Synthesis and molecular docking study of some novel 2,3-disubstituted quinazolin-4(3H)-one derivatives as potent inhibitors of urease

A new series of 2,3-disubstituted quinazolin-4(3H)-one compounds including oxadiazole and furan rings was synthesized. Their inhibitory activities on urease were assessed in vitro. All newly synthesized compounds exhibited potent urease inhibitory activity in the range of IC₅₀ = 1.55 ± 0.07–2.65 ± 0.08 µg/mL, when compared with the standard urease inhibitors such as thiourea (IC₅₀ = 15.08 ± 0.71 µg/mL) and acetohydroxamic acid (IC₅₀ = 21.05 ± 0.96 µg/mL). 2,3-Disubstituted quinazolin-4(3H)-one derivatives containing furan ring (3a-e) were found to be the most active inhibitors when compared with the compounds 2a-e bearing oxadiazole ring. Compound 3a, bearing 4-chloro group on phenyl ring, was found as the most effective inhibitor of urease with the IC₅₀ value of 1.55 ± 0.11 µg/mL. The molecular docking studies of the newly synthesized compounds were performed to identify the probable binding modes in the active site of the Jack bean urease (JBU) enzymes.     

Synthesis and molecular docking study of novel coumarin derivatives containing 4,5-dihydropyrazole moiety as potential antitumor agents

A series of novel coumarin derivatives containing 4,5-dihydropyrazole moiety as potential telomerase inhibitors were synthesized. The bioassay tests show that compound 3d exhibited potentially high activity against human gastric cancer cell SGC-7901 with IC₅₀ value of 2.69 ± 0.60 μg/mL. All title compounds were assayed for telomerase inhibition by a modified TRAP assay, the results show that compounds 3d and 3f can strongly inhibit telomerase with IC₅₀ values of 2.0 ± 0.07 and 1.8 ± 0.35 μM, respectively. Docking simulation was performed to position compound 3d into the telomerase (3DU6) active site to determine the probable binding model.     

LC-ESI-MS/MS Chemical Characterization,Antioxidant and Antidiabetic Properties of Propolis Extracted with Organic Solvents from Eastern Anatolia Region

Geographic conditions (altitude, climate, and local flora) lead to significant differences in the chemical composition of propolis. Therefore, more research is needed for propolis in different geographical regions. So, the aim of this study was to evaluate the phenolic profile, total phenolic content, antioxidant, and antidiabetic properties of Pülümür propolis from Turkey. Methanol (MeOH), chloroform (CHCl₃), and hexane extracts of propolis were analyzed. LC-ESI-MS/MS analysis of the extracts showed that the most abundant phenolic compound is caffeic acid in the MeOH extract (2943.12±11.12 μg phenolics/g extract), while on the other hand, CHCl₃ extract had the highest total phenolic content (125.75±1.02 mg GAE/g extract). Antioxidant activity was measured using ABTS and DPPH assays, whereas CHCl₃ extract (IC₅₀=6.35±0.11 and 28.84±0.10 μg/mL, respectively) and MeOH extracts (IC₅₀=5.04±0.07 and 28.80±0.09 μg/mL, respectively) showed relatively high antioxidant activity. The MeOH extract showed better antidiabetic activity than the standard compound, acarbose (IC₅₀=0.544 and 0.805 mg/mL, respectively).     

Tetrahydroindolocarbazoles (THICZs) as new class of urokinase (uPA) inhibitors: Synthesis,anticancer evaluation,DNA-damage determination,and molecular modelling study

Tetrahydroindolocarbazoles (THICZs) with versatile substituents, have been designed, synthesized, structure characterized, then investigated for their in-vitro anticancer screening, urokinase inhibition (uPA) evaluated, DNA-damage determination was further explored. Compounds 5, 8, 10 and 17 displayed the most promising antitumor activities against the breast cancer cell line as compared to the standard drug, doxorubicin with IC₅₀ = 5.24 ± 0.37, 4.00 ± 0.52, 7.20 ± 0.90 and 9.60 ± 1.10 µg/ml (versus 3.30 ± 0.48 µg/ml for doxorubicin). Compounds 5, 8, 10 and 17 represents the most significant uPA inhibitors of our study with IC₅₀ of 3.80, 2.70. 4.75, 10.80 (ng/ml) respectively. The expression levels of CDKN2A gene were decreased in 8, 10 and 17 cell lines as compared to those in positive control samples. Cell lines treated with 5, 8, 10 and 17 clearly observed a high score of damaged DNA cells. A deeper examination revealed that our hetroaromatics showed an extensive hydrogen bonding interactions that is required in the S pocket which is important for activity Arg 217, Gly 219, Gly 216, Lys 143 and Ser 190. So we present THICZs as promising uPA inhibitors expected as significant promise for further development as anti-invasiveness drugs.     

