Diodia sarmentosa (Sw) leaves were collected from the Federal University of Technology, Owerri, Nigeria (Latitude 5^o 23.5617ˈN, Longitude 6^o 59.1758ˈE). The leaves were identified by a botanist in the Department of Crop Science, Federal University of Technology, Owerri. The leaves were washed with distilled water, air-dried, and then dried in a laboratory oven (DIGITAL TT 9083, Techmel & Techmel USA) at a regulated temperature of 40°C. The dried plant material was grinded, then 800 g each of the powder was weighed and soaked separately in 4 L of ethanol for 48 h on an orbital shaker. The extracts were filtered using a Buckner funnel and Whatman No.1 filter paper and concentrated to dryness at 40^oC using a rotary evaporator.

Synthetic monosodium glutamate (Ajinomoto Co., Inc., Tokyo, Japan) was used for this study. All other chemicals were of analytical grade.

Twenty-one nine-week-old, adult female wistar albino rats were acclimatized for ten days in an animal house before being classified into three groups of seven animals each. The animals were bred in the animal house of the Department of Biochemistry, Michael Okpara University of Agriculture, Umudike under standard conditions (25–27 ⁰C, 12 h natural light-dark cycle) and fed with commercially available rat pellets and water ad libitum. The three experimental groups are as follows:

Group A (normal control (NC)): without any induction, provided with water and normal rat chew.

Group B (positive control (PC)): uterine leiomyoma induced in rats using a single daily dose of 200 mg/kg MSG, without treatment.

Group C (treatment group (TG)): uterine leiomyoma induced in rats using a single daily dose of 200 mg/kg MSG, then treated with ethanol extracts of Diodia sarmentosa (Sw) leaves.

Uterine fibroids were induced with a single daily dose of 200 mg monosodium glutamate (MSG)/kg over 30 days using an oral gavage tube, with slight modifications to the protocol described by Olowofolahan et al.29. At the end of the induction period, representative samples of the uterus were harvested from the three experimental groups and processed using histological techniques and routine Hematoxylin and Eosin (H&E) staining to confirm successful induction of tumors before the commencement of treatment. Presence of densely packed, spindle-shaped fibrous tissue and multifocal tumor cells in the endometrium of the uterus confirmed successful induction of uterine fibroid.

The LD₅₀ of the plant extract was determined using Lorke’s method30. Nine rats (100 – 110 g) were divided into three treatment groups treated with (1600, 2900, and 5000) mg/kg of the extract, respectively. Observations were made at 24 h and symptoms of toxicity and mortality were recorded. Two mortalities were recorded in the highest (5000 mg/kg) and medium (2900 mg/kg) dose groups after 24 hours, but none were recorded in the lowest (1600 mg/kg) dose group. Based on this, a safe dose of 400 mg/kg of the plant extract was chosen for treatment.

Animals were treated with 400 mg/kg ethanol extract of Diodia sarmentosa (Sw) leaves once per day from week 5 to week 8 after tumor induction. The dried ethanol extracts of Diodia sarmentosa leaves were weighed and fresh stock solutions prepared every two days by concentrating 8 g of the dried extract in 50 ml of distilled water.

Rats were fasted overnight after the experiment and then sacrificed. Blood was drawn from the rats’ orbital sinus using 0.5 ml syringes. Representative samples of the uterus were harvested from the three treatment groups and preserved in Bouin’s fluid. The samples were processed using histological techniques and stained using routine Hematoxylin and Eosin stain for histopathological examinations, adhering strictly to standard protocol.

Serum electrolytes (HCO₃, Cl^-, K⁺, Na⁺) were determined using Sood’s spectrometric method31. Berthelot’s method was used in measuring serum urea, while serum creatinine was determined using alkaline picrate method32. Uric acid was determined with the enzymatic method described by Fôssati et al.33. Total protein was assayed using a colorimetric method based on the Biuret reaction34. 17β-oestradiol and progesterone were assayed using the NoviWell™ assay kit (HySkill Diagnostics, Bahlingen, Germany), which employs the sandwich enzyme immunoassay (SIA) microtiter method.

