SRINIVASA RAO, G(MAJOR)RAVI KUMAR, PSATHEESH, KMURALIDHAR, MAFROZ, JAHAN2019-07-162019-07-162019-05http://krishikosh.egranth.ac.in/handle/1/5810114914THESESDysmenorrhea 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.ennullThesis