Unroasted Green Coffee Extract-Loaded Solid Lipid Nanoparticles for Enhancing Intestinal Permeation.

Green coffee bean extract (GCBE) provides diversified health benefits. However, its reported low bioavailability impeded its utilization in various applications. In this study, GCBE-loaded solid lipid nanoparticles (SLNs) were prepared to improve the bioavailability through enhanced intestinal absorption of GCBE. During the preparation of promising GCBE-loaded SLNs, the lipid concentration, surfactant concentration, and co-surfactant amount are crucial that were optimized using the Box-Behnken design, while particle size, polydispersity index (PDI), ζ-potential, entrapment efficiency, and cumulative drug release were the measured responses. GCBE-SLNs were successfully developed by a high shear homogenization technique using geleol as a solid lipid, tween 80 as a surfactant, and propylene glycol as Co-SAA. The optimized SLNs contained 5.8% geleol, 5.9% tween 80, and 80.4 mg PG resulting in a small particle size of 235.7 ± 12.5 nm, reasonably acceptable PDI of 0.417 ± 0.023, and ζ-potential of -15 ± 0.14 mV, with a high entrapment efficiency of 58.3 ± 0.85% and cumulative release of 7575 ± 0.78%. Furthermore, the performance of the optimized GCBE-SLN was evaluated using an ex vivo everted sac model where the intestinal permeation of GCBE was improved due to nanoencapsulation using SLN. Consequently, the results enlightened the auspicious potential of exploiting oral GCBE-SLNs for boosting intestinal absorption of chlorogenic acid.

Phytoecdysteroids and Anabolic Effect of Atriplex dimorphostegia: UPLC-PDA-MS/MS Profiling, In Silico and In Vivo Models.

Atriplex dimorphostegia (Saltbush) is an annual halophytic shrub that is widely distributed across various parts of Asia. The current study is the first to report the metabolites profile of the total ethanol extract of the aerial parts of A. dimorphostegia (TEAD), and its anabolic activity together with the isolated 20-hydroxyecdysone (20-HE) in orchidectomized male rats. TEAD was analyzed and standardized utilizing UPLC-PDA-ESI−MS/MS and UPLC-PDA-UV techniques, resulting in tentative identification of fifty compounds including polyphenols, steroids and triterpenoids. In addition, 20-HE was quantified, representing 26.79 µg/mg of the extract. Phytochemical investigation of TEAD resulted in the isolation of 20-HE from the ethyl acetate fraction (EFAD) and was identified by conventional spectroscopic methods of analysis. Furthermore, the anabolic effect of the isolated 20-HE and TEAD was then evaluated using in silico and in vivo models. Molecular docking experiments revealed in vitro selectivity of 20-HE towards estrogen receptors (ERs), specifically ERß over ERα and androgenic receptor (AR). The anabolic efficacy of TEAD and 20-HE was studied in orchidectomized immature male Wistar rats using the weight of gastrocnemius and soleus muscles. The weights of ventral prostate and seminal vesicles were used as indicators for androgenic activity. Rats administered 20-HE and TEAD showed a significant increase (p = 0.0006 and p < 0.0001) in the net muscle mass compared to the negative control, while the group receiving TEAD showed the highest percentage among all groups at p < 0.0001. Histopathological investigation of skeletal muscle fibers showed normal morphological structures, and the group administered 20-HE showed an increase in cross sectional area of muscle fibers comparable to methandienone and testosterone groups at p > 0.99. A. dimorphostegia exhibited promising anabolic activity with minimal androgenic side effects.

Cheminformatics Application in the Phytochemical and Biological Study of Eucalyptus globulus L. Bark as a Potential Hepatoprotective Drug.

