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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 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 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 ENDOCRINE DISRUPTING ACTIONS OF CADMIUM AND EXPERIMENTAL EVALUATION OF PROTECTION BY GREEN TEA EXTRACT(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2014-05) SHIVAKUMAR, PABBATHI; GOPALA REDDY, A(MAJOR); SRINIVASA RAO, G; ANJANEYULU, Y; RAMANA REDDY, Y; UDAYA KUMAR, MABSTRACT : An experimental study was conducted to evaluate the neuro-endocrine disrupting actions of cadmium and the effect of cadmium on the progeny that were born to cadmium exposed rats and to evaluate the protective role of green tea on neuro-endocrine disrupting actions of cadmium in Sprague dawley rats. Rats were randomly divided into 4 groups of 30 rats in each (male rats =12, female rats=18).Group 1 served as Sham control Group 2 treated with CdCl2, Group 3 treated with Green tea extract treatment and Group 4 Cd + green tea extract treatment. Blood was collected from all the groups at monthly intervals for analyzing sero-biochemistry (blood glucose, total cholesterol, HDL-cholesterol, triglycerides, total protein and albumin, biomarkers of cardiovascular, hepatic and renal pathology, and hormonal profile (thyroid profile, sex hormones). The key enzymes concerned with metabolism were assayed. Immune status was studied at the end of 3rd month by phytohaemagglutinin assay. Rats were subjected to neuro-behavioural studies at the end (Elevated plus maze and Morris water maze). Epididymal sperm count in males and estrous cycle pattern in females were studied. At the end of 3 months, 12 rats (6 males and 6 females) from each group were sacrificed to collect various organs and endocrine glands and subjected them to biochemical, histological and electron microscopic studies. Cadmium concentration was estimated in all the treated groups in kidney, testes, liver and brain at the end of 3 months. In all the groups, twelve (12) females were mated at the end of three months with male rats belonging to respective groups/treatments and the treatment was continued till 17th day of gestation. 50% of the pregnant rats in the respective groups were sacrificed on day 19 to study skeletal and soft tissue developmental anomalies and the rest were allowed to normal delivery. The pups of F1 generation from all the groups were kept till weaning (post-natal day 21) and were subjected to sero biochemical, neurobehavioural studies andthyroid hormone profile were estimated. There were significant alterations in sero-biochemistry biomarkers of cardiovascular, hepatic and renal pathology and hormonal profile thyroid profile, group 2 as compared to group 1.Treatment group revealed significant improvement in all the parameters as compared to group 2, while the combination treatment group 4 was found better The histological studies in group 2 revealed marked changes in all the organs studied, while groups 4 revealed moderate changes and groups 1 and 3 revealed no pathologically significant changes. The electron microscopy of kidney, testis and thyroid revealed marked alterations in architecture in group 2, while groups 4 revealed better architecture. There were no significant alteration in the TEM samples of the offspring and there were no skeletal abnormalities in the offspring as evidenced by skeletal staining. The results of the study revealed neuro-endocrine disrupting actions of cadmium and protctive role of green tea in cadmium toxicity. Further studies are warranted to know in detail on the endocrine disrupting actions of cadmium and protective role of green tea at various concentrations.ThesisItem Open Access INTERACTION STUDIES ON GYMNEMA SYLVESTRE WITH GLIMEPIRIDE AND INSULIN IN EXPERIMENTAL DIABETES MELLITUS IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2013-12) Srikanth, M.K; GOPALA REDDY, A(MAJOR); BHARAVI, K; MADHAVA RAO, T; KONDAL REDDY, K; ANAND KUMAR, AABSTRACT: An experimental study was conducted to evaluate the interaction of Gymnema sylvestre extract with insulin and glimepiride in diabetic