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2015 - 201932020 - 20211
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Subject: Veterinary Pharmacology and Toxicology × Start date: 2011 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    ANTINEOPLASTIC ACTIVITY OF BIOSYNTHESISED BAICALEIN AND PIPERLONGUMINE NANOPARTICLES
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-07-29) PREETHY JOHN; Nisha A. R.
    The research was undertaken with the objective of studying the antineoplastic activity of biosynthesised baicalein and piperlongumine nanoparticles in Daltons Lymphoma Ascitis (DLA) cells. The study was carried out in three phases. In Phase 1, silver nanoparticles (AgNPs) synthesised using baicalein and piperlongumine were characterised by UV-Vis spectroscopy, X Ray Diffraction (XRD) and Field emission Scanning Electron Microscopy (FESEM). The characterisation revealed that the particles synthesised were within the range of nanoscale (1-100 nm) and were of crystalline nature and had spherical shape. In Phase II, both the pure compounds and nanoparticles were screened for in vitro cytotoxic activity in DLA cell lines-by MTT assay. Based on the per cent inhibition assayed using MTT, IC50 value for baicalein (BCLN) was 59.41 µg/ mL whereas for AgNPs synthesised from BCLN (B-AgNPs), the value reduced to 50.22 µg/ mL. Similarly, the IC50 value for piperlongumine (PPLM) was 4.028 µg/ mL while the value reduced to 1.25 µg/ mL for AgNPs synthesised from PPLM (P-AgNPs). But reference drug, 5-FU and chemically synthesised C-AgNPs (C-AgNPs) imparted minimal cytotoxicity, with IC50 values calculated as 326.2 and 743.6 µg/ mL respectively. Cell viability assessed after incubation with IC50 of test compounds for three hours using trypan blue exclusion assay revealed that all test substances showed an average of 49.07 per cent cell viability in three hours. Acridine orange/Ethidium bromide (AO/EB) staining was done to assess the apoptotic changes in cells exposed to IC50 of test compounds for 24 h. The cells collected after exposure to IC50 of test compounds for 24 h were subjected to DCF DA assay for intracellular ROS generation, DNA fragmentation assay and JC-1 staining to analyse mitochondrial transmembrane potential (MMP). The relative expression of Bcl-2, Caspase-3 and p53 was also assayed in the cells exposed to treatments keeping GAPDH as reference gene. In vitro studies revealed that among the treatments, PPLM and P-AgNPs showed significant apoptotic changes, ROS generation, DNA fragmentation and MMP changes. BCLN and B-AgNPs also showed in vitro antineoplastic activity. In both the cases, biosynthesised nanoparticles showed remarkable in vitro antineoplastic potential than pure compounds. In Phase II, acute oral toxicity test of biosynthesised nanoparticles as per OECD guidelines-420 was performed and in vivo antineoplastic properties of nanoparticles was evaluated in DLA induced solid tumour in mice. No toxicity symptoms or loss in body weight were observed in animals that undergone acute toxicity test. Solid tumour was induced by injecting viable DLA cells (1 × 106 cells/mouse) subcutaneously into the right hind limb of Swiss albino mice. Fifty four tumour positive mice were selected and randomly allocated into nine groups with six animals in each group. Normal control (Group I) also comprised of six animals. Group II served as tumour control. Group III was administered with 5- FU at 20 mg/kg. Group IV and V received B-AgNPs at 50 mg/kg and 100 mg/kg respectively. Piperlongumine nanoparticle (P-AgNPs) was given at 50 mg/kg and 100 mg/kg to Group VI and VII respectively. Group VIII and IX received pure compounds, BCLN and PPLM respectively at 100 mg/kg. Chemically synthesised nanoparticles (C-AgNPs) at 100 mg/kg was administered to Group X. All the treatments were given orally for 10 days. Progression of the tumour was assessed by measuring the tumour volume once in four days. Animals were sacrificed and tumour masses were collected on day 11 to assess ratio of tumour weight to body weight, tumour volume, to estimate levels of lipid peroxidation and reduced glutathione. Standard staining using Haematoxylin and Eosin, special staining using AO/EB, relative gene expression studies of Bc-l2, Caspase-3 and p53 using Real time PCR were also carried out in tumour masses. The outcome of the tumour growth response studies suggested that biosynthesised nanoparticles (P-AgNPs and B-AgNPs) exerted a pronounced inhibitory effect on the tumour growth than that produced by the respective pure compounds, PPLM and BCLN. All treatment groups showed an increase in lipid peroxidation and decrease in reduced glutathione levels. There was a down regulation of Bcl-2 and upregulation of Caspase-3 and p53 in treated groups compared to tumour control group. As revealed in in vitro study, PPLM and P AgNPs showed more significant in vivo antineoplastic property than other treatments. From the study, it can be concluded that biosynthesised nanoparticles produced more antineoplastic activity than the respective pure compounds from which they were synthesised and in the current study, P-AgNPs at 100 mg/kg was showing the most potent antineoplastic action against DLA cells.
