INTRODUCTION:

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age; clinical characteristics of which mainly include menstrual disorders, secondary amenorrhea, serum hormonal disorders, hair loss, acne, obesity, and infertility^1,2. Studies indicate that in PCOS, some endocrine disorders reinforce and exacerbate each other^1,3. These disorders include defects in the hypothalamic-pituitary axis function and ovarian and adrenal function⁴.

PCOS is associated with abnormal gonadotropin secretion, increased ovarian steroid secretion, and insulin resistance^1,5. Oxidative stress (OS) is another contributing factor in some reproductive diseases such as endometriosis, PCOS, or unexplained infertility caused by an imbalance between oxidants and antioxidants^6,7. OS is effective in increasing the production of androgens, disrupting the development of ovarian follicles and damage of ovarian tissue in patients with PCOS. Even environmental factors that produce OS such as smoking, alcohol, malnutrition, and obesity are also effective in reducing fertility through this mechanism^6,8.

Letrozole (LTZ) is a non-steroidal aromatase inhibitor that was used recently for the induction of PCO in rats. In female rats, continuous LTZ treatment causes disrupted estrous cyclicity, larger ovarian weight, ovarian cysts, atretic follicles, and lack of corpora lutea, Besides, rats treated with LTZ develop increased levels of LH and Testosterone levels as occurs in PCOS indicating anovulation. Besides those reproductive abnormalities of PCOS, LTZ treatment produces many metabolic features of PCOS such as increased body weight^9-11.

DAFLON Belongs to the class of bioflavonoids containing purified micronized diosmin, hesperidin, and daflon used for the Preservation of venous valve structures Daflon helped attenuate postoperative pain and improve the quality of life¹², Protection of the microcirculation increase capillary resistance and reduce capillary filtration, resulting in the prevention of capillary leakage. Amelioration of lymphatic drainage Daflon may help treat lymphedema by reducing protein and extracellular fluid accumulation^13,14, Potent anti-inflammatory effect Daflon-treated animals, markers of inflammation were decreased in a dose-dependent manner. Daflon also served to significantly reduce parenchymal cell death and leukocyte rolling, adhesion to postcapillary venules, and migration^15,16.

Myoinositol (MYO) is a cyclitol naturally present in animal and plant cells, either in its free form or as a compound structure of phospholipids or inositol phosphate derivatives. These inositol phosphate derivatives act as secondary messengers for many hormones (including insulin) and mediators inside the eukaryotic cells, particularly the inositol triphosphates (IP3), phosphatidylinositol phosphate lipids (PIP2/PIP3), and possibly inositol glycans. MYO can increase glucose utilization by tissues and increase insulin sensitivity. MYO is a precursor for inositol phosphoglycans (IPGs) which provoke phosphorylation of insulin receptor substrates (IRS), besides, phosphoinositide 3 kinase (PI3K) and protein kinase B/Akt (PKB/Akt) that account for most, but not all, intracellular actions of insulin ¹⁷. For these reasons, Myo is important for the regulation of a wide range of cell functions, including cell growth and survival, development, and function of peripheral nerves, osteogenesis, and reproduction; It was found that MYO could restore normal ovulatory activity and Increase oocyte and egg quality causing improvement in fertilization rates¹⁸. Also, in the PCOS-endometrium, MYO could activate the AMPK pathway, increase GLUT-4 levels and, subsequently, increase glucose uptake by human endometrial cells. Therefore, MYO may be used as an effective treatment option in insulin-resistant PCOS women. the In the present study, regarding the role of OS and inflammation in the pathogenesis of PCOS and vice versa, a positive relationship between decreased OS and increased oocyte maturation in infertile women with PCOS is aimed to determine the effect of Daflon on serum levels of sex hormones and gonadotropins, inflammatory factors and OS indexes in ovarian tissue of PCOS control group.

MATERIALS AND METHODS:

Animals:

40 female albino Wister rats used in this study were classified as the following:

1-Group (1): 8 fertile female rats received normal feeding, and no drugs for 49 days and this group represent the control negative group.

2-Group (2): 8 female rats received letrozole (1mg/kg) with a high-fat diet for 21 days and this group represented the control positive of PCOS induction¹⁹.

3-Group (3): eight female rats received letrozole (1m/kg) for (21 days)¹⁹then treated with daflon (50mg/kg) for (2 weeks) (2,3).

