Microemulsions and Nanoemulsions for Topical Delivery of Tripeptide-3: From Design of Experiment to Anti-Sebum Efficacy on Facial Skin.

The targeted delivery of a hydrophilic Tripeptide-3 to the skin using microemulsions or nanoemulsions for facial oil reduction was the focus of this study. The impact factors affecting oil/water transparent dispersion formation, such as the surfactant system, HLB value, and co-solvent, were identified through the water titration method and pseudoternary phase diagram plots. The interfacial tension between caprylic/capric triglyceride (CCT oil) and water was significantly reduced by the surfactant/co-surfactant combination (Smix) of Cremophore® RH40 and a double-tails co-surfactant, polyglycerol-3-diisostearate, at an HLB of 13 together with a water-to-co-solvent (PG) ratio of 1:1. A two-level full factorial design of experiment (FFD-DoE) emphasized the independent variables of the HLB value, co-solvent, and CCT oil contents affecting the optimal compositions for micro- or nanoemulsion formation. The low-energy spontaneous emulsification of the optimized combination at a low Smix content (10%) yielded the translucent oil-in-water Tripeptide-3 nanoemulsions with an internal droplet size of 25.7 ± 1.20 nm, a narrow polydispersity index of 0.237 ± 0.129, and 70.6 ± 0.58% transmittance. The in vitro skin permeation study revealed a significantly higher skin penetration and retention of the Tripeptide-3 nanoemulsions compared to the high surfactant microemulsions and coarse emulsions. Skin irritation and oil control efficacy were evaluated in healthy volunteers before and after product application for 28 days. The obtained nanoemulsions not only decreased sebum production but also enhanced skin moisture levels. In conclusion, the meticulously designed nanoemulsions, incorporating suitable excipients, show a promising delivery system for hydrophilic peptides to control sebum overproduction in oily facial skin.

Natural Stabilizers and Nanostructured Lipid Carrier Entrapment for Photosensitive Compounds, Curcumin and Capsaicin.

Capsaicin and curcumin, the active components of chili and turmeric, are prone to instability when exposed to light. Therefore, this research aimed to enhance the photostability of both extracts via the use of antioxidants, natural sunscreen, and nanostructured lipid carriers (NLCs). NLCs were chosen for this this study due to their advantages in terms of stability, drug loading capacity, occlusive effect, skin penetration, and controlled release. The photostability of each extract and extracts mixed with antioxidants, including grape seed extract, tea extract, and chlorogenic acid, were determined. Chlorogenic acid can enhance the photostability of capsaicin from 6.79 h to 16.50 h, while the photostability of curcumin increased from 9.63 h to 19.25 h. In addition, the use of natural sunscreen (sunflower oil) also increased the photostability of capsaicin and curcumin. The mixed extracts were then loaded into NLCs. The particle size of the formulation was 153.73 nm with a PDI value of 0.25. It exhibited high entrapment efficiency (more than 95%). In addition, it effectively reduced the decomposition of capsaicin and curcumin. Importantly, the natural stabilizers chosen for NLC fabrication significantly improved the photostability of curcumin and capsaicin by 600% and 567% compared to the unstabilized counterparts. This improvement contributes to the sustainability and bioavailability of these compounds in both cosmeceutical and pharmaceutical products.

The in vitro effects of black soldier fly larvae (Hermitia illucens) oil as a high-functional active ingredient for inhibiting hyaluronidase, anti-oxidation benefits, whitening, and UVB protection.