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    ThesisItemOpen Access
    IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF POTENTIAL ACARICIDAL MOLECULE(S) FROM PLANT EXTRACT AND STUDIES ON THEIR MODE OF ACTION
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2015-09-30) SREELEKHA K. P.; Sanis Juliet
    The present study was conducted to isolate active biomolecule(s) from Blumea mollis plant extract having potent acaricidal activity against R. (B.) annulatus and to study their mode of action. Blumea mollis (D. Don) Merr., Asteraceae family is an annual aromatic herb indigenous to tropical South India. The plant materials were collected from identified phytogeographical zones. The ethanolic extract of different accessions of the plant was assayed for acaricidal activity using adult immersion test and larval packet test. Further, isolation and characterization of bioactive fractions / sub-fractions were performed using standard chromatographic and spectral techniques. The accession CAS 01 of B. mollis was selected as the best accessions with more than 80-100% efficacy against R.(B.) annulatus.The freeze dried crude extract of elite accession of CAS-01 was tested for safety using OECD guidelines. Phytochemical analysis revealed the presence of high content of terpenoids in the accession CAS 01. The hexane fraction was found to be active and HPTLC profiling showed marker concentration comparable to that of ethanolic extract of CAS 01. Phytochemical analysis of hexane fraction revealed the presence of high content terpenoids further confirmed by GC-MS. The GC-MS analysis of active sub-fractions revealed the presence of several components ranging from monoterpenes, diterpenes to sesquiterpenoids. Among the four molecules selected based on their chemical nature and area percent, ethyl palmitate, VP5 and marker compound VP1 elicited the acaricidal activity varying from 95 - 100 %. The histology and transmission electron microscopic analysis of active biomolecules exhibited marked changes comparable to that of synthetic acaricides deltamethrin and amitraz. The β octopamine receptor gene expression studies using whole tick and ovary showed significant down regulation comparable to amitraz in case of VP1 whereas ethyl palmitate and VP5 showed no significant change. Structural analogy results revealed similarity with compounds mediating action through GPCR receptors. The isolated molecules can be used as template for the development of promising acaricides.
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    ThesisItemOpen Access
    EVALUATION OF ANTITUMOUR PROPERTIES OF SIMAROUBA GLAUCA (‘LAKSHMI TARU’) AND THESPESIA POPULNEA (‘POOVARASU’) IN EXPERIMENTAL MAMMARY TUMOUR MODELS IN RATS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-30) ANU G; Usha P. T. A.
    The present study was undertaken to evaluate the antitumour properties of Simarouba glauca (‘Lakshmi taru’) and Thespesia populnea (‘Poovarasu’) in in vitro cancer cell line and in DMBA (7, 12 dimethyl benz[a]anthracene) induced mammary tumours in rats. The crude alcoholic extracts and different fractions of S. glauca leaves and T. populnea bark were screened for their cytotoxic property in MCF-7 human breast carcinoma cell line. Based on the IC50 value, chloroform soluble fractions (CSF) of both S. glauca and T. populnea were found to be superior in cytotoxic action than crude extracts and other fractions, and hence CSFs of both the plants were selected for further in vitro and in vivo studies. In in vitro haemolytic assay, CSF of S. glauca did not produce haemolysis in any of the tested concentrations whereas CSF of T. populnea showed haemolytic activity in a dose dependent manner starting from the concentration 40 µg/mL. Chloroform soluble fractions of S. glauca and T. populnea exhibited a superior in vitro antioxidant effect in a dose dependent manner when compared with vitamin C standard. On phase contrast microscopy and Acridine orange/ ethidium bromide (AOEB) dual staining, MCF-7 cells treated with CSFs of S. glauca and T. populnea exhibited marked morphological and nuclear alterations which were characteristics of apoptosis. DNA ladder assay and TUNEL assay was conducted to detect the apoptotic DNA fragmentation if any, occurred in MCF-7 cells after treatment with the plant fractions. A typical ladder pattern characteristic of apoptosis was not observed in DNA ladder assay whereas, there was an increase in positive reactions noticed in TUNEL assay which indicates an increase in DNA fragmentation after the plant fraction treatments. In acute toxicity study (OECD Guidelines no. 423) in rats, there was no apparent toxic symptoms or mortality observed in both S. glauca and T. populnea treatment groups up to an oral dose of 2000 mg/kg body weight. For in vivo antitumour studies, DMBA induced rat mammary tumour model was used. Experimental study was conducted for 14 days with 12 animals in each group. 