Antileishmanial diterpenoid alkaloids from Aconitum spicatum (Bruhl) Stapf

Abstract The crude extracts of tubers of Aconitum spicatum (Bruhl) Stapf were investigated for in vitro antileishmanial activity against Leishmania major. The dichloromethane extract at pH 2.5 showed antileishmanial activity with IC50 value of 27.10 ± 0.0 μg/mL. Chromatographic purification of the dichloromethane extract led to isolation of three C-19 norditerpenoid alkaloids indaconitine (1), chasmaconitine (2) and ludaconitine (3). Compounds 3 and 2 showed antileishmanial activity with IC50 = 36.10 ± 3.4 and 56.30 ± 2.1 μg/mL, respectively. Compound 1 was less effective (IC50 > 100 μg/mL). The cytotoxicity of compounds 1, 2 and 3 studied against MCF7, HeLa and PC3 cancer cell lines and 3T3 normal fibroblast cell line did not show cytotoxicity at 30 μM. Graphical abstract


Introduction
Leishmaniasis, a complex of parasitic tropical diseases caused by Leishmania species, is responsible for a growing public health problem worldwide. Based on World Health Organization epidemiological report, more than 350 million are at risk of leishmaniasis with 12 million ABSTRACT The crude extracts of tubers of Aconitum spicatum (Bruhl) Stapf were investigated for in vitro antileishmanial activity against Leishmania major. The dichloromethane extract at pH 2.5 showed antileishmanial activity with IC 50 value of 27.10 ± 0.0 μg/mL. Chromatographic purification of the dichloromethane extract led to isolation of three C-19 norditerpenoid alkaloids indaconitine (1), chasmaconitine (2) and ludaconitine (3). Compounds 3 and 2 showed antileishmanial activity with IC 50 = 36.10 ± 3.4 and 56.30 ± 2.1 μg/mL, respectively. Compound 1 was less effective (IC 50 > 100 μg/mL). The cytotoxicity of compounds 1, 2 and 3 studied against MCF7, HeLa and PC3 cancer cell lines and 3T3 normal fibroblast cell line did not show cytotoxicity at 30 μM. people affected (WHO 2010). Medications currently used in treatment of leishmaniasis are mainly associated with limitations such as serious side effects and high expenditure. In recent years, an intense search for antileishmanial compounds obtained from natural sources has led to the identification of several classes of active plant metabolites (Mishra et al. 2011). The diterpenoid alkaloids isolated from Aconitum, Delphinium and Consolida species have shown antileishmanial activity (González et al. 2005).
Aconitum spicatum (Bruhl) Stapf is a poisonous plant species belonging to family Ranunculaceae (Ghimire et al. 2008). Because of its toxicity, tubers of A. spicatum are used in the traditional medicine systems like Ayurveda and Traditional Chinese Medicines only after proper processing (Singhuber et al. 2009). A. spicatum is rich in C-19 norditerpenoid alkaloids (Gao et al. 2005). Recently, bioactivities of some new diterpenoid alkaloids have been reported from genus Aconitum (Yuan & Wang 2012;Guo et al. 2014;Yin et al. 2014). The structure activity relationship of diterpenoid alkaloids of Aconitum were carried out in order to design drugs for analgesic, anti-inflammatory, anti-epileptiform, arrhythmogenic and anti-arrhythmic activity (Friese et al. 1997;Ameri 1998;Yunusov 2011).
The dichloromethane extract at pH 2.5 had displayed more antileishmanial activity as compared to the norditerpenoid alkaloids 1-3 isolated from the same extract (Figure 1). From comparative structure-activity relationships, interestingly, hydroxyl group at C-8 position in compound 3 showed better antileishmanial activity with IC 50 = 36.10 ± 3.4 μg/mL than compounds 1 (IC 50 > 100 μg/mL) and 2 (IC 50 = 56.30 ± 2.1 μg/mL) consisting acetyl group at C-8. On the other hand, comparing the hydroxyl group at C-3 position, compound 2 showed less activity while compound 1 was not active (IC 50 > 100 μg/mL). It may be suggested that some other compounds present in the extract could be playing an important role in antileishmanial activity. Furthermore, ursolic acid (7), which has been reported for antileishmanial activity, was isolated from the same extract (Peixoto et al. 2011).
The crude methanolic extract showed Brine shrimp cytotoxicity (LD 50 = 86.30 μg/mL). However; n-hexane, dichloromethane, ethyl acetate and n-butanol extracts did not show cytotoxicity at 1000 μg/mL. The cytotoxicity of compounds 1-3 against MCF7, HeLa and PC3 cancer cell lines and 3T3 normal fibroblast cell line did not show cytotoxicity at 30 μM. Selectivity index was considered of interest when higher than 3 (Béziyin et al. 2003). Selectivity index (36.9) for the dichloromethane extract at pH 2.5 was found well as antileismanial activity and cytotoxicity testing on brine shrimp.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The work was supported by the World Academy of Sciences for Developing World (TWAS) [grant number 11-010-RG/CHe/AS_G UNeSCO FR: 3240262700]