The whole plant of Elephantopus mollis was collected in May 2022 at Diang (East region of Cameroon). Botanical identification was performed at the national herbarium, Yaoundé-Cameroon where the plant sample was compared to the voucher specimens N˚18231 SRF/cam. The sample was cleaned, sliced into small pieces, shade dried, and powdered with a grinder. Thereafter, the powder (300 g) was introduced into boiling water (3 L) for 5 hours. The mixture was filtered using Whatman paper N˚3 and dehydrated to yield 31.5 g of dry dark aqueous extract.

Phytochemical screening of this extract for its active biological principles was conducted using the standard methods found in the literature.

Male albino Wistar rats (6 - 8 weeks old) were used in this experiment. They were raised under natural environmental conditions of light and temperature in the animal house of the Laboratory of Human Metabolism and Non-Communicable Diseases of the Institute of Medical Research and Medicinal Plants Studies (IMPM), Yaoundé, Cameroon. The animals were kept in polypropylene cages with a metal mesh cover at room temperature, with adequate ventilation, and then were allowed to acclimatize to laboratory environmental conditions for two weeks. Food and water were given ad libitum. Animal handling and experiments were performed according to the European Union directives on ethical evaluation of animal experiments [11] adopted by the Cameroon institutional national ethics committee, Ministry of Scientific Research and Innovation (N˚: FWA-IRD 0001954).

All the ingredients of the diet were obtained from a local market in Yaoundé. Induction of obesity was done according to the method described by Kamgang et al. [12]. The high-fat diet was used to induce obesity in rats over 16 weeks [13]. In fact, after a two-week acclimatization period, rats were randomly subjected to a high-fat diet (Table 1). Food and water intakes were recorded every two days, and the weight variation was measured weekly during a 16-week period. To determine obese rats at the end of this treatment period, the Lee index (Li) was calculated using the body weight (bw in g) and naso-anal length (Lna in cm) as follows:

The animals with Lee index ≥ 0.31 were considered obese, selected, and randomly divided into 5 groups of five rats each for the next stage of the experiment.

Table 1. Diet composition per 1000 g [12].

ND: normal diet; HFD: high-fat diet.

The equivalent dose was calculated using the NOAEL calculation method [15] based on the dose administered to humans by traditional healers.

The rats were divided into six (6) groups of five animals each as follows:

Normal control (NC) group: rats fed with a normal diet [12], receiving distilled water (10 mL/kg);Obese control (OC) group: obese rats fed with HFD, treated with distilled water (10 mL/kg);Elephantopus mollis(Em) treated groups: obese rats fed with HFD, treated with E.mollis extract, respectively, at 50, 100, or 200 mg/kg body weight (Em50, Em100, or Em200, respectively);Atorvastatin (AVAS) group: obese rats fed with HFD, treated with the reference drug atorvastatin at 10 mg/kg body weight.

Rats once daily received the respective treatments, which were administered by intra-gastric gavage for 28 consecutive days. During this period, the body weight was recorded every week, and the food and water intakes were recorded every two days. At the end of treatment, all rats were fasted overnight for 12 hours with free access to water, then sacrificed under anesthesia (diazepam, 10 mg/kg bw, and ketamine, 50 mg/kg bw). The blood was collected in dried tubes, centrifuged, and the serum obtained was used to determine the serum lipid profile. White adipose tissues (visceral, subcutaneous, and testicular fats), carcass, and the liver were collected and weighed. The liver was immediately washed with an ice-cold saline solution (NaCl 0.9%), and a portion (200 mg) was homogenized in 1 mL of Tris-HCl (0.2 M, pH 7.4) buffer solution, then centrifuged (2500 g, 25 min). The supernatant obtained was used for biochemical estimations of total cholesterol and triglycerides.

BiochemicalAnalysis: Determination ofLipid Parameter Content

Total cholesterol (TC), HDL-cholesterol (HDL-C), and triglycerides (TG) were assayed through colorimetric methods with commercially available test kits according to the manufacturer’s recommendations (Lab Kit brand).

