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ThesisItem Open Access TOXICOLOGICAL INVESTIGATIONS OF LEAD ACETATE AND ARSENIC TRIOXIDE FOLLOWING SIMULTANEOUS EXPOSURE IN ZEBRAFISH 3097(JAU, JUNAGADH, 2020-09) KHADAYATA ANIKETKUMAR VINODBHAI; H. B. PatelHeavy metal contamination of aquatic environment is one of the most common problem which affects the health of aquatic organisms including fish. Assessment of heavy metals induced oxidative damage and antioxidant defenses in fish can reflect the toxic effects of metals to the aquatic organisms and it can be helpful to mitigate further human exposure. Hence, present study was carried out to investigate the effects of lead acetate (PbAc, 20 µg/L) and arsenic trioxide (As2O3, 50 µg/L) alone as well as combined exposure for 21 days on anatomical confirmation, behavioral and oxidative stress parameters, mRNA expression of antioxidant genes and histopathological changes in adult female zebrafish. In the present study, 224 adult female zebrafish were randomly divided into four groups. The zebrafish of control group were maintained in normal condition without any treatment (R.O water with standard range of temperature and pH). The zebrafish of second group were maintained in R.O. water containing lead acetate at the strength of 20 µg/L. The zebrafish of third group were maintained in R.O. water containing arsenic trioxide at the strength of 50 µg/L of water. The zebrafish of fourth group were exposed to both lead acetate and arsenic trioxide at above mentioned strength. Noticeable signs of toxicity were not observed except freezing and hiding behavior in all toxicity groups. The exposure of lead acetate and arsenic trioxide alone and in combination did not affect the physical parameters like body weight, body width at middle and body length. Light-dark preference test was carried out at the day 7 and day 21 during the experiment. The mean values of time (sec.) spent in light compartment by zebrafish of control, Pb, As and Pb + As groups at day 21 were 316.17 ± 6.32, 230.00 ± 17.08, 261.50 ± 6.63, 211.50 ± 13.91 sec., respectively and time (sec.) spent in dark compartment were 283.83 ± 6.32, 370.00 ± 17.08, 338.50 ± 6.63 and 388.50 ± 13.91 sec., respectively. At 21st day, the zebrafish exposed to Pb, As and Pb + As had spent significantly (p < 0.05) less time in light compartment and more time in dark compartment as compared zebrafish of control group. At 21st day, the zebrafish exposed to Pb, As and Pb + As had significantly (p < 0.05) decrease in no. of entries in light compartment as compared to control group. Findings of present study indicates that lead and arsenic exposure has induced anxiety in zebrafish. Novel tank test was carried out at the day 7 and day 21 during the experiment. Novel tank test findings revealed that at 21st day, the zebrafish exposed to Pb, As and Pb + As had significantly (p < 0.05) decrease in time spent in upper zone (sec.) and increase in time spent in lower zone (sec.) of test tank as compared to zebrafish of control group. In present study, SOD activity in brain tissue of zebrafish was significantly (p < 0.05) decreased after 21 days of combined exposure of Pb + As compared control group zebrafish. After 21 days of Pb, As and Pb + As combined exposure, CAT activities in brain were non-significantly decreased, whereas levels of GSH in brain were significantly (p < 0.05) reduced and MDA levels were significantly (p < 0.05) increased as compared to control group zebrafish. In intestine, SOD activities were significantly (p < 0.05) decreased in Pb, As and Pb + As treated groups as compared to control group, whereas CAT activities and GSH levels were slightly decreased (non-significantly) as compared to control group. The MDA levels of zebrafish intestine were significantly (p < 0.05) increased in zebrafish of all treatment groups as compared to control group. Alone Pb exposure for 21 days had shown to produce significantly (p < 0.05) increased SOD activity in ovary of zebrafish as compared to control group. The CAT activities and GSH levels were slightly decreased (non significantly) and MDA levels were significantly (p < 0.05) increased in ovary of zebrafish exposed to Pb, As and Pb + As groups as compared to control group. The SOD mRNA expression level in brain was significantly down regulated by 12.96 fold (p < 0.05) in alone Pb exposure group as compared to control. After Pb, As and Pb + As exposure, CAT mRNA expression levels in brain were non significantly down regulated by 12.42, 2.25 and 4.05 fold, respectively as compared to control. Similarly, Nrf2 mRNA expression levels in brain after Pb alone and Pb + As combination group were non-significantly down regulated by 19.04 and 1.04 fold, respectively. Findings of mRNA expressions were concomitant with activities of respective enzymes in brain of zebrafish. In intestine, after 21 days of Pb and As alone exposure, SOD and CAT mRNA expressions were down regulated. Alone As exposure showed up regulation of Nrf2 mRNA expression in intestine of zebrafish. In ovary, SOD mRNA expression level was significantly (p < 0.05) up regulated by 0.03 fold in Pb + As exposure group. After Pb and As alone exposure, CAT mRNA expression levels in ovary were non-significantly down regulated by 4.89 and 1.47 fold, respectively. The Nrf2 mRNA expression levels in ovary were non-significantly up regulated by 0.63, 0.14 and 0.10 fold in Pb, As and Pb + As exposure group, respectively as compared to control. In the present study, combined exposure of Pb and As (Pb + As) had increased SOD and Nrf2 mRNA expressions in ovary of zebrafish, but the extent of effect was much lower as compared to Pb and As alone exposure. After Pb, As and Pb + As exposure, microscopic examination revealed mild to moderate degree of histopathological changes in brain, intestine, ovary, liver, kidney, gill and retinal layer of eye in adult female zebrafish. In conclusion, lead acetate and arsenic trioxide alone and in combination exposure for 21 days had shown to produce anxiety like behavior and oxidative stress mediated alterations