Coumarins from Edgeworthia chrysantha

A new coumarin, edgeworic acid (1), was isolated from the flower buds of Edgeworthia chrysantha, together with the five known coumarins umbelliferone (2), 5,7-dimethoxycoumarin (3), daphnoretin (4), edgeworoside C (5), and edgeworoside A (6). Their structures were established on the basis of spectral data, particularly by the use of 1D NMR and several 2D shift-correlated NMR pulse sequences (1H-1H COSY, HSQC and HMBC), in combination with acetylation reactions.


Introduction
The genus Edgeworthia (Thymelaeaceae) consists of five species distributed around the World, which are native to China, India, Japan, and southeast of America [1]. E. chrysantha is widely distributed and is endemic to South and East China [1]. The bark of E. chrysantha is used as "Zushima" in some local areas in China for the treatment of traumatic injury and rheumatism [2,3], and the fiber of the stem bark is a raw materials for making high quality paper [1]. The flower buds are often used as the traditional Chinese medicine "Mimenghua" for the treatment of ophthalmalgia and delacrimation [1,4].
The 1 H-NMR spectrum of 1 (Table 1)  The aromatic H-atom at δ H 7.46, which correlated with 154.8 (C-7), 148.8 (C-9), 145.1 (C-4) in the HMBC spectrum (Figure 2), could be assigned to H-5. Since it coupled with the H-6, the substitution site at the coumarin skeleton was established at C-7 and C-8.  According to the molecular formula of 1, there were only two phenolic hydroxyl groups left. Unfortunately, no phenolic hydroxyl groups signals appeared in the 1 H-NMR spectrum, so the locations of the phenolic hydroxyl groups were confirmed by acetylation. The acetylated derivative of 1 (compound 1a) was obtained as a white powder. The molecular formula C 20 H 16 O 7 was determined by ESI-MS ([M+H] + peak at m/z 367.0), indicating 13 degrees of unsaturation. By comparing the MS data of 1a with those of 1, it was presumed that a six membered lactone ring was formed during the acetylation reaction. The comparison of the 13 C-NMR data of 1 with those of 1a (Table 1) revealed that the signals of C-6 and C-8 were shifted downfield in the range of δ 5-6 ppm, the signal of C-7 was shifted upfield by δ 8.7 ppm, and the signal of C-3' was shifted upfield by δ 3.6 ppm. According to the analysis mentioned above, one of the phenolic hydroxyl groups was deduced to be at C-7 [21], and another one was located at C-3', which was confirmed by the HMBC spectrum. (Figure 2).

General
All chemical solvents used were of analytical grade.

Plant Material
The flower buds of E. chrysantha were collected in a garden of Lishui, Zhejiang Province, China, in February 2008. The plants were authenticated by Dr. Chu Chu, Zhejiang University of Technology, China. A voucher specimen (TCM 2008-026) was deposited in College of Pharmaceutical Science, Zhejiang University of Technology.

Acetylation of Edgeworic acid (1)
A mixture of compound 1 (20 mg), Ac 2 O (5 mL), and pyridine (5 mL) was stirred at room temperature overnight. The resulting solution was concentrated under vacuum. The residue was dissolved in ethyl acetate and washed with water (5 mL). The product was purified on a silica gel column (n-hexane/ethyl acetate, 3:2 v/v) to afford 1a (15 mg).  Table 1.