Quali-quantitative determination of triterpenic acids of Ziziphus jujuba fruits and evaluation of their capability to interfere in macrophages activation inhibiting NO release and iNOS expression

Abstract

Ziziphus jujuba Mill fruits are recognized as an outstanding source of biologically active compounds including triterpenic acids. In order to evaluate the triterpenic profile of these edible and little commercial fruits and to contribute to the improvement of their potential value as food, a quali-quantitative study along with biological assays were carried out.The phytochemical investigation of the triterpenic fraction yielded nine triterpenic acids, ceanothic acid (1), alphitolic acid (2), maslinic acid (3), 3-O-cis-coumaroyl alphitolic acid (4), 3-O-trans-coumaroyl alphitolic acid (5), 3-O-trans-coumaroyl maslinic acid (6), 3-O-cis-coumaroyl maslinic acid (7), betulinic acid (8), and oleanolic acid (9). In order to perform the quantitative determination of compounds 1–9 in Z. jujuba fruits, an analytical method based on liquid chromatography coupled to mass spectrometry with ESI source and triple quadrupole analyzer (LC-ESI(QqQ)MS), using the very sensitive and selective mass tandem experiment called Multiple Reaction Monitoring (MRM), was developed and validated.Furthermore, the antiproliferative activity of compounds 1–9 was determined on three different cultured cell lines, namely J774A.1, MCF7 and A459 cells. For compounds 1, 2, 5 and 9, exhibiting no significant antiproliferative activity, the effects on nitrite production by LPS-stimulated J774.A1 cultured macrophages were evaluated (10–100μM), showing the ability of alphitolic acid (2), and 3-O-trans-coumaroyl alphitolic acid (5) to reduce significantly (P<0.001) and in a concentration related manner NO production. The capability of compounds 2 and 5 to influence the expression of the inducible nitric oxide synthase (iNOS) was also investigated, showing that both compounds reduced iNOS expression in a concentration related manner.