Anti-Obesity Evaluation of Averrhoa carambola L. Leaves and Assessment of Its Polyphenols as Potential α-Glucosidase Inhibitors

Averrhoa carambola L. is reported for its anti-obese and anti-diabetic activities. The present study aimed to investigate its aqueous methanol leaf extract (CLL) in vivo anti-obese activity along with the isolation and identification of bioactive compounds and their in vitro α-glucosidase inhibition assessment. CLL improved all obesity complications and exhibited significant activity in an obese rat model. Fourteen compounds, including four flavone glycosides (1–4) and ten dihydrochalcone glycosides (5–12), were isolated and identified using spectroscopic techniques. New compounds identified in planta included (1) apigenin 6-C-(2-deoxy-β-D-galactopyranoside)-7-O-β-D-quinovopyranoside, (8) phloretin 3′-C-(2-O-(E)-cinnamoyl-3-O-β-D-fucopyranosyl-4-O-acetyl)-β-D-fucopyranosyl-6′-O-β-D fucopyranosyl-(1/2)-α-L arabinofuranoside, (11a) phloretin3′-C-(2-O-(E)-p-coumaroyl-3-O-β-D-fucosyl-4-O-acetyl)-β-D-fucosyl-6′-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside, (11b) phloretin3′-C-(2-O-(Z)-p-coumaroyl-3-O-β-D-fucosyl-4-O-acetyl)-β-D-fucosyl-6′-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside. Carambolaside M (5), carambolaside Ia (6), carambolaside J (7), carambolaside I (9), carambolaside P (10a), carambolaside O (10b), and carambolaside Q (12), which are reported for the first time from A. carambola L. leaves, whereas luteolin 6-C-α-L-rhamnopyranosyl-(1-2)-β-D-fucopyranoside (2), apigenin 6-C-β-D-galactopyranoside (3), and apigenin 6-C-α-L-rhamnopyranosyl-(1-2)-β-L-fucopyranoside (4) are isolated for the first time from Family. Oxalidaceae. In vitro α-glucosidase inhibitory activity revealed the potential efficacy of flavone glycosides, viz., 1, 2, 3, and 4 as antidiabetic agents. In contrast, dihydrochalcone glycosides (5–11) showed weak activity, except for compound 12, which showed relatively strong activity.


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

No. Measured Parameters Tested Groups
Normal Obese Orly CLL    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, Obese rats are also reported to exhibit increment in the plasma levels of plasma glucose, insulin, insulin resistance, leptin, and α-amylase [22]. A significant elevation in plasma levels of glucose (1.5-fold increase), insulin (two-fold increase), and insulin resistance (three-fold increase) was noted in obese rats compared to normal rats.
Oral administration of Orly and CLL improved plasma levels of glucose (89.9 and 80 mg/dL, respectively), insulin (10.1 and 9.4 µg/L, respectively), and insulin resistance (2.2 and 1.9, respectively) with different degrees ( Figure 1C,G, Table 1). Obese rats exhibited significantly elevated levels of plasma leptin, a key hormone in the control of food intake and body weight and a target in obesity management [23,24] (two-fold increase) comparable to those in normal rats. Orly and CLL significantly reduced plasma levels of leptin (21.1 and 18.0 ng/mL, respectively) compared to obese control (24.1 ng/mL). Another drug target in obesity is the inhibition of the digestive enzyme α-amylase [25]. In this study, significant increase in α-amylase activity in obese rats (15.3 U/L) was dramatically reduced upon administration of both Orly and CLL (13.7 and 12.4 U/L, respectively) ( Figure 1C, Table 1). Hence, CLL is significantly excelling over Orly in decreasing leptin, insulin, glucose, and α-amylase levels (p < 0.05).