Objective: Larvae of Hermitia illucens, or black soldier fly larvae (BSFL), have been recognized for their high lipid yield with a remarkable fatty acid profile. BSFL oil (SFO) offers the added value of a low environmental footprint and a sustainable product. In this study, the characteristics and cosmetic-related activities of SFO were investigated and compared with rice bran oil, olive oil and krill oil which are commonly used in cosmetics and supplements. Methods: The physicochemical characteristics were determined including acid value, saponification value, unsaponifiable matter and water content of SFO. The fatty acid composition was determined using GC-MS equipped with TR-FAME. The in vitro antioxidant properties were determined using DPPH, FRAP and lipid peroxidation inhibition assays. Antihyaluronidase (anti-HAase) activity was measured by detecting enzyme activity and molecular docking of candidate compounds toward the HAase enzyme. The safety assessment towards normal human cells was determined using the MTT assay and the UVB protection upon UVB-irradiated fibroblasts was determined using the DCF-DA assay. The whitening effect of SFO was determined using melanin content inhibition. Results: SFO contains more than 60% polyunsaturated fatty acids followed by saturated fatty acids (up to 37%). The most abundant component found in SFO was linoleic acid (C18:2 n-6 cis). Multiple anti-oxidant mechanisms of SFO were discovered. In addition, SFO and krill oil prevented hyaluronic acid (HA) degradation via strong HAase inhibition comparable with the positive control, oleanolic acid. The molecular docking confirmed the binding interactions and molecular recognition of major free fatty acids toward HAase. Furthermore, SFO exhibited no cytotoxicity on primary human skin fibroblasts, HaCaT keratinocytes and PBMCs (IC50 values > 200 µg/mL). SFO possessed significant in-situ anti-oxidant activity in UVB-irradiated fibroblasts and the melanin inhibition activity as effective as well-known anti-pigmenting compounds (kojic acid and arbutin, p < 0.05). Conclusion: This study provides scientific support for various aspects of SFO. SFO can be considered an alternative oil ingredient in cosmetic products with potential implications for anti-skin aging, whitening and UVB protection properties, making it a potential candidate oil in the cosmetic industry.

Targeting Spike Glycoprotein S1 Mediated by NLRP3 Inflammasome Machinery and the Cytokine Releases in A549 Lung Epithelial Cells by Nanocurcumin.

Chronic inflammation and tissue damage can result from uncontrolled inflammation during SARS-CoV-2 or COVID-19 infections, leading to post-acute COVID conditions or long COVID. Curcumin, found in turmeric, has potent anti-inflammatory properties but limited effectiveness. This study developed nanocurcumin, a curcumin nanoparticle, to enhance its physical and chemical stability and investigate its in vitro anti-inflammatory properties upon CoV2-SP induction in lung epithelial cells. Nanocurcumin was prepared by encapsulating curcumin extract in phospholipids. The particle size, polydispersity index, and zeta potential of nanocurcumin were measured using dynamic light scattering. The encapsulated curcumin content was determined using HPLC analysis. The encapsulation efficiency of curcumin was 90.74 ± 5.35% as determined by HPLC. Regarding the in vitro release of curcumin, nanocurcumin displayed a higher release content than non-nanoparticle curcumin. Nanocurcumin was further investigated for its anti-inflammatory properties using A549 lung epithelial cell line. As determined by ELISA, nanocurcumin showed inhibitory effects on inflammatory cytokine releases in CoV2-SP-stimulated conditions, as evidenced by a significant decrease in IL-6, IL-1ß and IL-18 cytokine secretions compared with the spike-stimulated control group (p < 0.05). Additionally, as determined by RT-PCR, nanocurcumin significantly inhibited the CoV2-SP-stimulated expression of inflammatory genes (IL-6, IL-1ß, IL-18, and NLRP3) compared with the spike-stimulated control group (p < 0.05). Regarding the inhibition of NLRP3 inflammasome machinery proteins by Western blot, nanocurcumin decreased the expressions of inflammasome machinery proteins including NLRP3, ASC, pro-caspase-1, and the active form of caspase-1 in CoV2-SP-stimulated A549 cells compared with the spike-stimulated control group (p < 0.05). Overall, the nanoparticle formulation of curcumin improved its solubility and bioavailability, demonstrating anti-inflammatory effects in a CoV2-SP-induced scenario by inhibiting inflammatory mediators and the NLRP3 inflammasome machinery. Nanocurcumin shows promise as an anti-inflammatory product for preventing COVID-19-related airway inflammation.

Pharmacokinetics and analytical determination of acyclovir in Asian elephant calves (Elephas maximus).