4-Group (4): eight female rats received letrozole (1mg/kg) for (21 days)¹⁹ then daflon (100mg/kg) for (2 weeks)^6,9.

5-Group (5): eight female rats received letrozole (1mg/kg)for (21 days)^19,20then myoinositol (420mg/kg) for (3 weeks)²¹.

At the end of the experimental timeline, each group was sacrificed after being anesthetized by diethyl ether inhalation. Blood was collected to obtain serum for hormonal evaluation all rats were dissected and ovaries were cleaned from fats and rinsed with 9% normal saline and anchored in 10% formalin to be sent for histopathological examination.

Sample Collection:

At the end of the experimental period of each group, blood was collected from the jugular vein of euthanized rats and placed in a gel tube for 20 min. to clot and then centrifuged at 3000RPM for 15 min. the serum was separated and placed as 500µl in labeled Eppendorf tubes and stored at -20Cº until use²²for estimation of FSH (follicular stimulation hormone), LH(luteinizing hormone), T (testosterone) and catalase.

Biochemical assay for gonadotropins and sex hormones:

ELISA kits specific to rodents (bt-labrotary, shanghai, China) were used for hormonal assays. Testosterone Elisa kits E0259Ra, with detection range (0.12-25.6ng/ml).Catalase ELISA kit E-BC-K031-S with a detection range of 0.27-155.4 U/ml. Estimation of serum levels of these hormones were done as described in the manufacturer protocols.

Bodyweight assessment:

Bodyweight was weekly measured for assessment of obesity.

Vaginal Cytology:

The Estrus cycle was evaluated from the first day of the incubation period to the last day of the study via vaginal smear at 9:00 am and only rats with normal estrus cycle were included in this study²³smears were collected by insertion of ear stick dipped in normal saline and inserted gently into the vaginal opening²⁴as shown in the picture bellow Rats grasped as shown in the picture ²⁵ the smear stained with methylene blue and examined microscopically²⁶.

Histological Examination

Ovaries that were harvested from the rats were cleaned from fat using fine scissors and forceps and fixed in 10 % formaldehyde solution to be examined for histopathological changes according to the method of Junqueira LC²⁷. after fixation dehydrated using a raising concentration of ethanol, tissue was cleaned using xylene then infused with paraffin wax, heated, and poured into embedded models. blocks sectioned into 5um thickness by microtome washed in a water bath then left in the oven for dewaxing finally stained with hematoxylin and eosin (H and E) stain and examined by light microscope²⁷.

Statistical analysis:

Data analysis was done by using SPSS statistical software (version 25) data expressed as mean values, mean±standard deviation of samples (SD) the differences between the mean values were tested by one way analysis of variance (ANOVA), post-hoc test. Differenceswere considered statistically significant for p-value less than 0.05. visual assessments were used in the determination of significant differences.

RESULTS

Effect On Testosterone Level (T):

The PCOS-induced group (positive control) shows a significant increase in serum level of T compared to the negative control group (p<0.05).

While, serum levels of T were significantly decreased (p<0.05) in groups of animals treated with DAF-H (100mg/kg), DAF-L (50/kg), Myoinositol (420mg/kg) in contrast to the induction group. The mean level was illustrated in figure1. Moreover, there was no significant difference (p>0.05) in T levels among Daflon (100mg/kg) and MI (420mg/kg) when compared to each other and the negative control group. Interestingly the results of table-1 showed that Daflon treated group of doses (50mg/kg) was significantly increased compared to a negative control group.

Effect of different treatments On Serum Level of Catalase.

The induction group (positive control) shows a significant decrease (p>0.05) compared to the control group. while the DAF-H and MI treated group showed a significant increase in serum level of catalase (p<0.05) compared to the positive control group.

Additionally, there was a significant difference in DAF-L treated group compared to DAF-H (p<0.05) with the mean value of DAF-L which is significantly lower than the mean value of DAF-H but no significant difference was seen between the DAF-H treated group and MI-treated group (p<0.05) all the details demonstrated in table -1 and figure -2.

