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.