Catalase was determined using the modified method by Hadwan35. Serum glutathione peroxidase was determined using Paglia and Valentine’s method36 . Lipid peroxidation was estimated using Wallin et al.'s method37. Arthur and Boyne’s method was used to determine superoxide dismutase activity38. Total antioxidant capacity was evaluated using Prieto et al.'s method39, with slight modifications. Vitamin E content was estimated using Pearson and Cox's method40. Glutathione reduction was performed using Moron et al.'s method41. Ascorbic acid (vitamin C) was assayed using the micro techniques of clinical chemistry developed by Samuel Natelson in 196142.

Data obtained from the experiment was analyzed using Statistical Package for Social Sciences (SPSS) software, version 23. The data was expressed as mean ± standard deviation. Statistical significance was determined using a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. Statistical significance was defined as 𝑃 < 0.05.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Serum levels of urea, creatinine, chloride, sodium, and uric acid were significantly (P < 0.05) elevated in the positive control (PC) compared to the normal control (NC) (Figure 1). This suggests a possible relationship between uterine fibroids and renal impairment. Significant (P < 0.05) reductions in the serum levels of urea and creatinine were observed in the treated group (TG). No significant (P < 0.05) reduction was recorded in the serum levels of chloride, sodium, and uric acid in the treated group compared with the positive control. No significant (P < 0.05) difference was observed in the serum levels of bicarbonate (HCO₃) or total protein of the positive control or treated group when compared with the normal control (Figure 1 ). This could possibly indicate non-involvement of these two parameters in the development of uterine fibroids. Significant (P < 0.05) elevated levels of serum estradiol and progesterone were observed in the positive control compared with the normal control. Estradiol and progesterone are key players in the formation of fibrotic tissues, and significant (P < 0.05) reductions in the serum levels of estradiol and progesterone were observed in the treated group compared with the positive control.

Serum levels of malondialdehyde were significantly (P < 0.05) elevated in the positive control (PC) compared with the normal control (NC) (Figure 2). Elevated levels of malondialdehyde in the serum indicate the presence of oxidative stress. Significant (P < 0.05) reductions in the serum levels of malondialdehyde were observed in the treated group (TG) compared to the positive control. Significant (P < 0.05) reductions were also observed in the serum levels of most of the assayed antioxidants (superoxide dismutase, glutathione peroxidase, vitamin E, catalase, reduced glutathione, vitamin C, and total antioxidant capacity) in the positive control compared with the normal control (Figure 2). However, there was no significant (P < 0.05) difference in the serum levels of vitamin E and vitamin C in the treated group compared with the positive control. Significant (P < 0.05) increases were observed in the serum levels of superoxide dismutase, glutathione peroxidase, catalase, reduced glutathione, and total antioxidant capacity in the treated group compared with the normal control. Antioxidants play a key role in mitigating oxidative stress levels in tissues.

The photomicrograph of a portion of the uterus of the normal control (NC) showed a normal histologic architecture of the uterus, revealing an absence of any form of induction (Figure 3). The endometrium (En) and the epithelium (Ep) of the endometrial mucosa could be seen to be attached to a relatively straight basement membrane, with its mucosal surface facing the uterine lumen (L) and thrown into the longitudinal folds. The extensive lamina propia bears the glands (Gl) (Figure 3), and the blood vessels (BV) are also indicated. A photomicrograph of a portion of the uterus of the positive control (PC) revealed densely packed, spindle-shaped fibrous tissue (F) and multifocal tumor cells (arrow) in the endometrium (Figure 4). This is the group that was induced with uterine leiomyoma using monosodium glutamate but not given treatment. Reduced fibrous tissues and smooth muscle cells, which led to a reduced endometrial and myometrial wall thickness, could be seen in the photomicrograph of the uterus of the treated group (TG) (Figure 5).