Garcixanthone E and Garcimangophenone C: New Metabolites from Garcinia mangostana and Their Cytotoxic and Alpha Amylase Inhibitory Potential

Garcinia mangostana (Clusiaceae) is a rich pool of metabolites with diversified bioactivities. A new xanthone, garcixanthone E (1), and a new benzophenone, rhamnoside, as well as garcimangophenone C (9) together with garcinone E (2), α-mangostin (3), γ-mangostin (4), garcinone C (5), garcixanthone C (6), gartanin (7), and 2,4,6,3′,5′-pentahydroxybenzophenone (8) were purified from G. mangostana EtOAc extract. Their structural verification was accomplished utilizing assorted spectral tools and relating to the literature. The in vitro cytotoxic potential versus MCF-7, A549, and HCT-116 cell lines demonstrated the moderate potential of 1 (IC50s 8.5, 5.4, and 5.7 µM, respectively) in comparison to doxorubicin (IC50s 0.18, 0.6 and 0.2 µM, respectively) using a sulforhodamine B (SRB) assay. Additionally, 1 and 9 had AAI (α-amylase inhibition) with IC50s 17.8 and 12.9 µM, respectively, compared to acarbose (IC50 6.7 µM). Further, their AAI mechanisms were inspected utilizing molecular-docking evaluation by employing the crystal structure of the human α-amylase (PDB-ID: 5EOF). Compound 9 possessed a reasonable docking score of −7.746 kcal/mol compared with the native ligand 7JR which had a docking score of −9.932 kcal/mol. These results could further provide new insight into the potential usage of G. mangostana as a functional food for regulating postprandial hyperglycemia via suppressing AA.

Cancer is one of the major serious illnesses that has a high, unacceptable mortality rate and incidence [13]. In total, 19.3 million new cancer cases and ≈10 million deaths because of cancer worldwide were estimated in 2020 [14]. Breast cancer with 2.3 million new cases (11.7%) has transcended lung cancer (11.4%) as the most frequently pinpointed cancer, followed by colorectal, prostate, and stomach (10.0%, 7.3%, and 5.6%, respectively) cancers. On the other hand, lung cancer with 1.8 million deaths (18%) continued to be the dominant reason of cancer death, following colorectal (9.4%) and breast (6.9%) cancers [14].
So far, the majority of anticancer agents have failed to accomplish the expected results. Therefore, there is an intensive research reorientation towards the discovery of new chemopreventive agents from natural sources [15,16]. Many natural metabolites are known to have chemoprotective potential towards various types of cancers worldwide [15]. These metabolites are widely found in fruits, vegetables, and plants [17]. It is a fact that consuming vegetables and fruits lowers carcinogenesis incidence [16]. Fruits and vegetables contain vitamins, fiber, minerals, and various bioactive metabolites, such as flavonoids, carotenoids, sterols, and phenolics, and all of them could be responsible for this protective potential [18].
The objective of this work was to discover new AAIs (α-amylase inhibitors) and anticancer agents from G. mangostana pericarps.

Plant Material
G. mangostana fruits were secured in December 2019 from a Saudi local market. Its attestation was accomplished as earlier stated [6] and a voucher specimen (no. GM_1424) was kept in the herbarium at the Faculty of Pharmacy, KAU.

a-Amylase Inhibitory Assay
The AAI potential of 1 and 9 was assessed utilizing Enz-Chek ® Ultra-Amylase Assay Kits as formerly stated [7].

Protein Preparation
To perform the docking studies, the crystal structure of the alpha amylase (PDB ID: 5E0F) was imported from the available online protein databank. Before docking, the protein was prepared by employing the Schrödinger suite protein preparation wizard tool [19]. The hydrogen and heavy atoms were subjected to optimization by restrained minimization. Additionally, missed H atoms were added, and the correct charges were assigned using the OPSL4 force field. H 2 O molecules from HET groups beyond 5 Å were removed.

Ligand Preparation
Lig Prep was used to convert the compounds from 2D to 3D structures [20]. Strained minimization was carried out by employing the OPLS4 force field, the optimization of H-bonds was accomplished at pH 7.0 utilizing PROPKA, and water molecules beyond 3 Å were removed from the HET groups, Additionally, at 7.0 ± 2.0 pH, the metals' HET cofactors and states were generated.

