Effects of supplementing freeze-dried Mitsuokella jalaludinii phytase on the growth performance and gut microbial diversity of broiler chickens.

Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate pens were used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3 weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p < .05) and T2 diets (p < .05), demonstrating the efficiency of MJ as a supplement to low aP diet. Nevertheless, MJ did not significantly affect the microbial population and diversity in broilers' caeca, which mainly consists of members from Bacteroidetes, Firmicutes, and Proteobacteria. Regardless, significant variations in the caecal bacterial composition were observed over time, probably due to succession as the broilers aged. This is the first reported study on the effect of MJ on the microbial diversity of broiler's caeca.

Clausenidin Induces Caspase 8-Dependent Apoptosis and Suppresses Production of VEGF in Liver Cancer Cells

Clausena excavata Burm f. is used by traditional healers to treat cancer patients in South East Asia. The use of the plant and its compounds is based on Asian folklore with little or no scientific evidence supporting the therapeutic efficacy The current study aimed to determine the effect of pure clausenidin isolated from C. excavata on caspase-8-induced cell death as well as angiogenesis in the HepG2 hepatocellular carcinoma cell line. Caspase-8 and extrinsic death receptor protein expression was determined using spectrophotometry and protein profile arrays, respectively. Ultrastructural analysis of clausenidin-treated cells was conducted using transmission electron microscopy. In addition, anti-angiogenic effects of clausenidin were investigated by Western blot analysis. Clausenidin significantly (p<0.05) increased the activity of caspase-8 and expression of protein components of the death inducing signaling complex (DISC) in HepG2 cells. Ultrastructural analysis of the clausenidin-treated HepG2 cells revealed morphological abnormalities typical of apoptosis. Furthermore, clausenidin significantly (p<0.05) decreased the expression of vascular endothelial growth factor (VEGF). Therefore, clausenidin is a potential anti-angiogenic agent which may induce apoptosis of hepatocellular carcinoma cells.

Edible bird's nest modulate intracellular molecular pathways of influenza A virus infected cells.

BACKGROUND: Edible Bird's Nest (EBN) as a popular traditional Chinese medicine is believed to have health enhancing and antiviral activities against influenza A virus (IAV); however, the molecular mechanism behind therapeutic effects of EBN is not well characterized. METHODS: In this study, EBNs that underwent different enzymatic preparation were tested against IAV infected cells. 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50) of the EBNs against IAV strain A/Puerto Rico/8/1934(H1N1) were determined by HA and MTT assays. Subsequently, the sialic acid content of the used EBNs were analyzed by fluorometric HPLC. Western Blotting and immunofluorescent staining were used to investigate the effects of EBNs on early endosomal trafficking and autophagy process of influenza virus. RESULTS: This study showed that post inoculations of EBNs after enzymatic preparations have the highest efficacy to inhibit IAV. While CC50 of the tested EBNs ranged from 27.5-32 mg/ml, the IC50 of these compounds ranged between 2.5-4.9 mg/ml. EBNs could inhibit IAV as efficient as commercial antiviral agents, such as amantadine and oseltamivir with different mechanisms of action against IAV. The antiviral activity of these EBNs correlated with the content of N-acetyl neuraminic acid. EBNs could affect early endosomal trafficking of the virus by reducing Rab5 and RhoA GTPase proteins and also reoriented actin cytoskeleton of IAV infected cells. In addition, for the first time this study showed that EBNs can inhibit intracellular autophagy process of IAV life cycle as evidenced by reduction of LC3-II and increasing of lysosomal degradation. CONCLUSIONS: The results procured in this study support the potential of EBNs as supplementary medication or alternative to antiviral agents to inhibit influenza infections. Evidently, EBNs can be a promising antiviral agent; however, these natural compounds should be screened for their metabolites prior to usage as therapeutic approach.

Combination of cisplatin and bromelain exerts synergistic cytotoxic effects against breast cancer cell line MDA-MB-231 in vitro.

