Background: Colorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy. Evaluation of natural product-based anticancer drugs as adjuvant treatment with fewer side effects is largely unexplored research fields. Herbal melanin (HM) is an extract of the seed coats of Nigella sativa that modulates an inflammatory response through toll-like receptor 4 (TLR4). This TLR4 receptor is also involved in the modulation of apoptosis. We therefore explored the anticancer potential of HM and specifically its effect on the molecular mechanisms underlying adenocarcinoma and metastatic colorectal cancer (mCRC) cell death in vitro. Methods: Cell viability was evaluated using the MTT assay. Cellular reactive oxygen species (ROS), glutathione levels, and apoptotic status were assessed using fluorometric and colorimetric detection methods. HM-induced apoptotic and other signaling pathways were investigated using Western blot technology and mitochondrial transition pore assay kit. TLR4 receptor downregulation and blockade were performed using siRNA technology and neutralizing antibody, respectively. Results: Our results showed that HM inhibited the proliferation of the colorectal adenocarcinoma HT29 and mCRC SW620 cell lines. Furthermore, HM enhanced ROS production and decreased glutathione levels. HM-induced apoptosis was associated with mitochondrial outer membrane permeability and cytochrome c release, inhibition of the Bcl2 family proteins, and activation of caspase-3/-7. In addition, HM modulated MAPK pathways by activating the JNK pathway and by inhibiting ERK phosphorylation. TLR4 receptor downregulation enhanced HM-induced apoptosis while TLR4 receptor blockade partially alleviated HM-inhibited ERK phosphorylation. Conclusion: Altogether, these findings indicate that HM exerts pro-apoptotic effects and inhibits MAPK pathway through TLR4 in mCRC and colorectal adenocarcinoma cells, suggesting HM as a promising natural-based drug for the treatment of colorectal cancer.

The prevalence of nosocomial infections due to multidrug resistant (MDR) bacterial strains is associated with high morbidity and mortality. Folk medicine and ethnopharmacological data can provide a broad range of plants with promising antimicrobial activity. Triphala, an Ayurvedic formula composed of three different plants: Terminalia chebula Retz., Terminalia bellirica (Gaertn.) Roxb. (Combretaceae), and Phyllanthus emblica L. (Phyllanthaceae), is used widely for various microbial infections. Various extraction techniques were applied in the extraction of the biologically active constituents of Triphala in order to compare their efficiency. Microwave‐assisted extraction (MAE) was shown to be the most efficient method based on yield, extraction time, and selectivity. The Triphala hydroalcoholic extract (TAE) has been chemically characterized with spectroscopic and chromatographic techniques. Triphala hydroalcoholic extract was evaluated alone or with carvacrol. Different drug formulations including cream and nanoemulsion hydrogel were prepared to assess the antimicrobial activity against selected microorganism strains including Gram‐positive and Gram‐negative bacteria and fungi. We used a lipophilic oil of carvacrol (5 mg/mL) and a hydrophilic TAE (5 mg/mL) ingredient in a dosage form. Two solutions were created: hydrogel containing nanoemulsion as a lipophilic vector dispersed in the gel as a hydrophilic vehicle and a cream formulation, an oil‐in‐water emulsion. In both cases, the concentration was 250 mg of active ingredient in 50 mL of final formulation. The formulas developed were stable from a physical and chemical perspective. In the nanoemulsion hydrogel, the oil droplet size ranged from 124 to 129 nm, with low polydispersity index (PdI) 0.132 ± 0.013 and negative zeta potential −46.4 ± 4.3 mV. For the cream, the consistency factor (cetyl alcohol and white wax) induced immobilization of the matrix structure and the stability. Triphala hydroalcoholic extract in drug nanoformulation illustrated might be an adjuvant antimicrobial agent for treating various microbial infections.

E. coli is an Enterobacteriaceae that could develop resistance to various antibiotics and become a multi-drug resistant (MDR) bacterium. Options for treating MDR E. coli are limited and the pipeline is somewhat dry when it comes to antibiotics for MDR bacteria, so we aimed to explore more options to help in treating MDR E. coli. The purpose of this study is to examine the synergistic effect of a liposomal formulations of co-encapsulated azithromycin and N-acetylcysteine against E. coli. Liposomal azithromycin (LA) and liposomal azithromycin/N-acetylcysteine (LAN) were compared to free azithromycin. A broth dilution was used to measure the MIC and MBC of both formulations. The biofilm reduction activity, thermal stability measurements, stability studies, and cell toxicity analysis were performed. LA and LAN effectively reduced the MIC of E. coli SA10 strain, to 3 μg/ml and 2.5 μg/ml respectively. LAN at 1 × MIC recorded a 93.22% effectiveness in reducing an E. coli SA10 biofilm. The LA and LAN formulations were also structurally stable to 212 ± 2 °C and 198 ± 3 °C, respectively. In biological conditions, the formulations were largely stable in PBS conditions; however, they illustrated limited stability in sputum and plasma. We conclude that the formulation presented could be a promising therapy for E. coli resistance circumstances, providing the stability conditions have been enhanced.

