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ThesisItem Open 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 JulietThe 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.ThesisItem Open 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.ThesisItem Open 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.ThesisItem Open Access HEPATOPROTECTIVE EFFECT OF ECLIPTA PROSTRATA (L.) L. LEAVES ON EXPERIMENTALLY INDUCED AFLATOXICOSIS IN BROILER CHICKEN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, 2018-11-30) PRIYA. K; Preethy JohnThe study was aimed to investigate the hepatoprotective effect of E. prostrata (Kayyonni) leaf powder on experimentally induced aflatoxicosis in broiler chicken. The leaf powder was subjected to preliminary phytochemical screening to find out the active principles present in it. Aflatoxin was produced in maize using the culture Aspergillus flavus NRRL 6513. The maize culture powder yielded 143.48 ppm of aflatoxin. This mouldy maize was incorporated in experimental feed to arrive 500 ppb of aflatoxin. Sixty Cobb400 day old broiler chicks weighing 50 ± 5 g were randomly divided into six groups comprising 10 birds in each group. The birds were maintained under deep litter system and provided with ad libitum water and feed throughout the experimental period. All the birds were vaccinated as per the standard schedule. Aflatoxicosis was experimentally induced in all groups except T1 and T3 by giving 500 ppb of aflatoxin B1 (AFB1) from eighth day of age onwards. The group T1 was kept as normal control and T2 as toxic control. T3 was fed with E. prostata leaf powder at 0.2 per cent level. The leaf powder of E. prostrata was given to T4, T5 and T6 at dose rates of 0.05, 0.1 and 0.2 per cent respectivelyBody weight was recorded at weekly intervals and the blood was collected from the wing vein on days 7, 21 and 42. Serum was separated and used for the estimation of biochemical parameters such as aspartate transaminase (AST), creatine kinase (CK), cholesterol and total proteins. On the day 42, all the birds were sacrificed; detailed post- mortem examination was conducted. Liver samples were taken to estimate antioxidant parameters such as lipid peroxidation (LPO) and reduced glutathione (GSH). Representative liver samples were also taken and preserved with 10 per cent neutral buffered formalin for histopathological examinationThe preliminary phytochemical screening of E. prostrata leaf powder revealed the presence of steroid, tannins, flavonoids, diterpenes, tripterpenes and saponin.Treatment with E. prostrata leaves powder revealed hepatoprotection in dose dependent manner which is indicated by significant (P<0.05) reduction in the level of serum AST and increase in the level of cholesterol and total protein. The oxidative stress induced by aflatoxin in liver was reduced to a great extend as indicated by the increased level of reduced glutathione and decrease in the lipid peroxidation. Histopathological examination of liver showed regenerative changes in a dose dependent manner when compared with that of normal control group. Thus, it could be concluded that E .prostrata leaf powder had marked antioxidant and hepatoprotective effect on experimentally induced aflatoxicosis in broiler chickenThesisItem Open Access TOXICOKINETICS OF AMITRAZ FOLLOWING SINGLE DERMAL APPLICATION IN GOATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2014-05-26) N, SATHISH; Juliet, SanisAmitraz is a formamidine derivative widely used for the control of ticks and mange mites in animals. The work reported here describes the toxicokinetics of amitraz in Malabari goats after single dermal application at 0.25 per cent. Blood samples were collected at predetermined time intervals up to 168 h and milk samples were collected every morning and evening up to eight days post dermal application. Analysis of amitraz was done by High Performance Liquid Chromatography. Amitraz was detected in blood within 0.5 h attaining a peak concentration of 7.24 ± 0.79 at 6 h followed by a decline and persisted till 168 h. The various kinetic parameters were calculated using a two compartment open model. The absorption rate constant (Ka), apparent volume of distribution (V / F), elimination half-life (t1/2β), ratio of k12 and K21, tissue-blood ratio and mean residence time (MRT) were 0.58 h -1 , 33.29 µl / mg, 172.59 h, 0.63, 0.71 and 240.10 h respectively. Amitraz was rapidly absorbed from the site of application, widely distributed and slowly eliminated from the body. The lower tissue-blood ratio suggested its minimum affinity for accumulation in the tissues. The concentration of amitraz detected in milk ranged from 0.37-0.44 mg / L until the duration of the study period. Absorption of the retained drug from the application site contributed to the persistent concentration detected in the blood and milk till 168 h.ThesisItem Open Access IN VITRO HEPATIC METABOLISM OF THE PHYTOBIOTIC 1,8- CINEOLE IN DOMESTIC FOWL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018-08-13) D. M., MAHESH; Juliet, SanisChickens reared under intensive systems are likely to be exposed to feed additives and various xenobiotics. With the ban of antibiotics as in-feed growth promoters (AGPs), there is requirement of alternative method to improve the bird’s performance capacity and also cope well with harsh conditions during rearing period. Phytobiotics, especially essential oils (EOs), are a new class of feed additives that have attracted attention due to their attributed antimicrobial and growth promoter properties. 