A therapeutic regimen that includes antiviral drugs is critical for the survival of Asian elephant (Elephas maximus) calves infected with elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD), with acyclovir showing considerable promise. The purpose of this study was to determine the pharmacokinetics and bioavailability of acyclovir following intravenous (IV) and oral (PO) administration in Asian elephants. A single dose of acyclovir (15 mg/kg, IV or 45 mg/kg, PO) was administered to four healthy elephant calves, with a minimum 2-week washout period between treatments. Serial plasma samples were collected after each injection for acyclovir analysis using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Maximum plasma acyclovir concentrations were 27.02 ± 6.79 µg/mL at 0.94 ± 0.31 h after IV administration, and 1.45 ± 0.20 µg/mL at 3.00 ± 0.70 h after PO administration. The half-life of the elimination phase (T1/2) was 5.84 ± 0.74 and 8.74 ± 2.47 h after IV and PO administration, respectively. After IV administration, acyclovir concentrations were higher than the half-maximal inhibitory concentration (IC50) of those found for herpes simplex virus (HSV) 1 and 2 in humans, and equid alpha herpesvirus-1 (EHV-1) for at least 12 h. By contrast, the bioavailability of oral administration was low, only 6.03 ± 0.87%, so higher doses by that route likely are needed to be effective. Due to the high concentration of plasma acyclovir after IV administration, the dose may need to be adjusted to prevent any negative side effects.

Successful treatment of elephant endotheliotropic herpesvirus infection in an Asian elephant (Elephas maximus) calf by oral acyclovir medication: Case report.

Elephant endotheliotropic herpesvirus (EEHV) is a major cause of death in Asian elephant (Elephas maximus) calves. A 2-year, 11-month-old female, captive Asian elephant presented with facial edema and a mild fever. Blood samples were collected and showed EEHV1A positivity with a high viral load by real time PCR. Heterophil toxicity also was reported for the first time in this case. The calf was treated orally with acyclovir, 45 mg/kg tid for 28 days, which reduced the EEHV1A viral load to undetectable levels within 9 days and the calf survived. A successful outcome with oral acyclovir administration provides another and affordable option to treat EEHV hemorrhagic disease in Asian elephants, and one that is easier to administer in untrained calves.

Nanomaterial Lipid-Based Carrier for Non-Invasive Capsaicin Delivery; Manufacturing Scale-Up and Human Irritation Assessment.

Capsaicin is an active compound in chili peppers (Capsicum chinense) that has been approved for chronic pain treatment. The topical application of high-strength capsaicin has been proven to reduce pain; however, skin irritation is a major drawback. The aim of this study was to investigate an appropriate and scalable technique for preparing nanostructured lipid carriers (NLCs) containing 0.25% capsaicin from capsicum oleoresin (NLC_C) and to evaluate the irritation of human skin by chili-extract-loaded NLCs incorporated in a gel formulation (Gel NLC_C). High-shear homogenization with high intensity (10,000 rpm) was selected to create uniform nanoparticles with a size range from 106 to 156 nm. Both the NLC_C and Gel NLC_C formulations expressed greater physical and chemical stabilities than the free chili formulation. Release and porcine biopsy studies revealed the sustained drug release and significant permeation of the NLCs through the outer skin layer, distributing in the dermis better than the free compounds. Finally, the alleviation of irritation and the decrease in uncomfortable feelings following the application of the Gel NLC_C formulation were compared to the effects from a chili gel and a commercial product in thirty healthy volunteers. The chili-extract-loaded NLCs were shown to be applicable for the transdermal delivery of capsaicin whilst minimizing skin irritation, the major noncompliance cause of patients.

Enhanced Transdermal Delivery of Concentrated Capsaicin from Chili Extract-Loaded Lipid Nanoparticles with Reduced Skin Irritation.

The aim of this study was to develop lipid-based nanoparticles that entrapped a high concentration of capsaicin (0.25%) from a capsicum oleoresin extract. The solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were strategically fabricated to entrap capsaicin without a hazardous solvent. Optimized nanosize lipid particles with high capsaicin entrapment and loading capacity were achieved from pair-wise comparison of the solid lipid mixtures consisting of fatty esters and fatty alcohols, representing small and large crystal-structure molecules combined with a compatible liquid lipid and surfactants (crystallinity index = 3%). This report was focused on selectively captured capsaicin from oleoresin in amorphous chili extract-loaded NLCs with 85.27% ± 0.12% entrapment efficiency (EE) and 8.53% ± 0.01% loading capacity (LC). The particle size, polydispersity index, and zeta potential of chili extract-loaded NLCs were 148.50 ± 2.94 nm, 0.12 ± 0.03, and -29.58 ± 1.37 mV, respectively. The favorable zero-order kinetics that prolonged capsaicin release and the significantly faster transdermal penetration of the NLC attributed to the reduction in skin irritation of the concentrated capsaicin NLCs, as illustrated by the in vitro EpiDermTM three-dimensional human skin irritation test and hen's egg test chorioallantoic membrane assay (HET-CAM).

