Identification of biological raw materials in Chinese patent medicine Gejie Dingchuan Wan using high-throughput...

Gejie Dingchuan Wan (GDW), a classical prescription comprising fourteen ingredients, including twelve biological materials and two mineral components, is used to nourish yin, clear the lung, and relieve asthma and vulnerable to adulteration, yet a systematic molecular method for simultaneously authenticating all prescribed biological ingredients is lacking.

To optimize key technical steps, including DNA extraction and barcode selection, for mixed samples containing both animal- and plant-derived medicinal materials; and to establish an HTS-based method for identifying the raw medicinal materials in GDW.

Total DNA was extracted from three reference and nine commercial GDW samples. An integrated DNA barcoding strategy (16S rRNA for animal-derived materials and ITS2 for plant-derived materials) combined with morphological identification, and all sequences were deposited in GenBank to establish a dedicated reference database. OTUs were assigned taxonomically against custom reference databases. Diversity indices and statistical analyses were calculated.

41,918,610 high-quality reads were generated, yielding a total of 5103 operational taxonomic units (OTUs), including 2798 assigned to animal-derived materials and 2305 assigned to plant-derived materials. Assessment of potential technical biases showed that all samples had coverage values exceeding 0.9995, while the rarefaction, Shannon-Wiener, and species accumulation curves all reached plateaus, indicating that the sequencing depth was sufficient to capture most of the species diversity. All twelve prescribed biological components were detected in both reference and commercial samples. Gecko was the dominant component, accounting for 50.5%-98.07% relative abundance, while Ophiopogonis Radix was rarely detectable in most samples, with relative abundance ranging from 0.001% to 0.003%. In addition to the prescribed ingredients, HTS also detected sequences corresponding to potential adulterants and contaminants, highlighting the utility of this approach for adulteration surveillance. These non-prescribed taxa included Calotes wangi (a known adulterant of Gecko), Astragalus membranaceus, Bothus robinsi, and various fungal species, including Aspergillus and Penicillium. Notably, some of these taxa were detected not only in commercial samples but also in self-prepared reference samples formulated from raw materials that had been previously authenticated by Sanger sequencing. While 16S rRNA and ITS2 were effective for verifying the identity of individual raw materials, HTS revealed a broader spectrum of DNA sequences present in the finished products, including both prescribed and non-prescribed taxa.

The integrated barcoding-HTS pipeline provides a sensitive framework for molecular profiling of complex Chinese patent medicines and highlights the need for empirically defined interpretive criteria before detections of non-prescribed sequences can be translated into regulatory or safety conclusions.