1. Introduction
Shenling Baizhu San (SLBZS), composed of ten herbal materials for the treatment of gastrointestinal diseases, was first recorded in theTaiping Huimin Heji Jufang in the Song Dynasty. This product is a blended powder of ten herbal materials (The national pharmacopoeia committee of China, 2020b), nine of them are plant ingredients including roots, fruits, and seeds and another one is the sclerotium of a fungus. Each ingredient needs to go through certain processing steps before use to achieve the purpose of purity, flavor, lower toxicity, and drying without deterioration. They are then mixed in certain proportion and crushed into a fine powder. Finally, it is dispensed in certain doses for people to use. Modern clinical and pharmacological studies have verified that SLBZS can regulate the composition of intestinal flora (Feng et al., 2020; Shi et al., 2019; Zhang et al., 2019), exert significant anti-acid capacity (Wu et al., 2010), and improve the symptoms of gastrointestinal diseases (Zhang et al., 2019). At the same time, it also showed certain anti-inflammatory effect (Yang et al., 2014b)to reduce inflammation in the lung, liver, and can reduce liver and lung damage (Feng et al., 2020; Yang et al., 2014a). Besides, it also exerted the functions to improve metabolism and immunity (Chen & Xue, 2014b). Each ingredient of SLBZS is thought to combine and work together to achieve the effects of nourishing the spleen and stomach, and profit the lungs based on the theory of traditional Chinese medicine (TCM). Therefore, ensuring the accuracy of the herbal materials is the first step to ensure the efficacy. Currently, in the 2020 edition of the Pharmacopoeia of the People’s Republic of China, the identification method of SLBZS contains the microscopic identification and thin layer chromatography (TLC). Under the microscopic identification for the item of SLBZS, only the microscopic characteristics of nine herbal materials other than Coicis Semen were described, and TLC was only for the detection of the characteristic components of Ginseng Radix et Rhizoma (the roots of Panax ginseng ) and Glycyrrhizae Radix et Rhizoma (the roots and rhizomes with or without the periderm of Glycyrrhiza uralensis , G. glabra, or G. inflata ). In addition, there are researches conducted to quantify the active components of the four ingredients of SLBZS, i.e. Ginseng Radix et Rhizoma, Poria (the sclerotium of Wolfiporia cocos ), Atractylodis Macrocephalae Rhizoma (the rhizomes with or without periderm of Atractylodes macrocephala ), Platycodonis Radix (the roots with or without periderm of Platycodon grandiflorus ), using Ultra Performance Liquid Chromatography (UPLC) to provide a basis for quality control of their prescriptions (Li et al., 2019). A more comprehensive method for monitoring the quality of all herbal ingredients is urgently to be developed.
High throughput sequencing (HTS) has been becoming a hot spot in the identification of biological ingredients of mixtures. A study by Vangay et al., published on the journal of Cell using HTS to test stool samples from Thai immigrants in the US and US-born European American individuals, revealed that the gut microbiome diversity and function of non-Western immigrants westernises after immigration to the US (Vangay et al., 2018). A study used four eDNA barcoding assays to detect the biological components (plants, vertebrates, and arthropods) in five common terrestrial substrates, demonstrated that the ability of HTS-based eDNA metabarcoding could be used as a powerful tool for terrestrial biomonitoring (Van Der Heyde et al., 2020). Another study applied the method of combining capture enrichment and HTS to test two freshwater macrozoobenthos mock communities with different biodiversity, and found that capture enrichment provides a more reliable and accurate representation of species occurrences and relative biomasses in comparison with polymerase chain reaction (PCR) enrichment for bulk DNA (Gauthier et al., 2020). At the same time, the HTS also realizes the identification of ingredients and the detection of pollutants in food. A recent study by Haiminen developed a bioinformatics pipeline that used HTS to detect several samples of high protein powders contained not only in chicken but also pork and beef that were not expected (Haiminen et al., 2019). Another study on the identification of food ingredients showed that HTS could detect the ingredients in sausages, as well as the presence of sequences of microorganisms, plants, and other animal species (Ripp et al., 2014). The successful application of HTS in the identification of mixed species also provides a new idea for the identification of biological ingredients of compound traditional herbal patent medicine. The DNA metabarcoding technique has been successfully used to identify the species of different dosage forms of traditional herbal patent medicine such as Yimu Wan (Jia et al., 2017), Jiuwei Qianghuo Wan (Xin et al., 2018b), and Ruyi Jinhuang San (Shi et al., 2018). Although it is feasible to use PCR-based methods such as DNA metabarcoding to identify traditional herbal patent medicine (Shi et al., 2018; Xin et al., 2018b), PCR bias may lead to false-negatives in certain species (Piñol et al., 2015), and universal primer design is also a challenge for such methods. Researchers used shotgun sequencing technology combined with DNA barcoding region to analyze the species composition of traditional herbal patent medicine Wuhu San and proposed the concept of “shotgun metabarcoding” (Liu et al., 2021a). This method did not depend on PCR amplification, and could successfully detected all labeled ingredients in the mock samples of Wuhu San prescription, which proved the feasibility of the method, and the applicability of the method was verified with pharmaceutical samples (Liu et al., 2021a). In the same year, the researcher demonstrated that the method was also applicable to the quality control of the traditional herbal patent medicine Qingguo Wan, which has a different dosage form from Wuhu San (Liu et al., 2021b).
Here, this study obtained four barcode regions of ITS2,psbA-trnH , matK , and rbcL through shotgun sequencing and bioinformatics analysis, and analyzed the biological ingredients in SLBZS. Mock samples of SLBZS were used to establish a standard analytical method and verify its feasibility. Subsequently, the method was successfully used in the biological ingredients analysis of pharmaceutical samples to verify the applicability of the method, and the biological ingredients of the pharmaceutical samples were evaluated.