A new simple preparation method for a hippurate-intercalated zinc-layered hydroxide (ZLH) nanohybrid has been established, which does not need an anion-exchange procedure to intercalate the hippurate anion into ZLH interlayers.
1'-(S)-1'-Acetoxychavicol acetate (ACA) isolated from the Malaysian ethno-medicinal plant Alpinia conchigera Griff. was investigated for its potential as an anticancer drug. In this communication, we describe the cytotoxic and apoptotic properties of ACA on five human tumour cell lines. Data from MTT cell viability assays indicated that ACA induced both time- and dose-dependent cytotoxicity on all tumour cell lines tested and had no adverse cytotoxic effects on normal cells. Total mortality of the entire tumour cell population was achieved within 30 hrs when treated with ACA at 40.0 µM concentration. Flow cytometric analysis for annexin-V and PI dual staining demonstrated that cell death occurred via apoptosis, followed by secondary necrosis. The apoptotic effects of ACA were confirmed via the DNA fragmentation assay, in which consistent laddering of genomic DNA was observed for all tumour cell lines after a 24 hrs post-treatment period at the IC(50) concentration of ACA. A cell cycle analysis using PI staining also demonstrated that ACA induced cell cycle arrest at the G(0)/G(1) phase, corresponding to oral tumour cell lines. In conclusion, ACA exhibits enormous potential for future development as a chemotherapeutic drug against various malignancies.
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.
CYP450 enzymes are key determinants in drug toxicities, reduced pharmacological effect and adverse drug reactions. Mitragynine, an euphoric compound was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4 and protein expression and resultant enzymatic activity. The mRNA and protein expression of CYP450 isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis. CYP1A2 and CYP3A4 enzyme activities were evaluated using P450-Glo™ assays. The effects of mitragynine on human CYP3A4 protein expression were determined using an optimized hCYP3A4-HepG2 cell-based assay. An in silico computational method to predict the binding conformation of mitragynine to the active site of the CYP3A4 enzyme was performed and further validated using in vitro CYP3A4 inhibition assays. Mitragynine was found to induce mRNA and protein expression of CYP1A2. For the highest concentration of 25 μM, induction of mRNA was approximately 70% that of the positive control and was consistent with the increased CYP1A2 enzymatic activity. Thus, mitragynine is a significant in vitro CYP1A2 inducer. However, it appeared to be a weak CYP3A4 inducer at the transcriptional level and a weak CYP3A4 enzyme inhibitor. It is therefore, unlikely to have any significant clinical effects on CYP3A4 activity.
Eight novel N'-(2-oxoindolin-3-ylidene)-2-propylpentane hydrazide-hydrazone derivatives 4a-h were synthesized and fully characterized by IR, NMR ((1)H-NMR and (13)C-NMR), elemental analysis, and X-ray crystallography. The cyto-toxicity and in vitro anti-cancer evaluation of the prepared compounds have been assessed against two different human tumour cell lines including human liver (HepG2) and leukaemia (Jurkat), as well as in normal cell lines derived from human embryonic kidney (HEK293) using MTT assay. The compounds 3e, 3f, 4a, 4c, and 4e revealed promising anti-cancer activities in tested human tumour cells lines (IC50 values between 3 and 7 μM) as compared to the known anti-cancer drug 5-Fluorouracil (IC50 32-50 μM). Among the tested compounds, 4a showed specificity against leukaemia (Jurkat) cells, with an IC50 value of 3.14 μM, but this compound was inactive in liver cancer and normal cell lines.
The development of reliable and ecofriendly approaches for the production of nanomaterials is a significant aspect of nanotechnology nowadays. One of the most important methods, which shows enormous potential, is based on the green synthesis of nanoparticles using plant extract. In this paper, we aimed to develop a rapid, environmentally friendly process for the synthesis silver nanoparticles using aqueous extract of sumac. The bioactive compounds of sumac extract seem to play a role in the synthesis and capping of silver nanoparticles. Structural, morphological and optical properties of the nanoparticles were characterized using FTIR, XRD, FESEM and UV-Vis spectroscopy. The formation of Ag-NP was immediate within 10 min and confirmed with an absorbance band centered at 438 nm. The mean particle size for the green synthesized silver nanoparticles is 19.81 ± 3.67 nm and is fairly stable with a zeta potential value of -32.9 mV. The bio-formed Ag-NPs were effective against E. coli with a maximum inhibition zone of 14.3 ± 0.32 mm.
