Two new antidiabetic xanthones from the twigs of Garcinia oblongifolia

Abstract Two new xanthones, oblongixanthones I (1) and J (2), and seven known compounds (3–9), were isolated from an EtOAc extract of the twigs of Garcinia oblongifolia. Their structures were elucidated using spectroscopic methods, mainly 1 D and 2 D NMR. The antidiabetic effects of the two new compounds were evaluated using α-glucosidase and PTP1B inhibition assays. Both compounds displayed strong inhibition towards α-glucosidase with IC50 values of 258.7 ± 49.3 and 187.1 ± 27.5 μM, respectively (compared with acarbose, IC50 = 900.0 ± 3.0 μM) and moderate effects against PTP1B with IC50 values of 93.9 ± 12.3 and 64.1 ± 5.8 μM, respectively (compared with RK682, IC50 = 4.4 ± 0.3 l μM). Graphical Abstract


Introduction
According to the International Diabetes Federation, diabetes has spread with epidemic proportions. In 2021, 537 million adults (20-79 years old) were diagnosed with diabetes, and this number is predicted to rise to 643 million by 2030 and 784 million by 2045 (International Diabetes Federation 2021). One well-established therapeutic approach to maintain blood glucose level is by inhibiting a-glucosidase in the digestive system (Chiba 1997). Another approach is by inhibiting protein tyrosine phosphatase-1B (PTP1B), which is responsible for catalyzing the dephosphorylation of the activated insulin receptor, resulting in down regulation of the insulin-signaling pathway (Zhang and Lee 2003;Montalibet and Kennedy 2005). However, side effects of the existing a-glucosidase drugs (i.e. acarbose, miglitol, and voglibose) (Fowler 2008;Marcovecchio et al. 2011) and the lack of clinical drugs targeting PTP1B have been a concern (Zhang and Zhang 2007). Thus, demand of new a-glucosidase and PTP1B inhibitors is still needed.
Garcinia oblongifolia Champ. ex Benth. (Clusiaceae), called 'bua la tron dai' in Vietnamese, is a medium-sized tree growing from 8-10 metres tall. It has been used as folk medicine in Vietnam for the treatment of stomach and duodenal ulcers, allergies and burns (Pham 1999;Do 2004). Recent studies on chemical constituents have showed that the plant species biosynthesizes mainly xanthones and polyisoprenylated benzophenones (Hamed et al. 2006;Shan et al. 2012;Feng et al. 2014), which exhibit a wide range of bioactivities such as antitumor (Xia et al. 2018), anti-inflammatory , antienteroviral (Zhang et al. 2014;Wang et al. 2016), and anticancer effects (Huang et al. 2009;Shi et al. 2014;Li et al. 2016). In our previous work, a series of antidiabetic xanthones including three new ones, oblongixanthones F-H, were isolated from an ethyl acetate extract of the twigs of G. oblongifolia collected in south Vietnam, and one of which, norcowanin, significantly inhibited a-glucosidase and PTP1B enzymes (Trinh et al. 2017). Antidiabetic activities of other xanthones have been studied recently and the results show that many compounds possess strong inhibitory effects (Seo et al. 2007;Li et al. 2018;Trinh et al. 2021). In our efforts to identify more new bioactive compounds from G. oblongifolia, the remaining fractions were further investigated. We report herein the isolation and structure elucidation of two new isolates, 1 and 2, together with seven known compounds (3-9). a-Glucosidase and PTP1B inhibitory activities of 1 and 2 were also evaluated.