Effect of CLL on Oxidative Stress and Lipid Peroxidation
In the current study, elevated plasma levels of butyrylcholinesterase (BChE) were observed in the obese control (415.7 U/L) in agreement with [22], compared to different experimental groups (250.7, 274.8, and 285.2 U/L in normal, CLL and Orly treated groups, respectively). High plasma BChE activity is associated with aberrant lipid profiles, insulin resistance, and hypertension [23], suggestive for BChE role in many metabolic functions [24]. Oral administration of Orly as well as CLL reduced BchE plasma elevation significantly at different levels (285.2 and 274.8 U/L, respectively). Malondialdehyde (MDA), a biomarker used for assessing oxidative stress, was significantly enhanced in obese rats (15.1 nmol/mL) compared to those of normal ones (5.6 nmol/mL), as an indicator of lipid peroxidation, while catalase enzyme activity, an indicator of antioxidant status, showed reduction by 1.9 fold. Rats treated with Orly and CLL exhibited improved oxidative stress markers at different levels ( Figure 1D,E, Table 1). CLL revealed significant improvement in oxidative stress markers and lipid peroxidation profiles, better than Orly (p < 0.05).

Effect of CLL on Kidney and Liver Functions
Kidney function indicators (creatinine, urea, and uric acid) as well as plasma transaminases (AST and ALT) revealed significant elevation in obese rats compared to normal rats, in agreement with [25]. Treatment with Orly and CLL significantly improved kidney and liver functions, except for the significant elevation of uric acid content in the case of CLL, which is most probably attributed to the high oxalate level in the leaves [26] ( Figure 1F,H, Table 1). Hence, CLL was significantly better than Orly in terms of kidneyand liver-function improvement, except for an elevated uric acid level (p < 0.05).
Overall, despite the better effect of Orly in reducing body weight gain compared to CLL, the latter revealed better improvement in mostly all tested biochemical parameters, except for an elevated uric acid level.

Structure-Activity Relationship Assessment of Isolated Compounds as α-Glucosidase Inhibitors
To further confirm potential efficacy of CLL compounds, isolated compounds were tested for their in vitro α-glucosidase inhibitory activity, to assess their efficacy. Considering the limitation of yield, in vivo assay was not possible to be performed. The efficacy of the isolated compounds was measured and discussed in relationship to the flavonoid structures, as discussed in the next subsections for each class separately, to identify the most crucial motifs within each for activity.
Among glycosides, compound 2, identified as luteolin 6-C-α-L-rhamnopyranosyl-(1-2)-β-D-fucopyranoside, showed the highest inhibitory activity among all isolated flavone glycosides in line with its aglycone, suggestive for the improved efficacy of C-glycosyl flavone against α-glucosidase enzyme, which is in agreement with reports that sugar moiety attached at C-6 position improved efficacy against pancreatic lipase inhibitory activity [40], extended herein to include the α-glucosidase inhibition effect ( Figure 4B).
In contrast, compound 1, identified as apigenin 6-C-(2-deoxy-β-D-galactopyranoside)-7-O-β-D-quinovopyranoside, showed the weakest inhibitory activity among all isolated flavone glycosides, with IC 50 612.9 µM, likely attributed to the glycosylation of hydroxy group at the C-7 position [41] ( Figure 4B) and absent in compounds 2, 3, and 4. Compounds 3 and 4 exhibited strong inhibition with an IC 50 value of 439.2 µM and 390.4 µM, respectively, in line with previously published data on the efficacy of apigenin 6-C-(2 -O-α-rhamnopyranosyl)-β-fucopyranoside in lowering the glucose level in hyperglycemic rats [40]. Standard apigenin and luteolin showed the strongest inhibitory activity, with IC 50 values at 85.6 and 48.2 µM, respectively, compared to that of acarbose (661.6 µM), with luteolin showing the stronger inhibitory activity compared to that of acarbose, which is in accordance with the previously reported α-glucosidase inhibitory activity [42]. In line with our findings, hydroxylation at C-3 of the B-ring of apigenin, in particular, was reported to enhance the α-glucosidase inhibition activity [41], whereas glycosylation affected it negatively compared to the aglycones [43]. Among glycosides, compound 2, identified as luteolin 6-C-α-L-rhamnopyranosyl-(1-2)-β-D-fucopyranoside, showed the highest inhibitory activity among all isolated flavone glycosides in line with its aglycone, suggestive for the improved efficacy of C-glycosyl flavone against α-glucosidase enzyme, which is in agreement with reports that sugar moiety attached at C-6 position improved efficacy against pancreatic lipase inhibitory activity [40], extended herein to include the α-glucosidase inhibition effect ( Figure 4B).
In contrast, compound 1, identified as apigenin 6-C-(2-deoxy-β-D-galactopyranoside)-7-O-β-D-quinovopyranoside, showed the weakest inhibitory activity among all isolated flavone glycosides, with IC50 612.9 μM, likely attributed to the glycosylation of hydroxy group at the C-7 position [41] ( Figure 4B) and absent in compounds 2, 3, and 4. Compounds 3 and 4 exhibited strong inhibition with an IC50 value of 439.2 μM and 390.4 μM, respectively, in line with previously published data on the efficacy of apigenin 6-C-(2''-O-α-rhamnopyranosyl)-β-fucopyranoside in lowering the glucose level in hyperglycemic rats [40]. Standard apigenin and luteolin showed the strongest inhibitory activity, with IC50 values at 85.6 and 48.2 μM, respectively, compared to that of acarbose (661.6 μM), with luteolin showing the stronger inhibitory activity compared to that of acarbose, which is in accordance with the previously reported α-glucosidase inhibitory activity [42]. In line with our findings, hydroxylation at C-3' of the B-ring of apigenin, in particular, was reported to enhance the α-glucosidase inhibition activity [41], whereas glycosylation affected it negatively compared to the aglycones [43].
Regarding standard dihydrochalcones, phloretin was reported as a strong α-glucosidase inhibitor [44] and as a glucose transporter inhibitor [45], with a measured IC 50 value at 110.4 µM ( Figure 4A). Further, phloridzin revealed moderate inhibitory activity, with an IC 50 of 853.1 µM ( Figure 4A), in accordance with the reported dose-dependent α-glucosidase inhibition [46] and confirming that the inhibitory activity of monoglycosyl chalcones is lower than its aglycones [41] ( Figure 4C). These results suggests that α-glucosidase inhibitory activity of A. carambola L. extract is mainly mediated by flavone glycosides composition, with a smaller contribution coming from dihydrochalcone glycosides being less active, except for compound 12.