Synthesis,in vitro urease inhibition and molecular docking studies of some novel quinazolin-4(3H)-one derivatives containing triazole,thiadiazole and thiosemicarbazide functionalities

A new series of quinazolinone derivatives containing triazole, thiadiazole, thiosemicarbazide functionalities was synthesized and then screened for their in vitro urease inhibition properties. Most of the compounds showed excellent activity with IC₅₀ values ranging between 1.88 ± 0.17 and 6.42 ± 0.23 µg/mL, compared to that of thiourea (IC₅₀ = 15.06 ± 0.68) and acetohydroxamic acid (IC₅₀ = 21.03 ± 0.94), as reference inhibitors. Among the synthesized molecules, compounds 5c, 5e and 5a showed the best inhibitory effect against urease enzyme with IC₅₀ values of 1.88 ± 0.17 µg/mL, 1.90 ± 0.10 and 1.96 ± 0.07 µg/mL, respectively. Moreover in order to give better understanding of the inhibitory activity of synthesized compounds, molecular docking studies were applied at the target sites of jack bean urease enzyme (JBU). Their binding poses and energy calculations were analyzed using induced fit docking (IFD) and prime-MMGBSA tool. Binding poses of studied compounds were determined using induced fit docking (IFD) algorithms.     

Synthesis,biological evaluation,and molecular modelling of new naphthalene-chalcone derivatives as potential anticancer agents on MCF-7 breast cancer cells by targeting tubulin colchicine binding site

Abstract

A series of naphthalene-chalcone derivatives (3a–3t) were prepared and evaluated as tubulin polymerisation inhibitor for the treatment of breast cancer. All compounds were evaluated for their antiproliferative activity against MCF-7 cell line. The most of compounds displayed potent antiproliferative activity. Among them, compound 3a displayed the most potent antiproliferative activity with an IC₅₀ value of 1.42?±?0.15?µM, as compared to cisplatin (IC₅₀?=?15.24?±?1.27?µM). Additionally, the promising compound 3a demonstrated relatively lower cytotoxicity on normal cell line (HEK293) compared to tumour cell line. Furthermore, compound 3a was found to induce significant cell cycle arrest at the G₂/M phase and cell apoptosis. Compound 3a displayed potent tubulin polymerisation inhibitory activity with an IC₅₀ value of 8.4?µM, which was slightly more active than the reference compound colchicine (IC₅₀?=?10.6?µM). Molecular docking analysis suggested that 3a interact and bind at the colchicine binding site of the tubulin.     

Treatment with 50000 IU Vitamin D2 Every Other Week and Effect on Serum 25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, and Total 25-Hydroxyvitamin D in Aclinical Setting

ObjectiveTo examine the effect of 50 000 IU-vitamin D₂ supplementation in a clinical setting on serum total 25-hydroxyvitamin D (25[OH]D), 25-hydroxyvitamin D₂ (25[OH]D₂), and 25-hydroxyvitamin D₃ (25[OH]D₃).MethodsThis retrospective cohort study was performed in an urban tertiary referral hospital in Boston, Massachusetts. Patients who had been prescribed 50 000 IU vitamin D₂ repletion and maintenance programs were identified through a search of our electronic medical record. Baseline and follow-up total serum 25(OH)D, 25(OH)D₂, and 25(OH)D₃ levels were compared.ResultsWe examined the medical records of 48 patients who had been prescribed 50 000 IU vitamin D₂ in our clinic. Mean ± standard deviation baseline total 25(OH) D was 31.0 ± 10.6 ng/mL and rose to 48.3 ± 13.4 ng/mL after treatment (P <.001). 25(OH)D₂ increased from 4.2 ± 4.3 ng/mL to 34.6 ± 12.3 ng/mL after treatment (P <.001), for an average of 158 days (range, 35-735 days). Serum 25(OH)D3 decreased from 26.8 ± 10.8 ng/mL to 13.7 ± 7.9 ng/mL (P <.001).ConclusionsFifty thousand IU vitamin D₂ repletion and maintenance therapy substantially increases total 25(OH)D and 25(OH)D2 despite a decrease in serum 25(OH)D3. This treatment program is an appropriate and effective strategy to treat and prevent vitamin D deficiency.(Endocr Pract. 2012;18:399-402)     