1,8-cineole is a naturally occurring monocyclic monoterpene ether with an aromatic and camphor like odour and is one of the essential oil (EO) components used in poultry feed as phytobiotic. Even though the metabolism of 1,8-cineole is well studied in rats, rabbits, koala and brush tail possum and humans, its fate in poultry is not yet reported. Cytochrome P450 enzymes (CYP) are a group of monooxygenases playing a significant role in the biotransformation of several kinds of xenobiotics. CYP3A enzymes have been reported as responsible for metabolism of 1,8-cineole in rat and human liver microsomes. Avian and other mammalian species have distinctly different potential capacities of metabolizing drugs. The difference in the metabolic pathway and metabolic enzymes involved in biotransformation of drugs is one of the factors contributing for species variability in pharmacological responses. Although mammalian and avian CYPs are not strictly orthologs, some substrates were found to work for both groups. Hence, the present study was conducted in vitro to identify the cytochrome P450 enzyme orthologs involved in the biotransformation of 1,8-cineole in chicken hepatic S9 and microsomal fractions. The approaches used included the use of specific cytochrome P450 (CYP3A4) inhibitors and the correlation of prototype substrate activities with the formation of the hydroxylated metabolite of 1,8-cineole. Nifedipine and phenacetin were used as specific substrates for the CYP3A and CYP1A isoforms and ketoconazole was used as an inhibitor. Twelve day old Gramasree layer chicks were purchased from hatchery unit, Instructional Livestock Farm Complex, College of Veterinary and Animal Sciences, Pookode and were reared under standard management practices for 12 weeks. The chicks were fed with standard layer feed diet for starter (0-8 weeks) and grower (8-12 weeks) as per Bureau of Indian Standards, 2007 and had free access to water ad libitum until the experiment. A high performance liquid chromatography method was validated and applied for the determination of nifedipine, phenacetin and their formed metabolites nifedipine oxide and acetaminophen in hepatic microsomes and S9 fractions. Determination of nifedipine and nifedipine oxide were carried out at a flow rate of 1 mL/min using the mobile phase consisting of methanol and water in the ratio of 65:35. Phenacetin and its metabolite acetaminophen were determined at a flow rate of 1 mL/min by using the mobile phases consisting of methanol: water (70:30) and water: acetonitrile (85:15) respectively. Analysis of 1,8-cineole and its metabolite was done by gas chromatography mass spectrophotometry(GC-MS). The extraction efficiency was also determined for nifedipine, nifedipine oxide, phenacetin, acetaminophen and 1,8- cineole by comparing the peak areas from drug free samples spiked with known quantities of drug in the range of concentration of calibration curves and standard solutions with suitable solvent in which it is soluble, injected directly into analytical column. Chickens of ninety days age were euthanized by complete cranial decapitation followed by exsanguination. The liver was immediately collected and washed with icecold 1.15 per cent of potassium chloride solution. Both the body and liver weight were recorded. A portion of the collected livers (major lobe) was processed at 4 ºC for the generation of S9 and microsomal fractions. In vitro incubation studies were carried out in S9 and for with and without NADPH generating system with nifedipine, phenacetin and 1,8-cineole in presence and absence of the inhibitor ketoconazole at predetermined time intervals. The enzyme activities for the CYP isoforms were correlated with the formation of the metabolites. Analysis of various drug metabolisms were done using Graphpad prism 5 software and nonlinear regression analysis of drug disappearance versus time was done with mycurvefit. No mortality of the birds was recorded when reared strictly under intensive system. The birds at the end of 12th week attained an average body weight of. 771.33 ± 64.96 g. The nifedipine and its metabolite were best separated with retention times of 8.02 and 6.01 min respectively. The drug phenacetin and its metabolite had a retention time of 4.2 and 5.5 min respectively. Ketoconazole was detected at 220 nm with a retention time of 1.9 minute using the mobile phase mixture of methanol: 0.1% formic acid (90:10, v/v). The retention time of 1,8-cineole and its metabolite were 7.9 and 13.1 min respectively. An average obtained liver weight of 19.79 ± 2.88 g was obtained which was approximately 2.56 per cent