Acute toxicity, brine shrimp cytotoxicity and relaxant activity of fruits of callistemon citrinus curtis

<p>Abstract</p> <p>Background</p> <p><it>Callistemon citrinus </it>Curtis belongs to family Myrtaceae that has a great medicinal importance. In our previous work, fruits of <it>Callistemon citrinus </it>were reported to have relaxant (antispasmodic) activity. The current work describes the screening of fractions of the crude methanol extract for tracing spasmolytic constituents so that it shall help us for isolation of bioactive compounds. Acute toxicity and brine shrimp cytotoxicity of crude methanol extract are also performed to standardize it.</p> <p>Methods</p> <p>The crude methanol extract was obtained by maceration with distilled water (500 ml) three times and fractionated successively with <it>n-</it>hexane, chloroform, ethyl acetate and <it>n-</it>butanol (300 ml of each solvent). Phytochemical analysis for crude methanol extract was performed. Acute toxicity studies were performed in mice. Brine shrimp cytotoxicity studies were performed to determine its cytotoxicity and standardize it. In other series of experiments, rabbits' jejunum preparations were used in screening for possible relaxant activities of various fractions. They were applied in concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml on spontaneous rabbits' jejunum preparations. In similar fashion, fractions were also tested on KCl (80 mM) -induced contractions. Calcium chloride curves were constructed in K-rich Tyrode's solution. The effects of various fractions were tested on calcium chloride curves at concentrations 1.0, 3.0, 5.0 and 10.0 mg/ml. Curves of verapamil used as reference drug at concentration 0.1 μM and 0.3 μM were also constructed. The curves were compared with their respective controls for possible right shift.</p> <p>Results</p> <p>Methanol extract tested strongly positive for saponins and tannins. However, it tested mild positive for presence of proteins, amino acids, carbohydrates and phenolic compounds. LD₅₀value for crude methanol extract is 476.25 ± 10.3 (470-481, n = 4) mg/ml. Similarly, EC₅₀value for brine shrimp cytotoxicity is 65.5 ± 7.28 (60.8- 69.4, n = 4) mg/ml. All the fractions relaxed the spontaneous and KCl-induced contractions. EC₅₀values (mg/ml) for effects of ethyl acetate fraction on spontaneous and KCl induced contractions are 2.62 ± 0.78 (2.15-3.0, n = 4) and 3.72 ± 0.86 (3.38-4.28, n = 4) respectively. Respective EC₅₀values (mg/ml) for <it>n-</it>butanol fraction are 3.59 ± 0.2(3.07-3.9, n = 4) for spontaneous, and 5.57 ± 0.2 (5.07-6.11, n = 4) for KCl- induced contractions. EC₅₀value for control calcium chloride curve (without extract) is -2.73 ± 0.19 (-2.6 - -2.81, n = 4) while EC₅₀for curves treated with 5.0 mg/ml of chloroform is -2.22 ± 0.02 (-2.16 - -2.3, n = 4). EC₅₀value for ethyl acetate treated (1.0 mg/ml) tissues is -1.95 ± 0.10 (-1.88 - -2.0, n = 4) <it>vs</it>. control EC₅₀= -2.71 ± 0.08 (-2.66 - -2.76, n = 4). All the fractions, except <it>n-</it>hexane, showed a right shift like that of verapamil (EC₅₀= -1.72 ± 0.15 (-1.62 - -1.8, n = 4) vs. Control EC₅₀= -2.41 ± 0.06 (-2.38 - - 2.44, n = 4), a standard drug that blocks voltage operated calcium channels.</p> <p>Conclusion</p> <p>Relaxant constituents were more concentrated in ethylacetate fraction followed by chloroform, <it>n -</it>butanol and aqueous fractions that warrant for its isolation. The crude methanol extract is safe at concentration 250 mg/ml or below and results of brine shrimp cytotoxicity assay imply the plant specie may be a source of cytotoxic agents.</p

