Alkaloids of Buxus colchica, B. sempervirens, and B. balearica growing in Georgia

Abstract

The introduced species Buxus sempervirens and B. balearica grow in Georgia in addition to the endemic B. colchica Pojark. We studied leaves and stems of these plants collected during flowering and fruiting near Tbilisi, in Batum Botanical Garden, and on Shaor Pass. Pharmacological tests of total preparations that were performed at the Kutateladze Institute of Pharmaceutical Chemistry showed that total alkaloids soluble in ether, CHCl3, and water exhibited spasmolytic activity. The strength of the activity of the aqueous extracts of the Buxus species could be placed in the following order [1]: B. balearica Lam > B. sempervirens > B. colchica Pojark. Plant material was extracted beforehand with EtOAc followed by extraction of alkaloids by diethylether and CHCl3 after making the raw material basic in order to obtain total alkaloids. Total alkaloids soluble in water were obtained as an extract from leaves and stems of B. colchica, B. sempervirens, and B. balearica according to requirements of the SP, XIth Ed. (1982, Vol. II, p. 160). Seasonal variations of alkaloids in vegetative organs were studied by obtaining total alkaloids in a Soxhlet apparatus using extraction by CHCl3 and basic raw material with subsequent acid–base purification and separation into ether and CHCl3 fractions. Total alkaloids soluble in ether were separated using citrate–phosphate buffers in pH steps 6.6, 6.0, 5.0, 4.0, and 3.0 with 0.1 M citric acid. Separation into pure bases was carried out over Al2O3 (activity II, III, VI, and neutral) and SiO2 (100/160 μm) columns. The ratio of compounds to sorbent was 1:100. Elution used ether, benzene:ether (1%), benzene:EtOH (1–5%), benzene:CHCl3, CHCl3:CH3OH with increasing MeOH content. Fractions of 25–50 mL were collected. The yield of total alkaloids was determined by acid–base titration calculated as molecular weight of buxamine; the buxamine content, by a chromatographic titration method using B. colchica and B. sempervirens. Qualitative analysis was performed in thin layers of SiO2 (Merck) on plates using MeOH:hexane:NH4OH (25%) (40:5:3). The yield of total alkaloids from B. balearica was determined gravimetrically [2]. Alkaloids were identified based on a determination and analysis of physicochemical (mp, [α]D) and spectral data (UV, IR, PMR, mass) in addition to comparison with the literature [1-9]. Reference samples for TLC mobility were authentic alkaloids. Table 1 shows the variations in alkaloid contents with development phases in the vegetative organs. B. balearica, yield of total alkaloids from leaves and stems during flowering, 6%. The fraction from pH 0.6; 6.5 of the ether total was separated over an Al2O3 column with elution by benzene:CHCl3 and benzene:EtOH (1→5%). Fractions from EtOH (72–126) afforded 1, mp 199–201°C (EtOH), [α]D –100.3° (CHCl3). Mass spectrum (m/z): 414 [M]+, 386, 371, 356, 343, 340, 84, 72 (100), 58. PMR spectrum (CDCl3, δ, ppm): 0.74 (3H, s, CH3), 0.88 (6H, s, 2 × CH3), 0.92 (6H, s, 2 × CH3), 2.2 [6H, s, N(CH3)2], 2.40 [6H, s, N(CH3)2]. The experimental results allowed us to identify the isolated compound as cycloprotobuxin-A [4, 5]. B. sempervirens, yield of total alkaloids, 2.2%. Amorphous base 2 was isolated from the fraction with pH 5.0 of the ether total alkaloids by separation over a column of Al2O3 with elution by benzene:ether (1%) from effluents (9–12).