Evaluation of In vivo Reversible Antiovulatory Activity of Ursolic Acid after Cupric Acetate Induced Ovulation in Wistar Female Rats
Jainik Khamar1, Indermeet Singh Anand2*
1Research Scholar, Department of Pharmaceutical Sciences,
Gujarat Technological University, Chandkheda, Ahmedabad, Gujarat - 382424, India.
2Professor and Head, Department of Pharmacology and Pharmacy Practice,
Shri Sarvajanik Pharmacy College, Mehsana, Gujarat - 384001, India.
*Corresponding Author E-mail: jainikkhamar17@gmail.com
ABSTRACT:
This study investigates the reversible antiovulatory activity of ursolic acid in Wistar albino female rats, using cupric acetate to induce ovulation. Ursolic acid solubility was optimized for uniform distribution in a carboxymethyl cellulose (CMC) vehicle. Dosages were derived from the LD50 of ursolic acid, with low and high doses selected as 100mg/kg and 250mg/kg, correspondingly. After being isolated for 21 days, 24 of 30 rats were randomized to one of four groups: recovery high dosage, low dose, high dose, or vehicle control. Ursolic acid was administered orally via gavage for three days, followed by intravenous cupric acetate (4 mg/mL) to induce ovulation. Mortality, morbidity, clinical signs, body weight (BW), absolute and relative ovary weights, and histopathological parameters were assessed. All animals were euthanized 24 hours post-cupric acetate administration. No mortality or morbidity was observed. High-dose ursolic acid significantly reduced body weight and ovary weight compared to controls. Histopathological analysis showed fewer corpora lutea in the high-dose group and restored follicular formation in the recovery group. These findings suggest that ursolic acid exhibits reversible antiovulatory activity in Wistar female rats under the conditions of this study.
KEYWORDS: Ursolic Acid, Antiovulatory Activity, Cupric Acetate, Female wistar rats.
INTRODUCTION:
Hormonal contraceptives, while effective, often come with severe side effects and are not suitable for long-term use1. Alternative methods, including local contraceptives, offer less certainty in preventing conception2. Permanent sterilization is typically achieved through surgical means. Modern emergency contraceptives primarily delay ovulation, highlighting the need for effective, reversible, and safer alternatives3.
Ursolic acid, a pentacyclic triterpene found in various plants such as apples, bilberries, and Saraca indica, demonstrates multiple pharmacological activities for example, antioxidant, anti-inflammatory, antibacterial, as well as antifungal properties4. This has been shown to ameliorate ethanol-induced hepatotoxicity by reducing lipid peroxidation and increasing antioxidant levels5. Saraca indica, rich in ursolic acid, is traditionally used for menstrual and uterine disorders6. To produce the best contraceptive medications with few side effects and efficient reversibility, this research intends to assess the antiovulatory potential of ursolic acid7,8.
MATERIALS AND METHOD:
Animal Subject:
Twenty-four Wistar female rats were isolated for 21 days to guarantee they were not pregnant. Following isolation, the rats were divided into 4 categories at random: vehicle control, low dose, high dose, and recovery high dose. Randomization was based on BW, with minimal variation across groups9,10
The rationale for Selection of Dose:
The dose has been selected based on the arbitrary division of the ursolic acid’s LD50 by 1/4th and 1/10th to get the best low dose and high dose respectively. Hence, the final dose levels selected for the evaluation were high dose (250mg/Kg B.wt./day) and low dose (100mg/kg b.wt./day).
Rationale of vehicle selection:
Aqueous Carboxymethyl Cellulose (CMC) 0.5% w/v has been selected as a vehicle on basis of clear suspension formulation and the formulation passed syringebility test, which was performed before the dose administration.
Dose Formulation Preparation:
The dose formulations were prepared daily for all dose groups before dosing. Ursolic acid was mixed with aqueous Carboxymethyl Cellulose (CMC) for high-dose and low-dose groups. Homogeneity of the ursolic acid in the vehicle was maintained by continuous stirring utilizing a magnetic stirrer.
Dose Administration:
The BW of each animal was used to determine the necessary dose volume and administered using oral gavage attached with a suitably calibrated syringe for three consecutive days for low dose, high dose, and recovery higher dose group animals, whereas vehicle alone was administered to vehicle group animals. The dosage volume was kept at 10mL/Kg B.wt. Animals in Group 2 received ursolic acid at a dose level of 100mg/kg BW. Animals in Group 3 received ursolic acid at a dose level of 250mg per kg BW. Ursolic acid was administered to animals in Group 4 (High Dosage Recovery) at a dose level of 250mg per kilogram of BW. Each animal received an intravenous dosage of 4mg/kg BW of a freshly made 0.4percent Cupric acetate solution to induce ovulation around half an hour after the last treatment was given11,12.
