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1.  Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile 
PLoS ONE  2015;10(6):e0127441.
Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.
doi:10.1371/journal.pone.0127441
PMCID: PMC4457850  PMID: 26047480
2.  Induction of cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 cells by Dillenia suffruticosa root extract via multiple signalling pathways 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1472-6882-14-197
PMCID: PMC4096536  PMID: 24947113
Dillenia suffruticosa; Dichloromethane extract; Cell cycle; Apoptosis; Oxidative stress
3.  Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1472-6882-14-55
PMCID: PMC3927215  PMID: 24524627
Dillenia suffruticosa; Breast cancer; Cytotoxic; Apoptosis; Oxidative stress pathway
4.  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 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1472-6882-13-177
PMCID: PMC3726323  PMID: 23866310
Germinated brown rice; Antioxidant; Oxidative stress; Neuroprotective; SH-SY5Y
5.  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.
doi:10.1155/2013/549705
PMCID: PMC3626181  PMID: 23606884
6.  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.
doi:10.1155/2012/816501
PMCID: PMC3529503  PMID: 23304216
7.  Neuroprotective Effects of Germinated Brown Rice against Hydrogen Peroxide Induced Cell Death in Human SH-SY5Y Cells 
The neuroprotective and antioxidative effects of germinated brown rice (GBR), brown rice (BR) and commercially available γ-aminobutyric acid (GABA) against cell death induced by hydrogen peroxide (H2O2) in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H2O2-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP) and prevented phosphatidylserine (PS) translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.
doi:10.3390/ijms13089692
PMCID: PMC3431823  PMID: 22949825
germinated brown rice; γ-aminobutyric acid; hydrogen peroxide; neuroprotective; SH-SY5Y cell death
8.  Formulation optimization of galantamine hydrobromide loaded gel drug reservoirs in transdermal patch for Alzheimer’s disease 
Galantamine hydrobromide (GH) is an effective drug for Alzheimer’s disease. It is currently delivered via the oral route, and this might cause nausea, vomiting, and gastrointestinal disturbance. In the present work, GH was formulated in a gel-type drug reservoir and then optimized by using response surface methodology (RSM) based on central composite design. This optimization study involved three independent variables (carbopol amount, triethanolamine amount, and GH amount) and two dependent variables (cumulative drug release amount at 8 hours and the permeation flux of drug). Two models using expert design software were fitted into a quadratic polynomial model. The optimized gel was formulated with 0.89% w/w carbopol, 1.16% w/w triethanolamine, and 4.19% w/w GH. Optimization analysis revealed that the proposed formulation has the predicted cumulative drug release amount at 8 hours of 17.80 mg·cm−2 and the predicted permeation flux of 2.27 mg·cm−2/h. These predicted values have good agreement to actual cumulative drug release amount at 8 hours (16.93±0.08 mg·cm−2) and actual permeation flux (2.32±0.02 mg·cm−2/h). This optimized reservoir formulation was then fabricated in the transdermal patch system. This patch system has moderate pH, high drug content, and controlled drug-release pattern. Thus, this patch system has the potential to be used as the drug carrier for the treatment of Alzheimer’s disease.
doi:10.2147/IJN.S80253
PMCID: PMC4462858  PMID: 26089664
response surface methodology; central composite design; analysis of variance; Franz diffusion cell
9.  Biocompatibility of Bio Based Calcium Carbonate Nanocrystals Aragonite Polymorph on NIH 3T3 Fibroblast Cell Line 
Background
Currently, there has been extensive research interest for inorganic nanocrystals such as calcium phosphate, iron oxide, silicone, carbon nanotube and layered double hydroxide as a drug delivery system especially in cancer therapy. However, toxicological screening of such particles is paramount importance before use as delivery carrier. In this study we examine the biocompatibility of CaCO3 nanocrystal on NIH 3T3 cell line.