BACKGROUND: Bromelain, which is a cysteine endopeptidase commonly found in pineapple stems, has been investigated as a potential anti-cancer agent for the treatment of breast cancer. However, information pertaining to the effects of combining bromelain with existing chemotherapeutic drugs remains scarce. This study aimed to investigate the possible synergistic cytotoxic effects of using bromelain in combination with cisplatin on MDA-MB-231 human breast cancer cells. METHOD: MDA-MB-231 cells were treated with different concentrations (0.24-9.5 µM) of bromelain or cisplatin alone, as well as four different combinations of these two agents to assess their individual and combination effects after 24 and 48 h. Cell viability was analyzed using an MTT assay. The induction of apoptosis was assessed using cell cycle analysis and an Annexin V-FITC assay. The role of the mitochondrial membrane potential in the apoptotic process was assessed using a JC-1 staining assay. Apoptotic protein levels were assessed by western blot analysis and proteome profiling using an antibody array kit. RESULTS: Single-agent treatment with cisplatin or bromelain led to dose- and time-dependent decreases in the viability of the MDA-MB-231 cells at 24 and 48 h. Furthermore, most of the combinations evaluated in this study displayed synergistic effects against MDA-MB-231 cells at 48 h, with combination 1 (bromelain 2 µM + cisplatin 1.5 µM) exhibiting the greatest synergistic effect (P = 0.000). The results of subsequent assays indicated that combination 1 treatment induced apoptosis via mitochondria-mediated pathway. Combination 1 also resulted in significant decreases in the levels of several apoptotic proteins such as Bcl-x and HSP70, compared with bromelain (P = 0.002 and 0.000, respectively) or cisplatin (P = 0.000 and 0.001, respectively) single treatment. Notably, MDA-MB-231 cells treated with combination 1 showed increased levels of the pro-apoptotic proteins Bax compared with those treated with bromelain (P = 0.000) or cisplatin single treatment (P = 0.043). CONCLUSION: Bromelain in combination with cisplatin synergistically enhanced the induction of apoptosis in MDA-MB-231 cells.

Clausenidin from Clausena excavata induces apoptosis in hepG2 cells via the mitochondrial pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Clausena excavata Burm.f. is used locally in folk medicine for the treatment of cancer in South East Asia. AIM OF THE STUDY: To determine the mechanism of action of pure clausenidin crystals in the induction of hepatocellular carcinoma (hepG2) cells apoptosis. MATERIALS AND METHODS: Pure clausenidin was isolated from Clausena excavata Burm.f. and characterized using 1H and 13C NMR spectra. Clausenidin-induced cytotoxicity was determined by MTT assay. The morphology of hepG2 after treatment with clausenidin was determined by fluorescence and Scanning Electron Microscopy. The effect of clausenidin on the apoptotic genes and proteins were determined by real-time qPCR and protein array profiling, respectively. The involvement of the mitochondria in clausenidin-induced apoptosis was investigated using MMP, caspase 3 and 9 assays. RESULTS: Clausenidin induced significant (p<0.05) and dose-dependent apoptosis of hepG2 cells. Cell cycle assay showed that clausenidin induced a G2/M phase arrest, caused mitochondrial membrane depolarization and significantly (p<0.05) increased expression of caspases 3 and 9, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, clausenidin caused decreased expression of the anti-apoptotic protein, Bcl 2 and increased expression of the pro-apoptotic protein, Bax. This finding was confirmed by the downregulation of Bcl-2 gene and upregulation of the Bax gene in the treated hepG2 cells. CONCLUSION: Clausenidin extracted from Clausena excavata Burm.f. is an anti-hepG2 cell compound as shown by its ability to induce apoptosis through the mitochondrial pathway of apoptosis. Clausenidin can potentially be developed into an anticancer compound.

Clausenidin induces caspase-dependent apoptosis in colon cancer.