Design,synthesis, docking study, α-glucosidase inhibition,and cytotoxic activities of acridine linked to thioacetamides as novel agents in treatment of type 2 diabetes

A novel series of acridine linked to thioacetamides 9a–o were synthesized and evaluated for their α-glucosidase inhibitory and cytotoxic activities. All the synthesized compounds exhibited excellent α-glucosidase inhibitory activity in the range of IC₅₀ = 80.0 ± 2.0–383.1 ± 2.0 µM against yeast α-glucosidase, when compared to the standard drug acarbose (IC₅₀ = 750.0 ± 1.5 µM). Among the synthesized compounds, 2-((6-chloro-2-methoxyacridin-9-yl)thio)-N-(p-tolyl) acetamide 9b displayed the highest α-glucosidase inhibitory activity (IC₅₀ = 80.0 ± 2.0 μM). The in vitro cytotoxic assay of compounds 9a–o against MCF-7 cell line revealed that only the compounds 9d, 9c, and 9n exhibited cytotoxic activity. Cytotoxic compounds 9d, 9c, and 9n did not show cytotoxic activity against the normal human cell lines HDF. Kinetic study revealed that the most potent compound 9b is a competitive inhibitor with a K_i of 85 μM. Furthermore, the interaction modes of the most potent compounds 9b and 9f with α-glucosidase were evaluated through the molecular docking studies.     

Citrus Honeys from Three Different Regions of Turkey: HPLC-DAD Profiling and in Vitro Enzyme Inhibition,Antioxidant, Anti-Inflammatory and Antimicrobial Properties with Chemometric Study

The objectives of the present study are to compare the phenolic profiles and biological activities of 15 citrus honey samples from three different locations in Turkey using a chemometric approach. The HPLC-DAD analysis was used to determine phenolic profiles. Nineteen phenolic compounds were identified. Gallic acid (107.14–717.04 μg/g) was recorded as the predominant compound. AF (Antalya-Finike) had the highest antioxidant activity in ABTS⋅⁺ (IC₅₀: 18.01±0.69 mg/mL), metal chelating (IC₅₀: 6.20±0.19 mg/mL) and CUPRAC (A_0.50: 12.05±0.68 mg/mL) assays, while it revealed the best anti-inflammatory activity against COX-2 (17.28±0.22 %) and COX-1 (43.28±0.91 %). AM (Antalya-Manavgat) was the most active in β-carotene-linoleic acid (IC₅₀: 10.05±0.19 mg/mL), anti-urease (38.90±0.69 %), anti-quorum sensing and antimicrobial activities. AKO1 (Adana-Kozan-1) in DPPH⋅ (IC₅₀: 34.25±0.81 mg/mL) assay, AKU1 (Antalya-Kumluca-1) in tyrosinase inhibition activity (37.73±0.38 %) assay, AKU2 (Antalya-Kumluca-2) in AChE (10.55±0.63 %) and BChE (9.18±0.45 %) inhibition activity assays showed the best activity. Chemometric tools were applied to the phenolic compositions and biological properties. PCA and HCA ensured that 15 citrus honey samples were grouped into 3 clusters. The results showed that myricetin, kaempferol, vanillin, protocatechuic acid, rosmarinic acid, rutin, vanillic acid, gallic acid, catechin and p-hydroxyphenyl acetic acid are phenolic compounds that can be used in the classification of citrus honeys.     

Chemical Composition and Antioxidant,Antiparasitic, Cytotoxicity and Antimicrobial Potential of the Algerian Limonium Oleifolium Mill. Essential Oil and Organic Extracts

