteins are involved in various biological processes, such as signal transduction, metabolism, transcription and translation. Venn diagram analysis of the differentially expressed proteins was performed in six selected mouse groups, including negative control, positive control mice induced by AOM/DSS, the AOM/DSS groups receiving preventive or therapeutic bMO diets and their bMO-supplemented control groups. This analysis identified 7 proteins; 60S acidic ribosomal protein P1 (Rplp1), fragile X mental retardation, cystatin 9, round spermatids protein, zinc finger protein 638, protein phosphatase 2C (Ppm1g) and unnamed protein product as being potentially associated with the preventive and therapeutic effects of bMO in AOM/DSS-induced mouse colon cancer. Analysis based on the search tool for interactions of chemicals (STITCH) database predicted that Rplp1 interacted with the apoptotic and inflammatory pathways, whereas Ppm1g was associated only with inflammatory networks. This proteomic analysis revealed candidate proteins that are responsible for the effects of bMO supplementation, potentially by regulating apoptotic and inflammatory signaling networks in colorectal cancer prevention and therapy.

Bioinformatics and experimental studies of anti-leukemic activity from 6-gingerol demonstrate its role in p53 mediated apoptosis pathway.

6-gingerol is a traditional medicine that possesses anti-cancer activity against several types of cancer. However, the mechanism of action still remains unclear. Therefore, this study explored the effects of 6-gingerol on anti-leukemic mechanisms in NB4, MOLT4, and Raji leukemic cell. Results indicated that 6-gingerol inhibited cell proliferation and induced cell apoptosis in these 3 cell lines. Moreover, 6-gingerol was shown to increase the mRNA expression of the caspase family thereby suggesting that 6-gingerol induced apoptosis through the caspase-dependent pathway. To explore the signaling pathway regulating 6-gingerol induced apoptosis, we utilized and integrated the network pharmacology approach together with experimental investigations. Targets of 6-gingerol were identified from ChEMBL and STITCH databases, which were used for constructing the protein-protein interaction (PPI) network. Results from the PPI network indicated that p53 was a key regulator. Moreover, it was found that 6-gingerol could increase the levels of p53 mRNA in all leukemic cell lines. Thus, 6-gingerol has shown to have anti-cancer activity. In addition, p53, BAX and BCL2 could be involved in the apoptosis pathway of these leukemic cells. This study is anticipated to be useful for the development of 6-gingerol as an anti-leukemic drug in the future.

Cold plasma treatment to improve germination and enhance the bioactive phytochemical content of germinated brown rice.

The changes in the bioactive phytochemicals of six cultivars of Thai germinated brown rice (GBR) were monitored in parallel to those of cold plasma-treated GBR (PGBR). After treatment with the optimal plasma conditions, the germination percentage, root length, and seedling height measurements of the most sensitive rice cultivar increased by 84%, 57%, and 69%, respectively. For all of the rice cultivars, there were no significant differences in the antioxidant activities of the GBRs and PGBRs. Conversely, higher contents of γ-oryzanols were observed in the PGBR group than in the GBR group during the 2-day germination period. Certain cultivars in the PGBR group reached their maximum values for total phenolic compounds, total vitamin E, certain simple phenolics, phytosterols, triterpenoids, and anthocyanins one day earlier than the same values for GBR. In contrast, the concentrations of 2-acetyl-1-pyrroline in both the GBR and PGBR samples were reduced significantly with increased germination time.

Novel bioactive peptides demonstrating anti-dengue virus activity isolated from the Asian medicinal plant Acacia Catechu.

The therapeutic activities of food-derived bioactive proteins and peptides are attracting increased attention within the research community. Medicinal plants used in traditional medicines are an excellent source of bioactive proteins and peptides, especially those traditionally prepared by water extraction for use as tea or food supplement. In this study, novel bioactive peptides were isolated from enzymatic digests of 33 Thai medicinal plants. The inhibitory activity of each against dengue virus (DENV) infection was investigated. Of 33 plants, peptides from Acacia catechu extract demonstrated the most pronounced anti-DENV activity. Half maximal inhibitory concentration of 0.18 µg/ml effectively inhibited DENV foci formation. Treatment with 1.25 µg/ml crude peptide extract could reduce virus production less than 100-fold with no observable cell toxicity. Peptide sequences were determined by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Two bioactive peptides isolated from Acacia catechu inhibited DENV foci formation >90% at the concentration of 50 µM; therefore, they are recommended for further investigation as antiviral peptides against DENV infection.

Comparative proteomics and protein profile related to phenolic compounds and antioxidant activity in germinated Oryza sativa 'KDML105' and Thai brown rice 'Mali Daeng' for better nutritional value.

