Novel phthalide derivatives identified from Ligusticum chuanxiong (Chuanxiong)

Background Ligusticum chuanxiong Hort. (Chuanxiong) is a well-known Chinese medicine, and studies on its chemical constituents are important for explaining its mechanism of action and quality control. This study aims to investigate the chemical constituents of the dried rhizome of. L. chuanxiong. Methods The dried rhizome of L. chuanxiong was extracted with 60 % ethanol, and the concentrated extract was isolated by silica gel, octadecyl silane, and Sephadex LH-20 columns, followed by preparative/semipreparative high-performance liquid chromatography (HPLC) to obtain the pure chemical constituents. The structures of the constituents were elucidated by HR-ESI-MS, UV, IR, 1D NMR, and 2D NMR methods. Enantiomeric separation was achieved by a chiral HPLC method. The absolute configuration was determined by the modified Mosher’s method. Results Six novel phthalide derivatives, (+)/(−)-chuanxiongins A–F (1–6), together with four known phthalides (7–10) were isolated from Chuanxiong. All of the new compounds (1–6) were present as pairs of enantiomers. Enantiomeric separation of 1 was successfully achieved by HPLC on a chiral column. The absolute configuration of (−)-1 was determined by a modified Mosher’s method. Conclusion The six novel phthalide derivatives (1–6) isolated from Chuanxiong were phthalide fatty acid esters that were structurally analogous and characterized by fatty acid acylation at 6-OH or 7-OH.


Background
The dried rhizome of Ligusticum chuanxiong Hort. (Chuanxiong) has been used for the treatment of menstrual disorders in Chinese medicine [1]. Chuanxiong has the functions of activating blood flow, expelling wind, and relieving pain associated with blood (xue) and qi stagnation [2]. Previous phytochemical studies on Chuanxiong isolated some phthalides (monomeric and dimeric phthalides) [3][4][5][6], alkaloids [7], phenolic acids [8], ceramides, and cerebrosides [9]. Among them, phthalides as the characteristic constituents had bioactivities including vasodilatation [10], decreased platelet aggregation [11], analgesia [12], anti-inflammation [13,14], and butyrylcholine esterase inhibition [15]. The chemical studies on the constituents of Chuanxiong and identification of new constituents are beneficial for explaining how it exerts its effects and form the foundation for its quality control. This study aims to investigate the chemical constituents of Chuanxiong and identify new constituents.

Plant material
The dried rhizome of L. chuanxiong Hort. was supplied by Sichuan Neautus Traditional Chinese Medicine Co. Ltd. (China) in 2012, and identified by Yue-Cheng Li (Sichuan Institute for Food and Drug Control) by characteristic identification, microscopic identification, and the physics and chemistry identification according to Chinese pharmacopoeia [1]. A voucher specimen (LICH-2012-SC) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University (China).

Results
Six novel phthalide derivatives, (+)/(−)-chuanxiongins A-F (1-6), a new natural product (9), and three known phthalides (7, 8, and 10) were isolated from the 60 % ethanol extract of Chuanxiong (Fig. 1). Compounds 1-8 were obtained as pairs of enantiomers. Compound 1 was successfully resolved by chiral HPLC to afford (+)-1 and (−)-1. Their structures and absolute configuration were elucidated on the basis of spectroscopic data, circular dichroism (CD) analyses, and the modified Mosher's method.     (Fig. 2). On the basis of the mass spectral information, the fatty acid moiety was deduced as palmitic acid. The above spectral data suggested that 1 might be the product of 7 acylated at 6-OH or 7-OH with a long-chain fatty Hz, H-7)] in comparison with 7, the palmitoyl group was assigned to 7-OH via an ester bond. This was supported by the key HMBC correlation from H-7 to the ester carbonyl at δ C 174.0 of the palmitoyl group. The relative stereochemistry of 1 was found to be the same as 7 (cis-oriented) owing to the small J 6,7 value of 3.9 Hz, as well as the carbon chemical shifts of C-6 and C-7 [16]. Thus, the structure of 1 was elucidated as (4R*,5S*,Z)-1-butylidene-5-hydroxy-3-oxo-1,3,4,5,6,7hexahydroisobenzofuran-4-yl palmitate, and designated chuanxiongin A. The optical rotation value of 1 was almost zero, indicating a racemic nature, which was in accordance with the appearance of two peaks in the chiral HPLC analysis (Fig. 3). The chiral resolution of 1 by chiral HPLC afforded a pair of anticipated enantiomers, (+)-1 and (−)-1, which were opposite in terms of their optical values and CD curves (Fig. 4). The absolute configuration of the enantiomer (−)-1 was established by the modified Mosher's method [17]. A solution of (−)-1 (0.5 mg) in C 5 D 5 N (0.5 mL) was treated with  (Tables 1 and 2) to those of 1, except for the large upfield shift of H-7 and downfield shift of H-6, indicating the position of esterification at 6-OH instead of 7-OH. This deduction was further confirmed by the key HMBC correlation from H-6 to the ester carbonyl at δ C 173.3. Therefore, the structure of 2 was deduced as senkyunolide H 6-palmitate, with the systematic name of (4R*,5S*,Z)-1-butylidene-4-hydroxy-3-oxo-1,3,4,5,6,7hexahydroisobenzofuran-5-yl palmitate. It was designated with the common name chuanxiongin B.
Compounds 7 and 8 were identified as senkyunolide H (7) [5] and senkyunolide I (8) [5] by comparisons of their physiochemical properties and spectral data with those reported in the literature.
Similarly, the optical rotation values of compounds 2-8 were almost zero, suggesting that they were also present as pairs of enantiomers, as further confirmed by the observations of two separated peaks in the chiral chromatographic analysis experiment (Fig. 3).