Mangostanaxanthone VIII, a new xanthone from Garcinia mangostana and its cytotoxic activity

Abstract A new prenylated xanthone, mangostanaxanthone VIII (7) and six known metabolites: gartanin (1), 1,3,8-trihydroxy-2-(3-methyl-2-butenyl)-4-(3-hydroxy-3-methylbutanoyl)-xanthone (2), rubraxanthone (3), 1,3,6,7-tetrahydroxy-8-prenylxanthone (4), garcinone C (5), and xanthone I (9-hydroxycalabaxanthone) (6) were separated from the EtOAc-soluble fraction of the air-dried pericarps of Garcinia mangostana (Clusiaceae). Their structures have been verified on the basis of spectroscopic data analysis as well as comparison with the literature. The cytotoxic activity of 7 was assessed against MCF7, A549, and HCT116 cell lines using sulforhodamine B (SRB) assay. Compound 7 showed significant cytotoxic potential against MCF7 and A549 cell lines with IC50s 3.01 and 1.96 μM, respectively compared to doxorubicin (0.06 and 0.44 μM, respectively). However, it exhibited moderate activity towards HCT116 cell line.


Introduction
Recently, increasing evidence has supported that diet plays an important role in preventing the development of cancer Di Francia et al. 2016). Fruits are one of the main dietary components for daily consumption. It is popularly believed that increasing fruit consumption will contribute to the reduced risk of oral cavity, pharynx, esophagus, larynx, stomach, and lung cancers due to the intake of some specific active substances as we consume fruit every day (Grundy et al. 2016). The genus Garcinia (Clusiaceae) includes up to 800 species, the fruits of many of which are edible and serve as a substitute for tamarinds in curries (Chen et al. 2008). Garcinia mangostana L. (mangosteen, the queen of fruits) is famous for its flavorful and nutritious values in the Southeast Asian countries (Khumsupan and Gritsanapan 2014). It is known to be a prosperous source of oxygenated and prenylated xanthones (Zhao et al. 2012;Bui et al. 2014;Zhou et al. 2015;Mohamed et al. 2017;Ibrahim et al. 2018aIbrahim et al. , 2018b. These metabolites possessed a wide variety bioactivities: anti-leishmanial, anti-HIV, antimicrobial, antimicrobial, antitumor, anti-inflammatory, antimalarial, anti-quorum sensing, antioxidant, advanced glycation end-products inhibitory, cytotoxic, larvicidal, and antihypertensive (Ee et al. 2006;Zhao et al. 2010;Auranwiwat et al. 2014;Mahamodo et al. 2014;Mohamed et al. 2014;Abdallah et al. 2016aAbdallah et al. , 2016bAbdallah et al. , 2017Wang et al. 2017). Its pericarp or ripe fruit has been utilized for hundreds of years in Chinese and Ayurvedic medicines for treating various ailments as dysentery, gonorrhea, urinary tract infections, typhoid, sprains, chronic ulcers, wounds, suppurations, hyperkeratosis, eczema, psoriasis, and leucorrhoea (Upaganlawar and Badole 2012;Dharmaratne et al. 2013;Mohamed et al. 2017;Wang et al. 2017). Moreover, its root decoction is helpful in treating menstrual disorders. The leaves' infusion incorporated with unripe banana and a little benzoin is applied to the wound of circumcision (Khumsupan and Gritsanapan 2014;Ibrahim et al. 2018aIbrahim et al. , 2018b. Our previous phytochemical study of G. mangostana revealed the existence of xanthones, flavonoids, and phenolics (Mohamed et al. 2014;Abdallah et al. 2016aAbdallah et al. , 2017Ibrahim et al. 2018aIbrahim et al. , 2018b. In the course of our search for bioactive metabolites from G. mangostana, the EtOAc-soluble fraction of the fruit pericarps was subjected to a phytochemical investigation, leading to the isolation and structural characterization a new xanthone: mangostanaxanthone VIII (7) and six known metabolites (1-6) ( Figure 1). Their structures were determined unambiguously by one and two dimensional NMR techniques in conjunction with the comparison with the data for the known related compounds. Moreover, the cytotoxic capacity of 7 towards MCF7 (hormone-dependent breast carcinoma), A549 (lung carcinoma), and HCT116 (colon carcinoma) was evaluated using sulforhodamine B (SRB-U) assay.

