Safety evaluation of diester diterpenoid alkaloids and calcium oxalate needle crystals in Huafeng Dan Yaomu and the detoxification mechanism of fermentation.

ETHNOPHARMACOLOGICAL RELEVANCE: Huafeng Dan is a traditional Chinese medicinal formula with over 370 years of clinical use and is listed as a National Intangible Cultural Heritage. Its core component, "Yaomu" (medicinal precursor), is prepared through a distinctive fermentation process from four toxic raw herbs recorded in the Chinese Pharmacopoeia, namely Aconitum carmichaelii, Pinellia ternata, Arisaema erubescens, and Typhonium giganteum. The safety of this unconventional manufacturing paradigm featuring fermentation of raw herbs remains scientifically uncharacterized, posing a critical barrier to its modernization.

AIM OF THE STUDY: To systematically evaluate the safety-relevant properties of Huafeng Dan Yaomu through integrated exposure assessment, quantitative tracking of toxicity components during fermentation, and in-vivo toxicological verification, and to explore putative detoxification mechanism of the fermentation process.

MATERIALS AND METHODS: Daily human exposure to each toxic raw herb via Yaomu was calculated based on the national drug standard and compared with Pharmacopoeia recommended doses for processed products. This comparison serves only as a reference metric; therapeutic doses for processed herbs are not toxicological safety thresholds, and raw-herb exposure cannot be directly equated to the safety of orally administered processed herbal products. HPLC methods were employed to quantify diester diterpenoid alkaloids (DDAs) and their hydrolysis products in raw Aconiti Radix and Yaomu at different fermentation stages. A validated HPLC method was established to determine acid-releasable oxalate in raw herbs, processed products, and Yaomu. Acute toxicity (200-fold the clinical human dose) and 8-week subchronic toxicity (1.6-, 3.2-, and 4.9-fold rat clinical equivalent dose based on body-surface-area scaling) studies were conducted in mice and rats, respectively.

RESULTS: Daily exposure to each of the four toxic raw herbs via Huafeng Dan was only 0.039 to 0.073 g, accounting for 0.43 % to 4.87 % of the Pharmacopoeia recommended daily doses for processed products. Fermentation resulted in near-complete hydrolysis of hypaconitine and aconitine in all 22 Yaomu batches, with only trace mesaconitine detected in partial batches; mesaconitine was detectable but not quantifiable (LOQ = 0.0010 %) in most positive samples and only one one-month-fermentation sample exceeded LOQ. The total DDAs were far below the limit for processed Aconiti Radix (0.040 %). HPLC chromatographic peaks with matching retention times to benzoyl-monoester alkaloid reference standards were observed; identification was based solely on retention-time matching without definitive structural confirmation. Acid-releasable oxalate contents in Yaomu (0.60 %-1.01 %) were comparable to those in processed A. erubescens and T. giganteum, with no significant reduction during fermentation. No mortality or treatment-related adverse findings were observed in the acute limit-test at 200-fold clinical dose, nor in the 8-week subchronic study up to 4.9-fold rat clinical equivalent dose using finished Huafeng Dan.

CONCLUSIONS: Inferred safety-relevant properties of Yaomu are underpinned by a dual mechanism. Extremely low daily exposure constitutes the primary safety barrier, while fermentation-mediated near-complete hydrolysis of DDAs is hypothesised to generate low-toxicity benzoyl-monoester alkaloids and provides a secondary qualitative safety enhancement, though direct confirmatory experimental evidence remains absent. Despite stable acid-releasable oxalate levels, their minimal daily intake suggests minimal irritancy risk. This integrated evaluation framework offers a transferable paradigm for safety assessment of fermented traditional medicines containing toxic ingredients.