LDL-C (Low-density lipoprotein-cholesterol) was calculated with the standard formula [16]:

The atherogenic index (AI) was calculated as follows [17]:

The results were expressed as mean ± standard error of the mean. The statistical analyses were performed by one-way analysis of variance (ANOVA) associated with Tukey’s test, followed by the Dunnett test for non-repeated measures. Two-way ANOVA was used, followed by Bonferroni’s multiple comparisons test, to compare body weight, food and water intakes. GraphPad Prism 8.0.1 was used for analyses. The difference between and within various groups was significant at p < 0.05.

The phytochemical screening of E.mollis aqueous extract revealed the presence of different classes of chemical compounds such as phenols, tannins, anthraquinones, saponins, sterols, flavonoids, anthocyanidins, coumarins, triterpenes, alkaloids, and polysaccharides.

During the treatment, food and water intake of obese control rats (OC) did not significantly vary compared to normal control rats (NC) (Figure 1). The administration of E.mollis aqueous extract decreased the food and water intakes. From the 21^st day of the treatment, the extract at 100 mg/kg bw significantly (p < 0.05) decreased food intake compared to OC (Figure 1(a)). This dose of extract at the same period also significantly decreased water intake compared to OC (Figure 1(b)).

Body weight of obese control rats (OC) remained significantly (p < 0.01) high during the treatment (+31.6% at day 28) compared to normal control rats (NC). E.mollis aqueous extract at 200 mg/kg bw, as well as atorvastatin, slightly decreased the body weight from the 14^th day of treatment (Figure 2, Table 2).

The MACAPOS 2 high-fat diet significantly (p < 0.01) increased visceral (+197.19%), peri-testicular (+90.60%), and subcutaneous (+333.57%) white adipose tissues in non-treated obese rats compared to the normal control group (NC). Administration of E.mollis aqueous extract moderately decreased visceral (−4.87% Em50, −16.04% Em100, or −12.58% Em200) and testicular fat (−3.86% Em50, −13.68% Em100, or −2.46%% Em200). The plant extract at different doses, as well as the reference drug, remarkably (p < 0.01) reduced subcutaneous fat (−38.55% Em50; −38.39% Em100, or −37.07% Em200) compared to obese control rats (Table 2).

Figure 1. Effects of E.mollis aqueous extract, atorvastatin, or distilled water on food (a) and water (b) intakes (expressed as % compared to initial values) of high-fat diet-induced obese rats during 28 days of treatment. NC: normal control rats; OC: obese control rats; Em50, Em100, Em200: obese rats treated with E.mollis extract, respectively, at 50, 100, or 200 mg/kg bw; AVAS: obese rats treated with atorvastatin 10 mg/kg bw. Significant difference: *p < 0.05, **p < 0.01 compared to NC; ^ap < 0.05, ^bp < 0.01 compared to OC; n = 5.

Figure 2. Body weight variation (expressed as % of initial values) of obese rats during 28 days of treatment. NC: normal control rats; OC: obese control rats; Em50, Em100, Em200: obese rats treated with E.mollis extract, respectively at 50, 100, or 200 mg/kg bw; AVAS: obese rats treated with atorvastatin 10 mg/kg bw. Significant difference: ^αp < 0,05; ^βp < 0,01 compared to initial value; *p < 0.05, **p < 0.01 compared to NC; ^ap < 0.05, ^bp < 0.01 compared to OC; n = 5.

Table 2. Body weight (expressed in g) of obese rats during 28 days of treatment.

NC: normal control rats; OC: obese control rats; Em50, Em100, Em200: obese rats treated with E.mollis extract, respectively, at 50, 100, or 200 mg/kg bw; AVAS: obese rats treated with atorvastatin 10 mg/kg bw. Significant difference: *p < 0.05, **p < 0.01 compared to NC; n = 5.

