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ThesisItem Open Access INFLUENCE OF SODIUM HYPOCHLORITE ON PHARMACOKINETICS OF ENROFLOXACIN IN BROILER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) CHELSIA, YATHATI; RAVI KUMAR, P (MAJOR); BHARAVI, K; RAMA DEVI, VAn experimental study was conducted on broiler chicken weighing around 2 kg to know the influence of sodium hypochlorite in drinking water on the oral pharmacokinetics of enrofloxacin. The birds were divided into three groups with eight birds in each. Group I birds that were on normal drinking water received enrofloxacin orally at the dose of 10 mg/kg body weight. Group II birds received sodium hypochlorite in drinking water (10 ml/100L) for seven days followed by enrofloxacin orally (10 mg/kg) on the seventh day. Group III birds also received sodium hypochlorite for seven days but enrofloxacin was given orally (10 mg/kg) 12 hours post withdrawal of sodium hypochlorite containing water. Blood samples from all the treated groups were collected from either left or right tarsal veins at 0 (blank), 0.16, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24 and 48 h post enrofloxacin dosing and plasma was separated and analyzed by HPLC method. It was observed that t1/2 and tmax did not differ significantly (p<0.05) in all the three groups under study. Elimination rate constant, β was observed significantly (p<0.05) increased in Group III (0.077±0.005 l/h) and Group II (0.071±0.009 l/h) birds compared to that in Group I (0.040±0.002 l/h) birds. There was a noticeable decline in Cmax of enrofloxacin in Group II (1.793±0.160 μg/ml) and Group III (1.958±0.147μg/ml) birds over Group I (2.153±0.245 μg/ml) birds, although not statistically significant (p<0.05). The AUC0-t was apparently decreased though statistically not significant (p<0.05) in both Groups II (31.587±4.241 μg/ml.h) and Group III (33.669±3.593 μg/ml.h) birds. AUC0-α recorded in Group II (32.978±4.087 μg/ml.h) birds was significantly (p<0.05) low when compared to that in Group I (50.648±6.111 μg/ml.h) birds. Thus, the influence of sodium hypochlorite exposure on total exposure to enrofloxacin across time is evident. Likewise, administration of enrofloxacin, 12 hours post withdrawal of sodium hypochlorite (Group III) also resulted in reduced AUC0-α (34.751±3.681 μg/ml.h) of enrofloxacin, though statistically not significant (p<0.05). The area under first moment curve (AUMC) and mean residence time (MRT) recorded in Group III (525.468±61.695 μg/ml.h2 and 15.088±0.431 h) and Group II (538.396±61.064 μg/ml.h2 and 16.484±0.748 h) birds were significantly (p<0.05) lower than those recorded in Group I (1407.185±216.254 μg/ml.h2 and 27.076±1.375 h) birds. Sodium hypochlorite exposure or its exposure till 12 hours before the administration of enrofloxacin could not retain the enrofloxacin molecules for a period similar to that observed in control Group I. Although there was no influence of sodium hypochlorite on the volume of distribution (Vd/F), the total body clearance of enrofloxacin observed in Group III (0.310±0.032 l/kg/h) and Group II (0.329±0.031 l/kg/h) birds was significantly (p<0.05) higher when compared to that in Group I (0.216±0.022 l/kg/h) birds. It can be attributable to altered environment in the renal mechanisms involved in elimination of enrofloxacin or it metabolite(s). The study revealed that, sodium hypochlorite administration altered the pharmacokinetics of the enrofloxacin and the effect of sodium hypochlorite persisted even after its withdrawal 12 hours before the administration of enrofloxacin.ThesisItem Open Access ROLE OF COMMON EFFLUX PROTEIN INHIBITORS ON PHARMACODYANMICS AND PHARMACOKINETICS OF ENROFLOXACIN IN BROILER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) SRIVIDYA, GULLAPUDI; SRINIVASA RAO, G (MAJOR); RAVIKUMAR, P; RAMA DEVI, V; MURALIDHAR, MEnrofloxacin, an antimicrobial fluoroquinolone is most commonly used against majority of gram negative bacterial and mycoplasma infections in majority of livestock including poultry. Due to indiscriminate usage of enrofloxacin in the poultry farming and clinical practice, resistance had been developed to this quinolne drug. Microbes develop resistance to antibiotics by various pathways. Among them, efflux pump mediated drug resistance is one of the important pathways identified in the recent past. Phytochemicals namely, theobromine, glycyrrhetenic acid and glycyrrhizic acid and capsaicin were identified as efflux pump inhibitors. It was described that phytochemicals which possess efflux pump inhibitory activity if combined with classical antimicrobial agents reduces the development of resistance and also improves their therapeutic efficacy. In the present study, pharmacokinetic and pharmacodynamic interaction between enrofloxacin, a fluoroquinolone antibacterial agent and efflux protein inhibitors capsaicin, theobromine, glycyrrhetenic acid and glycyrrhizic acid were investigated using chicken as model system. Broiler chickens weighing about 1.5-2.0 kg were randomly divided into six groups with six birds in each group. Group I birds did not receive any treatment and is primarily meant for standardization of HPLC assay for determination of enrofloxacin/ciprofloxacin