This work aimed to investigate, for the first time, the chemical composition, antioxidant, antiparasitic, cytotoxicity, and antimicrobial activities of the aromatic plant Limonium oleifolium Mill. essential oil (EO) and organic extracts. L. oleifolium aerial parts essential oil was analyzed by GC-FID and GC-MS, and 46 constituents representing 98.25±1.12 % of the oil were identified. γ-Muurolene (10.81±0.07 %), cis-caryophyllene (7.71±0.06 %), o-cymene (7.07±0.01 %) and α-copaene (5.02±0.05 %) were the essential oil main compounds. The antioxidant activity of L. oleifolium EO and organic extracts (MeOH, CHCl₃, AcOEt, BuOH) was explored using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS, β-carotene/linoleic acid, cupric reducing antioxidant capacity (CUPRAC), and ferric reducing power assays. The results showed that L. oleifolium EO exhibit antioxidant capacity (IC₅₀=17.40±1.32 μg/mL for DPPH assay, IC₅₀=29.82±1.08 μg/mL for β-carotene assay, IC₅₀=25.23±1.01 μg/mL for ABTS assay, IC₅₀=9.11±0.08 μg/mL for CUPRAC assay and IC₅₀=19.41±2.06 mg/mL for reducing power assay). Additionally, the EO showed significant activity against trophozoite form of Acanthamoeba castellanii (IC₅₀=7.48±0.41 μg/mL) and promastigote form of Leishmania amazonensis (IC₅₀=19.36±1.06 μg/mL) and low cytotoxicity on murine macrophages (LC₅₀ 90.23±1.09 μg/mL), as well as good antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella oxytoca, and Pseudomonas aeruginosa. These results suggest that L. oleifolium essential oil is a valuable source of bioactive compounds presenting antioxidant, antiparasitic, and antimicrobial activities. Furthermore, it is considered nontoxic.     

In Vitro Anticancer Activity of Staphyloxanthin Pigment Extracted from <Emphasis Type="Italic">Staphylococcus gallinarum</Emphasis> KX912244, a Gut Microbe of <Emphasis Type="Italic">Bombyx mori</Emphasis>

The present study reports the in vitro biological nature of the pigment produced by Staphylococcus gallinarum KX912244, isolated as the gut microflora bacterium of the insect Bombyx mori. The purified pigment was characterized as Staphyloxanthin based on bio-physical characterization techniques like Fourier transform infrared spectroscopy, high performance liquid chromatography, Proton nuclear magnetic resonance spectroscopy (¹H NMR), Liquid chromatography-Mass spectroscopy and Gas chromatography-Mass spectroscopy. The Staphyloxanthin pigment presented considerable biological properties including in vitro antimicrobial activity against pathogens Staphylococcus aureus, Escherichia coli and Candida albicans; in vitro antioxidant activity by % DPPH free radical scavenging activity showing IC₅₀ value of 54.22 µg/mL; DNA damage protection activity against reactive oxygen species and anticancer activity evaluated by cytotoxicity assay against 4 different cancer cell lines like the Dalton’s lymphoma ascites with IC₅₀ value 6.20?±?0.02 µg/mL, Ehrlich ascites carcinoma having IC₅₀ value 6.48?±?0.15 µg/mL, Adenocarcinomic human alveolar basal epithelial cells (A549 Lung carcinoma) bearing IC₅₀ value 7.23?±?0.11 µg/mL and Mus mucus skin melanoma (B16F10) showing IC₅₀ value 6.58?±?0.38 µg/mL and less cytotoxicity towards non-cancerous human fibroblast cell lines (NIH3T3) with IC₅₀ value of 52.24 µg/mL. The present study results suggest that Staphyloxanthin acts as a potential therapeutic agent especially due to its anticancer property.     

Multi-targeted dihydrazones as potent biotherapeutics

Hydrazone compounds were considered as a useful moiety in drug design development. Therefore, these studies were aimed at the synthesis of new dihydrazones and were screened for their in vitro H⁺/K⁺-ATPase and anti-inflammatory activities. The results revealed that compounds 9 (22 ± 0.62 µg/mL), 10 (26 ± 0.91 µg/mL), 15 (24 ± 0.44 µg/mL), 16 (28 ± 0.63 µg/mL), 17 (12 ± 0.38 µg/mL), 18 (14 ± 0.47 µg/mL), 19 (26 ± 0.54 µg/mL), 20 (16 ± 0.41 µg/mL), 25 (06 ± 0.68 µg/mL) and 26 (08 ± 0.43 µg/mL) showed excellent H⁺/K⁺-ATPase activity and their IC₅₀ value were lower than the standard drug Omerazole (48 ± 0.12 µg/mL). Compounds 5 (28 ± 0.65 µg/mL), 6 (24 ± 0.61 µg/mL), 7 (28 ± 0.64 µg/mL), 8 (26 ± 0.45 µg/mL), 11 (30 ± 0.74 µg/mL), 12 (28 ± 0.40 µg/mL), 13 (32 ± 0.24 µg/mL), 14 (30 ± 0.55 µg/mL) and 21 (08 ± 0.47 µg/mL), 22 (12 ± 0.47 µg/mL), 23 (10 ± 0.51 µg/mL) and 24 (14 ± 0.84 µg/mL) showed better anti-inflammatory activity compared to standard indomethacin (44 ± 0.15 µg/mL). The structure activity relationship (SAR) showed that, electron donating groups (OH, OCH₃) favored the H⁺/K⁺-ATPase and antioxidants activity, whereas, electron withdrawing groups (F, Cl, Br and NO₂) favored the anti-inflammatory activity. Furthermore, molecular docking study was performed to investigate the binding interactions of the most active analogs with the active site of H⁺/K⁺-ATPase enzyme. Compounds 25 (G-score = −9.063) and 26 (G-score = −8.977) showed the highest docking G-scores for H⁺/K⁺-ATPase inhibition activity.     