Natural products are considered as a good source of antifibrotic agents, but identifying and isolating bioactive molecule(s) is still challenging. Fortunately, numerous computational techniques have evolved to save time and efforts in this field. The aim of the current study was to utilize several cheminformatics software to study the chemical and biological features of the bark of Eucalyptus globulus cultivated in Egypt. Sirius software, with the aid of online databases, was used to process liquid chromatography-mass spectrometry (LC-MS) chemical profiling and predict precise molecular formulae, chemical classes, and structures. Accordingly, 37 compounds were tentatively identified, including 15 reported here for the first time from this species. Also, the BioTransformer tool was successfully applied for in silico virtual study of the human metabolism of these compounds, and 1960 different products were obtained through various metabolic pathways. Finally, an electronic library of the identified compounds and their metabolites were developed and docked in silico against eight different protein targets that are involved in the liver fibrosis process. The results revealed that the extract may have a potential hepatoprotective effect through several mechanisms and that the metabolites have the highest binding affinities to the relevant enzymes than their parent compounds. The extract was found to show potent cytotoxic activity against the liver cancer cell lines HEPG2 and HUH-7, and its absorption was enhanced through nanoformulation, as proved using the ex vivo everted gut sac method.

Effects of Metformin Combined With Antifolates on HepG2 Cell Metabolism and Cellular Proliferation.

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancers worldwide, continues to maintain high levels of resistance to standard therapy. As clinical data revealed poor response rates, the need for developing new methods has increased to improve the overall wellbeing of patients with HCC. Furthermore, a growing body of evidence shows that cancer metabolic changes are a key feature of many types of human malignancies. Metabolic reprogramming refers to cancer cells' ability to change their metabolism in order to meet the increased energy demand caused by continuous growth, rapid proliferation, and other neoplastic cell characteristics. For these reasons, metabolic pathways may become new therapeutic and chemopreventive targets. The aim of this study was to investigate the metabolic alterations associated with metformin (MET), an anti-diabetic agent when combined with two antifolate drugs: trimethoprim (TMP) or methotrexate (MTX), and how metabolic changes within the cancer cell may be used to increase cellular death. In this study, single drugs and combinations were investigated using in vitro assays including cytotoxicity assay (MTT), RT-qPCR, annexin V/PI apoptosis assay, scratch wound assay and Seahorse XF analysis, on a human HCC cell line, HepG2. The cytotoxicity assay showed that the IC50 of MET as single therapy was 44.08 mM that was reduced to 22.73 mM and 29.29 mM when combined with TMP and MTX, respectively. The co-treatment of both drugs increased p53 and Bax apoptotic markers, while decreased the anti-apoptotic marker; Bcl-2. Both combinations increased the percentage of apoptotic cells and halted cancer cell migration when compared to MET alone. Furthermore, both combinations decreased the MET-induced increase in glycolysis, while also inducing mitochondrial damage, altering cancer cell bioenergetics. These findings provide an exciting insight into the anti-proliferative and apoptotic effects of MET and anti-folates on HepG2 cells, and how in combination, may potentially combat the aggressiveness of HCC.

Efficacy of pomegranate extract loaded solid lipid nanoparticles transdermal emulgel against Ehrlich ascites carcinoma.

The purpose of this work was to incorporate an optimized pomegranate extract loaded solid lipid nanoparticles (PE-SLNs) formula in a transdermal emulgel to evaluate its anticancer effect. The prepared emulgel formulae were evaluated for their physicochemical properties. An ex vivo permeation study was done through mouse skin and the kinetic parameters were determined. Kinetic data showed that the ex vivo permeation of PE from SLNs transdermal emulgel through mouse skin followed non-Fickian diffusion transport. Further, in vivo study was done by applying the optimized PE-SLNs transdermal emulgel on mice skin bearing a solid form of Ehrlich ascites carcinoma (EAC) as well as free PE, control, placebo, and standard groups for comparison. In addition, histopathological examinations of the samples obtained from the EAC mice model were performed. The results proved that application of the selected PE-SLNs emulgel formulation on the mice skin bearing solid tumor revealed statistically significant anticancer effects.

Anti-estrogenic and anti-aromatase activities of citrus peels major compounds in breast cancer.

