Niaz Ali

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

BackgroundSaponins 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 Achillea Wilhelmsii and Teucrium Stocksianum as these plants are rich with saponins.MethodsBrine 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 vitro anthelmintic activity was determined against roundworms, tapeworms and earthworms. Albendazole and piperazine citrate at concentration 10 mg/ml were used as standard anthelmintic drugs.ResultsCrude saponins of Achillea wilhelmsii (CSA) and Teucrium stocksianum (CST) had, respectively, cytotoxic activity with LC50 values 2.3 ± 0.16 and 5.23 ± 0. 34 μg/ml. For in vitro anthelmintic activity, time for paralysis and death of parasites (parasiticidal activity) was noted. At concentration 40 mg/ml, crude saponins of Achillea wilhelmsii are 1.96 and 2.12 times more potent than albendazole against Pheretima posthuma and Raillietina spiralis, respectively. Similarly, at concentration 40 mg/ml, crude saponins of Teucrium stocksianum (CST) has 1.89, 1.96 and 1.37 times more parasiticidal activity than albendazole against Pheretima posthuma, Raillietina spiralis and Ascardia galli, respectively.ConclusionCrude saponins of Achillea wilhelmsii and Teucrium stocksianum 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.

Antioxidant and relaxant activity of fractions of crude methanol extract and essential oil of Artemisia macrocephala jacquem

AbstractBackgroundThe current work is an attempt to know about additional chemical profile of Artemisia macrocephala. Antioxidant activity is performed as the plant is reported to contain flavonoids, which have antioxidant activity in general. Relaxant activity of fractions of crude methanol extract is performed to know in which fraction(s) the relaxant constituents concentrate as we have already reported that its crude methanol has relaxant activity. Antispasmodic activity of essential oil is also performed as the plant is rich with essential oil.MethodsPhytochemical profile of the plant is performed. Free radical scavenging activity was performed using 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Relaxation activity tests of fractions and essential oil of Artemisia macrocephala were performed on sections of rabbits’ jejunum. Calcium chloride curves were constructed to investigate the mode of action of plant extracts and its essential oil.ResultsWe detected carbohydrates, flavonoids and saponins in A. macrocephala. At concentration 0.005 mg/ml, free radical scavenging activity of ethyl acetate fraction was 121.5 ± 2.02% of ascorbic acid. n- hexane fraction relaxed spontaneous activity with EC50 0.74 ± 0.04 mg/ml. Essential oil relaxed spontaneous activity with EC50 0.8 ± 0.034 mg/ml. Chloroform and ethylacetate fractions relaxed both spontaneous and KCl-induced contractions suggesting its possible mode through calcium channels. Constructing calcium chloride curves, the test fractions showed a right shift in the EC50. Essential oil at concentration 0.1 mg/ml produced right shift with EC50 (log [Ca++]M) -2.08 ± 0.08 vs. control with EC50 -2.47 ± 0.07. The curve resembled the curves of verapamil, which caused a right shift at 0.1 μM, with EC50 -1.7 ±0.07 vs. control EC50 (log [Ca++]M) -2.45 ± 0.06.ConclusionsCrude methanol and its fractions (ethyl acetate, chloroform and butanol) are rich sources of antioxidant constituents. The relaxing constituents following calcium channel blocking mechanisms are more concentrated in n-hexane, chloroform and ethyl acetate fractions that warrant isolation.

Anthelmintic and relaxant activities of Verbascum thapsus Mullein.

BackgroundVerbascum 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.MethodsV. 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 rabbits 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.ResultsWe 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).ConclusionsOur results suggest that V. thapsus, which is currently used by some tribes in the Malakand region of Pakistan, has anthelmintic and antispasmodic value.

Spasmolytic Activity of Fruits of Tamarindus indica L.

