Thesis

Browse

Search Results

Now showing 1 - 7 of 7

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 John
The 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 chicken
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, Sanis
Chickens 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.
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 Juliet
Chickens 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.
Open Access
PROTECTIVE EFFECT OF Tribulus terrestris (NJERINJIL) IN CYCLOPHOSPHAMIDE TOXICITY ON REPRODUCTIVE SYSTEM OF MALE RATS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) KARTHIKA P. R.; Sujith. S
The present study was aimed to evaluate the protective effect of Tribulus terrestris (Njerinjil) in cyclophosphamide toxicity on reproductive system of male rats. The fruits of T. terrestris were procured locally, identified, dried in shade and pulverized. Ethanolic extract was prepared by soxhlet extraction and qualitative phytochemical analysis of the extract was performed to determine the active components. Forty adult male Wistar rats were procured from Small Animal Breeding Station, Mannuthy and divided into five groups of eight animals each. Group I served as normal control, while group II, III, IV and V received cyclophosphamide (CP) orally at the rate of 15 mg/ kg twice weekly for 30 days. Groups III, IV and V were supplemented with ethanolic extract of T. terrestris fruit daily at the dose rates of 100, 250 and 500 mg/kg respectively orally for 30 days along with cyclophosphamide. Body weight of all the animals were recorded on days 0, 15 and 30. On day 30, all the rats were sacrificed and dissected. After taking testes weight, size and volume, the left testis and epididymis from each animal were used for semen collection and for histopathological samples. The semen samples from cauda epidiymis were used for estimation of semen parameters like mass activity, sperm progressive motility, count, morphology, vital staining and acrosome integrity. The other testis and epididymis were collected immediately on ice cooled bags for the assessment of antioxidant assay of superoxide dismutase (SOD), reduced glutathione (GSH) and lipid peroxidation (LPO) and functional marker enzyme levels such as acid phosphatase (ACP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Representative samples of testes, epididymis, liver, kidney and heart were fixed in 10 per cent neutral buffered formalin and were stained with hematoxylin and eosin for histopathological screening. The preliminary phytochemical analysis of the ethanolic extract of T. terrestris revealed the presence of various active principles like steroids, alkaloids, glycosides, phenolic compounds, tannins, flavonoids, terpenes and saponins. Administration of cyclophosphamide significantly reduced body weight, relative testes weight, testes size, testes volume, mass activity, sperm motility, sperm count, sperm viability and acrosome integrity, SOD, GSH, ACP and ALP. There was also significant increase in per cent of abnormal sperms, LDH, SDH and LPO levels. Histopathological studies in testis and epididymis confirmed reproductive toxicity in the form of degeneration and loss of germinal epithelium within the seminiferous tubules giving a washed out appearance. Liver showed centrilobular necrosis, fatty degeneration and hepatocellular cytoplasmic vacuolation. Tubular epithelial degeneration, interstitial haemorrhages, atrophy of glomerular tufts and formation of casts within the lumen were observed in kidney. Sections of heart in GII animals revealed myocardial degeneration, disruption and separation of cardiac muscle fibres. Treatment with ethanolic extract of T. terrestris at various doses significantly increased body weight, relative testes weight, size and volume, mass activity, sperm progressive motility, sperm count, acrosome integrity, SOD, GSH, ACP and ALP. There was also a significant dose dependant decrease in per cent of abnormal sperms, LDH, SDH and LPO levels with maximum protective effect at 500 mg/kg of the extract. Histopathological examination showed a dose dependant regeneration and restoration of seminiferous tubular epithelium in testes and ciliated lining epithelium in epididymis. Reversion of hepatic and renal architecture to normal were observed in T. terrestris extract supplemented groups. GIII, GIV and GV revealed normal cardiac myocytes with centrally located nuclei having abundant cytoplasm outlined by distinct and intact cell walls which was comparable to GI . Therefore, it could be concluded that ethanolic extract of T. terrestris has protective effect and antioxidant potential in cyclophosphamide induced reproductive toxicity in rats.
Open Access
HEPATOPROTECTIVE EFFECT OF ECLIPTA PROSTRATA (L.) L. LEAVES ON EXPERIMENTALLY INDUCED AFLATOXICOSIS IN BROILER CHICKEN
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) PRIYA. K; Preethy John
The 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 respectively. Body 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 examination. The 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 chicken.
Open Access
MODULATION OF PROSTAGLANDIN E2 (PGE2) MEDIATED PATHWAY IN THE ECLOSION BLOCKING EFFECT OF TERPENOID FRACTION OF ARTEMISIA NILAGIRICA IN RHIPICEPHALUS (BOOPHILUS) ANNULATUS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018) PANICKER DEVYANI RAMACHANDRAN; Sanis Juliet