Body weight assessment:

The BW of rats with induced PCOS and treatment groups rats are shown in (Table 1 and figure 3). In the positive control group, the BW was significantly increased by elevating abdominal fat tissue compared to the negative control group (p<0.05). Treatment with (daflon 100mg/kg) and (MI 420mg/kg) in rats with PCOS induced weight loss and significantly decreased compared with the positive control group(p<0.05) and no significant changes (p>0.05)when compared to the negative control group.

Additionally, there was no significant change in BW between DAF-L (50mg/kg) when compared to the induction group as seen in (table-1and figure 3).

Figure 1: Effect of DAF-H, DAF-L, MI on Testosterone serum level, were values considered significantly different when (p<0.05), * =denote significant difference from the control +ve group, a= denote significant difference from the induction group (control +ve), #= means there is significant difference from the DAF-H group.

Figure 2: Effect of DAF-H, DAF-L, MI on Catalase serum level, were values considered significantly different when (p<0.05), * =denote significant difference from the control +ve group, a= denote significant difference from the induction group (control +ve), #= means there is significant difference from the DAF-H group.

Figure 3. Effect of DAF-H, DAF-L, MI on effect on body weight, were values considered significantly different when (p<0.05), * =denote significant difference from the control +ve group, a= denote significant difference from the induction group (control +ve), #= means there is significant difference from the DAF-H group.

Evaluation of vaginal cytology smears for induced Poly Cystic Ovarian Syndrome in mice:

There was a need for a more reliable noninvasive method for defining the stages and determining the time of ovulation in a laboratory setting. The changes occurred in the vaginal smear during the estrous cycle while inducing mice (except the control group) with Letrozole and were detected microscopically. The vaginal lumen has characteristic cell content that could be discovered.

Vaginal smear for all study groups (except the control group) showed persistence of estrous phase for ten consecutive days, this was determined through large non-nucleated epithelial cells.

Ovarian tissue examination:

Light microscopic examination of the ovarian tissue of the control shows there were numerous follicles at different developmental stages occupying the cortex (primary, secondary and luteal follicles). numerous corpus luteum were frequently observed.

While the PCOS induction group the cortical tissue revealed multiple follicles cysts with an irregular thickness of granulose layer and hypertrophied thickened theca layer. corpora lutea were absent indicating anovulation.

The DAF-H treated group showed signs of improvement in the ovary structure as decreased number of cystic follicles, numerous corpora lutea indicating ovulation, and multiple follicles at different stages of development.

As regards the DAF-L treated PCO group, the ovarian cortex also contained multiple corpora lutea, however, they were less frequent than those observed in the DAF-H group. Follicles of different developmental stages: primary follicles and numerous mature follicles could be observed in addition to numerous atretic follicles (Figure-4)The MYO group showed the same picture of improvement, however with more corpora lutea and fewer cystic follicles.

Figure-4: Histopathological features of ovary in Letrozole induced PCOS in rats. Representative photographs of section of ovary showing various developing follicles, cystic follicle (c.f), corpus luteum (c.l), secondary follicle (red arrow), primodial follicles (pink arrow),granulose layer (black arrow)theca layer (white arrow): (a) Control group (b) PCOS group (c) DAF-H group (d) DAF-L group and MI-group. (40× magnification)

DISCUSSION:

PCOS is a common disease among females in their reproductiveperiod, characterized by hyperandrogenemia, frequentanovulatory cycles and infertility. Impaired glucose tolerance arepresent in about 70% of lean and 95% of overweight womendiagnosed with PCOS²⁸, in thisstudy PCOS induced by daily oral administration of LTZ of a dose (1mg/kg) for 21 days and the induction of the syndrome were confirmed by daily vaginal smear examination which revealedthe loss of the cyclicity and arrested on the diestrus phase, besides the clinical examination showed hair loss and virilization and the serum hormone test revealed elevation in the testosterone level compared to the control (13) Letrozole also reduced the levels of catalase (CAT)²⁹.

Follicular development is the result of tightly regulated activities of FSH, estrogen, androgen and luteinizing hormone receptor (LHR) in granulosa and theca cells,chronically androgenized rats, exhibited increased body weight, disrupted estrus cyclicity, decreased insulin sensitivity³⁰.

Oxidative stress is defined as the imbalance between the overproduction of oxidants and the limited reserves of antioxidant defenses. PCOS is also considered as an oxidative state. Oxidative stress is partly associated with the various characteristics of this disease including IR, obesity, hyperandrogenism, and abdominal adiposity. These conditions lead towards the development of oxidative stress²⁹ Previously, increased oxidative status has been determined in the systemic circulation and follicular fluids of PCOS rats³¹.

