Tracing the Edible and Medicinal Plant Pueraria montana and Its Products in the Marketplace Yields Subspecies Level Distinction Using DNA Barcoding and DNA Metabarcoding.

Gaixia Zhang,Linchun Shi,Qing Zhao,Mei Gao,Jinxin Liu,Qiuling Wang,Weijun Kong

doi:10.3389/fphar.2020.00336

Abstract

Increased public awareness of nutritional and health issues has resulted in the increasing consumption of food and herbal products made from the root of Pueraria montana var. lobata (Willd.) Maesen & S. M. Almeida ex Sanjappa & Predeep (kudzu vine) and P. montana var. thomsonii (Benth.) M. R. Almeida. The famous herbal medicine Yufeng Ningxin, which is used to treat cardiovascular diseases, can be legally produced only using P. montana var. lobata. However, precise identification at the subspecies level is usually challenging when these products’ ingredients lose their morphological characteristics after deep processing. Here, six herbarium specimens, 21 expert-identified original plant samples, 30 raw material samples, 10 food products and 12 herbal products were collected to test the subspecies-level authentication abilities of ITS2 sequences. The results showed that ITS2 sequences can distinguish P. montana var. lobata from P. montana var. thomsonii with stable single nucleotide polymorphism (SNP) sites. A total of 93.3% of raw material samples were consistent with the markings on their labels, but only 50% of Gegen Powder samples were made from P. montana var. lobata. High-quality ITS2 sequences were successfully obtained from nine of the 12 herbal products using Sanger sequencing. Substitution and fungal contamination were detected in 3 herbal products by further DNA metabarcoding, even though thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) tests verified that the products met existing quality standards. This study demonstrated that DNA barcoding is a powerful tool for the identification of P. montana var. lobata and P. montana var. thomsonii at the subspecies level, and we conclude that DNA barcodes can be broadly applied to trace the raw materials of food and herbal products.

Highlights

Pueraria montana is a species in the plant family Fabaceae (Wu et al, 2010)
No single nucleotide polymorphism (SNP) sites were found in the sequence of P. montana var. thomsonii, whereas one SNP site was found in the sequence of P. montana var. lobata
One insertion/deletion variation was found at 29 bp (T/-), and three SNPs at 52 bp (A/G), 201 bp (C/T), and 244 bp (T/G) were found between the sequences of P. montana var. thomsonii and P. montana var. lobata

Summary

Introduction

Pueraria montana is a species in the plant family Fabaceae (Wu et al, 2010). It is native to China and has spread throughout many tropical regions, including those in Europe and the Americas. The root, with or without periderm, of P. montana var. Lobata is used as a type of herbal medicine, called Puerariae lobatae radix ( known as Gegen) in Chinese Thomsonii is popularly used as a food and for extracting starch, sometimes the root can be used as another herbal medicine called Puerariae thomsonii radix ( known as Fenge) in Chinese P. Commission, 2015) and European (E.P. Commission, 2014) pharmacopoeias. P. Commission, 2015) for the puerarin content in Puerariae thomsonii radix (Fenge) is no less than 0.3%, whereas that in Puerariae lobatae radix (Gegen) is no less than 2.4%. The roots of the two plants have different functions, and Puerariae thomsonii radix (Fenge) is often substituted for Puerariae lobatae radix (Gegen) due to ecological cost

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Results

Discussion

Conclusion