The leaves of Gnetumafricanum Welw. and Gnetumbuchholzianum Engl. were harvested from Dibombari, Coastal Region of Cameroon (Lat: 4.3179; Lon: 9.7209) and Limbe Southwest Region of Cameroon (Lat: 4.1796; Lon: 9.2825) respectively. They were identified at the National Herbarium Center (HNC), Cameroon and voucher specimen number assigned; NA 55068HNC for Gnetumafricanum and NE 52338HNC for Gnetumbuchholzianum.

After harvest, they were transported to the laboratory where they underwent successive tap water washing, dripping and drying in the shade for three weeks. They were then ground until a fine powder was obtained. The resulting powder was stored in a glass container and stored away from air and moisture until the extraction stage.

The powder (2.72 Kg) of G. africanum and G. buchholzianum leaves was used to extract bioactive compounds by ethanol-distilled water (70-30) maceration for 48 hours. After filtration, the residue was then rinsed with the same solvent and extracted again by the previous system and under the same conditions. The different filters were evaporated each time to the Heidolph brand rotary rotavapoor under reduced pressure and each hydroethanolic extract was obtained and then preserved.

A fraction of each hydroethanolic extract was used to obtain combinations of extracts in proportions (m/m) of (25-75), (50-50) and (75-25) referred to respectively as E1, E2, E3, 25%, 50% and 75% of the extract of G. africanum Welv. and 75%, 50%, 25% the extract of G. buchholzianum Engl.

The purpose of phytochemical screening is to detect the presence of large classes of secondary metabolites present in each extract. Thus, the presence of secondary compounds such as flavonoids, coumarines, anthraquinones, tannins, terpenoids, saponosides and alkaloids was sought using the methods described by [11].

42 non-gravitated males and females Wistar albino rats weighing an average of 140 ± 35 g were enrolled. These rats were obtained in August 2019 from the Research Laboratory in Food and Nutrition Sciences of the Faculty of Science of the University of Douala. They are raised in cages lined with renewable wood chips every two days, with free access to water and food in an environment subject to 12 hours of light and 12 hours of alternating darkness. During the acclimatization and multiplication period, they are fed a reference food called the whole food of the food sells with a supplement of 50 g/kg of protein from the fish Ethmalosa sp. and vitamin C supplementation to be able to withstand experimentation (Table 1) [12].

This phase was conducted in line with OECD Guidelines 407 [14]. It was conducted on 42 Wistar albino rats divided into seven equal batches of 3 males and 3 females as follows: lot 1 (controls), receiving distilled water at a rate of 1 ml/100

mg of body weight (control lot), lot 2 (E2-100), lot 3 (E2-100) lot 4 (E2-400), lot 5 (E3-100), lot 6 (E3-200), and lot 7 (E3-400), receiving a solution of the E2 and E3 extract combinations at a rate of 100, 200, 400 mg/kg body weight. The treatment lasted 32 days. Water intake, food intake and weight growth were assessed every 2 days.

The data was entered into a 2010 Excel sheet—USA and subsequently exported to GraphPad version 7.0.1 (GraphPad, San Diego, USA) for statistical analysis. The data were presented as an average ± Average Standard Error (ESM) in graphs. Non-parametric tests of Mann-Whitney and Kruskal-Wallis were used to make comparisons. The significance threshold has been set at p-value < 0.05.

Ethically, an application for ethical clearance was filed and obtained from the Institutional Ethics Committee of the University of Douala (IEC-UD) under the registration code No 1748CEI-UDo/08/2019/T.

The different ethanol extracts obtained after maceration were dark green in colour and patous in appearance. The combined extracts revealed the presence of groups of chemical compounds such as saponins, sterols and flavonoides while alkaloids were absent (Table 2). The yield on the extract was 40.2%.

Caption: −: Negative; Positive: Positive.

The extraction of the leaves of Gnetumafricanum Welw. and Gnetumbuchholzianum Engl. showed a high yield. This high yield could be explained by the choice of solvent used and the amount of plant matter. This result is, respectively, contrary to those found by [21] [22] for methanol extraction [6] for methanol extraction of the same leaves. The phytochemical screening (Table 2) of the extracts studied highlighted groups of chemical compounds such as saponins, cathechic tannins, steroids and Flavonoids. These contain more secondary metabolites than ethanolic extract or watery extract from the roots of Caricapapaya Linn [23]. Flavonoids are phenolic phytochemicals that represent substantial components of the human diet and are believed to keep the individual healthy. They are endowed with various biological activities, including antioxidant effects, lipid peroxidation inhibitors, protective anti-inflammatory [24] [25] and hepatotoxic effects.

