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Identifying plant, fungal, and animal ingredients in a specific mixture remains challenging during the limitation of PCR amplification and low specificity of traditional methods. Genomic DNA was extracted from mock and pharmaceutical samples. Four type of DNA barcodes were generated from shotgun sequencing dataset with the help of a local bioinformatic pipeline. Taxa of each barcode was assigned by blast to TCM-BOL, BOLD, and GenBank. Traditional methods including microscopy, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC) were carried out according to Chinese pharmacopoeia. On average, 6.8 Gb shotgun reads were sequenced from genomic DNA of each sample. Then, 97, 11, 10, 14, and one operational taxonomic unit (OTU) were generated for ITS2, psbA-trnH, rbcL, matK, and COI, respectively. All the labeled ingredients including eight plant, one fungal, and one animal species were successfully detected in both the mock and pharmaceutical samples, in which Chebulae Fructus, Poria, and Fritilariae Thunbergia Bulbus were identified via mapping reads to organelle genomes. In addition, four unlabeled plant species were detected from pharmaceutical samples, while 30 genera of fungi, such as Schwanniomyces, Diaporthe, Fusarium were detected from mock and pharmaceutical samples. Furthermore, the microscopic, TLC, and HPLC analysis were all in accordance with the standards stipulated by Chinese Pharmacopoeia. This study indicated that shotgun metabarcoding could simultaneously identified plant, fungal, and animal ingredients in herbal products, which has the ability to serve as a valuable complement to traditional methods.

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. 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. 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. 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. Furthermore, four commercial FKDSW samples were collected to verify the practicality of the shotgun metabarcoding approach. Then, a total of 52.16 Gb raw data for 174 million paired-end reads was generated using the Illumina NovaSeq sequencing platform. Meanwhile, 228, 23, and 14 operational taxonomic units (OTUs) were obtained for the ITS2, matK, and rbcL regions, respectively, after bioinformatic analysis. 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. and Bupleurum chinense DC. with 1–6 different bases. 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. or Bupleurum scorzonerifolium Willd.) was found in all the commercial samples, namely Bupleurum falcatum L. Here, 25 weed species representing 16 genera of ten families were detected. Moreover, 26 fungal genera belonging to 17 families were found in both lab-made and commercial FKDSW samples. This study demonstrated that the shotgun metabarcoding approach could overcome the biased PCR amplification and authenticate the biological ingredients of traditional herbal patent medicine with a distinct variance in the dosage of the herbal materials. Therefore, this provides an appropriate evaluation method for improving the safety and efficacy of traditional herbal patent medicine.

As one of the high-incidence diseases in the world, pharyngitis seriously affects the lives of those with the condition. Qingguo Wan is a herbal medicine used for treating pharyngitis, and its quality evaluation is currently only accomplished via traditional identification. However, precise identification becomes challenging with fake products on the market or fungal contamination during the production process. This study used the Illumina NovaSeq platform for targeting the ITS2, psbA-trnH, matK, and rbcL sequences to survey the species composition of lab-made and commercial samples. The results showed that a total of 34.56 Gb of raw data that was obtained represented more than 0.23 billion reads. After assembly, annotation, and operational taxonomic unit clustering, 103, 12, 10, and 12 OTUs were obtained, which belonged to the ITS2, psbA-trnH, matK, and rbcL sequences of the mock lab-made and commercial samples. The analytical results indicated that the sequences of all the prescription ingredients were successfully obtained in the two lab-made samples. The positive control medicinal Panax quinquefolius L. sequence was obtained in HSZY175, while Scutellaria baicalensis Georgi, Lonicera japonica Thunb. Menispermum dauricum DC. and Paeonia lactiflora Pall. were detected in the three commercial samples. The detection results of the other four herbs in different fragments were not all the same. In addition, a total of 28 fungi OTUs, representing 19 families and 20 genera, were obtained from both the commercial and mock lab-made samples. Aspergillus, Cladosporium, and Penicillium dominated among the 20 genera. This study demonstrated that the shotgun metabarcoding method is a powerful tool for the molecular identification of the biological ingredients in Qingguo Wan. It can be used to effectively supplement traditional methods while providing a new technique for the quality evaluation of Qingguo Wan.

Traditional herbal patent medicine typically consists of multiple ingredients, making it challenging to supervise contamination by impurities and the improper use of raw materials. This study employed shotgun metabarcoding for the species identification of biological ingredients in traditional herbal patent medicine, Wuhu San. The five prescribed herbal materials found in Wuhu San were collected, and their reference sequences were obtained by traditional DNA barcoding using Sanger sequencing. Two lab-made and three commercial Wuhu San samples were collected, and a total of 37.14 Gb of shotgun sequencing data was obtained for these five samples using the Illumina sequencing platform. A total of 1,421,013 paired-end reads were enriched for the Internal Transcribed Spacer 2 (ITS2), psbA and trnH intergenic spacer region (psbA-trnH), maturase k (matK), and ribulose-1, 5-bisphosphate carboxylase (rbcL) regions. Furthermore, 80, 11, 9, and 8 operational taxonomic units were obtained for the ITS2, psbA-trnH, matK, and rbcL regions, respectively, after metagenomic assembly, annotation, and chimeric detection. In the two lab-made mock samples, all labeled ingredients in the Wuhu San prescription were successfully detected, and the positive control, Panax quinquefolius L., was detected in the HSZY172 mock sample. Three species, namely Angelica sinensis (Oliv.) Diels, Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., and Carthamus tinctorius L., belonging to three labeled ingredients, Angelicae Sinensis Radix (Danggui), Saposhnikoviae Radix (Fangfeng), and Carthami Flos (Honghua), were detected in the three commercial samples. Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav., the original Angelicae Dahuricae Radix (Baizhi) species, was only detected in WHS003. Arisaema erubescens (Wall.) Schott, Arisaema heterophyllum Blume, or Arisaema amurense Maxim., the original Arisaematis Rhizoma (Tiannanxing) species, were not detected in any of the commercial samples, which could be attributed to the fact that this medicinal material underwent extensive processing. In addition, the Saposhnikovia divaricata adulterant was detected in all the commercial samples, while 24 fungal genera, including Aspergillus, were identified in both the lab-made and commercial samples. This study showed that shotgun metabarcoding provided alternative strategy and technical means for identifying prescribed ingredients in traditional herbal patent medicine and displayed the potential to effectively complement traditional methods.