Observations:
Mortality and morbidity were monitored twice daily. BW and clinical signs were recorded. Necropsy included measurements of absolute and relative ovary weights and histopathological examination of ovarian tissues 13,14,15.
Gross pathology:
At the time of termination, all animals were euthanized by the co2 asphyxiation followed by the exsanguination except for group 4 animals. All animals in group 4 received treatment with a high dose of cupric acetate at the conclusion. After completion of the treatment all the animals were kept in the recovery period of 21 days and then after terminated terminal parameters were performed. All the ovaries of the animals were excised, and histopathology was performed.
Statistical analysis:
The impact of ursolic acid in comparison to the vehicle control was examined using ANOVA (one-way analysis of variance) and Tukey's test; a P<0.05, significance level was established.
RESULT:
No mortality or morbidity occurred in any of the animals (Table 1). There was no abnormality observed in the detailed clinical observation (Table 2). The absolute and relative ovarian weights of the animals in the high-dose group were considerably (P< 0.05) reduced contrasted to those in the vehicle group (Table 3 and 4, Fig: 1, Fig: 2 and Fig: 3). The low-dose and recovery high-dose group animals didn’t display any significant changes in any of the parameters when compared with vehicle group animals.
Table No.1: Mortality and Morbidity Record
|
Group |
Treatment |
Animal No. |
Observation |
|
G1 |
Vehicle Control |
1 |
No Mortality and Morbidity observed during the treatment period |
|
2 |
|||
|
3 |
|||
|
4 |
|||
|
5 |
|||
|
6 |
|||
|
G2 |
Low Dose |
7 |
No Mortality and Morbidity observed during the treatment period |
|
8 |
|||
|
9 |
|||
|
10 |
|||
|
11 |
|||
|
12 |
|||
|
G3 |
High Dose |
13 |
No Mortality and Morbidity observed during the treatment period |
|
14 |
|||
|
15 |
|||
|
16 |
|||
|
17 |
|||
|
18 |
|||
|
G4 |
Recovery High Dose |
19 |
No Mortality and Morbidity observed during the treatment period |
|
20 |
|||
|
21 |
|||
|
22 |
|||
|
23 |
|||
|
24 |
Table No. 2: Clinical Sign Record
|
Group |
Treatment |
Animal No. |
Observation |
|
G1 |
Vehicle Control |
1 |
No abnormal clinical sign observed during the treatment period |
|
2 |
|||
|
3 |
|||
|
4 |
|||
|
5 |
|||
|
6 |
|||
|
G2 |
Low Dose |
7 |
No abnormal clinical sign observed during the treatment period |
|
8 |
|||
|
9 |
|||
|
10 |
|||
|
11 |
|||
|
12 |
|||
|
G3 |
High Dose |
13 |
No abnormal clinical sign observed during the treatment period |
|
14 |
|||
|
15 |
|||
|
16 |
|||
|
17 |
|||
|
18 |
|||
|
G4 |
Recovery High Dose |
19 |
No abnormal clinical sign observed during the treatment period |
|
20 |
|||
|
21 |
|||
|
22 |
|||
|
23 |
|||
|
24 |
Table No.3: Individual Body Weight Record
|
Group |
Treatment |
Animal No. |
Prior treatment Body weight (g) |
Terminal treatment Body weight (g) |
|
G1 |
Vehicle Control |
1 |
228.40 |
232.54 |
|
2 |
240.64 |
243.87 |
||
|
3 |
240.10 |