Anthelmintic and relaxant activities of <it>Verbascum Thapsus </it>Mullein

Abstract Background Verbascum thapsus is used in tribal medicine as an antispasmodic, anti-tubercular agent and wormicide. In this study, we investigated the antispasmodic and anthelmintic activities of crude aqueous methanolic extract of the plant. Methods V. thapsus extracts were tested against roundworms (Ascaridia galli) and tapeworms (Raillietina spiralis). Each species of worm was placed into a negative control group, an albendazole treatment group, or a V. thapsus treatment group, and the time taken for paralysis and death was determined. In addition, relaxation activity tests were performed on sections of rabbit's jejunum. Plant extracts were tested on KCl-induced contractions and the relaxation activities were quantified against atropine. V. thapsus calcium chloride curves were constructed to investigate the mode of action of the plant extracts. Results We detected flavonoids, saponins, tannins, terpenoids, glycosides, carbohydrates, proteins, fats and fixed oils in V. thapsus. For both species of worm, paralysis occurred fastest at the highest concentration of extract. The relative index values for paralysis in A. galli were 4.58, 3.41 and 2.08, at concentrations of 10, 20 and 40 mg/ml of plant extract, respectively. The relative index for death in A. galli suggested that V. thapsus extract is wormicidal at high concentration. Similarly, the relative indexes for paralysis and death in R. spiralis suggested that the extract is a more potent wormicidal agent than albendazole. The mean EC50 relaxation activity values for spontaneous and KCl induced contractions were 7.5 ± 1.4 mg/ml (6.57-8.01, n = 6) and 7.9 ± 0.41 mg/ml (7.44-8.46, n = 6), respectively. The relaxation activity of the extract was 11.42 ± 2, 17.0 ± 3, 28.5 ± 4, and 128.0 ± 7% of the maximum observed for atropine at corresponding concentrations. The calcium chloride curves showed that V. thapsus extracts (3 mg/ml), had a mean EC50 (log molar [calcium]) value of -1.9 ± 0.06 (-1.87 - -1.98, n = 6) vs. control EC50 = -2.5 ± 0.12 (-2.37 - -2.56, n = 6), whereas the verapamil (0.1 μM) EC50 was -1.7 ± 0.1 (-1.6 - -1.8, n = 6) vs. control EC50 = -2.4 ± 0.09 (-2.3 - -2.47, n = 5). Conclusions Our results suggest that V. thapsus, which is currently used by some tribes in the Malakand region of Pakistan, has anthelmintic and antispasmodic value.</p

In Vivo Antistress Effects of Synthetic Flavonoids in Mice: Behavioral and Biochemical Approach

Natural flavonoids, in addition to some of their synthetic derivatives, are recognized for their remarkable medicinal properties. The present study was designed to investigate the in vitro antioxidant and in vivo antistress effect of synthetic flavonoids (flavones and flavonols) in mice, where stress was induced by injecting acetic acid and physically through swimming immobilization. Among the synthesized flavones (F1&ndash;F6) and flavonols (OF1&ndash;OF6), the mono para substituted methoxy containing F3 and OF3 exhibited maximum scavenging potential against DPPH (2,2-diphenyl-1-picrylhydrazyl) with IC50 of 31.46 &plusmn; 1.46 &mu;g/mL and 25.54 &plusmn; 1.21 &mu;g/mL, respectively. Minimum antioxidant potential was observed for F6 and OF6 with IC50 values of 174.24 &plusmn; 2.71 &mu;g/mL and 122.33 &plusmn; 1.98 &mu;g/mL, respectively, in comparison with tocopherol. The ABTS scavenging activity of all the synthesized flavones and flavonols were significantly higher than observed with DPPH assay, indicating their potency as good antioxidants and the effectiveness of ABTS (2,2&prime;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) assay in evaluating antioxidant potentials of chemical substances. The flavonoids-treated animals showed a significant (* p &lt; 0.05, ** p &lt; 0.01 and *** p &lt; 0.001, n = 8) reduction in the number of writhes and an increase in swimming endurance time. Stressful conditions changed plasma glucose, cholesterol and triglyceride levels, which were used as markers when evaluating stress in animal models. The level of these markers was nearly brought to normal when pre-treated with flavones and flavonols (10 mg/kg) for fifteen days in experimental animals. These compounds also considerably reduced the levels of lipid peroxidation (TBARS: Thiobarbituric acid reactive substances), which was significant (* p &lt; 0.05, ** p &lt; 0.01 and *** p &lt; 0.001, n = 8) compared to the control group. A significant rise in the level of catalase and SOD (super oxide dismutase) was also observed in the treated groups. Diazepam (2 mg/kg) was used as the standard drug. Additionally, the flavonoids markedly altered the weight of the adrenal glands, spleen and brain in stress-induced mice. The findings of the study suggest that these flavonoids could be used as a remedy for stress and are capable of ameliorating diverse physiological and biochemical alterations associated with stressful conditions. However, further experiments are needed to confirm the observed potentials in other animal models, especially in those with a closer resemblance to humans. Toxicological evaluations are also equally important

