Xanthones from the latex and twig extracts of Garcinia nigrolineata Planch. ex T. Anderson (Clusiaceae) and their antidiabetic and cytotoxic activities

Abstract A new geranylated xanthone, nigrolineaxanthone AA (1) together with 18 known compounds (2–19) were isolated from latex and twig extracts of Garcinia nigrolineata Planch. ex T. Anderson. Some of the isolated compounds were assessed for their antidiabetic activities and cytotoxicity against three cancer cell lines. Of these, compounds 12 (IC50 value of 25.8 ± 0.2 µM), 16 (IC50 value of 124.8 ± 0.7 µM), and 17 (IC50 value of 44.4 ± 1.1 µM) exhibited the highest α-glucosidase inhibitory, α-amylase inhibitory, and glycation inhibition activities, respectively. Compound 11 showed glucose consumption and glucose uptake with IC50 values of 14.2 ± 0.8 µM and 3.1-fold. Compound 10 displayed cytotoxic activity against colon cancer (SW480) with an IC50 value of 4.3 ± 0.1 µM), while compound 2 showed cytotoxicity against leukemic cancer (K562) with IC50 value of 4.4 ± 0.3 µM. Graphical Abstract


Introduction
Garcinia nigrolineata Planch. ex T. Anderson is an evergreen tree belonging to the Clusiaceae family and is known as a rich source of xanthones derivatives (Negi et al. 2013;Santo et al. 2020). This plant has been used as a local Thai food ingredient and is involved in Thai traditional medicine preparations as an antipyretic, to balance body elements, control blood glucose levels, and improve excretory functions (Yapwattanaphun et al. 2002). The phytochemical evaluation of leaves, stem barks, and twigs showed the presence of xanthones along with minor amounts of benzophenones, benzoquinones, biphenyl, and isoflavones (Rukachaisirikul et al. 2003a(Rukachaisirikul et al. , 2003b(Rukachaisirikul et al. , 2005Raksat et al. 2019). From previous studies, the xanthone derivatives have been found to have interesting pharmacological effects such as anti-inflammatory (Feng et al. 2020), antioxidant (Zhao et al. 2010), antidiabetic (Miura et al. 2001), and anticancer activities (Shan et al. 2011). However, only antibacterial activity has been reported for compounds from G. nigrolineata (Rukachaisirikul et al. 2003b;Raksat et al. 2019). In ongoing research, the chemical constituents from latex and twigs of G. nigrolineata and their antidiabetic and cytotoxic activities against cancer cell lines are reported. The isolated compounds, including a new geranylated xanthone (1) along with 18 known compounds (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) were evaluated for in vitro assays based on enzyme and cells mechanisms to find bioactive compounds which showed potential for antidiabetic and anticancer activities.
The human body normally has several dynamic carbohydrates digestive enzymes; a-amylase is present in saliva and helps to degrade polysaccharides into glucose. Hence, inhibition of these digestive enzymes notably delays the conversion of polysaccharides into blood glucose, which serves as an effective process to control the blood glucose level in diabetic patients (Cheng et al. 2017). In this work, all isolated compounds (1-19) were determined for their a-amylase inhibition activity (Table S2). Of these, only xanthones 9 and 16 showed a-amylase inhibition activity with IC 50 values of 137.1 ± 2.2 and 124.8 ± 0.7 mM, respectively, and better than the standard controls, voglibose (198.3 ± 0.8 mM) and quercetin (180.1 ± 1.4 mM), but they were slightly less active than that of acarbose (105.8 ± 1.1 mM). The remaining compounds were inactive at a concentration of 200 mM.
Advanced glycation end products (AGEs) are harmful complex compounds obtained from nonenzymatic glycation between carbonyl groups of reducing sugars and free amino groups of proteins, lipids, or nucleic acids (Chen et al. 2018). AGEs contribute to the development and progression of different diabetic complications, including nephropathy, retinopathy, and neuropathy (Singh et al. 2014). The inhibition of glycation by compounds 1-19 is summarized in Table S2. Xanthones 17 and 10 displayed the greatest glycation inhibition activity with IC 50 values of 44.4 ± 1.1 and 59.1 ± 0.9 mM, respectively, which was better than that of the positive control (quercetin, IC 50 value of 62.4 ± 1.5 mM). Xanthone 11 also showed good glycation inhibition activity with an IC 50 value of 70.1 ± 1.1 mM, while all remaining compounds showed weak activity or were inactive. It should be noted that the slight structural difference of 1,3,6,7-tetraoxygenated xanthones 10, 11, and 17 had no impact on the inhibition of glycation. These findings may lead to further investigation and clarification in other mechanisms of the anti-AGE properties of xanthones.
Compounds 1-19 were also evaluated for their glucose consumption and glucose uptake in 3T3-L1 and L6 myotubes cells (Table S3). Only xanthones 11 and 15 displayed glucose consumption with IC 50 values 14.2 ± 0.8 and 57.5 ± 1.3 mM, respectively, and better than that of the positive control (metformin, IC 50 ¼ 50.3 ± 0.9 mM). These results are consistent with our previous report . To confirm the activity of promoting glucose consumption, xanthones 11 and 15 were further evaluated for glucose uptake induced by L6 myotube cells (Table S3). Xanthones 11 and 15 enhanced the glucose uptake stimulation in adipocyte L6-myotube cells by 3.1 and 1.2-fold, respectively, compared to the positive control (metformin, 3.8-fold). This suggests that xanthones 11 and 15 show potential glucose transportation into cells and provide energy in the form of adenosine triphosphate (ATP) and play a vital part in the variety of other cellular operations.
Cytotoxicity against several cancer cell lines of prenylated and geranylated xanthones isolated from Garcinia genus have been reported (Sukandar et al. 2019). Currently, all isolated compounds were evaluated for cytotoxicity against three cancer cell lines, including lung (A549), colon (SW480), and leukemic cancer (K562) using the MTT assay with doxorubixin used as a positive control (Table S4). Of these, only xanthones 10 and 11 were cytotoxic against A549 cancer cell lines with IC 50 values of 18.9 ± 0.1 and 87.3 ± 1.2 mM, respectively. The only difference between these two structures is the isoprenyl unit. Compound 10 has an isoprenyl unit while xanthone 11 contains a modified isoprenyl unit (4-hydroxy-3-methylbut-2-enyl unit), which clearly plays an important role in reducing cytotoxicity against lung cancer (A549). In the case of the SW480 cancer cell lines, xanthones 1-4, 6, 9-13, 16, and 17 showed IC 50 values in the range of 4.3 ± 0.1 À 83.7 ± 0.7 mM, and xanthone 10 had the highest cytotoxicity (IC 50 value of 4.3 ± 0.1 mM). Similar to lung cancer (A549), the modified isoprenyl unit of xanthones 11 and 12 seems to reduce their cytotoxicity against colon cancer (SW480) compared to that of xanthone 10. Xanthones 1, 2, 4, 11, 12, 14, and 18 also displayed cytotoxicity against K562 cell lines with IC 50 value ranging from 4.4 ± 0.3 À 99.1 ± 1.6 mM, and compound 2 displayed the highest cytotoxicity. Xanthones 1 and 4 have a similar structure to that of xanthone 2. However, the selective O-methylation at 7-OH in 4 or the loss of 6-OH in 1 are critical to the loss of cytotoxicity against the leukemic cancer cell line (K562).

