Polarity guided extraction,HPLC based phytochemical quantification,and multimode biological evaluation of Otostegia limbata (Benth.) Boiss

Purpose of studyOtostegia limbata (Benth.) Boiss. (Family: Lamiacae) is an important underexplored ethnomedicinal plant that has been used as antinflammatory, anticancer and antibacterial herbal remedy previously. The present work was aimed to evaluate the antioxidant, antimicrobial, antileishmanial, and anticancer prospective of O. limbata stem and leaf extracts.ResultsThe highest amount of phenolic and flavonoid content was obtained in the methanol-acetone and methanol stem extracts i.e., 53.29 ± 1.33 and 28.64 ± 1.16, respectively with highest DPPH scavenging in MeH stem extract (IC₅₀ = 34.5 ± 1.34 μg/ml). Significant amount of catechin, gallic acid, apigenin and rutin was quantified. A moderate antibacterial and substantial antifungal activity was observed. Cytotoxicity against brine shrimps categorized 21% of stem (3 out of 14 extracts) and 57% (8 out of 14 extracts) of leaf extracts as potent. Substantial cytotoxicity against THP-1 cell line (IC₅₀ = 3.46 ± 0.25 μg/ml) and Leishmania (IC₅₀ = 1.50 ± 0.23 μg/ml) was exhibited by methanol-distilled water leaf extract while noteworthy antiproliferative activity against Hep-G2 (IC₅₀ = 0.44 ± 0.45 μg/ml) was manifested by n-hexane stem extract. Absence of hemolysis in normal RBCs signified plant’s selective cytotoxicity. Methanol-distilled water and chloroform stem extracts displayed prominent protein kinase inhibition and antidiabetic potential of plant.ConclusionThe results of present study recommend O. limbata as a potential source of antifungal, antileishmanial, anticancer, and α-amylase inhibitory agents.     

Simultaneous determination of four sedative-hypnotics in human urine based on dendritic structured magnetic nanomaterials

A magnetic solid-phase extraction technique based on magnetic dendritic structured nanoparticles (Fe₃O₄@SiO₂-NH₂-G5) as adsorbent coupled with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) has been developed to detect diazepam, midazolam, zolpidem, and zaleplon in human urine. With Fe₃O₄@SiO₂-NH₂ as the central core, dendrimer (G5) grafted alternately with cyanuric chloride and imidazole were bonded to the surface of the core to synthesize Fe₃O₄@SiO₂-NH₂-G5. The morphology and structure of the magnetic materials were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), and dynamic light scattering (DLS). The key parameters affecting the extraction efficiency were optimized. A satisfactory performance was obtained under the optimum extraction conditions. The proposed method was validated, and the limits of detection of zaleplon, diazepam, zolpidem, and midazolam were 0.05, 0.05, 0.02, and 0.02 ng mL^?1, respectively. The linear correlation coefficients r of the four analytes were > 0.996, the intra-day precision was between 2.4 % and 9.4 % with the recoveries between 88.3 % and 104.8 %, and the inter-day precision ranged from 3.9 % to 15.2 % with the recovery in the range of 94.1 %?108.3 %. The magnetic dendritic structure nanomaterial Fe₃O₄@SiO₂-NH₂-G5 was successfully used to extract sedative-hypnotic drugs from human urine samples. The Fe₃O₄@SiO₂-NH₂-G5-based magnetic solid-phase extraction method eliminates centrifugation and filtration steps as in conventional extraction. Only one step of vortex dispersion extraction could achieve the separation and purification of the target compounds. The proposed method was simple, rapid, environment-friendly, and suitable for the analysis of sedative-hypnotic drugs in human urine.     

Atomistic De-novo Inhibitor Generation-Guided Drug Repurposing for SARS-CoV-2 Spike Protein with Free-Energy Validation by Well-Tempered Metadynamics

Computational drug design is increasingly becoming important with new and unforeseen diseases like COVID-19. In this study, we present a new computational de novo drug design and repurposing method and applied it to find plausible drug candidates for the receptor binding domain (RBD) of SARS-CoV-2 (COVID-19). Our study comprises three steps: atom-by-atom generation of new molecules around a receptor, structural similarity mapping to existing approved and investigational drugs, and validation of their binding strengths to the viral spike proteins based on rigorous all-atom, explicit-water well-tempered metadynamics free energy calculations. By choosing the receptor binding domain of the viral spike protein, we showed that some of our new molecules and some of the repurposable drugs have stronger binding to RBD than hACE2. To validate our approach, we also calculated the free energy of hACE2 and RBD, and found it to be in an excellent agreement with experiments. These pool of drugs will allow strategic repurposing against COVID-19 for a particular prevailing conditions.     

An Integrative Approach of an In Vitro Measurement of the Digestibility of Triacylglycerols of Human Milk

Several studies have been published regarding the effect of different factors on the digestion of milk lipids, considering their natural structural arrangement as milk fat globules and the efficiency of the digestive enzymes in the lipolysis of such complex structures. During digestion, the lipolytic products are dispersed in vesicles and micelles, which are the source for absorption of digested lipids. Therefore, it is necessary to consider the isolation of the micellar phase from the digesta to appropriately determine the amounts and classes of lipids which are bioaccessible. This study presents an integrative approach that included an isolation procedure to separate the micellar fraction from undigested and non-micellar parts, and the distribution of digested milk lipids in micelles determined directly through chromatographic techniques. Four groups of five full term mothers donated colostrum or mature milk. Two sets of samples were analyzed directly (raw), and two sets were pasteurized and then analyzed. Our data revealed that the profile of digested milk lipids is different depending on the lactation period and processing stage, while the carbon atom number distribution of the digested triacylglycerols in the micellar fraction provides a substantial information regarding the acylglycerols species that are less available for absorption.     