Hepatitis B virus reactivation is an important medical issue in cancer patients who undergo systemic chemotherapy. Up to half of CHB carriers receiving chemotherapy develop hepatitis and among these cases a notable proportion are associated with HBV reactivation. However, the molecular mechanism(s) through which various chemotherapeutic agents induce HBV reactivation is not yet fully understood. In this study, we investigated the role of the cell cycle regulator p21 (Waf1/Cip1) in the modulation of HBV replication when a common chemotherapeutic agent, doxorubicin, is present. We showed that p21 expression was increased by doxorubicin treatment. This elevation in p21 expression enhanced the expression of CCAAT/enhancer-binding protein α (C/EBPα); such an increase is likely to promote the binding of C/EBPα to the HBV promoter, which will contribute to the activation of HBV replication. Our current study thus reveals the mechanism underlying doxorubicin modulation of HBV replication and provides an increased understanding of HBV reactivation in CHB patients who are receiving systemic chemotherapy.
One of the current developments in drug research is the controlled release formulation of drugs, which can be released in a controlled manner at a specific target in the body. Due to the diverse physical and chemical properties of various drugs, a smart drug delivery system is highly sought after. The present study aimed to develop a novel drug delivery system using magnetite nanoparticles as the core and coated with polyvinyl alcohol (PVA), a drug 5-fluorouracil (5FU) and Mg-Al-layered double hydroxide (MLDH) for the formation of FPVA-FU-MLDH nanoparticles. The existence of the coated nanoparticles was supported by various physico-chemical analyses. In addition, the drug content, kinetics, and mechanism of drug release also were studied. 5-fluorouracil (5FU) was found to be released in a controlled manner from the nanoparticles at pH = 4.8 (representing the cancerous cellular environment) and pH = 7.4 (representing the blood environment), governed by pseudo-second-order kinetics. The cytotoxicity study revealed that the anticancer delivery system of FPVA-FU-MLDH nanoparticles showed much better anticancer activity than the free drug, 5FU, against liver cancer and HepG2 cells, and at the same time, it was found to be less toxic to the normal fibroblast 3T3 cells.
This study aims to evaluate the bioactive components, in vitro bioactivities, and in vivo hypoglycemic effect of P. frutescens leaf, which is a traditional medicine-food homology plant. P. frutescens methanol crude extract and its fractions (petroleum ether, chloroform, ethyl acetate, n-butanol fractions, and aqueous phase residue) were prepared by ultrasound-enzyme assisted extraction and liquid-liquid extraction. Among the samples, the ethyl acetate fraction possessed the high total phenolic (440.48 μg GAE/mg DE) and flavonoid content (455.22 μg RE/mg DE), the best antioxidant activity (the DPPH radical, ABTS radical, and superoxide anion scavenging activity, and ferric reducing antioxidant power were 1.71, 1.14, 2.40, 1.29, and 2.4 times higher than that of control Vc, respectively), the most powerful α-glucosidase inhibitory ability with the IC50 value of 190.03 μg/mL which was 2.2-folds higher than control acarbose, the strongest proliferative inhibitory ability against MCF-7 and HepG2 cell with the IC50 values of 37.92 and 13.43 μg/mL, which were considerable with control cisplatin, as well as certain inhibition abilities on acetylcholinesterase and tyrosinase. HPLC analysis showed that the luteolin, rosmarinic acid, rutin, and catechin were the dominant components of the ethyl acetate fraction. Animal experiments further demonstrated that the ethyl acetate fraction could significantly decrease the serum glucose level, food, and water intake of streptozotocin-induced diabetic SD rats, increase the body weight, modulate their serum levels of TC, TG, HDL-C, and LDL-C, improve the histopathology and glycogen accumulation in liver and intestinal tissue. Taken together, P. frutescens leaf exhibits excellent hypoglycemic activity in vitro and in vivo, and could be exploited as a source of natural antidiabetic agent.