in brain, intestine and ovary of zebrafish. Both lead acetate and arsenic trioxide alone and in combination affected the SOD, CAT and Nrf2 mRNA expressions in brain, intestine and ovary of zebrafish. The both metals alone and in combination resulted in significant histopathological changes in brain, intestine, ovary, liver, kidney, gills and retinal layer of eye in zebrafish. However, exposure of lead acetate (20 µg/L) and arsenic trioxide (50 µg/L) for 21 days did not show the synergistic toxicological effects upon simultaneous exposure in adult female zebrafish.ThesisItem Open Access “EVALUATION OF ANTIOXIDANT AND IMMUNOMODULATORY ACTIVITIES OF DAUCUS CAROTA L. ROOTS ON CYCLOPHOSPHAMIDE INDUCED IMMUNOSUPPRESSED RATS” 3066(JAU, JUNAGADH, 2020-09) VAJA RAHULKUMAR KARSHANBHAI; C. M. MODIThe present study was carried out to evaluate the antioxidant and immunomodulatory activities of Daucus carota L. polysaccharide (DCP) on cyclophosphamide induced immunosuppressed rats. In this study, forty-two male SD rats were divided into seven different groups, namely normal control group (C1), vehicle control group (C2), toxic control group (C3), standard treatment group (ST), treatment groups (T1, T2 and T3). All rats were treated with cyclophosphamide (CYP) at the dose rate of 100 mg/kg, i.p on 9th and 16th day of experiment except normal control group (C1). In this study, levamisole was taken as standard drug and given at the dose rate of 2.5 mg/kg b.w, p.o every 2 day for 21 days in rats. Rats of T1, T2 and T3 groups were treated with polysaccharide extracts of Daucus carota L., respectively at the dose rate of 50 mg/kg, 100 mg/kg and 150 mg/kg p.o for 21 days. Rats of C2 group was treated with carboxymethyl cellulose (1%, p.o for 21 days). The efficacy of Daucus carota L. polysaccharide (DCP) extract was assessed based on clinical signs, organ indices, hematological, biochemical alteration, alteration in humoral and cell mediated immune response, oxidative stress parameters, changes in hepatic mRNA expression of antioxidant gene, gross and histopathological examination of major functional organs. Rats of T1, T2, T3 and ST groups did not show any clinical signs. The clinical signs observed in toxicity group (C3) were exhibited in appetence, hair loss, decrease body weight and lower food consumption throughout the period of the experiment. Phytochemical analysis of polysaccharide extract of Daucus carota L. showed presence of sugar. The total carbohydrate content of the polysaccharide extract of Daucus carota L. was estimated to be 94.08 ± 2.03 % of extract. The FI-IR fingerprint of the polysaccharide extract of Daucus carota L. shown presence of functional group like O-H, C-H, C=O and C-O. Treatment with DCP increased the liver, kidney and spleen indices in a dose dependent manner as compared to the cyclophosphamide group. DCP administration (150 mg/kg) showed higher indices, indicating the protective effect of DCP against the cyclophosphamide induced myelosuppression. The toxicity effect of CYP on hematological parameter was characterized by significant (p<0.05) decrease in hemoglobin, packed cell volume, platelets count, total erythrocytes count and total leukocyte count. The elevation of those parameters values restored to normal level when CYP treated rats with different dose of DCP in treatment groups (T1, T2 and T3). Serum total protein, albumin and globulin were decreased in toxicity group (C3) while in treatment groups T1, T2 and T3 were restored the biochemical parameter alterations by administration DCP polysaccharide extract. Cell mediated immune response evaluated by DTH observed increase in paw skin thickness in groups treated with DCP (T1, T2 and T3) as compared to toxicity group (C3). Neutrophil adhesion (%) was modified toward the normal level at a higher dose of DCP as compared to the toxicity control group. The DCP treatmentsignificantly stimulated splenic T and B-lymphocyte mediated proliferation as compared to the toxicity control group. The DCP treatment significantly improved the HA titre in treatment groups (T1, T2 and T3) as compared to that of the toxicity control group (C3) which is an indication of antibody production. In CYP-treated rats, the DCP restored the levels of TNF-α and IL-10 at dose-dependent manner. The DCP treatment (T1, T2 and T3) significantly enhanced the antioxidant activity in CYP-treated rats, as shown by the evaluation of SOD, CAT and GSH activities, as well as MDA levels in liver, kidney, spleen and intestine. Moreover, hepatic CAT, SOD, GST and GPx mRNA expression of genes for the antioxidant enzymes, were down-regulated by CYP treatment which was reversed by DHA. Upon gross examination of all major organs (liver, spleen, kidney and intestine) mild to moderate changes in gross pathological lesions in all treatment groups have been observed while the rats of C3 group showed severe changes as compared to normal control group. Histopathological findings revealed alterations in different organs (liver, kidney, spleen and intestine) of toxicity group (C2), which was markedly improved by treatment of DCP in dose-dependent manner. In conclusion, daily oral administration of polysaccharide fraction from Daucus carota L. roots at the dose rate of 100 mg/kg b.w have ameliorated cyclophosphamide induced alterations in rats.ThesisItem Open Access “TOXICOLOGICAL INVESTIGATIONS OF CADMIUM CHLORIDE AND MERCURY CHLORIDE FOLLOWING SIMULTANEOUS EXPOSURE IN ZEBRAFISH” 3064(JAU, JUNAGADH) PATEL UTSAV NARENDRAKUMAR; U. D. PatelThe present study was carried out to investigate the toxicological interaction of cadmium chloride (CdCl2, 1 mg/L) and mercury chloride (HgCl2, 30 µg/L) following simultaneous exposure for 21 days in adult female zebrafish. In the present study, 224 adult female zebrafish (56 fish in each group) were randomly divided into four groups. The zebrafish of control group were maintained in normal condition without any treatment (R.O water with standard range of temperature and pH). The zebrafish of second group were maintained in R.O. water containing cadmium chloride at the