Antileukemic Cell Proliferation of Active Compounds from Kaffir Lime (Citrus hystrix) Leaves.

Kaffir lime (Citrus hystrix) is a plant member of family Rutaceae, and its leaves are commonly used in folk medicine. The present study explores antileukemic effects of the extracts and purified active compounds from the leaves. The antileukemic activity was investigated via inhibition of Wilms' tumor 1 (WT1), which is a protein that involves in leukemic cell proliferation. In addition, the compounds were investigated for their effects on WT1 gene expression using real time RT-PCR and Western blotting. Cell cycle arrest and total cell number were investigated using flow cytometry and trypan blue exclusion method, respectively. The results demonstrated that the hexane fractionated extract had the greatest inhibitory effect on WT1 gene expression of many leukemic cell lines and significantly decreased WT1 protein levels of K562 cells (representative of the leukemic cells), in a dose- and time-dependent manner. Subfraction No. 9 (F9) after partial purification of hexane fractioned extract showed the highest suppression on WT1 protein and suppressed cell cycle at G2/M. The organic compounds were isolated from F9 and identified as phytol and lupeol. The bioassays confirmed antiproliferative activities of natural products phytol and lupeol. The results demonstrated anticancer activity of the isolated phytol and lupeol to decrease leukemic cell proliferation.

Dermal targeting of Centella asiatica extract using hyaluronic acid surface modified niosomes.

The work aimed to develop Centella asiatica extract-loaded niosomes (CAE-Nio) and surface modified niosomes by hyaluronic acid (CAE-Nio-HA) to enhance transdermal penetration. Niosome formulations were prepared by film hydration method using Tween 60 and Span 60 as nonionic surfactants, cholesterol and various CAE contents. Various HA concentrations were investigated to obtain optimized CAE-Nio enhancing further skin penetration. Results showed that niosomes prepared from Tween 60 yielded suitable CAE encapsulated niosomes with mean particle size and zeta-potential of 155 nm and -15 mV, respectively. The niosomes exhibited high encapsulation efficiency (%EE) and drug loading capacity (%DL) of 71-77% and 3-7%, respectively. Incorporating HA to niosome decreased %DL and caused larger particle size and increased zeta-potential in a dose dependent manner while %EE remained unaffected. The sustained-release behaviour of CAE from all niosomes was under a diffusion controlled mechanism. Asiaticoside, a relatively polar compound from CAE-Nio-HA could penetrate through the stratum corneum and dermis in a larger amount than from CAE-Nio and CAE solution. CAE-Nio-HA formulations showed good stability under low temperature (4 °C and 25 °C) for periods longer than 4 months. In conclusion, the developed Nio-HA is a promising delivery system for asiaticoside to enhanced dermal absorption, permeation and accumulation in viable epidermis and dermis layers. This system can also be applied to other hydrophilic natural active compounds.

FLT3, a prognostic biomarker for acute myeloid leukemia (AML): Quantitative monitoring with a simple anti-FLT3 interaction and flow cytometric method.

BACKGROUND: Overexpression of fms-like tyrosine kinase 3 (FLT3) protein in leukemia is highly related to poor prognosis and reduced survival rate in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Simple but efficient quantification of FLT3 protein levels on the leukemic cell surface using flow cytometry had been developed for rapid determination of FLT3 on intact cell surface. METHODS: Quantitation protocol for FLT3 biomarker in clinical samples was developed and validated. Cell model selection for calibration curve construction was identified and evaluated. Selected antibody concentrations, cell density, and incubation time were evaluated for most appropriate conditions. Comparison of the developed FLT3 determination protocol with the conventional Western blot analysis was performed. RESULTS: EoL-1 cell line was selected for using as positive control cells. Calibration curve (20%-120% of FLT3 positive cells) and quality control (QC) levels were constructed and evaluated. The results demonstrated good linearity (r2  > 0.99). The intra- and inter-day precision and accuracy, expressed as the coefficient of variation (%CV) and % recovery, were <20% and fell in 80%-120% in all cases. When compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend. CONCLUSIONS: The effective, reliable, rapid, and economical analytical technique using the developed flow cytometric method was demonstrated for FLT3 protein determination on leukemic cell surface. This method provided a practical analysis of FLT-3 biomarker levels which is valuable for physician decision in acute leukemia treatment.