Histological examination showed presence of multiple cystic follicles with thin granulose layer and absent corpora lute in the induction group³¹.

Myo-inositol (MI) belongs to the vitamin B family. MI isinvolved in follicular gonadotropin pathways which orchestrate ovulation³². In this study, MI (420mg\kg) was given orally, afterinduction of the PCO syndrome for 3 weeks.Serum analysis of this group showed improvementof the condition as there was a significant decrease in thelevels of testosterone and body weight in addition to a significant increase in CAT level, compared to LTZtreated group^33,34.

Daflon used in this study at two doses 100mg/kg and 50mg/kg orally in CMC solution for 21 days after the induction of PCOS by LTZ in group 3 and 4,DAF-H treated group showed better effect in reducing testosterone level and body weight along with elevating CAT serum level this is attributed to the nature of daflon which is consist of bioflavonoid combination of 90% diosmin and 10% hesperidin flavonoids, Diosmin is a flavone glycoside In fact, this flavone glycoside is obtained by dehydrogenation of hesperidin,Clinical evidence suggests that diosmin is a safe, nontoxic drug, and well-tolerated drug³⁵. Also the histopathological examination revealed improvement in the follicles development and showed multiple corpora lutea which indicate ovulation return this effects exploited due to the anti-oxidant anti-inflammatory and antihyperglycemic efficiency of diosmin by stimulating insulin production from the existing β-cells of the pancreas³⁵ an other Studies claimed that diosmin alleviated the oxidative stress by enhanced the antioxidant status in the plasma, liver, and kidneys of³⁶ these effect seen with DAF-H dose (100mg/kg) more then with DAF-L (0mg/kg )that means there is a dose dependent effect of Daflon treatment for PCOS, DAF-L also had beneficial effects on the reproductive parameters of PCO but its effect was less than those of MI according to the level histologic examination, however, Daflon 100mg/kg was best on improving the signs and symptoms in the pathogenesis of PCOS.

CONCLUSION:

Current study showed that the Daflon had the potential to ameliorate polycystic ovarian syndrome at the dose of 100mg/kg which is statistically comparable with myo-inositol. This property might be attributed to antioxidant activity of Daflon.

REFRENCES:

1. Mhaibes SH, Taher MA, Badr AH. A comparative Study of Blood Levels of Manganese, Some Macroelements and Heavy Metals in Obese and Non-Obese Polycystic Ovary Syndrome Patients. Iraqi J Pharm Sci. 2017;26(2). doi:10.31351/vol26iss2pp85-94

2. Shoukath U, Shoukath U, Sultana S, Uddin MN. Polycystic Ovary Syndrome (PCOS): A Thief of Womenhood. Asian J. Res. Pharm. Sci. 2018; 8(2):68-72. doi: 10.5958/2231-5659.2018.00014.0

3. Kumar DB, Subash C, Bharat P, Sharma D, Hitesh D. Effect of Obesity on Fertility in Women with Polycystic Ovary Syndrome. Asian J. Res. Pharm. Sci. 1(4): Oct.-Dec. 2011; Page 127-130.

4. Guzar SH, Jawad ES, Altahan MA, Hameed NN. The Effects of Trace Element levels on Polycystic Ovary Syndrome in human female Sat Thi-Qar governorate/Iraq. Research J. Pharm. and Tech 2019; 12(9):4447-4453. doi: 10.5958/0974-360X.2019.00767.4

5. Julius RR. A. Effect of LH/FSH Ratio and its Correlation with Insulin Resistance in PCOS Obese woman of Reproductive age Group. Research J. Pharm. and Tech 2018; 11(6): 2217-2219. doi: 10.5958/0974-360X.2018.00410.9

6. Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol. 2012;10(1):1–31. doi:10.1186/1477-7827-10-49

7. Gade A, Sawant R, Parkar S, Kegade P. Polycystic ovary syndrome: An Overview, Diagnosis and Treatment of PCOS. Asian J. Pharm. Tech. 2020; 10(4):265-272. doi: 10.5958/2231-5713.2020.00044.6

8. Devi SB, Susila C. Diagnosis, causal factors and associated diseases of PCOS: A Mini-review. Asian Journal of Nursing Education and Research. 2022; 12(1):138-3. doi: 10.52711/2349-2996.2022.00030

9. Darabi P, Khazali H, Mehrabani Natanzi M. Therapeutic potentials of the natural plant flavonoid apigenin in polycystic ovary syndrome in rat model: via modulation of pro-inflammatory cytokines and antioxidant activity. Gynecol Endocrinol. 2020;36(7):582-587. doi: 10.1080/09513590.2019.1706084.