154 After treatment with S. glauca and T. populnea CSFs at the dose rates 50 mg/kg and 100 mg/kg, no significant changes were noticed on tumour volume, tumour weight and ratio of tumour weight to body weight. The elevated mean serum LDH levels decreased significantly in the treatment groups on days 7 and 14. When compared with normal control, SOD, CAT and GSH levels were noticed to increase whereas lipid peroxidation level reduced in the mammary tumour tissues after the plant fraction treatments. In AO/EB dual staining, an increased apoptotic and necrotic cell density was observed in both the plant fraction treatment groups in dose dependent manner. After treatment with CSFs of S. glauca and T. populnea, the antiapoptotic gene Bcl2 expression was downregulated both in in vitro and in vivo studies. On histopathological examination of the mammary tumour masses using H & E staining, a progressive reduction in cellularity and apoptotic changes were noticed in the plant fraction treated groups. Thus in the present study, CSF of ethanolic extract of S. glauca leaf and CSF of methanolic extract of T. populnea bark exhibited a dose dependent cytotoxic and antitumour activity both in vitro and in vivo. The plant fractions were effective in inducing apoptotic cell death and may be considered as potent sources for isolating therapeutic molecules for cancer treatment.
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    ThesisItemOpen Access
    SCREENING AND EVALUATION OF SELECTED HERBAL EXTRACT FOR MANAGEMENT OF OBESITY AND ASSOCIATED METABOLIC DISORDERS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2015-12-30) J. JOSHUA ALLAN; Usha P.T.A.
    The global prevalence of obesity and its associated metabolic comorbidities, especially dyslipidemia are at present escalating alarmingly, which necessitates urgent implementation of earnest preventive and therapeutic measures. Identifying the medications that target obesity, dyslipidemia and the underlying oxidative stress with less or negligible side effects, hence seem to be an excellent strategy. The present study was undertaken to identify a safe herbal substance possessing anti-obese and anti-dyslipidemic potentials along with targeting the underlying oxidative stress. The plants were shortlisted based on history of food use, usage in daily life, mentioned in texts of Ayurveda for medicinal use, not classified as endangered species and present in normally traded as commodities list. Accordingly, six plant extracts were initially screened for acute oral toxicity and hypotriglyceridemic effect in oral lipid load test. The screening tests indicated aqueous extract of Zingiber officinale rhizome as the safe and potent extract, which was further fractionated to improve the solubility. The fraction showing highest solubility i.e., water soluble fraction of Zingiber officinale extract (WFZE) was further subjected to acute oral toxicity and oral lipid load tests to ensure the safety and efficacy. The WFZE was found to be safe upto 5000 mg/kg body weight in acute oral toxicity study in rat and its triglyceride lowering activity was found to be better than the parent extract. Consequently, WFZE was evaluated for anti-obese and anti-dyslipidemic potentials. The anti-obesity effect of WFZE was established in high fat diet induced obese male rats by the significant reduction in body weight gain, adiposity and triglyceride levels along with the amelioration of metabolic perturbations such as hyperleptinemia, increase in the FFA, glucose and insulin. Moreover, the total hepatic lipids, serum AST and ALT were reduced while atherogenic index and insulin resistance indices such as HOMA and QUIKI were controlled and the severity of histopathological lesions in liver, pancreas and adipose tissue were reduced after treatment with WFZE. In addition, WFZE treatment significantly reduced the oxidative stress induced by high fat feeding as evident from the significant decrease in hepatic MDA and increase in SOD, catalase and glutathione reductase levels, indicating the anti-oxidant potential of WFZE. The anti-dyslipidemic activity of WFZE was further demonstrated in Triton WR-1339 induced and cholesterol cholic acid induced dyslipidemic models. A significant reduction in serum triglycerides and cholesterol levels following the administration of Triton WR-1339 and chronic feeding of cholesterol and cholic acid in rats established the anti-dyslipidemic activity of WFZE. To elucidate the mechanism of action, WFZE was investigated for its activity against pancreatic lipase, which indicated promising pancreatic lipase inhibitory activity in vitro. The findings of the present study suggest that WFZE is a safe, effective therapeutic agent for the management of obesity and dyslipidemia acting via the inhibition of pancreatic lipase and anti-oxidant mechanisms.