FLT3-specific curcumin micelles enhance activity of curcumin on FLT3-ITD overexpressing MV4-11 leukemic cells.

Curcumin, a major active compound in the turmeric rhizome, has many biological properties, especially anti-leukemia activity. The overexpression of FMS-like tyrosine kinase 3 protein with internal tandem duplication (FLT3-ITD) mutation protein was related to the poor prognosis and disease progression of leukemia. In this study, the cytotoxicity and inhibitory effect of curcumin on cell cycle of FLT3-ITD overexpressing MV4-11 leukemic cells were evaluated. Moreover, curcumin polymeric micelles conjugated with FLT3-specific peptide (FLT3-Cur-micelles) were prepared using a film hydration method to increase curcumin solubility and the inhibitory effect on MV4-11 cells was evaluated. Cytotoxicity and cell cycle analysis were performed using an MTT assay and flow cytometry, respectively. Physical properties of FLT3-Cur-micelles, including particle size, size distribution, morphology, and entrapment efficiency (EE), were evaluated. Cellular uptake of the micelles on MV4-11 cells was determined by flow cytometry and fluorescence microscopy. FLT3-Cur-micelles were observed with size less than 50 nm and high EE of >75%. In addition, FLT3-Cur-micelles demonstrated excellent internalization and increased curcumin accumulation in leukemic cells when compared to free curcumin. Furthermore, FLT3-Cur-micelles exhibited a strong cytotoxic effect on MV4-11 cells with IC50 value of 1.1 µM, whereas the blank micelles showed no effect. Furthermore, FLT3-Cur-micelles showed no significant effect on normal human PBMCs with IC50 value >25 µM. In summary, FLT3-Cur-micelles are a promising nanocarrier system for enhancing anti-leukemic activity of curcumin and suitable for further preclinical studies.

An integrated molecular modeling approach for the tryptase monomer-curcuminoid recognition analysis: conformational and bioenergetic features.

Human mast cell tryptase has been shown as an activating enzyme in matrix degradation process. The previous study suggest that tryptase either alone or in joining with activation of metalloproteinases, can associate in extra cellular matrix damage and the possible destruction of the basement membrane resulting in photoaging. Therefore the inhibition of tryptase activity is one of the most important therapeutic strategies against the photoaging. Curcumin has been shown to be a potential agent for preventing and/or treating the photoaging induced by UV radiation. However, the protective effect of curcumin against the photoaging through the tryptase inhibition is still inadequately understood. In this work, computational methods to characterize the structural framework and define the atomistic details of the determinants for the tryptase inhibition mechanism by curcuminoids were performed. By molecular docking, three putative binding models able to efficiently bind all curcuminoids were identified. Analysis of molecular dynamics simulations revealed that cyclocurcumin, curcumin glucuronide, and curcumin, the most effective inhibitors from the three models, modified significant tryptase monomer rigidity by binding in all the possible sites. The result of these binding events is the suppression of the functional enzymatic motions involving the binding of substrates to the catalytic site. On the basis of this finding may thus be beneficial for the development of new natural inhibitors for the therapeutic remedy of photoaging, targeting and modulating the activity of tryptase.

Stable curcumin-loaded polymeric micellar formulation for enhancing cellular uptake and cytotoxicity to FLT3 overexpressing EoL-1 leukemic cells.

The present study aims to develop a stable polymeric micellar formulation of curcumin (CM) with improved solubility and stability, and that is suitable for clinical applications in leukemia patients. CM-loaded polymeric micelles (CM-micelles) were prepared using poloxamers. The chemical structure of the polymers influenced micellar properties. The best formulation of CM-micelles, namely CM-P407, was obtained from poloxamer 407 at drug to polymer ratio of 1:30 and rehydrated with phosphate buffer solution pH 7.4. CM-P407 exhibited the smallest size of 30.3±1.3nm and highest entrapment efficiency of 88.4±4.1%. When stored at -80°C for 60days, CM-P407 retained high protection of CM and had no significant size change. In comparison with CM solution in dimethyl sulfoxide (CM-DMSO), CM kinetic degradation in both formulations followed a pseudo-first-order reaction, but the half-life of CM in CM-P407 was approx. 200 times longer than in CM-DMSO. Regarding the activity against FLT3 overexpressing EoL-1 leukemic cells, CM-P407 showed higher cytotoxicity than CM-DMSO. Moreover, intracellular uptake to leukemic cells of CM-P407 was 2-3 times greater than that of CM-DMSO. These promising results for CM-P407 will be further investigated in rodents and in clinical studies for leukemia treatment.

