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1.  Nordamnacanthal potentiates the cytotoxic effects of tamoxifen in human breast cancer cells 
Oncology Letters  2014;9(1):335-340.
Tamoxifen (TAM) is the mainline drug treatment for breast cancer, despite its side effects and the development of resistance. As an alternative approach, in the present study a novel combination therapy was established through combining TAM with nordamnacanthal (NDAM) in order to investigate the additive effect of these drugs in MCF-7 human breast cancer cells. A significant dose-dependent reduction in cell viability and an increase in apoptosis were observed in the MCF-7 cells cotreated with TAM and NDAM compared with the untreated control cells or the cells treated with TAM and NDAM alone (P<0.05). The cytotoxic influence of the combination of TAM and NDAM was found to be two-fold that of the individual agents. Annexin V/propidium iodide double-staining revealed the typical nuclear features of apoptosis. Furthermore, an increase in the proportion of apoptotic, Annexin V-positive cells was observed with the combination therapy. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and the generation of reactive oxygen species. To the best of our knowledge, the findings of the present study are the first to suggest that combining TAM with NDAM may be a potential combination therapy for the treatment of breast cancer and may have the potential to minimize or eliminate the side effects associated with high doses of TAM.
doi:10.3892/ol.2014.2697
PMCID: PMC4247001  PMID: 25435988
tamoxifen; nordamnacanthal; MCF-7; breast cancer; combination treatment; apoptosis
2.  Anti-Inflammatory and Antinociceptive Activities of Untreated, Germinated, and Fermented Mung Bean Aqueous Extract 
Evaluation of anti-inflammatory and antinociceptive activities of untreated mung bean (MB), germinated mung bean (GMB), and fermented mung bean (FMB) was performed on both in vitro (inhibition of inflammatory mediator, nitric oxide(NO)) and in vivo (inhibition of ear oedema and reduction of response to pain stimulus) studies. Results showed that both GMB and FMB aqueous extract exhibited potent anti-inflammatory and antinociceptive activities in a dose-dependent manner. In vitro results showed that GMB and FMB were potent inflammatory mediator (NO) inhibitors at both 2.5 and 5 mg/mL. Further in vivo studies showed that GMB and FMB aqueous extract at 1000 mg/kg can significantly reduce ear oedema in mice caused by arachidonic acid. Besides, both 200 mg/kg and 1000 mg/kg concentrations of GMB and FMB were found to exhibit potent antinociceptive effects towards hotplate induced pain. With these, it can be concluded that GMB and FMB aqueous extract exhibited potential anti-inflammatory and antinociceptive effects.
doi:10.1155/2014/350507
PMCID: PMC4089844  PMID: 25045389
3.  Immunomodulatory effects of Potentilla indica and Dendrophthoe pentandra on mice splenocytes and thymocytes 
Immunomodulators are agents that are able to stimulate or inhibit the immune response. The leaf extracts from Potentilla indica and Dendrophthoe pentandra were analyzed in vitro for immunomodulatory activity and an MTT colorimetric assay was conducted to determine the proliferation of mice splenocytes and thymocytes. A bromodeoxyuridine assay was performed to analyze DNA synthesis and the Trypan blue exclusion method was conducted to evaluate the changes in total cell population. The results indicated that treatment with P. indica and D. pentandra produced a time- and dose-dependent increase in cell viability and proliferation. Following 72 h of treatment with P. indica and D. pentandra, thymocyte proliferation was augmented by 18 and 41%, respectively and splenocyte proliferation increased by 35 and 42%, respectively, when compared with untreated cells. The present study demonstrated that these extracts may act as potential immunostimulants and, thus, represent an alternative source of immunomodulatory compounds for the treatment of human immune-mediated diseases.
doi:10.3892/etm.2014.1657
PMCID: PMC4043603  PMID: 24926376
immunomodulatory; Rosaceae; mistletoe; bromodeoxyuridine assay; MTT; spleen; thymus
4.  Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells 
Oncology Letters  2014;7(5):1479-1484.
