**1. Introduction**

The radical shift from malnutrition to overnutrition, as well as the increase in sedentary behaviour, has led to the increasing incidence of obesity, a complex chronic nutritional disorder characterized by an energy expenditure and intake imbalance. With estimates of 2.3 billion overweight individuals and 700 million obese adults, obesity with its comorbidities is considered the fifth-largest cause of death worldwide [1,2]. Insulin resistance is one of the most prevalent obesity-related changes [3] and hence, obesity is a key predisposal to type 2 diabetes [1]. Furthermore, some white fat storage areas in the body

**Citation:** Ramadan, N.S.; El-Sayed, N.H.; El-Toumy, S.A.; Mohamed, D.A.; Aziz, Z.A.; Marzouk, M.S.; Esatbeyoglu, T.; Farag, M.A.; Shimizu, K. Anti-Obesity Evaluation of *Averrhoa carambola* L. Leaves and Assessment of Its Polyphenols as Potential α-Glucosidase Inhibitors. *Molecules* **2022**, *27*, 5159. https:// doi.org/10.3390/molecules27165159

Academic Editor: Nour Eddine Es-Safi

Received: 25 July 2022 Accepted: 7 August 2022 Published: 12 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are more directly associated to metabolic consequences of obesity, such as diabetes, than others [2]. Obesity-related problems, viz., diabetes has been associated with decreased life expectancy [4] as well as imparting various clinical disorders such as renal disease, blindness, and amputation of lower limbs, among others [5].

Moreover, obesity and associated symptoms are predicted to cost the global economy USD 2 trillion per year nearly as much as smoking, armed conflict, and terrorism [4]. The common synthetic anti-obesity medicine orlistat is successful in treating obesity, however, it exerts serious gastrointestinal side effects [6].

Likewise, α-glucosidase inhibitors as typical anti-diabetic medicines reported to possess side effects despite their important function in lowering blood glucose levels [7]. Only three α-glucosidase inhibitors are currently used in clinical practice including acarbose, miglitol, and voglibose [5,7], warranting for the development of natural medicines that comprise medicinal herbs, either as pure components or as extracts, as an alternative therapy for obesity [3,7].

For decades, *Averrhoa carambola* L., commonly known as starfruit, a member of the Oxalidaceae family, indigenous to the tropical southeast, is planted across the tropics for its edible fruit as well as its ornamental traits, and it was recently domesticated in other countries, including Ecuador and Egypt [8]. *A. carambola* L. flesh is reported for its potential in the treatment of diabetes [9] as well as its confirmed hypoglycemic and porcine pancreatic lipase inhibitory effects [10,11].

In spite of being edible with several health benefits, starfruit is contraindicated in uremic patients owing to its high oxalate content in addition to its negative inotropic and chronotropic effects [8].

Besides, *A. carambola* leaves were reported for their traditional uses in treatment of hyperglycemia, diabetes, and its related diseases [12,13]. Biological studies further confirmed the hypoglycemic activities of leaves and some of its isolated compounds [13,14] Moreover, leaves were reported to possess potential antioxidant activity [15]. Further, leaf decoction are reported to be used for treatment of aphthous stomatitis and angina [16].

With regards to chemical composition and compared to fruits, *A. carambola* leaves are less investigated. Flavone *C*-glycosides have been previously isolated from leaves, viz., isovitexin, carambolaflavones A and B, and apigenin 6-*C*-(2-O-α-L-rhamnopyranosyl)-*β*-D-glucopyranoside [15]. Both carambolaflavones were reported for their hypoglycemic effect in rats [12]. Recently, 12 dihydrochalcone *C*-glycosides were reported from leaves [17]. Dihydrochalcones are natural phenolics with a C6–C3–C6 skeleton structure, where two aromatic rings are connected via a C3 chain [18] and abundant in *A. carambola*.

In the context of the overall strategy to control obesity and its complications using functional foods, an *A. carambola* crude methanol-leaf extract (CLL) anti-obese effect was assessed using an in vivo high fat diet (HFD)-induced obesity rat model. CLL extract as well as Orly (as reference drug) were orally administered as interventions for the management of obesity showing significant improvement in obesity and its associated complications.