in chicken plasma. Enrofloxacin alone (10 mg.kg-1, PO) was administered to birds in group II that served as control. Birds in groups III, IV, V and VI were received enrofloxacin (10 mg.kg-1, PO) after one hour of oral administration of efflux protein inhibitors, capsaicin (15 mg.kg-1), theobromine (125 mg.kg-1), glycyrrhetenic acid(100 mg.kg-1) and glycyrrhizic acid(100 mg.kg-1) respectively. Blood samples were collected from tarsal vein into heparinized tubes, at predetermined time intervals prior to and 0.166, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24 and 48 h post-enrofloxacin administration. Plasma was separated and used for HPLC analysis to determine plasma concentrations of enrofloxacin. Based on plasma concentrations of enrofloxacin, pharmacokinetic parameters for enrofloxacin were determined using non-compartmental method. Detectable concentrations of enrofloxacin in birds persisted upto 48h in all groups. The mean Cmax, AUC(0-t), AUMC(0-t) , (Vd/F), MRT and (ClB/F) in enrofloxacin control group were 2.17 μg/ml, 36.28+4.80 μg.h.mL-1, 633.04+85.53 μg.h2.mL-1, 4.47+0.43 L.kg-1, 16.60+0.42 h and 0.28+0.03 L.kg-1.h-1. Results obtained from the study revealed that upon coadministration of theobromine, glycyrrhetenic acid and glycyrrhizic acid, the maximum plasma concentration (Cmax) increased to 2.62+0.14, 2.94+0.03 and 2.96+0.04 μg.mL-1 , area under the plasma drug concentration time profile (AUC0-t) enhanced to 48.93+1.29, 63.35+1.08 and 43.37+0.98 μg.h.mL-1, mean residence time (MRT) were increased significantly to 19.05+0.42, 17.70+0.25 and 17.27+0.69 h suggesting that enrofloxacin residence time was prolonged in birds. The phytochemical, capsaicin co-administration significantly shortened the half life and increased the elimination rate constant, tmax of enrofloxacin in chicken. Synergisitic interaction of efflux protein inhibitory phytochemicals capsaicin, theobromine, glycyrrhetenic acid and glycyrrhizic acid with enrofloxacin were noticed in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against S. aureus ATCC 25923, E.coli ATCC25922, K. pneumoniae ATCC700603 and P.aureginosa ATCC27853 in vitro. MIC (μg/ml) of enrofloxacin against E. coli, S. aureus, K. pneumoniae and P. aureginosa were 0.02, 0.20, 1.65 and 2.43 respectively. The MIC (μg/ml) of enrofloxacin in the presence of capsaicin was decreased to 0.090 (55%) against S.aureus, 0.012 (40%) against E.coli , 0.266 (84%) against K. pneumoniae, 0.404 (83%) against P. aureginosa. The MIC (μg/ml) of enrofloxacin in the presence of theobromine was reduced to 0.110 (45%) against S.aureus, 0.012 (66%) against E.coli, 0.258 (84%) against K. pneumoniae, 0.450 (81%) against P. aureginosa. The MIC (μg/ml) of enrofloxacin in the presence of glycyrrhetenic acid was reduced to 0.041(80%) against S. aureus, 0.012 (40%) against E. coli, 0.404 (76%) against K. pneumoniae, 0.450 (81%) against P. aureginosa. The MIC (μg/ml) of enrofloxacin in the presence of glycyrrhizic acid was reduced to 0.110 (45%) against S. aureus, 0.012 (40%) against E. coli, 0.404 (76%) against K. pneumoniae, 0.45 (81%) against P. aureginosa. In vitro results of revealed that efflux protein inhibitors potentiated the antibacterial activity of enrofloxacin against the selected bacterial strains. The PK-PD mathematical integration of pharmacokinetic and pharmacodynamic data obtained in the study was attempted. The surrogate markers commonly applied for optimization of the dose with pharmacokinetic and pharmacodynamic parameters were Cmax/MIC>10, AUC/MIC>125. The Cmax/MIC in group II, group III, group IV, group V and group VI against E.coli ATCC25922 were 86.92, 80.2, 104.72, 117.36, and 118.48 respectively. The Cmax/MIC in group II, group III, group IV, group V and group VI against S.aureus ATCC25923 were 10.86, 10.02, 13.09, 14.67, and 14.81 respectively. The Cmax/MIC in group II, group III, group IV, group V and group VI against K. pneumonia ATCC 700603 were 1.31, 1.21, 1.58, 1.77, 1.79 respectively. The Cmax/MIC in group II, group III, group IV, group V and group VI against P.aureginosa ATCC 27853 were 0.89, 0.82, 1.07, 1.20, 1.21 respectively. The ratio of AUC/MIC>125 will prevent the development of resistance against the antibacterial agent. The AUC/MIC in group II, group III, group IV, group V and group VI against E.coli ATCC25922 were 1813.93, 1859.15, 2446.45, 3167.30, and 2165.40 h respectively. The AUC/MIC in group II, group III, group IV, group V and group VI against S.aureus ATCC25923 were181.39, 185.91, 244.64, 316.73 and 216.54 h respectively. The AUC/MIC in group II, group III, group IV, group V and group VI against K. pneumoniae ATCC 700603 were 21.98, 22.53, 29.65, 38.39 and 26.24 h respectively. The AUC/MIC in group II, group III, group IV, group V and group VI against P.aureginosa ATCC 27853 were14.92, 15.30, 20.13, 26.06 and 17.82 h respectively. Based on the PK-PD integration values obtained in the present study it appears that enrofloxacin at the dose rate of 10 mg.Kg-1 is effective against E. coli and S. aureus but not effective against K. pneumoniae and P. aureginosa. Administration of efflux pump inhibitors namely theobromine, glycyrrhetenic acid and glycyrrhizic acid increases bioavailability, maximum