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1.  Antioxidative Effects of Germinated Brown Rice-Derived Extracts on H2O2-Induced Oxidative Stress in HepG2 Cells 
The antioxidant properties of germinated brown rice (GBR) are likely mediated by multiple bioactives. To test this hypothesis, HepG2 cells pretreated with GBR extracts, rich in acylated steryl glycoside (ASG), gamma amino butyric acid GABA), phenolics or oryzanol, were incubated with hydrogen peroxide (H2O2) and their hydroxyl radical (OH•) scavenging capacities and thiobarbituric acid-reactive substances (TBARS) generation were evaluated. Results showed that GBR-extracts increased OH• scavenging activities in both cell-free medium and posttreatment culture media, suggesting that the extracts were both direct- and indirect-acting against OH•. The levels of TBARS in the culture medium after treatment were also reduced by all the extracts. In addition, H2O2 produced transcriptional changes in p53, JNK, p38 MAPK, AKT, BAX, and CDK4 that were inclined towards apoptosis, while GBR-extracts showed some transcriptional changes (upregulation of BAX and p53) that suggested an inclination for apoptosis although other changes (upregulation of antioxidant genes, AKT, JNK, and p38 MAPK) suggested that GBR-extracts favored survival of the HepG2 cells. Our findings show that GBR bioactive-rich extracts reduce oxidative stress through improvement in antioxidant capacity, partly mediated through transcriptional regulation of antioxidant and prosurvival genes.
PMCID: PMC4241308  PMID: 25431609
2.  Induction of cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 cells by Dillenia suffruticosa root extract via multiple signalling pathways 
Dillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells.
Dillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes.
DCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 μg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 μg/mL) and high concentration (50 μg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells.
DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.
PMCID: PMC4096536  PMID: 24947113
Dillenia suffruticosa; Dichloromethane extract; Cell cycle; Apoptosis; Oxidative stress
3.  In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals 
BioMed Research International  2014;2014:391869.
Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system.
PMCID: PMC4084513  PMID: 25028650
4.  A randomised controlled trial on hypolipidemic effects of Nigella Sativa seeds powder in menopausal women 
The risk of cardiovascular diseases (CVD) is increased tremendously among menopausal women, and there is an increasing demand for alternative therapies for managing factors like dyslipidemia that contribute to CVD development.
In this study, Nigella sativa was evaluated for its hypolipidemic effects among menopausal women. In a randomised trial, hyperlipidemic menopausal women were assigned to treatment (n = 19) or placebo groups (n = 18), and given N. sativa or placebo for two months after their informed consents were sought. At baseline, blood samples were taken and at one month intervals thereafter until one month after the end of the study.
The results showed that N. sativa significantly improved lipid profiles of menopausal women (decreased total cholesterol, low density lipoprotein cholesterol and triglyceride, and increased high density lipoprotein cholesterol) more than the placebo treatment over 2 months of intervention. One month after cessation of treatment, the lipid profiles in the N. sativa-treated group tended to change towards the pretreatment levels.
N. sativa is thought to have multiple mechanisms of action and is cost-effective. Therefore, it could be used by menopausal women to remedy hypercholesterolemia, with likely more benefits than with single pharmacological agents that may cause side effects. The use of N. sativa as an alternative therapy for hypercholesterolemia could have profound impact on the management of CVD among menopausal women especially in countries where it is readily available.
PMCID: PMC4013060  PMID: 24685020
Clinical trial; Complementary medicine; Hyperlipidemia; Menopause; Nigella sativa; Plant bioresources
5.  Protective Effects of Nigella sativa on Metabolic Syndrome in Menopausal Women 
Purpose: This study was conducted in menopausal women to determine the metabolic impact of Nigella sativa.
Methods: Thirty subjects who were menopausal women within the age limit of 45-60 were participated in this study and randomly allotted into two experimental groups. The treatment group was orally administered with N. sativa seeds powder in the form of capsules at a dose of 1g per day after breakfast for period of two months and compared to control group given placebo. Anthropometric and biochemical parameters were measured at baseline, 1st month, 2nd month and a month after treatment completed to determine their body weight, serum lipid profile and fasting blood glucose (FBG).
Results: The treatment group showed slight reduction with no significant difference in body weight changes of the respondents. However, significant (p<0.05) improvement was observed in total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), and blood glucose (p<0.05).
Conclusion: These results suggested that treatment with N. sativa exert a protective effect by improving lipid profile and blood glucose which are in higher risk to be elevated during menopausal period.
