Uncovering the poisonous aconitine containing plants in homemade herbal liquor using a convergent approach

Abstract

Human exposure to toxic plants is a global concern, with numerous reported cases of accidental poisoning. In this study, a patient experienced poisoning after inadvertently consuming an herbal preparation preserved in alcohol. The patient exhibited characteristic electrocardiogram abnormalities, prompting further investigation into the toxic plant responsible. A relative provided the suspected herbal sample for identification. Symptomatic treatment was administered, successfully stabilizing the patient. Given the suspicion of aconitine toxicity-despite the absence of naturally occurring Aconitum species in Thailand-a multi-approach methodology was employed to identify the plant source. Macroscopic and microscopic analyses were performed to characterize the morphological and histological features of the specimens. Organoleptic assessment revealed blackish-brown, shrunken surfaces with longitudinal wrinkles and a pale alcoholic-like odor. Microscopic examination identified three major structural layers: metaderm and cortex, vascular tissues, and a parenchyma-rich central pith, consistent with Aconitum storage roots. Chemical identification using thin-layer chromatography (TLC) compared the patient samples (SX1 and SX2) with standard aconitine and Aconitum crude drugs (AC1-AC5). The TLC chromatograms confirmed the presence of aconitine in SX1 and SX2, as evidenced by matching Rf values and characteristic color reactions. Further molecular analysis utilizing DNA barcoding targeted the trnH-psbA region to determine the genetic origin of the specimens. PCR successfully amplified DNA from SX1, SX2, and Aconitum reference samples, generating approximately 150 bp amplicons. BLAST analysis revealed a 99.07% sequence identity with Aconitum species, and phylogenetic analysis clustered the patient specimens with authenticated Aconitum species. Given that Aconitum, Delphinium, and Consolida species are not naturally distributed in Thailand, this case highlights the risks associated with imported medicinal plants containing aconitine. The integration of macroscopic, microscopic, chemical, and molecular techniques provided definitive identification of the toxic plant material. These findings underscore the importance of public health initiatives to raise awareness of aconitine poisoning and the need for regulatory measures to ensure the safe use of medicinal plants.

Keywords: Aconitine; DNA barcode; Identification; Monkshood; Toxicity.

Fig. 1

The electrocardiogram (ECG) upon arrival at the emergency department demonstrated wide complex tachycardia consistent with bidirectional ventricular tachycardia (bidirectional VT), characterized by alternating left bundle branch block (LBBB) and right bundle branch block (RBBB) patterns.

Fig. 2

Microscopic characters of the unknown specimens from the patient (SX1 and SX2).

Fig. 3

The TLC chromatogram of the samples from the patient (SX1 and SX2) and commercial Aconitum crude drugs (AC1–AC5) was compared to the aconitine standard (STD). The mobile phase used consisted of hexane, ethyl acetate, and methanol in a ratio of 6:5:1. The results were examined under UV light at a wavelength of 365 nm.

Fig. 4

A neighbor-joining (NJ) tree was constructed based on the trnH-psbA region, incorporating samples from the patient (SX1 and SX2) and commercial Aconitum crude drugs (AC1–AC5) (represented in purple), alongside nucleotide sequences from the GenBank database. These sequences included species containing aconitine, such as Aconitum (represented in red), Consolida (orange), and Delphinium (blue) from the Ranunculaceae family, as well as Spiraea (green) from the Rosaceae family. The NJ tree was generated using MEGA software version 11.0.10, applying the Kimura-2 Parameter (K2P) model of evolution with 10,000 bootstrap replications to ensure reliability.

Fig. 5

Suspected plant specimens obtained from patient: SX1 and SX2 (A) and the reference Aconitum samples (B–F) (B) Root tubers of Aconitum carmichaelii Debeax from Medicinal plant garden, Kanazawa University, Japan (AC1), (C) Unprocessed Aconitum crude drugs from Sichuan, China (AC2), (D) Unprocessed Aconitum crude drugs from Bangkok, Thailand (AC3), (E) Processed Aconitum crude drugs from Sichuan, China (AC4) and (F) Processed Aconitum crude drugs from Sichuan, China (AC5).