Damnacanthal, an anthraquinone compound, is isolated from the roots of Morinda citrifolia L. (noni), which has been used for traditional therapy in several chronic diseases, including cancer. Although noni has long been consumed in Asian and Polynesian countries, the molecular mechanisms by which it exerts several benefits are starting to emerge. In the present study, the effect of damnacanthal on MCF-7 cell growth regulation was investigated. Treatment of MCF-7 cells with damnacanthal for 72 h indicated an antiproliferative activity. The MTT method confirmed that damnacanthal inhibited the growth of MCF-7 cells at the concentration of 8.2 μg/ml for 72 h. In addition, the drug was found to induce cell cycle arrest at the G1 checkpoint in MCF-7 cells by cell cycle analysis. Damnacanthal induced apoptosis, determined by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) dual-labeling, acridine-orange/PI dyeing and caspase-7 expression. Furthermore, damnacanthal-mediated apoptosis involves the sustained activation of p21, leading to the transcription of p53 and the Bax gene. Overall, the present study provided significant evidence demonstrating that p53-mediated damnacanthal induced apoptosis through the activation of p21 and caspase-7.
doi:10.3892/ol.2014.1898
PMCID: PMC3997671  PMID: 24765160
damnacanthal; MCF-7; anticancer; caspase; p53; p21; apoptosis
5.  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.
doi:10.1111/jcmm.12188
PMCID: PMC3930417  PMID: 24266867
vitamin C; tamoxifen; breast cancer; ROS; free radical; apoptosis; lipid peroxidation
6.  Hepatoprotective Effect of Fermented Soybean (Nutrient Enriched Soybean Tempeh) against Alcohol-Induced Liver Damage in Mice 
Recently, soybean tempeh has received great attention due to many advantages such as higher nutritional value, lower production cost, and shorter fermentation time. In this study, the in vivo hepatoprotective and antioxidant effects of nutrient enriched soybean tempeh (NESTE) were determined. NESTE fermentation process which involved anaerobic incubation was previously proclaimed to increase the content of amino acids and antioxidant properties remarkably. The evaluation of histological sections, serum biochemical markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cholesterol and triglycerides (TG)), liver immune response level (nitric oxide (NO)) and liver antioxidant level (superoxide dismutase (SOD), ferric reducing antioxidant power (FRAP), and malondialdehyde (MDA)) was conducted in order to compare the effects of nonfermented soybean extract (SBE) and fermented soybean extract (NESTE) on alcohol-induced liver damage in mice. Results demonstrated that 1000 mg/kg of NESTE can significantly reduce the levels of AST, ALT, cholesterol, TG, MDA, and NO. On the other hand, it also raised the level of SOD and FRAP. Furthermore, the histological examination on 1000 mg/kg NESTE treatment group showed that this extract was capable of recovering the damaged hepatocytes to their normal structures. Thus, it can be concluded that NESTE produced through fermentation process was able to enhance hepatoprotective and antioxidant effects in vivo.
doi:10.1155/2013/274274
PMCID: PMC3766609  PMID: 24058369
7.  The Possible Potential Therapeutic Targets for Drug Induced Gingival Overgrowth 
Mediators of Inflammation  2013;2013:639468.
Gingival overgrowth is a side effect of certain medications. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin, the least fibrotic lesions are caused by cyclosporin A, and the intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Fibrosis is one of the largest groups of diseases for which there is no therapy but is believed to occur because of a persistent tissue repair program. During connective tissue repair, activated gingival fibroblasts synthesize and remodel newly created extracellular matrix. Proteins such as transforming growth factor (TGF), endothelin-1 (ET-1), angiotensin II (Ang II), connective tissue growth factor (CCN2/CTGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) appear to act in a network that contributes to the development of gingival fibrosis. Since inflammation is the prerequisite for gingival overgrowth, mast cells and its protease enzymes also play a vital role in the pathogenesis of gingival fibrosis. Drugs targeting these proteins are currently under consideration as antifibrotic treatments. This review summarizes recent observations concerning the contribution of TGF-β, CTGF, IGF, PDGF, ET-1, Ang II, and mast cell chymase and tryptase enzymes to fibroblast activation in gingival fibrosis and the potential utility of agents blocking these proteins in affecting the outcome of drug-induced gingival overgrowth.
doi:10.1155/2013/639468
PMCID: PMC3652200  PMID: 23690667
8.  Influence of Mast Cells in Drug-Induced Gingival Overgrowth 
Mediators of Inflammation  2013;2013:275172.