Design,synthesis, anticancer evaluation,molecular docking and cell cycle analysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine derivatives as potent histone lysine demethylases (KDM) inhibitors and apoptosis inducers

A novel series of pyrazolo[1,5-a]pyrimidines were synthesized and proved by their spectral and elemental analysis, some elected of the newly synthesized compounds were examined for their cytotoxic activity employing MTT assay on two cancer cell lines (Breast and Hela cancers). Compounds 5, 7e and 7i showed the higher cytotoxicity against two cancer cell lines with (IC₅₀ = 13.91 ± 1.4 and 22.37 ± 1.8 μM/L), (IC₅₀ = 6.56 ± 0.5 and 8.72 ± 0.9 μM/L) and (IC₅₀ = 4.17 ± 0.2 and 5.57 ± 0.4 μM/L) for two cancer cell lines breast and hela respectively, using doxorubicin as a reference drug. The most potent cytotoxic active compounds 5, 7e and 7i presented inhibitory activity against KDM (histone lysine demethylases) with IC₅₀ = 4.05, 1.91 and 2.31 μM, respectively. The most potent KDM inhibitor 7e (IC₅₀ = 1.91 μM) showed to cause cell cycle arrest at G2/M phase by 4 folds than control and induce total apoptotic effect by 10 folds more than control. In silico studies performed on the more potent cytotoxic active compounds 5, 7e and 7i included lipinisk's rule of five. Moreover, molecular docking study was utilized to explore the binding mode of the most active compounds to the target enzyme (PDB-ID: 5IVE). Also, some bioinformatics studies were carried out for compounds 7e and 7i using Swiss ADME (Swiss Institute of bioinformatics 2018).     

Synthesis and molecular docking studies of novel 2-chloro-pyridine derivatives containing flavone moieties as potential antitumor agents

A series of novel 2-chloro-pyridine derivatives containing flavone, chrome or dihydropyrazole moieties as potential telomerase inhibitors were synthesized. The bioassay tests showed that compounds 6e and 6f exhibited some effect against gastric cancer cell SGC-7901 with IC₅₀ values of 22.28 ± 6.26 and 18.45 ± 2.79 μg/mL, respectively. All title compounds were assayed for telomerase inhibition by a modified TRAP assay, the results showed that compound 6e can strongly inhibit telomerase with IC₅₀ value of 0.8 ± 0.07 μM. Docking simulation was performed to position compound 6e into the active site of telomerase (3DU6) to determine the probable binding model.     

Synthesis and biological evaluation of some thiazole derivatives as new cholinesterase inhibitors

In the present study, some thiazole derivatives were synthesized via the ring closure reaction of 1-[2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetyl]thiosemicarbazide with various phenacyl bromides. The chemical structures of the compounds were elucidated by ¹H NMR, ¹³C NMR and mass spectral data and elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using a modification of Ellman’s spectrophotometric method. The compounds were also investigated for their cytotoxic properties using MTT assay. The most potent AChE inhibitor was found as compound 4e (IC₅₀?=?25.5?±?2.12 µg/mL) followed by compounds 4i (IC₅₀?=?38.50?±?2.12 µg/mL), 4c (IC₅₀?=?58.42?±?3.14 µg/mL) and 4g (IC₅₀?=?68?±?2.12 µg/mL) when compared with eserine (IC₅₀?=?0.025?±?0.01 µg/mL). Effective compounds on AChE exhibited weak inhibition on BuChE (IC₅₀ > 80 µg/mL). MTT assay indicated that the cytotoxic dose (IC₅₀?=?71.67?±?7.63 µg/mL) of compound 4e was higher than its effective dose.     

