Phylattrin, a New Cytotoxic Xanthone from Calophyllum soulattri

Our continuing studies on secondary metabolites from the stem bark of Calophyllum soulattri has led to the isolation of another new diprenylated xanthone, phylattrin (1), in addition to five other xanthones and two common sterols. The xanthones are soulattrin (2), caloxanthone C (3), macluraxanthone (4), brasixanthone B (5) and trapezifolixanthone (6) while the sterols are stigmasterol (7) and β-sitosterol (8). The structures of these compounds were determined on the basis of spectroscopic analyses such as 1D and 2D-NMR, HRESIMS, IR and UV. Compounds 1–7 exhibited moderate cytotoxic activities against SNU-1, HeLa, Hep G2, NCI-H23, K562, Raji, LS174T, IMR-32 and SK-MEL-28 cells.


Introduction
Calophyllum belongs to the Clusiaceae family and is mainly distributed in tropical areas around the World, primarily in the Indo-Pacific region. It has many local names such as "bintangor" in Asia and "tamanu" in Hawaii. Calophyllum is well known for its biological activities which are the result of the

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presence of a variety of secondary metabolites. Xanthones [1][2][3][4], coumarins [5], triterpenoids [6] and flavonoids [7,8] are examples of the most reported secondary metabolites. Among all the biological activities, anti-HIV [9,10] and anti-cancer [11] were the most reported. Besides, some of these compounds are widely used in industry such as antiseptics, astringents, diuretics and purgatives [12]. This present paper reports the isolation and structural elucidation of a new compound, phylattrin (1) from the stem bark of Calophyllum soulattri. The cytotoxicities of the new compound and seven known compounds isolated at the same time will also be reported.

General
EIMS were recorded on a Shimadzu GC-MS model QP2010 Plus spectrophotometer. NMR spectra were obtained using a JEOL FT-NMR 500 MHz spectrophotometer using tetramethylsilane (TMS) as internal standard. Ultraviolet spectra were recorded in EtOH on a Shimadzu UV-160A, UV-Visible Recording Spectrophotometer. Infrared spectra were measured using the universal attenuated total reflection (UATR) technique on a Perkin-Elmer 100 Series FT-IR spectrometer. Melting points were measured using a Leica Galen III microscope, equipped with a Testo 720 temperature recorder.

Plant Material
The stem bark of Calophyllum soulattri was collected from the Sri Aman district in Sarawak, Malaysia by Prof. Dr. Jegak Uli. This plant was identified by Dr. Rusea Go from the Department of Biology, Faculty of Science, Universiti Putra Malaysia where a voucher specimen was deposited.

Cytotoxicity (MTT Assay)
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed according to a previously described method [18]. The tests were performed in a sterile 96-well flat bottom plate. Stock solutions of each pure compound were prepared by dissolving them in DMSO to a concentration of 20 mg/mL. A six-point serial dilution was developed to obtain six different sub-stocks with different concentrations. For suspension cells, concentrations needed were 50.00, 25.00, 12.50, 6.25, 3.13 and 1.56 μg/mL. Meanwhile, 100.00, 50.00, 25.00, 12.50, 6.25 and 3.13 μg/mL were the essential concentrations for anchorage-dependant cells. Each pure compound was tested in triplicate together with the controls.
After 72 h incubation at 37 °C and 5% of CO 2 , MTT solution (20 μL) was added into all the filled wells and incubated again for 3 h. The plate was spun at 1500 rpm for 10 min followed by discarding approximately 80% of supernatant carefully. The volume of supernatant discarded was the same as the volume of DMSO added into the wells. The absorbance of each well was determined by a microplate reader at 550 nm after the purple crystal formazan fully dissolved in DMSO. Three independent experiments for both suspension and anchorage-dependant cell lines were conducted. The average of the absorbance values was used in the calculation of percentage of cell viability. The cytotoxicity index used was IC 50 , which is the concentration that yields 50% inhibition of the cell compared with the untreated control.