Results and discussion
The powdered fruit pericarps were extracted with acetone. The acetone extract was mixed with water and partitioned with n-hexane and EtOAc. The EtOAc soluble-fraction was successfully chromatographed over Sephadex LH-20, SiO 2 , and RP 18 columns to furnish one new (7) and six known metabolites (1-6) ( Figure 1).
Compound 7 was assessed for its cytotoxic activity using sulforhodamine B (SRB) assay towards A549, MCF7, and HCT116 cancer cell lines. It possessed significant cytotoxic potential against MCF7 and A549 cell lines with IC 50 s 3.01 and 1.96 μM, respectively compared to doxorubicin (IC 50 0.06 and 0.44 μM, respectively). While it exhibited moderate activity towards HCT116 cell line with an IC 50 11.06 μM compared to doxorubicin (IC 50 0.027 μM). The cytotoxic activity of 1-6 towards these cancer cell lines was previously reported (Fu et al. 2013;Xu et al. 2014;Ruan et al. 2017).

Plant material
The fruits of G. mangostana were purchased from a Saudi local market in November 2016. The plant sample was authenticated by associate Professor Emad Al-Sharif (Plant Ecology, Dept. of Biology, Faculty of Science & Arts, Khulais, King Abdulaziz University, KSA). A specimen (No. GM-2016/2) was prepared and deposited at the Herbarium of the Faculty of Pharmacy, King Abdulaziz University.

In vitro cytotoxic activity
Compound 7 was tested for its in vitro cytotoxic activity against MCF7 (hormone-dependent breast carcinoma cell line), A549 (lung carcinoma), and HCT116 (colon carcinoma) cell lines, that were purchased from King Fahd Research Center (Jeddah, KSA). Stock culture was grown in T-75 flasks, containing 10 mL of RPMI-1640 medium and DMEM for MCF7, A549, and HCT116, respectively supplemented with bicarbonate, glutamine, and 10% fetal bovine serum (Gibco, USA). The medium was changed every 2 days and cells were detached, using 0.25% trypsin-EDTA solution (Gibco, USA) and then plated in 96 wells sterile microtiter plates, containing 100 μL RPMI/well at densities of 30,000-100,000 cells/well. The test compound was routinely tested at serial 2-fold dilutions, starting at the upper limit of 200 μM. The culture was incubated at 37 °C for 48 h in the existence of 5% CO 2 ).

Cell fixation and dying
The culture was washed with phosphate buffered saline prior to fixation to get rid of serum protein, which commonly caused cell detachment and loss. The culture was then fixed with cold 50% trichloroacetic acid (TCA) (BDH Chemical Ltd., England) and kept at 4 °C for 1 h. Following fixation, the cells were stained by SRB stain (SRB 0.4% in 1% acetic acid, Sigma-Aldrich, USA) for 30 min as mentioned previously (Skehan et al. 1990). After staining, the cells were washed three times with 1% acetic acid solution to remove unbound dye then air-dried. The bound dye was solubilized with 10 mM Tris buffer (pH 10.5) for 5 min. The optical density (OD) of both treated and untreated cells was read on an automated spectrophotometric plate reader (ELx808, BioTek, Fisher Scientific, USA) at λ max 490 nm. The IC 50 was calculated as: (OD control wells -OD treated wells)/(OD control wells). Doxorubicin was used as a positive control.

Conclusion
In conclusion, the present study described the separation and structural determination of one new (7) and six known (1-6) xanthones from the acetone extract of G. mangostana pericarps. Their structures were verified based on various spectroscopic techniques and comparison with literature. Compound 7 exhibited significant cytotoxic activity towards MCF7 and A549 cell lines.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S5.