Isolation of a new bioactive cinnamic acid derivative from the whole plant of Viola betonicifolia

A new cinnamic acid derivative was isolated from the whole plant of Viola betonicifolia as off white needle. On the basis of various modern spectroscopic techniques including HREI–MS and 1D and 2D NMR, its structure was elucidated as 2,4-dihydroxy, 5-methoxy-cinnamic acid. It showed marked inhibition against DPPH (diphenyl-2-picryl hydrazyl) free radicals with IC₅₀ = 124?±?5.76 µM. The antioxidant property of the compound was compared with α-tocopherole and vitamin C having IC₅₀ values 96?±?0.46 and 90?±?0.56 µM, respectively. In case of antiglycation assay, the compound exhibited moderate activity (IC₅₀ = 355?±?7.56 µM) similar to standard compound, rutin (IC₅₀ = 294?±?0.56 µM). However, it was non-toxic to PC-3 cell line. It is concluded that 2,4-dihydroxy, 5-methoxy-cinnamic acid has antiglycation potential which was further augmented by its antioxidant activity and thus offered an ideal natural therapeutic option for the effective management of diabetes.     

Evaluation of in vitro antioxidant and anticancer activity of <Emphasis Type="Italic">Allophylus cobbe</Emphasis> leaf extracts on DU-145 and PC-3 human prostate cancer cell lines

Allophylus cobbe (L.) Raeusch. belonging to the family Sapindaceae, is a commonly distributed small shrub in Western Ghats of India previously reported for its traditional medicinal properties. It is used for the treatment of various ailments. The present study is aimed at investigating preliminary phytochemicals, inducing the determination of the total phenolic contents, antioxidant assays and anticancer activity of A. cobbe leaf extracts on (DU-145) and (PC-3) cell lines. Preliminary phytochemical screening showed a broad spectrum of secondary metabolites. Highest amount of phenolic content was present in aqueous extract (91.96 ± 0.61 mg/g GAE) and it also proved to have the most potent antioxidant activity at a concentration of 100 mg/ml (64.71 ± 0.15%). IC₅₀ value was (431.10 ± 15.05 µg/mL) for DU-145 and (362.08 ± 24.17 µg/mL) for PC-3 cell lines while the standard drug paclitaxel showed an IC₅₀ value of 0.3 µM/mL. Morphological changes was observed in cancerous cells undergoing apoptosis in human prostate cancer cell lines (DU-145 and PC-3) while the extract showed no cytotoxicity towards normal cells (MEF-L929). It can be concluded that the tested extracts holds significant antioxidant and anticancer activities. However further investigation on lead compounds of A. cobbe will enable its therapeutic use.     

The improvement of an anti-CD22 immunotoxin

HA22-LR is a recombinant immunotoxin for the treatment of B-cell malignancies that contains the Fv portion of an anti-CD22 antibody fused to a functional portion of Pseudomonas exotoxin A. In the present study, we attempted to improve this molecule. First, we produced a single-chain version of HA22-LR (scdsFv-HA22-LR) in which a peptide linker was introduced between the disulfide-linked light and heavy chains to enable production via single fermentation. No difference in cytotoxic activity was observed between scdsFv-HA22-LR and prototype HA22-LR. Next, we attempted to increase the affinity of scdsFv-HA22-LR by using alanine scanning mutagenesis of complementarity determining regions (CDRs) to assess the specific contribution of each CDR residue to the antigen binding. We found that mutation of asparagine 34 in V_LCDR1, which is located at the V_L/V_H interface, to alanine (N34A) caused a substantial increase in affinity and activity. Estimated K_D values measured by fluorescence-activated cell sorting were lowered by 10-fold: 0.056 nM in the N34A mutant compared to 0.58 nM in wild type (WT). Cell viability assays of CD22-positive B-cell lymphoma and leukemia cell lines showed that the N34A mutant had increased cytotoxicity ranging from ~2 (HAL-1, IC₅₀(WT): 2.37 ± 0.62 ng/ml, IC₅₀(N34A): 1.32 ± 0.41 ng/ml) to 10 (SUDHL-6, IC₅₀(WT): 0.47 ± 0.090 ng/ml, IC₅₀(N34A): 0.048 ± 0.018 ng/ml)-fold compared to WT immunotoxin. The present study suggests that the N34A mutant of scdsFv-HA22-LR could have important consequences in a clinical setting.     

