*3.6. 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 IC50 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).

#### **4. 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**–**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.

**Figure 5.** Collective scheme for extraction, isolation, and biological activities performed on *A. carambola* leaves.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/molecules27165159/s1. HRESIMS: 1H NMR: 13C NMR, 1H-1H COSY, HSQC, and HMBC spectra of compound **1** (Figures S1–S6); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **2** (Figures S7–S11); HRESIMS, 1H NMR, and 13C NMR spectra of compound **3** (Figures S12–S14); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **4** (Figures S15–S19); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **5** (Figures S20–S24); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **6** (Figures S25–S29); HRESIMS, 1H NMR, 13C NMR, DEPT-135, 1H-1H COSY, HSQC, and HMBC spectra of compound **7** (Figures S30–S36); HRESIMS, 1H NMR, 13C NMR, 1H-1H COSY, DEPT-135, HSQC, and HMBC spectra of compound **8** (Figures S37–S43); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **9** (Figures S44–S48); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **10** (Figures S49–S54); HRESIMS, 1H NMR, 13C NMR, HSQC, and HMBC spectra of compound **11** (Figures S55–S60); HRESIMS, 1H NMR, 13C NMR, HSQC, 1H-1H COSY, DEPT-135, and HMBC spectra of compound **12** (Figures S61–S67); Scheme of isolation (Figure S69); UV spectra of isolated compounds (Figure S70); IR spectra of new compounds **1**, **8**, **11** (Figure S71).

**Author Contributions:** Conceptualization, N.S.R., N.H.E.-S., S.A.E.-T. and M.A.F.; data curation, N.S.R. and M.A.F.; formal analysis, N.S.R.; funding acquisition, N.S.R., K.S., M.A.F. and T.E.; investigation, N.S.R., D.A.M., M.S.M., M.A.F. and T.E.; methodology, N.S.R., K.S., S.A.E.-T. and M.A.F.; project administration, N.S.R., K.S., N.H.E.-S., S.A.E.-T. and M.A.F.; resources, N.S.R. and K.S.; software, N.S.R. and K.S.; supervision, K.S., N.H.E.-S., S.A.E.-T., D.A.M., Z.A.A. and M.A.F.; validation, N.S.R., D.A.M. and M.A.F.; visualization, N.S.R., M.S.M. and M.A.F.; writing—original draft, N.S.R.; writing—review and editing, N.S.R., K.S., N.H.E.-S., S.A.E.-T., M.S.M., Z.A.A., M.A.F. and T.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a fellowship supported by the Egyptian Cultural Affairs and Missions sector, Ministry of Higher Education, Egypt. The publication of this article was funded by the Open Access Fund of Leibniz Universität Hannover.

**Institutional Review Board Statement:** All animal experiments were carried out according to the research protocols established by Research Ethics Committee in Faculty of Pharmacy, Cairo University, and by Medical Research Ethics Committee (MREC) in NRC, which follow the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals Ethical Approval Certificate No. MP (1959).

**Acknowledgments:** The Egyptian Cultural Affairs and Missions sector, Ministry of Higher Education, Egypt, is acknowledged for the fellowship and financial support offered to N.S.R.

**Conflicts of Interest:** The authors declare no conflict of interest.