Plant material
The latex (32.4 g) and twigs (690.4 g) of G. nigrolineata (specimen number MFU-NPR0187) were collected in January 2019 from Chiang Rai Province, Thailand. Herbarium specimens were deposited at the Natural Products Research Laboratory, School of Science, Mae Fah Luang University.

Determination of antidiabetic activities
3.4.1. a-Glucosidase inhibitory activity The inhibitory assay for the a-glucosidase enzyme was carried out using the same protocol as in our prior study Raksat et al. 2020). After preparing various concentrations of samples and positive controls (acarbose, voglibose, and quercetin) in 5% DMSO with phosphate buffer saline (PBS) (pH 6.8), 50 mL was combined with 50 mL of the a-glucosidase enzyme (0.35 U/mL) in a 96-well microplate. Then, the samples were incubated at 37 C for 10 min. After that, 50 mL of 4-nitrophenyl-a-D-glucopyranoside (p-NPG) (1.5 mM) was added into the mixture and incubation was continued at 37 C for 20 min. The reaction was terminated by adding 100 mL of Na 2 CO 3 (1 M) and the absorbance at 405 nm was measured with a microplate reader. The procedure was carried out in triplicate.

a-Amylase inhibitory activity
The a-amylase inhibitory procedure was performed by a slight modification of previous work (Kusano et al. 2011). First, the substrate was prepared by dissolving soluble starch (500 mg) in 25 mL of 0.4 M NaOH. The solution was heated for 5 min at 100 C, and immediately cooled in ice. Then, the solution was adjusted to pH 7 with 2 M of HCl. Samples and positive controls (acarbose, voglibose, and quercetin) were prepared by dissolving the samples in acetate buffer (pH 6.5) at various concentrations. The reaction was started by incubating the mixture of the substrate (40 mL) and sample (20 mL) at 37 C for 3 min. Then, 20 mL of amylase solution (50 mg/mL) was added into each well and further incubated for 15 min. After that, 80 mL of 0.1 M HCl was pipetted to stop the reaction. The remaining starch was determined by adding 100 mL of iodine solution (1 mM) and the absorbance was measured by a microplate reader at 650 nm. The experiment was performed in triplicate.