This study was conducted with the aim of evaluating the toxicological effect in order to prevent human exposure to potential risks associated with the use of extract combinations of G. africanum and G. buchholzianum leaves. In order to assess the effects that could result from the repeated use of these combinations of plant extracts, subacute toxicity was performed, body weight was assessed and decreases were less observed in both sexes. This parameter is important because it can control the health of an animal. Weight loss is frequently used as a first indicator of the harmful effects of drugs [26]. A substance is considered toxic if it results in a mass reduction of more than 10%, and this condition can be considered a sign of toxicity even if other changes do not occur [27]. This weight loss of animals during the experiment could be attributed to antinutritional substances such as tannins and saponins although highlighted in phytochemical screening. The work carried out by [28] has reported that these substances cause a malabsorption of nutrients in the body of animals. These antinutrients are thought to be responsible for the low consumption of food and water and thus the reduction of the body weight of animals treated at different doses of plant extracts compared to controls. These results cannot corroborate for those of Onyekwe et al. [29] who showed a weight loss in animals treated with ethanolic extract of P. guava roots for 90 days. However, in this study, no weight loss was recorded (Table 3), showing that these combinations of leaf extracts of G. africanum and G. buchholzianum were not toxic. Transaminases are enzymatic biomarkers that may indicate tissue damage caused by secondary metabolites that can be observed before structural damage could be demonstrated by histological techniques [30]. These enzymes are synthesized in the cytoplasm and released into the general circulation when cells are damaged [31] and at some level can be used to check the extent of hepatocellular damages [32]. In females, daily administration of leaves hydroethanolic extract combinations selectively altered transaminase activities at 400 mg/kg body weight (b.w) ALAT and ASAT levels increased significantly at the respective probability thresholds of 0.026 and 0.027 compared to p < 0.05, this rate is higher for ALAT activity because more sensitive to hepatocytes [33], This increase in the level of transaminases in the serum may be due to alteration of another organ as the levels of MDA and CAT being raised non-statically significantly in control rats and those with 400 mg/kg and significant between controls and all other concentrations. However, in male animals, there was no statistically significant difference at the probability threshold of 0.05. Rather, a decrease in ALT and AST activities at the respective concentrations of E2-400 and E3-400 mg/kg of b.w. This decline suggests that these combinations of leaf extracts have an effect on the liver. Investigations conducted by Uboh et al. [30] showed a decrease in transaminase levels in males treated with P. guajava leaf extract and an increase in AST levels in females; they also reported a non-significant effect of watery P. guajava leaf extract on liver function in male animals. The decrease in transaminase levels may also reflect the hepatoprotective function of these leaf extract combinations. The work of Roy et al. [34] also showed the hepatoprotective effect of the watery solution of P. guajava leaf extract, with a more pronounced effect at a dose of 500 mg/kg in both sexes. The liver and kidneys are the target organs of toxic chemicals because of their essential functions in the detoxification and excretion process; these organs are very useful in toxicity studies because of their sensitivity to dangerous compounds [35]. In this work, the parameters of evaluated kidney function were creatinine and serum urea. The kidney regulates the excretion of urea, creatinine and the reabsorption of electrolytes in the blood; in cases of glomerular alteration, these substances accumulate in bodily fluids [36]. Abnormally high serum levels of creatinine, uric acid and urea are biomarkers of possible kidney dysfunction [37]. In our study, although serum levels of urea and creatinine increased, urinary levels of these two parameters are normal compared to those of control. In addition, on the histological level, sections of the animal’s kidneys showed no alteration of this organ, indicating its proper functioning. On the other hand, leukodist infiltrations were observed in female animals followed by a sharp rise in ALAT activity. These leukotic infiltrations would indicate acute hepatocellular insufficiency by centro-lobular hepatic necrosis. These results corroborate with those found by [33] and [32] (Figure 18) at higher doses of 400 mg/kg of extracts combinations.