The study was carried out following the “Guide for the Care and Use of Laboratory Animals” as assessed by the National Institute of Health and the protocols endorsed by the Ethic Committee of Laboratory Animals of the University of Granada (Spain) (Ref. No. 28/03/2016/030). 5-week-old C57BL/6J male mice (Janvier, St Berthevin, Cedex, France) were housed in standard conditions (12-h light/dark cycle, temperature 22°C ± 1°C, 55% ± 10% relative humidity) with free access to food and water. Mice were randomly assigned to one of the following groups: lean control (SD) (n = 8), obese (HFD) (n = 8), obese treated with S. aethiopicum (HFD-S. aethiopicum) (n = 8) and obese treated with metformin (HFD-Metformin) (n = 8). Control mice received a standard chow diet (SD group) (13% calories from fat, 20% calories from protein, and 67% calories from carbohydrate; Global diet 2014; Harlan Laboratories, Barcelona, Spain), whereas obese mice were fed a high-fat diet (HFD group) in which 60% of its caloric content was derived from fat (47% monosaturated, 37% saturated, 16% polyunsaturated; Purified diet 230 HF, Scientific Animal Food and Engineering, Augy, France).

The obese-treated mice (HFD-S. aethiopicum group) were daily administered by oral gavage with the ethanolic extract of the Lucanian Solanum aethiopicum peel dissolved in water (25 mg/kg), for a total period of 5 weeks. The lean mice (SD-group) only received the vehicle, instead. Additionally, metformin (250 mg/kg/day) treated mice were used as a positive control group in macroscopic and plasma biochemical determinations. Animal body weight, water, and food intake were controlled regularly during the whole experiment.

A glucose tolerance test was carried out 1 week before the end of the experiment (see a detailed protocol in Supporting Information online). Before sacrifice, mice were fasted overnight and, under isoflurane anaesthesia, blood samples were collected to determine glucose, Low-Density Lipoprotein (LDL)-cholesterol, High-Density Lipoprotein (HDL)-cholesterol, and total cholesterol. Liver, adipose, and colonic tissue were extracted to evaluate gene expression by RT-q-PCR (Supplementary Table S1, Supporting Information).

The obesity-associated vascular dysfunction was performed in descending thoracic aortic rings by measuring acetylcholine vaso-relaxant ability and NADPH oxidase activity (see a detailed protocol in Supporting Information online).

The Lucanian S. aethiopicum peel extract was tested in an experimental model of obese mice fed with a high-fat diet (HFD). As expected, the body weight gain of untreated HFD-mice was significantly higher when compared with that of non-obese mice (Figure 3A). However, the administration of S. aethiopicum peel extract or metformin to HFD-mice significantly reduced this weight gain without modifying total energy intake, thus lowering energy efficiency (Figure 3B). This effect was associated with a significant decrease in the adipose fat mass, since the increased weight of abdominal and epididymal adipose tissues observed in the control obese group was significantly reduced in those obese mice treated with the Lucanian eggplant extract or metformin (Figure 3C).

Some previous obesity and diabetes preclinical studies in mice and rats have reported the effects of S. aethiopicum leaves and fruits extracts to promote a reduction in weight gain (Akinwunmi and Ajibola, 2018; Tuem et al., 2021; Asuquo et al., 2022). This ability of eggplant extracts to reduce weight gain has been mainly attributed to the high presence of polyunsaturated fatty acids together with the ability of different phenolic compounds to inhibit key enzymes involved in lipogenesis and cholesterol synthesis, carbohydrates digestion and glucose absorption (such as α-amylase and α-glucosidase), as previously demonstrated in the in vitro assays. Similarly, a decreased activity of α-amylase and α-glucosidase activities had been also confirmed in rats receiving a fruit African S. aethiopicum extract (Nwanna et al., 2016). Additionally, the inhibition of these enzymatic activities has been shown to be associated with reduced glycemic values in diabetes (Tuem et al., 2021). In this regard, S. aethiopicum extract positively impacted glucose metabolism, as evidenced in the glucose tolerance test performed 1 week before mice sacrifice (Figure 4A). Concisely, it was able to reduce glycemic levels in obese mice, which resulted in lower values of the area under the curve (AUC) (Figure 4A). As expected, and according to these results, plasma glucose concentrations from HFD control animals were markedly increased compared to SD mice, but Lucanian S. aethiopicum extract significantly decreased them (Figure 4B). Of note, although insulin blood levels were the same in the three experimental groups, the administration of S. aethiopicum peel extract to HFD-fed mice resulted in a reduction of the HOMA-IR index (Figure 4C), thus indicating an amelioration in obesity-associated insulin resistance. Similarly, metformin administration showed a significant amelioration of the parameters evaluated.

