This research was carried out at multiple institutions. Laboratory work was conducted at the Microbiology and Food Quality Control Laboratory of the Faculty of Food Science and Technology (ESTBA), University of Lomé, and at the Regional Center of Excellence in Poultry Sciences, both located in Togo. Additional analyses were performed at the Biomedical Research Centre of the University of Granada (Granada, Spain).

Various chemicals and reagents such as DNEM (Dulbecco’s Modified Eagle Medium, FBS (Foetal calf serum), 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Ether were procured from SIGMA (USA).

The herbal preparation used in this study was composed of bark from Bridelia ferruginea, roots of Sansevieria liberica, and the whole plant of Phyllanthus amarus. The plant materials were collected in June 2023 from Hahotoé, a village located northeast of Lomé, Togo. Botanical identification and authentication were performed at the Laboratory of Botany and Plant Ecology, Faculty of Sciences, University of Lomé. The following voucher specimens were deposited: Sansevieria liberica Hort. ex Gérôme & Labroy (TG201529), Bridelia ferruginea Benth. (7382 FDS/UL), and Phyllanthus amarus Schumach. & Thonn (TG201530).

The animals used in this study were obtained from the Laboratory of Microbiology and Food Quality Control (LAMICODA) of ESTBA. All animal procedures were approved by the Ethics Committee for Animal Experimentation of ESTBA, University of Lomé, in accordance with international guidelines (ARRIVE and Directive 2010/63/EU). The number of animals used was minimized to ensure both ethical compliance and scientific validity.

Healthy female Wistar albino rats, weighing between 100 and 120 g and free from any visible signs of illness or injury, were used for the acute and subchronic toxicity evaluations. The animals were fed wheatgrass and had free access to water. They were housed in standard laboratory conditions: temperature 26˚C ± 4˚C, relative humidity around 60%, and a 12-hour light/dark cycle. All experimental procedures were conducted between April and May 2024.

The collected plant parts were shade-dried at the Laboratory of Microbiology and Food Quality Control (LAMICODA), University of Lomé, at ambient temperatures ranging from 26˚C to 30˚C. Once fully dried, the materials were finely ground into powder. Equal amounts (100 g) of powder from each plant part were mixed and macerated in 3 liters of a 70:30 ethanol-water solution. The mixture was left to macerate for 72 hours, with gentle stirring every 24 hours. After maceration, the mixture was filtered using Whatman No. 1 filter paper. The filtrate was concentrated at 45˚C using a Heidolph rotary evaporator, then frozen at −30˚C and lyophilized at −45˚C. The resulting dry extract was stored in airtight, opaque containers at a refrigerated temperature between 4˚C and 8˚C until use.

The experiment followed OECD guideline 423 for the testing of chemicals [7] .

A single-limit dose of 5000 mg/kg body weight of the plant extract was administered to assess its acute toxicity. Two groups of three healthy female Wistar rats were formed: the test group received the extract, while the control group was given physiological water. Food was withheld for 12 hours prior to administration, although water was available ad libitum. Rats were weighed immediately before dosing, and the extract was administered orally via gavage following a sequential dosing procedure.

Following administration, animals were observed individually during the first 30 minutes, continuously during the first 24 hours and then once daily for a total of 14 days. Observations focused on general behavior and clinical signs of toxicity, including changes in skin and fur, locomotor activity, posture, and any symptoms such as tremors, convulsions, salivation, diarrhea, lethargy, sleep, or coma. Body weight was recorded daily throughout the 14-day observation period.

The 28-day oral subchronic toxicity of the herbal extract was assessed in both male and female Wistar rats, in accordance with OECD Guideline 407 for the testing of chemicals [8] .

Three groups of 10 rats each (5 males and 5 females), weighing between 100 and 120 g, were used for the study. Group 1 served as the control and received physiological water. Group 2 and Group 3 received daily oral doses of the herbal extract at 500 mg/kg body weight and 1000 mg/kg body weight, respectively. Dosing was carried out by oral gavage using an esophageal probe, administered at the same time each day for 28 consecutive days.

Animals were observed at least twice daily for signs of morbidity or mortality. Body weight was measured daily prior to dosing to monitor changes over the treatment period.

At the end of the 28-day treatment, animals were fasted for 12 hours, anesthetized with ether, and blood samples were collected via retro-orbital puncture. Blood was drawn into dry tubes for biochemical analyses (including urea, glucose, creatinine, ALT, AST, GGT, total cholesterol, and triglycerides) and into EDTA tubes for complete blood count (CBC). Following blood collection, the animals were euthanized, and a full necropsy was performed. Organs, including the heart, lungs, liver, kidneys, spleen, testes, and ovaries, were carefully excised, weighed, and used to calculate relative organ-to-body weight ratios.

General Behavior and Mortality: Before treatment began, all animals were individually handled and examined to detect any pre-existing abnormalities in behavior or appearance. Throughout the study, animals were monitored twice daily for signs of illness, behavioral changes, abnormal posture, or alterations in skin, fur, eyes, or mucous membranes. Gait and locomotor activity were assessed weekly by allowing the animals to move freely in an observation area.

Hematological and Biochemical Parameters: Blood samples collected in dry tubes were centrifuged at 3000 rpm for 15 minutes using a CENTRIFUGE 80-3. The resulting serum was transferred into cryotubes and analyzed using a MINDRAY BS-280 chemistry analyzer for biochemical markers. Hematological parameters: red blood cell count (RBC), white blood cell count (WBC), hemoglobin concentration (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) were measured from EDTA-treated blood using a MINDRAY BC-10 automatic hematology analyzer.

Cytotoxicity was evaluated using the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) [9] .

