The anti­hepatitis B virus activity of sea buckthorn is attributed to quercetin, kaempferol and isorhamnetin.

The present study assessed the in vitro anti-hepatitis B virus (HBV) effects of cold-adapted sea buckthorn (Hippophae rhamnoides). Sea buckthorn leaf ethanol extracts subjected to chloroform (SB-Chl), ethyl acetate (SB-Eac), n-butanol (SB-But) and aqueous (SB-Aqu) fractionation were first examined (MTT assay) for their toxic effects on HepG2 cells. While SB-Chl (IC50, 32.58 µg/ml) exhibited high cytotoxicity, SB-Eac, SB-But SB-Aqu were non-toxic at up to 150 µg/ml. High performance liquid chromatography analysis led to the identification of the anti-HBV active flavonols, quercetin (93.09 µg/g), kaempferol (44.19 µg/g) and isorhamnetin (138.75 µg/g) in the extract. The analysis of the anti-HBV effects of SB-Eac, SB-But and SB-Aqu (50 µg/ml, each) on HepG2.2.15 cells revealed the marked inhibition of HBsAg and HBeAg expression levels. At the concentration of 10 µg/ml, quercetin and kaempferol exerted potent inhibitory effects on HBsAg (60.5 and 62.3%, respectively) and HBeAg synthesis (64.4 and 60.2%, respectively), as compared to isorhamnetin (30.5 and 28.4%, respectively). The HBV-polymerase inhibitor drug, lamivudine (2 µM), inhibited HBsAg and HBeAg expression by 87.4 and 83.5%, respectively. The data were in good agreement with a previous in vitro and in silico molecular docking analysis performed by the authors where quercetin, kaempferol and lamivudine had formed stable complexes with HBV-polymerase binding-pocket amino acids. On the whole, to the best of our knowledge, the present study provides the first report of the anti-HBV therapeutic potential of sea buckthorn, attributed to quercetin, kaempferol and isorhamnetin.

Response Surface Methodology (RSM)-Based Optimization of Ultrasound-Assisted Extraction of Sennoside A, Sennoside B, Aloe-Emodin, Emodin, and Chrysophanol from Senna alexandrina (Aerial Parts): HPLC-UV and Antioxidant Analysis.

In this study, ultrasound-assisted extraction conditions were optimized to maximize the yields of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol from S. alexandrina (aerial parts). The three UAE factors, extraction temperature (S1), extraction time (S2), and liquid to solid ratio (S3), were optimized using response surface methodology (RSM). A Box-Behnken design was used for experimental design and phytoconstituent analysis was performed using high-performance liquid chromatography-UV. The optimal extraction conditions were found to be a 64.2 °C extraction temperature, 52.1 min extraction time, and 25.2 mL/g liquid to solid ratio. The experimental values of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol (2.237, 12.792, 2.457, 0.261, and 1.529%, respectively) agreed with those predicted (2.152, 12.031, 2.331, 0.214, and 1.411%, respectively) by RSM models, thus demonstrating the appropriateness of the model used and the accomplishment of RSM in optimizing the extraction conditions. Excellent antioxidant properties were exhibited by S. alexandrina methanol extract obtained using the optimized extraction conditions with a DPPH assay (IC50 = 59.7 ± 1.93, µg/mL) and ABTS method (47.2 ± 1.40, µg/mL) compared to standard ascorbic acid.

Nephroprotective effect of persimmon leaves (Diospyros kaki L.f.) against CCl4-induced renal toxicity in Swiss Albino rats.

Diospyros kaki L.f. fruit and leaves are traditionally used for the treatment of hypertension, angina, internal hemorrhage, antithrombotic and anti-inflammatory effects.In the current study, the protective effects of ethyl acetate (Per-1), n-butanol (Per-2), and aqueous (Per-3) fractions of Diospyros kaki leaves against carbon tetrachloride (CCl4) induced nephrotoxicity in Swiss albino rats were tested. Animal were divided into nine groups; each group consists of six animals. The groups were : group I was untreated and kept as control, group II was treated with CCl4 only, group III (silymarin with CCl4); group IV (Per-1 100 mg/kg with CCl4);group V (Per-1 200 mg/kg with CCl4); group VI (Per-2 100 mg/kg with CCl4); group VII (Per-2 200 mg/kg with CCl4); group VIII (Per-3 100 mg/kg with CCl4); and group IX (Per-3 200 mg/kg with CCl4). Silymarin was used as standard drug. All tested fractions were found active (except Per-1 at low dose of 100 mg/kg) with significant value (p < 0.001) compared to CCl4 only group. Serum creatinine, malondialdehyde (MDA), and uric acid were significantly (p < 0.001) lowered in group VII-IX as compared to CCl4 only group. Similarly, total protein (TP) and non-protein sulfhydryls(NP-SH) level in kidney tissues were significantly (p < 0.001) elevated in the same groups compared to CCl4 only group. Further to check the cardio-protective potential, biochemical parameters such as LDH, creatine kinase, TP, MDA, and NP-SH levels in myocardial tissues were also estimated.These findings confirmed that the n-butanol and aqueous fractions are active and recommended for further bioactive phytoconstituents screening. Repeated column chromatography on silica gel G and sephadex-LH-20 of the active n-butanol fraction, four flavonoids were isolated. Based on the spectroscopic NMR data, compounds were identified as kaempferol (1), quercetin (2), astragalin (3), and rutin (4).