These results support previous in vivo studies reporting that the administration of three different African eggplant fruits (Solanum kumba, Solanum gilo, and Solanum aethiopicum) to diabetic rats significantly reduced blood glucose levels (Nwanna et al., 2016). Similarly, three additional assays revealed that the administration of either leaves or fruits extracts from African S. aethiopicum showed hypoglycemic activity in rats (Okafor et al., 2016; Akinwunmi and Ajibola, 2018; Tuem et al., 2021). In consequence, the presence of polyphenolic compounds in the extract, not only downregulates the hyperglycemia in obese mice by inhibiting α-amylase and α-glucosidase activities but also ameliorates the obesity-associated insulin resistance, as previously proposed for this crude drug (Aryaeian et al., 2017). Furthermore, different phenolic acids like caffeic acid, ellagic acid, or chlorogenic acid, which are present in the Lucanian S. aethiopicum peel extract, have been shown to improve glucose metabolism and insulin activity in experimental diabetes, by modulating GLUT expression and promoting its translocation via AMP-activated protein kinase (AMPK) and PI3K/Akt pathways (Vinayagam et al., 2016). AMPK is a serine/threonine kinase considered one of the most important indicators of cellular energy status. This enzyme promotes glucose uptake through the translocation of GLUT-4 to the plasmatic membrane and inhibits energy-consuming pathways such as cholesterol synthesis (Habegger et al., 2012); these processes are reduced in obesity and contribute to the development of insulin resistance. In the present study, decreased expression of Ampk and Glut-4 in both hepatic and adipose tissues was observed in control HFD-fed mice, thus explaining the observed impairment in glucose metabolism and insulin sensitivity (Figure 5). When obese mice were treated with the Lucanian S. aethiopicum peel extract, there was a significant amelioration in the expression of both Glut-4 and Ampk in tissue levels, being associated with the improved glucose homeostasis and insulin resistance previously discussed (Figure 5).

Insulin resistance may also drive the development of dyslipidemia, typically characterised by increased levels of triglycerides and low-density lipoprotein cholesterol (LDL), as well as by reduced levels of high-density lipoprotein cholesterol (HDL) (Vekic et al., 2019). The present study showed an increase in total cholesterol, LDL/HDL ratio, LDL, and HDL concentration in control HFD-fed mice (Figure 6A). However, the administration of S. aethiopicum peel extract did not reduce these parameters, showing no differences in LDL/HDL ratio compared to the SD-fed group (Figure 6A).

The beneficial effects of this eggplant extract on dyslipidemia confirmed previous reports in which the administration of other eggplant extracts obtained from fruits (S. kumba, S. aethiopicum, and S. gilo) to HFD-fed rats resulted in a significant improvement in the lipid profile (Akinwunmi and Ajibola, 2018; Nwanna et al., 2019b). It is well known that lipoprotein metabolism is mainly regulated by the lipoprotein lipase (LPL), an essential enzyme involved in the hydrolysis of triglycerides-rich lipoproteins, being its downregulation linked to a hypertriglyceridemia condition (Noh et al., 2006; Xiao et al., 2017). Specifically, LPL breaks down triglycerides into two free fatty acids and one monoacylglycerol molecule, which can enter the cell and be oxidised by β-oxidation (skeletal and cardiac muscle) or resynthesized into triglycerides in adipose tissue and then accumulated. Due to its important function in adipose storage, LPL has also been known as the “gatekeeper of the adipocyte”. Several data reported that body weight gain is inversely associated with LPL mRNA levels, while only a slight reduction was seen in the case of insulin resistance (Kern et al., 1990; Clemente-Postigo et al., 2011). Accordingly, in the present study, although no differences were observed in the concentration of triglycerides (Figure 6B), a significant reduction in Lpl expression was shown in HFD-mice while the extract significantly increased the expression of this marker (Figure 6C). Of note, it has been demonstrated that weight loss is linked to increased Lpl expression and activity (Kern et al., 1990). The beneficial effects exerted by S. aethiopicum peel extract on lipid metabolism in obesity can be associated with the presence of phenolic acids like caffeic acid or gallic acid. In particular, gallic acid regulates AMPK activity by activating the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) (Doan et al., 2015). On the other hand, caffeic acid, through the activation of AMPK, can inhibit the nucleus translocation of Sterol Regulatory Element-Binding Protein-1c (SREBP-1c), leading to lipogenesis and fatty acid synthesis impairment (Ajiboye et al., 2019; Lela et al., 2023). In line with these results, our recent study with the Caco-2 and HepG2 cell lines showed that the Lucanian S. aethiopicum peel extract was able to improve lipid absorption and reduce lipid accumulation through the modulation of SREBP-1c and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Lela et al., 2023).