Structure elucidation
During the course of an antidiabetic screening program of natural products, the EtOAc extract of G. oblongifolia was found to exhibit potential inhibitory activities of both a-glucosidase and PTP1B enzymes with IC 50 values of 4.0 ± 0.9 lg/mL and 8.0 ± 1.1 lg/ mL, respectively. Therefore, it was subjected to successive chromatographic separations to afford 9 compounds including two new (1-2) and seven known xanthones (3-9). This is a continuation of the previous work published in 2017(20 Trinh et al. 2017. Compound 1 was isolated as yellow needles, mp. 178-179 C, [a] þ53.4 (c 0.6, MeOH), and giving a positive test with FeCl 3 in EtOH. The UV spectrum showed four maxima at 244, 260 (sh), 318 and 360 (sh) nm, reminiscent of a polyoxygenated xanthone (Trinh et al. 2017 (m, H 2 -20)]. Consistent with the 1 H NMR data, the 13 C NMR spectrum showed resonances for a methoxy group [d C 61.0 (7-OCH 3 )], a 4-hydroxy-3-methyl-2butenyl side chain [d C 135.1 (C-13), 125.1 (C-12), 61.5 (C-14), 21.7 (C-15) and 21.4 (C-11)] and a 6-hydroxy-3,7-dimethyl-2,7-octadienyl side chain [d C 149.1 (C-22), 135.5 (C-18), 124.5 (C-17), 110.1 (C-23), 75.1 (C-21), 36.3 (C-19), 34.4 (C-20), 26.6 (C-16), 17.6 (C-25) and 16.5 (C-24)]. Resonances for a conjugated carbonyl carbon [d C 182.6 (C-9)] and 12 aromatic carbons, six of which were oxygenated, were also observed. The spectral data were similar to those of the xanthone cowanol previously isolated from G. cowa (Pattalung et al. 1994) and also from fraction 8.5 of the same EtOAc extract of this plant (Trinh et al. 2017), and the main difference lay in the structure of the C 10 side chain. The presence of the 4-hydroxy-3-methyl-2-butenyl unit was confirmed by analysis of the HMBC correlations. In the HMBC spectrum ( Figure S7), the allylic methyl at d H 1.73 (H 3 -15) correlated with a trisubstituted carbon-carbon double bond and an oxymethylene, indicating that the fully substituted olefinic carbon (d C 135.1, C-13) was the carbon to which the methyl (d C 21.7, C-15) and the oxymethylene groups (d C 61.5, C-14) were attached. Proton H-12 showed cross-peaks with a benzylic methylene (d C 21.4, C-11), which in turn correlated with three aromatic carbons (d C 163.2, 161.4, 110.0), confirming the presence of the C 5 unit in the molecule and revealing the attachment of the C 5 unit to the xanthone skeleton. In the NOESY spectrum ( Figure  S8), NOE interactions observed between H 2 -11 (d H 3.43)/H 2 -14 (d H 4.29) and H-12 (d H 5.35)/H 3 -15 (d H 1.73) indicating the E configuration of the double bond between C-12 and C-13. The presence of the 6-hydroxy-3,7-dimethyl-2,7-octadienyl group was also identified using HMBC experiment. In the HMBC spectrum, the allylic methyl protons at d H 1.64 (H 3 -25) correlated with a carbon-carbon double bond carrying a methylene (d C 149.1, C-22; 110.1, C-23) and to an oxymethine carbon (d C 75.1, C-21). The methylene protons at d H 1.58 (d C 34.4) showed correlations to C-21, C-22, another methylene (d C 36.3) and a fully substituted olefinic carbon (d C 135.5) whilst the allylic methylene proton at d H 2.03 (d C 36.3) gave cross-peaks with C-21, the