strength of 1 ppm. The zebrafish of third group were maintained in R.O. water containing mercury chloride at the strength of 30 µg/L of water. The zebrafish of fourth group were exposed to both cadmium chloride and mercury chloride at above mentioned strength. Noticeable signs of toxicity were not observed except freezing and hiding behavior in all toxicity groups. The exposure of cadmium chloride and mercury chloride alone and in combination did not affect the physical parameters like body weight, body width at middle and body length. Light-dark preference test and novel tank test were also carried out for the assessment of changes in behavior of zebrafish at the day 7 and day 21. The total time spent in light and total no. of entries in light by zebrafish were significantly lower in all toxicity groups as compared to control group at the day 7. Among all toxicity groups, Cd-exposed group significantly spent less time in the light compartment with less number of entries in the light compartment as compared to that observed in Hg and Cd + Hg-exposed zebrafish at the day 7. Total time spent in the dark compartment and total number of entries in the dark compartment by zebrafish of all toxicity groups were significantly higher and lower, respectively, as compared to that of control group. The Cd-exposed zebrafish spent significantly more time in the dark compartment with less number of entries in the dark compartment as compared to Hg and Cd + Hg-exposed zebrafish at day 7. The latency time to white side was significantly higher in all toxicity groups as compared to that of control group. Among all groups, latency to white was found significantly higher in Hg-exposed group as compared to Cd and Cd + Hg-exposed groups at day 7. Similar pattern of alterations in behavioral parameters were observed in all toxicity groups at day 21. However, Cd and Cd + Hg-exposed groups were at par with all behavioral parameters except latency to white side at day 21. The latency to white side was non-significantly higher in all toxicity groups as compared to that of control group at day 21. In context to the novel tank test, total time spent in lower zone by zebrafish of all toxicity groups were significantly higher as compared to that of control group at day 7 and 21. The changes in behavior clearly indicates anxiety in zebrafish after exposure to Cd, Hg and Cd + Hg. In the brain, SOD activity was significantly lower in all toxicity groups as compared to that of control group. The SOD activity in Hg-exposed group was found lower as compared to Cd and Cd + Hg exposed groups. The CAT activity in brain of zebrafish of all toxicity groups were non-significantly lower as compared to that of control group. In Cd and Cd + Hg-exposed groups, GSH level was found significantly lower as compared to that of control group. However, the GSH level in Hg-exposed group was non-significantly lower as compared to control group. The alteration in activity of SOD or CAT or GSH resulted in higher level of MDA in brain of zebrafish in all toxicity groups. As compared to group exposed to Cd and Cd + Hg, Hg-exposed group displayed significantly more lipid peroxidation in brain. The SOD activity in intestine was significantly increased in all toxicity groups as compared to that of control group. The Hg-exposed group exhibited significantly higher SOD activity as compared to Cd and Cd + Hg-exposed groups. The CAT activity in the intestine of zebrafish of all toxicity groups were non-significantly lower as compared to that of control group. The GSH level in the intestine of zebrafish in all toxicity groups were significantly lower as compared to that of control group. The MDA level in the intestine of zebrafish in all toxicity groups were significantly higher as compared to that of control group. Exposure to Hg resulted in significantly more lipid peroxidation in intestine of zebrafish as compared to that produced by exposure to Cd alone and Cd + Hg. In the ovary, the SOD and CAT activities were non-significantly lower in all toxicity groups as compared to that of control group. The levels of GSH and MDA were significantly lower and higher, respectively in Cd + Hg-exposed group as compared to other groups. The GSH and MDA levels in Cd and Hg exposed groups were non-significantly lower and higher, respectively, as compared to that of control group. The zebrafish exposed to Cd, Hg and Cd + Hg exhibited non-significantly down-regulation of SOD, CAT and NrF2 mRNA expression level in the brain. In Cd + Hg exposed group, NrF2 mRNA expression level in the brain was found to be significantly down-regulated as compared to that observed in Cd and Hg-exposed groups. In intestine, SOD and CAT mRNA expression levels were non-significantly up-regulated. However, the NrF2 mRNA expression level was to be found non significantly down-regulated in all toxicity groups. The SOD mRNA expression level in the ovary of zebrafish exposed to Hg was significantly up-regulated as compared to that of Cd and Cd + Hg-exposed groups. The CAT and NrF2 mRNA expression levels in the ovary were non-significantly lower in all toxicity groups. The exposure of cadmium (1 mg/L), mercury (30 µg/L) and cadmium (1 mg/L) + mercury (30 µg/L) in combination resulted in various histopathological changes in brain, intestine, ovary, liver, kidney, gills and retinal layer of zebrafish. In conclusion, cadmium chloride and mercury chloride alone and in combination produced anxiety like behavior, oxidative stress mediated alterations in brain, intestine and ovary of zebrafish. Both cadmium chloride and mercury chloride alone and in combination affected the SOD, CAT and NrF2 mRNA expression in brain, intestine and ovary of zebrafish. The both metals alone and in combination resulted in significant histopathological changes in brain, intestine, ovary, liver, kidney, gills and retinal layer. However, exposure to cadmium chloride for 21 days did not show the toxicological interaction upon simultaneous exposure with mercury chloride in adult female zebrafish.ThesisItem Open Access “GASTROINTESTINAL AND HEPATORENAL