Development and Characterization of FLT3-Specific Curcumin-Loaded Polymeric Micelles as a Drug Delivery System for Treating FLT3-Overexpressing Leukemic Cells.

This study aimed at developing a curcumin (CM) nanoparticle targeted to Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3) protein on the surface of leukemic cells and at evaluating their properties, specificity, cytotoxicity, and inhibitory effect on FLT3 protein level in FLT3-overexpressing leukemic cells, EoL-1, and MV-4-11 cells. FLT3-specific peptides were conjugated onto modified poloxamer 407 using the copper-catalyzed azide-alkyne cycloaddition reaction. The thin film hydration method was performed for FLT3-specific CM-loaded polymeric micelles (FLT3-CM-micelles) preparation. Flow cytometry and fluorescence microscopy were used to determine rate of cellular uptake. 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to test the cytotoxicity of the micelles on leukemic cells. FLT3-CM-micelles demonstrated a mean particle size less than 50 nm, high entrapment efficiency, and high rate of CM uptake by leukemic cells. The intracellular CM fluorescence is related to FLT3 protein levels on the leukemic cell surfaces. Moreover, FLT3-CM-micelles demonstrated an excellent cytotoxic effect and decreased FLT3 protein expression in the leukemic cells. The FLT3-CM-micelles could enhance both solubility and cytotoxicity of CM on FLT3-overexpressing leukemic cells. These promising nanoparticles may be used for enhancing antileukemic activity of CM and developed as a targeted drug delivery system in the future.

Effect of Oral Coadministration of Ascorbic Acid with Ling Zhi Preparation on Pharmacokinetics of Ganoderic Acid A in Healthy Male Subjects: A Randomized Crossover Study.

The objective of this randomized, open-label, single-dose, two-phase crossover study was to determine the effect of ascorbic acid on pharmacokinetics of ganoderic acid A, an important biologically active triterpenoid compound with anticancer activities, following oral administration of water extract of fruiting bodies of Ling Zhi in 12 healthy male subjects. Each subject was randomized to receive either one of the two regimens: (1) a single dose of 3,000 mg of the Ling Zhi preparation or (2) a single dose of 3,000 mg of the Ling Zhi preparation in combination with 2,500 mg of ascorbic acid. After a washout period of at least two weeks, subjects were switched to receive the alternate regimen. Blood samples were collected in each phase immediately before dosing and at specific time points for 8 hours after dosing. Plasma ganoderic acid A concentrations were quantified using liquid chromatography-mass spectrometry (LC-MS). The pharmacokinetic parameters analyzed were maximal plasma concentration (Cmax), time to reach peak concentration (Tmax), area under the plasma concentration-time curve (AUC), and half-life (t1/2). An oral coadministration of ascorbic acid with Ling Zhi preparation did not significantly alter the pharmacokinetic parameters of ganoderic acid A in healthy male subjects.

Down-regulatory mechanism of mammea E/BB from Mammea siamensis seed extract on Wilms' Tumor 1 expression in K562 cells.

BACKGROUND: Wilms' tumor 1 (WT1) is a biological marker for predicting leukemia progression. In this study, mammea E/BB, an active compound from Saraphi (Mammea siamensis) seed extract was examined for its effect on down-regulatory mechanism of WT1 gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell line. METHODS: M. siamensis seeds were obtained from the region of Chiang Mai (North of Thailand). Mammea E/BB was extracted from seeds of M. siamensis. WT1 protein expression and stability were evaluated by Western blot analysis. WT1 mRNA stability was assessed by qRT-PCR. WT1-DNA binding and WT1 promoter activity were assayed by ChIP assay and luciferase-reporter assay, respectively. Cell cycle arrest was studied by flow cytometry. RESULTS: Treatment with mammea E/BB led to down-regulation of WT1 expression. The suppression of WT1 expression did not involve protein and mRNA degradation. Rather, WT1 protein was down-regulated through disruption of transcriptional auto-regulation of the WT1 gene. Mammea E/BB inhibited WT1-DNA binding at the WT1 promoter and decreased luciferase activity. It also disrupted c-Fos/AP-1 binding to the WT1 promoter via ERK1/2 signaling pathway and induced S phase cell cycle arrest in K562 cells. CONCLUSION: Mammea E/BB had pleotropic effects on kinase signaling pathways, resulting in inhibition of leukemia cell proliferation.