Material and Methods
Transmission and field emission scanning electron microscopy (TEM and FESEM) were used for the characterisation of CaCO3 nanocrystals. Cytotoxicity and genotoxic effect of calcium carbonate nanocrystals in cultured mouse embryonic fibroblast NIH 3T3 cell line using various bioassays including MTT, and Neutral red/Trypan blue double-staining assays. LDH, BrdU and reactive oxygen species were used for toxicity analysis. Cellular morphology was examined by scanning electron microscopy (SEM) and confocal fluorescence microscope.
Results
The outcome of the analyses revealed a clear rod-shaped aragonite polymorph of calcium carbonate nanocrystal. The analysed cytotoxic and genotoxicity of CaCO3 nanocrystal on NIH 3T3 cells using different bioassays revealed no significance differences as compared to control. A slight decrease in cell viability was noticed when the cells were exposed to higher concentrations of 200 to 400 µg/ml, while increase in ROS generation and LDH released at 200 and 400 µg/ml was observed.
Conclusions
The study has shown that CaCO3 nanocrystal is biocompatible and non toxic to NIH 3T3 fibroblast cells. The analysed results offer a promising potential of CaCO3 nanocrystal for the development of intracellular drugs, genes and other macromolecule delivery systems.
PMCID: PMC4202393  PMID: 25392577
Biocompatibility; Calcium carbonate; nanocrystals; drugs and Cockle shells
10.  Formulation and Evaluation of Galantamine Gel as Drug Reservoir in Transdermal Patch Delivery System 
The Scientific World Journal  2015;2015:495271.
Galantamine hydrobromide is formulated in tablets and capsules prescribed through oral delivery for the treatment of Alzheimer's disease. However, oral delivery of drugs can cause severe side effects such as nausea, vomiting, and gastrointestinal disturbance. Transdermal delivery of galantamine hydrobromide could avoid these unwanted side effects. In this work, galantamine hydrobromide was formulated in gel drug reservoir which was then fabricated in the transdermal patch. The in vitro drug release studies revealed that the drug release from the donor chamber to receptor chamber of Franz diffusion cell was affected by the amount of polymer, amount of neutralizer, amount of drug, types of permeation enhancer, and amount of permeation enhancer. Visual observations of the gels showed that all formulated gels are translucent, homogeneous, smooth, and stable. These gels have pH in the suitable range for skin. The gel also showed high drug content uniformity. Hence, this formulation can be further used in the preparation of transdermal patch drug delivery system.
doi:10.1155/2015/495271
PMCID: PMC4374332  PMID: 25853145
11.  In vitro bioaccessibility and antioxidant properties of edible bird’s nest following simulated human gastro-intestinal digestion 
Background
Edible birds’ nest (EBN) is reported to be antioxidant-rich. However, the fate of its antioxidants after oral consumption is not yet reported. To explore this, we hypothesized that EBN antioxidants are released from their matrix when subjected to in vitro simulated gastrointestinal digestion.
Methods
EBN samples were extracted using hot water (100°C) with or without subsequent sequential enzymatic digestion using pepsin (10,000 units), pancreatin (36 mg) and bile extracts (112.5 mg). Additionally, pH changes (8.9 to 2 and back to 8.9) similar to the gut were applied, and a 10 KDa dialysis tubing was used to simulate gut absorption. The antioxidant capacities of the water extracts of EBN before and after digestion were then determined using ABTS and oxygen radical absorbance capacity (ORAC) assays, while the protective effects of the EBN samples against hydrogen peroxide-induced toxicity in HEPG2 cells were determined using MTT assay and acridine orange (AO)/propidium iodide (PI) staining.
Results
Antioxidant assays (ABTS and ORAC) showed that the undigested EBN water extract had little antioxidant activity (1 and 1%, respectively at 1000 μg/mL) while at similar concentrations the digested samples had significantly (p < 0.05) enhanced antioxidant activities, for samples inside (38 and 50%, respectively at 1000 μg/mL) and outside (36 and 50%, respectively at 1000 μg/mL) the dialysis tubing, representing absorbed and unabsorbed samples, respectively. Cell viability and toxicity assays also suggested that the EBN extracts were non-toxic to HEPG2 cells (cell viabilities of over 80% at 1000 μg/mL), while AOPI showed that the extracts protected HEPG2 cells from hydrogen peroxide induced-toxicity.