245.41 |
||
|
4 |
238.60 |
241.98 |
||
|
5 |
242.65 |
246.78 |
||
|
6 |
246.16 |
250.14 |
||
|
G2 |
Low Dose |
7 |
230.61 |
234.15 |
|
8 |
238.91 |
240.17 |
||
|
9 |
237.92 |
241.74 |
||
|
10 |
243.14 |
248.19 |
||
|
11 |
244.66 |
246.72 |
||
|
12 |
245.81 |
248.65 |
||
|
G3 |
High Dose |
13 |
233.60 |
226.17 |
|
14 |
230.49 |
226.47 |
||
|
15 |
238.27 |
237.46 |
||
|
16 |
240.61 |
232.67 |
||
|
17 |
245.67 |
240.22 |
||
|
18 |
240.64 |
233.87 |
||
|
G4 |
Recovery High Dose |
19 |
230.01 |
244.47 |
|
20 |
241.53 |
250.98 |
||
|
21 |
238.24 |
246.78 |
||
|
22 |
237.94 |
251.32 |
||
|
23 |
238.91 |
254.14 |
||
|
24 |
239.37 |
256.56 |
||
|
G1 |
Vehicle Control |
Mean |
239.43 |
243.45 |
|
SD |
6.00 |
6.02 |
||
|
N |
6 |
6 |
||
|
G2 |
Low Dose |
Mean |
240.18 |
243.27 |
|
SD |
5.64 |
5.66 |
||
|
N |
6 |
6 |
||
|
G3 |
High Dose |
Mean |
238.21 |
232.81 * |
|
SD |
5.44 |
5.69 |
||
|
N |
6 |
6 |
||
|
G4 |
Recovery High Dose |
Mean |
237.67 |
250.71 |
|
SD |
3.96 |
4.49 |
||
|
N |
6 |
6 |
* Indicates that the significant decrease (P<0.05) has been observed in the group 3 animal body weight in comparison of group 1 animal body weight (Fig – 1).
Fig: 1 – Comparison of terminal body weight of all four groups of animals using one way ANOVA followed by post – hoc tuckey’s test.
Table No. 4: Individual Absolute and Relative Ovary weight
|
Group |
Treatment |
Animal No. |
Absolute Ovaries weight (g) |
Terminal treatment Body weight (g) |
Relative Ovaries weight (%) |
|
G1 |
Vehicle Control |
1 |
0.9737 |
232.54 |
0.4187 |
|
2 |
0.9826 |
243.87 |
0.4029 |
||
|
3 |
1.0892 |
245.41 |
0.4438 |
||
|
4 |
0.9912 |
241.98 |
0.4096 |
||
|
5 |
1.0986 |
246.78 |
0.4452 |
||
|
6 |
1.0756 |
250.14 |
0.4300 |
||
|
G2 |
Low Dose |
7 |
0.8272 |
234.15 |
0.3533 |
|
8 |
0.8845 |
240.17 |
0.3683 |
||
|
9 |
0.9426 |
241.74 |
0.3899 |
||
|
10 |
0.9776 |
248.19 |
0.3939 |
||
|
11 |
1.0621 |
246.72 |
0.4305 |
||
|
12 |
0.9945 |
248.65 |
0.4000 |
||
|
G3 |
High Dose |
13 |
0.6625 |
226.17 |
0.2929 |
|
14 |
0.5746 |
226.47 |
0.2537 |
||
|
15 |
0.7810 |
237.46 |
0.3289 |
||
|
16 |
0.4654 |
232.67 |
0.2000 |
||
|
17 |
0.8842 |
240.22 |
0.3681 |
||
|
18 |
0.8801 |
233.87 |
0.3763 |
||
|
G4 |
Recovery High Dose |
19 |
1.0612 |
244.47 |
0.4341 |
|
20 |
0.9781 |
250.98 |
0.3897 |
||
|
21 |
1.0889 |
246.78 |
0.4412 |
||
|
22 |
1.0757 |
251.32 |
0.4280 |
||
|
23 |
1.0627 |
254.14 |
0.4182 |
||
|
24 |
0.9845 |
256.56 |
0.3837 |
||
|
G1 |
Vehicle Control |
Mean |
1.04 |
243.45 |
0.43 |
|
SD |
0.06 |
6.02 |
0.02 |
||
|
N |
6 |
6 |
6 |
||
|
G2 |
Low Dose |
Mean |
0.95 |
243.27 |
0.39 |
|
SD |
0.08 |
5.66 |
0.03 |
||
|
N |
6 |
6 |
6 |
||
|
G3 |
High Dose |
Mean |
0.71*** |
232.81 |
0.30 *** |
|
SD |
0.17 |
5.69 |
0.07 |
||
|
N |
6 |
6 |
6 |
||
|
G4 |
Recovery High Dose |
Mean |
1.04 |
250.71 |
0.42 |
|
SD |
0.05 |
4.49 |
0.02 |
||
|
N |
6 |
6 |
6 |
*** Indicates that the significant decrease (P<0.05) has been observed in the group 3 absolute weight of ovary in comparison of group 1 absolute weight of ovary (Fig – 2).