Receptor Grid Generation and Docking
By using Glide, both ligands docking and grid generation were accomplished [21]. The grid box was defined by selecting the cocrystalized peptide inhibitor of 5E0F, and the binding region was specified using Glide's Receptor-Grid-Generation tool. The generated grid was utilized for the prepared ligands docking using Glide software. The selected protocol was SP (standard precision). The default 0.25 potential charge cutoff and 1.0 radii scaling factor (vdW) were set. Compounds 1 and 9, in addition to the cocrystalized ligand, 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)-4H-chromen-3-yl-6-deoxy prop-2-enoyl]-beta-D-glucopyranosyl}-alpha-L-mannopyranoside (code:5JZ) and acarbose were redocked using the XP (extraprecision) protocol. All other settings were retained as the default.
Compound 9 was obtained as a brown powder. Its IR spectrum revealed bands at 3365, 1638, and 1605 cm −1 , which signalized the existence of hydroxyl, carbonyl, and C=C functionalities in 9. Additionally, it had UV bands at 310, 282, and 213 nm [7,8]. The HRESIMS demonstrated a pseudomolecular peak at m/z 393.1180 (calcd for 393.1186 for C19H21O9), corresponding to the molecular formula C19H20O9, which required 10 unsaturation degrees for two benzene, one carbonyl, and hexose moieties. Further, the HRESIMS 246.0535 [M+H-hexose moiety] + fragment peak indicated that 9 possessed a hexose sugar. In the HSQC and 13 C, nineteen carbons were noticed, consisting of one methyl, eleven methines, and seven quaternary carbons for the oxygen-bonded aromatics at δC 163. 8  characterized a rhamnose moiety in 9, which was ensured by the COSY and HMBC correlations [27]. Its attachment at C-6 was confirmed by the HMBC crosspeak of H-1″ to C-6 (δC 159.5). Based on these data, 9 was specified and named garcimangophenone C. It is noteworthy that this was the first report of isolating benzophenone rhamnosides from G. mangostana. Compound 9 was obtained as a brown powder. Its IR spectrum revealed bands at 3365, 1638, and 1605 cm −1 , which signalized the existence of hydroxyl, carbonyl, and C=C functionalities in 9. Additionally, it had UV bands at 310, 282, and 213 nm [7,8]. The HRESIMS demonstrated a pseudomolecular peak at m/z 393.1180 (calcd for 393.1186 for C 19 H 21 O 9 ), corresponding to the molecular formula C 19 H 20 O 9 , which required 10 unsaturation degrees for two benzene, one carbonyl, and hexose moieties. Further, the HRESIMS 246.0535 [M+H-hexose moiety] + fragment peak indicated that 9 possessed a hexose sugar. In the HSQC and 13 C, nineteen carbons were noticed, consisting of one methyl, eleven methines, and seven quaternary carbons for the oxygen-bonded aromatics at δ C 163. .0 (C-2 ) characterized a rhamnose moiety in 9, which was ensured by the COSY and HMBC correlations [27]. Its attachment at C-6 was confirmed by the HMBC crosspeak of H-1 to C-6 (δ C 159.5). Based on these data, 9 was specified and named garcimangophenone C. It is noteworthy that this was the first report of isolating benzophenone rhamnosides from G. mangostana.

Cytotoxic and AAI (Alpha-Amylase Inhibitory) Activities
The cytotoxic potential of 1 and 9 was assessed towards MCF-7, A549, and HCT-116 cell lines using a sulforhodamine B (SRB) assay. Compound 1 had moderate activity towards A549, MCF-7, and HCT-116 with IC 50 s 5.4, 8.5, and 5.7 µM, respectively, compared with doxorubicin (IC 50 s 0.18, 0.6, and 0.2 µM, respectively). Unfortunately, 9 had weak cytotoxic potential versus the tested cancer cell line. It is mentionable that GM pericarp extracts revealed a significant glucose-decreasing and insulin-sensitization capacity [28]. It also revealed the antihyperglycemic effectiveness through boosting insulin-forming β-cell populations, which referred to its antioxidative phenolic constituents [29]. Moreover, it Life 2022, 12, 1875 7 of 11 amended β-cells and pancreatic glands impairment caused by STZ in diabetic mice via promoting insulin production and modulating the sensitivity to the decreased insulin [30]. The treatments of diabetic mice with GM xanthones remarkably amended the antioxidant and biochemical parameters, reformed the kidney and liver histological changes, and lessened the kidney tissue cellular apoptosis [31]. Further, GM xanthones and benzophenones were proved to display α-amylase and α-glucosidase inhibitory capacities; therefore, they could minimize postprandial hyperglycemia via the prohibition of glucose absorption [7].
Accordingly, the new metabolites 1 and 9 were assessed for their AAI capacity. They demonstrated AAI potential (IC 50 17.8 and 12.9 µM, respectively) in comparison to acarbose (IC 50 6.7 µM).