BACKGROUND: Clausena excavata Burm.f. is a shrub traditionally used to treat cancer patients in Asia. The main bioactive chemical components of the plant are alkaloids and coumarins. In this study, we isolated clausenidin from the roots of C. excavata to determine its apoptotic effect on the colon cancer (HT-29) cell line. METHOD: We examined the effect of clausenidin on cell viability, ROS generation, DNA fragmentation, mitochondrial membrane potential in HT-29 cells. Ultrastructural analysis was conducted for morphological evidence of apoptosis in the treated HT-29 cells. In addition, we also evaluated the effect of clausenidin treatment on the expression of caspase 3 and 9 genes and proteins in HT-29 cells. RESULT: Clausenidin induced a G0/G1 cell cycle arrest in HT-29 cells with significant (p < 0.05) dose-dependent increase in apoptotic cell population. The DNA fragmentation assay also showed apoptotic features in the clausenidin-treated HT-29 cells. Clausenidin treatment had caused significant (p < 0.05) increases in the expression of caspase 9 protein and gene in HT-29 cells and mitochondrial ROS and mitochondrial membrane depolarization. The results suggest the involvement of the mitochondria in the caspase-dependent apoptosis in clausenidin-treated colon cancer cells. CONCLUSION: Clausenidin induces a caspase-dependent apoptosis in colon cancers through the stimulation of the mitochondria. The study demonstrates the potential of clausenidin for use in the treatment of colon cancers.

In vitro and in vivo mechanism of immunomodulatory and antiviral activity of Edible Bird's Nest (EBN) against influenza A virus (IAV) infection.

ETHNOPHARMACOLOGICAL RELEVANCE: For centuries, Edible Bird Nest (EBN) has been used in treatment of variety of respiratory diseases such as flu and cough as a Chinese natural medicine. AIM OF THE STUDY: This natural remedy showed the potential to inhibit influenza A virus (IAV). However, little is known about the mechanism of this process and also the evaluation of this product in an animal model. Hence, the current study was designed to elucidate the antiviral and immunomodulatory effects of EBN against IAV strain A/Puerto Rico/8/1934 (H1N1). MATERIALS AND METHODS: First, influenza infected MDCK cells treated with EBNs from two locations of Malaysia (Teluk Intan and Gua Madai) that prepared with different enzymatic preparations were analyzed by RT-qPCR and ELISA for detection of viral and cytokines genes. The sialic acid composition of these EBNs was evaluated by H-NMR. Subsequently, after toxicity evaluation of EBN from Teluk Intan, antiviral and immunomodulatory effects of this natural product was evaluated in BALB/c mice by analysis of the viral NA gene and cytokine expressions in the first week of the infection. RESULTS: EBN showed high neuraminidase inhibitory properties in both in vitro and in vivo, which was as effective as Oseltamivir phosphate. In addition, EBN decreased NS1 copy number (p<0.05) of the virus along with high immunomodulatory effects against IAV. Some of the immune changes during treatment of IAV with EBN included significant increase in IFNγ, TNFα, NFκB, IL2, some proinflammatory cytokines like IL1ß, IL6, and cytokines with regulatory properties like IL10, IL27, IL12, CCL2 and IL4 depends on the stage of the infection. EBNs from two locations contained different composition of sialic acid and thymol derivatives, which gave them different antiviral properties. EBN from Gua Madai that contained more acetylated sialic acid (Neu2,4,7,8,9 Ac6) showed higher antiviral activity. CONCLUSION: The findings of this study support the antiviral activity of EBN against influenza virus and validate the traditional usage of this natural remedy by elucidation of toxicity and the molecular mechanism of action.

Flavokawain B induced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB231 and inhibited the metastatic potential of MDA-MB231 via the regulation of several tyrosine kinases In vitro.