PMCID: PMC3885365  PMID: 24409406
Nigella sativa; Menopause; Metabolic syndrome; Hyperlipidemic; Hyperglycemia
6.  Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa 
Breast cancer is one of the most dreading types of cancer among women. Herbal medicine has becoming a potential source of treatment for breast cancer. Herbal plant Dillenia suffruticosa (Griff) Martelli under the family Dilleniaceae has been traditionally used to treat cancerous growth. In this study, the anticancer effect of ethyl acetate extract of D. suffruticosa (EADs) was examined on human breast adenocarcinoma cell line MCF-7 and the molecular pathway involved was elucidated.
EADs was obtained from the root of D. suffruticosa by using sequential solvent extraction. Cytotoxicity was determined by using MTT assay, mode of cell death by cell cycle analysis and apoptosis induction by Annexin-FITC/PI assay. Morphology changes in cells were observed under inverted light microscope. Involvement of selected genes in the oxidative stress-mediated signaling pathway was explored using multiplex gene expression analysis.
The treatment of EADs caused cytotoxicity to MCF-7 cells in a dose- and time-dependent manner at 24, 48 and 72 hours with IC50 of 76 ± 2.3, 58 ± 0.7 and 39 ± 3.6 μg/mL, respectively. The IC50 of tamoxifen-treated MCF-7 cells was 8 ± 0.5 μg/mL. Induction of apoptosis by EADs was dose- and time- dependent. EADs induced non-phase specific cell cycle arrest at different concentration and time point. The multiplex mRNA expression study indicated that EADs-induced apoptosis was accompanied by upregulation of the expression of SOD1, SOD2, NF-κB, p53, p38 MAPK, and catalase, but downregulation of Akt1.
It is suggested that EADs induced apoptosis in MCF-7 cells by modulating numerous genes which are involved in oxidative stress pathway. Therefore, EADs has the potential to act as an effective intervention against breast cancer cells.
PMCID: PMC3927215  PMID: 24524627
Dillenia suffruticosa; Breast cancer; Cytotoxic; Apoptosis; Oxidative stress pathway
7.  Synthesis, characterization, controlled release, and antibacterial studies of a novel streptomycin chitosan magnetic nanoantibiotic 
This study describes the preparation, characterization, and controlled release of a streptomycin-chitosan-magnetic nanoparticle-based antibiotic in an effort to improve the treatment of bacterial infections. Specifically, chitosan-magnetic nanoparticles were synthesized by an incorporation method and were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, and vibrating sample magnetometry. Streptomycin was incorporated into the nanoparticles to form a streptomycin-coated chitosan-magnetic nanoparticle (Strep-CS-MNP) nanocomposite. The release profiles showed an initially fast release, which became slower as time progressed. The percentage of drug released after 350 minutes was around 100%, and the best fit mathematical model for drug release was the pseudo-second order model. The Strep-CS-MNP nanocomposite showed enhanced antibacterial activity against methicillin-resistant Staphylococcus aureus. This study forms a significant basis for further investigation of the Strep-CS-MNP nanocomposite in the treatment of various bacterial infections.
PMCID: PMC3897325  PMID: 24549109
magnetic nanoparticles; streptomycin; nanoantibiotics; chitosan; release; antimicrobial activity; methicillin-resistant Staphylococcus aureus
8.  Novel kojic acid-polymer-based magnetic nanocomposites for medical applications 
Iron oxide magnetic nanoparticles (MNPs) were synthesized by the coprecipitation of iron salts in sodium hydroxide followed by coating separately with chitosan (CS) and polyethylene glycol (PEG) to form CS-MNPs and PEG-MNPs nanoparticles, respectively. They were then loaded with kojic acid (KA), a pharmacologically bioactive natural compound, to form KA-CS-MNPs and KA-PEG-MNPs nanocomposites, respectively. The MNPs and their nanocomposites were characterized using powder X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, vibrating sample magnetometry, and scanning electron microscopy. The powder X-ray diffraction data suggest that all formulations consisted of highly crystalline, pure magnetite Fe3O4. The Fourier transform infrared spectroscopy and thermogravimetric analysis confirmed the presence of both polymers and KA in the nanocomposites. Magnetization curves showed that both nanocomposites (KA-CS-MNPs and KA-PEG-MNPs) were superparamagnetic with saturation magnetizations of 8.1 emu/g and 26.4 emu/g, respectively. The KA drug loading was estimated using ultraviolet–visible spectroscopy, which gave a loading of 12.2% and 8.3% for the KA-CS-MNPs and KA-PEG-MNPs nanocomposites, respectively. The release profile of the KA from the nanocomposites followed a pseudo second-order kinetic model. The agar diffusion test was performed to evaluate the antimicrobial activity for both KA-CS-MNPs and KA-PEG-MNPs nanocomposites against a number of microorganisms using two Gram-positive (methicillin-resistant Staphylococcus aureus and Bacillus subtilis) and one Gram-negative (Salmonella enterica) species, and showed some antibacterial activity, which could be enhanced in future studies by optimizing drug loading. This study provided evidence for the promise for the further investigation of the possible beneficial biological activities of KA and both KA-CS-MNPs and KA-PEG-MNPs nanocomposites in nanopharmaceutical applications.
