New Isoflavanes from Spatholobus suberectus and Their Cytotoxicity against Human Breast Cancer Cell Lines

The rattans of Spatholobus suberectus Dunn are a traditional Chinese medicine activating blood circulation and removing stasis. They have often been used for the traditional Chinese medicinal treatment of breast cancer in modern China. In this study, four novel isoflavanes (1–3 and 5) and four known analogues (4 and 6–8) were isolated from an ethanolic extract of the rattans of S. suberectus. Their structures were elucidated by extensive spectroscopic analyses and electronic circular dichroism studies. MCF-7 and MDA-MB-231 human breast cancer cell lines were used to evaluate the cytotoxic effects of the isolates. Interestingly, compounds 1 and 2 only inhibited the proliferation of MCF-7 cells, while compound 6 showed a selective cytotoxicity against MDA-MB-231 cells. However, compound 4 had significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines.


Introduction
Spatholobus suberectus Dunn (Leguminosae) is a representative traditional Chinese medicine historically used to promote blood circulation and remove stasis. As traditional Chinese medicine theory considers cancer as a kind of disease most possibly related to severe blood stasis, S. suberectus is commonly used for the treatment of cancer in China [1]. Modern studies on S. suberectus have indicated that its extracts and compounds have cytotoxic effects against human cancer cell lines, especially breast cancer cell lines [1][2][3]. Phytochemical investigations have found that S. suberectus contains flavonoids, sterols, lignans, anthraquinones, phenolic acids, terpenoids, and their glycosides [4][5][6][7][8]. Flavonoids are reported to be the main active secondary metabolites, including flavones, flavanones, isoflavanes, isoflavones, and chalcones [2,4,9,10]. Some of them, such as genistein and gallocatechin, have been demonstrated to be effective for cancer prevention or treatment [11]. In our previous study, a series of chalcones and flavanones were isolated from S. suberectus and synthesized. The cytotoxicity against two breast cancer cell lines (MCF-7 and MDA-MB-231) and the structure-cytotoxicity relationship were studied. A methoxylated chalcone, 3 ,4 ,5 ,4"-tetramethoxychalcone, was found to be most effective [9]. To further explore the cytotoxic components of S. suberectus against breast cancer cells, the same ethanolic extract of the rattans of S. suberectus was continuously investigated.
As the extracts and the main compounds of S. suberectus have been reported to have cytotoxic activities against human breast cancer cells [1][2][3]9], all the isolates in this study were assayed for their cytotoxicity using an estrogen-dependent breast cancer cell line (MCF-7) and an estrogen-independent breast cancer cell line (MDA-MB-231). Four compounds (1, 2, 4, and 6) showed cytotoxic effects, with IC 50 values less than 100 µM (Table 3) 1, 2, and 4) had cytotoxicity against the MCF-7 cell line, and such cytotoxicity was lost when the OMe-2 transformed to the OH-2 ; all isoflavanes without the OMe-8 group (4 and 6) had cytotoxicity against the MDA-MB-231 cell line. Thus far, isoflavanes have been reported to have a wide range of activities, but few studies have been conducted on their anti-breast cancer effects. (3S)-3 -Hydroxy-8-methoxyvestitol [14], possessing a similar structure to compounds 1-4, showed better cytotoxicity against MCF-7 cells (IC 50 = 17.87 ± 0.30 µM) than 1-4. It can be speculated that the substituents on the aromatic rings in isoflavanes play an important role in inhibiting the proliferation of breast cancer cells. Although several natural isoflavones showed strong antiproliferative effects on breast cancer cells [15][16][17], the two isoflavones 7 and 8 isolated from S. suberectus were inactive in this study (IC 50 > 100 µM). It is worth investigating whether other isoflavanes and isoflavones of S. suberectus have anti-breast cancer effects. In addition, the previous studies of S. suberectus on the cytotoxic effects and mechanisms against human breast cancer cells mainly focused on the total flavonoid extract [1,3]. Thus, further studies of the cytotoxic chemical components of S. suberectus, especially various types of flavonoids, are necessary.

Plant Material
The rattans of Spatholobus suberectus were purchased from Sichuan Neautus Traditional Chinese Medicine Co., Ltd.

Cytotoxic Activity Assay
The cytotoxic activity of the isolates against the MCF-7 and MDA-MB-231 human breast cancer cells were determined by a colorimetric MTT assay, as described in the literature [9,28]. Paclitaxel was used as a positive control. The IC 50 values represented the mean of three independent replicates.

Conclusions
Spatholobus suberectus mainly contains flavonoids, including flavones, flavanones, chalcones, isoflavanes, and isoflavones, some of which have a cytotoxic effect against breast cancer cell lines. In this study, four novel isoflavanes (1-3 and 5) and four known analogues (4 and 6-8) were isolated from the rattans of S. suberectus. Compounds 1, 2, and 6 showed selective cytotoxicity against the estrogen-dependent breast cancer cell line (MCF-7) and the estrogen-independent breast cancer cell line (MDA-MB-231) (1 and 2 against MCF-7; 6 against MDA-MB-231), while compound 4 had cytotoxicity against both cell lines. Preliminary analysis of the structure-cytotoxicity relationship suggested that C-2 and C-8 substituents may affect the cytotoxicity and selectivity.