Chemical Constituuents Of Five Liverworts And Their Anticancer Activities

The bryophytes are taxonomically placed between the algae and the pteridophytes. Morphologically, they are divided into three classes, liverworts, mosses, and hornworts. The bryophytes have been used as medicinal plants to cure cuts, burns, scalds, pneumonia, pulmonary tuberculosis, neurasthenia, convulsions, edema, and so on. Generally, almost all species of bryophytes are not attacked by insects, snails, and mammals, nor infected by bacteria, fungi, and viruses. In recent years, studies on bryophytes have revealed various diterpenoids and aromatic compounds with interesting chemical structures, many of which show significant biological activity.In this research, five Chinese liverworts, Heteroscyphus tener, Chandonanthus hirtellus, Jungermannia fauriana, and J. hyalina collected from Guangxi Zhuang Autonomous Region, and H. coalitus collected from Guizhou Province were phytochemically investigated. A total of78secondary metabolites,41of which were new compounds, including60diterpenoids, four triterpenoids, and six sterides, were obtained following separation and purification by column chromatography (CC) on silica gel, MCI, reversed-phase C18silica gel, and Sephadex LH-20, as well as by HPLC. Their structures were elucidated on the basis of NMR, MS, CD, and X-ray. This work represents the first phytochemical study on H. tener and J. fauriana.Five ent-labdane diterpenoids (1-5), four of which were new ones (1-4), and three known fusicoccane-type diterpenoids (6-8) were isolated from the Chinese liverwort Heteroscyphus tener. The absolute configuration of compound1was defined by single-crystal X-ray diffraction using Cu Ka radiation. Cytotoxicity tests of compounds1-8revealed that compounds3and5exhibited weak activity against nine cell lines (PC3, DU145, K562, A549, NCI-H292, NCI-H1299, NCI-H446, HBE, and RWPE1). Further research found that the apoptotic effect of compound5was induced through ROS-mediated DNA damage and cell cycle arrest at the G0/G1phase in both DU145and PC3cells.From the ether extract of the liverwort H. coalitu, sixteen compounds, including a new dihydroisocoumarin derivative, R-(-)-heteroscyphide (9) and a known retinane-type diterpenoid (10), which was the first one isolated from bryophytes. Cytotoxic test found that they exhibited moderate inhibitory activity to three human tumor cell lines (PC3, DU145, and HepG-2). Chemotaxonomy analysis of Heteroscyphus species found that H. tener and H. coalitus were different chemically, and the liverwort H. tener more like belongs to the suborder Jubulineae.Seven cembrane-type diterpenoids (25-31), five of which were new ones (25-29), a known fusicoccane-type diterpenoid (6), and three aromatic compounds (32-34) were obtained from the liverwort Chandonanthus hirtellus. All isolated compounds were tested for their cytotoxic activities against six tumor cell lines (PC3, A549, NCI-H292, NCI-H1299, A172, and PC12). Unfortunately, they all exhibited week activity. Cembrane-type diterpenoid could be the chemical marker of the genus Chandonanthus.Twenty-four ent-kaurane diterpenoids (35-58), twenty of which were new (35-54), were isolated from the liverwort Jungermannia fauriana, while twenty-three ent-kaurane diterpenoids (56-78), of which compounds59-70were new, were obtained from J. hyalina. Chemotaxonomy analysis found that these two plants were rich in ent-kaurane diterpenoids, which could be used as the chemical marker of the genus Jungermannia. Containing three same compounds demonstrated they may have a certain kinship. Three ent-kaurane derivatives (80-82) were prepared from stevioside. All of the compounds were tested for their cytotoxic activities against twenty-four human cell lines, suggesting that these compounds have relatively potent toxicity and poor selectivity of cell types. We performed a systematic structure-activity relationship analysis and found that the a,(3-unsaturated carbonyl function is a structural requirement for cytotoxicity in these compounds. The mercapto group was detected by a fluorescent probe. The fluorescence intensity of PC3cells treated with these compounds was significantly reduced, which indicated that these compounds were widely distributed in the cells. However, compound82with a basic group, only partially reduced the intensity of the fluorescence. The lysosomal staining found that82could act on lysosomes, which were observed to be swelled after2h. The basic group modified compound may selectively locate in lysosomes. This modification changed the cell distribution and mode of action of this kind compound.To investigate whether ROS are involved in apoptosis, we measured cellular changes in the ROS levels using a ROS-sensitive fluorometric probe, DCFH-DA. Our results found that compound81could elevate the ROS levels of treated cells. NAC, a ROS scavenger, blocked the cell death induced by81. However, another antioxidant VC could not reverse the cytotoxicity, although could reverse the rise of ROS. By HPLC-MS/MS method, we found that when compound81was added to the medium containing NAC, the prototype compound was hardly detected, but the detection of the addition product81-NAC. These results showed that the reason for the reversal of cytotoxic of NAC was not scavenging ROS, but reacting with compounds, so that loss of efficacy group. Therefore, it is not accurate that many scholars use NAC as a reducing agent to prove the role of ROS. ROS are probably not the major factor causing cell death.In this manuscript, phytochemical investigation of five Chinese liverworts led to41new compounds. Cytotoxic test found a group of ent-labdane diterpenoids, of which compound5induced cell cycle arrest at G0/G1and apoptosis through ROS pathway, and ent-kaurane ones, the structure-activity relationships of which were summarized. It is the first report to use a small molecule fluorescent probe to detect the intracellular distribution of these compounds. By HPLC-MS/MS method we proved that the reason for the reversal of cytotoxic of NAC was not scavenging ROS, but reacting with ent-kaurane diterpenoids. Chemotaxonomy analysis of these plants was performed according to the chemical research.