Obesity is associated with a low-grade inflammation of white adipose tissue (WAT), resulting from chronic activation of the innate immune system (Bastard et al., 2006). Macrophage infiltration into WAT, along with hyperplasia and hypertrophy of adipocytes, results in a local and chronic inflammatory state, evidenced by increased production and secretion of a variety of inflammatory molecules, including tumour necrosis factor-α (TNF-α), interleukin 6 (IL-6), inflammasome-activated IL-1𝛽, and the monocyte chemotactic protein 1 (MCP-1) among others (Cancello et al., 2005; Rodríguez-Hernández et al., 2013). All these mediators work tightly to establish a vicious circle that activates different inflammatory pathways in both the liver and fat tissues. Accordingly, control HFD-fed mice showed increased mRNA expression of different pro-inflammatory cytokines such as Tnf-α, Il-1β, and Il-6, as well as of the chemokine Mcp-1 in both hepatic and adipose tissues (Figure 7). With regard to the latter, MCP-1 is normally secreted by adipocytes during obesity-associated inflammation, which typically results in the recruitment of monocytes from the bloodstream, where they differentiate into macrophages (Engin and Engin, 2017). After administration of S. aethiopicum peel extract to HFD-mice, the expression of all these inflammatory markers was significantly reduced (Figure 7). The amelioration of the obesity-associated inflammatory state was in accordance with the data discussed above and related to the increased expression of the Glut-4 receptor (Figure 5). Indeed, it is known that pro-inflammatory cytokines are involved in the development of obesity-related systemic insulin resistance by interfering with insulin receptor function and reducing the expression and activation of glucose transporters such as GLUT-4 (Shepherd and Kahn, 1999). This anti-inflammatory activity exerted by eggplant extracts was also demonstrated in previous in vitro and in vivo studies performed with the Lucanian S. aethiopicum peel extract and the African fruit extract, respectively (Anosike et al., 2012; Nwanna et al., 2019b; Lela et al., 2023), and may be very likely associated with its content in carotenoids, flavonoids and other polyphenols, that among others, target the Toll-like receptor (TLR)-4/NFkB signalling pathway (Lela et al., 2023). In fact, the presence of lycopene and β-carotene in S. aethiopicum peel extract might contribute to the beneficial effects on the systemic inflammatory response showed in HFD-fed mice by lowering the release of the pro-inflammatory cytokines, as demonstrated in other studies on inflammatory-associated conditions, like LPS-induced septic shock or obesity (Novoselova et al., 2009; Fenni et al., 2017). Similarly, polyphenols have demonstrated their ability to regulate the immune system through the modulation of gene expression and the subsequent synthesis of pro-inflammatory cytokines (Yahfoufi et al., 2018). In addition, TLR signalling also plays a role in the development of obesity-related low-grade inflammation. In fact, TLR signalling regulates the innate immune response in the presence of free fatty acids, which are known to bind either TLR-2 or TLR-4, thus representing a molecular link between hyperlipidemia and the immune system (O'Rourke, 2009). TLR-2 and TLR-4 have also been implicated in glucose metabolism and type 2 diabetes. Mice deficient in these genes were found to be protected against obesity and diet-induced insulin resistance, whereas in humans, TLR-4 gene polymorphisms were associated with a lower risk of type 2 diabetes (Strodthoff et al., 2015). Increased expression of Tlr-4 in the liver and adipose tissue of obese HFD-fed mice compared to the control group was observed in this study (Figure 8). In agreement with the reduction of the above-mentioned inflammatory parameters and hyperlipidemia, the administration of S. aethiopicum peel extract treatment to HFD-fed mice significantly reduced the expression of TLR-4 in both liver and adipose tissue (Figure 8).