PROTECTIVE EFFECTS OF QUERCETIN AND CURCUMIN AGAINST CADMIUM INDUCED TOXICITY IN RATS” 2827(JAU, JUNAGADH, 2019-06) SHREESHA RAO S; U. D. PatelThe present experiment was carried out to evaluate the gastrointestinal and hepatorenal protective effect of quercetin and curcumin alone as well as in combination against cadmium-induced toxicity in rats. The study was performed on 36 male rats which were randomly divided into six groups based on their body weights at the age of 8-9 weeks. Rats of group C1 were kept as normal control. Rats of toxic control group (C2), vehicle group (C3), quercetin treatment group (T1), curcumin treatment group (T2) and quercetin and curcumin in combination treatment group (T3) were administered with cadmium in drinking water (100 ppm) for 28 days. Rats of vehicle group (C3) were administered with corn oil (vehicle). Rats of group T1, T2 and T3 were orally administered with quercetin (50 mg/kg, P.O.), curcumin (100 mg/kg, P.O.) and both quercetin and curcumin in combination, respectively for 28 days. Noticeable signs of toxicity were not observed in rats of any groups except hair fall and diarrhea in toxicity control and vehicle groups, which were less in treatment groups (T1, T2 and T3). Cadmium exposure in rats did not affect the feed consumption. Body weight of rats of all groups were also not altered during the experimental period. Cadmium treatment resulted in significant (P<0.05) decrease in kidney weight and kidney body weight ratio. The weight of kidney and kidney body weight ratio was partially improved by curcumin treatment (T2). Cadmium exposure to rats for 28 days did not produce significant effect on hematological parameters. However, significantly (P<0.05) higher level of ALT, AST and ALP in the serum of cadmium exposed rats were observed suggesting of Cd related injury to the liver. Rats treated with quercetin and curcumin alone as well as in combination of both significantly (P<0.05) lowered the levels of AST and ALP as compared to cadmium-exposed and vehicle-treated group (C1 and C2). The total bilirubin level was significantly (P<0.05) increased in cadmium-exposed and vehicle treated group (C2 and C3) as compared to that of control group (C1). Quercetin treated group (T1), curcumin-treated group (T2) and the group treated with quercetin and curcumin in combination (T3) significantly (P<0.05) decreased total bilirubin level as compared to that of animals of toxic control group (C2). The blood glucose level was significantly (P<0.05) higher in cadmium-exposed group (C2) as compared to that of control group (C1) and it was lowered in quercetin treated group and group treated with combination of quercetin and curcumin. Exposure to cadmium caused significant increase in MDA and non-significant decrease in SOD, catalase and GSH in blood of rats. Quercetin and curcumin in combination non-significantly increased SOD activity, catalase activity and GSH level. GSH level was well improved in quercetin treatment group (T1). All treatments (T1, T2 and T3) significantly decreased the cadmium induced lipid peroxidation indicating low level of MDA. In intestine, MDA level and SOD enzyme activity were significantly (P<0.05) increased upon cadmium treatment due to lipid peroxidation. Non-significant decrease in catalase activity and GSH level were observed upon cadmium exposure in intestine tissue. Quercetin and curcumin alone as well as in combination resulted in reduction of SOD activity, lipid peroxidation and improvement in the catalase activity and GSH level in intestine. However, significantly (P<0.05) higher level of GSH was observed in curcumin-treated group (T2) as compared to that of animals of toxicity control group (C2). In liver, significant increase in MDA level, non-significant increase in SOD activity and decrease in catalase activity caused the lipid peroxidation and damage to the hepatocytes. The quercetin treatment (T1) reduced the increased MDA level and SOD activity in liver, simultaneously increased catalase and GSH activity. Curcumin treatment (T2) significantly decreased the SOD activity, MDA level and significantly increased the GSH level and partially improved the catalase activity However, quercetin when administered along with curcumin was able to reduce the MDA level significantly by stimulating the GSH level and higher catalase activity as compared to that of toxicity control group (C2). In kidney, cadmium exposure resulted in increase in lipid peroxidation as indicated by increased MDA level. Non-significant increase in SOD activity, significant decrease in catalase activity with no significant effect on GSH level was observed upon cadmium exposure. Quercetin and curcumin alone as well as in combination non-significantly reduced the SOD activity and improved the catalase activity and also stimulated the GSH level which reduced the lipid peroxidation. Sub-acute cadmium exposure at 100 ppm level altered normal architecture of stomach, intestine, liver and kidney in rats. Administration of quercetin and curcumin alone as well as in combination partially protected the stomach, intestine and liver from cadmium-induced oxidative damage. However, quercetin has more protective effect against cadmium-induced histopathological changes in kidney as compared to curcumin. As compared to individual treatment of quercetin and curcumin alone, the combination of both agents significantly prevented the histopathological changes caused by cadmium-induced oxidative stress. Quercetin and curcumin alone as well in combination may be useful for prevention of oxidative stress caused by continuous exposure to low level of xenobiotics. However, further study is needed to confirm the efficacy and the pathways of action of quercetin and curcumin against oxidative stress caused by different environmental toxicants.ThesisItem Open Access “AMELIORATING POTENTIAL OF PHYLLANTHUS EMBLICA AND TRIBULUS TERRESTRIS AGAINST MERCURIC CHLORIDE INDUCED TOXICITY IN RATS” 2825(JAU, JUNAGADH, 2019-06) LADUMOR VIPULKUMAR CHANABHAI; C. M. MODIThe present study was carried out to evaluate the ameliorating