Sprague dawley rats. Rats were randomly divided into 7 groups of 6 rats in each and blood glucose was estimated to ascertain group differences, if any. Group 1 was kept as normal control. Remaining 6 groups were induced diabetes by intraperitoneal injection of streptozotocin @ 40 mg/kg body weight. After 72 h, rats with blood glucose value of >200 mg/dl were included in the study (n=6). Treatment protocols were initiated 48 hrs post-confirmation of diabetes and continued for 2 months. Group 1: non-diabetic control, group 2: streptozotocin (40 mg/Kg i/p single dose)-induced diabetic (DM) control, group 3: Insulin treatment (4 U/kg b. wt. subcutaneously once daily), group 4: glimepiride treatment (4 mg/kg b. wt. orally once daily), group 5: Gymnema sylvestre methanolic leaf extract treatment ( 400 mg/kg b.wt. orally once daily), group 6: Insulin + Gymnema sylvestre methanolic leaf extract treatment (once daily) and group 7: glimepiride + Gymnema sylvestre methanolic leaf extract treatment (once daily). Blood glucose, body weights, sero-biochemical parameters, antioxidant profile in liver, kidney, brain and testis, ATPases, glucose 6 phosphate dehydrogenase (G6PD), cytochrome P450 (CYP450) activity and glycogen in liver, electron microscopy and histopathology of various tissues were studied at different time intervals. Also, pharmacokinetic interaction of glimepiride with Gymnema sylvestre extract was assessed. There were significant alterations in blood glucose, body weights and other biochemical parameters in diabetic control group 2 as compared to group 1. All the treated groups revealed significant improvement in all the parameters as compared to group 2, while the combination treatment in groups 6 and 7 was found better as compared to single agent-treated groups 3, 4 and 5. The histological studies revealed marked changes in group 2 in all the organs studied, while groups 3 to 5 revealed moderate changes and groups 6 and 7 revealed either minor changes or no pathologically significant changes. Group 1 was devoid of any histological alterations. The electron microscopy of kidney, pancreas and aorta revealed marked alterations in group 2, while groups 6 and 7 revealed better architecture. The pharmacokinetic study revealed the values of T1/2 (h), Ka (h-1), Ke (h-1) and Tmax (h) of glimepiride were siginificantly varied in Gymnema sylevestre pre-treated rats compared to normal rats administered with glimperide In conclusion, the study revealed that addition of Gymnema sylvestre leaf extract to insulin and glimepiride had positive pharmacodynamic interaction in improving the patho-biochemical alterations due to streptozotocin-induced diabetes mellitus in rats, which was evident from greater improvement in sero-biochemical and organ parameters in the groups that were treated using a combination of Gymnema sylvestre with either insulin or glimepiride as compared to individual agent-treated groups. Important pharmacokinetic parameters did not vary significantly when glimepiride was used in combination with Gymnema sylvestre leaf extract.ThesisItem Open Access TOXICODYNAMIC INTERACTION OF LEAD WITH CADMIUM AND THERAPEUTIC EVALUATION OF N-ACETYL L-CYSTEINE IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2011-07) ANlL KUMAR, B; GOPALA REDDY, A(MAJOR); RAVl KUMAR, P; MADHAVA RAO, T; ANAND KUMAR, AABSTRACT: An experimental study was conducted to evaluate the molecular mechanisms of lead and cadmium toxicity and their toxicodynamic interaction, and to evaluate therapeutic potential of N-Acetyl L-cysteine (NAC) against the toxicity in Wstar rats. After an acclimatization period of 2 weeks, rats were randomly divided into 8 groups comprising of 6 rats in each. Group 1 was kept as normal control throughout the experimental period, 2 was given NAC @ 300 mg per kg body weight administered by oral gavage, 3 was given lead (lead acetate @ I000 ppm in feed), 4 was given cadmium (cadmium chloride @ 300 ppm in feed), 5 was given lead + cadmium as per above doses in feed, 6 was given lead + NAC as per above schedule, 7 was given cadmium + NAC as per above schedule, and group 8 was