Mast cells (MCs) are multifunctional effector cells that were originally thought to be involved in allergic disorders. Now it is known that they contain an array of mediators with a multitude of effects on many other cells. MCs have become a recent concern in drug-induced gingival overgrowth (DIGO), an unwanted outcome of systemic medication. Most of the studies have confirmed the significant presence of inflammation as a prerequisite for the overgrowth to occur. The inflammatory changes within the gingival tissue appear to influence the interaction between the inducing drug and the fibroblast activity. The development of antibodies to MC-specific enzymes, tryptase and chymase, has facilitated the study of mast cells in DIGO. Many immunohistochemical studies involving MCs have been conducted; as a result, DIGO tissues are found to have increased the number of MCs in the gingiva, especially in the area of fibrosis. At the cellular level, gingival fibrogenesis is initiated by several mediators which induce the recruitment of a large number of inflammatory cells, including MCs. The purpose of this paper is to access the roles played by MCs in gingival overgrowth to hypothesize a relationship between these highly specialized cells in the pathogenesis of DIGO.
doi:10.1155/2013/275172
PMCID: PMC3569901  PMID: 23431239
9.  Antioxidant and Hepatoprotective Effect of Aqueous Extract of Germinated and Fermented Mung Bean on Ethanol-Mediated Liver Damage 
BioMed Research International  2012;2013:693613.
Mung bean is a hepatoprotective agent in dietary supplements. Fermentation and germination processes are well recognized to enhance the nutritional values especially the concentration of active compounds such as amino acids and GABA of various foods. In this study, antioxidant and hepatoprotective effects of freeze-dried mung bean and amino-acid- and GABA-enriched germinated and fermented mung bean aqueous extracts were compared. Liver superoxide dismutase (SOD), malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), nitric oxide (NO) levels, and serum biochemical profile such as aspartate transaminase (AST), alanine transaminase (ALT), triglycerides (TG), and cholesterol and histopathological changes were examined for the antioxidant and hepatoprotective effects of these treatments. Germinated and fermented mung bean have recorded an increase of 27.9 and 7.3 times of GABA and 8.7 and 13.2 times of amino acid improvement, respectively, as compared to normal mung bean. Besides, improvement of antioxidant levels, serum markers, and NO level associated with better histopathological evaluation indicated that these extracts could promote effective recovery from hepatocyte damage. These results suggested that freeze-dried, germinated, and fermented mung bean aqueous extracts enriched with amino acids and GABA possessed better hepatoprotective effect as compared to normal mung bean.
doi:10.1155/2013/693613
PMCID: PMC3591205  PMID: 23484140
10.  Betulinic Acid Inhibits Growth of Cultured Vascular Smooth Muscle Cells In Vitro by Inducing G1 Arrest and Apoptosis 
Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC) is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC50 of 3.8 μg/mL significantly (P < 0.05). Nevertheless, betulinic acid exhibited G1 cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G1 cell cycle arrest and dose-dependent DNA damage on VSMC.
doi:10.1155/2012/251362
PMCID: PMC3463985  PMID: 23056140
11.  Hepatoprotective Activity of Elephantopus scaber on Alcohol-Induced Liver Damage in Mice 
Elephantopus scaber has been traditionally used as liver tonic. However, the protective effect of E. scaber on ethanol-induced liver damage is still unclear. In this study, we have compared the in vivo hepatoprotective effect of E. scaber with Phyllanthus niruri on the ethanol-induced liver damage in mice. The total phenolic and total flavanoid content of E. scaber ethanol extract were determined in this study. Accelerating serum biochemical profiles (including AST, ALT, ALP, triglyceride, and total bilirubin) associated with fat drop and necrotic body in the liver section were observed in the mice treated with ethanol. Low concentration of E. scaber was able to reduce serum biochemical profiles and the fat accumulation in the liver. Furthermore, high concentration of E. scaber and positive control P. niruri were able to revert the liver damage, which is comparable to the normal control. Added to this, E. scaber did not possess any oral acute toxicity on mice. These results suggest the potential effect of this extract as a hepatoprotective agent towards-ethanol induced liver damage without any oral acute toxicity effect. These activities might be contributed, or at least in part, by its high total phenolic and flavonoid contents.
doi:10.1155/2012/417953
PMCID: PMC3437691  PMID: 22973401
12.  In Vitro and In Vivo Toxicity of Garcinia or Hydroxycitric Acid: A Review 
Obesity is one of the pandemic chronic diseases commonly associated with health disorders such as heart attack, high blood pressure, diabetes or even cancer. Among the current natural products for obesity and weight control, Garcinia or more specifically hydroxycitric acid (HCA) extracted from Garcinia has been widely used. The evaluation of the potential toxicity of weight control supplement is of the utmost importance as it requires long term continuous consumption in order to maintain its effects. Majority of reports demonstrated the efficacy of Garcinia/HCA without any toxicity found. However, a few clinical toxicity reports on weight-loss diet supplements of which some were combinations that included Garcinia/HCA as an active ingredient showed potential toxicity towards spermatogenesis. Nonetheless, it cannot be concluded that Garcinia/HCA is unsafe. Those products which have been reported to possess adverse effects are either polyherbal or multi-component in nature. To date, there is no case study or report showing the direct adverse effect of HCA. The structure, mechanism of action, long history of the use of Garcinia/HCA and comprehensive scientific evidence had shown “no observed adverse effect level (NOAEL)” at levels up to 2800 mg/day, suggesting its safety for use.