BACKGROUND: The kava-kava plant (Piper methysticum) is traditionally consumed by the pacific islanders and has been linked to be involved in several biological activities. Flavokawain B is a unique chalcone, which can be found in the roots of the kava-kava plant. In this study, the operational mechanism of the anti-cancer activity of a synthetic Flavokawain B (FKB) on two breast cancer cell lines, MCF-7 and MDA-MB231 was investigated. METHOD: Several in vitro assays were attempted such as MTT, flow cytometry of cell cycle analysis, annexin V analysis, and JC-1 analysis to detect apoptosis. Moreover, in vitro metastasis assays were also performed such as transwell migration assay, invasion assay, rat aorta ring and HUVEC tube formation. Molecular analysis of related genes and proteins were conducted using real-time PCR and proteome profiler analysis. RESULTS: Based on our results, apoptosis was induced when both MCF-7 and MDA-MB231 were treated with FKB. A significant G2/M arrest was seen in MDA-MB231 cells. Additionally, FKB also inhibited the in vitro migration and invasion in MDA-MB231 cells in a dose dependent manner. Moreover, FKB can be a potential inhibitor in angiogenesis as it suppressed the formation of vessels in HUVEC cells as well as in the ex-vivo rat aortic ring assay. CONCLUSION: Our findings suggested that FKB also regulated several receptor tyrosine kinases. Overall, FKB is not only a potential candidate to be an anti-cancer agent, but as an anti-metastatic agent as well.

In vitro Toxicity and in vivo Immunomodulatory Effects of Flavokawain A and Flavokawain B in Balb/C Mice.

Flavokawains are chalcones that can be found in the root extracts of the kava-kava (Piper methysticum) plant. Flavokawain A and flavokawain B are known to possess potential anti-inflammation and anti-cancer activities. Nevertheless, the effects of both these compounds on the normal function of the host have not been studied. There is a need to find agents that can enhance the functionality of the immune system without disturbing the homeostatic balance. This study aimed to determine the toxicity and immunomodulatory effects of flavokawain A and flavokawain B on Balb/c mice. Several assays were conducted, the MTT viability assay, cytokine detection (IL-2 and TNF-α), immunophenotyping of important immune markers, serum biochemical analysis and detection of nitric oxide levels. Based on our results, flavokawain A and B did not cause mortality and all mice were observed normal after the treatment period. Both flavokawains stimulated splenocyte proliferation, the secretion of IL-2 and TNF-α and raised the population of T cell subsets without significantly altering the level of several serum biochemical parameters. Overall, flavokawain A and B could serve as potential immune-modulator drugs without causing any toxicity, however further in vivo evidence is needed.

Development of enhanced antibody response toward dual delivery of nano-adjuvant adsorbed human Enterovirus-71 vaccine encapsulated carrier.

This study introduces a new approach for enhancing immunity toward mucosal vaccines. HEV71 killed vaccine that is formulated with nanosize calcium phosphate adjuvant and encapsulated onto chitosan and alginate delivery carriers was examined for eliciting antibody responses in serum and saliva collected at weeks 0, 1, 3, 5, 7 and 9 for viral-specific IgA & IgG levels and viral neutralizing antibody titers. The antibody responses induced in rabbits by the different formulations delivered by a single (buccal) route were compared to those of dual immunization (intradermal / mucosal) and un-immunized control. Chitosan-loaded vaccine adjuvant induced elevated IgA antibody, while Alginate-adjuvant irreversible bonding sequestered the vaccine and markedly reduced immunogenicity. The induced mucosal and parenteral antibody profiles appeared in an inverse manner of enhanced mucosal IgA antibody accompanied by lower systemic IgG following a single oral immunization route. The combined intradermal and oral dual-immunized group developed an elevated salivary IgA, systemic IgG, and virus neutralizing response. A reduced salivary neutralizing antibody titer was observed and attributed to the continual secretion exchanges in saliva. Designing a successful mucosal delivery formulation needs to take into account the vaccine delivery site, dosage, adjuvant and carrier particle size, charge, and the reversibility of component interactions. The dual immunization seems superior and is a important approach for modulating the antibody response and boosting mucosal protection against HEV71 and similar pathogens based on their transmission mode, tissue tropism and shedding sites. Finally, the study has highlighted the significant role of dual immunization for simultaneous inducing and modulating the systemic and mucosal immune responses to EV71.

