The plant material was lyophilized using the Antipalu phytomedicine obtained by freeze-drying from a commercial solution sold in the District of Abidjan. This phytomedicinal had been collected during the ethnomedicinal survey carried out between July and September 2020 in three municipalities of the Autonomous District of Abidjan.

The animal selection was in accordance with the Organization of Economic Cooperation and Development (OECD) guidelines no. 423 [10] . Healthy, young, and nulliparous, non-pregnant Wistar rats that weigh 100 - 120 mg, were 8 - 10 weeks old, and were obtained from the animal house of pharmaceutical science, Abidjan (Côte d’Ivoire) was selected. The animals are picked randomly, and marked to permit individual identification. Animals were kept in plastic cages with wood shavings that were changed every other day for 5 days before dosing. This allows animals to acclimatise to laboratory conditions (ambient temperature 25˚C ± 3˚C; humidity ranged from 35% to 60%; light and dark period, 12/12 hours, bedding cleaned and sterilized). All animals had a regular supply of drinking water and food [11] .

Vero cells derived from African green monkey Certopithecus aethiops cell line of renal origin: cell Vero (ATCC number, CCL-81) and have been found in various research as toxicology [12] [13] . The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and were kept in a 5% CO₂ incubator at 37˚C. The culture medium was replaced two times per week and the cells were sub-cultured using trypsin-ethylenediamine tetra acetic acid (EDTA) once a week.

Briefly, Vero cells were seeded in 96-well culture microplates (Costar; Fisher Scientific Labosi SAS) at a density of 1 × 10⁴ cells per 200 μl culture media and incubated at 37˚C in a humidified atmosphere containing 5% CO₂. Non-exposed cells were used as negative controls and Triton X-100 (2%, v/v)-exposed cells as positive controls. Accordingly, 16 replicates were chosen at random as control cells (i.e., incubated only with cultured media), eight replicates per Antipalu concentration as exposed cells (i.e., incubated with culture media containing 3.9, 7.81, 15.62, 31.25, 62.5, 125, 250, 500 or 1000 μg Antipalu/ml)) and eight replicates as positive controls. After 24, 48 or 72 h of incubation, Antipalu cytotoxicity in Vero cells was studied using two methods: the determination of the activity of extracellular LDH in cell-free culture supernatants (Cytotoxicity Detection Kit LDH, Roche Diagnostics) and MTT tetrazolium salt-based colorimeric assay with pertinence for the estimation of metabolic activity of viable cells. Both the effect concentrations (EC) at 10% (EC10) and at 50% (EC50) values were calculated based on the results arising from the most MTT cytotoxicity test

A repeated oral dose of toxicity study was carried out according to the OECD Guideline 407 [8] . The rats were divided into four groups of 10 animals each (5 males and 5 females). Group 1 received 1 ml/100g body weight of distilled water and served as the control group. Groups I, II, and III received extract doses of 200, 400, and 800 mg/kg body weight, respectively. Mortality, body weight, food and water consumption, as well as observation for general toxicity signs of the animals were evaluated daily for 28 days.

At the end of each week, the animals were anesthetized with diethyl ether. The blood was drawn through cardiac puncture and collected in sterile tubes without anticoagulant. Plasma was obtained in one set by centrifuging the blood at 3000 revolutions/min for 10 min and stored at −20˚C in Eppendorf bottles until it required enzymatic activities and concentration of biochemical metabolites assays.

The liver was collected and fixed with 10% buffered formalin for further analysis [14] [15] .

Hepatic enzyme activities were determined using a Cobas C311® HITACHI biochemistry system (Roche Diagnostics, France). Tests were performed using commercial kits (Roche Diagnostics, France) based on the manufacturer’s instructions, summarized in Table 1 .

The hepatic tissues were cut into transverse blocks. An automatic processor (RH-12EP Sakura, Fine Technical Co. Ltd., Tokyo, Japan) was used to further process the blocks. About 12 hours were required for dehydration (96% alcohol for one hour × four changes, and 100% alcohol for one hour × one change).

Clearing was done in three changes of toluene for one hour each. Tissues were impregnated in two changes of paraffin wax with a melting point of 50˚C for a period of 2 hours. Embedding of tissue was done in paraffin using L-shaped metallic moulds. These blocks were put in the refrigerator for a period of 4 - 6 hours. Each block was cut on a rotary microtome (MicromGmbh, Waldorf, Germany). About 5-micrometer-thick tissue section was obtained and placed in the water bath with a temperature of 50˚C below the melting point of paraffin wax. The cut ribbons of tissues were placed on the albumenized glass slide. The sample slides were subsequently stained with haematoxylin-eosin (HE) and examined under a light microscope; photomicrographs of the samples were recorded [11] [16] .

The results are presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) with repeated measures was employed to compare the results according to the administered doses and times of treatment. ANOVA was considered significant when the level of probability (P) was < 0.05; if P < 0.01, this difference is considered as incredibly significant; if highly significant, P < 0.001.