Normal human colon epithelial cells (NCM 356) were obtained from the cell culture unit of the Centro de Instrumentación Científica (CIC) at the University of Granada, Spain. Cells were cultured in DMEM medium supplemented with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 1 mg/mL streptomycin, maintained at 37˚C in a humidified 5% CO₂ incubator.

A total of 3 × 10⁴ NCM 356 cells were seeded per well in 96-well plates and allowed to adhere for 24 hours. Cells were then treated with the extract at concentrations of 2, 5, 10, 50, and 100 µg/mL, or with control medium, for 48 hours. The medium was then replaced with 90 µL/well of fresh medium plus 10 µL/well of MTS solution and incubated for 3 - 4 hours at 37˚C with 5% CO₂. Absorbance was measured at 490 nm using a plate reader. The assay was repeated three times (n = 3).

All results are expressed as mean ± standard deviation (SD). Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test to identify significant differences. A p-value of less than 0.05 (p < 0.05) was considered statistically significant. Data analysis was carried out using R software, version 4.2.3.

Throughout the acute toxicity study, no mortality or clinical signs of toxicity were observed in the treated animals. The rats maintained normal behavior, with no alterations in responsiveness to pain, noise, or movement. Furthermore, macroscopic examination of internal organs revealed no visible abnormalities, suggesting that the extract did not induce acute toxic effects at the administered dose.

Over the 28-day treatment period, no deaths or overt signs of toxicity were recorded in any of the experimental groups, regardless of the dose administered (500 or 1000 mg/kg body weight). All animals appeared healthy and displayed normal behavior, with no notable differences in food or water intake between the control and treatment groups.

Body Weight

In female rats, both doses of the extract (500 and 1000 mg/kg bw) led to a statistically significant increase in body weight compared to controls. This increase was observed from day 2 to day 11 (p < 0.0001), and continued significantly from day 12 to day 19 (p < 0.001) ( Figure 1 ).

In male rats, no significant body weight change was observed at the 500 mg/kg dose. However, rats receiving 1000 mg/kg showed a sustained and significant increase in body weight throughout the study period, from day 2 to day 28 (p < 0.0001), as illustrated in Figure 1 .

Relative Organ Weights and Macroscopic Observations: No treatment related macroscopic abnormalities were observed in any of the animals, regardless of sex or dosage level (500 and 1000 mg/kg bw). All internal organs appeared normal upon visual inspection during necropsy.

In female rats, there were no statistically significant differences in the relative weights of key organs, including the liver, spleen, heart, lungs, kidneys, and ovaries when compared to the control group, indicating that the extract did not cause any overt organ specific toxicity ( Figure 2 ).

At 500 mg/kg bw, a significant decrease in the relative weight of the testes (p = 0.013) and liver (p < 0.001) was observed in male rats. Conversely, at 1000 mg/kg bw, a significant increase in the relative weight of the lungs (p = 0.0073), kidneys (p = 0.03), and heart (p > 0.01) was noted ( Figure 3 ).

Blood Biochemical Parameters: The serum biochemical profiles of female and male rats following 28 days of treatment are summarized in Table 1 and Table 2 , respectively.

Values are expressed as mean ± standard deviation (SD).

Values are expressed as mean ± standard deviation (SD).

In female rats, no significant changes were observed in urea, glucose, AST, GGT, total cholesterol, or triglycerides compared to controls. However, a significant increase in creatinine was observed at 500 mg/kg bw (p = 0.0083), and ALT increased significantly at 1000 mg/kg bw (p = 0.002) ( Figure 4 ).

In male rats, no significant changes in glucose, creatinine, ALT, or triglycerides were observed. However, AST was significantly reduced at both 500 mg/kg (p < 0.01) and 1000 mg/kg (p < 0.001). GGT also decreased at 500 mg/kg (p = 0.02), as did total cholesterol at 1000 mg/kg (p < 0.001) and urea at both doses (p < 0.001) ( Figure 5 ).

Hematological Parameters

Hematological data are presented in Table 3 and Table 4 .

Values are expressed as mean ± standard deviation (SD).

Values are expressed as mean ± standard deviation (SD).

In female rats, a significant decrease in hematocrit was observed at both 500 mg/kg (p < 0.001) and 1000 mg/kg (p = 0.029) compared to the control group. No significant differences were found in other hematological parameters including hemoglobin (HGB), MCV, MCH, MCHC, or platelet count (PLT) ( Figure 6 ).

In male rats treated with 500 and 1000 mg/kg, no statistically significant changes were observed in RBC, HGB, HCT, MCV, WBC, or PLT. However, MCH levels showed a significant reduction at 500 mg/kg (p < 0.01) and 1000 mg/kg (p < 0.001), while MCHC significantly decreased at 1000 mg/kg (p = 0.024) ( Figure 7 ).

Cytotoxicity was evaluated using the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) [9] . Normal human colon epithelial cells (NCM 356) were obtained from the cell culture unit of the Centro de Instrumentación Científica (CIC) at the University of Granada, Spain. Cells were cultured in DMEM medium supplemented with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 1 mg/mL streptomycin, maintained at 37˚C in a humidified 5% CO₂ incubator.

Cytotoxicity was recorded as CC₅₀ values (mg/mL). This corresponds to the extract concentration that kills or inhibits the growth of 50% of the cell population. The percentage of cell lethality at the highest concentration was 10.88% ( Figure 8 ). The obtained data show that the extract from the recipe does not have the potential to inhibit the growth of NCM in a time- and dose-dependent manner. The cytotoxic concentration for 50% of the cells (CC₅₀) is 440,457 µg/mL, equivalent to 440.46 mg/mL.