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Here, 25 weed species representing 16 genera of ten families were detected. or Bupleurum scorzonerifolium Willd.) was found in all the commercial samples, namely Bupleurum falcatum L. The identification results showed that all six prescribed ingredients were successfully detected and that the non-authentic ingredient of Bupleuri Radix (Chaihu, Bupleurum chinense DC. Moreover, no differences were evident between the assembly sequences obtained via shotgun metabarcoding and their corresponding reference sequences of the same species obtained via Sanger sequencing, except for part of the ITS2 and matK assembly sequences of Paeonia lactiflora Pall., Saussurea costus (Falc.) Lipsch. Meanwhile, 228, 23, and 14 operational taxonomic units (OTUs) were obtained for the ITS2, matK, and rbcL regions, respectively, after bioinformatic analysis.

Then, a total of 52.16 Gb raw data for 174 million paired-end reads was generated using the Illumina NovaSeq sequencing platform. Furthermore, four commercial FKDSW samples were collected to verify the practicality of the shotgun metabarcoding approach. Six herbal materials were collected, and a lab-made mock FKDSW sample was produced to establish a method for the authentication assessment of biological ingredients in traditional herbal patent medicine based on shotgun metabarcoding. This study utilized the shotgun metabarcoding approach to analyze the biological ingredients of Fuke Desheng Wan (FKDSW), which is an effective traditional herbal product for the treatment of dysmenorrhea. However, it is difficult to supervise the authenticity of herbal materials in mixed herbal products according to the current quality standards, especially for traditional herbal patent medicine, with a distinct variance in the dosage of herbal materials. With the widespread use of traditional medicine around the world, the safety and efficacy of traditional herbal patent medicine have become an increasing concern to the public. 4Department of Pharmacy, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China.3Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.