plasma concentration of enrofloxacin whereas capsaicin significantly reduced the tmax and half life and increased the elimination rate constant in broiler chicken. The MIC, MBC which were used as indicators of antibacterial effect of enrofloxacin were significantly increased in the presence of efflux protein inhibitors.ThesisItem Open Access EVALUATION OF THE AMELIORATIVE EFFECT OF NANOQUERCETIN IN ACUTE KIDNEY INJURY IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) ASHOK REDDY, KANDADI; ADILAXMAMMA, K(MAJOR); RAVI KUMAR, P; SATHEESH, kAcute kidney injury (AKI) is a sudden onset of potentially life-threatening kidney dysfunction and is a common disorder in dogs, cats and humans. The present study was conducted to evaluate the ameliorative effect of quercetin mediated zinc oxide nanoparticles (QZnO NPs) in AKI in rats. Rats were randomly categorised into seven groups, each group containing 3 males and 3 females. Rats of all groups were deprived of water for 24 hours before glycerol administration. Group I served as control group. In all other groups, AKI was induced by injecting 10 ml/kg body weight of 50% glycerol in sterile normal saline into the hind limbs. Half an hour after glycerol injection, the treatment groups III, IV, V, VI and VII received zinc oxide nanoparticles (ZnO NPs)@ 50 mg/kg, quercetin @ 50 mg/kg, quercetin mediated nanozinc (QZnO NPs) @ 10 mg/kg, QZnO NPs @ 25 mg/kg and QZnO NPs @ 50 mg/kg respectively. Rats of group II did not receive any treatment and served as AKI control. The treatments were given after every 24 hours for 3 consecutive days and the animals were sacrificed on the 4th day. Blood biochemical profile was studied by estimating RBC, WBC, haemoglobin, creatinine, urea, total protein, albumin and globulin. Antioxidant profile of renal tissue was studied by measuring TBARS, SOD and GPx levels. Histopathology of renal tissue was studied by H&E staining. Further the apoptotic changes were also studied for bcl-2 protein expression. The present study revealed that ZnO NPs and quercetin reduced glycerol induced nephrotoxicity and treatment with QZnO NPs exhibited better nephroprotective action at low and medium doses (10 mg/kg b.wt. and 25 mg/kg b.wt. respectively) tested when compared with quercetin, ZnO NPs and QZnO NPs at higher dose (50 mg/kg b.wt.). The possible mechanism behind nephroprotection by QZnO NPs could be attributed to their free radical scavenging property. However, to understand the exact mechanism of nephroprotection by QZnO NPs, further studies are warranted.ThesisItem Open Access IN VIVO AND EX VIVO STUDIES ON THERAPEUTIC POTENTIAL OF QUERCETIN IN THE TREATMENT OF EXPERIMENTAL DYSMENORRHOEA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-05) AFROZ, JAHAN; SRINIVASA RAO, G(MAJOR); RAVI KUMAR, P; SATHEESH, K; MURALIDHAR, MDysmenorrhea is one of the most common gynecological complaints among adolescent and young adult women which is painful and is the leading cause of absenteeism. Non-steroidal anti-inflammatory drugs (NSAIDs) or oral contraceptive pills are the main therapeutic agents in alleviating pain in dysmenorrhoea. However, ideal agent for the prevention and treatment of abnormal uterine contractility in dysmenorrhoea is elusive. Flavonoids are widely occurring polyphenols that are found in vegetables, fruits, wine and tea. They are recognized for their diverse physiological activity including antioxidant, anticancer, anti-inflammatory, anti-carcinogenesis, anti-diabetic, antiallergic and spasmolytics. The relaxant activity of flavonoids has been observed in different vascular and non-vascular smooth muscles. Quercetin is a flavonoid found in vegetables, nut, red wine, fruits and onion and has been reported for its inhibitory effect on uterine contractions in rats. Keeping all these facts as backdrop, the present study was carried out to explore the uterine relaxant activity of quercetin in rat and mice in normal and experimental dysmenorrhoea condition. Experimental dysmenorrhoea was induced in rats/mice by administering oxytocin after priming them with oestradiol benzoate for three days. In vitro study was conducted in two groups of rats. Group I consisted of normal rats whereas rats in Group II were oestrogen sensitized with oestradiol benzoate 24 hours prior to sacrifice. Rats were sacrificed and uterine horns were isolated for mechanistic functional studies. Anti-spasmodic effect of quercetin was evaluated on rat myometrium of control and oestrogen primed animals on both receptor and voltage gated ion channel mediated contractile response. Both Oxytocin (12.11) and PGF2α (7.29) elicited hypercontractility of uterine smooth muscle in oestrogen primed rats in comparison with control rats (OT: 12.04 and PGF2α: 7.15) as indicated by their respective pD2 values. It was observed that both oxytocin and PGF2α have failed to elicit contractile response in presence of both quercetin and indomethacin. Both quercetin and ritodrine produced dose dependent relaxation in normal and oestrogen primed uterine smooth muscle in rats. Ritodrine appears to be more potent than quercetin. It was also observed that quercetin and ritodrine failed to produce an appreciable dose- dependent relaxation in both the