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20192
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Subject: Veterinary Pharmacology and Toxicology × End date: 2019 × Start date: 2019 ×
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    ThesisItemOpen Access
    CHARACTERIZATION OF MUSCARINIC RECEPTOR SUBTYPES IN THE SMALL INTESTINE OF JAPANESE QUAIL (Coturnix coturnix japonica)
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-28) SANJAY, B M; Suresh N Nair
    Motility of small intestine which determine the efficacy of digestion and assimilation of nutrients and thus the rate of growth in the animals and birds is modulated mainly by muscarinic cholinergic receptor system. Among the five major subtypes of muscarinic receptors reported so far, from M1 to M5, M1, M2 and M3 are the major receptor subtypes in the intestine. The efficacy of agonists and antagonists in modulating the gastrointestinal motility is governed by subtypes of muscarinic receptors in the organs. So far, there are no reports of muscarinic receptors of quail intestine. The current study was conducted for identifying the presence of muscarinic receptor subtypes and its functional assessment, for better pharmacological management of dysfunctions of the small intestine. Eight healthy quails of either sex were raised under uniform management conditions. Birds were euthanized and two to three centimetres length ileum was separated from a region five centimetres away from the ileo-caecal junction and transferred to tyrode solution at 37.2 °C. The ileum tissue was mounted under 1 g tension in an organ bath chamber with constant aeration. The contractile responses to the agonist alone, agonist in presence of antagonists and relaxant effect of muscarinic receptor antagonists with submaximal contraction of ACh were recorded with isometric transducer connected to a recorder. The median effective concentration 50 (EC50), median inhibitory concentration 50 (IC50) and pD2 values were determined. From the results, it is evident that muscarinic acetylcholine receptors are present in the small intestine of Japanese quail. The EC50 values of acetylcholine alone in ileum of Japanese quail varied from 1.235 X 10-7 M to 2.344 X 10-7 M with mean value of 1.701 X 10-7 M and pD2 value of 6.769. The EC50 of ACh in presence of atropine varied from 4.19 X 10-7 M to 8.36 X 10-7 M with a mean value of 5.92 X 10-7 M and pD2 value of 6.23. In presence of pirenzepine, mean EC50 of 4.51 X 10-7 M with a range of 3.17 X 10-7 M to 6.42 X 10-7 M and pD2 values of 6.35 and in presence of solifenacin EC50 value varied from 2.05 X10-7 M to 3.44 X 10-7 M with a mean value of 2.65 X 10-7 M and pD2 values of 6.58. Muscarinic receptor antagonists mainly atropine, pirenzepine and solifenacin completely relaxed the contraction induced by submaximal dose of ACh. The IC50 of atropine varied from 5.21 X 10-8 M to 8.64 X 10-8 M with a mean value of 6.71 X 10-8 M and pD2 value of 7.173, the IC50 of pirenzepine varied from 1.573 X 10-6 M to 1.926 X 10-6 M with a mean value of 1.74 X 10-6 M and pD2 value of 5.759. For solifenacin, the IC50 value varied from 6.25 X 10-7 M to 8.20 X 10-7 M with a mean value of 7.16 X 10-7 M and pD2 value of 6.145. There was a significant increase in the EC50 values of ACh in presence of atropine and pirenzepine compared to EC50 value of ACh alone. Also, there was a significant increase in the EC50 value of ACh in presence of atropine when compared to EC50 of ACh in presence of solifenacin. This indicates that the muscarinic receptor subtypes responsible for contraction of small intestine in Japanese quail is contributed by both M2 and M3 muscarinic receptor subtypes since M1 receptor is absent in them just like other avian species as evidenced by a negative result in PCR. It was also being found that EC50 of ACh is increased 3.48 times in presence of atropine, 2.65 times in presence of pirenzepine and 1.56 times in presence of solifenacin. This indicates that the muscarinic receptor subtypes responsible for contraction of small intestine in Japanese quail is contributed by both M2 and M3 muscarinic receptor subtypes and molecular studies have revealed the absence of M1 receptor gene in Japanese quail.
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    ThesisItemOpen Access
    ANTICANCER ACTIVITY OF Hordeum vulgare AND Pergularia daemia IN TRIPLE NEGATIVE BREAST CANCER CELLS
    (College of Veterinary and animal Science,Mannuthy, 2019) AKHIL G.H.; Bibu John Kariyil
    The present study was aimed to evaluate the anticancer activity of methanol extract of germinated seeds of Hordeum vulgare (Barley) and leaves of Pergularia daemia (Veliparuthi) in MDA-MB-231 cell line. Qualitative phytochemical screening showed the presence of steroids, glycosides, diterpenes, triterpenes and saponins in H. vulgare whereas tannins and flavonoids were additionally detected in P. daemia. The RP-HPLC analysis showed the presence of hordenine and lupeol in H. vulgare and P. daemia respectively. In vitro antioxidant activity of the extracts by DPPH and superoxide anion radical scavenging assays showed a significant (p<0.05) concentration-dependent antioxidant activity for both the extracts. The IC50 values for DPPH assay were found to be 112.75±5.74 and 100.67±7.9 μg/mL for H. vulgare and P. daemia respectively and for superoxide radical scavenging assay, the IC50 values were found to be 28.90±1.85 and 39.63±1.41 μg/mL for H. vulgare and P. daemia respectively. Cytotoxicity assay revealed that both the extracts produced potent cytotoxicity with IC50 values of 41.28±0.30 and 35.95 35.95±3.57 μg/mL for H. vulgare and P. daemia respectively. Morphological evaluation produced similar cytotoxic morphological changes in cells treated with standard hordenine and lupeol when compared with their respective extracts exhibiting morphological alterations such as reduction in cell population, presence of apoptotic bodies, cell shrinkage and vacuole formation. Both the extracts produced early apoptosis as evident by acridine orange/ethidium bromide staining characterised by yellowgreen fluorescence. Hoechst staining revealed that both the extracts altered nuclear morphology indicative of apoptosis with features such as nuclear marginalisation and fragmentation. Mitochondrial membrane potential (MMP) assessed by JC-1 staining revealed that H. vulgare partially altered MMP whereas P. daemia completely altered MMP. The results of the comet assay revealed that P. daemia alone was able to cause DNA damage significantly (p<0.01). The results of western blotting showed a significant (p<0.01) downregulation of Bcl-2 expression in P. daemia treated cells whereas caspase-8 expression was significantly (p<0.01) upregulated in H. vulgare treated cells. Thus, the study revealed that H. vulgare and P. daemia produced apoptosis by extrinsic and intrinsic pathway respectively.