Abstract
Ethnopharmacological relevanceAconitum heterophyllum Wall. ex Royle is a traditionally important medicinal plant having numerous therapeutic actions as documented in Ayurveda. This plant is traditionally known for combating worm infestation, fever, respiratory tract disease, vomiting, diarrhoea, diabetes, skin disorders, anaemia, and joint disorders. Further, it has been used alone and in combination with other plants to prepare various anti-malarial formulations. However, there is no report on the assessment of its anti-plasmodial activity, and the metabolite(s) responsible for this activity. Aim of the studyThe 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. Materials and methodsHydroalcoholic 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 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 of marker compounds and quality assessment of this commercially important Himalayan medicinal plant. ResultsPhytochemical investigation of A. heterophyllum root led to the isolation of six specialized metabolites viz. 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 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). The 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 mg/g) than in all other fractions (<7 mg/g). 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. ConclusionThis study provides the scientific rationale for the traditional use of this plant in treating malaria. Further, this study revealed that the anti-malarial potential of this plant might be due to the presence of diterpenoid alkaloids.
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