groups when it was pre-contracted with KCl (40mM). In vivo study was conducted in six groups of mice. Group I consisted normal mice whereas mice from group II to VI were subjected to the experimental dysmenorrhoea. Group II mice did not receive any treatment. The animals in group III received meloxicam (5 mg/kg, P.O) for 28 days prior to induction of dysmenorrhoea. Similarly the animals in group IV, V and VI received quercetin (20, 40 and 80 mg/kg, P.O), respectively for 28 days followed by induction of dysmenorrhoea. Blood samples collected from mice of all groups were immediately subjected for analysis of haematological parameters. Plasma was harvested from collected blood and stored at - 20ºC for estimation of PGF2α, PGE2, 6-keto PGF1α and TXB2, Ca2+ and NO by ELISA. At the end of the study, mice from all groups were sacrificed and uterine horns were isolated for mechanistic functional studies. In addition, ovary and uterus were collected for histopathological and ultra-structural studies. A part of uterus collected in chilled buffer was preserved for western blotting and the remaining portion was immediately processed and 10% tissue homogenate was prepared for estimation of oxidative stress parameters and hormone levels in uterine tissue homogenate by ELISA. In in vivo studies, writhing responses (Abdominal wall contraction, hind limb stretches, pelvic rotation and lag period) were observed for 30 min in mice. Per cent inhibition of writhing response, abdominal wall contraction and hind limb stretches were significantly (P<0.01) increased in experimental dysmenorrhoea group which was restored back to normal with quercetin treatment in a dose dependent manner whereas latency period for writhing response was increased with quercetin treatment. Quercetin treatment has no effect on haematological parameters however platelet count was significantly (P<0.01) increased with quercetin treatment in a dose dependent manner. Quercetin treatment has restored back the oxidative stress biomarkers (TBAR’S and SOD) to nearly normal values significantly (P<0.01) indicating protective effect of quercetin whereas it could only partially reversed back the altered GSH and total protein levels. Antispasmodic or uterine relaxant activity of quercetin was studied for functional changes in experimentally induced dysmenorrhoea in mice myometrium. The mean pD2 of oxytocin and PGF2α in experimental dysmenorrhea group (OT: 12.1 and PGF2α: 7.24) was significantly different from mean pD2 of control animals with amplitude (OT: 11.91 and PGF2α: 7.09) whereas with frequency the significant difference was observed only for PGF2α (Control: 7.16 and Experimental dysmenorrhoea: 7.27). It indicates that the myometrium from dysmenorrhea animals shows increase in uterine contractility in presence of oxytocin. This hyper uterine contractility was partially reversed by quercetin treatment in a dose dependent manner. Oxytocin and PGF2α failed to elicit a classical dose response after pre-incubation with quercetin and indomethacin, dose response curve and EC50 could not be calculated in all the groups. Quercetin treatment had no significant change on the hyper-contractility of oxytocin and PGF2α in uterine smooth muscle of whereas ritodrine produced significant dose dependent relaxation in all the experimental groups. Quercetin produced dose dependent relaxation on oxytocin and PGF2α induced contraction in all the experimental groups but its effect was less potent than ritodrine. It was also observed that quercetin failed to produce an appreciable dose dependent relaxation on KCl induced contractions in all the experimental groups except in group VI (5.08). Similarly ritodrine failed to produce dose dependent relaxation on KCl induced contractions in group VI. The histo-pathological and ultra-structural changes observed in ovary and uterus was restored back to normal in group treated with quercetin 80 mg/kg. The altered levels of different hormones in both plasma and uterine tissue homogenates were also restored back to normal in group treated with quercetin 80 mg/kg. The expression level of COX-2 was up-regulated and β-2 adrenergic receptor was down regulated in experimental dysmenorrhea group. The altered expression levels were nearly restored back to normal in group treated with quercetin 80 mg/kg. Quercetin treatment partially reversed the hyper contractility induced by oxytocin and PGF2α in uterine smooth muscle and augmented the relaxation of ritodrine in experimentally induced dysmenorrhoea model in mice. Further quercetin treatment modulated the oxidative stress biomarkers, histo-pathological and ultrastructural changes, various hormone levels and receptor expression levels in experimental dysmenorrhoea mice model. Further studies are needed to elucidate the molecular mechanism involved in partial reversal of uterine hypercontractility by quercetin. It can be concluded that from the present study, quercetin appears to be a promising molecule in alleviating dysmenorrheic pain induced by the PGF2α.ThesisItem Open Access EFFECT OF SINGLE AND SHORT-TERM ADMINISTRATION OF QUERCETIN ON THE PHARMACOKINETICS OF ENROFLOXACIN IN BROILER CHICKENS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-01) LAKSHMINARAYANA, ALAVALA; RAVI