PMCID: PMC3890966  PMID: 24453486
chitosan; polyethylene glycol; magnetic nanoparticle; kojic acid; controlled release; biological activity
9.  Vanillin Differentially Affects Azoxymethane-Injected Rat Colon Carcinogenesis and Gene Expression 
Journal of Medicinal Food  2012;15(12):1096-1102.
Vanillin is the substance responsible for the flavor and smell of vanilla, a widely used flavoring agent. Previous studies reported that vanillin is a good antimutagen and anticarcinogen. However, there are also some contradicting findings showing that vanillin was a comutagen and cocarcinogen. This study investigated whether vanillin is an anticarcinogen or a cocarcinogen in rats induced with azoxymethane (AOM). Rats induced with AOM will develop aberrant crypt foci (ACF). AOM-challenged rats were treated with vanillin orally and intraperitoneally at low and high concentrations and ACF density, multiplicity, and distribution were observed. The gene expression of 14 colorectal cancer-related genes was also studied. Results showed that vanillin consumed orally had no effect on ACF. However, high concentrations (300 mg/kg body weight) of vanillin administered through intraperitoneal injection could increase ACF density and ACF multiplicity. ACF were mainly found in the distal colon rather than in the mid-section and proximal colon. The expression of colorectal cancer biomarkers, protooncogenes, recombinational repair, mismatch repair, and cell cycle arrest, and tumor suppressor gene expression were also affected by vanillin. Vanillin was not cocarcinogenic when consumed orally. However, it was cocarcinogenic when being administered intraperitoneally at high concentration. Hence, the use of vanillin in food should be safe but might have cocarcinogenic potential when it is used in high concentration for therapeutic purposes.
PMCID: PMC3523249  PMID: 23216109
apoptosis; cell cycle; DNA repair; protooncogene; tumor suppressor gene; vanillin
10.  A pH-Sensitive, Biobased Calcium Carbonate Aragonite Nanocrystal as a Novel Anticancer Delivery System 
BioMed Research International  2013;2013:587451.
The synthesised biobased calcium carbonate nanocrystals had demonstrated to be an effective carrier for delivery of anticancer drug doxorubicin (DOX). The use of these nanocrystals displayed high levels of selectivity and specificity in achieving effective cancer cell death without nonspecific toxicity. These results confirmed that DOX was intercalated into calcium carbonate nanocrystals at high loading and encapsulation efficiency (4.8 and 96%, resp.). The CaCO3/DOX nanocrystals are relatively stable at neutral pH (7.4), resulting in slow release, but the nanocrystals progressively dissociated in acidic pH (4.8) regimes, triggering faster release of DOX. The CaCO3/DOX nanocrystals exhibited high uptake by MDA MB231 breast cancer cells and a promising potential delivery of DOX to target cells. In vitro chemosensitivity using MTT, modified neutral red/trypan blue assay, and LDH on MDA MB231 breast cancer cells revealed that CaCO3/DOX nanocrystals are more sensitive and gave a greater reduction in cell growth than free DOX. Our findings suggest that CaCO3 nanocrystals hold tremendous promise in the areas of controlled drug delivery and targeted cancer therapy.
PMCID: PMC3845482  PMID: 24324966
11.  Germinated brown rice and its bioactives modulate the activity of uterine cells in oophorectomised rats as evidenced by gross cytohistological and immunohistochemical changes 
Germinated brown rice (GBR) is gaining momentum in the area of biomedical research due to its increased use as a nutraceutical for the management of diseases. The effect of GBR on the reproductive organs of oophorectomised rats was studied using the gross, cytological, histological and immunohistochemical changes, with the aim of reducing atrophy and dryness of the genital organs in menopause.
