The Alkaloid-Enriched Fraction of Pachysandra terminalis (Buxaceae) Shows Prominent Activity against Trypanosoma brucei rhodesiense

Abstract

In the course of our studies on antiprotozoal natural products and following our recent discovery that certain aminosteroids and aminocycloartanoid compounds from Holarrhena africana A. DC. (Apocynaceae) and Buxus sempervirens L. (Buxaceae), respectively, are strong and selective antitrypanosomal agents, we have extended these studies to another plant, related to the latter—namely, Pachysandra terminalis Sieb. and Zucc. (Buxaceae). This species is known to contain aminosteroids similar to those of Holarrhena and structurally related to the aminocycloartanoids of Buxus. The dicholoromethane extract obtained from aerial parts of P. terminalis and, in particular, its alkaloid fraction obtained by acid–base partitioning showed prominent activity against Trypanosoma brucei rhodesiense (Tbr). Activity-guided fractionation along with extended UHPLC-(+)ESI QTOF MS analyses coupled with partial least squares (PLS) regression modelling relating the analytical profiles of various fractions with their bioactivity against Tbr highlighted eighteen constituents likely responsible for the antitrypanosomal activity. Detailed analysis of their (+)ESI mass spectral fragmentation allowed identification of four known constituents of P. terminalis as well as structural characterization of ten further amino-/amidosteroids not previously reported from this plant.

Antiprotozoal Nor-Triterpene Alkaloids from Buxus sempervirens L.

“…Moreover, cyclomicrophyllidine-A (6) was primarily obtained from the leaves of B. sempervirens L. Several of the isolated compounds displayed promising and selective in vitro activity against the protozoan parasites Pf and Tbr. Of the 25 structures isolated, five compounds (3,4,(6)(7)(8) showed auspicious antiplasmodial activity with IC 50 values of <1.0 µM. Against the causative agent of HAT, compound 2, 6, 8, and 24 exhibited interesting IC 50 values in the range of 1.1-1.5 µM.…”

“…In a recent study [8], it was determined that the alkaloid-enriched fraction of Pachysandra terminalis (Buxaceae) possessed promising activity against another protozoan parasite, Trypanosoma brucei rhodesiense (Tbr), the causative agent of East African sleeping sickness, a poverty-related neglected tropical disease. This plant contains aminosteroids structurally related to the nor-triterpene alkaloids of B. sempervirens L. Furthermore, an isolated compound of B. sempervirens L., cyclovirobuxeine-B, was found to be highly active and selective against Tbr in vitro [9].…”

Identification of Antiprotozoal Compounds from Buxus sempervirens L. by PLS-Prediction

“…The +ESI-QqTOF MS/MS spectra corresponding to the highlighted variables were examined in particular for the occurrence of prominent core fragments, which initially provide information about the substitution present and the occurrence of double bonds in the structures of the compounds. In previous studies, characteristic core fragments of Buxaceae alkaloids have already been identified for the amino-/amidosteroids in Pachysandra [8] and Sarcococca [13]. For the nor-triterpene alka-loids of the nor-cycloartane type, occurring in the genus Buxus, a few prominent fragments were also discovered, which can be used for structural assignment [14].…”

“…Correlations between analytical fingerprint data of complex mixtures, such as extracts and fractions and their bioactivity data can be investigated by PLS regression and the resulting models used to identify constituents mainly responsible for the biological effect under study [8][9][10]. In order to apply such an approach to the present set of fractions, they were all analyzed by LC/MS (Figure S1, Supplementary Materials) and the fingerprint data were processed using the software DataAnalysis 4.1 and ProfileAnalysis 2.1 (Bruker Daltonik GmbH, Bremen, Germany).…”

“…Partial least squares regression (PLS) was used to correlate the complex UHPLC/+ESI-QqTOF-MS/MS (henceforth termed "LC/MS") data of 20 different alkaloid fractions from a B. sempervirens L. leaf extract with their antiprotozoal bioactivity data. PLS regression has already been used in previous work by our group as well as others to make accurate predictions of constituents with antitrypanosomal [8][9][10] or antiplasmodial [11] activity, respectively, in plant extracts and fractions thereof. In this work, we present PLS models that highlight, besides those already isolated, further alkaloids of B. sempervirens L. with high predicted activity against Pf and Tbr.…”