Phenolic profile,antioxidant and cytotoxic properties of polar extracts from leaves and flowers of Isatis tinctoria L. (Brassicaceae) growing in Sicily

This study was designed to define and compare the antioxidant and cytotoxic properties of polar extracts obtained from basal leaves (It-BL), cauline leaves (It-CL) and flowers (It-F) of Isatis tinctoria L. growing wild around Acireale (Sicily, Italy). The phenolic profile was characterized by HPLC-PDA-ESI-MS analysis and the correlation between phenolic content and the observed biological effects was established. Further, LC/MS analysis showed that the extracts contain glucosinolates at very low concentrations. The antioxidant activity of the extracts was tested in vitro; It-F was the most effective in the DPPH test (IC₅₀ = 0.437 ± 0.003 mg/mL), whilst It-CL showed the best reducing power (1.546 ± 0.006 ASE/mL) and ferrous ions chelating activity (IC₅₀ = 0.564 ± 0.011 mg/mL). The extracts exhibited anti-proliferative effects against three different human thyroid carcinoma cell lines, and It-BL displayed the strongest activity; particularly, it markedly inhibited the growth of CAL-62 cells, causing nearly 85% reduction of viability at the highest tested dose. No cytotoxicity against Artemia salina was observed. The results of our investigations indicate that the polar extracts obtained from I. tinctoria are a potential source of antioxidant and anticancer compounds, which could be suitable for nutraceutical and therapeutic applications.     

In-vitro antioxidant,antimutagenic and cancer cell growth inhibition activities of Rhododendron arboreum leaves and flowers

In the current investigation, the active principles of the methanol extracts of Rhododendron arboreum leaves (MEL) and flowers (MEF) were investigated with the help of ultra-high performance liquid chromatography (UHPLC), amino acid analyzer and gas chromatography mass spectrometry (GC-MS). UHPLC revealed different polyphenols present in the extracts. GC-MS identified 20 phytochemicals in leaves and 17 in the flowers, whereas, amino acid analyzer confirmed 11 amino acids in leaves and 10 in the flowers. The extracts were subjected to the investigation of biological activity through analysis of antioxidant activity in different in vitro assays, antimutagenic activity in Ames assay and cancer cell growth inhibition activity by MTT (3-4,5 dimethylthiazol-2,5 diphenyltetrazolium bromide) assay. MEL showed higher antioxidant activity in lipid peroxidation inhibition assay (95.32 ± 0.37%) than MEF (77.09 ± 4.17%) with IC₅₀103.6 µg/ml for MEL and 271.17 µg/ml for MEF. In nitric oxide scavenging assay, an activity of 94.46 ± 0.32% (IC₅₀ 150.13) was observed in MEF followed by 83.71 ± 0.74% (IC₅₀ 179.52) in MEL. The antimutagenic activity of both the extracts was evaluated against sodium azide, 4-nitro-O-phenylenediamine and 2-aminofluorene mutagens in TA-98 and TA-100 strains of Salmonella typhimurium. The analysis was carried out using pre- and co-incubation modes. However, both extracts were observed to possess considerable antimutagenic activity against different known mutagens, flowers came out to be more effective than the leaves in terms of % inhibition. The extracts also exhibited significant cancer cell growth inhibition activity, when tested against 3 cancer cell lines namely, Human cervical cancer cell line (HeLa), Breast cancer cell line (MCF7) and Lung cancer cell line (A549). In case of HeLa and A549, MEL showed higher activity of 64.62 ± 2.65 and 75.08 ± 1.68% as compared to 53.11 ± 2.84 and 45.92 ± 2.43% in MEL, respectively. The EC₅₀ values for MEL in HeLa and A549 were noted to be 232.76 and 155.38 µg/ml, respectively, whereas, MEF had EC₅₀ of 395.50 µg/ml in HeLa and 660.26 µg/ml in A549. Further, MEF showed higher cytotoxicity in MCF7 cell line (84.93 ± 1.17%) followed by the MEL (73.57 ± 1.27%) with EC₅₀ value of 95.16 µg/ml for MEF followed by 172.19 µg/ml for MEL. The biological activities of the extracts can be attributed to the phyto-constituents identified by sophisticated instruments.The biological activities of the extracts can be attributed to the active principles identified by sophisticated instruments.