given lead + cadmium + NAC as per above schedule for 3 months. Body weights, haematology (TEC, TLC, Hb, PCV, MCH and MCHC), activity of 6-ALAD and erythrocytic SOD, sero-biochemical parameters (ALT, CPK, troponins, plasma TBARS and serum creatinine), antioxidant profile (GSH, GST, TBARS and protein carbonyls) in liver, kidney, heart, testis and brain, ATPases and tissue lipids in liver and brain, neurotransmitters (Ach and glutamate) in brain, CYP450, glycogen and G6PD in liver, weight of testes, testicular LDH and sperm count, electron microscopy of kidney in cadmium exposed groups and histopathology of liver, kidney, testis and heart were studied. Also, interaction of lead and cadmium with zinc and copper in liver, kidney, heart, testis and brain was assessed. The present study revealed significant alterations in body weights, haematology, sero-biochemical parameters, antioxidant profile, ATPases, tissue lipid profile, neurotransmitter, CYPd50, glycogen, GGPD, weights of testes, testicular LDH, sperm count, and concentration of zinc and copper in toxic control groups 3, 4 and 5 as compared to control and NAC-treated groups. The toxic combination (Pb + Cd) group 5 showed significant alterations in most of the parameters studied as compared to Pb alone and Cd alone administered groups. All the NAC-treated groups revealed significant improvement in all the parameters. The histological studies of liver, kidney, testis and brain revealed marked changes in toxic control groups, while therapeutic groups revealed mild changes or no pathologically significant changes. Groups 1 and 2 were devoid of any alterations. The electron microscopy of kidney revealed marked alterations in kidney architecture in groups 4 and 5, while groups 7 and 8 revealed better architecture. The results of the investigation revealed that lead, cadmium' and their combination induced toxicity to the biological system due to the excess generation of free radicals and impairment of antioxidant defenses. Toxic effects were more pronounced in the group that received a combination of lead and cadmium suggesting positive toxicodynamic interaction. Use of NAC countered the adverse effects of lead and cadmium induced toxicity to a major extent suggesting its antioxidant potential owing to replenishment of tissue pool of GSH. Further, NAC administration reduced the extent of accumulation of lead and cadmium in various tissues.ThesisItem Open Access INTERACTION OF TRIGONELLA FOENUM GRAECUM WITH INSULIN AND GLIMEPIRIDE IN EXPERIMENTAL DIABETES MELLITUS IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2011-01) HARITHA, C; GOPALA REDDY, A(MAJOR); SRINIVASA RAO, G; ANJANEYULU, Y; MADHAVA RAO, T; RAMANA REDDY, YABSTRACT: An experimental study was conducted to evaluate the interaction of fenugreek seed powder with insulin and glimepiride in diabetic Sprague dawley rats. Rats were randomly divided into 7 groups of 8 rats in each and blood glucose was estimated to ascertain group differences, if any. Group 1 was kept as normal control. Remaining 6 groups were induced diabetes by intraperitoneal injection of streptozotocin @ 40 mg/kg body weight. After 72 h, rats with blood glucose value of >250 mg/dl were included in the study (n=8). Treatment protocols were initiated from day 2 post-confirmation of diabetes and continued for 8 wks. Group 1: non-diabetic control, group 2: streptozotocin (40 mg/Kg i/p single dose)-induced diabetic (DM) control, group 3: Insulin treatment (4 U/kg once daily), group 4: glimepiride treatment (4 mg/kg orally once daily), group 5: fenugreek seed powder treatment (1 g/kg orally once daily), group 6: Insulin + fenugreek seed powder treatment (once daily) and group 7: glimepiride + fenugreek seed powder treatment (once daily). Blood glucose, body weights, sero-biochemical parameters, antioxidant profile in liver, kidney, brain and testis, ATPases in liver and brain, relative weights of kidney and testes, electron microscopy of kidney and histopathology of various tissues were studied at different time intervals. Also, pharmacokinetic interaction of glimepiride with fenugreek seed powder was