In vitro and in silico antioxidant and α-glucosidase inhibitory potential of compounds isolated from Garcinia gaudichaudii

Abstract Nine compounds including a new one, garcichaudiic acid (1), were isolated from the bark of G. gaudichaudii and their structures were characterized mainly by 1 D and 2 D NMR experiments. The antioxidant capacity of the isolated compounds was determined using DPPH radical scavenging assay and the anti-hyperglycemic activity was assessed by measuring the inhibitory effect against α-glucosidase. Among them, compound 4 showed higher antioxidant activity than the positive control, ascorbic acid, while both compounds 1 and 7 exhibited more significant α-glucosidase inhibitory activity than the reference drug acarbose. Molecular docking analysis of the bioactive compounds was also performed to examine the binding modes and key interactions with the catalytic site. Graphical Abstract


Introduction
The genus Garcinia, one of the largest genera of the Guttiferae or Clusiaceae family, has been proved to be a rich source of xanthones, benzophenones, biflavonoids, triterpenoids, and many of them display diverse biological properties such as antioxidant (Zhao et al. 2010;Aravind et al. 2016), ; antidiabetic (Tran et al. 2016;Nguyen et al. 2021), antibacterial (Wang et al. 2018), anti-inflammatory (Weng et al. 2004) and anticancer activities (Ibrahim et al. 2019). G. gaudichaudii Planch. & Triana is a mediumsized tree growing up to 10 m whose roots are used to rub on cuts and minor wounds (Vo 2012). A few studies have explored the chemical constituents and cytotoxicity (Cao et al. 1998a(Cao et al. , 1998bWu et al. 2000;Xu et al. 2000;Wu et al. 2001) but there has been no investigation on the antioxidant and a-glucosidase inhibitory activities of the species. Thus, we herein report the isolation, structure elucidation, in vitro antioxidant and a-glucosidase inhibition of nine compounds including a new one, from the ethyl acetate extract of the bark of G. gaudichaudii. Molecular docking analysis of the bioactive compounds was also conducted to acquire more structural insights of binding modes and critical interactions.

Structure elucidation
Chromatographic separation of an ethyl acetate extract of G. gaudichaudii led to the isolation of a new compound (1), together with eight known compounds (2-9).
Garcichaudiic acid (1)  The 1 D NMR data of 1 were similar to those of gambogic acid (2), the caged xanthone previously isolated from the bark of G. hanburyi (Lin et al. 1993) and also from the ethyl acetate extract of this plant extract (see Experimental Section). The main difference was that the 2-methyl-2-(4-dimethylpent-3-enyl)-pyran moiety in ring A of compound 2 were replaced by a 2-(1-hydroxy-1,5-dimethylhex-4-enyl)-2,3-dihydrofuran ring in compound 1. Analysis of the HSQC and HMBC spectra of 1 (SI, Figures S5 and S6) reconfirmed the presence of the caged skeleton in ring B and the side chains in ring A, whose positions were established using HMBC correlations. In the HMBC spectrum, the chelated hydroxy group (d H 12.73) correlated to an oxygenated aromatic carbon (d C 156.7, C-1) and two substituted aromatic carbons (d C 105.6 and 100.5, C-2 and C-9a). The benzylic methylene protons of the dihydrofuran ring (d H 3.02 and 2.96, H 2 -11) correlated to C-1 and the substituted aromatic carbon at d C 105.6, which had to be C-2. Thus, the substituted aromatic carbon at d C 100.5 was C-9a. Protons H 2 -11 also gave cross-peaks to an oxygenated aromatic carbon (d C 167.9), indicating that it was C-3. The 2,3-dihydrofuran ring therefore fused with ring A at C-2 and C-3 with C-3 being oxygenated. Observation of correlations of the methylene protons at d H 3.15 (H 2 -21) with C-3, C-4 (d C 102.3) and C-4a (d C 158.0) revealed the attachment of the isoprenyl side chain to C-4. The compound, named garcichaudiic acid, was thus established to have structure 1, which was consistent with the molecular formula of C 38 H 46 O 9 with 16 degrees of unsaturation. The COSY spectrum (SI, Figure S7) was also in agreement with the structure. Cross-peaks observed between Me-29 and H-27 in the NOESY spectrum (SI, Figure S8) indicated the protons to be on the same side, thus the D 27,28 double bond had the Z configuration. This is also in accordance with the 13 C chemical shift of C-29 (d C 20.5). If the D 27,28 double bond had the E configuration, C-29 should have d C $11-13 ppm, as in the cases of isogambogic acid and isomorelliol (Lin et al. 1993).

