Aconitum heterophyllum is a traditionally important medicinal plant having numerous therapeutic actions as documented in Ayurveda. This plant has been used alone as well as in combination with other plants for the preparation of different anti-malarial formulations. However, there is no report on the assessment of its anti-plasmodial activity, and the compound responsible for this activity. The main aim of this study was to conduct phytochemical investigation of A. heterophyllum roots for the preparation of extract, fractions and isolation of pure molecules to identify active fractions/molecules responsible for the anti-plasmodial activity, and development of UHPLC-DAD based analytical method which can be used for the quantification of marker compounds in the extracts and fractions. Hydro-alcoholic extract (1:1 v/v) and fractions (n-hexane, chloroform, ethyl acetate, n-butanol and water) were prepared from the dried powdered roots of A. heterophyllum. Fractions were further subjected to silica gel-based column chromatography to isolate pure specialized secondary metabolites from this plant. All extracts, fractions and pure molecules were evaluated against the chloroquine resistant Pf INDO and chloroquine sensitive Pf3D7 strains in culture for calculating their IC50 values. UHPLC-DAD based analytical method was also developed for the first time for the quantification and quality assessment of this commercially important Himalayan medicinal plant. Phytochemical investigation of A. heterophyllum root led to the isolation of six specialized metabolites named as 2-O-cinnamoyl hetisine (1), atisinium (2), 4-oxabicyclo [3.2.2] nona-1(7),5,8-triene (3), atisinium cinnamate (4), aconitic acid (5), and atisinium formate (6). Compound 1 is a new hetisine type diterpenoid alkaloid, compounds 4 and 6 are new counter ionic forms observed with atisinium ion, and compound 3 is being reported for the first time from this genus. Chloroform fraction was found to be the most active with IC50 (µg/mL) 1.01 (Pf INDO) and 1.32 (Pf3D7). The isolated molecule 2-O-cinnamoyl hetisine (1), a new diterpenoid alkaloid isolated from chloroform fraction, showed promising antiplasmodial activities with IC50 (µM) 1.92 (Pf INDO) and 10.8 (Pf 3D7). Activity of chloroform fraction was further validated by the developed UHPLC-DAD based method as the quantity of 2-O-cinnamoyl hetisine (1) was higher in the chloroform fraction (≅200 µg/mL) than in all other fractions (< 7µg/mL). Atisinium (2) and 2-O-cinnamoyl hetisine (1) were found to be the main marker compounds of this plant based on quantity and antiplasmodial activity, respectively. This study provides the scientific rational for the traditional use of this plant in treating malaria. Further, this study revealed that anti-malarial potential of this plant might be due to the presence of diterpenoid alkaloids.
Antiplasmodial diterpenoid alkaloid from Aconitum heterophyllum: Isolation, characterization, and UHPLC-DAD based quantification