potential of Phyllanthus emblica and Tribulus terrestris against mercuric chloride induced toxicity in rats. In this study, forty-two male SD rats were divided into seven different groups, namely normal control group (C1), toxic control group (C2), vehicle control group (C3), positive control group (C4), Phyllanthus emblica extract group (T1), Tribulus terrestris extract group (T2) and bi-herbal mixture group (T3). Rats of group C2 was treated with mercuric chloride at the dose rate of 2 mg/kg, PO for 28 days. In this study, vitamin E was taken as standard drug and given at the dose rate of 100 mg/kg, PO for 28 days to C4 group along with mercuric chloride in rats. Rats of T1 and T2 groups were treated with flavonoid rich fraction of the hydro-alcoholic extracts of P. emblica and saponin rich fraction of the hydro-alcoholic extracts of T. terrestris, respectively at the dose rate of 100 mg/kg, PO for 28 days along with mercuric chloride. Rats of T3 group was treated with bi-herbal mixture (100 mg/kg, PO for 28 days). The efficacy of treatment was assessed based on clinical signs, body weight, hematological, biochemical alteration, oxidative stress parameters, gross and histopathological examination of major functional organs. Rats of T1, T2, T3 and C4 groups given mercuric chloride along with P. emblica, T. terrestris, bi-herbal mixture and vitamin E, respectively did not show any clinical signs. The clinical signs observed in toxic rats (C2) were inappetance, hair loss, sluggish movement, depression and diarrhea throughout the period of the experiment. However, there was observed no mortality in all treatment groups. Phytochemical analysis of flavonoid rich extract of P. emblica and saponin rich extract of T. terrestris were showed presence of various phytoconstituents like flavonoid, saponin, tannin etc. TLC finger print of extracts revealed the presence of the gallic acid, quercetin and rutin in the P. emblica and protodioscin in the T. terrestris. The combination of P. emblica and T. terrestris extract shown 55.24 per cent maximum inhibition of free radical scavenging activity at 9 µg/mL concentration. The saponin rich fraction of T. terrestris showed maximum inhibition of albumin denaturation at the dose of 600 µg/ml which possess strong anti inflammatory activity. Total phenolic content and total flavonoid content were found highest in P. emblica extract followed by T. terrestris extract.The average feed consumed (g/day/rat) by the rats of all the treatment groups did not show significant difference during the experiment. The mean body weight (g) in rats of all the treatment groups increased steady throughout the experimental period. The sub-acute toxicity effect of mercury on hematological parameter was characterized by significant (p<0.05) decrease in hemoglobin, packed cell volume, total erythrocytes count and total leukocyte count. The elevation of those parameter values restored to normal level when mercuric treated rats with P. emblica, T. terrestris and their combination treatment. Non-significant change were observed in mean values of MCHC and MCH in all the treatment groups throughout the experimental period A significant (p<0.05) higher in serum AST, ALP, BUN and creatinine were recorded in mercury treated groups, On the other way there were significantly decreased serum total protein, albumin. The treatment groups T1, T2 and T3 were restored the biochemical parameter alterations induced by mercury. he exposure of rats to mercuric chloride induced oxidative damage caused significant a decrease in GSH level, CAT and SOD activities, significant an increase in MDA level of the different tissues, which were restored near to normal in rats treated with treatment groups (T1, T2 and T3). The mercuric chloride intoxicated rats showed significant decrease in AChE activity when compared to normal control group. The rats treated with treatment T3 was significantly increased in brain tissues AChE activity when compared to toxicity group. Upon gross examination of all major organ (liver, intestine, stomach and brain), no gross pathological lesions in all treatment groups have been observed while stomach of the rats of C2 group showed congested and ulcerated as compared to normal control group. Histopathological findings revealed alterations in different organs (liver, kidney, intestine and brain) of toxicity group (C2), which was markedly improved by treatment of P. emblica and T. terrestris and their combination extract. In conclusion, the administration of P. emblica, T. terrestris and their combination are effective in preventing hematological, biochemical alterations and oxidative stress caused by mercuric chloride in rats. Therefore, mercuric chloride induced toxicity is reduced by the extract of P. emblica, T. terrestris and their combination. Ameliorating effect of fruit extract of the P. emblica, T. terrestris might be due to the presence of the active constituents, which possess strong antioxidant activity and provoke free radical scavenging enzyme system. The administration of P. emblica, T. terrestris and their combination extract proved to be beneficial in ameliorating the mercuric chloride-induced toxicity.ThesisItem Open Access “AMELIORATION OF OXIDATIVE DAMAGE IN BRAIN, TESTES AND HEART OF RATS BY QUERCETIN AND CURCUMIN” 2822(JAU, JUNAGADH, 2019-06) MAKWANA CHANDRASINH NARANBHAI; U. D. PatelThe present experiment was carried out to evaluate the effect of quercetin and curcumin against cadmium induced oxidative damage in brain, testes and heart of rats. The study was conducted on 36 male rats which were randomly divided into six groups based on their body weights at the age of 8-9 weeks. Rats of group C1 were kept as normal control. Rats of toxic control group (C2), vehicle group (C3), quercetin treatment group (T1), curcumin treatment group (T2) and, quercetin and curcumin in combination treatment group were administered with cadmium in drinking water (100 ppm) for 28 days. Rats of vehicle group (C3) were administered with corn oil (vehicle). Rats of group T1, T2 and T3 were orally administered