Experimental rats were divided into eight groups of six rats per group. Groups 1, 2 and 3 (sham-operated (SH), oophorectomised without treatment (OVX) and oophorectomised treated with 0.2 mg/kg oestrogen, respectively) served as the controls. The groups 4,5,6,7 and 8 were treated with 20 mg/kg Remifemin, 200 mg/kg of GBR, ASG, oryzanol and GABA, respectively. All treatments were administered orally, once daily for 8 weeks. Vaginal smear cytology was done at the 7th week on all the rats. The weight and dimensions of the uterus and vagina were determined after sacrifice of the rats. Uterine and vaginal tissues were taken for histology and Immunohistochemical examinations.
GBR and its bioactives treated groups significantly increased the weight and length of both the uterus and the vagina when compared to Oophorectomised non-treated group (OVX-non-treated) (p < 0.05). Significant changes were observed in the ratio of cornified epithelial cells and number of leucocytes in the vaginal cytology between the oophorectomised non-treated and treated groups. There was also an increase in the luminal and glandular epithelial cells activity in the treated compared with the untreated groups histologically. Immunohistochemical staining showed specific proliferating cell nuclear antigen (PCNA) in the luminal and glandular epithelium of the treated groups, which was absent in the OVX-non-treated group. GBR improved the length and weight of the uterus and also increased the number of glandular and luminal cells epithelia of the vagina.
GBR and its bioactives could be a potential alternative in improving reproductive system atrophy, dryness and discomfort during menopause.
PMCID: PMC3750460  PMID: 23899096
Germinated brown rice; Menopause; Uterine atrophy; Vagina dryness; Cyto-histology; Immunohistochemistry
12.  Ethyl acetate extract of germinated brown rice attenuates hydrogen peroxide-induced oxidative stress in human SH-SY5Y neuroblastoma cells: role of anti-apoptotic, pro-survival and antioxidant genes 
There are reports of improved metabolic outcomes due to consumption of germinated brown rice (GBR). Many of the functional effects of GBR can be linked to its high amounts of antioxidants. Interestingly, dietary components with high antioxidants have shown promise in the prevention of neurodegenerative diseases like Alzheimer’s disease (AD). This effect of dietary components is mostly based on their ability to prevent apoptosis, which is believed to link oxidative damage to pathological changes in AD. In view of the rich antioxidant content of GBR, we studied its potential to modulate processes leading up to AD.
The total phenolic content and antioxidant capacity of the ethyl acetate extract of GBR were compared to that of brown rice (BR), and the cytotoxicity of both extracts were determined on human SH-SY5Y neuronal cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay. Based on its higher antioxidant potentials, the effect of the GBR extract on morphological changes due to hydrogen peroxide (H2O2)-induced oxidative damage in human SH-SY5Y neuronal cells was examined using inverted light microscope and fluorescence microscope by means of acridine orange-propidium iodide (AO/PI) staining. Also, evaluation of the transcriptional regulation of antioxidant and apoptotic genes was carried out using Multiplex Gene Expression System.
The ethyl acetate extract of GBR had higher total phenolic content and antioxidant capacity compared to BR. The cytotoxicity results showed that GBR extract did not cause any damage to the human SH-SY5Y neuronal cells at concentrations of up to 20 ppm, and the morphological analyses showed that the GBR extract (up to 10 ppm) prevented H2O2-induced apoptotic changes in the cells. Furthermore, multiplex gene expression analyses showed that the protection of the cells by the GBR extract was linked to its ability to induce transcriptional changes in antioxidant (SOD 1, SOD 2 and catalase) and apoptotic (AKT, NF-Kβ, ERK1/2, JNK, p53 and p38 MAPK) genes that tended towards survival.
Taken together, the results of our study showed that the ethyl acetate extract of GBR, with high antioxidant potentials, could prevent H2O2-induced oxidative damage in SH-SY5Y cells. The potential of GBR and its neuroprotective mechanism in ameliorating oxidative stress-related cytotoxicity is therefore worth exploring further.