Conclusions
Based on the findings, it is likely that EBN bioactives are released from their matrix when digested in the gut and then absorbed through the gut by passive-mediated transport to exert their functional effects. However, there is need to confirm these findings using in vivo systems to determine their clinical significance.
doi:10.1186/1472-6882-14-468
PMCID: PMC4289220  PMID: 25475744
Antioxidant; Edible bird’s nest; Gastro-intestinal digestion
12.  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.
doi:10.1155/2014/371907
PMCID: PMC4241308  PMID: 25431609
13.  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.
doi:10.1155/2014/391869
PMCID: PMC4084513  PMID: 25028650
14.  A randomised controlled trial on hypolipidemic effects of Nigella Sativa seeds powder in menopausal women 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1479-5876-12-82
PMCID: PMC4013060  PMID: 24685020
Clinical trial; Complementary medicine; Hyperlipidemia; Menopause; Nigella sativa; Plant bioresources
15.  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.
doi:10.5681/apb.2014.005
PMCID: PMC3885365  PMID: 24409406
Nigella sativa; Menopause; Metabolic syndrome; Hyperlipidemic; Hyperglycemia
16.  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.
doi:10.2147/IJN.S53079
PMCID: PMC3897325  PMID: 24549109
magnetic nanoparticles; streptomycin; nanoantibiotics; chitosan; release; antimicrobial activity; methicillin-resistant Staphylococcus aureus
17.  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.
doi:10.2147/IJN.S53847
PMCID: PMC3890966  PMID: 24453486
chitosan; polyethylene glycol; magnetic nanoparticle; kojic acid; controlled release; biological activity
18.  Vanillin Differentially Affects Azoxymethane-Injected Rat Colon Carcinogenesis and Gene Expression 
Journal of Medicinal Food  2012;15(12):1096-1102.
Abstract
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.
doi:10.1089/jmf.2012.2245
PMCID: PMC3523249  PMID: 23216109
apoptosis; cell cycle; DNA repair; protooncogene; tumor suppressor gene; vanillin
19.  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.
doi:10.1155/2013/587451
PMCID: PMC3845482  PMID: 24324966
20.  Germinated brown rice and its bioactives modulate the activity of uterine cells in oophorectomised rats as evidenced by gross cytohistological and immunohistochemical changes 
Background
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.
Methods
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.
Results
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.
Conclusion
GBR and its bioactives could be a potential alternative in improving reproductive system atrophy, dryness and discomfort during menopause.
doi:10.1186/1472-6882-13-198
PMCID: PMC3750460  PMID: 23899096
Germinated brown rice; Menopause; Uterine atrophy; Vagina dryness; Cyto-histology; Immunohistochemistry
21.  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.
doi:10.1155/2013/601838
PMCID: PMC3671281  PMID: 23762147
22.  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.
doi:10.1155/2013/134694
PMCID: PMC3647596  PMID: 23671850
23.  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.
doi:10.3390/nu5020468
PMCID: PMC3635206  PMID: 23389305
peroxisome proliferator activated receptor gamma; germinated brown rice; oryzanol; gamma amino butyric acid; acylated steryl glycoside
24.  Hippuric acid nanocomposite enhances doxorubicin and oxaliplatin-induced cytotoxicity in MDA-MB231, MCF-7 and Caco2 cell lines 
Background
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.
Methods
The hippuric acid nanocomposite (HAN) was prepared by the direct reaction of a HA solution with an aqueous suspension of ZnO.
Results
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.
Conclusion
The resulting combination HAN with drugs has exhibited higher inhibition in cells growth in all cancer cell lines.
doi:10.2147/DDDT.S37070
PMCID: PMC3549678  PMID: 23345969
hippuric acid nanocomposite; zinc-layered hydroxide; doxorubicin; oxaliplatin; MDA-MB231; MCF-7; Caco2 cell lines
25.  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.
doi:10.2147/IJN.S32267
PMCID: PMC3418079  PMID: 22904631
magnesium; aluminum; layered double hydroxide; perindopril erbumine; ion exchange; angiotensin-converting enzyme; Chang cells line

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