assessed. There were significant alterations in blood glucose, body weights and other biochemical parameters in diabetic control group 2 as compared to group 1. All the treated groups revealed significant improvement in all the parameters as compared to group 2, while the combination treatment groups 6 and 7 were found better as compared to single agent-treated groups 3 through 5. The histological studies revealed marked changes in all the organs studied, while groups 3 to 5 revealed moderate changes and groups 6 and 7 revealed either minor changes or no pathologically significant changes. Group 1 was devoid of any alterations. The electron microscopy of kidney revealed marked alterations in kidney architecture in group 2, while groups 6 and 7 revealed better architecture. Fenugreek seed powder treatment increased AUC and elimination half life of glimepiride in combination as compared to glimepiride-alone treated group, while the Cmax and tmax did not vary between groups 4 and 7. The results of the study revealed positive pharmacodynamic interaction between fenugreek and either insulin or glimepiride in improving the patho-biochemical alterations in diabetic rats. Further, there was a favourable pharmacokinetic interaction. Further studies are warranted to estimate P-gp and OATP activities along with CYP2C9 estimation for better understanding of pharmacokinetic interactions of fenugreek and glimepiride in diabetes mellitus.ThesisItem Open Access ANTICARCINOGENIC ACTIVITY OF CINNAMALDEHYDE AND NANO CINNAMALDEHYDE AGAINST MAMMARY CANCER IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-05) Ravi, Killi; Ravi Kumar, P(MAJOR); Bharavi, K; Rama Devi, V; VINOO, RABSTRACT: Cancer is a growing health problem in both developing and developed countries. Cancer chemoprevention is defined as the use of natural or synthetic agents that reverse, suppress or arrest carcinogenic and/or malignant phenotypic progression towards invasive cancer. The effectiveness of many anticancer drugs is limited due to their inability to reach the target site in sufficient concentrations and efficiently exert the pharmacological effect on affected cells without harming the healthy cells. Therefore, the search for new anticancer agents with better efficacy and fewer side effects is an ongoing phenomenon. Plant phenolic compounds are a group of secondary metabolites with wide pharmacological activities (Al-Rimawi et al., 2016). Plant polyphenols have drawn increasing attention due to their potent antioxidant properties and their marked effects in the prevention of different oxidative associated diseases such as cancer (Dai and Mumper, 2010). trans- Cinnamaldehyde is a phenolic compound found naturally in various species of the genus Cinnamomum and is used in preparing beverages, medicinal products, perfumes and cosmetics. Nanoparticles (NPs) are versatile agents with a variety of biomedical applications including drug delivery. Keeping this in view, the present study was undertaken to evaluate the anticancer potential of cinnamaldehyde (CNMA) and its nano preparation, the nano zinc cinnamaldehyde (CZN) in chemical induced rat breast cancer model. Further, cinnamaldehyde was also studied for its disposition kinetics in rats. For induction of mammary tumors, fifty days old virgin female Sprague-Dawley rats were administered with single oral dose of 20 mg dimethyl benz(a)anthracene (DMBA). Those rats positive for mammary tumors on 90th day were only selected for further studies. The study was carried out in seven experimental groups viz. normal control (C), DMBA control (DMBA-C), cinnamaldehyde prophylactic (CNMA-P), cinnamaldehyde treatment (CNMA-T), nano cinnamaldehyde control (CZN-C), nano cinnamaldehyde treatment (CZN-T) and tamoxifen (TAM). CNMA-P group rats received cinnamaldehyde (50 mg/kg b.wt) orally starting from the 45th day i.e. five days prior to the DMBA administration and continued to received the same until the end of the study on 120th day. CNMA-T, CZN-C and TAM treatment groups received the respective treatments for a period of 30 days beginning from the 90th day. All the animals were