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Chemical Compounds from the Twigs and Leaves of Caesalpinia cucullata Roxb

A new stilbene dimer, caesalstilbene A (1), along with twelve known compounds were isolated from the twigs and leaves of Caesalpinia cucullata Roxb by means of various chromatographic techniques. Their structures were identified on the basis of NMR spectral analysis and comparing their spectral data with those reported in the literatures. The absolute configuration of 1 was assigned by the comparison of the experimental and calculated electronic circular dichroism spectra. Compound 1 was evaluated for their cytotoxicity on HL-60, SMMC-7721, A-549, MCF-7 and SW-480 human cancer cell lines, but it was inactive. This is the first report of chemical investigation on Caesalpinia cucullata.


Introduction
Caesalpinia cucullata Roxb, belonging to the family Leguminosae, is distributed in south of Yunnan province of China, India, Nepal, Sikkim [1]. There are 17 Caesalpinia species widespread in China, and 14 species have long been used in Chinese traditional medicine to reduce swelling and alleviate pain, to treat rheumatism and inflammatory [2]. Because of their characteristic cassane diterpenoids and homoisoflavonoids, whose structures and bioactivities are diverse [2,3,4], the plants of Caesalpinia have drawn wide attention. As our continuous investigation on Caesalpinia plants [5,6], one new stilbene dimer, caesalstilbene A (1), along with twelve known compounds were isolated from the twigs and leaves of C. cucullata. All these compounds were isolated from C. cucullata for the first time. Here, we described the isolation and structural elucidation of these compounds.

Instrumentation and Reagents
NMR spectra were acquired on Bruker DRX-500 spectrometer. MS data were obtained using a Bruker microTOF spectrometers. Fractions were monitored by TLC on silica gel plates (GF254, Qingdao Puke separation material Co., Ltd., Qingdao, China). Column chromatography (CC) was performed on silica gel (100-200 mesh or 200-300 mesh; Qingdao Puke separation material Co., Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare) and MCI gel (75-150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan). All solvents used in column chromatography were of industrial grade and used after distillation under vacuum.

Plant Material
The twigs and leaves of Caesalpinia cucullata wer463e collected from Xishuangbanna, Yunnan, China in November 2015, and identified by Chunfen Xiao (Xishuangbanna Botanical Garden, Chinese Academy of Sciences) to be the same as the voucher specimen (HITBC 017509) deposited at Xishuangbanna Botanical Garden, Chinese Academy of Sciences.

Extraction
The powdered twigs and leaves (11.5 kg) of C.cucullata were extracted with EtOH at room temperature, which afforded a dark residue after evaporation under reduced pressure. The residue was dissolved in H2O and extracted by ethyl acetate (EtOAc). The EtOAc extract (360 g) was subjected to CC (SiO2, 100-200 mesh; petroleum ether/ EtOAc 10: ), to gain ten fractions (Fr. T1-T10), and then it was eluted with CH3OH to afford fraction T11.

Results and Discussion
Compound 1 was isolated as brown amorphous solid, with the molecular formula C28H22O7 (18 degrees of unsaturation), as determined by HRESIMS at m/z 471.1446 [M +H] + . The 1 H NMR and 13 C NMR spectra showed the presence of one set of ortho-coupled aromatic protons derived from a 4hydroxyphenyl group, one set of aromatic protons coupled in an AX2 system due to a 3,5dihydroxyphenyl group, one set of aromatic protons coupled in an AX system due to a 2,3,5substituted phenyl group, two singlet aromatic protons attributed to a 2,4,5-substituted phenyl group, and four aliphatic methines (H-7a, H-8a, H-7b and H-8b). This evidence, together with the limitation imposed by eighteen unsaturations, indicated the existence of two ring systems and four benzene rings in 1. These observations suggested that compound 1 was a stilbene dimer with a planar structure similar to that of cararosinol C [7], except that the location of the hydroxyl groups on ring B1 and B2 were not as same as that of cararosinol C. Ring B1 and ring B2 were 3,5-dihydroxy and 2,4,5substituted phenyl groups respectively, which was verified by the HMBC correlations of H-2b(6b)/C-7b, H-11b/C-7a, H-2a(6a)/C-7a, H-14a/C-8a, H-14b/C-8b, H-7b/C-11a (Figure 1). In its ROESY spectrum, the correlations between H-7b/H-8a, H-7b/H-14b, H-7a/H-8b，and H-7a/ H-11b indicated that the hydroxyphenyl groups at C-7a and C-7b were trans-oriented to each other, while they were cis-oriented towards H-8a and H-8b respectively. Therefore, the relative stereostructure of compound 1 was established as shown in Figure 1 (1a or 1b). The absolute configuration of 1 was determined by comparison of the experimentally obtained-, and simulated, electronic circular dichroism (ECD) spectra. The ECD spectra of 1a better matched the experimental ECD spectra of 1 (Figure 2), suggest the (7aR,8aR,8bR,8aS) absolute configuration for 1. Hence, the structure of 1 was elucidated to be 1a, and named as caesalstilbene A.
The cassane diterpenoids and homoisoflavonoids are the characteristic components of Caesalpinia plants [17]. However, our present study showed that the cassane diterpenoids were not found in Mezoneuron sub-genus [5,6]. This is the major difference between Caesalpinia sub-genus and Mezoneuron sub-genus. Thus the cassane diterpenoids might be a unique chemotaxonomic marker for Caesalpinia sub-genus. Depth studies need to be carried out.
Using the MTS method reported in the literature [5], compound 1 was tested for their cytotoxicity against the HL-60 (acute leukemia), SMMC-7721 (liver cancer), A-549 (lung cancer), MCF-7 (mammary cancer) and SW-480 (colon cancer) human tumor cell lines, but no activity was noted with IC50 values more than 40 μM.