The carcass weight of obese control rats (OC) significantly (p < 0.01) decreased compared to NC. Just like atorvastatin, plant extract at the doses used slightly increased the relative carcass weight (+4.82% Em50, +5.37% Em100, and +3.40% Em200) at the end of treatment compared to OC (Table 3).

Table 3. Relative weight of visceral fat, peri-testicular fat, subcutaneous fat, and carcass (expressed in %) of obese rats after 28-day treatment.

NC: normal control rats; OC: obese control rats; Em50, Em100, Em200: obese rats treated with E.mollis extract, respectively, at 50, 100, or 200 mg/kg bw; AVAS: obese rats treated with atorvastatin 10 mg/kg bw. Significant difference: *p < 0.05, **p < 0.01 compared to NC; ^bp < 0.01 compared to OC; n = 5.

High-fat diet significantly (p < 0.01) increased total cholesterol, triglycerides, and LDL-cholesterol (+115.29%, +44.77% and 592.47%, respectively) while decreasing HDL-cholesterol (−30.82 %) in obese control rats (OC) compared to normal control rats (Figure 3). Administration of E.mollis aqueous extract remarkably (p < 0.01) decreased the serum level of total cholesterol (−61.38% Em50; −19.38% Em100; and −28.96% Em200) in obese rats compared to OC (Figure 3(a)). Better than atorvastatin, the plant extract at the used doses significantly (p < 0.01) reduced serum triglycerides (−41.94% Em50, −8.63% Em100, and −36.42% Em200) compared to OC, bringing them to a level comparable to that of the normal control rats (NC) (Figure 3(b)). In a dose-dependent manner, plant extract led to a significant (p < 0.01) decrease in serum LDL-cholesterol level (−72.16% Em50, −79.43% Em100, and −87.08% Em200) compared to obese control rats, bringing it to a level comparable to that of the normal control rats (Figure 3(c)). The plant extract at 100 mg/kg bw increased (p < 0.01) serum level of HDL-cholesterol (+92.04%) compared to OC (Figure 3(d)).

The atherogenic index remarkably (p < 0.01) increased (+111%) in obese control rats compared to normal control rats. Better than atorvastatin, E.mollis aqueous extract, respectively, at 50 and 100 mg/kg bw, led to a remarkable (p < 0.01) decrease in the atherogenic index: +56.5% and +36.5%, bringing it into the normal range (Figure 3(e)).

Figure 3. Serum lipid: total cholesterol (a), triglycerides (b), LDL-cholesterol (c), HDL-cholesterol (d), and atherogenic index (e) of obese rats after 28 days of once daily treatment. NC: normal control rats; OC: obese control rats; Em50, Em100, Em200: obese rats treated with E.mollis extract, respectively, at 50, 100, or 200 mg/kg bw; AVAS: obese rats treated with atorvastatin 10 mg/kg bw. Significant difference: *p < 0.05, **p < 0.01 compared to NC; ^ap < 0.05, ^bp < 0.01 compared to OC; n = 5.

A high-fat diet significantly (p < 0.01) increased hepatic lipids, including total cholesterol and triglycerides, compared to normal control rats (NC). E.mollis aqueous extract at 200 mg/kg bw significantly (p < 0.01) decreased hepatic triglycerides (−52.63%) compared to obese control rats (Figure 4(a)). The extract also, in a dose-dependent manner, remarkably (p < 0.01) lowered hepatic total cholesterol, respectively: −25.13% (Em50), −37.23% (Em100), or −42.98% (Em200) to a level similar to that of the normal control rats (Figure 4(b)).

Figure 4. Hepatic lipid profile: triglycerides (a) and total cholesterol (b) of rats after 28 days of once daily treatment. NC: normal control rats; OC: obese control rats; Em50, Em100, Em200: obese rats treated with E.mollis extract, respectively, at 50, 100, or 200 mg/kg bw; AVAS: obese rats treated with atorvastatin 10 mg/kg bw. Significant difference: *p < 0.05, **p < 0.01 compared to NC; ^ap < 0.05, ^bp < 0.01 compared to OC; n = 5.