Drug metabolism involving cytochrome P450 (CYP) enzymes is a key determinant of significant drug interactions. Deoxyelephantopin was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4, and protein expression and resultant enzymatic activity. The mRNA and protein expression of cytochrome isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis, respectively. Human CYP3A4 protein expression was determined using an optimized hCYP3A4-HepG2 cell-based assay and the enzymatic activity was evaluated using P450-Glo™ CYP3A4 assay. The molecular interaction and possible inhibition of deoxyelephantopin of the CYP3A4 enzyme was determined in silico and further validated using substrate-specific CYP3A4 inhibition assays. Deoxyelephantopin produced no significant effect on the CYP1A2 and CYP2D6 mRNA and protein expression. However, it has a weak induction effect on CYP3A4 at the transcriptional level. In silico docking simulation showed that deoxyelephantopin has a weak interaction with CYP3A4 enzyme and it minimally affects the metabolism of CYP3A4 substrates. Deoxyelephantopin is not an in vitro CYP1A2 and CYP2D6 inducer. It is both a weak in vitro CYP3A4 inducer and inhibitor and is unlikely to elicit a clinically significant effect in human.
Glucoraphenin, a glucosinolate present in large quantities in radish is hydrolysed by myrosinase to form the isothiocyanate sulforaphene, which is believed to be responsible for its chemopreventive activity; however, the underlying mechanisms of action have not been investigated, particularly in human cell lines. The aim of the study is to assess the cytotoxicity of sulforaphene in HepG2 cells and evaluate its potential to enhance apoptosis. The cytotoxicity of sulforaphene in HepG2 cells was carried out ensuing an initial screening with two other cell lines, MFC-7 and HT-29, where sulforaphene displayed highest toxicity in HepG2 cells following incubation at 24, 48 and 72 h. In contrast, the intact glucosinolate showed no cytotoxicity. Morphological studies indicated that sulforaphene stimulated apoptosis as exemplified by cell shrinkage, blebbing, chromatin condensation, and nuclear fragmentation. The Annexin V assay revealed significant increases in apoptosis and the same treatment increased the activity of caspases -3/7 and -9, whereas a decline in caspase-8 was observed. Impairment of cell proliferation was indicated by cell cycle arrest at the Sub G₀/G₁ phase as compared to the other phases. It may be concluded that sulforaphene, but not its parent glucosinolate, glucoraphenin, causes cytotoxicity and stimulates apoptosis in HepG2 cells.
Cell-internalizing peptides (CIPs) can be used to mediate specific delivery of nanoparticles across cellular membrane. The objective of this study was to develop a display technique using hepatitis B virus (HBV) capsid-binding peptide as a "nanoglue" to present CIPs on HBV nanoparticles for cell-targeting delivery. A CIP was selected from a phage display library and cross-linked specifically at the tips of the spikes of the HBV capsid nanoparticle via the "nanoglue" by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Fluorescent oligonucleotides packaged in the nanoparticles and the fluorescein molecules conjugated on the nanoparticles were delivered to cells by using this display technique. This study demonstrated a proof of principle for cell-targeting delivery via "nanoglue" bioconjugation.
The development of nanocomposites has swiftly changed the horizon of drug delivery systems in defining a new platform. Major understanding of the interaction of nanocomposites with cells and how the interaction influences intracellular uptake is an important aspect to study in order to ensure successful utilisation of the nanocomposites. Studies have suggested that the nanocomposites' ability to permeate into biological cells is attributable to their well-defined physicochemical properties with nanoscale size, which is relevant to the nanoscale components of biology and cellular organelles. The functionalized graphene oxide coated with polyethylene glycol, loaded with protocatechuic acid and folic acid (GOP-PCA-FA) nanocomposite intracellular uptake was analysed using transmission electron microscope. The accumulation of fluorescent-labelled nanocomposites in the HepG2 cell was also analysed using a fluorescent microscope. In vitro cellular uptake showed that there was uptake of the drug from 24 h into the cells and the release study using fluorescently tagged nanocomposite demonstrated that release and accumulation were observed at 24 h and 48 h. Moreover, the migration ability of tumor cells is a key step in tumor progression which was observed 48 h after treatment. The GOP serves as a potential nanocarrier system which is capable of improving the therapeutic efficacy of drugs and biomolecules in medical as well as pharmaceutical applications through the enhanced intracellular release and accumulation of the encapsulated drugs. Nonetheless, it is essential to analyse the translocation of our newly developed GOP-PCA-FA, and its efficiency for drug delivery, effective cellular uptake, and abundant intracellular accumulation would be compromised by possible untoward side effects.