Plant Material
A. carambola, fresh leaf was collected from Groppy Arboretum, Giza, Egypt, in May 2021. The soil is of clay type with high humidity up to 90%. The tree grows in shade and is irrigated every 15 days. Shade-dried powdered sample of A. carambola leaf (2 kg) was repeatedly extracted with 70% MeOH of analytical grade (Sigma Aldrich, St. Louis, MO, USA) in a water bath at 40 • C (3 × 5 L, each 48 h) until exhaustion and then filtered off. The filtrate was concentrated under reduced pressure to dryness at 55 • C to yield 500 g (25%) crude extract of A. carambola leaves. The obtained extract was kept at 4 • C for further phytochemical and biological assessments.

Chemicals
Biodiagnostic kits were purchased from Biodiagnostic Co. (Dokki, Giza, Egypt) for measurement of AST, ALT, urea, uric acid, creatinine, total cholesterol, HDL, LDL, MDA, leptin, insulin, glucose, α-amylase, BChe, and CAT levels. The enzyme α-glucosidase was purchased from Oriental Yeast Co. (Tokyo, Japan), while HEPES for making buffer solution was purchased from EMD Millipore Corp (Billerica, MA USA). Phenolic standards, i.e., luteolin, apigenin, phloretin, and phloridzin, and 5-fluorouracil as reference cytotoxic drug were purchased from Wako Pure Chemical Industries (Osaka, Japan). Orly as a reference anti-obese drug for in vivo experiment was obtained from Eva Pharma, Egypt. Acarbose as a reference antidiabetic for in vitro experiments was purchased from Wako Pure Chemical Industries (Tokyo, Japan).