with quercetin (50 mg/kg, P.O.), curcumin (100 mg/kg, P.O.) and both quercetin and curcumin in combination, respectively for 28 days. The symptoms of toxicity, feed consumption, body weight gain, oxidative stress parameters, AChE activity in the brain cortex, plasma nitric oxide level, epididymal sperm parameters and gross and histopathological changes in brain, testes and heart were studied. Noticeable signs of toxicity were not observed in rats of any groups except hair fall which was less in other treatment groups. Cadmium exposure in rats had no significant (P>0.05) effect on feed consumption. The body weight gain was reduced during 4th week only which was prevented by the treatment of quercetin and curcumin in combination. In brain cortex, the SOD and CAT activity in brain cortex was slightly lowered with significant increased level of MDA in rats of cadmium-exposed and vehicle-treated groups. Quercetin treatment slightly improved SOD and catalase activity (non-significantly) with significant higher level of GSH in brain cortex which resulted in lower value of MDA level of brain cortex. Curcumin treatment also significantly improved SOD activity and GSH level of brain cortex. Animals treated with quercetin and curcumin in combination increased SOD and catalase activity along with improved GSH level in brain cortex which resulted in significantly lowered level of MDA. Quercetin in combination of curcumin showed more prevention to lipid peroxidation in brain cortex. In testes, cadmium exposure to animals caused slight increase in SOD activity, unaltered catalase activity and level of GSH as compared to normal control animals. The lipid peroxidation in testes was higher. Quercetin treatment was able to increase CAT activity which resulted in low level of MDA. Curcumin treatment did not improve oxidative stress parameters. Combined treatment of quercetin and curcumin resulted nearly normal activity of SOD, higher activity of CAT with lowest level of MDA amongst all groups. Improved SOD activity in heart with lower level of plasma nitric oxide by quercetin alone treatment might be responsible for reduction of MDA level (non significant) in the heart of rats. Curcumin treatment could not be able to alter the cadmium-induced lipid peroxidation in heart of rats. However, quercetin when administered along with curcumin were able to manage higher activity of SOD and CAT (non-significant) along with significant GSH stimulating effect resulted in decrease in MDA level (less lipid peroxidation) in the heart of rats (T3). Activity of AChE in the brain cortex was non-significantly (P>0.05) decreased in cadmium-exposed control groups. Group treated with quercetin (T1), curcumin (T2) and quercetin and curcumin in combination (T3) showed slight higher AChE activity (non-significant, P>0.05) as compared to that of animals of cadmium-exposed and vehicle-treated groups (C2 and C3). Plasma nitric oxide level was significantly (P<0.05) increased in cadmium-exposed and vehicle-treated groups (C2 and C3). Nitric oxide levels in plasma of rats treated curcumin alone (T2) as well as quercetin and curcumin (T3) were significantly higher. The oxidative damage following sub acute cadmium exposure at 100 ppm level through oral route was mainly due to increased level of nitric oxide in rats. In epididymis, mean values of total epididymal sperm count, epididymal sperm motility, total epididymal live sperm count were significantly altered in cadmium-exposed rats which were reversed by the treatment of quercetin, curcumin alone as well as in combination. Sperm deformities in rats of different groups were non-significantly differ from each other. Cadmium exposure for 28 days at 100 ppm caused histopathological alterations in brain cortex, testes and heart of rats under study. The quercetin and curcumin when given alone as well as in combination partially prevented the alterations caused by cadmium-induced oxidative stress. As compare to individual treatment of quercetin and curcumin alone, the combination of both agents produced more ameliorating effect against cadmium-induced histopathological changes.ThesisItem Open Access PHARMACOLOGICAL EFFECTS AND SAFETY PROFILE FOLLOWING ADMINISTRATION OF POLY-HERBAL EXTRACT IN DIABETIC RATS(JAU,JUNAGADH, 2018-10) SHAUL AHMED R.; Dr. U. D. PATELDiabetes mellitus describes a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. It is one of refractory diseases identified by Indian Council of Medical Research for which an alternative medicine is a need for the treatment. Looking to the pharmacological properties of different plant varieties like Allium cepa, Trigonella faenum-graecum, Tinospora cordifolia, Gymnema sylvestre, Syzigium cumini and Momordica charantia, the present study was carried out to evaluate the pharmacological effects and safety profile following administration of poly-herbal extract in streptozotocin induced diabetic rats. Thirty Srague-Dawley rats were randomly divided based on body weight in five groups (C1, C2, C3, T1 and T2). Rats of four groups (C2, C3, T1 and T2) were injected with streptozotocin to induce diabetes. Rats of group C1, C2, and C3 were kept as normal, diabetic and standard control, respectively. Rats of group C3 were administrated with glibenclamide (5 mg/kg, PO for 28 days). Rats of group T1 and T2 were treated with mixture of polyherbal extract at 100 and 200 mg/kg, respectively orally for 28 days. After induction of diabetes, clinical symptoms of diabetes were observed in rats of diabetic control group. These symptoms were mild to moderate in all other treatment groups compare to diabetic control group. Administration of poly-herbal extract significantly (P<0.05) reduced the blood glucose level in T1 and T2 experimental groups especially at 4th week of experiment. However the blood glucose level was maximally reduced in glibenclamide treated group. In oral glucose tolerance test (OGTT), the blood glucose levels in rats of different treatment groups were significantly (P<0.05) increased at 30 and 60 minutes after oral glucose administration at dose of 2 g/kg. The