The FOXE1 gene was screened for mutations in a cohort of 34 unrelated patients with congenital hypothyroidism, 14 of whom had thyroid dysgenesis and 18 were normal (the thyroid status for 2 patients was unknown). The entire coding region of the FOXE1 gene was PCR-amplified, then analyzed using single-stranded conformational polymorphism, followed by confirmation by direct DNA sequencing. DNA sequencing analysis revealed a heterozygous A>G transition at nucleotide position 394 in one of the patients. The nucleotide transition changed asparagine to aspartate at codon 132 in the highly conserved region of the forkhead DNA binding domain of the FOXE1 gene. This mutation was not detected in a total of 104 normal healthy individuals screened. The binding ability of the mutant FOXE1 protein to the human thyroperoxidase (TPO) promoter was slightly reduced compared with the wild-type FOXE1. The mutation also caused a 5% loss of TPO transcriptional activity.
Interleukin-6 (IL-6) is a major mediator of the acute phase response (APR) that regulates the transcription of acute phase proteins (APPs) in the liver. During APR, the plasma levels of negative APPs including retinol binding protein 4 (RBP4) are reduced. Activation of the IL-6 receptor and subsequent signaling pathways leads to the activation of transcription factors, including peroxisome proliferator-activated receptor alpha (PPARα) and CCAAT/enhancer binding protein (C/EBP), which then modulate APP gene expression. The transcriptional regulation of RBP4 by IL-6 is not fully understood. Therefore, this study aimed to elucidate the molecular mechanisms of PPARα and C/EBP isoforms in mediating IL-6 regulation of RBP4 gene expression. IL-6 was shown to reduce the transcriptional activity of RBP4, and functional dissection of the RBP4 promoter further identified the cis-acting regulatory elements that are responsible in mediating the inhibitory effect of IL-6. The binding sites for PPARα and C/EBP present in the RBP4 promoter were predicted at -1079 bp to -1057 bp and -1460 bp to -1439 bp, respectively. The binding of PPARα and C/EBPs to their respective cis-acting elements may lead to antagonistic interactions that modulate the IL-6 regulation of RBP4 promoter activity. Therefore, this study proposed a new mechanism of interaction involving PPARα and different C/EBP isoforms. This interaction is necessary for the regulation of RBP4 gene expression in response to external stimuli, particularly IL-6, during physiological changes.
Gallic acid (GA) is a natural phenolic compound with therapeutic effects that are often challenged by its rapid metabolism and clearance. Therefore, GA was encapsulated using gum arabic into nanoparticles to increase its bioavailability. The formulated nanoparticles (GANPs) were characterized for physicochemical properties and size and were then evaluated for antioxidant and antihypertensive effects using various established in vitro assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide scavenging (NO), β-carotene bleaching and angiotensin-converting enzyme (ACE) inhibitory assays. The GANPs were further evaluated for the in vitro cytotoxicity, cell uptake and cell migration in four types of human cancer cell lines including (MCF-7, MDA-MB231) breast adenocarcinoma, HepG2 hepatocellular cancer, HT-29 colorectal adenocarcinoma, and MCF-10A breast epithelial cell lines. The GANPs demonstrated potent antioxidant effects and have shown promising anti-cancer properties in a dose-dependent manner with a predilection toward HepG2 and MCF7 cancer cells. The uptake of GANPs was successful in the majority of cancer cells with a propensity to accumulate in the nuclear region of the cells. The HepG2 and MCF7 cancer cells also had a significantly higher percentage of apoptosis and were more sensitive to gallic acid nanoparticle treatment in the cell migration assay. This study is the first to confirm the synergistic effects of gum arabic in the encapsulation of gallic acid by increasing the selectivity towards cancer cells and enhancing the antioxidant properties. The formulated nanoparticles also had remarkably low toxicity in normal cells. Based on these findings, GANPs may have promising therapeutic applications towards the development of more effective treatments with a probable targeting precision in cancer cells.
Although there have been considerable advances in the study of dengue virus, no vaccines or anti-dengue drugs are currently available for humans. Therefore, new approaches are necessary for the development of potent anti-dengue drugs. Natural antimicrobial peptides (AMPs) with potent antiviral activities are potential hits-to-leads for antiviral drug discovery. We performed this study to identify and characterise the inhibitory potential of the latarcin peptide (Ltc 1, SMWSGMWRRKLKKLRNALKKKLKGE) against dengue virus replication in infected cells.