doi:10.1155/2012/197920
PMCID: PMC3424601  PMID: 22924054
13.  scFv Antibody: Principles and Clinical Application 
To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.
doi:10.1155/2012/980250
PMCID: PMC3312285  PMID: 22474489
14.  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.
doi:10.1155/2012/786487
PMCID: PMC3175394  PMID: 21941589
15.  Antioxidant effects of pineapple vinegar in reversing of paracetamol-induced liver damage in mice 
Chinese Medicine  2015;10:3.
Background
Pineapple (Ananas comosus) was demonstrated to be hepatoprotective. This study aims to investigate the reversing effects of pineapple vinegar on paracetamol-induced liver damage in murine model.
Methods
Pineapple juice was fermented via anaerobic and aerobic fermentation to produce pineapple vinegar. Male BALB/c mice (n = 70) were separated into 7 treatment groups (n = 10). Pineapple vinegar (0.08 and 2 mL/kg BW) and synthetic vinegar were used to treat paracetamol-induced liver damage in mice. The hepatoprotective effects were determined by serum biochemistry profiles (aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and triglyceride (TG)), liver antioxidant levels (ferric-reducing ability plasma (FRAP), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), and reduced glutathione assays (GSH)) and histopathological examination with hematoxylin and eosin (H&E) staining. The effects were further evaluated by the expression levels of iNOS, NF-κB, and cytochrome P450 2E1 by quantitative real-time PCR and Western blot analyses. Vinegar samples were also tested for in vitro antioxidant (FRAP, 2,2-diphenyl-2-picrylhydrazyl (DPPH), and total phenolic content (TPC)). Soluble phenolic acid contents in the samples were identified by HPLC.
Results
Pineapple vinegar contained 169.67 ± 0.05 μg GAE/mL of TPC, with 862.61 ± 4.38 μg/mL gallic acid as the main component. Oral administration of pineapple vinegar at 2 mL/kg BW reduced serum enzyme biomarker levels, including AST (P = 0.008), ALT (P = 0.006), ALP (P= 0.002), and TG (P = 0.006) after 7 days of paracetamol treatment. Liver antioxidant levels such as hepatic glutathione (P = 0.003), SOD (P < 0.001), lipid peroxidation (P = 0.002) and FRAP (P <0.001) were restored after the treatment. Pineapple vinegar reduced the expressions of iNOS (P = 0.003) and NF-kB (P = 0.003) and the level of NO (P = 0.003) significantly. Pineapple vinegar also downregulated liver cytochrome P450 protein expression.
Conclusions
Oral administration of pineapple vinegar at 0.08 and 2 mL/kg BW reduced serum enzyme biomarker levels, restored liver antioxidant levels, reduced inflammatory factor expressions, and down regulated liver cytochrome P450 protein expression in paracetamol-induced liver damage in mice.
doi:10.1186/s13020-015-0030-4
PMCID: PMC4333164
16.  Antistress and antioxidant effects of virgin coconut oil in vivo 
Virgin coconut oil (VCO) has been consumed worldwide for various health-related reasons and some of its benefits have been scientifically evaluated. Medium-chain fatty acids were found to be a potential antidepressant functional food; however, this effect had not been evaluated in VCO, which is rich in polyphenols and medium-chain fatty acids. The aim of this study was to evaluate the antistress and antioxidant effects of VCO in vivo, using mice with stress-induced injury. The antistress effect of VCO (administered per os, at a dose of 10 ml/kg body weight) was evaluated using the forced swim test and chronic cold restraint stress models. VCO was able to reduce immobility time and restore oxidative stress in mice post-swim test. Furthermore, mice treated with VCO were found to exhibit higher levels of brain antioxidants, lower levels of brain 5-hydroxytryptamine and reduced weight of the adrenal glands. Consequently, the serum cholesterol, triglyceride, glucose and corticosterone levels were also lower in VCO-treated mice. These results suggest the potential value of VCO as an antistress functional oil.
doi:10.3892/etm.2014.2045
PMCID: PMC4247320  PMID: 25452773
antioxidant; depression; medium-chain fatty acids
17.  Flavokawain A Induces Apoptosis in MCF-7 and MDA-MB231 and Inhibits the Metastatic Process In Vitro 
PLoS ONE  2014;9(10):e105244.