In vitro a-glucosidase inhibition assay
All isolated compounds were tested for their ability to inhibit a-glucosidase. At the concentration of 1000 lg/mL, compounds 1-4 and 7 showed more than 90% of inhibition, while other compounds exhibited low activity ( Figure S9). Thus, serial dilution of the compounds was performed to determine IC 50 values which were obtained from the dose-response curves (SI, Figure S10) and represented in Table S2. Compounds 1 and 7 showed significant a-glucosidase inhibition with IC 50 values of 52.4 ± 8.1 and 36.9 ± 1.0 lM, respectively, compared to that of the positive control acarbose (223.0 ± 2.6 lM). Compounds 3 and 4 were slightly more active than acarbose with IC 50 $200 lM, whereas compound 2 was less active (IC 50 ¼ 346.1 ± 10.7 lM). The activity of 1 was better than that of 2 and 3, suggesting that the replacement of the 2methyl-2-(4-dimethylpent-3-enyl)-pyran moiety in ring A by the 2-(1-hydroxy-1,5-dimethylhex-4-enyl)-2,3-dihydrofuran ring caused stronger inhibitory activity.

DPPH radical scavenging activity assay
In DPPH radical scavenging activity assay, all the isolated compounds were accessed at a concentration of 1000 lg/mL, and only compounds 4 and 7 were selected for IC 50 value determination for showing more than 90% inhibition ( Figure S9). The IC 50 values were obtained from the dose-response curves (SI, Figure S11) and represented in Table S2. Of these compounds, 4 exhibited a potent DPPH scavenging activity, showing an IC 50 value of 58.3 ± 0.5 lM, which is lower than that of the positive control ascorbic acid (86.5 ± 6.1 lM). Compound 7 displayed a strong effect (IC 50 value of 95.4 ± 2.4 lM) while other compounds were considered as inactive.

Molecular docking analysis
In order to validate the molecular docking model, three types of ligands (as described in the Materials and Methods Section) were successively docked into the catalytic site of the glucosidase (PDB ID: 3TOP) using the aforementioned procedure. The bestranked poses of these molecules obtained with GOLD 2021.1.0 deviated 0.84 Å, 1.42 Å and 1.36 Å from the true crystal pose deposited in the Protein Data Bank, respectively. This result showed that the docking procedure managed to correctly pose the ligands and could be employed for further investigation (SI, Figure S12).
The test-set compounds were successively docked into the catalytic site of the glucosidase using the same procedure as mentioned above. It was observed that the hydroxyl group of the molecule 1 participated in a hydrogen bond with the carboxylate function of Arg1526. Three other hydrogen bonds were also formed between the hydroxyl, ketone and carboxylate functions of the ligands with the side chains of Asp1157, Lys1490 and Glu1158, respectively. The phenol group of the molecule 7 engaged in a hydrogen bond with the carboxylate moiety of Arg1526. Additionally, the ketone and another phenol function of the ligand were involved in two other hydrogen bonds with the guanidine group of Arg1510 and the carboxylate function of Asp1279, respectively ( Figure S13). As Arg1526 is the key residue ensuring that the enzyme would exert its hydrolysis activity (Ren et al. 2011), its strong interaction with the ligands could help stabilize protein-ligand complexes and is expected to impair a-glucosidase's biological function in a similar manner to that observed with the native ligand a-acarbose.

Plant material
The bark of G. gaudichaudii was collected in Phu Quoc National Park, Kien Giang Province, Vietnam, and authenticated by Dr. Son V. Dang, Institute of Tropical Biology. A voucher specimen (Vang nghe-PQ) is stored at the Natural Product and Medicinal Chemistry Lab, VNUHCM-University of Science.