α-Mangostin has been reported to possess a broad range of pharmacological effects including potent cholinesterase inhibition, but the development of α-mangostin as a potential lead compound is impeded by its toxicity. The present study investigated the impact of simple structural modification of α-mangostin on its cholinesterase inhibitory activities and toxicity toward neuroblastoma and liver cancer cells. The dialkylated derivatives retained good acetylcholinesterase (AChE) inhibitory activities with IC50 values between 4.15 and 6.73 µM, but not butyrylcholinesterase (BChE) inhibitory activities, compared with α-mangostin, a dual inhibitor (IC50 : AChE, 2.48 µM; BChE, 5.87 µM). Dialkylation of α-mangostin produced AChE selective inhibitors that formed hydrophobic interactions at the active site of AChE. Interestingly, all four dialkylated derivatives of α-mangostin showed much lower cytotoxicity, being 6.4- to 9.0-fold and 3.8- to 5.5-fold less toxic than their parent compound on neuroblastoma and liver cancer cells, respectively. Likewise, their selectivity index was higher by 1.9- to 4.4-fold; in particular, A2 and A4 showed improved selectivity index compared with α-mangostin. Taken together, modification of the hydroxyl groups of α-mangostin at positions C-3 and C-6 greatly influenced its BChE inhibitory and cytotoxic but not its AChE inhibitory activities. These dialkylated derivatives are viable candidates for further structural modification and refinement, worthy in the search of new AChE inhibitors with higher safety margins.
Curcumin (CUR) has been formulated into a host of nano-sized formulations in a bid to improve its in vivo solubility, stability and bioavailability. The aim of this study was to investigate whether the encapsulation of CUR in nanocarriers would impede its biological interactivity, specifically its potential anti-cancer adjuvant activity via the modulation of CYP enzymes in vitro. NanoCUR, a micellar dispersion prepared via a thin film method using only Pluronic F127 as excipient, was amenable to lyophilization, and retained its nano-sized spherical dimensions (17-33 nm) upon reconstitution with water followed by dilution to 5 μM with HBSS or EMEM. NanoCUR was a weaker cytotoxic agent compared to CUR in solution (sCUR), affecting HepG2 cell viability only when the incubation time was prolonged from 4h to 48 h. Correlation with 2h uptake data suggests this was due to a lower cellular uptake rate of CUR from NanoCUR than from sCUR. The poorer CUR accessibility might also account for NanoCUR being a weaker inhibitor of CYP2C9 and CYP2D6 than sCUR. NanoCUR was, however, 1.76-fold more potent against the CYP3A4 (IC50 5.13 ± 0.91 μM) metabolic function. The higher activity against CYP3A4 might be attributed to the synergistic action of Pluronic F127, since the blank micellar dispersion also inhibited CYP3A4 activity. Both sCUR and NanoCUR had no effect on the CYP3A4 mRNA levels in the HepG2 cells. NanoCUR therefore, maintained most of the biological activities of CUR in vitro, albeit at a lower potency and response rate.
Plants are primary source of natural product drugs. However, with every new bioactive molecule reported from a plant source, there follows reports of endangered status or even extinction of a medicinally important plant due to over-harvesting. Hence, the attention turned towards fungi namely the endophytes, which reside within medicinally important plants and thus may have acquired their medicinal properties. Strobilanthes crispus is a traditional medicinal plant which has been used traditionally to treat kidney stones, diabetes, hypertension and cancer as well as having antimicrobial activities. In our efforts to bioprospect for anticancer and antimicrobial metabolites, two fungal endophytes most closely related to the Sordariomycetes sp. showed promising results. Sample (PDA)BL3 showed highest significant antimicrobial activity against 6 bacteria at 200 µg/disc whereas sample (PDA)BL5 has highest significant anticancer activity against all 5 cancer cell lines at concentrations ranging from 30 to 300 μg/ml. As for the gas chromatography coupled with mass spectrometry (GC-MS) results, a total of 20 volatile metabolites identified from sample (PDA)BL3 and 21 volatile metabolites identified from sample (PDA)BL5 having more than 1% abundance. Both GC-MS analysis showed that compound Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) has the highest abundance at 15.10% abundance for sample (PDA)BL3 and 19.00% abundance for sample (PDA)BL5 respectively. In conclusion, these results have shown bio-prospecting potential of endophytic fungi having antimicrobial and anticancer activities as well as its potential secondary metabolites of interest. Therefore, this work has further indicated the medicinal and industrial potential of endophytic fungi.