In Vivo Anti-Tumor Effects of Flavokawain A in 4T1 Breast Cancer Cell-Challenged Mice.

Flavokawain A is a chalcone that can be found in the kava-kava plant (Piper methsyticum) extract. The kava-kava plant has been reported to possess anti-cancer, anti-inflammatory and antinociceptive activities. The state of the immune system, and the inflammatory process play vital roles in the progression of cancer. The immunomodulatary effects and the anti-inflammatory effects of flavokawain A in a breast cancer murine model have not been studied yet. Thus, this study aimed to elucidate the basic mechanism as to how flavokawain A regulates and enhance the immune system as well as impeding the inflammatory process in breast cancer-challenged mice. Based on our study, it is interesting to note that flavokawain A increased the T cell population; both Th1 cells and CTLs, aside from the natural killer cells. The levels of IFN-γ and IL-2 were also elevated in the serum of flavokawain A-treated mice. Apart from that, flavokawain A also decreased the weight and volume of the tumor, and managed to induce apoptosis in them. In terms of inflammation, flavokawain A-treated mice had reduced level of major pro-inflammatory mediators; NO, iNOS, NF-KB, ICAM and COX-2. Overall, flavokawain A has the potential to not only enhance antitumor immunity, but also prevents the inflammatory process in a cancer-prone microenvironment.

Neural differentiation of human umbilical cord matrix-derived mesenchymal cells under special culture conditions.

Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.

Prophylactic effect of herbal-marine compound (HESA-A) on influenza A virus infectivity.

BACKGROUND: Influenza virus is still a severe respiratory disease affecting human and other species. As conventional drugs are not recommended for long time because of side effects and drug resistance occurrence, traditional medication has been focused as alternative remedy. HESA-A is a natural compound from herbal-marine origin. Previous studies have reported the therapeutic properties of HESA-A on psoriasis vulgaris and different types of cancers and we also showed its anti-inflammatory effects against influenza A infection. METHODS: This study was designed to investigate the potential properties of HESA-A as prophylaxis or treatment. To investigate the prophylaxis or treatment activities of HESA-A, Madin-Darby Canine Kidney (MDCK) cells were exposed to HESA-A and influenza A virus in different manners of exposure and different time intervals. The results were evaluated by MTT and HA assays. RESULTS: It was found that HESA-A is much more effective against influenza cytopathic effects when it is applied for prophylaxis and also in concurrent treatment (p ≤ 0.05) but not in post-infection treatment (p ≥ 0.05). CONCLUSION: In conclusion, HESA-A is significantly effective against influenza replication in prophylaxis application affecting the virus penetration/adsorption to the cell without any toxic effect on the cell viability.

Vitamin C suppresses cell death in MCF-7 human breast cancer cells induced by tamoxifen.

Vitamin C is generally thought to enhance immunity and is widely taken as a supplement especially during cancer treatment. Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial oestrogen receptor beta in MCF-7 cells and induces apoptosis by activation of pro-caspase-8 followed by downstream events, including an increase in reactive oxygen species and the release of pro-apoptotic factors from the mitochondria. In addition to that, TAM binds with high affinity to the microsomal anti-oestrogen-binding site and inhibits cholesterol esterification at therapeutic doses. This study aimed to investigate the role of vitamin C in TAM-mediated apoptosis. Cells were loaded with vitamin C by exposure to dehydroascorbic acid, thereby circumventing in vitro artefacts associated with the poor transport and pro-oxidant effects of ascorbic acid. Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by acridine-orange/propidium iodide (AO/PI) and Annexin V assay after treatment with TAM. Vitamin C dose-dependently protected cancer cells against lipid peroxidation caused by TAM treatment. By real-time PCR analysis, an impressive increase in FasL and tumour necrosis factor-α (TNF-α) mRNA was detected after TAM treatment. In addition, a decrease in mitochondrial transmembrane potential was observed. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

The flavokawains: uprising medicinal chalcones.

