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

Serum insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation status in subjects with and without ischaemic heart disease

Background: Insulin-like growth factor binding protein-1 (IGFBP-1) modulates the activity of IGF-I. It exists in serum as phosphorylated and less phosphorylated forms. We wished to measure serum levels of both these forms of IGFBP-1, using a novel assay, in subjects with, or without ischaemic heart disease (IHD). Methods: We measured serum concentrations of the phosphorylated and less phosphorylated forms of IGFBP-1 in 75 subjects (36 with and 39 without IHD). Two immunoassays were used, one which detects non-, and less-phosphorylated forms (LpIGFBP-1), and another which specifically detects the serine phosphorylated form of IGFBP-1 (pIGFBP-1). Results: LpIGFBP-1 concentrations were significantly higher in subjects without IHD than in those with IHD (5.3 +/- 0.5 mu g/L vs. 2.7 +/- 0.4 mu g/L, p < 0.001). pIGFBP-1 levels were also significantly higher in subjects without IHD compared to those with IHD (33.3 +/- 2.0 mu g/L vs. 25.3 +/- 2.2 mu g/L, p < 0.01). The correlation between LpIGFBP-1 and pIGFBP-1 for all subjects was (r = 0.71, p < 0.001). This association was stronger in subjects without IHD (r = 0.76, p < 0.001) than for those with IHD (r = 0.60, p < 0.001). A significant negative association was observed between IGF-I and the ratio between the two forms (r = -0.45, p < 0.0001). Receiver-Operating Characteristic (ROC) curve showed the highest area under the curve for LpIGFBP-1 (0.75) [95% CI: 0.63-0.86] and optimum cut-off value of 2.83 mu g/L with 75% sensitivity and 74% specificity. Conclusions: We propose that low serum concentrations of IGFBP-1 forms could be a marker of coronary risk, and the LpIGFBP-1: pIGFBP-1 ratio may be an index of biologically active IGF-

Cytotoxic and anthelmintic potential of crude saponins isolated from <it>Achillea Wilhelmsii </it>C. Koch and <it>Teucrium Stocksianum </it>boiss

<p>Abstract</p> <p>Background</p> <p>Saponins isolated from plant sources have a number of traditional and industrial applications. Saponins have pharmacological effects like anti-inflammatory, molluscicidal, antimicrobial, antispasmodic, antidiabetic, anticancer, anticonvulsant, anthelmintic, antitussive and cytotoxic activities. The current work describes the anthelmintic and cytotoxic activities of crude saponins of <it>Achillea Wilhelmsii </it>and <it>Teucrium Stocksianum </it>as these plants are rich with saponins.</p> <p>Methods</p> <p>Brine shrimp cytotoxic activity of crude saponins was determined by Meyer et al. (1982) at test concentrations of 1000 μg/ml, 100 μg/ml, 10 μg/ml, 7.5 μg/ml, 5.0 μg/ml, 2.5 μg/ml and 1.25 μg/ml. Percentage mortality of test concentrations was determined. Similarly, in <it>vitro </it>anthelmintic activity was determined against roundworms, tapeworms and earthworms. Albendazole and piperazine citrate at concentration 10 mg/ml were used as standard anthelmintic drugs.</p> <p>Results</p> <p>Crude saponins of <it>Achillea wilhelmsii </it>(CSA) and <it>Teucrium stocksianum </it>(CST) had, respectively, cytotoxic activity with LC₅₀values 2.3 ± 0.16 and 5.23 ± 0. 34 μg/ml. For in <it>vitro </it>anthelmintic activity, time for paralysis and death of parasites (parasiticidal activity) was noted. At concentration 40 mg/ml, crude saponins of <it>Achillea wilhelmsii </it>are 1.96 and 2.12 times more potent than albendazole against <it>Pheretima posthuma </it>and <it>Raillietina spiralis</it>, respectively. Similarly, at concentration 40 mg/ml, crude saponins of <it>Teucrium stocksianum </it>(CST) has 1.89, 1.96 and 1.37 times more parasiticidal activity than albendazole against <it>Pheretima posthuma, Raillietina spiralis </it>and <it>Ascardia galli</it>, respectively.</p> <p>Conclusion</p> <p>Crude saponins of <it>Achillea wilhelmsii </it>and <it>Teucrium stocksianum </it>have cytotoxic and anthelmintic activity. The crude saponins may be excellent sources of cytotoxic and anthelmintic constituents that warrant its isolation and purification for new drug development.</p