Impact of Different Extraction Methods on Furanosesquiterpenoids Content and Antibacterial Activity of Commiphora myrrha Resin.

The oleo-gum-resin of Commiphora myrrha is one of the most known natural antimicrobial agents, mainly due to its furanosesquiterpenes. A validated method based on sample extraction by matrix solid-phase dispersion (MSPD) followed by high-performance column chromatography (HPLC) determination is applied to analyze two furanosesquiterpenoids, namely, 2-methoxyfuranodiene (CM-1) and 2-acetoxyfuranodiene (CM-2), existing in C. myrrha. The trial parameters that controlled the extraction prospective were studied and optimized. These include the nature of dispersant, mass ratio of sample to the dispersant, and the volume of elution solvent. A comparative antimicrobial study that used the Minimum Inhibitory Concentration Assay (MIC) method between MSPD, ultrasonic, and Soxhlet of myrrh extracts was also conducted. The optimal MSPD parameters used were (i) 15 mL of methanol applied as elution solvent; (ii) silica gel/sample mass at a 2 : 1 ratio; and (iii) a dispersing sorbent selected as silica gel. Technique retrievals were regulated from 96.87% to 100.54%, with relative standard deviations (RSDs) from 1.24% to 4.45%. Commiphora myrrha-MSPD (CM-MSPD) extract showed the highest antibacterial activity against gram-positive and gram-negative bacteria (156.25 µg/mL and 312.5 µg/mL, respectively) and antifungal activity (156.25 µg/mL). Yields acquired through the MSPD technique were larger than yields from other extraction techniques (sonication and traditional reflux extraction methods) with less consumption of time, sample, and solvent. The mode of antibacterial action of CM-1 and CM-2 was elucidated by performing molecular docking with bacterial DNA gyrase. Both the compounds interacted with key residues of DNA gyrase.

Charge-Transfer Complex of Linifanib with 2,3-dichloro-3,5-dicyano-1,4-benzoquinone: Synthesis, Spectroscopic Characterization, Computational Molecular Modelling and Application in the Development of Novel 96-microwell Spectrophotometric Assay.

BACKGROUND: Linifanib (LFB) is a multi-targeted receptor tyrosine kinase inhibitor used in the treatment of hepatocellular carcinoma and other types of cancer. The charge-transfer (CT) interaction of LFB is important in studying its receptor binding mechanisms and useful in the development of a reliable CT-based spectrophotometric assay for LFB in its pharmaceutical formulation to assure its therapeutic benefits. PURPOSE: The aim of this study was to investigate the CT reaction of LFB with 2,3-dichloro-3,5-dicyano-1,4-benzoquinone (DDQ) and its application in the development of a novel 96-microwell spectrophotometric assay for LFB. METHODS: The reaction was investigated, its conditions were optimized, the physicochemical and constants of the CT complex and stoichiometric ratio of the complex were determined. The solid-state LFB-DDQ complex was synthesized and its structure was analyzed by UV-visible, FT-IR, and 1H-NMR spectroscopic techniques, and also by the computational molecular modeling. The reaction was employed in the development of a novel 96-microwell spectrophotometric assay for LFB. RESULTS: The reaction resulted in the formation of a red-colored product, and the spectrophotometric investigations confirmed that the reaction had a CT nature. The molar absorptivity of the complex was linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9526 and 0.9459, respectively. The stoichiometric ratio of LFB:DDQ was 1:2. The spectroscopic and computational data confirmed the sites of interaction on the LFB molecule, and accordingly, the reaction mechanism was postulated. The reaction was utilized in the development of the first 96-microwell spectrophotometric assay for LFB. The assay limits of detection and quantitation were 1.31 and 3.96 µg/well, respectively. The assay was successfully applied to the analysis of LFB in its bulk and tablets with high accuracy and precision. CONCLUSION: The assay is simple, rapid, accurate, eco-friendly as it consumes low volumes of organic solvent, and has high analysis throughput.