Glycation inhibitory assay
The glycation inhibition assay was evaluated according to the same procedure as in previous research (Justino et al. 2016). Concisely, samples or the positive control (quercetin) were prepared at various concentrations in DMSO. Then 50 mL of the samples were added a mixture consisting of BSA (100 mL, 50 mg/mL) and fructose (100 mL, 50 mg/mL). The solutions were incubated at 37 C in the dark for 72 hr. After that, 20% trichloroacetic acid was added and the solution centrifuged at 10000 RPM for 10 min. The residue was resuspended in a PBS buffer and fluorescence intensity at 350 nm ex/ 420 nm em was measured with a microplate reader. The experiment was performed in triplicate.

Glucose uptake assay
The glucose uptake assay followed the protocol from a prior study with a slightly adapted procedure (Sharma et al. 2019). Briefly, L6 myotubes (1 Â 10 4 cells/well) were seeded into 96-well microplates together with samples or a positive control (metformin) at various concentrations. Then, the samples were incubated at 37 C in a 5% CO 2 incubator for 24 hr. After removing the supernatant liquid and washing by PBS, treated cells were further incubated in Kreb-Ringer bicarbonate buffer for 1 hr. After that, serum-free phosphate buffer saline containing 0.2% bovine serum albumin was added and the reaction was incubated for 1 hr. The fluorescence intensity was measured at 485 nm ex/530 nm em after incubating cells with 2-deoxy-2-[(7-nitro-2, 1, 3benzoxadiazol-4-yl) amino]-D-glucose for 20 min. The experiment was carried out in triplicate.

Glucose consumption assay
The glucose consumption assay was determined following our previous report . Firstly, 100 mL of 3T3-L1 cells (density of 1 Â 10 5 cells/well) were cultured in 96 well microplates along with the various concentrations of the samples and the positive control (metformin) and incubated at 37 C in a 5% CO 2 incubator for 24 hr. After that, 10 mL of supernatant liquid was transferred to a new 96-well microplate. The illuminated reaction was used to evaluate glucose concentration in suspension after 30 min. The absorbance was measured by a microplate reader at 495 nm. The procedure was done in triplicate. Cell viability was carried out by 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, as previously reported (Kang et al. 2013).
3.5. Cytotoxic activity against lung cancer (A549), Colon cancer (SW480), and leukemic cancer (K562) cell lines The cells cytotoxicity of three cancer cell lines including lung cancer (A549), colon cancer (SW480), and leukemic cancer (K562) were evaluated following a previous method (Suthiphasilp et al. 2021). Briefly, cells were propagated into 96-well microplates and incubated at 37 C in 5% CO 2 . A549 and SW480 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) and the K562 cell line was maintained in supplemented PMI-1640. The medium containing 1% v/v antibiotic and 10% v/v fetal bovine serum (FBS). Subsequently, samples were treated and incubated at 37 C in 5% CO 2 for 24 hours. Cell cytotoxicity was determined using an MTT assay (Kang et al. 2013). The experiments were repeated in triplicate.

Calculation
All data are expressed as the mean of triplicate experiments ± standard deviation. Dose-response curves were plotted using the percentages of anti-diabetic, anticancer, and concentration (lg/mL). The concentration of treatment that inhibited a-glucosidase, a-amylase enzyme, glycation, glucose uptake, promoted glucose consumption, and cytotoxicity by 50% (IC 50 ) were determined from regression analysis using Microsoft Excel (Microsoft Corporation, USA) and Graph Pad Prism version 6 (Graphpad Software Inc., USA).

Conclusion
The phytochemical investigation of G. nigrolineata latex and twigs extracts led to the isolation and identification of the new xanthone, nigrolineaxanthone AA (1), together with 17 known xanthones (2-18) and one phloroglucinol (19). Compounds 4, 10, 11, and 15 were found in both latex and twigs extracts. Xanthone 13 showed the greatest a-glucosidase inhibitory activity, Meanwhile, xanthones 9 and 16 showed moderate a-amylase inhibitory activity. Xanthone 17 showed the greatest inhibition of glycation activity. In terms of glucose consumption and uptake activities, xanthone 11 displayed the best activities without cell toxicity. Moreover, xanthones 10 and 2 have good cytotoxicity against lung cancer (A549), colon cancer (SW480), and leukemic cancer (K562). The present findings concerning bioactive compounds from G. nigrolineata latex and twigs may be an advantage for future research into the application of medicinal plants to develop new drug candidates for diabetes mellitus and cancer treatment.