methylene at d C 34.4, and a trisubstituted carbon-carbon double bond (d C 135.5, 124.5). The correlations established the long range CH structural connectivities of C-25 and C-23/C-22/C-21/C-20 (d C 34.4)/C-19 (d C 36.3)/C-18 (d C 135.5)/C-17 (d C 124.5). This was inconsistent with crosspeaks observed between the allylic methyl at d H 1.84 (H 3 -24) and C-17, C-18 and C-19. Further correlations of the shielded benzylic methylene protons (d H 4.14, H 2 -16) with C-17 and three aromatic carbons (d C 157.6, 144.4, 137.9) established the substructure of the C 10 side chain and its bond to the xanthone skeleton. NOE  In the HMBC spectrum, the chelated hydroxy proton correlated to an oxygenated carbon (d C 161.4, C-1), and two substituted aromatic carbons (d C 110.0 and 103.3) which were C-2 and C-9a. Correlations of the methylene protons of the 4-hydroxy-3methyl-2-butenyl group (d H 3.43, H 2 -11) to C-1 and C-2 (d C 110.0) indicated that the side chain was attached to C-2. The protons also gave cross-peak to another oxygenated aromatic carbon (d C 163.2), which had to be C-3. The aromatic singlet proton at d H 6.37 was identified as H-4 based on its HMBC correlations to C-2, C-3, C-9a and an oxygenated aromatic carbon (d C 155.7, C-4a). The xanthone B ring therefore contained the second hydroxy group, the methoxy group and the 6-hydroxy-3,7-dimethyl-2,7octadienyl side chain. The attachment of this C 10 unit at the peri position (C-8) was suggested by its benzylic methylene proton signal (d H 4.14, H 2 -16), which experienced a downfield shift due to deshielding caused by the carbonyl group. The protons showed correlations to an oxygenated aromatic carbon (d C 144.4), which was C-7. The methoxy group was placed at C-7 since its resonance signal (d H 3.80) showed a correlation to this carbon. The second isolated aromatic proton (d H 6.83) was assigned to be H-5 (d C 102.5) in accordance with its HMBC correlations to C-6, C-7, C-8a and C-10a ( Figure S1). The structure of compound 1, named oblongixanthone I, was therefore established as shown in Figure 1. The absolute configuration of C-21 in 1 remains undetermined.
Compound 2 was obtained as yellow needles, mp. 161-162 C, giving a positive test with FeCl 3 /EtOH, and showing UV absorptions at k max 244, 260, 316 and 360 nm. HRESIMS displayed a an [M þ Na] þ ion peak at m/z 533.2166, corresponding to the molecular formula of C 29 H 34 O 8 . The 1 H and 13 C NMR spectra showed close similarities to those of 1, with the exception of significant differences in the chemical shifts of the C 10 unit attached to C-8 at C-19 to C-23 and C-25. The signals due to the carbon-carbon double bond carrying two methylene protons at d J ¼ 6.4 Hz, H 2 -19]; d C 43.1 (C-19)], and two methyls bonded to an oxygenated sp 3 carbon [d H 1.19 (6H, s, H 3 -23 and H 3 -15); d C 30.3 (C-23 and C-25). This finding indicated the occurrence of a 7-hydroxy-3,7-dimethyl-2,5-octadienyl side chain in 2 instead of the 6-hydroxy-3,7-dimethyl-2,7-octadienyl side chain in 1, which was confirmed by the HMBC correlations ( Figure S9). The structure of this compound, named oblongixanthone J, was thus identified as 2 (Figure 1).