Objective: To study the possible effects of methanolic extract of fruits of Tamarindus indica on rabbit’s jejunum preparations. Materials and Methods: The study was carried out on rabbit’s jejunum preparations. Methanolic extract of fruits of T. indica was applied in different doses. Potassium chloride (KCl 80 mM)-induced contractions were relaxed by the extract. For determination of possible mode of action of relaxing effects, calcium chloride curves were constructed in decalcified tissues using K+ normal and followed by K+ rich solution. The curves were then compared with verapamil. Results: Relaxant effects on KCl-induced contractions were prominent at a concentration of 5.0-10.0 mg/ml (P<0.05). A right shift at a concentration of 3.0 mg/ml (EC50 ± SEM = 1.98 ± 0.03) and 10.0 mg/ml (EC50 ± SEM = 1.79 ± 0.05) versus control (EC50 ± SEM = 2.33 ± 0.058) resembled the effects of verapamil on the calcium chloride curves. Conclusion: The results confirmed its possible mode of relaxing effects i.e. through calcium channel blockade.

Brine shrimp cytotoxicity of crude methanol extract and antispasmodic activity of α-amyrin acetate from Tylophora hirsuta Wall

BackgroundWe have previously reported that aerial parts of Tylophora hirsuta have antispasmodic profile. The current work is an attempt for isolation of pharmacologically active compound(s) that contribute for its antispasmodic activity.MethodsPreliminary phytochemical screening for crude methanol extract of Tylophora hirsuta (Th.Cr) is performed. Brine shrimp cytotoxicity of crude methanol extract is performed. Column chromatography was used for isolation of compounds. Mass spectroscopy, H1 NMR and C13 NMR were used for structural determination of compounds. α-amyrin acetate was tried for possible spasmolytic activity in rabbit’s jejunal preparations and KCl-induced contractions.ResultsTh.Cr tested positive for saponins, alkaloids, flavonoids and terpenoids. Compound 1 was isolated as α-amyrin acetate. Compound 2 was heptaeicosanol. Crude methanol extract tested positive for brine shrimp cytotoxicity with LC50 492.33± 8.08 mg/ml. Compound 1 tested positive for antispasmodic activity on spontaneous rabbits’ jejunum preparations with EC50 (60 ± 2) × 10-5M. The compound also tested positive on KCl induced contractions with EC50 (72 ± 3) × 10-5M.ConclusionsThe present work confirms that α-amyrin acetate is has antispasmodic profile and the relaxant effect may be attributed to α-amyrin acetate which is a major compound.

Preliminary Phytochemical Screening and Antispasmodic Activity of Artemisia macrocephala Jacquem

The current work describes the antispasmodic action of Artemisia macrocephala, which is achieved via blocking of the calcium channels. This explains its traditional use as an antispasmodic.The crude methanolic extract of A. macrocephala was studied for possible relaxant effect(s) on spontaneous rabbits′ jejunum preparations. Analytical-grade chemicals were used in the experimental protocols. A. macrocephala gave positive tests for flavonoids, saponins, glycosides, alkaloids, and terpenes. A. macrocephala caused relaxation of spontaneous rabbits′ jejunum preparations (n=6) at a dose of 10.0 mg/mL (EC 50 = 6.95 ± 0.20 mg/mL; 95% CI: 6.2 to 7.5). Contractions induced by 80 mM potassium chloride (KCl) were also relaxed by the A. macrocephala at dose of 10.0 mg/mL. Attempting to find an explanation for the possible mode of action, we found that, A. macrocephala at concentration of 1.0 mg/mL produced rightward shift in the calcium chloride curves, with EC 50 value of −1.65 ± 0.02 log [Ca ++ ] M vs control with EC 50 value of −2.44 ± 0.043 for calcium chloride curves. At a concentration of 1.0 mg/mL it could produce 52.4% of the control response at log [Ca ++ ] M = −1.6. Similarly, verapamil at a concentration of 0.1 μM produced a rightward shift, with EC 50 value of −1.74 ± 0.026 log [Ca ++ ] M (95% CI: -1.66 to -1.82; n=6) vs control with EC 50 value of −2.45 ± 0.05 log [Ca ++ ] M (95% CI: - 2.23 to -2.91; n=6). The right shift of the EC 50 values is justification for the folkloric use of A. macrocephala as an antispasmodic, suggesting that the possible mode of action is through calcium channel blockade.