BACKGROUND: Brown rice (BR) and germinated brown rice (GBR) are considered as prime sources of carbohydrate and bioactive compounds for more than half of the populations worldwide. Several studies have reported on the proteomics of BR and GBR; however, the proteomic profiles related to the synthesis of bioactive compounds are less well documented. In the present study, BR and GBR were used in a comparative analysis of the proteomic and bioactive compound profiles for two famous Thai rice varieties: Khao Dawk Mali 105 (KDML) and Mali Daeng (MD). RESULTS: The proteomes of KDML and MD revealed differences in the expression patterns of proteins after germination. Total phenolic compound content, anthocyanin contents and antioxidant activity of red rice MD was approximately 2.6-, 2.2- and 9.2-fold higher, respectively, compared to that of the white rice KDML. Moreover, GBR of MD showed higher total anthocyanin content and greater antioxidant activity, which is consistent with the increase expression of several proteins involved in the biosynthesis of phenolic compounds and protection against oxidative stress. CONCLUSION: Red rice MD exhibits higher nutrient values compared to white rice KDML and the appropriate germination of brown rice could represent a method for improving health-related benefits. © 2017 Society of Chemical Industry.

Hepcidin suppression in ß-thalassemia is associated with the down-regulation of atonal homolog 8.

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in ß-thalassemia. We found that inhibition of hepcidin expression in ß-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in ß-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with ß-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the ß-thalassemia patient sera. In conclusion, hepcidin suppression in ß-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in ß-thalassemia.

Activity of andrographolide against chikungunya virus infection.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has recently engendered large epidemics around the world. There is no specific antiviral for treatment of patients infected with CHIKV, and development of compounds with significant anti-CHIKV activity that can be further developed to a practical therapy is urgently required. Andrographolide is derived from Andrographis paniculata, a herb traditionally used to treat a number of conditions including infections. This study sought to determine the potential of andrographolide as an inhibitor of CHIKV infection. Andrographolide showed good inhibition of CHIKV infection and reduced virus production by approximately 3log10 with a 50% effective concentration (EC50) of 77 µM without cytotoxicity. Time-of-addition and RNA transfection studies showed that andrographolide affected CHIKV replication and the activity of andrographolide was shown to be cell type independent. This study suggests that andrographolide has the potential to be developed further as an anti-CHIKV therapeutic agent.

Assessment of flavaglines as potential chikungunya virus entry inhibitors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that recently caused large epidemics in islands in, and countries around, the Indian Ocean. There is currently no specific drug for therapeutic treatment or for use as a prophylactic agent against infection and no commercially available vaccine. Prohibitin has been identified as a receptor protein used by chikungunya virus to enter mammalian cells. Recently, synthetic sulfonyl amidines and flavaglines (FLs), a class of naturally occurring plant compounds with potent anti-cancer and cytoprotective and neuroprotective activities, have been shown to interact directly with prohibitin. This study therefore sought to determine whether three prohibitin ligands (sulfonyl amidine 1 m and the flavaglines FL3 and FL23) were able to inhibit CHIKV infection of mammalian Hek293T/17 cells. All three compounds inhibited infection and reduced virus production when cells were treated before infection but not when added after infection. Pretreatment of cells for only 15 minutes prior to infection followed by washing out of the compound resulted in significant inhibition of entry and virus production. These results suggest that further investigation of prohibitin ligands as potential Chikungunya virus entry inhibitors is warranted.

Molecular mechanism of anti-inflammatory activity of Pluchea indica leaves in macrophages RAW 264.7 and its action in animal models ofinflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: PLUCHEA INDICA LESS.: (Asteraceae) is a Thai medicinal plant used in traditional medicine for the treatment of hemorrhoids, lumbago, leucorrhoea and inflammation. This study investigated the molecular mechanism of anti-inflammatory activity of Pluchea indica leaf extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and also determined its action in acute inflammation animal models. MATERIALS AND METHODS: The inhibitory effect of Pluchea indica leaf extract on LPS-induced nitric oxide (NO) production was evaluated by Griess reaction. Protein and mRNA expressions were determined by real time RT-PCR and Western blotting analysis, respectively. Inducible nitric oxide synthase (iNOS) promoter activity was evaluated by iNOS promoter based reporter gene assay. In vivo anti-inflammatory effect was examined in ethylphenylpropiolate (EPP)-induced ear edema and carrageenan-induced paw edema in rat models. RESULTS: Ethyl acetate fraction of ethanol extract of Pluchea indica leaves (EFPI) exhibited the potent inhibitory effect on NO production in LPS-induced macrophages and also inhibited PGE2 release. EFPI reduced iNOS mRNA and protein expression through suppressed iNOS promoter activity and nuclear translocation of subunit p65 of nuclear factor-κB, but did not inhibit phosphorylation of the mitogen-activated protein kinases (MAPKs). Moreover, EFPI possessed anti-inflammatory activities on acute phase of inflammation as seen in EPP-induced ear edema and carrageenan-induced paw edema inrats. CONCLUSIONS: These data support the pharmacological basis of Pluchea indica plant as a traditional herbal medicine for treatment of inflammation.