Darunavir: A comprehensive profile.

Darunavir: (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl [(2S,3R)-4-{[(4-aminophenyl)sulfonyl] (isobutyl)amino}-3-hydroxy-1-phenyl-2-butanyl]carbamate is a synthetic non-peptide protease inhibitor. On June 2006, it was first approved by the Food and Drug administration (FDA) for treatment of resistant type-1 of the human immunodeficiency virus (HIV). In July 2016, the FDA expanded the approval for use of darunavir in pregnant women with HIV infection. Darunavir prevents the replication of HIV virus by inhibiting the catalytic activity of the HIV-1 protease enzyme, and selectively inhibits the cleavage of HIV encoded Gag-Pol polyproteins in virus-infected cells, which prevents the formation of mature infectious virus particles. Darunavir is unique among currently available protease inhibitors because it maintains antiretroviral activity against a variety of multidrug-resistant HIV strains. This article discusses, by a critical extensive review of the literature, the description of darunavir in terms of its names, formulae, elemental composition, appearance, and use in the treatment of HIV-infected patients. The article also discusses the methods for preparation of darunavir, its physical-chemical properties, analytical methods for its determination, pharmacological properties, and dosing information.

In Vitro Antioxidant, Cytotoxic Activities, and Phenolic Profile of Senecio glaucus from Saudi Arabia.

Current treatments for complex diseases have remarkable side effects that negatively impact patients' quality of life. Thus, natural compounds with fewer side effects represent a promising source for safe drugs. The genus Senecio is widely used in folk medicine due to its various pharmacological properties. In the present study, the total phenolic content of Senecio glaucus, which is grown in Saudi Arabia, was assessed using the Folin-Ciocalteau colorimetric method. Scavenging DPPH and ABTS assays were utilized to determine the antioxidant properties of S. glaucus fractions, and MTT assay was used to screen the cytotoxic activity of S. glaucus against various cancer cells. In addition, HPLC-UV was utilized to detect the presence of two phenolic acids, namely, vanillic acid (VA) and gallic acid (GA). Among all fractions tested, S. glaucus chloroform fraction (SGCF) yielded the highest value (125.3 mg·GA/g) in terms of total phenolic content. SGCF also exhibited the highest scavenging activities (76.7 and 74.1%) on both DPPH and ABTS assays, respectively. Similarly, SGCF also possessed the most potent cytotoxic activity against the MCF-7 cell line, with an IC50 value of 41.8 µg/ml. The validated HPLC method confirmed the presence of VA (4.8 µg/mg DW) and GA (3.9 µg/mg DW) in SGCF. Overall, our data show that S. glaucus had antioxidant and cytotoxic properties. A developed validated HPLC method which could be helpful for quantifying phenolic compounds in S. glaucus was established.

Preparation and Characterization of Two Immunogens and Production of Polyclonal Antibody with High Affinity and Specificity for Darunavir.

Darunavir (DRV) is a potent antiviral drug used for treatment of infections with human immunodeficiency virus (HIV). Effective and safe treatment with DRV requires its therapeutic drug monitoring (TDM) in patient's plasma during therapy. To support TDM of DRV, a specific antibody with high affinity is required in order to develop a sensitive immunoassay for the accurate determination of DRV in plasma. In this study, two new and different immunogens were prepared and characterized. These immunogens were the DRV conjugates with keyhole limpet hemocyanin (KLH) protein. The first immunogen (DRV-KLH) was prepared by zero-length direct linking of DRV via its aromatic amino group with the tyrosine amino acid residues of KLH by diazotization/coupling reaction. The second immunogen (G-DRV-KLH) was prepared by conjugation of the N-glutaryl derivative of DRV (G-DRV) with KLH. The 5-carbon atoms-spacing G-DRV hapten was synthesized by reaction of DRV via its aromatic amino group with glutaric anhydride. The reaction was monitored by HPLC and the chemical structure of G-DRV was confirmed by mass, 1H-NMR, and 13C-NMR spectroscopic techniques. The hapten (G-DRV) was linked to the KLH protein by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling procedure. The pertinence of the coupling reactions of haptens to protein was confirmed, and the immunogens were characterized by ultraviolet (UV) spectrophotometry. Both DRV-KLH and G-DRV-KLH were used for the immunization of animals and the animal's antiserum that showed the highest affinity was selected. The collected antiserum (polyclonal antibody) had very high affinity to DRV (IC50 value = 0.2 ng mL-1; defining IC50 as the DRV concentration that can inhibit antibody binding by 50% of its maximum binding) and high specificity to DRV among other drugs used in the combination therapy with DRV. Cumulative results from direct and competitive enzyme-linked immunosorbent assay (ELISA) using this polyclonal antibody proved that the immunogens were highly antigenic and elicited a specific polyclonal antibody. The produced polyclonal antibody is valuable for the development of highly sensitive and selective immunoassays for TDM of DRV.