Chromatographic and Spectroscopic Techniques
Polyamide 6S, Silica Gel 60 (60-120 mesh), and Sephadex LH-20 (Riedel-de Haën AG, Seelze, Germany) were used for column chromatography (CC). Medium-pressure liquid chromatography (MPLC) was performed using Reveleris Prep System set (Buchi, Flawel, Switzerland) with a UV-ELSD detector, a C-18 flash column (FP ID C18, 35-45 µM, 40 g). Celite No. 545 from Wako (Japan) was used for loading sample. Analytical pre-coated Silica Gel 60 F245 plates (NP-TLC), preparative reversed-phase silica gel 60 RP-18 F254S (RP 18 -PTLC) thin layer chromatography plates (Merck, Germany), and preparative normal phase silica Gel 70 FM (NP-PTLC) thin layer chromatography plates (Wako, Japan) were used for the final purification of compounds. Thin layer chromatography (TLC) plates were visualized under UV light at (254 and 365 nm) and sprayed with 10% MeOH-H 2 SO 4 reagent, followed by heating for 2-3 min. Methanol used for extraction in CC was of analytical grade. Methanol and formic acid for MS and HPLC analyses were of HPLC grade. HPLC analysis was employed using an Agilent 1220 Infinity LC system, equipped with ELSD detector, a binary solvent delivery system, and an autosampler and connected to YMC column (5 µM, 4.6 × 150 mm, Japan). Aqueous formic acid (0.1%) and acetonitrile were used as mobile phases A and B, respectively, with the total flow rate at 1.0 mL/min for 35 min.
Detection of UV absorption of isolated compounds was done using a Shimadzu ultraviolet-visible (UV-Vis) 1601 recording spectrophotometer (P/N 206-67001, Kyoto, Japan) over the range of 190-500 nm was used for all measurements. Path length of cuvettes used was 1 cm. Manipulation of spectra was performed using UVProbe 2.42 software.
Optical rotation was measured on a Jasco DIP-370 polarimeter.
The NMR 1D and 2D spectra were recorded in CD 3 OD, using TMS as internal standard, and chemical shift values were recorded in δ ppm on a Bruker DRX 600 NMR spectrometer. Sample was completely dried to remove any residual solvent, resuspended in 600 µL deuterated methanol (CD 3 OD), and centrifuged prior to NMR analysis.
The HR-ESI-MS was acquired on an Agilent 6545 Q-TOF LC-MS system with dual electrospray ionization (ESI) (Santa Clara, CA, USA) in negative ionization mode, as it is more sensitive for the detection of phenolics, due to their acidic nature making it easier for them to lose protons. IR was recorded on an FTIR-6700 (JASCO, Tokyo, Japan). The sample was ground with KBr in a ratio of (1:10); the mixture is then pressed in disc form and placed into the sample hold, and the IR spectrum was run.

In Vivo Assay Experimental Design
Twenty-four rats were randomized into two groups and received either standard chow diet (8% fat, n = 6), as a normal control, or an HFD (30% saturated fat, n = 18) to induce obesity for 8 weeks [47]. Body weight and food intake were recorded every week. After induction of obesity, rats were divided into three subgroups and still fed on HFD. For subgroup 1, rats were fed on HFD and given the vehicle as obese control. For subgroup 2, rats were fed on HFD and given oral dose of Orly (10 mg/kg RBW/day) for 4 weeks as anti-obesity drug group. Rats in subgroup 3 were fed on HFD and given oral administration of crude methanol extract of A. carambola leaf (CLL), prepared as described in Section 3.1 (300 mg/kg RBW/day) for four weeks. Normal control rats were continued to be fed on standard chow diet for four weeks. Body weight and food intake were recorded every week.
The animal experiment has been carried out according to Ethics Committee, National Research Centre, Cairo, Egypt, following the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

Statistical Analysis
Statistical analyses were done using SPSS version 22. The results were expressed as mean ± standard error (SE) and analyzed statistically using one-way analysis of variance (ANOVA) followed by Duncan test. The statistical significance of difference was taken as p ≤ 0.05.

