Accumulation dynamics of alkaloids in Aconitum talassicum

Abstract

The medicinal plant Aconitum talassicum is indigenous to mountainous regions of Central Asia at heights of 2,5003,000 m in damp meadows, ancient moraines, river valleys, and in juniper thickets [1, 2]. The plant has been used since antiquity in folk medicine as treatment for rheumatism and malaria [3] and in veterinary practice to treat flesh wounds and ulcers [4, 5]. The principal stands are located in the Talas river valley (Kyrgyzstan). All previously studied plant specimens contained talatisamine as the principal alkaloid constituent [6–9]. The plant grows in Uzbekistan on slopes of the Turkestan (Kulsai river gorge) and Chatkal (near Pskem village, on Kamchik pass, Chadaksai river gorge) ridges. The alkaloid composition of plants growing in Uzbekistan has not been studied before. A derivative of practical medical interest was prepared from talatisamine. In order to obtain a raw material source for preparing the drug, we studied roots and the aerial part of A. talassicum collected during various vegetative periods on Kamchik pass (Tashkent Oblast) (Table 1). Air-dried raw material was moistened with Na2CO3 solution (5%) and extracted with CHCl3 in order to extract fully the alkaloids. The condensed CHCl3 extracts were worked up with aqueous H2SO4 (5%) until the reaction for alkaloids was negative. The resulting acidic solutions were washed with CHCl3, made basic with Na2CO3, and extracted exhaustively with CHCl3. The resulting CHCl3 extracts were evaporated to afford total alkaloids (Table 1). According to TLC, all studied samples contained talatisamine. Table 1 shows that the alkaloid content was greatest in the aerial parts of the plant in the early period. Keeping in mind that the aerial part of the plant is used as a source of talatisamine, we separated the total alkaloids obtained from the sample collected during the early period. Total alkaloids (3.55 g) were separated over a column of KSK silica gel (sorbent:compound ratio 50:1) with elution by CHCl3:MeOH with gradually increasing fraction of the latter. Fractions obtained using CHCl3 and CHCl3:MeOH (100:1) afforded talatisamine (0.81 g) upon work up with acetone and recrystallization from MeOH. It was identified by comparison of its TLC, mixed melting point with an authentic sample, and comparison of IR and PMR spectra. Elution by CHCl3:MeOH (50:1 and 25:1) isolated isotalatisidine (0.17 g). Fractions obtained upon elution by CHCl3:MeOH (1:1) contained talatisine (0.08 g). The mother liquor of these fractions was rechromatographed over a column of silica gel (sorbent:compound ratio 20:1) with elution by the aforementioned solvents to afford isotalatisidine (0.08 g) and talatisidine (0.03 g). Mother liquors obtained upon elution by CHCl3 were rechromatographed over a column of silica gel (sorbent:compound ratio 20:1) using CHCl3 and CHCl3:MeOH as eluents. Elution by CHCl3 isolated 14-O-acetyltalatisamine (0.04 g). Subsequent fractions eluted by CHCl3:MeOH (100:1, 50:1) afforded talatisamine (0.27 g). The talatisamine content was 0.18% of the dry plant weight. Talatisamine (0.12% of the dry plant weight) and isotalatisidine and talatisine were isolated using an analogous separation method from total alkaloids of the aerial part of the plant collected during flowering. Thus, the aerial part of A. talassicum in the early period can be used as a raw material source for talatisamine production.