Prostaglandins are a group of important cell signaling molecules involved in the the regulation of ovarian maturation, oocyte development, egg laying and associated behaviours in invertebrates. However, the presence of prostaglandin E2 (PGE2), the key enzymes for biosynthesis and its interference by drugs have not been investigated in the ovary of ticks. The present study was undertaken to assess the modulation of PGE2 mediated pathway in the eclosion blocking effect of terpenoid fraction isolated from Artemisia nilagirica in Rhipicephalus (Boophilus) annulatus ticks. The dried hexane fraction of ethanolic extract of aerial parts of A. nilagirica was used for isolation of terpenoid fraction by Herz-Hogenauer method. The acaricidal activity of the terpenoid fraction was evaluated by adult immersion test. The chemical profiling of the extract/fraction was done by HPTLC and GCMS analysis. Localization of the cyclooxygenase1 and prostaglandin E synthase enzymes in the ovaries of control and treated ticks were done by immunohistochemistry. The vitellogenin concentration in hemolymph also was assayed by indirect ELISA. The terpenoid fraction of A.nilagirica elicited a concentration dependent percentage inhibition of fecundity and hatching rate in the laid eggs of R (B.) annulatus. The HPTLC fingerprint profiling of terpenoid fraction revealed the presence of nine polyvalent compounds. Twenty two terpenoid compounds were identified in the terpenoid fraction based on mass spectral matching with commercial libraries (NIST, Wiley). Presence of PGES in stage III oocytes in control ticks was confirmed by immunohistochemistry whereas immunereactivities were absent in the vitellogenic oocytes of flumethrin and terpenoid fraction treated ticks. The intensity of immunoreactivities were too low to be detected in vitellogenic oocytes of both control and treated ticks. The levels of PGE2 were below detection limit in in the ovaries of flumethrin treated ticks. The levels were significantly lower in ovaries of terpenoid fraction than control ovaries. No significant difference in the concentration of Vg was observed in the hemolymph of treated and control ticks.
Open Access
EFFECT OF PIPERINE AS AN EFFLUX PUMP INHIBITOR FOR FLUROQUINOLONES AND TETRACYCLINES IN ESCHERICHIA COLI ISOLATES FROM MASTITIS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018) VANISHREE; Nisha. A. R.
‘Antimicrobial resistance’ is a global epidemic issue nowadays. In the recent past, role of efflux pumps in development of resistance is intervened so as to overcome bacterial resistance to wide class of chemotherapeutic agents. In the current study an attempt has been made to investigate the ability of ‘piperine’, a phytochemical to overcome the efflux pump (AcrAB-TolC) mediated resistance in E. coli isolates from bovine mastitis. Antibiotic disc diffusion assay was carried out to determine the minimum inhibitory concentration (MIC) for enrofloxacin, ciprofloxacin (fluoroquinolones) tetracycline and oxytetracycline (tetracyclines) in the presence of piperine (20 ppm, 40 ppm, 60 ppm, 80 ppm, 100 ppm). Further, piperine alone at various concentrations was also tested for its antibacterial activity if any. Gene expression of acr A, acr B and tol C genes were studied by reverse transcription and real time PCR. Biofilm formation was examined in the resistant colonies in the presence of each of the above antibiotics and piperine by congo red assay. In silico analysis to determine piperine with acr B of efflux pump was carried out by using autodock software. The study revealed that piperine exposure in combination with enrofloxacin, ciprofloxacin, tetracycline or oxytetracycline resulted in dose-dependent significant (p < 0.05) increase in ‘zone of inhibition’ in the disc diffusion assay. Further, piperine reduced the MIC of enrofloxacin, ciprofloxacin, tetracycline or oxytetracycline against E.coli isolates from mastitis and showed synergistic action at a concentration of 100 ppm. A 16 fold reduction in MIC of enrofloxacin and ciprofloxacin, 5.2 fold reductions in the MIC of tetracycline and 4.6 fold reduction in the MIC of oxytetracycline were observed upon coexposure at concentration of 100 ppm. Gene expression studies indicated down regulation of expression of acr A, acr B and tol C efflux pump in E. coli isolates resistant to enrofloxacin, ciprofloxacin and oxytetracycline, while tetracycline inhibited expression of only acr B. Piperine downregulated acrB gene in both fluoroquinolones and tetracyclines resistance E.coli isolates. Relatively piperine showed more efflux pump inhibitory activity in ciprofloxacin resistant E. coli isolates. However fluoroquinolones and tetracycline tested failed to prevent biofilm formation in E. coli isolates. In silico studies revealed that piperine bind with acr B protein of efflux pump of E coli. To conclude, piperine co-exposure with enrofloxacin, ciprofloxacin, tetracycline or oxytetracycline potentiate the antibacterial activity against E. coli isolates from mastitis through inhibition of AcrAB-TolC pump. Thus, piperine can be used as an adjuvant with fluoroquinolones or tetracyclines against resistant E.coli isolates causing mastitis.

Thesis

Browse

Thesis Type

Type

Author

Date

Subject

Search Results