KUMAR, P(MAJOR); SRINIVASA RAO, G; RAMA DEVI, VAn experimental study on thirty broiler chicken weighing about 2.0 kg was conducted to evaluate the pharmacokinetics of enrofloxacin along with quercetin either as co- administration or as short - term pre - treatment. The birds were divided into 3 groups of 10 birds each and were randomly administered enrofloxacin orally at dose rate of 10 mg/kg body weight (group1), quercetin followed by enrofloxacin sixty minutes later (group 2) and quercetin for 10 days with enrofloxacin sixty minutes after the quercetin on the 10th day. Both enrofloxacin and quercetin were given orally at the dose rate of 10 and 15 mg/kg body weight (group 3). Blood samples from the treated groups were collected from either left (or) right tarsal veins at 0 (blank), 0.166, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36 and 48 h post dosing and plasma was separated and analysed for enrofloxacin by HPLC method. The HPLC method applied was found table accurate and precise and exhibited acceptable recovery statistics from plasma spiked samples. Enrofloxacin was detected in the birds of all three groups at all the tested time points The pharmacokinetic parameters observed in group 1 birds were in accordance with the previous reports on the pharmacokinetics of enrofloxacin in broiler chickens. Elimination rate constant, β in group 2 (0.047±0.004 1/h) birds, in which quercetin was co-administrated with enrofloxacin, was significantly lower when compared to group 1(0.061±0.001 1/h) and 3 (0.079±0.004 1/h) birds. This was further reflected in singnificaly increased elimination half-life, t1/2 (15.642±1.085 h) in group 2 when compared with group 1(11.437±0.248 h) and 3 (9.105±0.641 h) birds. This could be attributed to the ability of quercetin to inhibit the hepatic metabolizing enzymes like CYP3A4 and efflux proteins like P-glycoprotein. Slowdown in the metabolic inactivation of enrofloxacin with subsequently lowered elimination from the body might have resulted in increased half-life. AUC0-∞ observed in group 2 (44.437±2.729 μg/ml.h) was significantly higher than that of group 3 (30.393±2.111 μg/ml.h), which indicated that the extent of absorption of enrofloxacin was high when co-administered orally with quercetin compared to enrofloxacin oral administration after short-term quercetin pre-treatment. The area under first moment curve (AUMC) and mean resident time (MRT) recorded in group 2 (1067.707±81.605 μg/ml.h2 and 23.876±1.030 h) was significantly higher than those recorded in groups 1 (654.193±65.964 μg/ml.h2 and 17.077±0.364 h) and 3 (480.580±53.213 μg/ml.h2 and 15.636±1.076 h). The clearance observed in group 2 (0.234±0.016 L/kg/h) was significantly lower when compared to group 3 (0.347±0.030 L/kg/h) but not with group 1(0.282±0.024 L/kg/h). It was observed from the study that enrofloxacin co-administered with quercetin in group 2 has exhibited higher t1/2, AUC0-∞, AUMC and MRT values. On the other hand, short – term treatment with quercetin in group 3 resulted in significantly higher elimination rate constant (β), significantly lower (t1/2), apparently lower Cmax, apparently lower AUC0-t, significantly lower AUC0-∞, significantly lower AUMC, significantly lower MRT and significantly increased ClB when compared to those observed in quercetin co-administrated birds. These results indicate that though co-administration of single dose quercetin increased the half-life, area under curve and mean resident time of enrofloxacin, repeated administration of quercetin actually reduced the above kinetic parameters of enrofloxacin. Hence in the present study, it is possible that increased bioavailability of enrofloxacin with single dose quercetin might have resulted from inhibition of P-gp in the intestine. On the other hand the decreased bioavailability of enrofloxacin with repeated quercetin treatment might have resulted from induction of CYP3A activity. It can be concluded from the present study that single dose co – administration of quercetin enhances the bioavailability of enrofloxacin, while repeated administration of quercetin reduces the same.ThesisItem Open Access IN VIVO AND EX VIVO STUDIES ON AMELIORATIVE EFFECT OF QUERCETIN ON STRUCTURAL AND FUNCTIONAL CHANGES INDUCED BY CADMIUM IN UTERUS OF MICE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-01) JOSE, GISSA M.; BHARAVI, K(MAJOR); SRINIVASA RAO, G; RAMA DEVI, VUterine abnormalities either hereditary or acquired are the major cause of infertility among reproductive females that leads to recurrent miscarriages, spontaneous abortions, pre-term labour, infertility etc. Cadmium is an important endocrine disruptor and reproductive toxicant causing uterine abnormalities. Quercetin, a powerful antioxidant flavonoid has the ability to scavenge the free radicals and also has heavy metal chelating property, smooth muscle relaxant property and estrogenic effect. The present study evaluated the structural and functional changes caused by cadmium in mice myometrium and also evaluated the ameliorative effect of quercetin on cadmium induced myometrial changes. The study was conducted in four groups. Group I considered as control with apparently healthy animals, group II