Estrogen signaling is crucial for breast cancer initiation and progression. Endocrine-based therapies comprising estrogen receptor (ER) modulators and aromatase inhibitors remain the mainstay of treatment. This study aimed at investigating the antitumor potential of the most potent compounds in citrus peels on breast cancer by exploring their anti-estrogenic and anti-aromatase activities. The ethanolic extract of different varieties of citrus peels along with eight isolated flavonoids were screened against estrogen-dependent breast cancer cell lines besides normal cells for evaluating their safety profile. Naringenin, naringin and quercetin demonstrated the lowest IC50s and were therefore selected for further assays. In silico molecular modeling against ER and aromatase was performed for the three compounds. In vivo estrogenic and anti-estrogenic assays confirmed an anti-estrogenic activity for the isolates. Moreover, naringenin, naringin and quercetin demonstrated in vitro inhibitory potential against aromatase enzyme along with anticancer potential in vivo, as evidenced by decreased tumor volumes. Reduction in aromatase levels in solid tumors was also observed in treated groups. Overall, this study suggests an antitumor potential for naringenin, naringin and quercetin isolated from citrus peels in breast cancer via possible modulation of estrogen signaling and aromatase inhibition suggesting their use in pre- and post-menopausal breast cancer patients, respectively.

Comparing the protective effects of resveratrol, curcumin and sulforaphane against LPS/IFN-γ-mediated inflammation in doxorubicin-treated macrophages.

Doxorubicin (DOX) chemotherapy is associated with the release of inflammatory cytokines from macrophages. This has been suggested to be, in part, due to DOX-mediated leakage of endotoxins from gut microflora, which activate Toll-like receptor 4 (TLR4) signaling in macrophages, causing severe inflammation. However, the direct function of DOX on macrophages is still unknown. In the present study, we tested the hypothesis that DOX alone is incapable of stimulating inflammatory response in macrophages. Then, we compared the anti-inflammatory effects of curcumin (CUR), resveratrol (RES) and sulforaphane (SFN) against lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-mediated inflammation in the absence or presence of DOX. For this purpose, RAW 264.7 cells were stimulated with LPS/IFN-γ (10 ng/mL/10 U/mL) in the absence or presence of DOX (0.1 µM). Our results showed that DOX alone is incapable of stimulating an inflammatory response in RAW 264.7 macrophages. Furthermore, after 24 h of incubation with LPS/IFN-γ, a significant increase in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) mRNA levels was observed. Similarly, nitric oxide (NO) production and TNF-α and IL-6 protein levels were significantly upregulated. Moreover, in LPS/IFN-γ-treated macrophages, the microRNAs (miRNAs) miR-146a, miR-155, and miR-21 were significantly overexpressed. Interestingly, upon testing CUR, RES, and SFN against LPS/IFN-γ-mediated inflammation, only SFN was able to significantly reverse the LPS/IFN-γ-mediated induction of iNOS, TNF-α and IL-6 and attenuate miR-146a and miR-155 levels. In conclusion, SFN, at the transcriptional and posttranscriptional levels, exhibits potent immunomodulatory action against LPS/IFN-γ-stimulated macrophages, which may indicate SFN as a potential treatment for DOX-associated inflammation.

Development of Pomegranate Extract-Loaded Solid Lipid Nanoparticles: Quality by Design Approach to Screen the Variables Affecting the Quality Attributes and Characterization.

