Quality control of the traditional Chinese medicine Ruyi jinhuang powder based on high-throughput sequencing and real-time PCR

Qiang Li,Na Zheng,Liran Xu,Hongyu Ma,Ying Sun,Huijun Guo,Hai Peng,Feng Sang

doi:10.1038/s41598-018-26520-3

Abstract

Traditional Chinese medicine (TCM) has been practiced for thousands of years, although concerns about the efficacy, legality, and safety of TCM continue to be raised. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCM preparations. However, chromatography is not able to identify all of the compounds of TCM, particularly those items that are not clearly labeled on the packaging. The present study aimed to establish a supplemental method that better assesses the ingredient components of TCM preparations.We established an effective approach to screen the biological and toxical composition of TCM based on high-throughput sequencing (HTS), as well as fast detection and validation of the toxical species by real-time PCR, based on ITS2 DNA barcoding. Ruyi jinhuang powder (RHP), a classical herbal prescription containing the toxical herb Arisaematis rhizoma, was chosen to test the method. This method could determine whether the Arisaematis Rhizoma had been replaced by Pinellia pedatisecta in the RHP. The results were validated by real-time PCR. 90% compositions of RHP were identified by ITS2 DNA barcoding, suggesting that more DNA barcoding markers are needed for TCM identification. The strategy of high-throughput sequencing has the potential for comprehensive ingredient profiling for TCM preparations. Real-time PCR provides a expeditious metehod for monitoring the safety and legality of TCM preparations.

Highlights

Traditional Chinese medicine (TCM) is an important tool for syndrome differentiation, treatment and the clinical application of modern Chinese Medicine
One reference sample (REF) and one commercial sample of Ruyi jinhuang powder (RHP) were used as the study materials, and the data were acquired through DNA extraction, PCR amplification, library construction and high-throughput sequencing
This study demonstrates the application of high-throughput sequencing combined with real-time PCR to detect the biological ingredients in herbal preparations

Summary

Introduction

Traditional Chinese medicine (TCM) is an important tool for syndrome differentiation, treatment and the clinical application of modern Chinese Medicine. The market of Chinese medicinal materials has been found to contain many counterfeits and substitutes, which can affect the healthy development of traditional Chinese medical industry, harm the safety and effectiveness of TCM, and have an impact on the international reputation of TCM. Included in the British and Chinese pharmacopoeias[9], DNA barcoding technology has become the international standard for TCM identification. DNA barcoding technology can perform high-throughput identification of multiple species in mixed samples, and has been used successfully in the study of species composition of animal and plant communities in mixed samples[10,11]. We attempted to use high-throughput sequencing (HTS) with DNA barcording to determine the biological composition of a traditional herbal preparation, with the specific goal of detecting the mixed components of traditional patent medicines during industrial preparation. The 5.8 S and 28 S rRNA regions, which are located at the two ends of the ITS2 sequence, are conserved enough for primer design[13]

Methods

Results

Conclusion