PMCID: PMC3726323  PMID: 23866310
Germinated brown rice; Antioxidant; Oxidative stress; Neuroprotective; SH-SY5Y
13.  Antidiabetic and Hypolipidemic Activities of Curculigo latifolia Fruit:Root Extract in High Fat Fed Diet and Low Dose STZ Induced Diabetic Rats 
Curculigo latifolia fruit is used as alternative sweetener while root is used as alternative treatment for diuretic and urinary problems. The antidiabetic and hypolipidemic activities of C. latifolia fruit:root aqueous extract in high fat diet (HFD) and 40 mg streptozotocin (STZ) induced diabetic rats through expression of genes involved in glucose and lipid metabolisms were investigated. Diabetic rats were treated with C. latifolia fruit:root extract for 4 weeks. Plasma glucose, insulin, adiponectin, lipid profiles, alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), urea, and creatinine levels were measured before and after treatments. Regulations of selected genes involved in glucose and lipid metabolisms were determined. Results showed the significant (P < 0.05) increase in body weight, high density lipoprotein (HDL), insulin, and adiponectin levels and decreased glucose, total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL), urea, creatinine, ALT, and GGT levels in diabetic rats after 4 weeks treatment. Furthermore, C. latifolia fruit:root extract significantly increased the expression of IRS-1, IGF-1, GLUT4, PPARα, PPARγ, AdipoR1, AdipoR2, leptin, LPL, and lipase genes in adipose and muscle tissues in diabetic rats. These results suggest that C. latifolia fruit:root extract exerts antidiabetic and hypolipidemic effects through altering regulation genes in glucose and lipid metabolisms in diabetic rats.
PMCID: PMC3671281  PMID: 23762147
14.  Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29) Cell Lines 
Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β-sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.
PMCID: PMC3626181  PMID: 23606884
15.  The Hypocholesterolemic Effect of Germinated Brown Rice Involves the Upregulation of the Apolipoprotein A1 and Low-Density Lipoprotein Receptor Genes 
Journal of Diabetes Research  2013;2013:134694.
Germinated brown rice (GBR) is rich in bioactive compounds, which confer GBR with many functional properties. Evidence of its hypocholesterolemic effects is emerging, but the exact mechanisms of action and bioactive compounds involved have not been fully documented. Using type 2 diabetic rats, we studied the effects of white rice, GBR, and brown rice (BR) on lipid profile and on the regulation of selected genes involved in cholesterol metabolism. Our results showed that the upregulation of apolipoprotein A1 and low-density lipoprotein receptor genes was involved in the hypocholesterolemic effects of GBR. Additionally, in vitro studies using HEPG2 cells showed that acylated steryl glycoside, gamma amino butyric acid, and oryzanol and phenolic extracts of GBR contribute to the nutrigenomic regulation of these genes. Transcriptional and nontranscriptional mechanisms are likely involved in the overall hypocholesterolemic effects of GBR suggesting that it may have an impact on the prevention and/or management of hypercholesterolemia due to a wide variety of metabolic perturbations. However, there is need to conduct long-term clinical trials to determine the clinical relevance of the hypocholesterolemic effects of GBR determined through animal studies.
PMCID: PMC3647596  PMID: 23671850
16.  Effects of Germinated Brown Rice and Its Bioactive Compounds on the Expression of the Peroxisome Proliferator-Activated Receptor Gamma Gene 
Nutrients  2013;5(2):468-477.
Dysregulated metabolism is implicated in obesity and other disease conditions like type 2 diabetes mellitus and cardiovascular diseases, which are linked to abnormalities of peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ has been the focus of much research aimed at managing these diseases. Also, germinated brown rice (GBR) is known to possess antidiabetic, antiobesity and hypocholesterolemic effects. We hypothesized that GBR bioactive compounds may mediate some of the improvements in metabolic indices through PPARγ modulation. Cultured HEP-G2 cells were treated with 50 ppm and 100 ppm of extracts from GBR (GABA, ASG and oryzanol) after determination of cell viabilities using MTT assays. Results showed that all extracts upregulated the expression of the PPARγ. However, combination of all three extracts showed downregulation of the gene, suggesting that, in combination, the effects of these bioactives differ from their individual effects likely mediated through competitive inhibition of the gene. Upregulation of the gene may have therapeutic potential in diabetes mellitus and cardiovascular diseases, while its downregulation likely contributes to GBR’s antiobesity effects. These potentials are worth studying further.
PMCID: PMC3635206  PMID: 23389305
peroxisome proliferator activated receptor gamma; germinated brown rice; oryzanol; gamma amino butyric acid; acylated steryl glycoside
17.  Hippuric acid nanocomposite enhances doxorubicin and oxaliplatin-induced cytotoxicity in MDA-MB231, MCF-7 and Caco2 cell lines 
The aim of the current study is to design a new nanocomposite for inducing cytotoxicity of doxorubicin and oxaliplatin toward MDA-MB231, MCF-7, and Caco2 cell lines. A hippuric acid (HA) zinc layered hydroxide (ZLH) nanocomposite was synthesized under an aqueous environment using HA and zinc oxide (ZnO) as the precursors.