Determination of Cytotoxic Activity of Sanguinaria canadensis Extracts against Human Melanoma Cells and Comparison of Their Cytotoxicity with Cytotoxicity of Some Anticancer Drugs

Melanoma is an enormous global health burden, and should be effectively addressed with better therapeutic strategies. Therefore, new therapeutic agents are needed for the management of this disease. The aim of this study was the investigation of cytotoxic activity of some isoquinoline alkaloid standards and extracts obtained from Sanguinaria canadensis—collected before, during, and after flowering—against three different human melanoma cells (A375, G361, SK-MEL-3). The cytotoxicity of these extracts was not previously tested on these melanoma cell lines. Determination of alkaloid contents was performed by HPLC-DAD using Polar RP column and mobile phase containing acetonitrile, water, and 1-butyl-3-methylimidazolium tetrafluoroborate. The cytotoxicity of alkaloid standards was investigated by determination of cell viability and calculation of IC₅₀ values. Significant differences were observed in the alkaloids content and cytotoxic activity of the extracts, depending on the season of collection of the plant material. In the Sanguinaria canadensis extracts high contents of sanguinarine (from 4.8543 to 9.5899 mg/g of dry plant material) and chelerythrine (from 42.7224 to 6.8722 mg/g of dry plant material) were found. For both of these alkaloids, very high cytotoxic activity against the tested cell lines were observed. The IC₅₀ values were in the range of 0.11–0.54 µg/mL for sanguinarine and 0.14 to 0.46 µg/mL for chelerythrine. IC₅₀ values obtained for Sanguinaria canadensis extracts against all tested cell lines were also very low (from 0.88 to 10.96 µg/mL). Cytotoxic activity of alkaloid standards and Sanguinaria canadensis extracts were compared with the cytotoxicity of anticancer drugs—etoposide, cisplatin, and hydroxyurea. In all cases except the one obtained for cisplatin against A375, which was similar to that obtained for Sanguinaria canadensis after flowering against the same cell line, IC₅₀ values obtained for anticancer drugs were higher than the IC₅₀ values obtained for sanguinarine, chelerythrine, and Sanguinaria canadensis extracts. Our results showed that Sanguinaria canadensis extracts and isoquinoline alkaloids, especially sanguinarine and chelerythrine, could be recommended for further in vivo experiments in order to confirm the possibility of their application in the treatment of human melanomas.     

Searching for Potential Markers of Glomerulopathy in Urine by HS-SPME-GC×GC TOFMS

Volatile organic compounds (VOCs) exiting in urine are potential biomarkers of chronic kidney diseases. Headspace solid phase microextraction (HS-SPME) was applied for extraction VOCs over the urine samples. Volatile metabolites were separated and identified by means of two-dimensional gas chromatography and time of flight mass spectrometry (GC × GC TOF MS). Patients with glomerular diseases (n = 27) and healthy controls (n = 20) were recruited in the study. Different VOCs profiles were obtained from patients and control. Developed methodology offers the opportunity to examine the metabolic profile associated with glomerulopathy. Four compounds found in elevated amounts in the patients group, i.e., methyl hexadecanoate; 9-hexadecen-1-ol; 6,10-dimethyl-5,9-undecadien-2-one and 2-pentanone were proposed as markers of glomerular diseases.     

Garcinielliptone G from Garcinia subelliptica Induces Apoptosis in Acute Leukemia Cells

Cytotoxicity and apoptosis-inducing properties of compounds isolated from Garcinia subelliptica leaves were investigated. The hexane-soluble portion of MeOH extracts of G. subelliptica leaves that showed cytotoxic activity was separated to yield seven compounds 1–7. Chemical structure analysis using NMR spectroscopy and mass spectrometry confirmed that compound 1 was canophyllol, and compounds 2–7 were garcinielliptones N, O, J, G, F, and garcinielliptin oxide, respectively. Among them, garcinielliptone G (5) showed growth inhibition by causing apoptosis in THP-1 and Jurkat cells derived from human acute monocytic leukemia and T lymphocyte cells, respectively. Apoptosis induced by garcinielliptone G (5) was demonstrated by the detection of early apoptotic cells with fluorescein-labeled Annexin V and increases in cleaved caspase-3 and cleaved PARP protein levels. However, the addition of caspase inhibitor Z-VAD-FMK did not affect growth arrest or apoptosis induction. These results suggest that garcinielliptone G (5) can induce both caspase-3 activation and caspase-independent apoptosis. Therefore, garcinielliptone G (5) may be a potential candidate for acute leukemia treatment.     

Role of Gut Microbiota,Probiotics and Prebiotics in the Cardiovascular Diseases

In recent years, there has been a growing interest in identifying and applying new, naturally occurring molecules that promote health. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer health benefits on the host”. Quite a few fermented products serve as the source of probiotic strains, with many factors influencing the effectiveness of probiotics, including interactions of probiotic bacteria with the host’s microbiome. Prebiotics contain no microorganisms, only substances which stimulate their growth. Prebiotics can be obtained from various sources, including breast milk, soybeans, and raw oats, however, the most popular prebiotics are the oligosaccharides contained in plants. Recent research increasingly claims that probiotics and prebiotics alleviate many disorders related to the immune system, cancer metastasis, type 2 diabetes, and obesity. However, little is known about the role of these supplements as important dietary components in preventing or treating cardiovascular disease. Still, some reports and clinical studies were conducted, offering new ways of treatment. Therefore, the aim of this review is to discuss the roles of gut microbiota, probiotics, and prebiotics interventions in the prevention and treatment of cardiovascular disease.