little higher blood glucose levels in group T1 and T2 indicates inability of poly-herbal extract to reduce the increased blood glucose level at day 15. This result was supported by no effect of poly-herbal extract treatment on regulation of blood glucose level up to 3 weeks of our study period. In lipid profile, levels of total cholesterol, triglyceride and LDL-cholesterol were non-significantly (P>0.05) increased while levels of HDL-cholesterol were significantly (P<0.05) lower in diabetic control group compared to other groups. The mean levels of total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol in rats treated with poly-herbal extract were found comparable to normal control rats. In hematological parameters, mean values of PCV and TEC in rats of diabetic control were significantly (P<0.05) lower compared to other groups. Whereas, the mean value of above parameters in rats treated with poly-herbal extract was significantly (P<0.05) higher than other treatment groups. In biochemical parameters, mean values of ALT, AST, BUN and creatinine were non-significantly increased while, the mean values of total protein, albumin, globulin and total bilirubin were found non-significant in all groups compared to control rats. In case of antioxidant parameters evaluated from blood, liver, kidney and pancreas tissue samples, mean values of SOD, catalase and GSH in diabetic control group were non-significantly (P>0.05) decreased compared to control group. Whereas, the mean values of SOD, catalase and GSH were non-significantly (P>0.05) higher in glibenclamide as well as in poly-herbal treated groups compared to diabetic control group at the end of study period. Upon gross examination of pancreas, no appreciable gross lesions in all treatment groups have been observed. Macroscopic examination of liver and kidneys of experimental rats of diabetic control group shown congestion, paleness and mild to moderate enlargement. No appreciable macroscopic lesions have been observed in the spleen, heart and lung of rats in all treatment groups. The histopathological changes of pancreas of rats of diabetic control group (C2) revealed varying degree of structural changes as well as degenerative changes, vascular changes and infiltration of inflammatory cells with loss of normal architecture of parenchyma and changes in kidney were also observed in rats of diabetic control group. No appreciable histopathological lesions have been observed in the liver, spleen, heart and lung of rats in all treatment groups. In conclusion, administration of poly-herbal extract at the dose rate of 200 mg/kg, PO for 28 days have shown ameliorating effect on glucose, lipid profile and antioxidant levels and pathological lesions in diabetic rats. Though the findings of this short term study are encouraging, further detailed investigation is required to determine the effect of long term administration of poly-herbal extract in diabetic ratsThesisItem Open Access PHARMACOLOGICAL EFFECTS AND SAFETY PROFILE FOLLOWING ADMINISTRATION OF POLY-HERBAL EXTRACT IN DIABETIC RATS(JAU, JUNAGADH, 2018-10) SHAUL AHMED R.; Dr. U. D. PATELDiabetes mellitus describes a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. It is one of refractory diseases identified by Indian Council of Medical Research for which an alternative medicine is a need for the treatment. Looking to the pharmacological properties of different plant varieties like Allium cepa, Trigonella faenum-graecum, Tinospora cordifolia, Gymnema sylvestre, Syzigium cumini and Momordica charantia, the present study was carried out to evaluate the pharmacological effects and safety profile following administration of poly-herbal extract in streptozotocin induced diabetic rats. Thirty Srague-Dawley rats were randomly divided based on body weight in five groups (C1, C2, C3, T1 and T2). Rats of four groups (C2, C3, T1 and T2) were injected with streptozotocin to induce diabetes. Rats of group C1, C2, and C3 were kept as normal, diabetic and standard control, respectively. Rats of group C3 were administrated with glibenclamide (5 mg/kg, PO for 28 days). Rats of group T1 and T2 were treated with mixture of polyherbal extract at 100 and 200 mg/kg, respectively orally for 28 days. After induction of diabetes, clinical symptoms of diabetes were observed in rats of diabetic control group. These symptoms were mild to moderate in all other treatment groups compare to diabetic control group. Administration of poly-herbal extract significantly (P<0.05) reduced the blood glucose level in T1 and T2 experimental groups especially at 4th week of experiment. However the blood glucose level was maximally reduced in glibenclamide treated group. In oral glucose tolerance test (OGTT), the blood glucose levels in rats of different treatment groups were significantly (P<0.05) increased at 30 and 60 minutes after oral glucose administration at dose of 2 g/kg. The little higher blood glucose levels in group T1 and T2 indicates inability of poly-herbal extract to reduce the increased blood glucose level at day 15. This result was supported by no effect of poly-herbal extract treatment on regulation of blood glucose level up to 3 weeks of our study period. In lipid profile, levels of total cholesterol, triglyceride and LDL-cholesterol were non-significantly (P>0.05) increased while levels of HDL-cholesterol were significantly (P<0.05) lower in diabetic control group compared to other groups. The mean levels of total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol in rats treated with poly-herbal extract were found comparable to normal control rats. In hematological parameters, mean values of PCV and TEC in rats of diabetic control were significantly (P<0.05) lower compared to other groups. Whereas, the mean value of above parameters in rats treated with poly-herbal extract was significantly (P<0.05) higher than other treatment groups. In biochemical parameters, mean values of ALT, AST, BUN and creatinine were non-significantly increased while, the mean values of total protein, albumin, globulin and total bilirubin were found