consisted of animals that received cadmium @ 30ppm in drinking water for 30 days. The group III animals were treated with cadmium @30 ppm in drinking water and simultaneously with quercetin at 50 mg/kg bw orally for 30 days and group IV consisted of animals that received quercetin alone @ 50mg/Kg bw orally. Among each group, estrus was induced in half of the animals with estradiol benzoate whereas other animals were sacrificed when they were on non-estrus stage. After sacrifice uterine horn was isolated to use for functional studies using digital polygraph. The liver and kidney homogenate were used for estimation of tissue antioxidant markers like GSH and SOD and peroxidation marker like TBARS. The mice uterus, liver and kidney were collected for histopathological changes. The increased TBARS level and decreased SOD and GSH levels were observed in both liver and kidney of cadmium treated group compared to control indicating peroxidative damage by cadmium to various tissues of mice and it was reversed back in cadmium along with quercetin treated group compared to cadmium group that showed an antioxidant protective effect of quercetin on cadmium damage. The alerations in histological structure of liver, kidney and uterus in cadmium treated groups were rectified to normal architecture in cadmium along with quercetin treated animals. The mean EC 50 values of oxytocin contractile dose amplitude and frequency response in cadmium treated estrus mice were 9.344x10-13 and 8.864x10-13 respectively and it was significantly lower than control estrus mice. The simultaneous administration of cadmium along with quercetin to estrus mice increases the mean EC50 value of oxytocin induced amplitude of contraction from 9.344x10-13 to 1.306x10-12, but it was not comparable to control indicating protective effect of quercetin. The mean EC50 of oxytocin dose amplitude contractile response in cadmium along with quercetin treated non-estrus mice was lower than control and cadmium. The mean EC50 value of KCl induced amplitude and frequency of contractile response in cadmium along with quercetin treated estrus mice showed a higher value (8.81x10-4 and 8.401x10-4) compared to cadmium group (5.686x10-4 and 5.778x10-4) and also the KCl induced amplitude of contraction showed a significantly higher EC50 value than control group. Hence we can conclude that cadmium produces significant estrogen mimicking effect in estrus myometrium and quercetin was found to have non-uniform effects due to its contradictory actions on myometrium.ThesisItem Open Access EFFECT OF PIPERINE ON THE PHARMACOKINETICS OF ENROFLOXACIN IN BROILER CHICKENS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-11) NAGARJUNA, NALLAPANENI; DILIP REDDY, G(MAJOR); RAVI KUMAR, P; SATHEESH, KThe study was aimed to evaluate the effect of piperine on the pharmacokinetics of enrofloxacin. It was aimed to study the effect of piperine co-administration and pre-treatment on the pharmacokinetics of enrofloxacin in broiler chickens(Vencobb). Adult birds weighing around 2.0 kg were randomly assigned to their equal groups with 10 birds in each. The treatment protocol consisted of single oral dose of enrofloxacin (10 mg/kg b.wt) (group 1), single oral dose of piperine (15 mg/kg b.wt) followed by single oral dose of enrofloxacin (10 mg/kg b.wt) (group 2) and piperine (15 mg/kg b.wt) orally for ten days followed by enrofloxacin (10 mg/kg b.wt) on the 10th day (group 3). Blood samples were collected from either left (or) right tarsal vein at 0 (blank), 0.166, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36 and 48 h post dosing and plasma was separated for HPLC analysis. The plasma concentration-time data were analysed by non-compartmental pharmacokinetic analysis. There was no significant (p>0.05) difference in Cmax among the three groups. Elimination rate constant (β) observed in group 3 birds (0.044±0.0031/h) was significantly (p<0.05) lower when compared to group 1 (0.061±0.001 1/h) and 2 birds (0.066±0.001 1/h), which reflected the elimination half-life, t1/2 in group 3 (16.130±0.898 h), where significantly (p<0.05) higher value was observed when compared to groups 1 (11.437±0.248 h) and 2 (10.510±0.155 h). Tmax recorded in group 2 birds (7.600±0.267 h) was significantly (p<0.05) higher than that of group 1 (4.550±0.462 h). AUCs (AUC0-t and AUC0-∞) recorded in group 3 (56.551±2.035 µg/ml.h and 66.382±2.973 µg/ml.h) were significantly (p<0.05) high; implying more amount of drug was present for longer time in the body. The AUCs (AUC0-t and AUC0-∞) observed in group 2 (44.073±1.357 µg/ml.h and 46.294±1.457 µg/ml.h) were comparatively higher than group 1 (36.268±3.501 µg/ml.h and 38.104±3.637 µg/ml.h), indicating the effect of piperine in increasing the absorption over period of time. The area under first moment curve (AUMC) and mean resident time (MRT) recorded in group 3 (1711.716±140.298 µg/ml.h2 and 25.379±1.212 h) were significantly (p<0.05) higher than those recorded in groups 1 (654.193±65.964 µg/ml.h2 and 17.077±0.364 h) and 2 (808.749±38.688 µg/ml.h2 and 17.391±0.384 h). The increased values indicate that high concentration of enrofloxacin was present in the circulation for longer time in piperine pre-treated birds. The clearance