Abstract
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.
Highlights
In recent years, traditional herbal medicine has been widely used to prevent and treat clinical diseases
They were first identified using the morphological method and authenticated using DNA barcoding to ensure the accuracy of the mock samples
The Internal Transcribed Spacer 2 (ITS2) and psbA-trnH sequences obtained via Sanger sequencing were assigned to species by blasting to the TCM–BOL system, while the maturase k (matK) and rbcL DNA barcodes obtained using the same method were assigned to a species or genus using the barcode of life data system (BOLD) system and GenBank NT database
Summary
Traditional herbal medicine has been widely used to prevent and treat clinical diseases. With the development of high-throughput sequencing (HTS) technology, shotgun metagenomics based on the genetic information of species has been successfully applied to identify ingredients in mixed samples. This technique involves the untargeted sequencing of all biological ingredient genomes present in a sample (Quince et al, 2017) to break the metagenomic DNA into small fragments, after which bioinformatics methods are used for assembly without the need for PCR amplification. If the information is used to analyze the traditional DNA barcode region, it is known as shotgun metabarcoding This was applied here for the species identification of the biological ingredients in traditional herbal patent medicine. Shotgun metabarcoding can be a powerful supplement to the conventional identification method used for traditional herbal patent medicine
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