10. Pokale SB, Jadhav G. Piper longum (Linn.) restores ovarian function in Letrozole induced PCOS in Rats: Comparison with Metformin and Clomiphene citrate. Research Journal of Pharmacy and Technology. 2021; 14(10):5190-6. doi: 10.52711/0974-360X.2021.00903

11. Otuokere IE, AmakuFJ. Conformational analysis and excited – state properties of a highly potent and totally selective aromatase inhibitor, 4,4'-(1H-1,2,4-triazol-1-ylmethanediyl) dibenzo nitrile (letrozole). Research Journal of Pharmacology and Pharmacodynamics. 2015; 7(4): 176-180. doi: 10.5958/2321-5836.2015.00035.X

12. 4Veverková L, Jedlicka V, Wechsler J, Kalac J. Analysis of the various procedures used in great saphenous vein surgery in the Czech Republic and benefit of Daflon 500 mg to postoperative symptoms. Phlebolymphology. 2006;13(4):195.

13. Casley-Smith JR, Casley-Smith JR. The pathophysiology of lymphedema and the action of benzo-pyrones in reducing it. Lymphology. 1988;21(3):190-4.

14. Nardarajah D. Hesperidin-A short Review. Research J. Pharm. and Tech. 2014;7(1):78-80.

15. Takase S, Delano FA, Lerond L, Bergan JJ, Schmid-Schönbein GW. Inflammation in chronic venous insufficiency. Is the problem insurmountable? J Vasc Res. 1999;36 Suppl 1:3-10. doi: 10.1159/000054068.

16. Al-Rikabi RH,Al-Shmgani HS. Evaluation of Hesperidin Protective Effect on Lipopolysaccharide -Induced Inflammation and lipid Peroxidation in BALB/C Mail Mice. Research J. Pharm. and Tech 2018; 11(12): 5513-5516. doi: 10.5958/0974-360X.2018.01003.X

17. Abdelrahman A, Mahmoud AA, Lamie Fanous Y, Abd Elhaliem NG, Elalaf H. Impact of erythropoietin and myoinositol versus metformin on insulin resistance in a rat model of polycystic ovary syndrome. Arch Physiol Biochem. 2021:1-12. doi: 10.1080/13813455.2021.1949023.

18. Ciotta L, Stracquadanio M, Pagano I, Carbonaro A, Palumbo M, Gulino F. Effects of myo-inositol supplementation on oocyte’s quality in PCOS patients: a double blind trial. Eur Rev Med Pharmacol Sci. 2011;15(5):509–14.

19. Sun J, Jin C, Wu H, Zhao J, Cui Y, Liu H, Wu L, Shi Y, Zhu B. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats. PLoS One. 2013;8(11):e79382. doi: 10.1371/journal.pone.0079382.

20. Gouda E, Babiker F. Micronized flavonoid fraction Daflon 500 protects heart against ischemia–reperfusion injury: an old medicine for a new target. All Life. 2020;13(1):556–68. doi:10.1080/26895293.2020.1832921

21. Bevilacqua A, Dragotto J, Giuliani A, Bizzarri M. Myo-inositol and D-chiro-inositol (40:1) reverse histological and functional features of polycystic ovary syndrome in a mouse model. J Cell Physiol. 2019;234(6):9387-9398. doi: 10.1002/jcp.27623.

22. Greenfield EA. Sampling and Preparation of Mouse and Rat Serum. Cold Spring Harb Protoc. 2017;2017(11):pdb.prot100271. doi: 10.1101/pdb.prot100271.

23. Rajan RK, M SS, Balaji B. Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome (PCOS) model through anti-androgenic mechanism. Pharm Biol. 2017;55(1):242-251. doi: 10.1080/13880209.2016.1258425.