observed in group 3 (0.154±0.008 L/kg/h) was significantly (p<0.05) lower when compared to group 2 (0.218±0.007 L/kg/h) and further the clearance of group 2 was significantly (p<0.05) lower than that of group 1 (0.282±0.024 L/kg/h). It can be concluded from the study that enrofloxacin administered after pre- treatment with piperine has exhibited higher t1/2, AUC, AUMC, MRT values and lower clearance values. The increase in plasma concentration of enrofloxacin in birds pre-treated with piperine could be attributed to the ability of piperine to enhance the intestinal absorption, to inhibit the metabolism in liver and to inhibit P-glycoprotein mediated drug efflux of during intestinal absorption. The increased AUC and half-life and decreased elimination rate constant could be helpful in designing formulations, that can be used in the treatment of resistant infections.ThesisItem Open Access ROLE OF EUGENOL IN REVERSAL OF VASCULAR DYSFUNCTION INDUCED BY EXPERIMENTAL DIABETES AND HYPERTENSION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-10) Vamsikrishna, Bobba; Srinivasa Rao, G(MAJOR); Ravi Kumar, P; Rama Devi, V; Vinoo, RABSTRACT : Phenylpropanoids are a diverse group of phytochemicals with immense health benefits and found throughout the plant kingdom. Eugenol is a member of the phenylpropanoids and is remarkably versatile molecule which is present abundantly in clove, nutmeg, cinnamon, basil and bay leaf. Epidemiological evidence and clinical trial data indicates that due to presence of biologically active phytochemicals, the plant originated diets can reduce the risk of chronic disease conditions such as cardiovascular disease, hypertension, diabetes and cancer. Hypertension and diabetes are the lifestyle diseases which are considered to be main causes of mortality for decades in humans. Vascular dysfunction is the major change that is associated with diabetes and hypertension. It is well documented that both diabetes and hypertension occur together in most of the human beings that is an additive cause for increase in risk of vascular complications. Though there are standard treatments available at present for these complications, a look for an alternative approach that can better address the vascular problems is most wanted. Hence the present study was designed to know the effect of eugenol against vascular dysfunction associated with either diabetes or hypertension alone and diabetes with hypertension together. The study was carried out in rats that are divided into eight groups with ten rats in each. Group-I (normal control) received vehicle alone for eight weeks whereas group II (eugenol control) received eugenol orally and daily at the rate of 80 mg/kg for eight weeks. Group- III, IV and V constitute experimentally induced hypertension control, diabetic control and diabetic rats with hypertension. Hypertension was induced in rats with administration of L-NAME in drinking water (40 mg/kg/day). Whereas single dose of streptozotocin was injected intraperitoneally at 40 mg/kg for inducing diabetes in rats. Group V rats received both streptozotocin and L-NAME. Group- VI (eugenol treated hypertensive rats), VII (eugenol treated diabetic rats) and VIII (eugenol treated diabetic rats with hypertension) received eugenol 80 mg/kg orally from the day after the onset of diabetes, hypertension or both conditions experimentally. Development of hypertension and diabetes in rats was confirmed by decrease in total nitrate/nitrite levels in serum and high blood glucose levels (>300 mg/dl) respectively. At the end of the study, rats were sacrificed and thoracic aorta was collected for studying vascular reactivity and histopathology. In addition, effect of eugenol in ameliorating the oxidative stress induced by experimental diabetes, hypertension and diabetes associated hypertension was also studied. Moreover, liver and kidney function markers in plasma were estimated in different study groups to know the effect of eugenol on liver and kidney function. Total nitrate (NO3-) and nitrite (NO2-) levels in serum were significantly (P < 0.05) decreased in hypertension control, diabetic control and diabetes associated hypertensive rats. Eugenol treatment had no impact on reversing the nitrate and nitrite levels in diabetes and hypertension back to the normal values noticed in control rats. Hyperglycemia was observed both in diabetic and diabetic hypertensive rats. Eugenol treatment did not have any effect in restoring the blood glucose levels to normal. Eugenol treatment could not show any favorable effect on body weight that had reduced in diabetic and diabetic hypertensive rats. Eugenol treatment had no effect on increased oxidative stress noticed in diabetic, hypertensive and diabetic hypertensive rats. Levels of liver function markers were raised in diabetic and diabetic hypertensive rats indicating liver damage and eugenol had no protective effect on liver damage. But elevated plasma creatinine, blood urea nitrogen levels and reduced plasma total protein in diabetic and diabetic hypertensive rats were restored to normal by eugenol treatment indicating protective effect of eugenol on kidney. Vascular reactivity was studied in-vitro by taking myographic recordings of aorta as described here; 1. Contractile response to phenylephrine and 5-HT. 2. Ach relaxation on phenylephrine and 5-HT induced contraction. 