Characterization And Structure Elucidation Of Polyphenolic Compounds In The Leaves Of Perilla Frutescens Var. Acuta With Anticancer Bioactivity

Perilla is an annual herb originated from Asia. It has been widely cultivated all over the country as edible and medical plant. Taking full advantage of these natural perilla resources while having a clear knowledge of its composition and bioacitivity may have a significant influence in the field of food and pharmacy industries.Perilla frutescens var. acuta was used as our plant material. Anthocyanin extract from the leaves were isolated and characterized with advanced high mass accuracy and multi-dimensional fragmentation by means of ultra-performance liquid chromatography (UPLC) and electrospray ionization-ion trap-time of flight mass spectrometry analysis (ESI-IT-TOF-MSn). Seven anthocyanins including one feruloyl (Cyanidin 3-O-feruloylglucoside-5-O-glucoside), two caffeoyl (Cyanidin 3-O-caffeoylglucoside-5-O-glucoside, Cyanidin 3-O-caffeoylglucoside-5-O-malonylglucoside) and four coumaroyl substituted anthocyanins (Cis-shisonin, Malonyl-cis-shisonin, Shisonin, and Malonyl-shisonin) were identified. Malonyl-shisonin was confirmed to be the most abundant anthocyanin while the feruloyl acylated anthocyanin was identified for the first time in Perilla kind. Anthocyanins have ionization signals in positive ion mode and the cleavage in high dimention CID often occurs at C-O bond between aglycone and glucoside.Flavonoids were also isolated and identified by the advanced LC-MS method. A total of 7 flavonoids compounds were comfirmed as follows. Apigenin 6,8-glycopyranoside, Scutellarein 7-O-diglucuronide, Luteolin 7-O-diglucuronide, Apigenin 7-0-diglucuronide, Luteolin 7-O-glucoside, Scutellarin, Apigenin 7-O-glucuronide. Scutellarin was the most abundant flavonoids in Perilla while Apigenin 6,8-glycopyranoside was identified for the first time in Perilla kind. In positive ion mode flavonoids have similar cleavage patterns as anthocyanins, while in negative ion mode flavonoids tend to generate cross-ring cleavage.In order to determine the anticancer ability of Perilla anthocyanins, cell cytotoxicity experiments, cell morphological observation and apoptosis induction experiments were conducted. The results showed Perilla anthocyanins can inhibit the proliferation of Hela cell with an IC50 (12h)=229.3 μg·ml-1 and typical apoptotic cell morphological changes were observed in anthocyanin treated cells. In addition, the Annexin-V FITC/PI flow cytometric assay suggested that Perilla anthocyanins can induce Hela cell apoptosis by a dose dependent manner.