The hippuric acid nanocomposite (HAN) was prepared by the direct reaction of a HA solution with an aqueous suspension of ZnO.
The basal spacing of the nanocomposite was 21.3 Å, which is average of four harmonics at 2θ = 8.32°, 12.50°, 16.68°, and 20.84°. This result indicates that the hippurate anion was successfully intercalated into the interlayer space of ZLH. The combinations of HAN with chemotherapy (drugs) has inhibited the cell growth of the MDA-MB231, MCF-7, and Caco2 cancer cells when compared to drugs alone. An IC50 value for the combination of HAN with doxorubicin toward MCF-7 is 0.19 ± 0.15 μg/mL and toward MDA-MB231 is 0.13 ± 0.10 μg/mL. Similarly, the IC50 for the combination of HAN with oxaliplatin toward Caco2 is 0.24 ± 0.11 μg/mL. In the antiproliferative results, the equal combination of HAN (0.5 μg/mL) with doxorubicin (0.5 μg/mL) has reduced the cell proliferation in MCF-7 and MDA-MB-231 cells into 37.3% and 17.6%, respectively after 24 hours. Similarly, the antiproliferation percentage for equal combination HAN with oxaliplatin (5.00 μg/mL) toward Caco2 is 72.7% after 24 hours.
The resulting combination HAN with drugs has exhibited higher inhibition in cells growth in all cancer cell lines.
PMCID: PMC3549678  PMID: 23345969
hippuric acid nanocomposite; zinc-layered hydroxide; doxorubicin; oxaliplatin; MDA-MB231; MCF-7; Caco2 cell lines
18.  Antidiabetic Properties of Germinated Brown Rice: A Systematic Review 
Diet is an important variable in the course of type 2 diabetes, which has generated interest in dietary options like germinated brown rice (GBR) for effective management of the disease among rice-consuming populations. In vitro data and animal experiments show that GBR has potentials as a functional diet for managing this disease, and short-term clinical studies indicate encouraging results. Mechanisms for antidiabetic effects of GBR due to bioactive compounds like γ-aminobutyric acid (GABA), γ-oryzanol, dietary fibre, phenolics, vitamins, acylated steryl β-glucoside, and minerals include antihyperglycemia, low insulin index, antioxidative effect, antithrombosis, antihypertensive effect, hypocholesterolemia, and neuroprotective effects. The evidence so far suggests that there may be enormous benefits for diabetics in rice-consuming populations if white rice is replaced with GBR. However, long-term clinical studies are still needed to verify these findings on antidiabetic effects of GBR. Thus, we present a review on the antidiabetic properties of GBR from relevant preclinical and clinical studies, in order to provide detailed information on this subject for researchers to review the potential of GBR in combating this disease.
PMCID: PMC3529503  PMID: 23304216
19.  Comparative study of Mg/Al- and Zn/Al-layered double hydroxide-perindopril erbumine nanocomposites for inhibition of angiotensin-converting enzyme 
The intercalation of a drug active, perindopril, into Mg/Al-layered double hydroxide for the formation of a new nanocomposite, PMAE, was accomplished using a simple ion exchange technique. A relatively high loading percentage of perindopril of about 36.5% (w/w) indicates that intercalation of the active took place in the Mg/Al inorganic interlayer. Intercalation was further supported by Fourier transform infrared spectroscopy, and thermal analysis shows markedly enhanced thermal stability of the active. The release of perindopril from the nanocomposite occurred in a controlled manner governed by pseudo-second order kinetics. MTT assay showed no cytotoxicity effects from either Mg/Al-layered double hydroxide or its nanocomposite, PMAE. Mg/Al-layered double hydroxide showed angiotensin-converting enzyme inhibitory activity, with 5.6% inhibition after 90 minutes of incubation. On incubation of angiotensin-converting enzyme with 0.5 μg/mL of the PMAE nanocomposite, inhibition of the enzyme increased from 56.6% to 70.6% at 30 and 90 minutes, respectively. These results are comparable with data reported in the literature for Zn/Al-perindopril.