Rapid and Sensitive Method for Extraction of Plicosepalus acacia with Determination of Its Main Polyphenolic Compounds Using Validated HPLC.

Matrix solid phase dispersion (MSPD) trailed by HPLC is a quick and fruitful technique utilized for fortitude of flavonoids such as Catechin, Kaempferol, Quercetin, and Rutin existing in P. acacia. The trial parameters that influenced the extraction potential (comprising the mass ratio of sample to the dispersant, nature of dispersant, and the nature of elution solvent and its volume) were examined and optimized. These MSPD optimized parameters regulated are as follows: 8 mL of methanol was utilized as elution solvent, silica gel/sample mass ration was selected to be 2 : 1, and dispersing sorbent was silica gel. The technique retrievals were regulated to be "from 96.87 to 100.54%" and the RSDs from 1.24 to 4.45%. The product of extract obtained by MSPD method was larger than that of other methods, i.e., sonication extraction or traditional reflux with lessened necessities on time, sample, and solvent.

Spectroscopic and molecular docking studies reveal binding characteristics of nazartinib (EGF816) to human serum albumin.

The interactions of novel anti-cancer therapeutic agents with the different plasma and tissue components, specifically serum albumins, have lately gained considerable attention due to the significant influence of such interactions on the pharmacokinetics and/or -dynamics of this important class of therapeutics. Nazartinib (EGF 816; NAZ) is a new anti-cancer candidate proposed as a third-generation epidermal growth factor receptor tyrosine kinase inhibitor that is being developed and clinically tested for the management of non-small cell lung cancer. The current study aimed to characterize the interaction between NAZ and human serum albumin (HSA) using experimental and theoretical approaches. Experimental results of fluorescence quenching of HSA induced by NAZ revealed the development of a statically formed complex between NAZ and HSA. Interpretation of the observed fluorescence data using Stern-Volmer, Lineweaver-Burk and double-log formulae resulted in binding constants for HSA-NAZ complex in the range of (2.34-2.81) × 104 M-1 over the studied temperatures. These computed values were further used to elucidate thermodynamic attributes of the interaction, which showed that NAZ spontaneously binds to HSA with a postulated electrostatic force-driven interaction. This was further verified by theoretical examination of the NAZ docking on the HSA surface that revealed an HSA-NAZ complex where NAZ is bound to HSA Sudlow site I driven by hydrogen bonding in addition to electrostatic forces in the form of pi-H bond. The HSA binding pocket for NAZ was shown to encompass ARG 257, ARG 222, LYS 199 and GLU 292 with a total binding energy of -25.59 kJ mol-1.

Development of a highly sensitive ELISA for determination of darunavir in plasma samples using a polyclonal antibody with high affinity and specificity.

Aim: To support pharmacokinetic studies and therapeutic monitoring of darunavir (DRV), a highly sensitive ELISA was developed for the determination of DRV in plasma samples at picogram levels. Results: The assay LOD and LOQ were 15 and 30 pg ml-1, respectively. The working range of the assay was 20-2000 pg ml-1. Analytical recoveries of DRV from spiked plasma were in the ranges of 98.4-113.0 and 86.0-99.1% for intra-assay and inter-assay runs, respectively. The precision of the assay was satisfactory. Conclusion: The ELISA is characterized by high throughput and it is expected to significantly contribute to routine analysis of DRV in its pharmacokinetic studies and therapeutic monitoring.

Development and validation of a novel UPLC-MS/MS method for quantification of delafloxacin in plasma and aqueous humour for pharmacokinetic analyses.