*** Indicates that the significant decrease (P<0.05) has been observed in the group 3 relative weight of ovary in comparison of group 1 relative weight of ovary (Fig – 3).
Fig: 2 – Comparison of absolute ovaries weight of all four groups of animals using one way ANOVA followed by post – hoc tuckey’s test
Fig: 3 – Comparison of relative ovaries weight of all four groups of animals using one way ANOVA followed by post – hoc tuckey’s test
Histopathology:
A solitary layer of altered mesothelium, denoting the surface epithelium, envelops the ovarian tissue in the histogram. The cortical and medullary regions of the ovarian stroma were not separated. The majority of the stroma was made up of fibroblast-like spindle-shaped stromal cells, which were usually grouped in an uneven whorled pattern. Alongside corpora lutea and corpora albicantia, ovarian follicles at various stages of maturity were present, including primordial, maturing (secondary, primary, Graafian, and tertiary), and atretic follicles. The mature follicle comprised the granulosa layer, the oocyte, along with the theca layers, while the primordial follicles exclusively contained germ cells. Large hemorrhagic corpora lutea having new blood-filled cavities inside the corpus luteum body were also visible in the control group's ovaries. Additionally observed were mature corpora lutea, which are spherical, yellow entities that range in size from 1.5 to 2.5cm and have lobulated edges and a cystic center. The theca cells along with granulosa both had considerable luteinization. Developed Graphian follicle has been seen in the vehicle control group animals (Table 5, Fig:4 - G1 Vehicle Control). In group 2, only primary follicles have been found in the histograph (Table 5, Fig:5 - G2 Low Dose). The histogram of Group 3 indicates a substantial quantity of follicles in the initial phases of maturation; the count of ruptured corpora lutea in this group was markedly below compared to the control group. (Table 5, Fig:6- G3 high Dose). In group 4, recovery high dose treated animals formation of the follicles have been seen in the ovaries (Table 5, Fig:7- G4 recovery high Dose).
Table No.5: Histopathology
|
|
|
|
|
|
Fig: 4 - G1 (Vehicle Control) |
Fig: 5- G2 (Low Dose) |
Fig: 6 - G3 (High Dose) |
Fig: 7 - G4 (Recovery High Dose) |
DISCUSSION:
The methods commonly used for the screening of possible antiovulatory substances vary considerably16. In one test, the compound is fed to a group of mature female rats, and the fertility rate is observed. Some investigators followed the changes in the female rat and observed whether the estrous phase was suppressed by the test substance. These methods are not specific as ovulation cannot be predicted accurately in spontaneously ovulating mammals17. Other tests have been devised where the gonadotrophin activity of the pituitary is determined in immature male and female rats. Ovulation is also detected by the presence of corpora lutea in serial histological sections of the ovaries. The absence of corpora lutea is an indication of antiovulatory activity.
CONCLUSION:
Depending on the outcomes it could be decided that the ursolic acid has been screened as a revisable antiovulatory activity in Wistar female rats as it did show fewer corpora lutea in the ovaries.
ACKNOWLEDGEMENT:
We extend our sincere appreciation to Accuprec Research Labs Pvt. Ltd., Ahmedabad, Gujarat, India, for generously providing the essential resources and facilities crucial for the successful execution of our present animal research. Their unwavering support not only facilitated the progress of this study but also contributed significantly to the advancement of scientific understanding in our field. We are grateful to the management of the laboratory for their dedication to fostering research excellence and their commitment to promoting animal welfare.
ANIMAL ETHICS APPROVAL:
The (IAEC Institutional Animal Ethical Committee) of Accuprec Research Labs Pvt. Ltd. in Ahmedabad, Gujarat, India, gave its approval to the research (Protocol No. ARL/PT/691/2023 dated 12-05-2023). The animals had been housed in standard settings, which involved a 12-hour light/dark cycle, unrestricted access to water and food, and a climate that had been maintained at 22 ± 3°C with humidity of 30–70 %.
DECLARATION OF INTEREST:
None.
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Received on 08.09.2024 Revised on 14.01.2025 Accepted on 22.03.2025 Published on 01.10.2025 Available online from October 04, 2025 Research J. Pharmacy and Technology. 2025;18(10):4704-4708. DOI: 10.52711/0974-360X.2025.00676 © RJPT All right reserved
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