Introduction
The kava-kava plant (Piper methsyticum) is traditionally known as the pacific elixir by the pacific islanders for its role in a wide range of biological activities. The extract of the roots of this plant contains a variety of interesting molecules including Flavokawain A and this molecule is known to have anti-cancer properties. Breast cancer is still one of the leading diagnosed cancers in women today. The metastatic process is also very pertinent in the progression of tumorigenesis.
Methods
MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed.
Results
We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA's anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well.
Conclusions
FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.
doi:10.1371/journal.pone.0105244
PMCID: PMC4186755  PMID: 25286005
18.  Probiotic Potential of Lactobacillus Strains with Antimicrobial Activity against Some Human Pathogenic Strains 
BioMed Research International  2014;2014:927268.
The objective of this study was to isolate, identify, and characterize some lactic acid bacterial strains from human milk, infant feces, and fermented grapes and dates, as potential probiotics with antimicrobial activity against some human pathogenic strains. One hundred and forty bacterial strains were isolated and, after initial identification and a preliminary screening for acid and bile tolerance, nine of the best isolates were selected and further identified using 16 S rRNA gene sequences. The nine selected isolates were then characterized in vitro for their probiotic characteristics and their antimicrobial activities against some human pathogens. Results showed that all nine isolates belonged to the genus Lactobacillus. They were able to tolerate pH 3 for 3 h, 0.3% bile salts for 4 h, and 1.9 mg/mL pancreatic enzymes for 3 h. They exhibited good ability to attach to intestinal epithelial cells and were not resistant to the tested antibiotics. They also showed good antimicrobial activities against the tested pathogenic strains of humans, and most of them exhibited stronger antimicrobial activity than the reference strain L. casei Shirota. Thus, the nine Lactobacillus strains could be considered as potential antimicrobial probiotic strains against human pathogens and should be further studied for their human health benefits.
doi:10.1155/2014/927268
PMCID: PMC4106073  PMID: 25105147
19.  In Vivo Hypocholesterolemic Effect of MARDI Fermented Red Yeast Rice Water Extract in High Cholesterol Diet Fed Mice 
Fermented red yeast rice has been traditionally consumed as medication in Asian cuisine. This study aimed to determine the in vivo hypocholesterolemic and antioxidant effects of fermented red yeast rice water extract produced using Malaysian Agricultural Research and Development Institute (MARDI) Monascus purpureus strains in mice fed with high cholesterol diet. Absence of monacolin-k, lower level of γ-aminobutyric acid (GABA), higher content of total amino acids, and antioxidant activities were detected in MARDI fermented red yeast rice water extract (MFRYR). In vivo MFRYR treatment on hypercholesterolemic mice recorded similar lipid lowering effect as commercial red yeast rice extract (CRYR) as it helps to reduce the elevated serum liver enzyme and increased the antioxidant levels in liver. This effect was also associated with the upregulation of apolipoproteins-E and inhibition of Von Willebrand factor expression. In summary, MFRYR enriched in antioxidant and amino acid without monacolin-k showed similar hypocholesterolemic effect as CRYR that was rich in monacolin-k and GABA.
doi:10.1155/2014/707829
PMCID: PMC4083602  PMID: 25031606
20.  In Vivo Antistress and Antioxidant Effects of Fermented and Germinated Mung Bean 
BioMed Research International  2014;2014:694842.
Mung bean has been traditionally used to alleviate heat stress. This effect may be contributed by the presence of flavonoids and γ-aminobutyric acid (GABA). On the other hand, fermentation and germination have been practised to enhance the nutritional and antioxidant properties of certain food products. The main focus of current study was to compare the antistress effect of none-process, fermented and germinated mung bean extracts. Acute and chronic restraint stresses were observed to promote the elevation of serum biochemical markers including cholesterol, triglyceride, total protein, liver enzymes, and glucose. Chronic cold restraint stress was observed to increase theadrenal gland weight, brain 5-hydroxytryptamine (5-HT), and malondialdehyde (MDA) level while reducing brain antioxidant enzyme level. However, these parameters were found reverted in mice treated with diazepam, high concentration of fermented mung bean and high concentration of germinated mung bean. Moreover, enhanced level of antioxidant on the chronic stress mice was observed in fermented and germinated mung bean treated groups. In comparison between germinated and fermented mung bean, fermented mung bean always showed better antistress and antioxidant effects throughout this study.
doi:10.1155/2014/694842
PMCID: PMC4022108  PMID: 24877129
21.  Crude ethyl acetate extract of marine microalga, Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells 
Pharmacognosy Magazine  2014;10(37):1-8.