The aim of this work was to study the influence of process variables on the quality attributes of pomegranate extract loaded solid lipid nanoparticles (PE-SLNs) using Plackett-Burman design. PE-SLN formulations were prepared by hot homogenization followed by ultra-sonication technique and evaluated based on the dependent variables that were analyzed utilizing Statgraphics Centurion XV software. The lipid and surfactant (type and concentration), co-surfactant concentration, sonication time, and amplitude were selected as the independent variables (X 1-X 7). The dependent parameters were particle size, polydispersity index, zeta potential, entrapment efficiency, and cumulative drug release (Y 1-Y 5). Response surface plots, Pareto charts, and mathematical equations were generated to study the influence of independent variables on the dependent quality parameters. Out of seven variables, X 1, X 2, and X 6 have the main significant (p value < 0.05) effect on the entrapment efficiency, the cumulative drug release, the polydispersity index, respectively, while particle size was mainly affected by X 3, X 6 and zeta potential by X 1, X 3, and X 4. Consequently, this screening study revealed that stearic acid as lipid, Tween 80 as surfactant, as well as sonication with short time and high amplitude can be selected for the development of PE-SLN formulation with minimum particle size, maximum zeta potential, highest entrapment, and sustained drug release behavior. Meanwhile, concentrations of lipid, surfactant, and co-surfactant are planned to be scaled up for further optimization study. In conclusion, the Plackett-Burman design verified its influence and significance in determining and understanding both process and formulation variables affecting the quality of PE-SLNs.

Dual Targeting of VEGFR2 and C-Met Kinases via the Design and Synthesis of Substituted 3-(Triazolo-thiadiazin-3-yl)indolin-2-one Derivatives as Angiogenesis Inhibitors.

The vascular endothelial growth factor receptor 2 (VEGFR2) and c-mesenchymal epithelial transition factor (c-Met) are members of receptor tyrosine kinases which have a crucial role in the process of angiogenesis. Isatin moiety is a versatile group that is shared in many compounds targeting both c-Met and VEGFR2 kinases. In this study, we designed and synthesized different derivatives of substituted 3-(triazolo-thiadiazin-3-yl)indolin-2-one derivatives (6a-y) as dual inhibitors for c-Met and VEGFR2 enzymes. Eight compounds 6a, 6b, 6e, 6l, 6n, 6r, 6v, and 6y were assessed for their anticancer activities against a panel of 58 cancer cell lines according to the US-NCI protocol. Compound 6b revealed the most effective antiproliferative potency (GI %), with broad-spectrum activity against different subpanels of the most NCI 58 tumor cell lines. An in vivo hen's egg-chorioallantoic membrane (HET-CAM) angiogenic study was carried out for 21 compounds 6a, b, d, f, h, i, k-o, t, and 6x to check their mortality and toxicity. At 100 µM concentration, all compounds produced 100% mortality of the chick embryos. At 40 µM concentration, 13 compounds did not exhibit any detectable mortality (nontoxic) and revealed a potent antiangiogenic effect. Seven compounds 6b, 6d, 6f, 6n, 6o, 6t, and 6x significantly decreased the number of blood vessels, and compound 6b was the most effective antiangiogenic agent comparable to dexamethasone. Molecular docking studies were conducted for compound 6b to investigate its mode of interaction within the binding site of both c-Met and VEGFR2 kinases.

A Validated Reverse Phase-Ultra-Performance Liquid Chromatography Method for the Determination of Gemifloxacin Mesylate in Bulk and its Pharmaceutical Preparation.

OBJECTIVES: Gemifloxacin Mesylate is a fourth generation fluoroquinolone antibacterial agent. A simple, accurate, and precise reversed phase (RP)-ultra performance liquid chromatography (UPLC) method was developed and validated for short time analysis of Gemifloxacin Mesylate in its bulk and pharmaceutical preparation. MATERIALS AND METHODS: The optimum separation was achieved at 0.5±0.03 min using an AcclaimTM RSLC 120 C18 column 2.2 µm (2.1×100 mm) at 30°C by isocratic mobile phase at pH 3.0 composed of acetonitrile:phosphate buffer (25 mM) in a ratio of 75:25 (v/v). The column effluents were monitored at 276 nm using a photodiode array detector at a flow rate of 0.5 mL/min. The method was validated according to International Conference on Harmonization guidelines. RESULTS: The linearity of the calibration curve ranged from 0.5 µg/mL to 10 µg/mL and the square of the regression coefficient (r2) was 0.9991. The % relative standard deviation (RSD) of inter-day precision ranged from 0.081% to 1.233%, while for intra-day it ranged from 0.364% to 1.018%. The method was accurate with % recovery ranging from 93.71% to 100.29% and % RSD ranging from 1.054 to 2.722. The limit of detection and the limit of quantification were 0.066 and 0.2 µg/mL, respectively. CONCLUSION: The validated method proved its ability for the assay of Gemifloxacin Mesylate in its bulk and dosage form in a short time (less than 1 min). To the best of our knowledge, this is the first RP-UPLC method for the determination of Gemifloxacin Mesylate.