Where, rats were fed on high fat diet for eight weeks resulting in dyslipidemia, hyperglycemia, hyperleptinemia, insulin resistance, oxidative stress, and abnormalities in liver and kidney functions. To confirm development of the obesity model and to assess different treatment actions, both physical and biochemical parameters were monitored. Further, leaf extract was subjected to detailed phytochemical isolation to identify active agent(s) using NMR and MS spectroscopy, with pure compounds assessed for their αglucosidase inhibitory activity.

#### **2. Results and Discussion**

#### *2.1. In Vivo Assay of A. carambola Leaf Extract against HFD-Induced Obesity Model in Rats*

*A. carambola* L. flesh has been reported for the treatment of diabetes in folk medicine [9]. Besides, pharmacological assays confirmed its hypoglycemic effect [10] as well as its porcine pancreatic lipase inhibitory effect [11]. Starfruit is known for its richness in phenolics, especially flavonoids [19], its potential for preventing and curing metabolic disorders, viz., obesity and obesity-related metabolic syndrome [20]. The existence of bioactive phytochemicals, i.e., flavan-3-ols and 2-diglycosyloxybenzoates in carambola leaf, with reported lipase and α-glucosidase inhibitory activities [17], might participate in the anti-obese activity of leaves, warranting their assessment. The effect of CLL extract was assessed against different parameters in HFD-induced obese rats including body weight, dyslipidemia, effect on leptin, α-amylase, plasma glucose, insulin levels and insulin resistance, oxidative stress, and lipid peroxidation as well as the effect on liver and kidney functions, as detailed in the next subsections.

#### 2.1.1. Body Weight and Biochemical Markers Determination

CLL extracts showed a significant reduction in rat body weight (258 g) compared to obese rats (291 g), however, it was still higher than the normal rats group (246 g) (Figure 1A and Table 1). Although Orly caused a significant decrease in BW gain (*p* < 0.05), as compared with the CLL and obese groups, several biochemical markers were measured as the index for obesity status, revealing the excelling of CLL over Orly, as detailed in the next subsections.

**Table 1.** List of nutritional and biochemical parameters in obese, normal, and treated animal groups (*n* = 3).


Results are expressed as mean ± S.E.M. Values with different superscript letters in the same raw are significantly different at *p* < 0.05 levels. b,c,d and <sup>e</sup> are significantly higher than a.

**Figure 1.** Body weight and biochemical parameters in the 4th week of treatment of experimental animals with Orly and CLL compared to normal and obese groups. (**A**) The initial, final body weights (BW) and BW gain (g). (**B**) The level of plasma total cholesterol (TCh mg/dL), triglycerides (TG mg/dL), high-density lipoprotein cholesterol (HDL-Ch mg/dL), and low-density lipoprotein cholesterol (LDL-Ch mg/dL). (**C**) The level of leptin (ng/mL), insulin (μg/L), glucose (mg/dL), and α-amylase (U/L). (**D**) The level of plasma butyrylcholinesterase (BChE (U/L)) and plasma catalase activity (CAT (U/L)). (**E**) The level of malondialdehyde (MDA (nmol/mL) and TCh/HDL. (**F**) The level of plasma uric acid (mg/dL) and plasma creatinine (mg/dL). (**G**) Calculated insulin resistance (IR). (**H**) The activity of aspartate transaminase (AST (IU/L)), alanine transaminase (ALT (IU/L)), and urea (mg/dL). Each bar graph represents the mean replicate measurement (*n* = 6) expressed as mean ± S.E. The bar graphs with a similar lower-case letter (such as 'a') among experimental groups are not significantly different from each other (*p* > 0.05). The bar graphs with different lower-case letters (such as a, b, c, d, and e) are statistically different from each other (*p* < 0.05).

#### 2.1.2. Effect of CLL on Dyslipidemia

Dyslipidemia, a metabolic complication of obesity manifested by hypertriglyceridemia [21], was observed in obese rats compared to normal rats, as shown by the elevation of plasma total cholesterol (two-fold increase), triglycerides (1.7-fold increase), LDL cholesterol (fourfold increase), and the ratio of T-Ch/HDL-Ch (three-fold increase) (Figure 1A, Table 1) concurrent with the reduction in plasma level of HDL-Ch (1.5-fold decrease) (Figure 1E, Table 1). CLL significantly improved dyslipidemia compared to obese control rats as well as rats treated with Orly (*p* < 0.05), however, it was still higher than normal rats (Figure 1A,E, Table 1).