Plant-based compounds have been in the spotlight in search of new and promising drugs. Flavokawain A, B and C are naturally occurring chalcones that have been isolated from several medicinal plants; namely the piper methysticum or commercially known as the kava-kava. Multiple researches have been done to evaluate the bioactivities of these compounds. It has been shown that all three flavokawains may hold promising anti-cancer effects. It has also been revealed that both flavokawain A and B are involved in the induction of cell cycle arrest in several cancer cell lines. Nevertheless, flavokawain B was shown to be more effective in treating in vitro cancer cell lines as compared to flavokawain A and C. Flavokawain B also exerts antinociceptive effects as well as anti-inflammation properties. This mini-review attempts to discuss the biological properties of all the flavokawains that have been reported.

Rhaphidophora korthalsii modulates peripheral blood natural killer cell proliferation, cytokine secretion and cytotoxicity.

BACKGROUND: Rhaphidophora korthalsii (Araceae) is a root-climber plant which has been widely used in Chinese traditional medicine for cancer and skin disease treatment. Previous reports have recorded its immunomodulatory effects on mice splenocyte and human peripheral blood. This study investigated the potential immunostimulatory effect of Rhaphidophora korthalsii on human PBMC enriched NK cell. METHODS: PBMC was exposed to various concentrations of R. korthalsii extract and the T and NK cell population in the control and extract treated PBMC were identified by immunophenotyping. Intracellular perforin and granzyme B expressions were detected by flow cytometry and extra-cellular Granzyme B, IFN-γ and TNF-α production in the isolated NK cells were determined by ELISA. The cytotoxicity of effector NK cell towards target K562 cell was assessed by CytoTox 96 assay. RESULTS: Rhaphidophora korthalsii methanol extract significantly increased PBMC NK cell population and intracellular perforin and granzyme B expressions. Moreover, the extract also enhanced the secretion of IFN-γ and TNF-α which subsequently enhanced the cytotoxicity of NK cell against the NK sensitive target K562 cell line. NK cell enriched with extract treated PBMC showed better activation than NK cell directly treated with the extract. CONCLUSION: Our findings indicated a potential IL-2 free immunotherapy through direct and indirect R. korthalsii stimulation on NK cell activation.

Attenuation of influenza virus infectivity with herbal-marine compound (HESA-A): an in vitro study in MDCK cells.

BACKGROUND: The influenza virus is still one of the most important respiratory risks affecting humans which require effective treatments. In this case, traditional medications are of interest. HESA-A is an active natural biological compound from herbal-marine origin. Previous studies have reported that the therapeutic properties of HESA-A are able to treat psoriasis vulgaris and cancers. However, no antiviral properties have been reported. METHODS: This study was designed to investigate the potential antiviral properties of HESA-A and its effects in modulating TNF-α and IL-6 cytokine levels. HESA-A was prepared in normal saline as a stock solution (0.8 mg/ml, pH = 7.4). Percentages of cell survival when exposed to different concentrations of HESA-A at different time intervals was determined by MTT assay. To study the potential antiviral activity of HESA-A, Madin-Darby Canine Kidney (MDCK) cells were treated with the effective concentration (EC50) of HESA-A (0.025 mg/ml) and 100 TCID50/0.1 ml of virus sample under different types of exposure. RESULTS: Based on the MTT method and hemagglutination assay (HA), HESA-A is capable of improving cell viability to 31% and decreasing HA titre to almost 99% in co-penetration exposures. In addition, based on quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), it was found that HESA-A causes decrements in TNF-α and IL-6 cytokine expressions, which was significant for TNF-α (p ≤ 0.05) but not for IL-6. CONCLUSION: In conclusion, HESA-A was effective against influenza infection through suppressing cytokine expression.

Immunomodulatory Effect of Rhaphidophora korthalsii on Natural Killer Cell Cytotoxicity.

