Chemical Constituents from the Stems of Diospyros maritima

A new phenolic, bis(6-hydroxy-2,3,4-trimethoxylphen-1-yl)methane (1) and a new butanedioate, butylmethyl succinate (2), along with twenty-nine known compounds including one naphthoquinone derivative, two chromanones, eight benzenoids, one lignan, one tocopherol, and sixteen triterpenoids were isolated from the stems of Diospyros maritima. epi-Isoshinanolone (3) was isolated in pure form for the first time. In addition, 5,7-dihydroxy-2-methylchomanone (4) was isolated from a natural source for the first time. Their structures were established on the basis of spectroscopic data as well as direct comparison with authentic samples.


Results and Discussion
The EtOH extracts of the stems of D. maritima was concentrated to give a black residue which was suspended in water and partitioned successively with n-hexane and n-BuOH. The combined n-BuOH soluble layer was subjected to repeated chromatography using silica gel and further purification by semipreparative HPLC to furnish two new compounds, bis(6-hydroxy-2,3,4-trimethoxylphen-1yl)methane (1) and butylmethyl succinate (2), in addition to twenty-nine known compounds. The identification of the known compounds were performed by comparing their physical and spectral data (IR, UV, MS, and NMR) with literature values. This paper deals with the structural elucidation of compounds 1-4.
The IR spectrum of 2 exhibited an absorption band (1735 cm -1 ) for an ester functionalty. The 13 C-NMR and DEPT spectra indicated the presence of one methyl (δ C 13.7), one methoxy (δ C 51. ] was observed. The above features were almost identical to those of the known compound, epiisoshinanolone [15]. Thus, compound 3 was determined as epi-isoshinanolone. It was isolated in pure form for the first time. Analysis the IR spectrum of 4 suggested that it contains hydroxyl (3,400 cm -1 ), conjugated ketone (1,668 cm -1 ), and aromatic (1,620, 1,600 cm -1 ) functionalities.  1H, s, H-6)]. The above spectral information was similar to that of 5,7-dihydroxy-2-methylchromanone previously reported in the literature [17]. Thus, compound 4 was characterized as 5,7-dihydroxy-2-methylchromanone. Compound 4 was isolated from the natural product for the first time in the present investigation.

General
Melting points were determined with a Yanagimoto (MP500D) micromelting point apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 781 spectrophotometer. 1 H-and 13 C-NMR spectra were obtained in CDCl 3 at a constant temperature controlled and adjusted to around 300 K on a Bruker AM-300 spectrometer, and the residual proton resonance (CHCl 3 ) of CDCl 3 was used as internal shift reference. The 2D NMR spectra were recorded on a Bruker DMX-300 spectrometer by using standard pulse sequences. EI-MS, FAB-MS, HR-EI-MS, UV spectra, and specific rotations were recorded on a JEOL JMS-HX 300, a JEOL JMS-HX 110, a JEOL SX-102A, a Hitachi S-3210 spectrometer, and a JASCO DIP-1000 digital polarimeter, respectively. TLC was performed by using Si gel 60 F 254 plates (Merck). Column chromatography was performed on silica gel (Merck 9385, 70-230 mesh). HPLC was performed by using a Lichrosorb Si gel 60 (5μm) column (250 × 10 mm).

Plant material
The stems of D. maritima were collected in Lin-Ko, Taiwan, in 1993. The plant material was identified by Muh-Tsuen Gun, formerly a technician of the Department of Botany, National Taiwan University, and a voucher specimen has been deposited at the National Research Institute of Chinese Medicine, Taipei, Taiwan, R.O.C.

Extraction and isolation
The stems of D. maritima (16 kg) were extracted three times with EtOH (160 L) at 60 °C for 10 h each time. The EtOH extract was evaporated under reduced pressure, yielding a black residue, which was suspended in H 2 O (12 L), and then partitioned (×5) with 1 L of n-hexane. The aqueous layer was partitioned (×4) again with 1 L of n-BuOH. The combined n-BuOH extracts (180 g) was then subjected to column chromatography over silica gel (120 × 6 cm) eluted with a setpwise gradient mixture of hexane and EtOAc as eluent. Seven fractions were collected as follows: 1 [3000 mL, hexane], 2 [4000 mL, hexane-EtOAc

Conclusions
Thirty-one compounds were isolated from the stems of D. maritima. Among them, bis(6-hydroxy-2,3,4-trimethoxylphen-1-yl)methane (1) and butylmethyl succinate (2) are new compounds and epiisoshinanolone (3) was isolated in pure form for the first time. In addition, 5,7-dihydroxy-2methylchromanone (4) was isolated from the natural source for the first time. This investigation of secondary metabolites may contribute to better understanding on the chemical characteristics of D. maritima.