The aggressive triple-negative breast cancer (TNBC) is a challenging disease due to the absence of tailored therapy. The search for new therapies involves intensive research focusing on natural sources. Achillea fragrantissima (A. fragrantissima) is a traditional medicine from the Middle East region. Various solvent extracts from different A. fragrantissima plant parts, including flowers, leaves, and roots, were tested on TNBC MDA-MB-231 cells. Using liquid chromatography, the fingerprinting revealed rich and diverse compositions for A. fragrantissima plant parts using polar to non-polar solvent extracts indicating possible differences in bioactivities. Using the CellTiter-Glo™ viability assay, the half-maximal inhibitory concentration (IC₅₀) values were determined for each extract and ranged from 32.4 to 161.7 µg/mL. The A. fragrantissima flower dichloromethane extract had the lowest mean IC₅₀ value and was chosen for further investigation. Upon treatment with increasing A. fragrantissima flower dichloromethane extract concentrations, the MDA-MB-231 cells displayed, in a dose-dependent manner, enhanced morphological and biochemical hallmarks of apoptosis, including cell shrinkage, phosphatidylserine exposure, caspase activity, and mitochondrial outer membrane permeabilization, assessed using phase-contrast microscopy, fluorescence-activated single-cell sorting analysis, Image-iT™ live caspase, and mitochondrial transition pore opening activity, respectively. Anticancer target prediction and molecular docking studies revealed the inhibitory activity of a few A. fragrantissima flower dichloromethane extract-derived metabolites against carbonic anhydrase IX, an enzyme reported for its anti-apoptotic properties. In conclusion, these findings suggest promising therapeutic values of the A. fragrantissima flower dichloromethane extract against TNBC development.

Electron-rich, nitrogenous heteroaromatic compounds interact more with biological/cellular components than their non-nitrogenous counterparts. The strong intermolecular interactions with proteins, enzymes, and receptors confer significant biological and therapeutic properties to the imidazole derivatives, giving rise to a well-known and extensively used range of therapeutic drugs used for infections, inflammation, and cancer, to name a few. The current study investigates the anti-cancer properties of fourteen previously synthesized nitrogenous heterocycles, derivatives of imidazole and oxazolone, on a panel of cancer cell lines and, in addition, predicts the molecular interactions, pharmacokinetic and safety profiles of these compounds. Method: The MTT and CellTiter-Glo^® assays were used to screen the imidazole and oxazolone derivatives on six cancer cell lines: HL60, MDA-MB-321, KAIMRC1, KMIRC2, MCF-10A, and HCT8. Subsequently, in vitro tubulin staining and imaging were performed, and the level of apoptosis was measured using the Promega ApoTox-Glo^® triplex assay. Furthermore, several computational tools were utilized to investigate the pharmacokinetics and safety profile, including PASS Online, SEA Search, the QikProp tool, SwissADME, ProTox-II, and an in silico molecular docking study on tubulin to identify the critical molecular interactions. Results: In vitro analysis identified compounds 8 and 9 to possess the most significant potent cytotoxic activity on the HL60 and MDA-MB-231 cell lines, supported by PASS Online anti-cancer predictions with pa scores of 0.413 and 0.434, respectively. In addition, compound 9 induced caspase 3/7 dependent-apoptosis and interfered with tubulin polymerization in the MDA-MB-231 cell line, consistent with in silico docking results, identifying binding similarity to the native ligand colchicine. All the derivatives, including compounds 8 and 9, had acceptable pharmacokinetics; however, the safety profile was suboptimal for all the tested derivates except compound 4. Conclusion: The imidazole derivative compound 9 is a promising anti-cancer agent that switches on caspase-dependent apoptotic cell death and modulates microtubule function. Therefore, it could be a lead compound for further drug optimization and development.

Rhei rhizome (Rheum officinale Baill.) (RR) contains a large number of anthraquinone bioactive, yet little is known of the combined effect of these anthraquinones in a mixture. The goals of this study were: to determine the inhibitory potencies of individual anthraquinones and whole RR extract against human liver microsomal CYP1A2/3A4 activity, to predict the content of anthraquinones in RR using the concentration addition (CA) model, and to compare predicted and empirical contents in the same RR sample. Anthraquinone concentrations in the RR extract were determined using HPLC. The inhibitory potencies of individual anthraquinones were determined in incubations containing human liver microsomes. The study results were used to predict an effect-based dose measure of the anthraquinones in RR using the CA model. An empirical dose measure also was determined in the whole RR extract using the CYP1A2/3A4-based bioassay. For the CYP1A2-based studies, the predicted and empirical dose measures of anthraquinones were identical; they were 12.0 ± 1.80 and 12.20 ± 0.81 mg aloe-emodin equivalents/g RR, respectively. For the CYP3A4-based studies, the predicted and empirical dose measures were different; they were 2.80 ± 0.10 and 19.04 ± 0.41 mg aloe-emodin equivalents/g RR, respectively. Only the CYP1A2-based CA model which assumed additive effects of RR anthraquinones predicted an effect-based dose measure that was verifiable by empirical data. The CA model provides an alternative approach to the CYP1A2/3A4-based bioassay or empirical method to screen for the anthraquinones in RR. The CA model as described in this study is applicable to other botanical drugs, plant-based foods and dietary supplements.