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Chemical Constituents from the Roots of Calophyllum pisiferum Planch. & Triana and Their Cytotoxic and Antioxidant Activities

This is the first phytochemical investigation of the roots of Calophyllum pisiferum Planch. & Triana. A new 4-phenyl coumarin, calopisifuran (1), and a new xanthone, 1-hydroxy-4,5-dimethoxyxanthone (2), were isolated and identified, together with 11 known compounds, including 4-phenyl coumarin (37) and xanthones (813). Their structures were elucidated by 1Dand 2D-NMR spectra and by HR-ESI-QTOF mass spectra and were compared with the literature data for known compounds. The isolated compounds exhibited significant cytotoxic activity in the MDA-MB-231, MCF-7 and A-549 cell lines, with IC50 values of 14.87, 23.56 and 43.34 μg/mL, respectively, for 10 and IC50 values of 17.15, 45.76 and 85.17 μg/mL, respectively, for 1, while the DPPH assay revealed weak antioxidant activity.


Introduction
Calophyllum is one of the largest genera of the Clusiaceae family. The members of this genus are widely distributed throughout Asia, and some species exist in Africa, the Americas, Australasia, and the Pacific Islands [1]. This genus includes approximately 187 species, of which 17 occur in Thailand [2]. Calophyllum is a rich source of secondary metabolites and has been reported to contain many bioactive compounds, such as coumarins, xanthones, flavonoids, triterpenes, benzofuran, acylphloroglucinol and chromanone; in particular, coumarins and xanthones [3] are found in almost all species and are well recognized as phytochemical markers for this genus. The biological activities of these types of compounds have been reported in some of the extracted pathways, and some of the isolated compounds have also shown good pharmaceutical activities, including cytotoxic activity [4], anti-HIV activity [5], antiviral activity, antitumor-promoting activity, antimalarial activity, antibacterial activity [3], anti-inflammatory activity [6] and central nervous system (CNS) activity [7]. Calophyllum pisiferum Planch. & Triana, locally known as "Tung hon-bailek" in Thailand, is found only in lowland and swamp forests in southern Thailand [8], Cambodia, Indonesia, Malaysia, Myanmar and Vietnam. [9]. The wood of C. pisiferum has been used for the construction of dwellings, and there is no evidence for the utilization of this plant for folk medicine in Thailand.
A literature survey showed that the chemical constituents and biological activities of this plant have not been reported. Therefore, we report herein the first isolation and structural elucidation of a new 4-phenyl coumarin compound (1) and a new xanthone (2) together with 11 known compounds (313) from the root of C. pisiferum ( Figure 1). Some of the isolated compounds were tested for their cytotoxicity against the MCF-7, MDA-MB-231 and A-549 cell lines and for their antioxidant activities.

General Experimental Procedures
Melting points were determined by a Buchi melting point B-540 apparatus and were reported without correction. Optical rotations were measured on a JASCO P-1020 digital polarimeter. Ultraviolet spectra were measured in a methanol solution on a Biochrom Libra S50 UV/Vis spectrophotometer. IR spectra were measured for samples kept in the same holder with an IRTracer-100 (Shimadzu) spectrophotometer. 1 H and 13 C NMR spectra were recorded on Bruker FTNMR Ultra Shield TM 300 and 500 MHz instruments (300 and 500 MHz for 1 H NMR and 125 and 75 MHz for 13 C NMR); CDCl3 and acetone-d6 were used as solvents, and TMS was used as the internal standard.
Chemical shifts were reported in  [ppm], and the coupling constants (J) were expressed in hertz.
High-/low-resolution mass spectra were obtained by means of a mass spectrometer (Model: LCMS-IT-TOF, Shimadzu). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 GF254 (Merck) and silica gel 100 (Merck), respectively. Preparative thinlayer chromatography (PTLC) was performed on precoated silica gel 60 GF254 plates (Merck); spots were detected by UV and used for analysis. The solvents used for extraction and chromatography were distilled at their boiling points prior to use.

Cytotoxicity Assay
The cytotoxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [10]. The breast cancer cell line MCF-7, human breast adenocarcinoma cell line MDA-MB-231 and human lung carcinoma cell line A-549 were obtained from the American Type Culture Collection. Briefly, the cells were seeded at a density of 7 10 3 cells/well in 96-well plates for 24 h. The cells were then treated with various concentrations of the isolated compounds and incubated for 24 h, and then the MTT formazan performance was measured at 570 nm. Doxorubicin and ellipticine were used as standard drugs.

Antioxidant Assay
The antioxidant effect of the isolated compounds was determined using the 2,2′-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging assay [11]. Trolox was used as a positive control. The scavenging activity was estimated based on the percentage of DPPH radicals scavenged by using the following equation:

Structure Elucidation
Compound (1) was isolated as a white solid, and the HRESITOFMS spectrum exhibited an [M-H] + ion at m/z 361.0721, which corresponded to the molecular formula C22H18O5 and supported the identification as an unusual type of 4-phenylcoumarin chromophore [14]. The 1 H and 13 C NMR spectra of 1 ( Table 1)  respectively. Additional evidence to support the isomer structure of 1 was obtained from the difference between their 13 C NMR data and those of isodisparfuran (Table S1, supplementary data) [14]. Therefore, compound 1 was named calopisifuran.
Compound (2) was obtained as a yellow solid with a molecular ion peak [M+H] + at m/z 273.0149 in the HRESI−TOFMS spectrum, which suggested a chemical formula of C15H12O5 ; in addition, the UV and IR spectra of this newly isolated compound had characteristic signals of a xanthone chromophore [21]. The 1 H NMR spectrum (Table 1) showed a chelated hydroxyl group at H 12.81 (s, 1-OH), aided by the HMQC (Table 1) and COSY (Figure 2) spectra, which exhibited two ortho-doublet signals at H 7.06 (d, J = 9.0 Hz, C 106.0) and 7.33 (d, J = 9.0 Hz, C 120.9), and the positions of these ortho couples were placed at H-2 and H-3, respectively, due to the HMBC correlations ( Figure 2) (Table S2, supplementary data) [22]. Thus, from the above data, the structure of compound 2 was elucidated as 1-hydroxy-4,5-dimethoxyxanthone.

Conclusion
In this study, a new 4-phenyl coumarin (1) and a new xanthone (2), along with eleven known compounds (313), were isolated from the roots of C. pisiferum for the first time, and all the compounds were characterized by means of on spectroscopic methods and by comparison with previously reported data. New compound 1 and compound 10 showed significant cytotoxicity against the MDA-MB-231 cell line, whereas the remaining compounds showed weak antioxidant activity.