24. Schneider T, Popik P. An animal model of premenstrual dysphoric disorder sensitive to antidepressants. Curr Protoc Neurosci. 2009 Jan;Chapter 9:Unit 9.31. doi: 10.1002/0471142301.ns0931s46.

25. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62(4A):609-14. doi: 10.1590/s1519-69842002000400008.

26. Yener T, Turkkani Tunc A, Aslan H, Aytan H, Cantug Caliskan A. Determination of oestrous cycle of the rats by direct examination: how reliable? Anat Histol Embryol. 2007;36(1):75-7. doi: 10.1111/j.1439-0264.2006.00743.x.

27. Junqueira LC, Carneiro J. Histology and its methods of study. Junqueira, LC; Carneiro, J Basic Histol. 2013;11:1–18.

28. Mahdi KS, Al-Hady FNA. Effect of Repaglinide and Metformin as Anti-Diabetic Drugs on Fertility and Sex Hormonal Levels of Male Albino Rats. Ann Rom Soc Cell Biol. 2021;5874–95.

29. Younas A, Hussain L, Shabbir A, Asif M, Hussain M, Manzoor F. Effects of Fagonia indica on Letrozole-Induced Polycystic Ovarian Syndrome (PCOS) in Young Adult Female Rats. Evid Based Complement Alternat Med. 2022;2022:1397060. doi: 10.1155/2022/1397060.

30. Hossain MM, Cao M, Wang Q, Kim JY, Schellander K, Tesfaye D, Tsang BK. Altered expression of miRNAs in a dihydrotestosterone-induced rat PCOS model. J Ovarian Res. 2013;6(1):36. doi: 10.1186/1757-2215-6-36.

31. Atef MM, Abd-Ellatif RN, Emam MN, Abo El Gheit RE, Amer AI, Hafez YM. Therapeutic potential of sodium selenite in letrozole induced polycystic ovary syndrome rat model: Targeting mitochondrial approach (selenium in PCOS). Arch Biochem Biophys. 2019;671:245-254. doi: 10.1016/j.abb.2019.06.009.

32. Laganà AS, Garzon S, Casarin J, Franchi M, Ghezzi F. Inositol in Polycystic Ovary Syndrome: Restoring Fertility through a Pathophysiology-Based Approach. Trends Endocrinol Metab. 2018;29(11):768-780. doi: 10.1016/j.tem.2018.09.001.

33. Zhang Y, Li C, Zhang W, Zheng X, Chen X. Decreased Insulin Resistance by Myo-Inositol Is Associated with Suppressed Interleukin 6/Phospho-STAT3 Signaling in a Rat Polycystic Ovary Syndrome Model. J Med Food. 2020;23(4):375-387. doi: 10.1089/jmf.2019.4580.

34. Merviel P, James P, Bouée S, Le Guillou M, Rince C, Nachtergaele C, Kerlan V. Impact of myo-inositol treatment in women with polycystic ovary syndrome in assisted reproductive technologies. Reprod Health. 2021 Jan 19;18(1):13. doi: 10.1186/s12978-021-01073-3. PMID: 33468143; PMCID: PMC7816413.

35. Srinivasan S, Vinothkumar V, Murali R. Antidiabetic efficacy of citrus fruits with special allusion to flavone glycosides. In: Bioactive Food as Dietary Interventions for Diabetes. Elsevier; 2019: 335–46. doi: 10.1016/B978-0-12-813822-9.00022-9

36. Catarino MD, Alves-Silva JM, Pereira OR, Cardoso SM. Antioxidant capacities of flavones and benefits in oxidative-stress related diseases. Curr Top Med Chem. 2015;15(2):105-19.

Received on 11.09.2022 Modified on 27.09.2022

Research J. Pharm. and Tech 2023; 16(6):2765-2770.

DOI: 10.52711/0974-360X.2023.00454

Variable	-ve control	+ve control	DAF-H	DAF-L	MI
T ng/l	0.182±0.47	11.391±3.72^*	2.629±0.98	4.201±1.21^*,a	3.068±0.663
CAT U/ml	1543.87±224.24^a	730.25±416.42^*	1413.50±249.9^a	882.12±368.9^*,#	1234.37±322.2^a
Body weight	193.68±9.21^a	261.25±20^*	206.87±18.69^a	234.43±34.30^*	205±20.34^a