Acute bacterial skin and skin structure infections are one of the most frequent infectious disease requiring hospitalization for treatment. Delafloxacin is a clinically approved fluoroquinolone antibiotic for the treatment of ABSSSIs. In spite of being marketed since 2017, there is no published analytical method for quantification of delafloxacin in biological samples. Herein, a selective and sensitive UPLC-MS/MS method was developed and validated for quantitative analysis of delafloxacin in rat plasma and rabbit aqueous humour samples. The liquid liquid extraction (using ethyl acetate) was used for analyte extraction form rat plasma, whereas protein precipitation (acetonitrile) was used for aqueous humour samples preparations. An Acquity UPLC BEH C18 column was used for chromatographic separation of delafloxacin and internal standard (rivaroxaban). The mobile phase composition of acetonitrile (containing 0.1% formic acid) and 10 mM ammonium acetate in ratio of 60:40 were used for sample elution at 300 µL/min flow rate. The electrospray ionization operated in positive mode was used for sample ionization and detection of analyte and internal standard were performed by multiple reaction monitoring (MRM) mode. The MRM transitions were set to 441.14 > 379.09 and 436.89 > 144.87 for delafloxacin and internal standard, respectively. The method was validated as per USFDA guideline for bioanalytical method and all the evaluated parameters were within the acceptable ranges. The developed method in plasma was successfully used to analyze samples in pharmacokinetic study of newly developed stearic acid-chitosan solid lipid nanoparticles formulation of delafloxacin in rat.

Antioxidant activities and molecular docking of 2-thioxobenzo[g]quinazoline derivatives.

BACKGROUND: Oxidative stress and related diseases resulting from the overproduction of free radicals can be counteracted by designing and developing novel antioxidative agents that can protect the human body against the damage caused by free radicals. METHODS: The present study evaluated the antioxidant activities of 15 derivatives of 2-thioxobenzo[g]quinazoline using three different assays: 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging, reducing power capability, and ferric reduction antioxidant power. RESULTS: Some benzoquinazolines had good activity and had the capacity to deplete DPPH and free radicals compared to a positive control butylated hydroxyl toluene (BHT). A docking study identified the possible interactions between binding models and the antioxidant activities of the target compounds. CONCLUSIONS: The active compounds can be used as templates for further development of more potent antioxidative agents.

Ultra-performance hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for simultaneous determination of allopurinol, oxypurinol and lesinurad in rat plasma: Application to pharmacokinetic study in rats.

A fixed dose combination of lesinurad and allopurinol has been recently approved by USFDA and EMA for treatment of gout-associated hyperuricemia in patients who have not achieved target serum uric acid levels with allopurinol alone. In this study, an ultra-performance hydrophilic interaction liquid chromatography (UPHILIC) coupled with tandem mass spectrometry method was developed and validated for simultaneous determination of allopurinol, oxypurinol and lesinurad in rat plasma. Liquid liquid extraction using ethyl acetate as extracting agent was used for samples extraction procedure. Acquity UPLC HILIC column (100 mm x 2.1, 1.7µm) was used for separation of allopurinol, oxypurinol, lesinurad and internal standard (5-Florouracil). The mobile phase consisting of acetonitrile, water and formic acid (95:5:0.1, v/v/v), were eluted at 0.3 mL/min flow rate having total chromatographic run time of 3 min per sample. The analytes were detected on Acquity triple quadrupole mass spectrometer equipped with a Z-Spray electrospray ionization (ESI). The ESI source was operated in negative mode and multiple reaction monitoring was used for ion transition for all compounds. The precursor to product ion transition of m/z 134.94 > 64.07 for allopurinol, 150.89 > 41.91 for oxypurinol, 401.90 > 176.79 for lesinurad and 128.85 >41.92 for internal standard were used for identification and quantification. The calibration curves for all analytes were found to be linear with weighing factor of 1/x2 using regression analysis. The developed assay was successfully applied in an oral pharmacokinetic study of allopurinol, oxypurinol and lesinurad in rats.

Novel BTK inhibitor acalabrutinib (ACP-196) tightly binds to site I of the human serum albumin as observed by spectroscopic and computational studies.

Spectrofluorometric, UV-vis spectroscopic and theoretical tools were recruited to comprehend the interaction of acalabrutinib (ACP-196; ACLB) with human serum albumin (HSA). Fluorescence intensity determinations revealed that ACLB statically quenched the HSA-native fluorescence. Analysis of the observed fluorescence data resulting from the ACLB-HSA interaction presented binding constants in the range of 6.65-7.54 × 104 M-1 with the studied temperatures. Those constants showed steady decline with the rising temperatures that further signifies static interaction of the HSA and ACLB. Binding energetics were also interpreted using the fluorescence-recorded results that exhibited a spontaneous exothermic binding reaction with a negative change in Gibbs free energy as well as negative enthalpy and positive entropy changes. Those results suggested the involvement of electrostatic forces as discovered by further computational investigation. Those docking results verified that ACLB binds to domain IIA (site I) of the HSA as demonstrated experimentally by site markers displacement binding studies. Circular dichroism studies along with the synchronous and 3D fluorescence observations showed that ACL binding does not alter the HSA conformation.