sacrificed on 120th day. Plasma concentration of cinnamaldehyde and cinnamic acid were estimated in rats administered with cinnamaldehyde at a dose rate of 500 mg/kg b.wt. From the pharmacokinetic study it was evident that cinnamaldehyde is rapidly converted into cinnamic acid (CA). Apparent volume of distribution (Vd) and plasma clearance (Cl) were high for CNMA but its AUC0-t, MRT and t1/2 values were low when compared to CA. Tumor volume, tumor multiplicity, tumor burden, body weight, tissue antioxidant and peroxidation status and various plasma biochemical parameters including total sialic acid (TSA) levels were assessed in all experimental groups. In addition tumor latency period was recorded in CNMA-P group. Mammary tumor tissues were subjected to light and electron microscopic examination for pathological changes and immunohistological studies for the presence of estrogen and progesterone receptors. Prophylactic treatment with cinnamaldehyde increased the tumor latency period and decreased the tumor volume, tumor burden and tumor multiplicity. Compared to DMBA control group, cinnamaldehyde and its nano preparation showed favourable results in terms of tissue antioxidant profiles and plasma biochemical parameters. Mild to moderate regressive changes were observed in various treatment groups. However tamoxifen treated rats showed greater extent of favourable changes in terms of tumor histology. The study revealed that, cinnamaldehyde (CNMA) and nano zinc cinnamaldehyde (CZN) have mild degree of anticancer effects, with CZN being little more effective than CNMA. However CNMA was more effective when used prophylactically than when used as a therapeutic agent. Hence, it is likely that CNMA can reduce the possibility of breast cancer occurrence and severity when used prophylactically.ThesisItem Open Access PHARMACODYNAMIC AND PHARMACOKINETIC EVALUATION OF VERBENONE IN NORMAL AND OVALBUMIN INDUCED ASTHMATIC RATS.(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2017-05) SURESH N NAIR; SRINIVASA RAO, G (Major); BHARAVI, K; SATHEESH, K; VINOO, RABSTRACT: Verbenone is a naturally occurring anti-aggregation pheromone generated by bark beetles from a host tree resin precursor, -pinene. Asthma is a common chronic disorder of the airways that is complex and characterized by variable and recurring symptoms, airflow obstruction, bronchial hyper-responsiveness, and an underlying inflammation. In the current study, the acute toxicity at the rate of 2000 mg/kg body weight single oral dosing, sub acute toxicity at the rate of 200 mg/kg oral dosing for 30days, pharmacokinetics of verbenone in normal healthy as well as ovalbumin-induced asthmatic animals and pharmacodynamic activity of verbenone in healthy and asthmatic animals were done. It was found that the verbenone did not produce any toxicity at the dose rate of 2000 mg/kg single oral dose making it a practically non toxic compound. Besides, repeated dose administration for 30 days at the dose rate of 200 mg/kg orally also did not produce any appreciable toxicity. It was also found that the drug did not produce any central nervous system effects as evidenced by lack of variation in spontaneous locomotor activity. In single dose non-compartmental pharmacokinetics in rats, it was found that the mean terminal elimination rate constant was 0.133±0.030/ h, mean elimination t1/2 of 4.368±0.888 hours, observed mean AUC0-inf was 1.361 ± 0.0520 μg/ml*h, observed AUMC0- inf was 6.441± 0.6356 μg/ml*h2, mean residence time was 6.304± 0.385 hours, Cmax and Tmax were 0.497±0.049 μg/ml and h hour, respectively. Apparent volume of distribution during terminal phase was 1134.798± 161.65 (mg)/(μg/ml) and apparent total body clearance of the drug from plasma was 147.1769±5.645 (mg)/(μg/ml)/h. Comparing the pharmacokinetics in normal and asthmatic animals it was found that though the Tmax did not change in disease condition all other parameters were significantly altered. In asthmatic animals maximum plasma concentration changed from 0.497 ± 0.049 to 0.322 ± 0.015 μg/ml. The AUC0-inf had reduced from 1.361 ± 0.052 to 0.828 ± 0.012 μg/ml*h. The observed