The results of the determinations performed on cell culture supernatants ( Figure 1 ) show an increase in extracellular LDH activity on cells exposed to different concentrations and at different exposure times 24, 48 and 72 H. This increase was non-significant after 24 H incubation, except for Antipalu concentrations of 62.5 µg/ml and 1000 µg/ml, where a tendency to increase was observed (P < 0.05). On the other hand, highly significant increases (P < 0.001) in extracellular LDH activity on cells exposed to different concentrations were observed after 72 h of exposure.

Figure 2 shows MTT results. The results show a tendency towards a decrease in cell proliferation or viability of Vero cells exposed to Antipalu at incubation times of 24, 48 and 72 hours. This decrease is significant and is observed after 24 H for concentrations between 3.9 mg/mL and 125 µg/ml. Similarly, for concentrations between 250 mg/mL and 1000 µg/ml and from 7.81 mg/mL to 1000 µg/ml respectively after 48 h and 72 h exposure to Antipalu, cell viability decreased significantly (P < 0.01). However, for concentrations between 3.9 mg/mL and 125 µg/ml after 48 h exposure, the viability of Véro cells decreased, but not significantly (P > 0.05).

The proliferation of exposed Vero cells compared with controls measured by MTT assay enabled us to determine the inhibition concentrations 10% IC₁₀ and 50% IC₅₀ = 555 µg/mL of Antipalu ( Figure 3 ). These 10% and 50% inhibition concentrations are respectively (IC₁₀ = 111 µg/mL,) and 50% (IC₅₀ = 555 µg/mL).

Figure 4 shows the ALT activity of rats exposed to Antipalu extract ( Table 2 ). Mean ALAT activity values at the start of the experiment (J0) were 228.65 ± 21.85 IU/L in Lot T and 193 ± 38.48 IU/L to 326.20 ± 63.40 IU/L for Lots I to III respectively. After 14 days of treatment with Antipalu extract, a decrease in ALAT activity was observed in Lot I. Similarly, after 28 days of treatment, ALAT activity decreased in treated rats. However, this was not significant significantly (P > 0.05). Mean values for ALAT activity ranged from −15.59% to 42.66% (D0); −25.77% to 0.8% (D14); 35.51 to 84.45% (D28) compared with the control Lot.

Figure 5 shows the influence of Antipalu phytomedicinal extract on ASAT activity ( Table 2 ). The results of ANTIPALU’s influence on ASAT activity are shown in Figure 5 . At J0, mean ASAT activity values were 289.30 ± 9.40 IU/L in Lot T, while they ranged from 280.26 ± 35.29 IU/L to 383.88 ± 96.57 IU/L in treated lot animals. On day 14, ASAT activity increased in Lot II and Lot III treated animals and remained at 229.83 ± 11.21 IU/L and 309.65 ± 90.15 IU/L until the end of treatment. However, this increase was not significant (P > 0.05. Rates of change in ASAT activity were obtained from mean values and ranged from −3.12% to 32.69% (J0); −6.79% to 27.58% (J14); 3.43% to 39.35% (J28) compared with the control lot.

Figure 6 shows the influence of Antipalu extract on ALPs activity ( Table 2 ). ALPs activity did not vary significantly (P > 0.05) in the treated animals compared to the control animals. However, depending on the evolution over time, we observe a slight increase (P > 0.05) in the ALPs activity of Lot III 895.67 ± 82.67 (J14). Likewise, ALPs activity decreased in rats from batch III from 895.67 ± 82.67 (J14) to 530.33 ± 44.80 UI/L (J28), which corresponded to variations of −19.96%; −15.74%; −12.86% and −15.42% decrease.

Figure 7 shows the influence of Antipalu phytomedinal extract on GGT activity ( Table 2 ). Mean GGT activity values on treatment day 0 were 1.50 ± 1.5 IU/L in lot T, while they ranged from 2.25 ± 1.31 IU/L to 5.25 ± 1.49 IU/L in the treated lots. At J14, these values were 2.5 ± 1.5 IU/L in batch T, while they ranged from 2 ± 1.08 IU/L to 4.67 ± 1.86 IU/L in treated lots. At J28, GGT activity values were 2 ± 1 IU/L

in batch II and 5.33 ± 2.03 IU/L in batch III treated animals. These results show a non-significant decrease in the activity of GGT in Lot II rats from 5.25 ± 1.49 (J0) to 2 ± 1 (J28). Rates of change in GGT activity in all treated animals ranging from 50% to 250% (J0); −20% to 86.66% (J14); −8.33% to 77.77% (J28) compared with the control Lot T.

Figure 8 shows histological sections of the liver in animals. These sections show an almost identical normal anatomical structure in the liver of rats from Lot T to Lot III. Hepatic cells show no alteration in tissue structure compared with controls.