A novel sesquiterpene acid and an alkaloid from leaves of the Eastern Nigerian mistletoe with potent immunostimulatory activity on C57BL6 splenocytes
In our continued efforts to identify the immunoactive constituents of a local mistletoe species in Eastern Nigeria, a novel sesquiterpenoidal acid, 2, 3-dimethoxy-benzo [a, b] cyclopentenyl-3', 3', 5'-trimethyl pyran-4-carboxylic acid (1), and a known alkaloid, lupinine (2)  were isolated from a bioactive chloroform fraction of the leave extract of the mistletoes parasitic on kola tree, Cola acuminata (P.Beauv.) Schott & Endl.. These compounds were screened for immunostimulatory activities on isolated C57BL/6 mice splenocytes at concentrations of 10, 25 and 100µg/ml. Their effects on the expression of CD69, an early immune cells activation marker , were determined using flow cytometry techniques and compared to Lipopolysaccharide (LPS; 10µg/ml) and Concanavalin A (ConA; 2µg/ml) as standards. The compounds (1 and 2) at a concentration of 25µg/ml showed statistically significantly (p<0.05) stimulatory activity on the isolated splenocytes compared to the non-stimulated control cells with values of 56.34±0.26% and 69.84±0.19% respectively compared to 7.58±0.42% recorded for the control. Similarly, the CD69 expression assay at the above dose showed that the compounds were stimulatory with statistically significant values (p<0.05) of 2.31±0.07% and 2.71±0.03% respectively compared to 1.69±0.05% recorded for the non-stimulated control. The compounds were characterized using a combination of UV/visible, IR, NMR (13C-NMR and 1H-NMR) and DEPT, MS and 2-dimensional correlation (H-H COSY, HSQC, HMBC, NOE, and NOESY) studies. These compounds may be responsible in part, for the immunostimulatory activities already established for the Eastern Nigerian mistletoes.
Acknowledgement: The author wish to thank Mr Alfred Ozioko of BDCP Nsukka for identifying the plant material and Associate Professor Akira Kawamura of CUNY for the detailed spectral studies.
References: 1. Michael JP (1998) Nat Prod Rep 16: 675–696. (2) Borrego F et al. (1999) Immunol 97: 159–165