non-significant in all groups compared to control rats. In case of antioxidant parameters evaluated from blood, liver, kidney and pancreas tissue samples, mean values of SOD, catalase and GSH in diabetic control group were non-significantly (P>0.05) decreased compared to control group. Whereas, the mean values of SOD, catalase and GSH were non-significantly (P>0.05) higher in glibenclamide as well as in poly-herbal treated groups compared to diabetic control group at the end of study period. Upon gross examination of pancreas, no appreciable gross lesions in all treatment groups have been observed. Macroscopic examination of liver and kidneys of experimental rats of diabetic control group shown congestion, paleness and mild to moderate enlargement. No appreciable macroscopic lesions have been observed in the spleen, heart and lung of rats in all treatment groups. The histopathological changes of pancreas of rats of diabetic control group (C2) revealed varying degree of structural changes as well as degenerative changes, vascular changes and infiltration of inflammatory cells with loss of normal architecture of parenchyma and changes in kidney were also observed in rats of diabetic control group. No appreciable histopathological lesions have been observed in the liver, spleen, heart and lung of rats in all treatment groups. In conclusion, administration of poly-herbal extract at the dose rate of 200 mg/kg, PO for 28 days have shown ameliorating effect on glucose, lipid profile and antioxidant levels and pathological lesions in diabetic rats. Though the findings of this short term study are encouraging, further detailed investigation is required to determine the effect of long term administration of poly-herbal extract in diabetic rats.ThesisItem Open Access EFFECT OF PIPERINE AND QUERCETIN ADMINISTRATION ON PHARMACOKINETICS AND SAFETY PROFILE OF MARBOFLOXACIN IN BROILER CHICKENS(jau,junagadh, 2018-08) PATEL HARSHADKUMAR B.; Dr. U. D. PATELTherapeutic and prophylactic use of antibiotics has allowed poultry production to achieve significant improvements by enhancing growth rate, feed efficiency and reduce mortality. Marbofloxacin is a 3rd generation fluorinated quinolone having broad spectrum of antimicrobial activity against gram-negative, gram-positive bacteria and mycoplasma spp. Pharmacokinetics of marbofloxacin after oral administration in chickens, reveals poor absorption and reduced bioavailability, which limits its therapeutic effectiveness. Several dietary constituents like piperine and quercetin have improved the bioavailability of co-administered drug by inhibiting efflux pumps (P-gp) or drug metabolizing enzymes CYP450 at intestine and liver. Hence, present study was planned to generate valuable information about effect of piperine and quercetin pretreatment (10 mg/kg, p.o., for 3 days) on pharmacokinetics and safety profile of marbofloxacin after single and repeated (5mg/kg, for 5 days) intravenous and oral administration in broiler chickens; and effect of on CYP3A37 and MDR1 mRNA expression levels in liver and duodenum of broiler chickens. After repeated intravenous administration, the initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration he initial plasma concentration (Cp 0) marbofloxacin of 17.72 5.28 g/mL which was significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the significantly higher than the respective value of respective value of respective value of respective value of respective value of respective value of respective value of respective value of respective value of respective value of 6.78 0.28 g/mL after single administration. single administration.single administration. single administration.single administration. single administration. single administration. single administration. single administration. Following single dose oral administration of marbofloxacin, the mean peak plasma concentrations (Cmax) in normal, piperine pretreated, quercetin pretreated and both in combination pretreated broiler chickens were almost similar and achieved at Tmax 0.83 0.11, 0.67 0.11 h, 0.83 0.25 and 1.50 0.22 h, respectively. Thus, the piperine pretreatment has enhanced the marbofloxacin absorption after oral administration in chickens. Following single dose oral administration of marbofloxacin in combination pretreated broiler chickens, significantly higher elimination half-life (normal: 4.62 0.42; combination pretreatment: 7.71 0.59 h), volume of distribution (normal: 1.32 0.10; combination pretreatment: 2.30 0.27 L/kg), the mean residence time (normal: 7.03 0.33; combination pretreatment: 10.71 0.70 h) and bioavailability (normal: 60.22 8.07; combination pretreatment: 75.39 7.34 %) were observed as compared to normal chickens. After repeated oral administration of marbofloxacin, significantly higher area under curve (single: 18.60 1.31; repeated: 23.20 2.30 μg.h/mL) and bioavailability (single: 75.39 7.34; repeated: 89.60 9.06 %) were observed as compared to single dose in combination pretreated broiler chickens. Piperine and quercetin pretreatment had no any significant effect on body clearance rate of marbofloxacin after single and repeated oral administration. After single oral administration of marbofloxacin AUC/MIC90 ratios were higher in piperine (353.20 h), quercetin (367.20 h) and combination pretreatment group (372.00 h) as compared to normal chickens (302.30 h) at MIC value of 0.05 μg/mL. After piperine and quercetin combination pretreatment, CYP3A37 mRNA expression was significantly down regulated in liver and duodenum by 24.35 and 17.59 fold, respectively and MDR1 by 7.65 and 21.59 fold, respectively in broiler chickens as compared to normal. Following multiple intravenous and oral administration of marbofloxacin, some of hematological and biochemical parameters were altered, but they were in the normal range for broiler chickens. Piperine and quercetin combined pretreatment has improved the pharmacokinetic profile and efficacy of marbofloxacin after single and repeated oral administration by inhibition of drug efflux protein (MDR1) and drug metabolizing enzyme (CYP3A37).
