Immunomodulatory Constituents of Conocephalum conicum (Snake Liverwort) and the Relationship of Isolepidozenes to Germacranes and Humulanes

Abstract

Structural elucidation of three new sesquiterpenoids, namely, (1Z,4E)-lepidoza-1(10),4-dien-14-ol (1), rel-(1(10) Z,4S,5E,7R)-germacra-1(10),6 diene-11,14-diol (2), and rel-(1(10) Z,4S,5E,7R)-humula-1(10),5-diene-7,14-diol (3), isolated from the liverwort Conocephalum conicum, was accomplished by a combination of extensive NMR experiments, 1 H NMR simulation, and other means. Additionally, the change of the identity of bicyclogermacren-14-al, previously reported as a C. conicum constituent, to isolepidozen-14-al is proposed. Compounds 2 and 3 appear to be related to 1 via hydration involving a shared intermediate, a substituted cyclopropylmethyl cation, formed by a highly regio-and stereoselective protonation of 1, followed by a stereospecific fission of the three-membered ring. In other words, an isolepidozene derivative might be a branchpoint to humulanes and germacranes; this transformation could be of, up to now, unknown, biosynthetic and/or synthetic relevance. Multivariate statistical analysis of the compositional data of C. conicum extract constituents was used to probe the hypothesized biochemical relations. The immunomodulatory effect of 1−3 and conocephalenol (4) was evaluated in an in vitro model on both nonstimulated and mitogen-stimulated rat splenocytes. The compounds displayed varying degrees of cytotoxicity to nonstimulated splenocytes, whereas 2 and 3 were found to exert immunosuppressive effects on concanavalin A-stimulated splenocytes while not being cytotoxic at the same concentrations.

Revisiting the Effect of 3 Sesquiterpenoids From Conocephalum conicum (Snake Liverwort) on Rat Spleen Lymphocyte Viability and Membrane Functioning

“…10 Also, these compounds were found to inhibit cell proliferation in the presence of T and B cell mitogens. 10 In the mentioned study, we employed the MTT assay, which is considered to be one of the most sensitive methods for determining cell viability since it relies on cell mitochondrial enzymes to perform the reduction of MTT to blue formazan crystals. 10 In the present study, we found that lower concentrations of the tested compounds only slightly affected the cell's ability to metabolize MTT (Figure 2).…”

“…Our previous study revealed that the investigated compounds 1 , 2 , and 3 expressed cytotoxicity towards rat splenocytes only in either high concentrations (100 μM) or after prolonged incubation (48 h). 10 Also, these compounds were found to inhibit cell proliferation in the presence of T and B cell mitogens. 10 In the mentioned study, we employed the MTT assay, which is considered to be one of the most sensitive methods for determining cell viability since it relies on cell mitochondrial enzymes to perform the reduction of MTT to blue formazan crystals.…”

“…10 In the mentioned study, we employed the MTT assay, which is considered to be one of the most sensitive methods for determining cell viability since it relies on cell mitochondrial enzymes to perform the reduction of MTT to blue formazan crystals. 10 In the present study, we found that lower concentrations of the tested compounds only slightly affected the cell's ability to metabolize MTT (Figure 2). These findings indicate that in the tested concentrations compounds 1 , 2 , and 3 are not cytotoxic.…”

“…The tested compounds were isolated in a previous study. 10 Their identity and purity were checked by 1 H NMR and GC-MS, as described before, 10 prior to the performed in vitro assays. 10…”

“…Recently, we isolated 3 new sesquiterpenoids, compounds 1 - 3 (Figure 1), from Conocephalum conicum (L.) Dum., Conocephalaceae, (Snake liverwort) and in vitro screened them for immunomodulatory activities. 10 The sesquiterpenoids were shown to be cytotoxic to non-stimulated rat splenocytes to a certain extent, while 2 and 3 were immunosuppressive toward ConA-stimulated splenocytes, while not exerting cytotoxicity at the same concentrations. As these activities were all determined using the MTT assay, revealing the reducing capability of live cells, usually connected with the functioning of mitochondria, they may be subject to interpretation that is other than altered cell viability.…”