Pomegranate extract-loaded solid lipid nanoparticles: design, optimization, and in vitro cytotoxicity study.

BACKGROUND: Pomegranate extract (PE) is a natural product with potent antioxidant and anticancer activity because of its polyphenols content. The main purpose of this study was to maximize the PE chemotherapeutic efficacy by loading it in an optimized solid lipid nanoparticles (SLNs) formula. MATERIALS AND METHODS: The influence of independent variables, which were lipid concentration (X1), surfactant concentration (X2) and cosurfactant concentration (X3), on dependent ones, which were particle size (Y1), polydispersity index (Y2), zeta potential (Y3), entrapment efficiency (Y4) and cumulative % drug release (Y5), were studied and optimized using the Box-Behnken design. Fifteen formulations of PE-SLNs were prepared using hot homogenization followed by ultra-sonication technique. Response surface plots, Pareto charts and mathematical equations were produced to study the impact of independent variables on the dependent quality parameters. The anti-proliferative activity of the optimized formula was then evaluated in three different cancer cell lines, namely, MCF-7, PC-3 and HepG-2, in addition to one normal cell line, HFB-4. RESULTS: The results demonstrated that the particle sizes ranged from 407.5 to 651.9 nm and the entrapment efficiencies ranged from 56.02 to 65.23%. Interestingly, the 50% inhibitory concentration of the optimized formula had more than a 40-fold improved effect on the cell growth inhibition in comparison with its free counterpart. Furthermore, it was more selective against cancer cells than normal cells particularly in MCF-7 breast cancer cells. CONCLUSION: These data proved that nanoencapsulation of PE enhanced its anticancer efficacy. Therefore, our results suggested that a PE-loaded SLNs optimized-formula could be a promising chemo therapeutic agent.

Modulation of NAD(P)H:quinone oxidoreductase by vanadium in human hepatoma HepG2 cells.

Recent studies demonstrated the carcinogenicity and the mutagenicity of vanadium compounds. In addition, vanadium (V(5+)) was found to enhance the effects of other genotoxic agents. However, the mechanism by which V(5+) induce toxicity remain unknown. In the current study we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in human hepatoma HepG2 cells. Therefore, HepG2 cells were treated with increasing concentrations of V(5+) in the presence of two NQO1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V(5+) inhibited the TCDD- and SUL-mediated induction of NQO1 at mRNA, protein and activity levels. Investigating the effect of V(5+) at transcriptional levels revealed that V(5+) significantly inhibited the TCDD- and SUL-mediated induction of antioxidant responsive element (ARE)-dependent luciferase reporter gene expression. In addition, V(5+) was able to decrease the TCDD- and SUL-induced nuclear accumulation of nuclear factor erythroid 2-related factor-2 (Nrf2) without affecting Nrf2 mRNA or protein levels. Looking at the post-transcriptional level, V(5+) did not affect NQO1 mRNA stability, thus eliminating the possible role of V(5+) in decreasing NQO1 mRNA levels through this mechanism. In contrast, at post-translational level, V(5+) was able to significantly decrease NQO1 protein half-life. The present study demonstrates for the first time that V(5+) down-regulates NQO1 at the transcriptional and post-translational levels in the human hepatoma HepG2 cells via AhR- and Nrf2-dependent mechanisms.