of the average body weight for Gramasree birds. The microsomal protein in the present study was 28.42± 0.780 per gram of fresh weight tissue. Incubation of 1,8-cineole in the hepatic S9 fraction with a protein concentration of 7 mg/ml up to 180 min did not exhibit any significant decrease in the concentration of the parent compound compared to samples at ‘zero’ min. On the contrary, 1,8 cineole was significantly metabolized by the microsomal fraction with a linear decrease in the concentration of the drug. This could be due to the difference in the metabolic enzymes present in the cytosolic and microsomal fractions. No significant difference in metabolic activity was noted between the male and female birds. The formation of nifedipine oxide at different incubation times indicated the existence of chicken CYP ortholog of the human enzyme studied. Phenacetin, also metabolized by CYP1A2 isoenzyme was used as a specific substrate probe for determining its activity. Phenacetin was not metabolized by the hepatic cytosolic enzymes till 180 minutes. However, both phenacetin and 1,8-cineole was metabolised in the S9 fraction 6 hours post incubation indicating low CYP activity in the hepatic S9 fraction. . The study indicated that CYP3A isoform catalysed the hydroxylation of 1,8- cineole in chicken liver microsomes with the formation of 2α-hydroxy-1,8-cineole. The human isoenzyme CYP3A specific inhibitor ketoconazole significantly inhibited the metabolisms of nifedipine and 1,8-cineole in chicken liver microsomes. Since ketoconazole is also reported as a nonspecific inhibitor of CYP 1A in chickens, the role of CYP 1A2 in the metabolism of 1,8-cineole in chicken in the present study cannot be ruled out. Further, the rate of 1,8- cineole biotransformation is low in chickens when compared to biotransformation in humans and rats in vitro.ThesisItem Open 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 NairMotility 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.ThesisItem Open 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 KariyilThe 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.ThesisItem Open Access IN VITRO HEPATIC METABOLISM OF THE PHYTOBIOTIC 1,8- CINEOLE IN DOMESTIC FOWL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018) MAHESH D. M.; Sanis JulietChickens reared under intensive systems are likely to be exposed to feed additives and various xenobiotics. Phytobiotics, especially essential oils (EOs), are a new class of feed additives that have attracted attention due to their attributed antimicrobial and growth promoter properties. 1,8- cineole is a naturally occurring monocyclic monoterpene ether with an aromatic and camphor like odour and is one of the EO components used in poultry feed as phytobiotic. Even though the metabolism of 1,8-cineole is studied in rats, rabbits, koala and brushtail possum and humans, its fate in poultry is not yet reported. The present study was conducted in vitro to identify the cytochrome P450 enzyme orthologs involved in the biotransformation of 1,8-cineole in chicken hepatic S9 and microsomal fractions. The approaches used included the use of specific cytochrome P450 (CYP3A4) inhibitors and the correlation of prototype substrate activities with the formation of the hydroxylated metabolite of 1,8-cineole. Nifedipine and phenacetin were used as specific substrates for the CYP3A and CYP1A isoforms and ketoconazole was used as an inhibitor. A high performance liquid chromatography method was validated and applied for the determination of nifedipine, phenacetin and their formed metabolites nifedipine oxide and acetaminophen in hepatic microsomes and S9 fractions. Analysis of 1,8-cineole and its metabolite was done by gas chromatography mass spectrophotometry(GC-MS). No significant difference in metabolic activity of specific probes and 1,8-cineole was noted between the male and female birds. Incubation of 1,8-cineole in the hepatic S9 fraction with a protein concentration of 7 mg/ml up to 180 min did not exhibit any significant decrease in the concentration of the parent compound compared to samples at ‘zero’ minutes. On the contrary, 1,8 cineole was significantly metabolized by the microsomal fraction with a linear decrease in the concentration of the drug. The formation of nifedipine oxide at different incubation times indicated the existence of chicken CYP ortholog of the human enzyme studied. Phenacetin was not metabolized by the hepatic cytosolic enzymes till 180 minutes. However, both phenacetin and 1,8- cineole was metabolised in the S9 fraction 6 hours post incubation. The study indicated that CYP3A isoform catalyzed the hydroxylation of 1,8-cineole in chicken liver microsomes with the formation of 2α-hydroxy-1,8-cineole. The human isoenzyme CYP3A specific inhibitor ketoconazole significantly inhibited the metabolisms of nifedipine and 1,8-cineole in chicken liver microsomes. Since ketoconazole is also reported as a nonspecific inhibitor of CYP 1A in chickens, the role of CYP 1A2 in the metabolism of 1,8-cineole in chicken in the present study cannot be ruled out.