Ligands and Protein Preparation
Compounds 1, 9, and 5J7 (native inhibitor of 5EOF) were prepared using LigPrep to convert 2D structures into 3D; additionally, the ionization state at a pH of 7.0 ± 2.0 and tautomeric forms were created. Using the protein preparation wizard, the human αamylase's protein crystal structure (PDB ID: 5E0F) was prepared, whereby the hydrogens were added, the bond orders were specified, and the het states using an Epik at pH 7.0 ± 2.0 were generated. The H-bonds were optimized at pH 7.0 employing PROPKA in sample water orientation, and the restrained minimization was performed using the OPLS4 force field.

Receptor Grid Generation and Molecular Docking Studies
The grid box was created all over the protein's binding site of the minimized protein that contained the cocrystalized inhibitor utilizing the crystal structure (PDB-ID: 5E0F), and the binding area was specified by the 5J7 native inhibitor's selection. The nonpolar atoms were located and the Van der Waals radii scaling factor was set to 1, and 0.25 was the partial charge cutoff. The ligands docking was executed utilizing the Schrödinger suite "ligand docking" tool, the protocol was SP (standard precision), and all other settings were retained in their default form. The redocking of the ligand 5J7 (5,7-dihydroxy-4-oxo-2-(3,4,5trihydroxyphenyl)-4H-chromen-3-yl 6-deoxy-2-O-{6-O-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]-beta-D-glucopyranosyl}-alpha-L-mannopyranoside) was performed to evaluate the docking study. Compound 9 exhibited a reasonable docking score (−7.746 kcal/mol) compared with the native inhibitor 5J7 (−9.932 kcal/mol) as shown in Table 2. For the docking validation, the native inhibitor was prepared and redocked alongside compounds 9 and 1; then, the poses of the protein compound complexes were examined, and the RMS of the native inhibitor 5J7 was found to be in an acceptable range (1.0862). The investigation of the cocrystalized human AA with the native inhibitor 5J7 showed the formation of hydrogen bonds and hydrophobic interactions with many amino acids' residues. Hydrogen bonds formed between 5J7 and Gln63, Asp97, Glu233, and His305 while forming a hydrophobic interaction via pi-pi stacking with the residues Tyr62 and His 29 ( Figure 3). Compound 9 interacted with the AA through hydrogen bonds between its hydroxyl groups and the amino acids' residues Thr163, Asp197, Lys200, His 201, and His 299 in addition to the aromatic hydrogen bonds' hydroxyl groups and TRP 59 and Tyr151 (Figure 4). acids' residues. Hydrogen bonds formed between 5J7 and Gln63, Asp97, Glu233, and His305 while forming a hydrophobic interaction via pi-pi stacking with the residues Tyr62 and His 29 ( Figure 3). Compound 9 interacted with the AA through hydrogen bonds between its hydroxyl groups and the amino acids' residues Thr163, Asp197, Lys200, His 201, and His 299 in addition to the aromatic hydrogen bonds' hydroxyl groups and TRP 59 and Tyr151 (Figure 4).

Conclusions
G. mangostana is one of the most valuable tropical fruits and its usage as a functional product has been growing because of its bioactivities that are related to its xanthones' content. In the current study, two new metabolites, garcixanthone E (1) and garcimangophenone C (9), along with seven known compounds were separated from G. mangostana EtOAc extract using different chromatographic tools. Their structures were assigned based on various spectral analyses, including UV, IR, MS, and NMR. Compound 1 displayed moderate in vitro cytotoxic potential versus MCF-7, A549, and HCT-116 cell lines in the SRB assay. Additionally, 1 and 9 possessed moderate AAI potential. In the molecular docking study, 9 revealed a reasonable docking score compared to the native ligand

Conclusions
G. mangostana is one of the most valuable tropical fruits and its usage as a functional product has been growing because of its bioactivities that are related to its xanthones' content. In the current study, two new metabolites, garcixanthone E (1) and garcimangophenone C (9), along with seven known compounds were separated from G. mangostana EtOAc extract using different chromatographic tools. Their structures were assigned based on various spectral analyses, including UV, IR, MS, and NMR. Compound 1 displayed moderate in vitro cytotoxic potential versus MCF-7, A549, and HCT-116 cell lines in the SRB assay. Additionally, 1 and 9 possessed moderate AAI potential. In the molecular docking study, 9 revealed a reasonable docking score compared to the native ligand 7JR that agreed with the in vitro activity findings. These results could further prove a possible usage of G. mangostana as a functional food for treating diabetes and cancer. Certainly, more future in vivo and mechanistic studies are required to validate the activity of these interesting metabolites.
Further, to overcome the hazardous impacts and disadvantages of the conventional extraction of such metabolites by organic solvents such as MeOH, extraction using ecofriendly green solvents such as supercritical fluids, biobased solvents, and liquified gases could be applied. These solvents possess beneficial characteristics, including ease of preparation, biocompatibility, custom tunability, high selectivity, and low cost and volatility [32].