Alkaloid subfractions of Buxus sempervirens L. leaves with strong in vitro activity against Trypanosoma brucei rhodesiense

Buxus sempervirens L. is very rich in triterpene-alkaloids of different types. The 9β-19-cyclo-5α-pregnanes and (9-(10→19))abeo-5α-pregnanes with variable substitution patterns are described in literature as characteristic for B. sempervirens L. [1, 2]. In a previous study we reported on the antiplasmodial activity of a CH₂Cl₂ extract from the leaves and isolated as the main responsible alkaloid O-tigloylcyclovirobuxeine B [3]. In the course of this investigation, we also found specific antitrypanosomal activity in some subfractions obtained by ESI-MS/MS guided 'spiral coil-countercurrent chromatography' [3]. Their antitrypanosomal IC₅₀ values of the tested fractions, along with their cytotoxicity and selectivity indices, are reported in Table 1. Characterization of the complex pattern of triterpene-alkaloids in the fractions with a high antitrypanosomal activity and selectivity was performed by UHPLC/+ESI-QTOF MS analyses. The aim of this study is to identify the compounds with a selective antitrypanosomal activity. Especially subfraction E9 with an IC₅₀ of 0.18 µg/mL and a good selectivity index of 26.2 is remarkable in comparison with the neighboring fractions 146 and E53, from which it differs conspicuously by the occurrence of compounds with elemental formulas of C₂₄H₃₅N (337 m/z), C₂₆H₄₄N₂ (384 m/z), C₂₅H₄₁NO (371 m/z) and C₃₅H₄₈N₂O₃ (544 m/z). Isolation, identification and testing of these alkaloids with hitherto unknown antitrypanosomal activity is in progress.

Tab. 1: In vitro activity of main alkaloid fractions from B. sempervirens leaves against T. b. rhodesiense and cytotoxicity data against L6 rat skeletal myoblasts. Data represent represent IC₅₀ values in µg/mL Melarsoprol and Podophyllotoxin are positive controls.

	Trypanosoma brucei rhodesiense (STIB 900 strain, bloodstream trypomastigotes)	Cytotoxicity: rat skeletal myoblasts L6	Selectivity Index (SI)
Podophyllotoxin Melarsoprol	0.002	0.01
Fraction 62	1.29	10.75	8.33
Fraction 68	1.61	6.98	4.34
Fraction 82	1.28	7.47	5.83
Fraction98	0.252	2.65	io.5o
Fraction 116	0.197	2.53	12.84
Fraction 130	0.206	3.68	17.86
Fraction 142	0.216	3.73	17.25
Fraction 146	0.603	4.24	7.02
Fraction E9	0.183	4.80	26.20
Fraction E53	0.7	14.00	20.00
Fraction E73	0.485	12.90	26.60
Fraction E97	2.06	37.73	18.32
Fraction E105	2.32	55.50	23.92

Acknowledgements: This work is part of the activities of ResNetNPND: http://www.uni-muenster.de/ResNetNPND/

Keywords: Buxus sempervirens L., triterpene-alkaloid, antitrypanosomal activity, Trypanosoma brucei rhodesiense

References:

[1] Tomko J, Voticky Z. III. Structures and Chemical and Physicochemical Properties of Buxus alkaloids. In Manske RHF, editor. The Alkaloids. New York: Academic Press; 1973: 14, 32 – 82.

[2] Ata A, Andersh BJ. Steroidal Bases from B. sempervirens. In Cordell GA, Editor. The Alkaloids. Amsterdam: Elsevier; 2008: 66, 199 – 201.

[3] Althaus JB. Antiprotozoal Activity of Buxus sempervirens and Activity-Guided Isolation of O-tigloylcyclovirobuxeine-B as the Main Constituent Active against Plasmodium falciparum. Molecules, online publication 15 May 2014; doi:10.3390/molecules19056184