AUMC 0-inf reduced from 6.441 ± 0.636 to 2.5 ± 0.104 μg/ml*h2. The mean residence time reduced from 6.3036± 0.385h to 3.101± 0.11h and t1/2 had reduced from 4.368 ± 0.888 to 2.149 ± 0.109 hours in asthmatic animals. This experiment indicated that the elimination of verbenone from plasma was faster in asthma and as a result more frequent administration will be required in asthmatic condition. The major pharmacodynamic activities assessed were modulatory activity in contractile response of spasmogens like acetylcholine, histamine and 5-HT in isolated rat tracheal chains from normal, verbenone alone given rats, ovalbumin treated asthmatic rats, dexamethasone treated asthmatic rats and verbenone- treated asthmatic rat. It was found that verbenone reduced the airway hyperresponsiveness to the spasmogens in asthma as indicated by the respective EC50’s. The EC50 value of acetylcholine in control animals was 6.17 X 10- 7M whereas the EC50 of verbenone pre incubated group was 7.24 X 10-7 M. In case of verbenone treated control animals, mean EC50 of 7.24 X 10-7 M while the ovalbumin treated asthmatic model showed a significant reduction in EC50 of acetylcholine with a mean of 1.10 X 10-7M. In dexamethasone (5mg/kg) and the test group with verbenone (200 mg/kg) concomitantly with ovalbumin, EC50 of 6.76 X 10-7M and 7.24 X 10-7M were observed. Histamine failed to produce a consistent bronchospasm in rat trachea. The EC50 values of 5- HT in control animals was 2.88 X10-7 M while that of verbenone pre-incubated group was 8.51X10-7 M which was significantly less than control animals. In case of verbenone treated control animals, mean EC50 of 8.51X10-7 M; the effect being significantly less than that of ovalbumin as well as control animals. The ovalbumin treated asthmatic model showed a significant reduction in EC50 of 5-HT with a mean of 1.07 X10-7 M and a range from 8.61 X10-8M to 1.33 X10-7M. The two treatment groups of induced asthma with ovalbumin, namely the standard treatment group which were given dexamethasone (5mg/kg) and the test group with verbenone (200 mg/kg) concomitantly with ovalbumin, showed significant change from ovalbumin alone treated asthmatic animals. The efficacy was comparable with control group, indicating the efficacy of the drugs in alleviating asthma. This was indicated by the mean EC50 of 3.89 X10-7M in case of concomitant dexamethasone and ovalbumin treated animals while in case of verbenone-treated asthmatic animals a mean EC50 of 4.47 X10-7M was noted, which was significantly less than ovalbumin group and even the control group. Though verbenone was able to relieve the airway hyperresponsiveness in asthmatic animals akin to dexamethasone, it failed to produce any direct relaxant effect on precontracted trachea, indicating its inability to act through any bronchodilatory receptors or ion channels. But a non quantifiable relaxation was noted with histamine receptor, indicating the probability of acting on histamine receptors. This finding was supported by histopathology of lung and trachea wherein the verbenone administration reduced the inflammatory status of these organs to near normal stage. Also it was noted that the drug improved the antioxidant status of vital organs like liver, kidney, lung, heart and plasma in asthmatic animals, restoring it to the normal status. Even in disease condition, administration of verbenone did not alter the structural and functional status of the organs grossly as indicated by the normal relative organ weights of the vital organs. Besides, it was found that verbenone possesses significant anti inflammatory activity as assessed by carrageenan induced paw oedema and wound healing activity as evidenced by improved excisional wound healing. But it was found to lack any central analgesic activity. From this it can be concluded that the verbenone is practically non toxic compound which is having a better pharmacokinetic profile so as to enable its use as a drug and exerts anti-asthmatic activity by virtue of its anti inflammatory and anti oxidant activity, though its effect on bronchodilatory mechanisms are not predominant