PMCID: PMC3418079  PMID: 22904631
magnesium; aluminum; layered double hydroxide; perindopril erbumine; ion exchange; angiotensin-converting enzyme; Chang cells line
20.  Cationized dextran nanoparticle-encapsulated CXCR4-siRNA enhanced correlation between CXCR4 expression and serum alkaline phosphatase in a mouse model of colorectal cancer 
The failure of colorectal cancer treatments is partly due to overexpression of CXCR4 by tumor cells, which plays a critical role in cell metastasis. Moreover, serum alkaline phosphatase (ALP) levels are frequently elevated in patients with metastatic colorectal cancer. A polysaccharide, dextran, was chosen as the vector of siRNA. Spermine was conjugated to oxidized dextran by reductive amination process to obtain cationized dextran, so-called dextran-spermine, in order to prepare CXCR4-siRNAs/dextran-spermine nanoparticles. The fabricated nanoparticles were used in order to investigate whether downregulation of CXCR4 expression could affect serum ALP in mouse models of colorectal cancer.
Colorectal cancer was established in BALB/C mice following injection of mouse colon carcinoma cells CT.26WT through the tail vein. CXCR4 siRNA for two sites of the target gene was administered following injection of naked siRNA or siRNA encapsulated into nanoparticles.
In vivo animal data revealed that CXCR4 silencing by dextran-spermine nanoparticles significantly downregulated CXCR4 expression compared with naked CXCR4 siRNA. Furthermore, there was correlation between CXCR4 expression and serum ALP.
CXCR4 siRNA/dextran-spermine nanoparticles appear to be highly effective, and may be suitable for further in vivo applications. Further research evaluation will be needed to determine the effect of CXCR4 silencing on serum ALP levels, which may be a useful marker to predict liver metastasis in colorectal cancer.
PMCID: PMC3415322  PMID: 22888250
nanoparticles; cationized dextran; colorectal cancer; serum ALP enzyme; CXCR4; siRNA
21.  Controlled-release formulation of antihistamine based on cetirizine zinc-layered hydroxide nanocomposites and its effect on histamine release from basophilic leukemia (RBL-2H3) cells 
A controlled-release formulation of an antihistamine, cetirizine, was synthesized using zinc-layered hydroxide as the host and cetirizine as the guest. The resulting well-ordered nanolayered structure, a cetirizine nanocomposite “CETN,” had a basal spacing of 33.9 Å, averaged from six harmonics observed from X-ray diffraction. The guest, cetirizine, was arranged in a horizontal bilayer between the zinc-layered hydroxide (ZLH) inorganic interlayers. Fourier transform infrared spectroscopy studies indicated that the intercalation takes place without major change in the structure of the guest and that the thermal stability of the guest in the nanocomposites is markedly enhanced. The loading of the guest in the nanocomposites was estimated to be about 49.4% (w/w). The release study showed that about 96% of the guest could be released in 80 hours by phosphate buffer solution at pH 7.4 compared with about 97% in 73 hours at pH 4.8. It was found that release was governed by pseudo-second order kinetics. Release of histamine from rat basophilic leukemia cells was found to be more sensitive to the intercalated cetirizine in the CETN compared with its free counterpart, with inhibition of 56% and 29%, respectively, at 62.5 ng/mL. The cytotoxicity assay toward Chang liver cells line show the IC50 for CETN and ZLH are 617 and 670 μg/mL, respectively.
PMCID: PMC3405893  PMID: 22848164
cetirizine hydrochloric acid; nanocomposite; zinc-layered hydroxide; histamine release; rat basophilic leukemia (RBL-2H3) cells
22.  Controlled release and angiotensin-converting enzyme inhibition properties of an antihypertensive drug based on a perindopril erbumine-layered double hydroxide nanocomposite 
The intercalation of perindopril erbumine into Zn/Al-NO3-layered double hydroxide resulted in the formation of a host-guest type of material. By virtue of the ion-exchange properties of layered double hydroxide, perindopril erbumine was released in a sustained manner. Therefore, this intercalated material can be used as a controlled-release formulation.
Perindopril was intercalated into the interlayers and formed a well ordered, layered organic-inorganic nanocomposite. The basal spacing of the products was expanded to 21.7 Å and 19.9 Å by the ion-exchange and coprecipitation methods, respectively, in a bilayer and a monolayer arrangement, respectively. The release of perindopril from the nanocomposite synthesized by the coprecipitation method was slower than that of its counterpart synthesized by the ion-exchange method. The rate of release was governed by pseudo-second order kinetics. An in vitro antihypertensive assay showed that the intercalation process results in effectiveness similar to that of the antihypertensive properties of perindopril.