3. Eugenol relaxation of phenylephrine and 5-HT induced contraction. The lower mean log EC50 values of phenylephrine (-7.856 M) and 5-HT (-6.967 M) in hypertensive control and diabetic hypertensive rats (Phe: -7.960 M and 5-HT: -7.035 M) demonstrates hyper responsiveness of aortic smooth muscle to phenylephrine and 5-HT in comparison with normal control (Phe: -6.588 M and 5-HT: -5.700 M), and hyper-responsiveness of aorta to phenylephrine was partially reversed by eugenol treatment. But aorta from diabetic control rats showed hyper-responsiveness to phenylephrine (-7.137 M) and hypo reactivity to 5-HT (-5.247 M) compared to normal control rats. Eugenol treatment showed no impact on hyper-reactivity to phenylephrine or hypo-reactivity to 5-HT in diabetic rats. Maximum relaxation (% Emax) by acetylcholine in aorta on phenylephrine and 5-HT induced contractions was significantly reduced in diabetic, hypertensive and diabetic hypertensive rats. The effect was complete in hypertension and diabetic hypertension. Eugenol treatment had no significant change on acetylcholine induced relaxation in diabetic rats but significantly (P<0.001) improved relaxation in hypertensive and diabetic hypertensive rats. Eugenol produced dose dependent relaxation on phenylephrine and 5-HT induced contraction in all experimental groups but its effect was less potent than acetylcholine. Emax of eugenol on phenylephrine induced contraction was reduced in hypertensive, diabetic and diabetic hypertensive rats. Eugenol treatment to diabetic and diabetic hypertensive rats significantly improved Emax of eugenol. Eugenol relaxation on 5-HT induced contraction in diabetic control, hypertensive control and diabetic hypertensive rats were similar to control rats. The pathological changes observed in aorta, heart and kidney due to hypertension, diabetes and diabetic hypertension were not reestablished to normal with eugenol treatment. In conclusion, eugenol partially reversed phenylephrine and 5-HT induced vascular hyper-responsiveness in aorta and augmented the relaxation to acetylcholine in hypertensive and diabetic hypertensive rats but failed to produce a similar response in diabetic rats. However, eugenol had no role in maintaining blood glucose and serum nitrate levels indicating its inability to alleviate diabetes and hypertension. Further, eugenol treatment could not modulate oxidative stress and histopathological changes induced by diabetes and hypertension in plasma, heart and kidney. Further studies are needed to know the molecular mechanism involved in partial reversal of vascular dysfunction by eugenol.ThesisItem Open Access PHARMACOLOGICAL EVALUATION OF HERBAL METHIONINE IN METHIONINE DEFICIENCY AND IRON INDUCED STRESS IN BROILERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-12) SAI GOPAL, T; USHA RANI, M(MAJOR); GOPALA REDDY, A; ANAND KUMAR, AABSTRACT: A total of 120 sexed male broiler chicks of Vencobb strain of day-old age were randomly divided into 8 groups consisting of fifteen chicks in each group. Group 1 was maintained on methionine deficient diet and groups 3, 5 and 7 were supplemented with herbal methionine at level 1 and 2, and synthetic methionine, respectively. Group 2 was maintained as iron added methionine deficient diet and groups 4, 6 and 8 were supplemented with herbal methionine at level 1 and 2, and synthetic methionine, respectively. All the groups were maintained on iso-nitrogenous and iso-caloric diet for a period of 6 weeks. The performance parameters were recorded at weekly intervals. Antioxidant defense profile, biomarkers of hepatic damage, renal damage, protein profile and lipid profile were carried out at 2"d, 4'h and 6th week. At 5m week phytohaemagglutinin (PHA) index and at the end of 6th week histopathological studies were carried out. The methionine deficient and iron added methionine deficient diet groups had a significant (Pe0.05) reduction in body weight, GSH, activity of SOD and catalase, and PHA index, while FCR, and the concentration of TBARS, protein carbonyls and serum creatinine, and the activity of AST were significantly (Pc0.05) increased. Supplementation with herbal methionine at level 1 and 2 respectively in groups 3 and 5 resulted in a marked improvement in all the above parameters as compared to those of methionine deficient diet. Supplementation of herbal methionine at level 2 revealed the performance comparable with synthetic methionine supplementation. Histological abnormalities were also recorded in the liver, kidney, spleen and bursa in all groups, while the groups, 5 and 7 did not reveal any abnormalities on histopathology, while the treated groups 3, 5 and 7 revealed lesions of mild intensity or signs of regeneration. Thus, it is concluded that deficiency of methionine alone, and iron also induces biological damage by means of oxidative stress and the herbal methionine in test offered better performance. The beneficial effects of herbal methionine may be attributed to its antioxidant, anti-stress, hepato-protective principles and biological utilization was as good as synthetic methionine.