Background:
Marine brown diatom Chaetoceros calcitrans and green microalga Nannochloropsis oculata are beneficial materials for various applications in the food, nutraceutical, pharmaceutical and cosmeceutical industries.
Objective:
This study investigated cytotoxicity of different crude solvent extracts from C. calcitrans and N. oculata against various cancer cell lines.
Materials and Methods:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out to screen the cytotoxic effects of hexane (Hex), dichloromethane (DCM), ethyl acetate, and methanol extract from C. calcitrans and N. oculata toward various cancer cell lines. Flow cytometry cell cycle was used to determine the cell cycle arrest while the mode of cell death was investigated through acridine orange/propidium iodide (AOPI) staining, Annexin V-Fluorescein Isothiocyanate (FITC) and Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling (TUNEL) assays. Expression profile of apoptotic and proliferative-related genes was then determined using the multiplex gene expression profiler (GeXP).
Results:
Crude ethyl acetate (CEA) extract of C. calcitrans inhibited growth of MDA-MB-231 cells, with IC50 of 60 μg/mL after 72 h of treatment. Further studies were conducted to determine the mode of cell death at various concentrations of this extract: 30, 60 and 120 μg/mL. The mode of cell death was mainly apoptosis as shown through apoptosis determination test. The expression data from GeXP showed that caspase-4 was upregulated while B-cell leukemia/lymphoma 2(Bcl-2) was down regulated. Thus, caspase-4 induction endoplasmic reticulum death pathway is believed to be one of the mechanisms underlying the induction of apoptosis while Bcl-2 induced S and G2/M cell cycle phase arrest in MDA-MB-231 cells.
Conclusion:
CEA extract of C. calcitrans showed the highest cytotoxicity on MDA-MB-231 via apoptosis.
doi:10.4103/0973-1296.126650
PMCID: PMC3969653  PMID: 24696543
Apoptosis; Chaetoceros calcitrans; crude ethyl acetate extract; gene expression profiler; MDA-MB-231
22.  Immunomodulatory Effects of Newcastle Disease Virus AF2240 Strain on Human Peripheral Blood Mononuclear Cells 
Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.
doi:10.7150/ijms.8170
PMCID: PMC4196125  PMID: 25317070
Newcastle disease virus; AF2240; immunomodulation; PBMC; breast cancer; immunophenotyping
23.  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.
doi:10.1186/1475-2867-13-102
PMCID: PMC4015927  PMID: 24148263
Anti-cancer; Anti-inflammation; Apoptosis; Flavokawain; Kava-kava
24.  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.
doi:10.1186/1472-6882-13-145
PMCID: PMC3701493  PMID: 23800124
Rhaphidophora korthalsii; NK cell; Immunomodulation
25.  In Vivo Immunomodulation and Lipid Peroxidation Activities Contributed to Chemoprevention Effects of Fermented Mung Bean against Breast Cancer 
Mung bean has been reported to have antioxidant, cytotoxic, and immunomodulatory effects in vitro. Fermented products are reported to have enhanced immunomodulation and cancer chemopreventive effects. In this study, fermented mung bean treatments in vivo were studied by monitoring tumor development, spleen immunity, serum cytokine (interleukin 2 and interferon gamma) levels, and spleen/tumor antioxidant levels after injection with low and high risk 4T1 breast cancer cells. Pretreatment with fermented mung bean was associated with delayed tumor formation in low risk mice. Furthermore, this treatment was connected with higher serum anticancer cytokine levels, spleen T cell populations, splenocyte cytotoxicity, and spleen/tumor antioxidant levels. Histopathological evaluation of fermented mung bean treated tumor revealed lower event of mitotic division. On the other hand, antioxidant and nitric oxide levels that were significantly increased in the untreated mice were inhibited in the fermented mung bean treated groups. These results suggested that fermented mung bean has potential cancer chemoprevention effects through the stimulation of immunity, lipid peroxidation, and anti-inflammation.
doi:10.1155/2013/708464
PMCID: PMC3654717  PMID: 23710232

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