Intercalated perindopril showed better thermal stability than its free counterpart. The resulting material showed sustained-release properties and can therefore be used as a controlled-release formulation.
PMCID: PMC3356206  PMID: 22619549
perindopril erbumine; layered double hydroxides; ion-exchange; coprecipitation; sustained release; angiotensin-converting enzyme
23.  In Vitro Inhibition of Histamine Release Behavior of Cetirizine Intercalated into Zn/Al- and Mg/Al-Layered Double Hydroxides 
The intercalation of cetirizine into two types of layered double hydroxides, Zn/Al and Mg/Al, has been investigated by the ion exchange method to form CTZAN and CTMAN nanocomposites, respectively. The basal spacing of the nanocomposites were expanded to 31.9 Å for CTZAN and 31.2 Å for CTMAN, suggesting that cetirizine anion was intercalated into Layered double hydroxides (LDHs) and arranged in a tilted bilayer fashion. A Fourier transform infrared spectroscopy (FTIR) study supported the formation of both the nanocomposites, and the intercalated cetirizine is thermally more stable than its counterpart in free state. The loading of cetirizine in the nanocomposite was estimated to be about 57.2% for CTZAN and 60.7% CTMAN. The cetirizine release from the nanocomposites show sustained release manner and the release rate of cetirizine from CTZAN and CTMAN nanocomposites at pH 7.4 is remarkably lower than that at pH 4.8, presumably due to the different release mechanism. The inhibition of histamine release from RBL2H3 cells by the free cetirizine is higher than the intercalated cetirizine both in CTZAN and CTMAN nanocomposites. The viability in human Chang liver cells at 1000 μg/mL for CTZAN and CTMAN nanocomposites are 74.5 and 91.9%, respectively.
PMCID: PMC3382767  PMID: 22754339
(Zn/Al, Mg/Al)-layered double hydroxides; cetirizine; nanocomposite; controlled release; RBL2H3 cell; human Chang liver cells
24.  Effects of Brown Rice and White Rice on Expression of Xenobiotic Metabolism Genes in Type 2 Diabetic Rats 
Xenobiotics constantly influence biological systems through several means of interaction. These interactions are disturbed in type 2 diabetes, with implications for disease outcome. We aimed to study the implications of such disturbances on type 2 diabetes and rice consumption, the results of which could affect management of the disease in developing countries. In a type 2 diabetic rat model induced through a combination of high fat diet and low dose streptozotocin injection, up-regulation of xenobiotic metabolism genes in the diabetic untreated group was observed. Xenobiotic metabolism genes were upregulated more in the white rice (WR) group than the diabetic untreated group while the brown rice (BR) group showed significantly lower expression values, though not as effective as metformin, which gave values closer to the normal non-diabetic group. The fold changes in expression in the WR group compared to the BR group for Cyp2D4, Cyp3A1, Cyp4A1, Cyp2B1, Cyp2E1, Cyp2C11, UGT2B1, ALDH1A1 and Cyp2C6 were 2.6, 2, 1.5, 4, 2.8, 1.5, 1.8, 3 and 5, respectively. Our results suggest that WR may upregulate these genes in type 2 diabetes more than BR, potentially causing faster drug metabolism, less drug efficacy and more toxicity. These results may have profound implications for rice eating populations, constituting half the world’s population.
PMCID: PMC3430253  PMID: 22942722
rice; drug biotransformation; gene expression; cytochrome P450
25.  Antioxidant Property Enhancement of Sweet Potato Flour under Simulated Gastrointestinal pH 
Sweet potato is known to be rich in healthful antioxidants, but the stability of its antioxidant properties under gastrointestinal pH is very much unknown. Hence, this study aimed to evaluate the changes in antioxidant properties (total contents of phenolics and flavonoids as well as antioxidant activity) of sweet potato flour (SPF) under simulated gastrointestinal pH conditions. It was found that the yield of SPF crude phenolic extract increased from 0.29 to 3.22 g/100 g SPF upon subjection to gastrointestinal pH conditions (p < 0.05). Also elevated significantly were the total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity of SPF (p < 0.05). In summary, the antioxidant properties of SPF were enhanced under gastrointestinal pH conditions, suggesting that SPF might possess a considerable amount of bound phenolic and other antioxidative compounds. The antioxidant properties of SPF are largely influenced by pH and thus might be enhanced during the in vivo digestive process.
PMCID: PMC3430278  PMID: 22942747
sweet potato flour; antioxidant activity; phenolic content; flavonoid content; simulated gastrointestinal pH

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