Isolation and Structural Elucidation
An amount of 150 g from aqueous methanol leaf extract (CLL; see Section 3.1) was fractionated using a polyamide column ( Figure S69). Elution started with distilled H 2 O followed by H 2 O/MeOH, with gradual increase until reaching pure MeOH. The obtained fractions from the column (500 mL each) were examined using PC and TLC and observed under UV light. Similar fractions were pooled together, according to their TLC and PC profiles, to furnish 9 major fractions (A~I). Fraction B was the selected fraction for further purification based on TLC and PC detection. Fraction B (100% H 2 O, 40 g) was subjected to column chromatography (CC) on Sephadex LH-20 with aqueous MeOH for elution (30-100%). Similar fractions were pooled together, according to their TLC profiles, to furnish 7 fractions, B-1~B-7.

In Vitro α-Glucosidase Inhibitory Assay
The assay of α-glucosidase inhibitory activity of compounds was adopted from [63]. Briefly, 100 µL of DMSO and 100 µL of α-glucosidase enzyme (5 U/mL in 0.15 M HEPES buffer) were added to 100 µL substrate (0.1 M sucrose solution dissolved into 0.15 M HEPES buffer). The mixture was vortexed for 5 sec and then incubated at 37 • C for 30 min to allow for enzymatic reaction. After incubation, the reaction was stopped by heating at 100 • C for 10 min in a block incubator. The formation of glucose was determined by means of glucose oxidase method, using a BF-5S Biosensor (Oji Scientific Instruments, Hyogo, Japan). Mathematically, α-glucosidase inhibitory activity of each sample was calculated according to this equation: (Average value of control (Ac) − average value of the sample (As))/Ac × 100.
The IC 50 values were calculated from plots of log concentration of inhibitor concentration against the percentage inhibition curves, using Microsoft Excel 2016. The data were expressed as mean ± standard deviation (SD) of at least three independent experiments (n = 3).

Conclusions
The global quest for anti-obesity as well as anti-diabetic drugs is currently ongoing, as obesity and its complications continue to afflict the world's population, warranting the discovery of new therapeutic regimens. A high-fat diet induced obesity model in rats was used for the assessment of anti-obese activity of A. carambola leaf extract, in relation to its phenolic composition. To the best of our knowledge, this study presents the first comprehensive attempt to reveal the in vivo anti-obese activity of A. carambola leaf extract, leading to the isolation of new bioactive components. Oral administration of A. carambola leaf extract enhanced all obesity complications, viz., dyslipidemia, hyperglycemia, insulin resistance, and oxidative stress, and exhibited significant anti-obesity activity in obese rats ( Figure 5). Further, the effect of CLL was significantly better than Orly in almost all tested biochemical parameters, except for elevated uric acid level, although Orly revealed better reduction in body weight gain.
Multiple chromatographic approaches of the leaf extract led to the isolation of 14 compounds, including 4 flavone glycosides (1-4) and 10 dihydrochalcone glycosides (5)(6)(7)(8)(9)(10)(11)(12) with two non-separable mixtures, including four newly described compounds, i.e ., 1, 8, 11a, and 11b were reported for the first time in the literature. Further, in vitro α-glucosidase inhibitory activity assessment of isolated compounds revealed the strong potency of isolated flavone glycosides, viz., compounds 1, 2, 3, and 4, as α-glucosidase inhibitors, compared to dihydrochalcone glycosides, except for compound 12. These results suggest for the role of flavone glycosides in alleviation of the major obesity comorbidity, i.e., diabetes via α-glucosidase inhibition, and has yet to be confirmed for other action mechanisms. An extended approach utilizing detailed studies on the molecular mechanisms of A. carambola leaf effect should now follow, together with subclinical and clinical trials on leaf crude extract, to be more conclusive, especially considering the known negative impact of its fruit on kidney functions. Moreover, assessment of the isolated phytoconstituents for their anti-obese activity using in vivo model or targeting other enzymes, i.e., lipases, etc., should now follow to correlate for the extract's potential anti-obesity effect. This study poses A. carambola leaf as a new anti-obesity functional food and adds to its effects aside from its fruit's more explored uses.