Inhibitory effect of mammea E/BB from Mammea siamensis seed extract on Wilms' tumour 1 protein expression in a K562 leukaemic cell line.

Mammea siamensis is used in traditional Thai medicine. This study was designed to extract and isolate an active compound from the M. siamensis seeds and to investigate its activity on Wilms' tumour 1 (WT1) protein expression in K562 cells. WT1 is a transcription factor that stimulates cell proliferation. The ethanol saraphi seed (ESS) extract was fractionated using n-hexane, ethyl acetate, n-butanol and water to obtain n-hexane saraphi seed (HSS), ethyl acetate saraphi seed (EASS), n-butanol saraphi seed (BSS), and water saraphi seed (WSS) extracts, respectively. The ESS, HSS and EASS extracts had strong cytotoxic effects on K562 cells in the MTT assay. All three fractions decreased WT1 protein levels and decreased total cell numbers. The HSS extract decreased the WT1 protein levels in a time- and dose-dependent manner. HPLC and NMR analyses indicated that the active compound of HSS was mammea E/BB. M. siamensis seeds are thus identified as a promising source of bioactive compounds for potential inhibition of WT1 protein expression.

Inhibitory effect of turmeric curcuminoids on FLT3 expression and cell cycle arrest in the FLT3-overexpressing EoL-1 leukemic cell line.

Leukemia is a hematologic malignancy with a frequent incidence and high mortality rate. Previous studies have shown that the FLT3 gene is overexpressed in leukemic blast cells, especially in acute myeloid leukemia. In this study, a commercially available curcuminoid mixture (1), pure curcumin (2), pure demethoxycurcumin (3), and pure bisdemethoxycurcumin (4) were investigated for their inhibitory effects on cell growth, FLT3 expression, and cell cycle progression in an FLT3-overexpressing EoL-1 leukemic cell line using an MTT assay, Western blotting, and flow cytometry, respectively. The mixture (1) and compounds 2-4 demonstrated cytotoxic effects with IC50 values ranging from 6.5 to 22.5 µM. A significant decrease in FLT3 protein levels was found after curcuminoid treatment with IC20 doses, especially with mixture 1 and compound 2. In addition, mixture 1 and curcumin (2) showed activity on cell cycle arrest at the G0/G1 phase and decreased the FLT3 and STAT5A protein levels in a dose-dependent manner. Compound 2 demonstrated the greatest potential for inhibiting cell growth, cell cycle progression, and FLT3 expression in EoL-1 cells. This investigation has provided new findings regarding the effect of turmeric curcuminoids on FLT3 expression in leukemic cells.

Pharmacokinetics of ganoderic acids a and f after oral administration of ling zhi preparation in healthy male volunteers.

The objectives of this paper were to evaluate the pharmacokinetics of ganoderic acids A and F after a single oral dose of the water extract of MG2-strain Ling Zhi (MG2FB-WE) and to assess the influence of food on the pharmacokinetics in 12 healthy male volunteers. This study was a single-dose, open-label, randomized, two-phase crossover study with at least 2 wk washout period. Each subject was randomly assigned to receive a single oral dose of 3,000 mg of MG2FB-WE in granular formulation dissolved in 200 mL of warm water, either under a fasting condition, or immediately after a standard breakfast (fed condition). Blood samples were collected immediately before and at specific time points until 8 h after MG2FB-WE administration. Plasma ganoderic acids A and F concentrations were determined by using liquid chromatography-mass spectrometry (LC-MS) technique. In conclusion, the pharmacokinetic profile of both ganoderic acids under a fasting condition was characterized by rapid absorption from the gastrointestinal tract (T(max) at approximately 30 min) and a short elimination half-life (<40 min). Food significantly decreased C(max) and delayed T(max), but did not affect the extent of ganoderic acid A absorption. However, concomitant food intake markedly impeded both rate and extent of ganoderic acid F absorption.