X-ray characterization, Hirshfeld surface analysis, DFT calculations,in vitroandin silicolipoxygenase inhibition (LOX) studies of dichlorophenyl substituted 3-hydroxy-chromenones

This work reports the synthesis and X-ray characterization of three dichlorophenyl substituted 3-hydroxy-chromen-4-one derivatives,i.e., 2-(2,4-dichlorophenyl)-3-hydroxy-4H-chromen-4-one (1), 2-(2,3-dichlorophenyl)-3-hydroxy-4H-chromen-4-one (2), and 2-(2,6-dichlorophenyl)-3-hydroxy-4H-chromen-4-one (3). The solid-state architecture of these three isomers is quite different due to the large influence of the Cl-substituents on the dihedral angle between the phenyl and chromenone rings. The different assemblies and synthons have been studied using Hirshfeld surface analysis and by computing their interaction energies and different contributions (electrostatic, polarization, dispersion and repulsion). Furthermore, only one isomer forms directional halogen bonding interactions (C-Cl⋯O) that have been analyzed using MEP surface calculations and characterized using a combination of QTAIM and NCIplot index computational methods. The ability to form halogen bonds depending on the substitution has also been analyzed. The docking results showed that compound1forms strong binding interactions with soybean lipoxygenase followed by compounds2and3. The molecular docking results are in good agreement with the bioactivity data of1-3(IC50values) against lipoxygenase.Fil: Ahmed, Muhammad Naeem. The University of Azad Jammu and Kashmir Muzaffarabad; PakistánFil: Ghias, Mehreen. University of Malakand; PakistánFil: Shah, Syed Wadood Ali. University of Malakand; PakistánFil: Shoaib, Mohammad. University of Malakand; PakistánFil: Tahir, Muhammad Nawaz. University of Sargodha; PakistánFil: Ashfaq, Muhammad. University of Sargodha; PakistánFil: Ibrahim, Mahmoud A. A.. Minia University; EgiptoFil: Andleeb, Hina. No especifíca;Fil: Gil, Diego Mauricio. Universidad Nacional de Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Frontera, Antonio. Universidad de las Islas Baleares; Españ

Flavonoid Derivatives as Potential Cholinesterase Inhibitors in Scopolamine-Induced Amnesic Mice: An In Vitro, In Vivo and Integrated Computational Approach

Flavonoids are one of the most exciting types of phenolic compounds with a wide range of bioactive benefits. A series of flavone derivatives (F1&ndash;F5) were previously synthesized from substituted O-hydroxy acetophenone and substituted chloro-benzaldehydes. The titled compounds F1&ndash;F5 in the present study were evaluated for their anticholinesterase potential (against AChE and BuChE). The obtained results were then validated through a molecular docking approach. Compound F5 was found to be the most potent inhibitor of AChE (IC50 = 98.42 &plusmn; 0.97 &micro;g/mL) followed by compound F4, whereas compound F2 was found to be the most promising inhibitor of BuChE (IC50 = 105.20 &plusmn; 1.43 &micro;g/mL) among the tested compounds. The molecular docking analysis revealed a similar trend in the binding affinity of compounds with the targeted enzymes and found them to be capable of forming highly stable complexes with both receptors. The selected compounds were further subjected to in vivo assessment of cognitive function in a scopolamine-induced amnesic animal model, in which almost all compounds F1&ndash;F5 significantly attenuated the amnesic effects as evaluated through Y-Maze Paradigm and novel object discrimination (NOD) tasks, findings that were further supported by ex vivo experimental results. Among (F1&ndash;F5), F5 showed significant anti-amnesic effects in scopolamine-induced amnesic models and ameliorated the memory loss in behavioral model studies as compared to counterparts. In ex vivo study, noteworthy protection from oxidative stress in the brains of scopolamine-induced amnesic mice was also recorded for F5. These findings also confirmed that there were no significant differences among the in vivo and ex vivo results after administration of F1&ndash;F5 (7.5 or 15 mg/kg) or donepezil (2 mg/kg). These synthesized flavonoids could serve as potential candidates for new neuroprotective and nootropic drugs. However, further studies are needed to validate their observed potential in other animal models as well