Furthermore, obesity has also been associated with altered production of adipokines by adipose tissue, such as leptin and adiponectin. Leptin shows pro-inflammatory activity because of its capacity to stimulate the proliferation of monocytes and their differentiation into macrophages, modulating the activation of natural killer lymphocytes or inducing the production of pro-inflammatory cytokines such as TNF-α, IL-6, or IL-12. It is well known that leptin levels in adipose tissue and plasma are related to the amount of energy stored in fat tissue depots and the energy balance status, being these levels increased in obese subjects (Sáinz et al., 2015; Obradovic et al., 2021). Instead, adiponectin is an anti-inflammatory adipokine whose bloodstream concentration is strongly influenced by fat mass, since a decrease in obese individuals compared with lean individuals has been observed (Ouchi et al., 2011). As expected, in this study, the expression of both Leptin and Adiponectin in the adipose tissue was impaired in HFD-fed mice compared to those fed a standard diet (Figure 9). Furthermore, the increased expression of Leptin in HFD-fed mice was associated with a reduction in the expression of Leptin receptor (Leptin-R) (Figure 9), thus confirming the leptin signalling impairment and the consequently observed hyperleptinemia (Sáinz et al., 2015). However, the administration of S. aethiopicum peel extract to HFD-fed mice resulted in a significant improvement in the expression of these adipokines in the fat tissue, (Figure 9). This regulatory activity on adipokine levels could be related to the presence of phenolic acids, like chlorogenic acid, or flavonols, such as quercetin, which are known to reduce leptin levels and increase adiponectin release (Meng et al., 2013; Yahfoufi et al., 2018).

Obesity-associated inflammatory state is a leading risk factor for various cardiovascular disorders, which are considerably associated with impaired vascular homeostasis and endothelial dysfunction (Iantorno et al., 2014). Although previous studies have evaluated the cardiac risk ratio by the estimation of atherogenic coefficient and lee index in obese rats treated with an extract of the fruit of S. aethiopicum (Akinwunmi and Ajibola, 2018), our study evaluated the functionality of the endothelium, measuring the vasodilator response to acetylcholine of aortic rings, thus representing, as far as we know, the first study to address the effect of S. aethiopicum peel extract on endothelial dysfunction. According to previous studies, the HFD-fed group showed a decrease in acetylcholine-induced vasodilatory responses compared to SD-fed mice, thus evidencing an endothelial dysfunction (Figure 10). Interestingly, mice treated with the extract showed an enhancement of endothelial relaxation. When the aortic rings were previously incubated with the NADPH oxidase inhibitor VAS2870, no marked differences in the maximal relaxant response were observed among the experimental groups (Figure 10A).

Also, the activity of the NADPH enzyme, the main source of reactive oxygen species (ROS), was measured to analyse the oxidative stress. It is well reported that ROS are key regulators of vascular health and associated diseases (Chen et al., 2018). Indeed, physiological levels of ROS are responsible for normal smooth muscle cell contraction and endothelial homeostasis. Nevertheless, excessive ROS generation can promote lipid peroxidation, reduced nitric oxide (NO) availability (NO) (a potent vasodilator), the recruitment of inflammatory cells, and the consequent cardiovascular complications characterised by functional and structural vascular cell alterations. As expected, obese mice exhibited a more pronounced NADPH activation than the lean mice, while the treatment with the extract was able to downregulate it (Figure 10B). This suggested a restoration of NO bioavailability and a reduction of ROS production, which could justify the improvement of the impaired endothelium-dependent relaxation to acetylcholine.