In vitro a-glucosidase inhibition assay
a-Glucosidase inhibitory activity was evaluated following the method described by Kim et al. (Kim et al. 2008). Briefly, 100 mL tested sample dissolved in DMSO and 20 mL a-glucosidase (20 U/mL) were added to 1.9 mL phosphate buffer (0.01 mM, pH 7.0). All were incubated at 37 C for 10 min before adding 20 mL of p-nitrophenyl a-D-glycopyranoside (3 mM). The reaction mixture stood at 37 C for 30 min, and was then stopped by adding 1 mL of Na 2 CO 3 solution (0.1 M). The amount of the released p-nitrophenolate was recorded at 405 nm using a UV-Vis spectrometer. Acarbose was applied as the positive control and all measurements were triplicated. The concentration of the sample required for 50% inhibition (IC 50 ) was attained using GraphPad Prism 8.0.
3.5. DPPH radical scavenging activity assay DPPH radical scavenging activity was evaluated following the method of Espin et al. and Kang et al. (Espin et al. 2000;Kang et al. 2006). One mL of the sample dissolved in methanol was mixed with 3 mL of methanolic solution containing 0.075 mM DPPH. The reaction mixture was shaken thoroughly and was kept in the dark at room temperature for 1 h. The absorbance was monitored at 517 nm using a spectrophotometer. Acid ascorbic was used as the positive control and all measurements were triplicated. The concentration of the sample that could lower the DPPH radicals by 50% (IC 50 ) was attained using GraphPad Prism 8.0.

Molecular docking analysis
Protein preparation: The crystallographic tri-dimensional structure of human intestinal maltase-glucoamylase was downloaded from the Protein Data Bank (PDB entry 3TOP) (Ren et al. 2011;Burley et al. 2019). Water molecules that took part in at least three hydrogen bonds with the protein and/or the ligand, at least two of which were with the protein, were kept. Other molecules were deleted. Hydrogen atoms were added with GOLD 2021.1.0 (Cambridge, UK) using default settings, after which the protein was further processed to keep only one monomer by the same program (Verdonk et al. 2003). The apoprotein structure was saved as a mol2 file.
Ligand preparation: three types of ligands for redocking were prepared as follows: Co-crystallized ligand extracted from the complex structure with no changes in conformation and configuration: the ligand (after hydrogens were added) was saved as a mol2 file without the presence of the protein and any further modification. Energyminimized co-crystallized ligand extracted from the complex: the mol2 ligand structure was energetically minimized with the built-in MMFF94 forcefield of MOE 2020.09 and saved as another mol2 file. Energy-minimized reconstructed ligand: the structure of the ligand was rebuilt with ChemDraw Professional 16.0 , its 3D structure was then energetically minimized as described above. The ligand structure was saved as a mol2 file. All test-set molecules were built with ChemDraw Professional 16.0. Their 2 D structures were then converted into 3D, and energy minimization was carried out as mentioned above. All output ligands were saved as a sd file.
Molecular docking: A rigid molecular docking procedure was carried out using GOLD 2021.1.0. The binding site comprised all protein residues with at least one heavy atom located within 6 Å from the centroid of the co-crystallized ligand (HET code: PRD_90007) whose coordinates are À31.51, 34.73, 26.35. Hydrogen bond constraints were applied to Asp1526-OD1, Asp1526-OD2 and Asp1279-OD2. Two ring centre pharmacophore constraints were defined at À31. 31, 40.43, 23.21 and À29.68, 34.03, 27.85. The built-in CHEMPLP scoring function was used to rescore the output poses (10 best-scored poses were kept for each compound).

Conclusion
In this study, nine compounds including a new one, garcichaudiic acid (1), were isolated from an EtOAc extract of the bark of G. gaudichaudii, and their structures were elucidated using spectroscopic methods. The compounds were tested for their in vitro antioxidant and a-glucosidase inhibitory activities, followed by molecular docking analysis of the bioactive compounds to examine the binding modes and key interactions with the catalytic site. The results showed that macluraxanthone (4) and 12 b-hydroxydes-D-garcigerrin A (7) possess both activities. As free radicals are considered to play a vital role in developing diabetes mellitus and its complications, a-glucosidase inhibitors with potential antioxidant activity could be applicable in the development of suitable medication to manage T2D. This is the first report of in vitro antioxidant and a-glucosidase inhibitory effects of G. gaudichaudii, suggesting the study's findings might be useful for future research, especially to explore new antidiabetic drugs of natural origin.

Disclosure statement
The authors declare no conflict of interest.