Calyxins represent a rare class of natural products hybridized from a diarylheptanoid and a flavonoid (chalcone or flavanone) [1]. Interestingly, they have been exclusively reported from the plants of genus Alpinia Roxb. (family Zingiberaceae) to date. The first members of this fascinating group of molecules were initially reported by Kadota and coworkers from A. blepharocalyx in 1994 [2], and about 30 analogues have been described since then. Previous studies have revealed that calyxins possess a variety of biological activities especially antiproliferative [[3], [4], [5], [6], [7]] and nitric oxide (NO) production inhibitory [8] effects. Owing to their complex structures and attracting bioactivities, great efforts have been put into the core skeleton construction and total synthesis of these intriguing compounds [[9], [10], [11], [12], [13]]. Albeit the significant progress in the chemical studies on the calyxins, the mode of action by which they exert bioactivities have never been explored in the literature.

As the only genus of family Typhaceae, Typha is a very small plant tribe with only 16 species mainly growing in the temperate regions of Asia, Europe and North America [14]. Most of these herbal plants possess high economic values and have found their applications in many industrial areas [14]. Particularly, the pollen of several Typha species such as T. angustifolia and T. orientalis, has been used in traditional Chinese medicine (called Pu-huang) to treat a number of illnesses including hemoptysis, uterine bleeding, dysmenorrhea, and so on [15]. However, the reports on the bioactive constituents in Typha plants are very limited. For example, only several publications have reported few flavonoids, lipids and steroids from T. angustifolia [[16], [17], [18]], with anti-platelet aggregation [18] and antiproliferative [17] activities.

As an extension of our former work aiming to explore new chemotherapies from traditional herbal remedies for the treatment of cancer [[19], [20], [21], [22]], we have lately carried out a chemical study on the 70% EtOH extract of the pollen of T. angustifolia. This effort has been rewarded by the isolation and identification of four novel calyxin analogues, typhatifolins A−D (1−4, Fig. 1), whose structuration characterization was accomplished on the basis of chemical and spectroscopic approaches. Excitingly, all the four compounds displayed remarkable growth inhibitory activity toward MDA-MB231, MCF-7, HeLa and A549 cancer cells. Following our continuous focus on breast cancer, both the in vitro and in vivo antitumor evaluations and further mechanistic studies were performed on breast cancer cells in the subsequent work. Hence, details of the separation, structure elucidation and biological assessments will be presented below.