Acute toxicity, brine shrimp cytotoxicity, anthelmintic and relaxant potentials of fruits of Rubus fruticosus Agg

BackgroundRubus fruticosus is used in tribal medicine as anthelmintic and an antispasmodic. In the current work, we investigated the anthelmintic and antispasmodic activities of crude methanol extract of fruits of R. fruticosus on scientific grounds. Acute toxicity and brine shrimp cytotoxicity activity of the extract were also performed.MethodsAcute toxicity study of crude methanol extract of R. fruticosus was performed on mice. In vitro Brine shrimp cytotoxicity assay was performed on shrimps of Artemia salina. In vitro Anthelmintic activity was tested against Raillietina spiralis and Ascaridia galli. Relaxant activities were tested on spontaneous rabbits’ jejunal preparations. Calcium chloride curves were constructed to elucidate possible mode of action of the extract.ResultsLD 50 of the extract for acute toxicity studies was 887.75 ± 9.22 mg/ml. While CC 50 of the extract for Brine shrimps cytotoxicity assay was 13.28 ± 2.47 μg/ml. Test samples of crude methanolic extract of R. fruticosus (Rf.Cr) at concentration 20 mg/ml showed excellent anthelmintic activity against Raillietina spiralis. Anthelmintic activity was 1.37 times of albendazole against the Raillietina spiralis at concentration 40 mg/ml. At higher concentration (40 mg/ml), Rf.Cr has 89. 83% parasiticidal activity. The mean EC50 relaxation activity for spontaneous and KCl-induced contractions was 7.96 ± 0.1 and 6.45 ± 0.29 mg/ml, respectively. EC 50 (Log[Ca++]M) for control calcium chloride curves was −1.75 ± 0.01 vs. EC 50 −1.78 ± 0.06 in the presence of 3.0 mg/ml of Rf.Cr. Similarly, EC 50(Log[Ca++]M) in the absence and presence of verapamil (0.1 μM) were −2.46 ± 0.01 and −1.72 ± 0.02, respectively.ConclusionsThe anthelmintic and relaxant activities explained traditional uses of R. fruticosus on scientific grounds. Relaxant activity follows the inhibition of voltage gated channels. Although the plant extract has cytotoxic effects, yet it is evident from acute toxicity study that it is safe in concentration 100 mg/kg. Further work is required to isolate pharmacologically active compounds.

In Vitro Enzyme Inhibition Potentials and Antioxidant Activity of Synthetic Flavone Derivatives

Free radicals are produced by an important chemical process known as oxidation that in turn initiates chain reactions to damage the cells and originate oxidative stress. Flavones have got special position in research field of natural and synthetic organic chemistry due to their biological capabilities as antioxidant. The antioxidants are known to possess extensive biological effects that include antiviral, antibacterial, anti-inflammatory, antithrombotic, and vasodilatory activities. The simple flavone (F1) and substituted flavone derivatives (F2–F5) have been synthesized from o-hydroxyacetophenone and benzaldehyde derivatives in good yield. The structures have been established by different spectroscopic techniques like 1H NMR, 13C NMR, IR, and elemental analysis. Antioxidant profile of these compounds was established using DPPH and H2O2 free radical scavenging assay. The findings showed that halogenated flavones showed more enzyme inhibitions and antioxidant activities than simple flavones and are potential candidates for the treatment of wide range of diseases.

Sequence and apoptotic activity of VacA cytotoxin cloned from a Helicobacter pylori Thai clinical isolate.

The vacuolating cytotoxin VacA produced by Helicobacter pylori induces the formation of large cytoplasmic vacuoles in host gastric epithelial cells as well as a release of cytochrome C from mitochondria resulting in cell apoptosis. Considerable sequence diversity in VacA relating to different degrees of disease severity is observed with clinical samples from a multitude of geographic places. In this study we describe expression in Escherichia coli, purification to homogeneity and in vitro assay of its apoptotic activity of a VacA toxin from a H. pylori isolate of a Thai patient with gastrointestinal lymphoma. Sequencing revealed that the deduced amino acid sequence of the cloned Thai isolate VacA is similar to H. pylori s1/m2 type strains. The percent sequence similarity to the model strain 60190 was lower due to the presence of extra amino acids in the mid (m) region. The purified VacA toxin exhibited significant apoptotic activity on both T84 and MDCK epithelial cell lines, as revealed by DAPI staining, whereby the observed activity was significantly higher on MDCK cells. These findings could relate to a modulation of VacA activity on host cells in the Thai isolate-VacA toxin that may differ from those of the model strain.

Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U)

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (kcat) and a lower Km value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.