a-Glucosidase inhibitory activity
The two new compounds (1 and 2) were accessed for in vitro a-glucosidase and PTP1B inhibitory activities with acarbose and RK682 as positive controls, respectively. The compounds were dissolved in DMSO and dilution series were prepared for IC 50 determination. The dose-response curves are shown in Figure S16 and the IC 50 values of all tested compounds are given in Table S3. Compounds 1 and 2 showed strong a-glucosidase inhibitory activity with IC 50 values of 258.7 ± 49.3 and 187.1 ± 27.5 lM, respectively) compared to that of the positive control acarbose (900 ± 3 lM). In the PTP1B assay, they displayed moderate effects (IC 50 values of 93.9 ± 12.3 and 64.1 ± 5.8 lM, respectively) compared to that of the positive control RK682 (4.4 ± 0.3 lM). The two inhibitory activities of compound 2 were better than those of compound 1, suggesting that the presence of a 7-hydroxy-3,7-dimethyl-2,5-octadienyl side chain at C-8 caused stronger inhibitory activity than the 6-hydroxy-3,7-dimethyl-2,7-octadienyl side chain located at the same carbon.

Plant material
The twigs of G. oblongifolia were collected in Song Kon Plantation, Binh Dinh Province and identified by Dr. Dang Van Son, Institute of Tropical Biology, Ho Chi Minh City. A voucher specimen (Bua LTD-SongKon) is deposited in the Natural Product and Medicinal Chemistry Lab, VNUHCM-University of Science.

Spectrophotometric microplate-based in vitro a-glucosidase inhibition assay
Inhibition of yeast a-glucosidase was assessed by spectrophotometric measurement of the cleavage rate of p-nitrophenyl b-D-glucopyranoside (PNPG) to the strongly chromogenic p-nitrophenolate ion as described by Schmidt et al. (Schmidt et al. 2014). In brief, 90 lL of 0.1 M phosphate buffer (pH 7.5, 0.02% NaN 3 ), 10 lL test sample dissolved in DMSO, and 80 lL of enzyme solution (final well concentration 0.05 U/mL) were added to each well of a 96-well microplate. The mixture was incubated at 28 C for 10 min before adding PNPG to a final volume of 200 lL (final well concentration 1.0 mM). The hydrolysis rate of PNPG to p-nitrophenolate was monitored at 405 nm every 30 s for 35 min. Incubation and absorbance measurements were performed with a Multiscan FC microplate photometer with built-in incubator (Thermo Scientific, Waltham, MA), controlled by SkanIt ver. 2.5.1 software for data acquisition. The a-glucosidase inhibitory activity was expressed as percentage inhibition and was calculated using the following formula: Acarbose was used as positive control and all measurements were performed in triplicate. Dose-response curves and IC 50 values were obtained using GraFit Version 5 (Erithacus Software Ltd., Horley, UK).

Spectrophotometric microplate-based in vitro PTP1B inhibition assay
Inhibition of PTP1B was assessed by spectrophotometric measurement of the cleavage rate of p-nitrophenyl phosphate (pNPP) to the strongly chromogenic p-nitrophenolate ion as described by Tahtah et al. (Tahtah et al. 2016). The PTP1B inhibition assay was performed at 25 C in 96-well microplates using a two-component buffer, containing 50 mM Tris, 50 mM bis-Tris and 100 mM NaCl, adjusted to pH 7.0 with acetic acid. Final reaction volume was 180 lL, including 18 lL test sample dissolved in DMSO, 52 lL of buffer with 3.46 Mm EDTA (final well concentrations 10% DMSO and 1 mM EDTA), and 60 lL of buffer with 1.5 mM p-nitrophenyl phosphate (pNPP) and 6 mM DTT (final well concentrations 0.5 mM pNPP and 2 mM DTT). After preincubation at 25 C for 10 min, the reaction was started by addition 50 lL of 0.001 lg/lL PTP1B stock solution (final well concentration: 0.05 lg/well). The hydrolysis rate of pNPP to release the p-nitrophenolate ion was determined by measuring the absorbance at 405 nm every 30 s for 10 min. Preincubation and absorbance measurements were performed with a Multiscan FC microplate photometer with built-in incubator (Thermo Scientific, Waltham, MA) coupled to SkanIt version 2.5.1 software for data acquisition.
The PTP1B inhibitory activity was expressed as percentage inhibition and was calculated using the following formula: % inhibition ¼ Slope blank -Slope sample À Á =Slope blank Â 100 RK682 was used as positive control and all measurements were performed in triplicate. Dose-response curves and IC 50 values were obtained using GraFit Version 5 (Erithacus Software Ltd., Horley, UK).

Conclusions
In summary, two new xanthones, oblongixanthones I (1) and J (2), together with seven known compounds, were isolated from the twigs of G. oblongifolia. The new compounds were evaluated for their a-glucosidase and PTP1B inhibitory activities, and displayed strong inhibition towards a-glucosidase and moderate effect against PTP1B enzyme.