The in vivo immunomodulatory effect of ethanolic extracts from leaves of Rhaphidophora korthalsii was determined via immune cell proliferation, T/NK cell phenotyping, and splenocyte cytotoxicity of BALB/c mice after 5 consecutive days of i.p. administration at various concentrations. Splenocyte proliferation index, cytotoxicity, peripheral blood T/NK cell population, and plasma cytokine (IL-2 and IFN-γ) in mice were assessed on day 5 and day 15. High concentration of extract (350 µg/mice/day for 5 consecutive days) was able to stimulate immune cell proliferation, peripheral blood NK cell population, IL-2, and IFN- γ cytokines, as well as splenocyte cytotoxicity against Yac-1 cell line. Unlike rIL-2 which degraded rapidly, the stimulatory effect from the extract managed to last until day 15. These results suggested the potential of this extract as an alternative immunostimulator, and they encourage further study on guided fractionation and purification to identify the active ingredients that contribute to this in vitro and in vivo immunomodulatory activity.

Bioactive compounds and biological activities of Jatropha curcas L. kernel meal extract.

Defatted Jatropha curcas L. (J. curcas) seed kernels contained a high percentage of crude protein (61.8%) and relatively little acid detergent fiber (4.8%) and neutral detergent fiber (9.7%). Spectrophotometric analysis of the methanolic extract showed the presence of phenolics, flavonoids and saponins with values of 3.9, 0.4 and 19.0 mg/g DM, respectively. High performance liquid chromatography (HPLC) analyses showed the presence of gallic acid and pyrogallol (phenolics), rutin and myricetin (flavonoids) and daidzein (isoflavonoid). The amount of phorbol esters in the methanolic extract estimated by HPLC was 3.0 ± 0.1 mg/g DM. Other metabolites detected by GC-MS include: 2-(hydroxymethyl)-2 nitro-1,3-propanediol, ß-sitosterol, 2-furancarboxaldehyde, 5-(hydroxymethy) and acetic acid in the methanolic extract; 2-furancarboxaldehyde, 5-(hydroxymethy), acetic acid and furfural (2-furancarboxaldehyde) in the hot water extract. Methanolic and hot water extracts of kernel meal showed antimicrobial activity against both Gram positive and Gram negative pathogenic bacteria (inhibition range: 0-1.63 cm) at the concentrations of 1 and 1.5 mg/disc. Methanolic extract exhibited antioxidant activities that are higher than hot water extract and comparable to ß-carotene. The extracts tended to scavenge the free radicals in the reduction of ferric ion (Fe(3+)) to ferrous ion (Fe(2+)). Cytotoxicity assay results indicated the potential of methanolic extract as a source of anticancer therapeutic agents toward breast cancer cells.

Identification and characterisation of a novel anti-viral peptide against avian influenza virus H9N2.

BACKGROUND: Avian influenza viruses (AIV) cause high morbidity and mortality among the poultry worldwide. Their highly mutative nature often results in the emergence of drug resistant strains, which have the potential of causing a pandemic. The virus has two immunologically important glycoproteins, hemagglutinin (HA), neuraminidase (NA), and one ion channel protein M2 which are the most important targets for drug discovery, on its surface. In order to identify a peptide-based virus inhibitor against any of these surface proteins, a disulfide constrained heptapeptide phage display library was biopanned against purified AIV sub-type H9N2 virus particles. RESULTS: After four rounds of panning, four different fusion phages were identified. Among the four, the phage displaying the peptide NDFRSKT possessed good anti-viral properties in vitro and in ovo. Further, this peptide inhibited the hemagglutination activity of the viruses but showed very little and no effect on neuraminidase and hemolytic activities respectively. The phage-antibody competition assay proved that the peptide competed with anti-influenza H9N2 antibodies for the binding sites. Based on yeast two-hybrid assay, we observed that the peptide inhibited the viral replication by interacting with the HA protein and this observation was further confirmed by co-immunoprecipitation. CONCLUSION: Our findings show that we have successfully identified a novel antiviral peptide against avian influenza virus H9N2 which act by binding with the hemagglutination protein of the virus. The broad spectrum activity of the peptide molecule against various subtypes of the avian and human influenza viruses and its comparative efficiency against currently available anti-influenza drugs are yet to be explored.