Phenolic Compounds from the Leaves of Castanopsis fargesii

In the course of a phytochemical and chemotaxonomical investigation of Castanopsis species (Fagaceae), three new phenolic compounds, (3R,1′S)-[1′-(6″-O-galloyl-β-d-gluco-pyranosyl)oxyethyl]-3-hydroxy-dihydrofuran-2(3H)-one (1), (2R,3S)-2-[2′-(galloyl)oxyethyl]-dihydroxybutanoic acid (2), and (3S,4S)-3-hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(4′-hydroxy-3′-methoxyphenyl)-1H-[2]-benzopyran-1-one (3) were isolated from the fresh leaves of Castanopsis fargesii. In addition, a known phenolic glycoside, gentisic acid 5-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranoside (4) was also isolated and identified. Their structures were elucidated by means of spectroscopic methods including one- and two-dimensional NMR techniques.


Introduction
The genus Castanopsis belongs to the Fagaceae family and is commonly found in the evergreen forests of East Asia. There are about 120 species of Castanopsis, however, the classical plant taxonomy of the species is very complicated and sometimes confusing [1], thus, the application of other auxiliary methods and technologies, such as chemotaxonomy and cytotaxonomy, is necessary to identify species within this genus [2]. Previous phytochemical investigations on the plants of this genus C. fissa, C. cuspidata var. seiboldii, and C. hystrix have led to the isolation of triterpene hexahydroxydiphenoyl (HHDP) esters, HHDP glucoses, galloyl, acylated quinic acids, phenol glucosides, condensed tannins, and flavonol glycosides [3][4][5][6][7][8].
In this study, we investigated C. fargesii, which is widely distributed in southern China, where it is usually used as a traditional medicine for the treatment of diarrhea, hemorrhage, and chronic ulcers [1]. Our preliminary analysis by HPLC and TLC indicated that the leaves are rich in tannins. Subsequent chromatographic separation of the extract yielded two metabolites 1 and 2, which were identified as 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic acid derivatives, and two phenolic compounds 3 and 4. This paper reports the isolation and structural characterization of the new compounds 1-3 and an assessment of the cytotoxicity of these molecules.
Compound 3 was obtained as a brown amorphous powder, which gave a dark blue color with FeCl3. The molecular formula C17H16O8 was deduced from the [M − H] − peak at m/z 347.0768 in the LC-MS/IT-TOF (calcd. for C17H15O8, 347.0772). Comparison of the 1 H-and 13 C-NMR data of 3 (Table 2)

Materials
The leaves of C. fargesii were collected at Guangxi Institute of Botany, Guangxi, China, in August 2014, and were identified by Prof. Shi-Hong Lu. A voucher specimen (20140627) was deposited in the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, China.

Materials
The leaves of C. fargesii were collected at Guangxi Institute of Botany, Guangxi, China, in August 2014, and were identified by Prof. Shi-Hong Lu. A voucher specimen (20140627) was deposited in the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, China.

Preparation of MTPA Esters Derivatives
CH 3 I (30 mg) and K 2 CO 3 (15 mg) were added to a solution of 2 (10 mg) in DMF (5 mL). After stirring for 24 h at room temperature (r.t.), the reaction mixture was suspended in H 2 O and extracted with CHCl 3 . The CHCl 3 layer was vacuum dried to afford a residue (6.2 mg). Then, DMAP

Conclusions
In this study, we separated and identified three new compounds 1-3 and a known compound 4 from the leaves of C. fargesii. The 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic acid moiety in 1 and 2 is an unusual carboxylic acid in Nature, thus, these compounds might be recognized as chemotaxonomic markers. Our preliminary examination also suggested this plant contains triterpene HHDP esters, which are important chemotaxonomical markers of Castanopsis sp.; therefore, further phytochemical investigations of the leaves of C. fargesii are in progress.